Multiple Endocrine Neoplasia Type 1

ABSTRACT

Multiple Endocrine Neoplasia Type 1 (MEN1) is a rare autosomal dominantly inherited endocrine tumor predisposition syndrome, caused by mutations in the MEN1 gene. Cardinal manifestations are primary hyperparathyroidism (pHPT), pituitary adenomas (PA), and neuroendocrine tumors (NETs) of the pancreas (PanNET) and duodenum. Other manifestations are NETs of thymus, lung, and stomach, adrenal tumors, and an increased breast cancer risk in women. Malignant NETs are the most important cause of disease-related mortality, mainly NF-PanNETs, gastrinomas and thymus NETs. MEN1 can be diagnosed genetically and a clinical diagnosis in patients with negative comprehensive testing has been debated. Timely recognition of MEN1, referral for genetic testing and swift cascade screening is essential. MEN1-related pHPT (penetrance >95%) is a multiglandular disease and recurrence after initial operation is to be expected. Subtotal parathyroidectomy is the preferred initial operation. Prolactinomas are the most prevalent PA in MEN1, followed by non-functioning (NF) PAs. Treatment and treatment results do not differ from sporadic PAs. Life-time penetrance of duodenopancreatic NETs is >80%. NF-PanNETs are most frequent, followed by gastrinomas and insulinomas. Surgical resection is the mainstay of treatment, and is indicated in non-gastrinoma functional PanNETs and NF-PanNETs >2cm or with progression during follow-up. No consensus exists on the surgical treatment of MEN1-related gastrinoma. MEN1-related dpNETs are currently detected at earlier stages and more indolent small dpNETs are seen. The main challenge is to identify patients at risk for an aggressive disease course. Thymic NETs (2-8%) occur predominantly in males and have a poor prognosis. Bronchopulmonary NETs are more frequent than previously thought, occur in both sexes, and are usually indolent although cases with a deviant progressive course occur. Adrenal tumors are mostly indolent non-functioning adenomas, but adrenocortical carcinomas and pheochromocytomas do occur. Women with MEN1 have an increased (RR 2.8) risk of breast cancer, at a younger age than the general population. Given the complexity of the disease, it is strongly advised that patients, whenever possible, be followed and treated in centers of expertise.

INTRODUCTION

Multiple Endocrine Neoplasia Type 1 (MEN1) is an inherited endocrine tumor predisposition syndrome. The prevalence is estimated at 1 in 20.000 to 1 in 40.000, and is therefore considered a rare disease (1). The syndrome predisposes mutation carriers to develop several endocrine tumors (Figure 1) with a high lifetime incidence of primary hyperparathyroidism (pHPT), pituitary adenomas (PA), and neuroendocrine tumors (NETs) of the pancreas (PanNET) and duodenum (dNET) (2). These tumors are considered the cardinal manifestations of the syndrome. Besides the cardinal manifestations, patients with MEN1 are at a higher risk for developing NETs of the thymus, lung, and stomach. In addition, there is a higher risk for adrenal tumors and a higher risk for developing breast cancer in women (3, 4). Next to these endocrine manifestations, patients are at risk for developing several non-endocrine lesions of the skin and subcutaneous tumors such as lipomas.

Figure1. Manifestations of MEN1

After identification of the causative MEN1 mutation an intensive lifelong surveillance program follows, if possible, starting at childhood because of the high life-time risk for developing tumors (2). This intensive follow-up is aimed at early detection of tumors to enable timely interventions in order to prevent complications and metastases of tumors and thereby preventing premature death and improving the quality of the life of patients. In 2001 the first set of clinical practice guidelines was published by Brandi et al (5). Because of paucity of scientific evidence, these guidelines were mainly based on expert opinion. Although more evidence was available, the updated clinical guidelines of Thakker, et al. which were published in 2012 were also written in the absence of robust scientific evidence (2). However, publishing the clinical practice guidelines led to more structured care of patients, which facilitated studies of the natural course of the disease and the effect of follow-up and treatment strategies. Therefore, in the last two decades large and sometimes nation-wide MEN1 cohort studies were initiated which led to new insights into the course of the disease and knowledge about more optimal follow-up and treatment. However, to date there remains a paucity of prospective data from interventional trials designed specifically for MEN1 patients.

GENETICS

The MEN1 gene (OMIM 613733 (gene) and OMIM 131100 (phenotype)), identified in 1997 (6, 7), consists of 10 exons and is localized on chromosome 11q13. Exon 1, the 5’ region upstream of exon 2 and the 3’ region of exon 10 are non-coding, so there are 9 coding exons (exons 2 through 10). The MEN1 gen encodes the protein menin, a 610 amino-acid nuclear scaffold protein that regulates gene transcription by coordinating chromatin remodeling.

In 2008, Lemos and Thakker published an overview of the 459 different germline mutations reported in the first decade since the discovery of the gene (8). In 2016, Concolino, et al. identified an additional 208 novel germline variants, of which 76 were reported as Variant of Unknown Significance (VUS) (9). Around 40% of all identified mutations are frameshift mutations, 25% nonsense, 20% missense, 10% splice-site, and the remainder 5% are rarer mutations such as in-frame deletions/insertions and partial or whole gene deletions (10). Frameshift, nonsense, and splice-site mutations, which are the majority, are predicted to be loss of function mutations that lead to truncated forms of menin (8). The variants are scattered throughout the MEN1 gene with no evident hotspots, although some mutations are found in apparently unrelated families. This is considered to be a founder effect (11). Sequence analysis of the MEN1 gene will reveal most of the variants. Since 1-2% of the variants are (partial) deletions of the MEN1 gene (8), multiplex ligation-dependent probe amplification (MLPA) or Copy Number Variation (CNV) analysis should also be included in the diagnostic DNA testing.

When a sequence variant is identified in the MEN1 gene this is classified by the genetic laboratory as either benign or likely benign (which is considered a “negative” result i.e., no disease-causing variant is found, clinically similar to no sequence variant identified), as a VUS (uncertain if the variant is disease causing) or as pathogenic or likely pathogenic (which is considered a “positive” result, a disease-causing variant is found). Especially new non-truncating (mostly missense) mutations may be difficult to classify (12).

The approximate prevalence of MEN1 has been reported as 1 in 30,000 individuals with no apparent gender bias. MEN1 follows an autosomal dominant pattern of inheritance with >95% penetrance by age 40–50 years (11). The cardinal clinical manifestations of MEN1 are primary hyperparathyroidism (pHPT), anterior pituitary adenomas (PA), and NET of the duodenum and pancreas, the so called three P’s.

If a patient is diagnosed with MEN1, he or she should be advised to undergo screening to detect manifestations and remain under lifelong surveillance in a center of expertise where care is provided by multidisciplinary teams (MDTs) comprising relevant specialists with experience in the diagnosis and treatment of patients with endocrine tumors (2).

Single center cohorts have identified certain genotypes that are associated with a more aggressive course of the disease (especially related to duodenopancreatic NETs), such as mutation in the JUND (13) or the CHES1 (14)interacting domain or nonsense/frameshift versus missense mutations (15), but since none of these associations have been independently validated, genotype cannot be used to individualize surveillance.

Evaluation of 10 Dutch families suggest genetic anticipation (decreased age of disease onset or an increased disease severity in successive generations), a known phenomenon which to date cannot be explained in autosomal dominant inherited disease genes without trinucleotide repeat expansions (“growing genes”) (16). Somatic mosaicism with subsequent germline inheritance has been described (17).

Function of the MEN1 Gene

MEN1 is considered to act as a tumor suppressor gene which is demonstrated by the identification of inactivating mutations, together with loss of heterozygosity (LOH) in MEN1-related tumors. Biochemical, proteomics, genetics and genomics approaches have identified various potential roles, which converge on the regulation of gene expression. The most consistent findings show that menin connects transcription factors including JUND (OMIM 165162), NFKB (OMIM 164011), and SMAD3 (OMIM 603109) and modulates their activities. In the nucleus, menin acts as a scaffold protein to regulate gene transcription by coordinating chromatin remodeling interacting with chromatin regulatory proteins in the MLL1 /MLL2 complex. Menin is implicated in both histone deacetylase and histone methyltransferase activity (HMT), and via the latter it regulates the expression of cyclin-dependent kinase inhibitor (CDKI) and homeobox domain genes (18, 19). While the MEN1 gene functions as a tumor suppressor gene in MEN1, it has an oncogenic role in sporadic breast cancer cells (18). Some excellent reviews on the function of the MEN1 gene can be found elsewhere (11, 18, 20).

Potential Therapeutic Opportunities (18)

Loss of menin in MEN1-associated tissues, leads to the disruption of anti-proliferative gene expression programs and to the development of endocrine tumors. Restoration of the epigenetic perturbations or correction of the function of aberrantly expressed genes in the absence of menin hold promise for molecular mechanism-based means to treat or prevent MEN1-related tumors. The fate and function of a cell are determined by its gene expression signature. As menin is a transcriptional regulator, MEN1-related tumorigenesis is likely to be the result of aberrant tumor suppressive gene expression due to the loss of menin. Restoration of the expression of menin target genes in MEN1-affected tissues could therefore have therapeutic consequences. This has been shown in a preclinical study in mice, where MEN1 replacement in pituitary tumors of Men1 (+/-) mice led to a decrease in proliferation of the pituitary tumors (21).

DIAGNOSIS AND GENETIC TESTING

Diagnosis of MEN1

Patients with MEN1 are at risk to develop different endocrine and non-endocrine manifestations. The most important of which are (with approximate lifetime prevalence in parentheses):

Cardinal manifestations/Major criteria: pHPT (>95%), duodenopancreatic NETs (dpNETs) (>80%) and anterior pituitary tumors (50–65%)
Minor clinical criteria: adrenal adenomas (11–35%) and bronchopulmonary, thymic, and gastric NETs (20–30%) (22, 23).

Additionally, there might be an association with meningioma (<10%). Cutaneous manifestations such as subcutaneous lipomas (but visceral, pleural, or retroperitoneal lipomas have also been described), facial angiofibromas (22-88%) and collagenomas (0-72%) are also seen (2). Women with MEN1 have a 2-3-fold elevated risk of developing breast cancer (3, 4).

Presently, MEN1 can be diagnosed genetically by identifying the germline heterozygous (likely) pathogenic variant in the MEN1 gene through DNA analysis. According to the guidelines, a diagnosis of MEN1 can also be made on familial grounds in a patient with one of the cardinal MEN1 manifestations and a first-degree family member with MEN1 (2). Additionally, a clinical diagnosis can be made in individuals with two of the three cardinal manifestations (2). However, with modern-day sensitive DNA testing, the value of the clinical criterion in patients with negative DNA testing is under debate. There is mounting evidence that patients who have clinical MEN1, but negative DNA testing have a different clinical course from patients with positive DNA testing (24, 25). The same could also be argued for patients with a familial diagnosis with negative DNA testing for the family mutation, as these may have a sporadically occurring endocrine tumor. This subject is discussed in more detail in the paragraph on genetic heterogeneity.

Patients with MEN1 suffer from high morbidity and a decreased life expectancy. In the present day and age, MEN1-related malignancy is the main MEN1-related cause of death, particularly due to duodenopancreatic and thymic NETs (26, 27). A timely and accurate diagnosis of MEN1 is paramount to improve disease outcomes. This enables early identification of tumor manifestations allowing timely treatment to reduce morbidity and improve survival (28).

It is therefore important for clinicians to consider the diagnosis of MEN1 not only in those patients meeting clinical or familial criteria, but also in patients with a suspicious family or personal medical history, but not meeting clinical or familial diagnostic criteria. In patients presenting with an endocrine tumor within the MEN1 spectrum, taking a family history of MEN1-related tumors is very important. Additionally, a young age at presentation or multifocality of tumors within a single organ may point to a diagnosis of MEN1. The combination of a major and minor criterion or two minor criteria should also raise suspicion of MEN1. In all these cases of suspected MEN1, the patient should be referred to a clinical geneticist or genetic counselor for counseling and consideration of DNA testing. For patients presenting with sporadically occurring endocrine tumors, de Laat, et al. developed and validated a prediction rule to predict the presence of an MEN1 mutation (29). In this model, recurrent pHPT, non-recurrent pHPT, dpNETs, PA, NET of the stomach, lung and thymus, a positive family history for a NET and age, predicted the risk of having MEN1. The authors developed a nonogram for clinical practice, allowing the clinician to calculate the risk of MEN1 in patients suspected of MEN1 with sporadically occurring endocrine tumors (29).

DNA Testing

According to current practice guidelines diagnostic DNA testing for MEN1 should be offered to (2):

all patients fulfilling the diagnostic criteria of a clinical or familial MEN1 diagnosis
all patients with a pHPT under the age of 30 or multiple (synchronous) parathyroid adenomas under the age of 40 or recurrent parathyroid adenomas
patients with a gastrin-producing NET (irrespective the age of presentation)
patients with multiple PanNETs (irrespective the age of presentation)
patients with two different minor criteria
a patient with an MEN1-related tumor with a positive family history of MEN1-related tumors

Once MEN1 is diagnosed in the proband, genetic counseling and DNA testing should be offered to family members, preferably by means of cascade screening. Data from the DutchMEN Study Group (DMSG) emphasize the importance of timely genetic testing of family members and prompt clinical screening according to MEN1 guidelines. In a study determining lag time between MEN1 diagnosis in index cases and their non-index family members, they found a median lag time of 3.5 years (range 0-30) years, in which clinically significant manifestations occurred in the non-index family members with MEN1 (30). Genetic testing in asymptomatic family members of MEN1 patients is called pre-symptomatic or predictive genetic testing and involves testing the at-risk family members for the familial MEN1 mutation. This is single-site testing, and outcome is whether the family mutation is present or absent in this particular family member.

Genetic Heterogeneity

Some studies have reported that between 5% and 10% of patient who fulfill the clinical criteria for MEN1 will not harbor mutations in the coding region or adjacent splice sites. In some of these patients a (likely) pathogenic variant can be found in the CDKN1A (OMIM 600778 ), CDKN1B (OMIM 116899 ), CDKN2B (OMIM 600431 ) or CDKN2Cgene (OMIM 603369 ). Mutations in CDKN1B are the cause of the MEN4 syndrome, the latest of the MEN syndromes and most rare, with <50 cases reported in the literature to date (31-33). Rather than being a separate phenotype, (likely) pathogenic variants in these genes are more likely to cause a MEN1 phenotype, with pHPT, PA and gastroenteropancreatic NETs as the main features, and are best met with the same guidelines for surveillance, until more is known about the phenotype of this rare syndrome.

Recent data have shown that patients with a clinical diagnosis of MEN1, in whom no (likely) pathogenic variant in the MEN1 gene can be found (genotype-negative MEN1/GN-MEN1), and who do not have another known germline mutation, have a different phenotype and clinical course compared to mutation positive patients (24, 25). Genotype-negative patients develop MEN1 manifestations at higher age, rarely develop a third main MEN1 manifestation, and have a life expectancy comparable with the general population (24, 25).

Additionally, regarding the individual manifestations, it seems that GN-MEN1 patients have less recurrent or multigland pHPT, less multifocal PanNETs, and more somatotrophinomas and less prolactinomas compared to genotype-positive patients (25). Most patients with GN-MEN1 present with the combination of pHPT and PA, followed by pHPT/dpNET and dpNET/PA (25). The apparent differences in clinical course suggest that GN-MEN1 patients do not have true MEN1, but another MEN1-like syndrome or sporadic co-incidence of two NETs (2, 33). In these patients there is usually a negative family history for MEN1-related disease. Although not specified in the current guidelines, these patients may benefit from a separate classification with alternative surveillance recommendations based on the clinical picture, as has been suggested by Pieterman, et al. (25). Important baseline considerations for an alternative surveillance are genetic counseling, comprehensive genetic testing based on the personal and family history, and baseline screening to identify any unrecognized manifestations. In these patients, there is generally no cause for surveillance of the first-degree relatives, although these decisions should be individualized and discussed in multidisciplinary teams.

GN-MEN1 patients with a positive family history of clinical MEN1 or a foregut NET and those presenting with all three main MEN1-related tumors, should be followed according to MEN1 guidelines as should their relatives. In these patients, a “false-negative result” of DNA testing should also be considered. This may either be because deletion/duplication analyses are not performed, a sequence variant exists outside of the assayed region, or polymerase chain reaction primer selection led to selective amplification of wild-type DNA (25). Additionally, somatic mosaicism or alternative mechanisms of MEN1 gene silencing could lead to inactivation of normal menin (25).

Depending on the presenting clinical picture and the family history, other hereditary syndromes causing endocrine tumors should also be considered.

If pHPT is the primary phenotype, other genes associated with hereditary pHPT are for example CDC73, CASR and RET (MEN2). Germline CDC73 (formerly HRPT2) (OMIM 607393) analysis is recommended in individuals with (suspected) Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, familial isolated pHPT, atypical or malignant parathyroid histology, and young individuals with pHPT. These criteria would increase germline CDC73 mutation detection, enabling optimal clinical management of pHPT as well as genetic counseling and surveillance for family members at risk for developing CDC73-related disorders(34).

If PAs are the primary phenotype, mutations in the AIP gene (OMIM 605555) should be considered as these can cause (familial) pituitary adenomas. Predictors of a genetic cause of sporadic pituitary adenomas are young age of diagnosis, and also in AIP pathogenic variants there is an association with gigantism and macroadenomas (35).

PanNETs can also be seen in neurofibromatosis type 1 (NF1), Von Hippel-Lindau (VHL), and Tuberous Sclerosis Complex (TSC).

Pretest Counseling

Before DNA testing, pretest counseling is of utmost importance. The patient should be informed by genetic counseling about all aspects (medical, psychological, social, and familial implications) of the possible outcome of genetic testing. This should lead to an individual decision whether or not to opt for DNA testing. In case of DNA testing in minors the counseling should be offered to the parents, and include the minor if possible (which is obligatory over the age of 12 in the country where the authors practice (the Netherlands) to obtain informed consent for testing.

In case of diagnostic testing the patient must be informed about the possible outcomes of the DNA test (finding an (likely)pathogenic variant, finding a VUS, and not finding (likely) pathogenic variants) and the implications of these findings for the patient and family members.

In diagnostic DNA testing the MEN1 gene should be analyzed, for which Sanger sequencing can be used, or Next Generation Sequencing (NGS) techniques. To exclude deletions MLPA or CNV analysis must be performed. As mentioned earlier there is genetic heterogeneity and panel DNA diagnostics can be considered. In particular CDKN1A, CDKN1B, CDKN2B, CDKN2C, CDC73 and AIP can be added to the panel using NGS techniques, also to be completed with CNV analysis, depending on the clinical picture. In case of panel testing, the patient must be prepared for the possible findings in the different genes, differentiation of the consequences, and implications of these findings.

In case of testing for a familial (likely) pathogenic variant (so-called presymptomatic or predictive DNA testing) presence or absence of the familial mutation can be ascertained, but the differences in expression of the MEN1 syndrome, both within and between families must be emphasized.

In case of future pregnancy the possible options like invasive prenatal diagnostics and Preimplantation Genetic Diagnosis (PGD) should be discussed so the prospective parents can make an informed decision about the desired pregnancy.

Periodical Screening

The identification of an MEN1 mutation in patients and family members at risk is followed by the advice to remain under lifelong surveillance, with at least annual clinic visits including history, physical examination, biochemical screening, and radiological screening at specific intervals (2). This should preferably be carried out in centers of expertise with a dedicated multidisciplinary team well versed in management of patients with MEN1. In MEN1 there are no prophylactic treatments, so the goal of this screening & surveillance program is early detection of MEN1-related tumors to minimize morbidity by hormonal hypersecretion and to prevent malignant NETs by timely intervention. In the absence of known genotype-phenotype correlations and with a heterogeneous clinical course, even within families, the specific mutation or family history cannot solely guide the surveillance program. In the following sections screening and surveillance is discussed within each manifestation.

Screening at the Pediatric Age

The current clinical guidelines for MEN1 suggest starting clinical and biochemical screening at the age of 5 years (2), which is based on the earliest reported case of a patients with a clinical MEN1 manifestation (36). For radiologic screening in asymptomatic children, pituitary imaging is suggested from age 5 years onward (every 3 years), with abdominal imaging starting at age 10 years (every 1-3 years) and thoracic imaging starting at age 15 years (every 1-2 years) (2). However, this intensive surveillance at the pediatric age has been questioned by some groups who suggest postponing routine screening of asymptomatic patients until ages 15 or 16 years while counseling parents about typical clinical signs of MEN1 manifestations and contacting providers if they occur (37).

PRIMARY HYPERPARATHYROIDISM

Primary hyperparathyroidism (pHPT) (Figure 1) is one of the cardinal manifestations of MEN1 and has an almost complete lifetime penetrance (24, 38). It is often the first clinical manifestation of the disease and biochemical (asymptomatic) pHPT can be diagnosed several years before symptoms arise. The reported mean age of pHPT diagnosis in published MEN1 cohorts is in the fourth decade of life (39-45), with wide ranges. When interpreting these mean ages at diagnosis it is important to realize that these cohorts often span multiple decades, are made up of both index cases and family members, and contain patients who did and did not follow prospective screening programs. Recent studies reporting on MEN1 at the pediatric age show that in a screened population at least half of the pediatric patients already have primary hyperparathyroidism, although rarely seen before the age of 10 (46-49). In most cases, patients diagnosed at a pediatric age are asymptomatic and the diagnosis is made biochemically by screening (46-49). Clinical and symptomatic pHPT is usually seen in the third decade of life.

Primary hyperparathyroidism in MEN1 is a multiglandular disease, affecting all parathyroid glands, although often asymmetrically and asynchronously. Parathyroid tumors in adults with MEN1 usually represent mono- or oligoclonal proliferations that probably arise independently in each parathyroid gland (50). Tumorigenesis is initiated when the remaining normal allele of the MEN1 gene is lost (the second hit), and as this cumulative chance increases with age, normal parathyroid tissue is less often seen with increasing age (51). Supernumerary glands (that is, more than four parathyroid glands) are frequently seen in MEN1, with reported ranges between 12-30% (52). Parathyroid glands at ectopic locations are also not uncommon in MEN1, especially in the thymus.

The diagnosis of primary hyperparathyroidism can be made when there is hypercalcemia in combination with an elevated or inadequately normal parathyroid hormone (PTH). In patients with MEN1 who follow a prospective screening program from an early age, the diagnosis is often made while they are still asymptomatic. Classic objective symptoms of pHPT include polyuria and polydipsia, gastro-intestinal complaints (nausea, abdominal pain, constipation, pancreatitis), (symptomatic) urolithiasis, and decreased bone mineral density (BMD) which can lead to pathological fractures. Non-specific symptoms occurring in pHPT are fatigue, musculoskeletal complaints, neuropsychiatric symptoms such as anxiety, depression, concentration disturbance and sleep-disturbances, and a general decrease in quality of life.

The diagnosis of pHPT in patients with known MEN1 or from a known MEN1 kindred is straightforward. However, pHPT can also be the first clinical clue to an MEN1 diagnosis in a patient or family without prior MEN1 diagnosis or suspected history. The prevalence of pHPT in the general population can be up to 1% (53, 54) and among cohorts of patients with pHPT, depending on the characteristics, the incidence of MEN1 is 1-18% (2). Considering MEN1 in patients presenting with pHPT is extremely important, because the diagnosis alters the management and prognosis of pHPT, allows screening and surveillance for other endocrine tumors associated with MEN1, and allows for cascade screening within the family to identify MEN1 germline mutations carriers. Important clues to an MEN1 diagnosis in a patient presenting with pHPT are young age of onset, a family history of pHPT or other MEN1-related tumors, a personal history of other MEN1-related tumors, and multiglandular disease or persistent/recurrent pHPT (29). Recurrent pHPT is one of the strongest predictors for the presence of an MEN1 mutation (29). Compared to sporadic pHPT, patients with MEN1-related pHPT present at an earlier age, have an almost equal gender distribution compared to female predominance in sporadic pHPT, and present with lower levels of calcium and PTH (55, 56). Even though they have biochemically milder disease, BMD seems to be lower in patients with MEN1-related pHPT and renal involvement similar compared to patient with sporadic pHPT, which may reflect longer standing disease (55). MEN1-related pHPT is a multiglandular disease, as already stipulated, while sporadic pHPT is predominantly caused by single-gland adenomas (56, 57). This also affects recurrence rates which are much higher in MEN1-related pHPT (56, 57). The American Association of Endocrine Surgeons (AAES) guidelines advise genetic counseling for patients younger than 40 years with pHPT and multiglandular disease and to consider this for those with a family history or syndromic manifestations (57). The European guidelines slightly differ suggesting genetic testing for MEN1 in patients with pHPT before the age of 40, multiglandular disease, or persistent/recurrent pHPT (58).

When comparing, several studies show that patients with MEN1-related pHPT have lower BMD compared to patients with sporadic pHPT (55, 59, 60), although a Chinese study found no significant difference (61). In patients with MEN1-related pHPT, decreased BMD is frequently seen and already present at a young age (62-64). When measured the 1/3 distal radius seems most affected, so including this location in dual-energy X-ray absorptiometry (DEXA) should be considered in patients with MEN1 (62, 64). Parathyroidectomy improves BMD (59, 65), although in one small study improvement was less for patients with MEN1 compared to patients with sporadic pHPT (59). A factor contributing to the earlier and more severe bone involvement in MEN1-related pHPT may be the early-onset of the disease thereby also influencing peak bone formation. In addition, other MEN1-related diseases may also contribute to bone loss such as pituitary insufficiency caused by pituitary adenomas or their treatment, hypercortisolism (although infrequent in MEN1), and gastro-intestinal surgery (66).

Urolithiasis is also frequently seen and at a young age in patients MEN1-related pHPT (55, 62, 64). In addition, a recent study showed that patients with MEN1 age 20-59 had a higher prevalence of chronic kidney disease stage 3 compared to the general US population (67).

It is therefore important to perform Dual-energy X-ray absorptiometry (DEXA) to assess BMD as well as a renal ultrasound and 24-hour urine for calcium excretion to asses risk of urolithiasis in patients with MEN1 diagnosed with pHPT. And if initial observation is chosen, DEXA should be repeated every 2 years (68).

In patients with MEN1 and pHPT, the interplay with Zollinger-Ellison Syndrome (ZES; increased gastric acid section due to gastrinomas) is also relevant, as calcium can increase gastrin levels. In a study among 84 patients with MEN1-pHPT and ZES, successful parathyroidectomy resulted in biochemical cure of ZES without any resection of duodenal or pancreatic NETs in 20% of the patients (69). In a recent perspective paper Hackeng and colleagues propose a parathyroid-gut axis arguing that hypercalcemia may promote the gastrin-cell hyperplasia to neoplasia sequence through the calcium-sensing receptor (70). The reverse, a more severe form of pHPT among patients with MEN1-ZES has also been suggested, because in the aforementioned study of 84 patients with MEN1-pHTP and ZES, patients had a higher frequency of urolithiasis at presentation, higher serum PTH, and higher recurrences rates after initial subtotal parathyroidectomy compared to the literature (69).

Parathyroidectomy

The treatment of hyperparathyroidism in MEN1 is surgical. Intervention is aimed at achieving eucalcemia for as long as possible, while preventing permanent hypoparathyroidism and facilitating potential subsequent surgery.

The optimal timing of the initial operation is still a matter of debate, especially in (asymptomatic) children and young adults. The guidelines for the management of asymptomatic pHPT recommend surgical intervention in case of significant hypercalcemia (1 mg/dL or 0.25 mmol/L above the upper limit of normal), skeletal abnormalities (a T-score of < -2.5 at the Lumbar Spine, Total Hip, Femoral Neck or 1/3 Distal Radius or a vertebral fracture), risk of renal complications (creatinine clearance below 60 ml/min, 24-h urine calcium excretion of >400 mg/d (>10 mmol/L)), the presence of nephrolithiasis/nephrocalcinosis, or age below 50 (68). However, these guidelines are not intended for patients with MEN1 and most patients with MEN1 will meet the age-criterion regardless of other values. In patients with MEN1 surgery is indicated in case of symptoms, significant hypercalcemia, and renal or skeletal complications. In addition, concomitant gastrinoma may also provide an indication for surgical intervention of pHPT. For patients not meeting any of these criteria, there is no evidence to determine timing of surgery. Arguments have been made in favor of observation to avoid the risk of symptomatic hypoparathyroidism, multiple operations, and by allowing the disease to progress a little bit more, making the glands more easily identifiable upon intervention. However, on the other hand, data showing early bone and renal complications have made others suggest and prefer early intervention to prevent downstream disabilities (71).

For initial parathyroidectomy in patients with MEN1 there are theoretically four different strategies: focused parathyroidectomy (removing a single affected parathyroid gland), unilateral clearance (resection of all parathyroid tissue on one side, including unilateral cervical thymectomy), subtotal parathyroidectomy with concomitant cervical thymectomy, or total parathyroidectomy, cervical thymectomy and immediate auto-transplantation of parathyroid tissue (usually to the non-dominant forearm).

The initial operation recommended by most experts and guidelines is a bilateral cervical exploration, identifying all four parathyroid glands and performing a subtotal parathyroidectomy (leaving a vascularized remnant about 1.5-2 times the size of a normal gland) with concomitant cervical thymectomy (2, 57, 58, 71-73). The latter serves the dual purpose of removing any ectopic/supranumerary parathyroid glands and potentially decreases the risk of subsequent development of thymic NETs. This approach offers the best balance between persistence (persisting pHPT after operation or recurrence within 6 months after operation) and recurrence (recurrent pHPT 6 months or more after the operation preceded by a eucalcemic period) on the one hand and permanent (lasting >6 months after the operation) hypoparathyroidism on the other hand. Persistence is infrequent in subtotal (0-22%) and total parathyroidectomy (0-19%), but rates range from 0-53% in less than subtotal parathyroidectomy(58, 72). Recurrence rates are also significantly higher after less than subtotal parathyroidectomy (0-100%) compared to subtotal (0-65%) or total parathyroidectomy (0-56%) (58, 72) and occur earlier (74). Permanent hypoparathyroidism on the other hand is rarely seen after less than subtotal parathyroidectomy. When comparing subtotal with total parathyroidectomy, hypoparathyroidism is significantly more frequent after total parathyroidectomy (RR 1.61 (95%CI 1.12-2.31) (72).

Pre-operative imaging plays a limited role at initial parathyroidectomy in patients with MEN1, because the recommended initial operation always constitutes bilateral neck exploration( 58). In addition, data has shown that pre-operative imaging (consisting of neck ultrasound and sestamibi scan as first line and parathyroid computed tomography (CT) or magnetic resonance imaging (MRI) as second line) only identified 68% of the largest glands pre-operatively (75). Pre-operative imaging may have some use for identifying ectopic glands (7% of ectopic glands were identified by pre-operative imaging in one series) and for identifying concomitant thyroid abnormalities that need attention (71, 75). Similar, intra-operative PTH monitoring seems of little value during the initial parathyroidectomy (76).

Recently, several groups have advocated unilateral clearance as an initial operation, especially for young patients with MEN1 (74, 77-79). The rationale behind this approach is to provide several years of eucalcemia during acquisition of peak bone mass, while preventing hypoparathyroidism and allowing subsequent reoperations to be performed in a non-operated neck (the contra-lateral side). A prerequisite for this strategy is that pre-operative imaging concordantly shows unilateral disease. Intra-operative PTH monitoring should be used to ensure there is an adequate drop in PTH after the resection. Although persistence rates between 10-15% after unilateral clearance or single-gland excision have been reported by these groups (74, 77), others state that less than subtotal parathyroidectomy has an unacceptable failure rate (69% in one study) (80). Several remarks must be made when using retrospective studies to evaluate this strategy. The first being that intentional less than subtotal resection is a different entity from an intended subtotal or total resection in which not all glands were identified (78). Secondly, true unilateral clearance in which all parathyroid tissue on one side of the neck is removed including unilateral cervical thymectomy is a very different operative strategy from minimal invasive parathyroidectomy/single gland excision and in retrospective studies these are often lumped together under “less than subtotal” resections. Thirdly, the success of such an approach is dependent on the sensitivity of pre-operative imaging and in most retrospective studies, more sensitive imaging modalities such as 18F-fluorcholine positron emission tomography (PET)/CT have not been used. Finally, since in MEN1 inherently all parathyroids are affected, although asynchronously, such an approach may be more successful in younger patients, where there may still be normal parathyroid glands (51). Currently this approach is controversial. Therefore, prospective data are needed to determine if and when unilateral clearance can benefit patients with MEN1 at the time of their initial parathyroidectomy.

Currently, subtotal parathyroidectomy remains the initial procedure of choice, but total parathyroidectomy or unilateral clearance can be considered depending on individual circumstances. Single gland excision is generally not recommended.

After initial subtotal parathyroidectomy, the 10-year recurrence rate is approximately 50% (2). Reoperation is therefore a frequent necessity in patients with MEN1. Recurrence can be caused by parathyroid glands missed during the initial operation, parathyroid glands intentionally left in situ, growth of the remnant of a partially resected gland, supranumerary and/or ectopic glands, and hyperplasia of autotransplanted parathyroid tissue. As reoperations are more complex and have a higher risk of complications (12% not including hypoparathyroidism in one study of reoperative parathyroidectomy in MEN1 (81)), the timing of the reoperation is individualized and patients with mild biochemical recurrence are usually initially observed. When reoperation is indicated, careful examination of the operation notes and pathology reports of previous procedures, if available, is very important. In contrast to the initial surgery, pre-operative imaging is essential for surgical planning in reoperations. First-line imaging studies are neck ultrasound and Tc99m-sestamibi scan, although this may not show all enlarged glands. Second-line imaging studies are 4-dimentional CT or MRI and PET (18F-fluorocholine or 11C-methionine) (82, 83). In a small study 18F-fluorocholine PET-CT has also shown to be of added value in MEN1-related pHPT (84). If first- and second-line studies are inconclusive more invasive localization studies can be considered such as arteriography, venous sampling, and neck ultrasound with fine needle aspiration and PTH measurement (83). The exact operative strategy (bilateral or unilateral neck exploration or focused resection) is individualized based on previous operation(s) and results of preoperative imaging. If the thymus was not removed during the initial operation, its removal is recommended at reoperation (81). Intra-operative PTH monitoring is valuable for the reoperative setting in MEN1 as it can inform when the exploration can be ended (71, 81).

As a consequence of the extended initial operation necessary, as well as frequent reoperation, life-time risk of postoperative hypoparathyroidism is relatively high for patients with MEN1. Transient hypoparathyroidism, defined as lasting less than six months after parathyroidectomy, may be seen in more than 50% of patients and its absence after subtotal parathyroidectomy may even be associated with recurrence (85, 86). Rates of permanent hypoparathyroidism are dependent on the procedure performed and vary greatly between series. It is important to realize that, unless patients are truly aparathyroid, recovery of parathyroid function can occur after 6 months up to several years, and permanent hypoparathyroidism may therefore be more aptly termed “prolonged” hypoparathyroidism (86). To prevent hypoparathyroidism immediate autotransplantation is used when it is suspected that all parathyroid glands are resected or when there is concern about parathyroid tissue viability in situ (57). Cryopreservation with delayed autotransplantation can also be used as a rescue from permanent hypoparathyroidism, but is not available everywhere and its use has been under debate (57).

Non-Surgical Interventions

For those patients who require intervention, but who are not surgical candidates, cinacalcet, an allosteric agonist of the calcium receptor, can be used. It has been shown to reduce/normalize calcium and PTH in small studies in patients with MEN1, although it has no effect on bone and renal complications(87-89). Cinacalcet should be used with great caution in children, as a death from acute hypocalcemia has been reported in a 14-year-old (90). Another alternative may be ethanol ablation of enlarged parathyroid glands. A study from the Mayo Clinic reported results from 37 patients who had an average of 2.2 treatments and a mean duration of eucalcemia of 25 months. Complications were hypocalcemia in 8%, hoarseness in 5%, and cough in 1% (91).

Parathyroid Carcinoma

Parathyroid carcinoma is a very rare endocrine malignancy seen in <1% of all patients with pHPT (92). It is likewise very rare in patients with MEN1, with only 21 reported cases in the literature (based on a review published in 2020) (93). In three large series from The University of Texas MD Anderson Cancer Center, the Mayo Clinic, and The Peking Union Medical College Hospital the prevalence of parathyroid carcinomas was 2/242 (0.8%), 1/348 (0.3%) and 1/153 (0.7%) respectively and the prevalence of atypical parathyroid neoplasm was 1/242 (0.4%), 0, and 2/153 (1.3%) respectively (93-95).

Conclusion

In conclusion, pHPT in MEN1 has an almost complete penetrance and is responsible for most MEN1-related surgeries. It is a multiglandular disease and recurrence after initial operation is to be expected. End-organ damage (bone, renal) can occur early and in asymptomatic patients and should be systematically looked for. Recognizing MEN1 in a patient presenting with apparently sporadic pHPT has important consequences for both the patient and his/her family. Surgical decision making is complex both for initial and reoperations and patients with MEN1 should whenever possible be treated in centers of expertise by a high-volume endocrine surgeon. Treatment decisions are made by multidisciplinary teams in shared decision making with the patient taking into account not only medical information but also the patient’s individual situation, such as but not limited to, ability to adhere to follow-up and insurance issues.

PITUITARY ADENOMAS

In 1903, the first description of a case with MEN1 was published by Erdheim. The necropsy report of a patient with acromegaly revealed a pituitary adenoma and enlarged parathyroid glands (96). Pituitary adenomas (PAs) are one of the three cardinal features associated with MEN1 and part of the so-called ‘three Ps’ (Figure 1). PAs are in general benign lesions and do not seem to negatively affect survival in patients with MEN1(26), although cases of mortality due to PAs have been reported (27). However, they can cause significant morbidity due to mass effect on the optic chiasm or hormone secretion leading to functional symptoms or hormone deficiency.

As in other main manifestations of MEN1, loss of heterozygosity (LOH) at the MEN1 locus has been demonstrated in pituitary adenomas in patients with MEN1, confirming the role of MEN1 in the pathogenesis of these tumors (97-100). However, in contrast to PanNETs, the role of MEN1/menin in tumorigenesis of sporadic PAs seems to be limited. Although initially, before the identification of the MEN1 gene, 19-33% of sporadic PAs showed allelic loss on chromosome 11 (101, 102), subsequent studies investigating LOH, somatic mutations, and messenger mRNA expression found limited involvement of MEN1 in sporadic PAs (103-107).

As the prevalence of clinically relevant PAs is 68-98/100,000 in the general population and in general <3% of patients with a PA will have MEN1, the question is when to think of MEN1 in a patient presenting with a PA (2, 35). Obviously, MEN1 should be considered in a patient with a family history of MEN1-related tumors or presenting with other MEN1-related tumors. For patients with apparently sporadic PAs (no suspicious family history or syndromic features), a recent systematic review has shown that MEN1 mutation analysis is recommended in patients ≤ 30 years, although this was a weak recommendation based on low quality of evidence (35).

Characteristics of Pituitary Adenoma in MEN1

From the earliest descriptions of MEN1 in the 1950s PAs have been recognized as one of the main characteristics of the syndrome. However, since the original description of MEN1, the clinical picture of MEN1-related PAs has changed. In a summary of the first 85 reported cases of MEN1 (many of which were autopsy cases), Ballard found a very high prevalence of 65% of PA, with 42% being chromophobe adenomas and more than one in four being acromegaly/eosinophilic adenoma (108). With the discovery of prolactin, it was soon realized that in fact prolactinomas were the most frequently occurring PA in patients with MEN1. The discovery of the MEN1 gene in 1997 (6, 7), and more advanced genetic testing techniques such as NGS and MLPA, have allowed better identification of patients as having MEN1. This has led to the recognition that patients with a clinical diagnosis of MEN1 because they have two out of the three main MEN1-related tumors, but negative mutation analysis, have a different clinical course than mutation positive patients and arguably do not have true MEN1, but rather an MEN1-like syndrome or a co-occurrence of two sporadic tumors (24, 25). Most patients in this group have a clinical MEN1 diagnosis based on the combination PA and pHPT. As these patients may have been included in older MEN1 cohorts, before the widespread availability of genetic testing, and these patients seem to have macro-adenomas and somatotrophinomas more often, this can be one of the reasons of the changing clinical picture of MEN1-related PAs. Additionally, imaging techniques have markedly improved over the last decades and guidelines have been developed for the screening and surveillance of patients with MEN1 including regular pituitary imaging and biochemical screening using Insulin-like Growth Factor-1 (IGF-1) and prolactin (2, 5). All this has led to earlier identification of PAs in patients with MEN1 and more frequent detection of (small) non-functioning PA (NFPAs).

After the discovery of the MEN1 gene (1997), six cohorts of MEN1-PA have been published, the first two by the French multicenter Groupe d’étude des Tumeurs Endocrines (GTE) in 2002 (109) and 2008 (110), in 2015 the DutchMEN Study Group (DMSG) published the results from their national population-based database (111), which was followed by two single-center cohort from China (112) and the Mayo Clinic respectively (113). Recently, the GTE have published an update to their previous study, only including patients diagnosed since January 1^st 2000 (114).

As in sporadic PAs, PAs in patients with MEN1 show a slight female predominance (52-69%) (109, 111-114). With exception of the Chinese cohort, where the mean age of diagnosis was 54 years (112), the mean/median age of diagnosis of MEN1-related PAs is in the fourth decade. Lifetime prevalence of a PA in patients with MEN1 is 49-58% (38, 111).

Although not as frequent as pHPT, PAs are often the first clinical manifestation of the MEN1 syndrome. In the Dutch cohort, in 29% of the patients with a PA, it was the first manifestation (111). In the most recent GTE cohort, 88/202 patients with a PA were the index case in their family and in 84% of these patients a PA was (one of) the first manifestation(s) (114).

Prolactinomas are the most prevalent PA in patients with MEN1 and account for 30-80% of adenomas diagnosed in patients with clinically evident disease (42, 43, 109, 111-114). Second most prevalent are non-functioning PA comprising 36-48% in the most recent cohorts (111-114). Other functioning PAs are seen in <10%, and are in decreasing order of prevalence somatotropinomas, ACTH-producing adenomas (Cushing’s disease), and TSHomas and gonadotropinomas (the latter two being equally rare) (109, 111-114). Co-secreting tumors are seen in less than 10% (109, 111-114).

Multifocal PAs are rare in MEN1, and are found in 1.5% in the most recent GTE cohort (114) and in 4% in the 2008 GTE cohort of surgically resected MEN1-related PAs (110). In this latter cohort the prevalence of multifocal tumors was compared to that in non-MEN1 resected PAs and was found to be significantly larger. Additionally, MEN1-related resected PAs were more often plurihormonal on immunostaining (110).

Signs and symptoms in MEN1-related PAs (Table 1) are not different from those observed in sporadic PAs and are caused by size effects (chiasm compression, compression of nerves in the cavernous sinus, hypopituitarism) and effects of hormonal hypersecretion in functioning tumors.

Table 1. Signs and Symptoms of Pituitary Adenomas in MEN1
Related to tumor size/ growth	headache, visual field defects (usually bitemporal hemianopsia), diplopia, hypopituitarism
Prolactinoma	females: amenorrhea, galactorrhea, infertility males: hypogonadism, impotence, lack of libido, galactorrhea (rare), infertility
Somatotrophinoma	Acromegaly: local overgrowth of bone (most often mandible, skull), soft tissue growth (acral enlargement, coarse facial features), hyperhidrosis, fatigue, hyperglycemia, hypertension, sleep apnea, skin tags, hypogonadism.
Corticotrophinoma	Cushing syndrome: central obesity, hypertension, hyperglycemia, gonadal dysfunction, moon facies, plethora, osteoporosis, proximal muscle weakness, psychological disturbance, wide purple striae, easy bruising
Thyrotropinoma	heat intolerance, unintentional weight loss, anxiety, tremor, palpitations, frequent bowel movements
Gonadotropinoma	hypogonadism, ovarian hyperstimulation in women
Pediatric specific	delayed or halted pubertal development, primary amenorrhea (females), accelerated linear growth, poor growth velocity, decline in school performance

Presently, most non-functioning PAs in MEN1 are microadenomas detected by prospective screening. These micro-adenomas show indolent behavior during follow-up. In the Dutch series after a median follow-up of 5.3 yrs, 9.7% showed minimal tumor growth which was without clinical significance in all and none progressed to macro-adenoma (111). In the Mayo Clinic cohort, in those with asymptomatic non-functioning PA (size not specified) progression to surgery was seen only in 1.7/100yr (113). In the most recent GTE cohort, after a median follow-up of 2 years (IQR 0-4), progression in Hardy classification was only seen in 1 out of 63 patients with a non-functioning micro-adenoma (2%) (114). In the Chinese cohort, of the 19 patients with non-functioning micro-adenomas, no progression to macro-adenomas was seen during a median follow-up of 3 years (112).

Prolactinomas are also mostly micro-adenoma, while 30-38% are macro-adenomas (111, 113, 114). As in sporadic PAs, GH-secreting tumors are more often macro-adenomas and ACTH-secreting tumors are generally microadenomas.

Although the youngest patient with a clinical manifestation of MEN1 described in the literature is a 5-year-old boy with gigantism and a lactosomatotroph macro-adenoma (36), PAs are rare in patients with MEN1 below the age of 10 (37, 46-49). However, pediatric cohorts show that in children and adolescents who have clinical manifestations of MEN1 up to 1/3 have PAs (37, 47-49). As in adults, most PAs the pediatric and adolescent age are prolactinomas followed by non-functioning PAs and more rarely GH or ACTH producing tumors (37, 46-49, 115). In the two largest pediatric cohorts, PAs were symptomatic in 50% of the cases and were macro-adenomas in 33-51% (47, 48).

Treatment

The treatment of MEN1-related pituitary adenomas follows the same strategy as sporadic pituitary adenomas. Management is aimed at tumor reduction, normalization of hormone secretion, and preservation of pituitary function.

Dopamine agonists are the first line of treatment for patients with prolactinoma (116), in which cabergoline has proven to be most effective at restoring normal prolactin concentrations and achieving tumor shrinkage than other dopamine agonists. With regards to adverse effects, cabergoline shows fewer side effects than bromocriptine. In case of treatment resistance, or treatment intolerance, surgery or radiotherapy are considered as second-line treatment options (116).

In Cushing’s disease(117) and acromegaly(118) surgery is the first treatment option. In addition, non-functioning PAs with mass effect or rapid progressive adenomas will also benefit from surgery.

MEN1-related functional PAs were initially considered more resistant to medical treatment than those with sporadic disease (109). However, the latest reports do not confirm this (111, 114) and treatment results seem to be in line with what is reported in sporadic PAs. The latter cohorts consist of a population with meticulous surveillance and therefore PAs are detected in an early phase (111, 114).

Pituitary Carcinoma

Pituitary carcinoma is extremely rare, and this is equally so in patients with MEN1. Although at higher risk for PA than the general population, there does not seem to be an increased risk of pituitary carcinoma. Single cases of malignant, metastatic prolactinoma (119, 120), gonadotropinoma (121), thyrotropin secreting adenoma (122), and non-functioning PA (123) have been reported.

Surveillance for Pituitary Adenoma

Current guidelines recommend examination by MRI of the pituitary gland every three years from the age of five years, and an annual blood test of IGF-I and prolactin concentrations, together with a clinical assessment (2). The young starting age – which was based on a single case-report – has been disputed, given that PAs are rarely seen before the age of 10.

The aim of surveillance imaging is to detect the PAs in an early phase before clinical symptoms become apparent. In general, surveillance leads to detection of smaller non-functioning PAs (111, 113, 114). However, early diagnosis by surveillance is not associated with smaller prolactinomas, but treatment is required less frequently and a longer safe observation period can be conducted (111). There are currently no specific recommendations for the follow-up of MEN1-related (micro-)adenomas under observation, on medical treatment or after surgical resection.

DUODENOPANCREATIC NEUROENDOCRINE TUMORS AND GASTRIC NETS

General

Duodenopancreatic neuroendocrine tumors (dpNETs) (Figure 1) are one of the cardinal features of MEN1 and highly penetrant, with a prevalence of over 80% at the age of 80 in recent cohorts (24, 38, 124). Malignant dpNETs are the most important cause of MEN1-related death (26, 125).

Duodenopancreatic NETs in MEN1 can secrete hormones that produce a clinical syndrome or be functionally silent (non-functioning, NF). Due to improved imaging techniques in the past decades, including endoscopic ultrasound (EUS) and somatostatin receptor (SSTR) imaging, non-functioning pancreatic NETs (NF-PanNETs) are now recognized as the most frequent type of dpNET in patients with MEN1. Of the functional dpNETs, gastrinomas are the most frequent, seen in approximately 30% of patients with dpNETS. In patients with MEN1, gastrinomas are almost exclusively of duodenal origin (126). Insulinomas (pancreatic in origin) are the second most common functional dpNET and occur in approximately 10-15% of patients with MEN1. More rare functional PanNETs such as glucagonomas, vipomas, somatostatinomas (127) or even rarer PanNETs secreting GHRH (128), calcitonin or PTH-related peptide (129), can also occur. Upon histological examination of the duodenum in patients with MEN1, small somatostatin-positive tumors can also be found (130) although they do not seem to give rise to the somatostatinoma syndrome.

The hallmark of duodenopancreatic involvement in MEN1 is multifocality, with the pancreas usually containing multiple NETs <5mm, called micro-adenomas, combined with one or more macroscopic PanNETs (130). These micro-adenomas already have loss of heterogeneity (LOH) of the MEN1 locus and are considered precursors to PanNETs (130). Similarly, duodenal gastrinomas in MEN1 are usually multiple and accompanied by gastrin cell hyperplasia, although LOH was demonstrated in duodenal gastrinomas, but not in gastrin cell hyperplasia (131). This multiplicity sets MEN1-related dpNETs apart from sporadic duodenal and pancreatic NETs, which are usually single tumors.

For patients with MEN1, the cumulative probability of having a dpNET increases with age, however the age of onset varies somewhat per tumor type. In a recent study from the Dutch MEN1 cohort, the modeled cumulative probability of having developed a NF-PanNET was 8.6% (95%CI 0.8-15.3%) at age 15, 12% (95% CI 5.9-17.0) at age 18, 16.1% (11.2-21.5) at age 21 and rising to 80% at age 70 (72.2-97.0)(15). Insulinomas can also occur at a young age and the prevalence of insulinoma among the larger (n>50) cohorts describing pediatric and adolescent MEN1 ranges from 6-25% (37, 46-48). Data from a recent multicenter cohort study show that half of the patients with MEN1-related insulinoma were diagnosed before the age of 30 (96 patients who underwent surgery for MEN1-related insulinoma from 46 centers in Europe and North-America between 1990-2016) (132). The onset of gastrinomas is usually later, with a reported mean age of onset around 30-35 years in the National Institutes of Health (NIH) MEN1-ZES cohort (133, 134) to 51 years in the Dutch MEN1 cohort (135). The occurrence of MEN1-related gastrinoma in childhood or adolescence is rare.

Duodenopancreatic NETs can be the first manifestation of MEN1, both in patients from known MEN1 families but also in the index case. Approximately 20-25% of all patients with gastrinoma have MEN1 (136), a rate much lower for insulinomas (approximately 5%) (2). Therefore, genetic testing for MEN1 is recommended in all patients diagnosed with gastrinoma (137). For patients presenting with a non-gastrinoma dpNET without a family history of MEN1, referral for genetic testing should be guided by the individual clinical characteristics, such as patient age, concomitant other MEN1-related tumors, multifocality of dpNETs, and family history of endocrine tumors. If a new diagnosis of MEN1 is made in a family, cascade screening and subsequent screening and lifelong surveillance of affected family members is of utmost importance, as delays may lead to preventable morbidity and mortality in non-index cases in the family (30).

Distant metastases occur in approximately 15-30% of MEN1-related dpNETs and are the most important prognostic factor for disease-related survival (125, 134, 138, 139). In the Dutch MEN1 cohort, 5- and 10-year overall survival rates were 95% and 86% for patients with dpNETs without liver metastases, compared to 65% and 50% for those with liver metastases (139). Non-functional pancreatic NETs and duodenal gastrinomas are the most frequent cause of distant metastases. Regional lymph node metastases are seen more often, but the exact reported prevalence highly depends on the type of cohort, primary dpNET, and the manner of diagnosis (i.e., surgical cohorts versus observational cohorts, surgery with or without systematic lymph node dissection, imaging with or without SSTR-PET imaging, etc.). In a recent publication from the Dutch MEN1 cohort, in 350 patients with MEN1-related NF-PanNETs without metastases at diagnosis, metastases (regional and/or distant) developed in 18%, while the cumulative probability of having any PanNET-related metastases at the age of 70 was 41.2% (95%CI 31.3-50.3) (15). Since patients with MEN1 often have multiple concomitant dpNETs and most patients with duodenal gastrinomas have concomitant NF-PanNETs, it may be difficult to determine the primary tumor for regional and distant metastases.

Unlike in MEN2, in MEN1 there is no clear genotype-phenotype correlation. Several groups however have studied the association between MEN1 germline mutation and the disease course of dpNETs in their cohorts, to see if genotype might be able to identify a subset of patients with a more aggressive clinical course. This was in part fueled by the clinical observation that in some families dpNETs seem to be more prevalent, occur at a younger age and have a higher proportion of metastatic disease.

Several associations have been reported: in the French GTE cohort mutations in the JUND interacting domain were associated with death (13), in the German Marburg cohort CHES1 loss of interaction was associated with aggressive pNETs and pNET-related mortality (14), in the Italian Florence cohort mutations in exon 8 were associated with higher risk of progression and mortality (140), in the MD Anderson cohort mutations in exon 2 were associated with a higher risk of distant metastases (141), and in the Dutch MEN1 cohort nonsense/frameshift mutations were associated with a higher cumulative probability of developing metastases in NF-PanNET (regional and/or distant) compared to missense mutations 53.9 (37.8-74.3%) vs 10% (2.6-82.7%)) (15). However, these associations up until now have not been independently validated, either because associations were not confirmed in other cohorts or validation was not performed.

In patients with MEN1, dpNETs are usually diagnosed at an early stage, especially in patients from families with MEN1 or who have had predictive genetic testing. Additionally, even in index cases, benign MEN1 manifestations may lead to the diagnosis of MEN1 and dpNETs can be diagnosed early. In the French GTE cohort and the Dutch MEN1 cohort, both spanning multiple decades, synchronous metastases were seen in 6.5 and 6.4% of patients with a dpNET respectively (125, 139). In MEN1-related dpNETs the focus of care therefore lies before the onset of metastatic disease and with a younger population than is seen in sporadic dpNETs. The goals of follow-up and treatment are to prevent metastatic disease, cure hormonal hypersecretion, and prevent complication from hormonal hypersecretion, while minimizing treatment-related complications and preserving Quality of Life. It is therefore of utmost importance that whenever possible patients with MEN1 and MEN1-related dpNETs are treated in centers of expertise with a knowledgeable and experienced multidisciplinary team.

Staging and Grading

MEN1-related dpNET are graded according to the latest WHO classification (Table 2) of digestive system tumors (2019, 5^th edition) and the WHO Classification of Tumors of Endocrine Organs (2017, 4^th edition) (142). Where previously dpNET grading was only covered in the Classification of Tumors of Endocrine Organs, it is now included in the classification of digestive system tumors as well (142).

Table 2. WHO Classification of Digestive Neuroendocrine Tumors
Classification	Ki-67 proliferation index	Mitotic rate (mitoses/2mm²)
Well-differentiated Neuroendocrine Tumors (NET)
NET, G1	<3%	<2
NET, G2	3-20%	2-20
NET, G3	>20%	>20
Poorly-differentiated Neuroendocrine Carcinomas (NEC)
NEC (G3) Small-cell type Large-cell type	>20%	>20

Pancreatic NETs are staged according to the AJCC UICC 8^th edition Neuroendocrine tumors of the pancreas (Table 3a and b).

Table 3a. TNM Staging of Pancreatic Neuroendocrine Tumors (AJCC UICC 8^thedition)
Primary Tumor (T) For any T add (m) for multiple tumors e.g., T2(m).
TX	Tumor cannot be assessed
T1	Tumor limited to the pancreas*, <2 cm
T2	Tumor limited to the pancreas*, 2-4 cm
T3	Tumor limited to the pancreas*, >4 cm; or tumor invading the duodenum or CBD
T4	Tumor invading adjacent organs (stomach, spleen, colon, adrenal gland) or the wall of large vessels (celiac axis or the superior mesenteric artery)
Regional lymph Nodes (N)
NX	Regional lymph nodes cannot be assessed
N0	No regional lymph node involvement
N1	Regional lymph node involvement
Distant Metastases (M)
M0	No distant metastases
M1	Distant metastases
M1a	Hepatic metastases only
M1b	Extra-hepatic metastases only
M1c	Both hepatic and extra-hepatic metastases

* Limited to the pancreas means no invasion of adjacent organs or the wall of large vessels. Extension into peripancreatic adipose tissue is included in “limited to the pancreas”. CBD common bile duct

Table 3b Stage Grouping
Stage I	T1 N0 M0
Stage II	T2-3 N0 M0
Stage III	T4 N0 M0 Any T N1 M0
Stage IV	Any T Any N M1

Duodenal NETs are staged according to the AJCC UICC 8^th edition Neuroendocrine Tumors of the duodenum and ampulla of Vater (Table 4a and b).

Table 4a. TNM Staging of Duodenal Neuroendocrine Tumors (AJCC UICC 8^th edition)
Primary Tumor (T) If the number of tumors is known use T (#), if unavailable or too numerous T(m) e.g., T2(3) or T2(m)
TX	Tumor cannot be assessed
T1	Tumor invades the mucosa or submucosa only and is ≤ 1 cm (duodenal) Tumor ≤ 1 cm and confined within the sphincter of Oddi (ampullary)
T2	Tumor invades the muscularis propria or is >1 cm (duodenal). Tumor invades through sphincter into duodenal submucosa or muscularis propria, or is >1 cm (ampullary).
T3	Tumor invades the pancreas or peripancreatic adipose tissue
T4	Tumor invades the visceral peritoneum (serosa) or other organs
Regional lymph Nodes (N)
NX	Regional lymph nodes cannot be assessed
N0	No regional lymph node involvement
N1	Regional lymph node involvement
Distant Metastases (M)
M0	No distant metastases
M1	Distant metastases
M1a	Hepatic metastases only
M1b	Extra-hepatic metastases only
M1c	Both hepatic and extra-hepatic metastases

Table 4b. Stage Grouping
Stage I	T1 N0 M0
Stage II	T2-3 N0 M0
Stage III	T4 N0 M0 Any T N1 M0
Stage IV	Any T Any N M1

Non-Functioning Pancreatic NETs

In patients with known MEN1, screening is advised for early detection of NF-PanNETs. When NF-PanNETs are diagnosed and there is no immediate indication for intervention, surveillance should be performed at regular intervals to re-evaluate indications for intervention, as well as to detect newly developing dpNETs. Current guidelines suggest to start screening for NF-PanNETs in MEN1 below the age of 10 by a combination of biochemical tests and yearly imaging (either MRI, CT or EUS) (2).

Since the publication of the guidelines, it has become clear that the diagnosis of NF-PanNETs in patients with MEN1 relies heavily on imaging, since tumors markers chromogranin A, pancreatic polypeptide, and glucagon have low accuracy for the diagnosis of NF-PanNETs as summarized in a recent systematic review (143). Additionally, since the publication of the guidelines SSTR-PET-CT has emerged as a high-sensitive diagnostic imaging tool for dpNETs and its role within the screening and surveillance of MEN1-related dpNETs has yet to be determined.

There is currently no consensus regarding the best imaging modality and interval for screening and surveillance of MEN1-related NF-PanNETs (144). In a recent systematic review on the diagnosis of NF-PanNETs in MEN1, it was concluded that for lifelong screening and surveillance CT was probably least suitable given the inferior sensitivity compared to EUS and SSTR-PET-CT in combination with the cumulative exposure to ionizing radiation, although head-to-head comparisons with MRI are not available (143). This does not mean that a CT scan cannot still be indicated and be the best imaging for specific clinical situations (for example pre-operative imaging). EUS is the most sensitive method for the diagnosis of NF-PanNETs and offers the possibility of obtaining tissue for analysis pre-operatively. However, it is also invasive, operator dependent, clinically significant PanNETs can be missed in the pancreatic tail, and for the diagnosis of NF-PanNETs in MEN1 histological confirmation is usually not necessary given the high pre-test likelihood and the typical appearance on imaging. On the other hand, tissue-based analysis prior to intervention may become more relevant in MEN1 as more novel prognostic factors are identified. MRI has the advantage of performing more homogenously throughout the pancreas and the absence of ionizing radiation although a significant proportion of NF-PanNETs >2 cm is missed. The authors of the systematic review therefore suggest alternate use of EUS and MRI (143).

Given a reported growth rate of 0.1-1.32 mm/year for small NF-PanNETs, if initial screening imaging is negative, the next imaging can be performed with an interval of two to three years, providing no clinical reason for earlier imaging. For prevalent NF-PanNETs without an indication for intervention, the interval for active surveillance imaging should be individualized according to growth rate. Initial repeat imaging should be done after 6-12 months to assess growth rate, but afterwards in small stable NF-PanNETs the interval can be extended to once every 1-2 years. The imaging modality can be either MRI or alternating with EUS. EUS alone should always be combined with an imaging method for metastases detection, since it does not offer complete abdominal imaging. The exact role of SSTR-PET-CT in the screening and surveillance of MEN1-related NF-PanNETs is still to be determined, however, based on currently available evidence, it is best employed when results may change management such as in prevalent NF-PanNETs >10 mm for early detection of metastases, or as comprehensive staging before planned interventions (143, 145).

Another dilemma is when to start radiological screening for NF-pNET in children with MEN1. As mentioned before, current guidelines advise initiating screening before the age of 10 (2). However, others have advocated postponing until the age of 16, in the absence of signs and symptoms (37). Recently, modeled data from the Dutch population-based MEN1 cohort show that the estimated age at a 1%, 2,5% and 5% risk of having developed a clinically significant NF-PanNET (≥ 20mm or documented growth of ≥1.6 mm within one year above a baseline size of ≥ 15mm) is 9.5, 13.5 and 17.8 years respectively and they conclude that there is medical indication to initiate radiological screening during the second decade of life and that starting between 13-14 years of age is justifiable (15).

It is important to remember that each screening and surveillance schedule should be tailored to the needs of the individual patient in his or her unique circumstances, should be based on well-informed shared decisions making between providers and patients (and parents if applicable), with multidisciplinary team input when necessary.

The only curative treatment for NF-PanNETs in MEN1 is surgical resection, and the goal of surgical intervention in NF-PanNETs is to prevent metastases and thereby NF-PanNET-related mortality, while preserving as much pancreatic tissue as possible and limiting treatment-related morbidity and mortality. Although theoretically, total duodenopancreatectomy would prevent metastatic disease altogether, short-term morbidity associated with this complex major surgery is high and the subsequent life-long brittle diabetes that follows rarely justifies such major intervention when balanced against the risk of distant metastases and PanNET-related death.

Since the risk of future metastases and disease-related death must be balanced against short- and long-term treatment-related morbidity and mortality, information regarding prognosis in MEN1-related NF-PanNETs is of vital importance to make well-informed decisions regarding timing and extent of intervention. However, presently there is a paucity of prognostic factors on which to base these decisions (146). The most important factor to date is tumor size, with the risk of (distant) metastases increasing with increased size. Recent data from retrospective cohort studies have shown that small (<2cm) NF-PanNETs generally have an indolent course, that surgical resection of small NF-PanNETs does not seem to offer benefit over active surveillance, and that the risk of metastases and disease-related death is low, albeit not zero (124, 146-150). Most small NF-PanNETs are stable during follow-up, but there is a subset with progression in size (150). Generally, size progression is also considered to be a prognostic factor. An important tissue-based prognostic factor is tumor grade, with grade 2 tumors being more often associated with metastases (146). Grade 3 NF-PanNETs or NECs are rarely seen in patients with MEN1, but are associated with a worse prognosis. More recently, advancements in molecular techniques have identified several potential prognostic biomarkers for NF-PanNETs, mostly in sporadic NF-PanNETs, but limited data in MEN1-related NF-PanNETs is also available. Mutations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX), which lead to the alternative lengthening of telomeres (ALT) phenotype have been found to be associated with decreased disease-free survival and higher rates of distant metastases (146, 151). Mutations in DAXX and ATRX result in loss of nuclear expressions of their proteins by immunohistochemistry (IHC) and ALT can be identified in tissue-samples by telomere-specific fluorescence in situ hybridization (FISH). Next to DAXX/ATRX and ALT, the differential expression of transcription factors aristaless-related homeobox gene (ARX) and pancreatic and duodenal homeobox 1 (PDX1) as assessed by IHC was also found to be associated with risk of metastases (152, 153). In patients with MEN1-related NF-PanNETs, one study showed that liver metastases were only seen in ARX+ or ARX-/PDX1- tumors and that ALT positivity was only seen in ARX+ or ARX-/PDX1- tumors and significantly correlated with relapse rate (152). However, since the publication of these data, a large international cohort of 1322 NETs (not including MEN1-related NETs), was evaluated by immunolabelling for ARX/PDX1, ATRX/DAXX and by telomere-specific FISH for ALT and it was found that ATRX/DAXX and ALT, but not ARX/PDX1 were independent negative prognostic factors (151).

A recent study by Fahrmann, et al. identified a 3-marker polyamine signature that distinguished patients with metastatic dpNETs from controls and which yield an AUC of 0.84 (95% CI: 0.62-1.00) with 66.7% sensitivity at 95% specificity for distinguishing cases form controls in an independent test set (154). These results form the basis for prospective testing of plasma polyamines as a prognostic factor for MEN1-related dpNETs.

Further validation of these molecular markers in MEN1, may also change the role of pre-intervention EUS-guided aspiration or biopsy.

So, when to intervene in MEN1-related NF-PanNETs? Presently, these decisions are mostly based on tumor size and growth, with the current guidelines suggesting considering surgical resection in NF-PanNETs >10 mm or those that show significant growth during follow-up (doubling of tumor size over a 3–6-month interval and exceeding 10 mm) (2). Given the emerging evidence that most NF-PanNETs <20 mm are indolent in nature as described above, a more recent consensus statement states that surgical resection is indicated for NF-PanNETs >20 mm and those that progress during surveillance (155). Additionally, the presence of suspicious lymph nodes, or a higher grade on EUS-guided aspiration may guide intervention decisions. In all cases these decisions should be made in multidisciplinary teams and in shared decision making with the patient. The extent of resection depends on multiple patient-, tumor- and MEN1-related factors and should be individualized.

Gastrinoma

Gastrinomas, NETs secreting gastrin, cause the Zollinger-Ellison Syndrome (ZES). ZES is a syndrome characterized by tumor-related hypergastrinemia leading to gastric acid hypersecretion.

Sign and symptoms of ZES/Gastrinoma are gastro-esophageal reflux disease (GERD), (proton-pump inhibitor (PPI) responsive) diarrhea, abdominal pain, nausea/vomiting, weight loss, and peptic ulcer disease. Complications may arise from the peptic ulcer disease including upper gastro-intestinal bleeding, strictures, and bowel perforation.

Before the introduction of PPIs, complications from gastric acid hypersecretion were an important cause of death in patients with MEN1 (134). With the arrival of proton pump inhibitors, gastric acid hypersecretion can be effectively treated, although higher dosages are needed than for the treatment of non-ZES hyperacidity.

The diagnosis of gastrinoma in MEN1 is challenging at present. The gold standard for the diagnosis is the demonstration of inappropriate fasting hypergastrinemia without the use of antisecretory drugs. The diagnosis is established if the fasting serum gastrin (FSG) is more than tenfold the upper limit of normal with a gastric pH of less than two (after ruling out retained antrum) (156, 157). When gastric pH is low and FSG is <10-fold upper limit of normal, additional testing is needed to establish the diagnosis, such as a secretin provocative test or measuring basal acid output (156, 157). The latter situation occurs in 60% of ZES, and this might even be higher in MEN1, given the early detection through prospective screening programs. Due to unreliable gastrin assays, the limited availability of secretin and therewith the loss of expertise in performing the secretin provocative test, the wide-spread use of PPIs and the risk associated with cessation of PPI for proper testing, the diagnosis of gastrinoma is challenging (157, 158). Additionally, the value of non-biochemical tests in the diagnosis of gastrinoma (such as SSTR-PET-CT and EUS/ esophagogastroduodenoscopy (EGD)-guided cytology/biopsy) might also need re-evaluation (157). Recently experts have therefore suggested possible new criteria for diagnosis of ZES in patients with fasting hypergastrinemia with and without PPI use (157).

Patients with MEN1 are screened for the presence of a gastrinoma by at least annually assessing clinical symptoms and fasting serum gastrin (2). If a diagnosis of gastrinoma is established or suspected, EGD should be performed to assess the presence of complications of gastric acid hypersecretion, type II gastric NETs, and possibly to identify duodenal gastrinomas. Duodenal gastrinomas in MEN1 are small, but despite their small size 70-80% are metastatic to the regional lymph nodes at the time of diagnosis (159). However, these regional lymph node metastases do not seem to have a negative impact on overall survival (159). In MEN1, attributing locoregional lymph nodes to the correct primary dpNET is important for adequate treatment planning and prognostic inferences. It is also challenging however, given that most patients with MEN1 and duodenal gastrinoma(s) also have concomitant PanNETs, and the duodenal gastrinoma(s) may not be visible on imaging due to their small size. Hackeng, et al. studied 137 microscopic and macroscopic dpNETs and 36 matched metastases (lymph node and distant) in 10 patients with MEN1 to unravel the relationship between the multiple primary dpNETs in MEN1 and the metastases (160). They found that most patients had a single NET of origin for their metastases, but multiple metastatic primaries were also seen. In addition, and very important for MEN1-related gastrinomas, in 6 patients with MEN1 and hypergastrinemia, periduodenopancreatic lymph node metastases clustered with minute duodenal gastrinomas and not with larger pancreatic NETs. So a duodenal origin for periduodenopancreatic lymph node metastases in patients with MEN1 and hypergastrinemia should always be considered (160).

Although its role still needs to be delineated, in MEN1-gastrinoma next to EUS/EGD, SSTR-PET-CT may be very useful for staging to visualize duodenal gastrinomas, lymph node metastases, and concomitant PanNETs.

Presently, as in other MEN1-related dpNETs, surgery is the only potentially curative treatment. And although MEN1-related ZES has been historically been considered a surgically incurable disease, more recent small studies have shown, that when the correct target organ is addressed, namely the duodenum and not the pancreas, biochemical cure can be achieved after partial pancreaticoduodenectomy(PD), combined with regional lymph node dissection (159). However, this must be balanced against the risk of peri-operative and long-term complications and loss of quality of life. Overall survival is generally good in duodenum-preserving operations as well, but persistence or recurrence of ZES occurs in 6-100% (159). Most often, hyperacidity can be adequately controlled with PPIs, making the main goal of surgical resection the prevention of distant metastases and disease-related death. The majority of MEN1-ZES patients with associated small PanNETs have an indolent disease course with excellent overall survival even without surgical intervention (161). Still, in retrospective studies around a quarter of the patients develop liver metastases and around 15% shows aggressive growth (138), and presently there are no good markers to predict which patients with MEN1-ZES will have a more aggressive disease course. In a study from the NIH age at ZES diagnosis (≤33), FSG levels ≥10,000 pg/mL, pancreatic tumors >3 cm, presence of liver/bone metastases and presence of gastric carcinoids were associated with aggressive tumor growth (138). In a study from the DutchMEN Study Group, overall survival rates of MEN1-gastrinoma were 83% and 65% at 5 and 10 years respectively, which was significantly worse than age- and gender-matched patients without gastrinoma. FSG ≥ 20x upper limit of normal, PanNETs≥ 2cm, synchronous liver metastases, EGD suspicious for gastric NETs, and multiple concurrent NETs were associated with decreased overall survival (135). A recent study from the French GTE, ZES was independently associated with a higher risk of distant metastases, but did not significantly seem to be associated with decreased overall survival (125).

So presently, if surgical intervention should be performed, when surgical intervention should be performed and how (to what extent) surgical intervention should be performed for gastrinoma in MEN1 are all controversial topics. Treatment of patients in centers of expertise with a highly dedicated multidisciplinary team and experienced surgeons is therefore very important. Treatment decisions for MEN1-ZES should be made after MDT discussion in shared decision making with the patient.

As stands to reason, treatment of MEN1-ZES patients with (high-dose) PPI is mandatory and patients should not cease this treatment without consultation with their provider. If specific testing without PPI is needed this needs to be performed under close supervision in centers of expertise.

As mentioned in the section on parathyroid tumors, the interplay between pHPT and ZES in MEN1 is important to realize as hypercalcemia can increase gastrin levels. Additionally, a recent paper on the Tasmanian MEN1 cohort showed an association between H. pylori seropositivity and hypergastrinemia and severe ZES-range hypergastrinemia. Further work is needed to fully elucidate this relationship, but testing for H. Pylori and eradication if positive might be consider in patients with MEN1-ZES (162).

Insulinoma

As already stated, MEN1-related insulinomas occur at a young age and are the most frequent functional PanNET at the pediatric age. Early recognition of signs and symptoms of insulinoma is of extreme importance in both children and adults. Signs and symptoms may be erroneously attributed to epilepsy or behavioral or neurological disorders, especially if insulinoma is the presenting manifestation of MEN1 in an index case. In children this can lead to decline in school performance and in children and adults alike episodes of hypoglycemia can lead to accidents or irrational behavior.

As insulinomas secrete insulin inappropriately and lead to hypoglycemia the signs and symptoms are those of hypoglycemia; both adrenergic symptoms (such as fast heartbeat, jitteriness/shakiness, sweating and pale skin) as well as neuroglycopenic symptoms (such as mental status changes and irritability). Symptoms are relieved with food (glucose) intake. They usually occur during fasting, before meals or after exercise, but can occasionally occur at other times. In patients fulfilling Whipple’s triad (symptoms and/ or signs consistent with hypoglycemia, a low plasma glucose concentration, and resolution of symptoms/signs after plasma glucose concentration is raised) diagnosis can be established by a supervised fast (163). In patients with MEN1, screening for insulinoma is advised from the age of five by careful history taking and measurement of fasting insulin and – more importantly – glucose (2). However, almost all patients with MEN1 and insulinoma are symptomatic, therefore the history is probably the most important element.

When the diagnosis of insulinoma is made, localization in MEN1 can be challenging, if there are multiple PanNETs. These usually are concomitant NF-PanNETs, since in surgical series multiple insulin-positive PanNETs in patients operated for insulinoma were seen in 8-40% (132, 164-166).

For MEN1-related insulinoma, especially if conventional imaging shows multiple PanNETs and correctly identifying the insulinoma(s) among them would change surgical strategy, ⁸⁶Ga-Exendin-4 PET-CT is very promising. Although there is limited data in MEN1 patients, a recent meta-analysis showed a positive predictive value (PPV) of 94%, with a negative predictive value (NPV) of 67%; In MEN1 PPV was 95% with NPV 96%, although based on a limited number of patients (167, 168).

Surgical resection is the treatment of choice for MEN1-related insulinomas and is associated with a high cure rate. In a retrospective cohort study of 40 European and 6 North-American institutes 92 patients with MEN1-related insulinomas who underwent surgical resection were followed for a median of 8 years after surgery (132). Overall, after different surgical procedures, only 1 patient had persistence of hypoglycemia and six had recurrent hypoglycemia, four due to new primaries and 2 due to development of liver metastases, leading to a 10-year hypoglycemia-free survival of 91% (95% CI 80-96). For those with unifocal insulinoma based on pre- and intra-operative assessment (n=63), 1/46 (2.2%) undergoing pancreas resection had persistent disease, while among those who underwent enucleation 1/17 (6%) had recurrence of hypoglycemia based on a new primary insulinoma. For those with multifocal insulinoma (n=33), of whom 30 underwent pancreatic resection, mostly distal pancreatic resection, and three had multiple enucleations, 15% had recurrent hypoglycemia (9% based on new primaries and 6% based on liver metastases) (132).

Therefore, given the better outcomes of pancreatic function over the long-term and young age of the patients, if surgically feasible, enucleation seems the better option for solitary insulinomas in MEN1, provided of course that concomitant functional and non-functional tumors do not make a different strategy necessary (132).

Among MEN1-related dpNETs, insulinomas have the best oncological prognosis (26, 125, 169). Data from the international MEN1 Insulinoma Study Group and the DutchMEN Study Group show that for surgically resected insulinomas 10-yr liver-metastases free survival was 87% (72-91%) (169). Malignant insulinoma is rare, both in sporadic and MEN1-related insulinoma. In the two largest MEN1-insulinoma cohorts synchronous liver metastases were seen in 3.8-8.1% and metachronous liver metastases in 0-2.2% after a median follow-up of 8-9 years (132, 165).

Rare Functional dpNETS

Functional dpNETs besides gastrinomas and insulinomas, are rare in MEN1 and are seen in <1% of patients with dpNETs(2). These include PanNETs producing vaso-active intestinal peptide (VIPoma), somatostatin, glucagon and other (ectopic) hormones such as growth hormone releasing hormone (GHRH), calcitonin, or PTH-related peptide (PTHrP). A rare functional tumor is considered if there are elevated hormone levels in conjunction with a fitting clinical syndrome. Without a clinical syndrome, tumors are not considered functional but merely hypersecreting. This is relevant as for example glucagon can be elevated in patients with MEN1 and PanNETs without the patient having the glucagonoma syndrome. VIPomas lead to watery diarrhea, hypokalemia, achlorhydria and dehydration, the somatostatinoma syndrome consists of diabetes mellitus, diarrhea, steatorrhea and cholelithiasis, while glucagonomas give rise to necrolytic migratory erythema, diabetes mellitus, and weight loss. Tumors producing GHRH, calcitonin, and PTHrP lead to acromegaly, diarrhea and hypercalcemia, respectively. In these rare functional dpNETs without synchronous distant metastases surgery is generally indicated.

Non-Surgical Treatments of Non-Metastatic dpNETs in MEN1

Although for most non-metastatic functional dpNETs in MEN1 surgery is indicated, there may be a (temporary) need to control the hormonal syndrome medically. As such gastrinomas are treated with high-dose PPI, insulinomas with diazoxide or frequent feedings and in all cases somatostatin analogues might be considered if needed to control the functional syndrome.

Local resection of sporadic small dNETs is increasingly considered as an alternative to surgery (170), and current European Neuroendocrine Tumor Society (ENETS) guidelines recommend endoscopic management for dNETs ≤ 10mm in size, confined to the submucosal layer and without lymph node and distant metastases (171). However, since in MEN1 dNETs are usually multiple, may grow beyond the submucosa and, in case of gastrinomas, are associated with lymph nodes in up to 80% of the cases, this is not generally recommended (159).

Similarly, for PanNETs EUS-guided intervention using ethanol or radio-frequent ablation has been reported in around 80 and 70 cases respectively in the literature, with only a handful procedures performed in patients with MEN1 (172). Whether or not interventional EUS may play a role in treatment of MEN1-related PanNETs is therefore unclear at the present time.

There is much interest in chemoprevention in small NF-PanNETs using somatostatin analogues (SSA). SSA have proven anti-proliferative effect in advanced PanNETs (173, 174) and the question has been raised if SSA may be used to prevent progression and metastases of small NF-PanNETs in patients with MEN1. In mouse models of Men1PanNET lanreotide and pasireotide showed the ability to decrease tumor proliferation. In a retrospective non-controlled study of 20 patients with small NF-PanNETs who received long-acting octreotide for 12-75 months 10% had an objective tumor response, 80% stable disease, and 10% showed progression (175). In another small (n=8) prospective series patients with small NF-PanNETs were treated with SSA for up to 72 months, with stable disease in all, however again without a control group (176). In a recent observational cohort study lanreotide was compared with standard of care active surveillance in n=42 patients with pNETs <2 cm (N=23 lanreotide vs n=19 active surveillance) during a median follow-up of 6 years (177). The study showed improved RECIST-defined progression-free survival (PFS) in the lanreotide group. In both groups however, one patient developed distant (liver) metastases (177). Limitations include sample size, non-experimental and therefore non-randomized design, and non-blinded outcome evaluation. In addition, improved RECIST PFS is not yet known to predict longer overall survival for MEN1 patients with small NF-PanNETs. Ideally this is further evaluated in a randomized double-blind trial. The most important challenge in the design of such a study however, is the definition of appropriate surrogate endpoint for distant metastases and overall survival (144).

Metastatic dpNET in MEN1

The treatment of stage IV dpNET in patients with MEN1 is similar to that of patients with sporadic dpNETs(178). There is very limited evidence regarding MEN1-specific outcome data, and from the limited evidence available there seems to be no difference with sporadic NETs. In landmark studies leading to approval of lanreotide (173), everolimus (179), sunitinib (180) and peptide receptor radionuclide therapy (PPRT) (181), patients with MEN1 were either excluded, only single cases included or MEN1-status was not mentioned (182). With the advancing molecular understanding of MEN1-related NETs, MEN1-specific targeted therapies might be possible in the future, which in turn might benefit the almost 50% MEN1-mutated sporadic PanNETs (182).

Gastric NETs in MEN1 (Type II Gastric NETs)

NETs of the stomach (Figure 1), formerly called gastric carcinoids or carcinoids of the stomach, are classified into three different types(171):

Type I, associated with atrophic gastritis
Type II, associated with MEN1/ZES
Type III, without associated conditions

Gastric NETs are graded according to the latest WHO classification of digestive system tumors (2019, 5^th edition) as described above for dpNETs (Table 2) (142). TNM staging is shown in Table 5a and b.

Table 5a. TNM Staging of Neuroendocrine Tumors of the Stomach (AJCC UICC 8th edition)
Primary Tumor (T) For any T, add (m) for multiple tumors, for multiple tumors with different Ts, use the highest (e.g., if three tumors’ sizes 0.5, 0.5, and 1.5 cm, T stage should be T2(m).
TX	Tumor cannot be assessed
T0	No evidence of primary tumor
T1	Invades lamina propria or submucosa and ≤ 1 cm
T2	Invades muscularis propria or >1cm
T3	Invades through the muscularis propria into subserosal tissue without penetration of overlying serosa
T4	Invades visceral peritoneum (serosal) or other organs or adjacent structures
Regional lymph Nodes (N)
NX	Regional lymph nodes cannot be assessed
N0	No regional lymph node involvement
N1	Regional lymph node involvement
Distant Metastases (M)
M0	No distant metastases
M1	Distant metastases
M1a	Hepatic metastases only
M1b	Extra-hepatic metastases only
M1c	Both hepatic and extra-hepatic metastases

Table 5b. Stage Grouping
Stage I	T1 N0 M0
Stage II	T2-3 N0 M0
Stage III	T4 N0 M0 Any T N1 M0
Stage IV	Any T Any N M1

Gastric NETs are tumors of the gastric entero-chromaffin like (ECL) cells, which develop in MEN1 due to the trophic effect of gastrin on ECL-cells combined with the predisposing germline MEN1 mutation. Both components seem to be necessary for the development of gastric NETs in patients with MEN1. Gastric NETs are rarely seen in sporadic ZES patients and loss of heterogeneity was demonstrated in 75% of MEN1-related gastric NETs (183, 184), while in patients with MEN1 gastric NETs occur almost exclusively in patients with gastrinoma and regression of gastric NETs has been reported after normalization of hypergastrinemia (185, 186). ECL-cell hyperplasia is considered a precursor lesion for gastric NETs (187).

In the NIH-ZES cohort, 57 patients were extensively studied for gastric ECL-cell changes. All of the patients were found to have proliferative ECL-cell changes, with advanced changes in 53% and gastric NETs in 23% (188). More recently, data on ECL-cell changes in patients with MEN1 was reported from the Marburg MEN1 database (185). They reported on 38 MEN1 patients who underwent regular screening including EGD, regardless of gastrinoma status. Sixteen of these patients had a gastrinoma diagnosis, 13 of whom had biochemical ZES at the time of first EGD. They found that ECL-changes and gastric NETs were exclusively seen in patients with MEN1-gastrinoma albeit in a lower percentage than in the NIH. They found ECL hyperplasia in 62.5% of patients with a gastrinoma diagnosis, versus 0% in those without gastrinoma. No advanced ECL-cell changes were seen and gastric NETs were found in 12.5% of patients with a gastrinoma diagnosis (185). These differences might also reflect practice changes with earlier gastrinoma diagnosis due to screening and surveillance and more frequent surgical treatment of gastrinoma in the Marburg cohort compared to the NIH cohort.

In the NIH cohort, higher levels of FSG as well as longer duration of ZES were associated with a higher risk of advanced ECL-cells changes and gastric NETs (188). Higher levels of FSG as risk factor could not be confirmed in the Marburg cohort, however numbers of MEN1-ZES were small (185). As mentioned above, in the section on gastrinoma, the presence of gastric NETs in patients with MEN1-ZES was associated with a more aggressive disease course (138) and decreased overall survival (135).

Therefore, in the current MEN1 Clinical Practice Guidelines, EGD with biopsy, is recommend every 3 years in patients with MEN1 and hypergastrinemia. Although treatment of MEN1-related gastric NETs is not well established (2, 171), the guideline suggest that lesions <10 mm may remain under endoscopic surveillance, while larger tumors require endoscopic resection or local resection, which is analogue to the treatment of type I gastric NETs (2, 171).

The prognosis of MEN1-related gastric NETs is generally good, with metastases (regional and distant) reported in 10-30% and disease-related death <10% (171). Nevertheless, aggressive symptomatic and metastatic cases leading to mortality have been reported (189, 190).

Conclusion

In conclusion, dpNETs are highly prevalent in patients with MEN1 reaching a more than 80% penetrance at the age of 80. NF-PanNETs are most frequently seen, followed by gastrinomas and insulinomas. Most MEN1-related dpNETs are diagnosed at an early stage and NF-PanNETs <2 cm generally have an indolent course. However, distant metastatic dpNETs (mostly NF-PanNETs and gastrinoma) are the most important cause of MEN1-related mortality. Treatment goals for MEN1-related dpNETs are therefore to prevent metastatic disease, cure hormonal hypersecretion, and prevent complication from hormonal hypersecretion, while minimizing treatment-related complications and preserving Quality of Life. Surgical resection is the mainstay for treatment, and is indicated in non-gastrinoma functional PanNETs and NF-PanNETs >2cm or with progression during follow-up. No consensus exists on the surgical treatment of MEN1-related gastrinoma. With increasing awareness of MEN1, increasingly refined and defined screening and surveillance programs, and increasing sensitive imaging modalities, MEN1-related dpNETs are detected at earlier stage and more indolent small dpNETs are seen. The main challenge at this point is therefore identifying those patients who are at risk for a more aggressive disease course and distant metastases to be able to offer those patients close follow-up schedules and earlier and more aggressive treatment, while limiting treatment-related morbidity in patients with low risk. Novel prognostic indicators are therefore needed, ideally blood-based, so minimal invasive assessment is possible. Other future directions are the investigation of chemoprevention in small NF-PanNETs.

Gastric NETs in MEN1 are almost exclusively seen in patients with gastrinoma and usually have an indolent course. Screening with EGD should be performed in all patients with MEN1-ZES.

THORACIC NEUROENDOCRINE TUMORS

General

Thoracic NETs occurring as part of the MEN1 syndrome are thymic (thNET) and bronchopulmonary NETs (bpNET) (Figure 1), although recently it was suggested that thymomas may also be part of the MEN1-related tumor spectrum (191). These tumors are not considered main disease-defining manifestations. As in other MEN1-related tumors, loss of heterogeneity (LOH) at the MEN1 locus was demonstrated in bpNETs in patients with MEN1 (97). This in contrast to thymic NETs, where until recently no LOH at the MEN1 locus was found. However, in a recent publication by the NIH, LOH was seen at the MEN1 locus in both MEN1-related thymic NETs (8 out of 12) and two (out of two) thymomas in patients with MEN1 (191). MEN1 is also the most frequently mutated gene in sporadic well-differentiated bpNETs, this is not described in thymus NET (192). However, approximately 25% of patients with thNET have germline mutations in MEN1, therefore it is very important to consider the diagnosis of MEN1 in patients presenting with a sporadic thNET (193). Thymic and bronchopulmonary NETs in MEN1 generally develop in adults. In pediatric and adolescent series (age up to 21, 31 in one series), there is only one reported case of thNET (diagnosed at age 16) and two cases of bpNET (diagnosed at age 15 and 20 respectively) (37, 46-48).

Staging and Grading

bpNET and thNET are classified according to the WHO classification (Table 6). TNM staging for thymicNETs is shown in Table 7a and b. TNM staging of bronchopulmonary NETs follows the same classification as bronchogenic lung carcinomas (Table 8a and b).

Table 6. WHO Classification of Bronchopulmonary and Thymic Neuroendocrine Tumors
Classification	Mitotic Rate and Necrosis
Well-differentiated
Typical Carcinoid, NET G1	Mitotic rate <2 and absence of necrosis
Atypical Carcinoid, NET G2	Mitotic rate 2-10 and/or presence of necrosis
Poorly differentiated
Neuro-endocrine carcinomas Small-cell type Large-cell type	Mitotic rate >10

Table 7a. Staging of Thymic Neuroendocrine Tumors (AJCC UICC 8th edition)
Primary Tumor (T)
TX	Tumor cannot be assessed
T0	No evidence of primary tumor
T1 T1a T1b	Tumor encapsulated or extending into the mediastinal fat; may involve the mediastinal pleura Tumor with no mediastinal pleura involvement Tumor with direct invasion of mediastinal pleura
T2	Tumor with direct invasion of the pericardium (either partial or full thickness)
T3	Tumor with direct invasion into any of the following: Lung, brachiocephalic vein, superior vena cava, phrenic nerve, chest wall, or extrapericardial pulmonary artery or veins
T4	Tumor with invasion into any of the following: Aorta (ascending, arch, or descending), arch vessels, intrapericardial pulmonary artery, myocardium, trachea, esophagus
Regional lymph Nodes (N)
NX	Regional lymph nodes cannot be assessed
N0	No regional lymph node involvement
N1	Metastasis in anterior (perithymic) lymph nodes
N2	Metastasis in deep intrathoracic or cervical lymph nodes
Distant Metastases (M)
M0	No pleural, pericardial, or distant metastasis
M1	Pleural, pericardial, or distant metastasis
M1a	Separate pleural or pericardial nodule(s)
M1b	Pulmonary intraparenchymal nodule or distant organ metastasis

Table 7b. Stage Grouping
Stage I	T1 N0 M0
Stage II	T2 N0 M0
Stage IIIa	T3 N0 M0
Stage IIIb	T4 N0 M0
Stage IVa	Any T N1 M0 Any T N0-1 M1a
Stage IVb	Any T N2 M0-M1a
	Any T Any N M1b

Table 8a. TNM Staging of Bronchopulmonary Neuroendocrine Tumors (AJCC UICC 8th edition)
Primary Tumor (T) If the number of tumors is known used T (#), if unavailable or too numerous T(m) (e.g., T2a(2) or T2a(m)).
TX	Primary tumor cannot be assessed or tumor proven by presence of malignant cells in sputum or bronchial washings but not visualized by imaging or bronchoscopy
T0	No evidence of primary tumor
Tis	Tumor in situ
T1 T1a(mi) T1a T1b T1c	Tumor ≤3 cm in greatest dimension surrounded by lung or visceral pleura without bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the main bronchus)* Minimally invasive adenocarcinoma Tumor ≤1 cm in greatest dimension Tumor >1 cm but ≤2 cm in greatest dimension Tumor >2 cm but ≤3 cm in greatest dimension
T2 T2a T2b	Tumor >3 cm but ≤5 cm or tumor with any of the following features: Involves main bronchus regardless of distance from the carina but without involvement of the carina Invades visceral pleura Associated with atelectasis or obstructive pneumonitis that extends to the hilar region, involving part or all of the lung Tumor >3 cm but ≤4 cm in greatest dimension Tumor >4 cm but ≤5 cm in greatest dimension
T3	Tumor >5 cm but ≤7 cm in greatest dimension or associated with separate tumor nodule(s) in the same lobe as the primary tumor or directly invades any of the following structures: chest wall (including the parietal pleura and superior sulcus tumors), phrenic nerve, parietal pericardium
T4	Tumor >7 cm in greatest dimension or associated with separate tumor nodule(s) in a different ipsilateral lobe than that of the primary tumor or invades any of the following structures: diaphragm, mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, and carina
Regional lymph Nodes (N)
NX	Regional lymph nodes cannot be assessed
N0	No regional lymph node involvement
N1	Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extension
N2	Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s)
N3	Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)
Distant Metastases (M)
M0	No distant metastasis
M1	Distant metastases
M1a	Separate tumor nodule(s) in a contralateral lobe; tumor with pleural or pericardial nodule(s) or malignant pleural or pericardial effusion
M1b	Single extrathoracic metastasis
M1c	Multiple extrathoracic metastases in one or more organs

Table 8b. Stage Grouping
Stage 0	Tis N0 M0
Stage IA	T1 N0 M0
Stage IB	T2a N0 M0
Stage IIA	T2b N0 M0
Stage IIB	T1a-c N1 M0 T2 N1 M0 T3 N0 M0
Stage IIIA	T1a-c N2 M0 T2 N2 M0 T3 N1 M0 T4 N0-1 M0
Stage IIIB	T1a-c N3 M0 T2 N3 M0 T3 N2 M0 T4 N2 M0
Stage IIIC	T3-4 N3 M0
Stage IVA	Any T Any N M1a-b
Stage IVb	Any T Any N M1c

Thymic NET

ThNETs develop in 2.0 - 8.2% of MEN1 patients, with a median age at diagnosis of 43 years (range 16–72 years) (194-201). There is a strong male predominance (male to female ratio 4:1) in MEN1-related thNET, which is more pronounced in American and European cohorts compared to Asian series (194). Although one of the earliest studies on MEN1-related thNET suggested a higher prevalence of truncating MEN1 mutations in patients with thNET (202), no clear genotype-phenotype relationship has been described in later cohorts (196, 197, 199, 200). Furthermore, familial clustering of thNET within MEN1 families has been reported in a number of studies (193, 197, 199) but others could not find comparable results (196, 200). The suggested link between smoking and the occurrence of thNET in MEN1 remains controversial as well, as the portion of (heavy) smokers varied significantly among studies (193-196, 199, 201).

With the exception of a small subset of ACTH-producing tumors, most thNETs are functionally silent. As a result, the majority of patients only experiences symptoms when the tumor has reached an advanced stage, underlining the importance of periodic thoracic imaging for a timely detection.

MEN1-related thNET are characterized by their aggressive nature, illustrated by their frequent presentation with metastatic disease (53.5% of patients), usually located in lymph nodes, bones and lungs (194). Despite the low prevalence of thNETs among patients with MEN1, they are responsible for 19% of MEN1-related deaths (134). The poor prognosis of MEN1-related thNET has also been illustrated in a meta-analysis of 99 MEN-1 thNETs: median survival was 8.4 years, and the 10-year survival rate was 33%. An older age at diagnosis, a tumor diameter >5 cm and the presence of metastasis were associated with worse outcome (194).

Total (thoracic) thymectomy, including excision of the tumor, the entire thymus and perithymic fat, is the recommended treatment of choice (2, 203). Additional radiotherapy and chemotherapy may be used in patients with unresectable or metastatic disease. Data from the earlier mentioned meta-analysis suggested that adjunctive therapy after surgery tended to result in a better survival compared to surgery alone (after adjusting for gender, age at diagnosis, tumor size and smoking), but this effect did not reach statistical significance (HR 0.557, 95%CI: 0.110–2.817) (194). Prophylactic cervical thymectomy, generally performed during parathyroid surgery for primary hyperparathyroidism, may decrease the chance of the occurrence of thNET. However, several cases of thNET have been reported in patients after this procedure, indicating that surveillance imaging is still required in these patients (197, 202).

Bronchopulmonary NET

Histopathologically proven bpNETs occur in 4.7-6.6% of MEN1 patients, but a much higher proportion of MEN1 patients may be diagnosed with lesions radiologically suspect of bpNET (22.9%, 26.0% and 29.3% in the Dutch, Tasman and German cohort respectively) (195, 204-207). BpNETs are diagnosed at a median age of ± 45 years and the reported age at bpNET diagnosis ranges between 20 and 69 years. Although the earliest report suggested a female predominance among MEN1 patients with bpNET (205), later studies could not find a relationship between the occurrence of bpNET and sex (200, 204, 206, 207). Likewise, genotype (the type of mutation) or smoking status does not seem to influence the development of bpNET in MEN1 patients (204, 206, 207).

Only a minority of patients experience symptoms (dyspnea, cough, hemoptysis), which explains the high rate of bpNET (77–100%) diagnosed through periodic thoracic imaging surveillance (195, 206, 207). Growth analysis of lung lesions highly suspect of bpNET have demonstrated their overall indolent course, illustrated by a tumor doubling time of ±12 years at long-term follow-up in a Dutch national cohort study (204). However, a very small number of lesions showed sudden aggressive tumor growth. Unfortunately, no prognostic factors for tumor growth have been identified to date.

The vast majority of MEN1-related bpNETs are well-differentiated NETs (typical and atypical carcinoids); only five cases of poorly differentiated neuroendocrine carcinomas have been identified in MEN1 patients until now, all in the French Groupe d’étude des Tumeurs Endocrines (GTE) cohort (206). Considering the large cohort size (n=1023 MEN1 patients), long-term follow-up, high frequency of smokers and lack of molecular analyses confirming a causal relationship with the MEN1 syndrome, a sporadic coincidental occurrence of neuroendocrine carcinomas in MEN1 patients might also be a possible explanation for the manifestation of these carcinomas in this particular study. The overall benign histopathological characteristics of MEN1-related bpNET may explain their usually good prognosis: large cohort studies have shown that bpNETs do not significantly affect survival in MEN1 patients (204, 206), although a few (eight) aggressive cases with fatal outcome have been described (206, 207). A recent comparison between patients with MEN1-related and sporadic bpNET with comparable histopathological features showed a significantly higher disease-specific mortality in sporadic bpNET, however this has not yet been confirmed in other cohorts (208).

Data from the largest cohort of histologically proven bpNETs in MEN1 patients (n=51) suggested that patients with distant metastasis at diagnosis and non-operated patients had a significantly worse survival (206). Additionally, females, patients with a typical carcinoid (compared to atypical carcinoid) and those without lymph node involvement tended to have a better survival (p=0.07, p=0.08 and p=0.08, respectively (206). However, the most recent Dutch cohort study could not find any prognostic factors (204).

Surgical resection is considered the first treatment of choice, and should be done as lung-sparing as possible, including considering endobronchial resection if feasible (2, 203). Given their usually indolent course, watch-and-wait policy may be considered in patients with small, non-central, slow-growing lesions (203). Parallel to treatment regimens in sporadic bpNET, additional radiotherapy and/or chemotherapy could be used in case of persistent or metastasized disease, although data on the effect of these regimens in aggressive MEN1-related bpNET is very limited.

Surveillance

Current clinical guidelines recommend thoracic imaging (CT or MRI) every 1-2 years for detection of thymic and bronchopulmonary NETs (2). The optimal imaging modality still remains to be elucidated, although CT scans are used in the far majority of cases. A direct comparison between MRI and CT scans among MEN1 patients is lacking, and the role of nuclear imaging in screening programs for thoracic NETs has to be determined yet (209-212). Furthermore, the frequency of periodic surveillance is subject of debate; on one hand, the overall indolent course of bpNET and rareness of thNET might argue for less frequent screening – thereby diminishing radiation exposure, physical and psychological distress for patients, and health care costs –, but the lack of predictors for (sudden) aggressive tumor growth in bpNET and the aggressive nature of thNET plead for frequent thoracic imaging in order to enable timely intervention if necessary. Therefore, treating physicians should inform their patients about the benefits and disadvantages of a strict surveillance program, in order to come to a personalized surveillance strategy based on shared decision-making.

ADRENAL TUMORS

Adrenal involvement (Figure 1) is frequently seen in patients with MEN1 and considered to be part of the syndrome though not one of the cardinal manifestations. Mice with heterozygous inactivation of the Men1 gene develop adrenocortical lesions to a greater proportion than Men1 wild-type controls (213, 214) and the adrenal tumors show loss of heterogeneity (LOH) and loss of menin staining. In humans with MEN1, LOH is rarely seen in benign adrenocortical tumors (215-217). It has been hypothesized that the development of adrenal tumors in MEN1 might be related to PanNETs and hyperinsulinemia, because in some cohorts an association was seen between the occurrence of PanNETs, hyperinsulinemia, and adrenal lesions. However, in the largest series to date, no difference was found in the prevalence of main MEN1 manifestations between those with and without adrenal lesions (217).

In retrospective cohorts on adrenal involvement in MEN1 the reported prevalence greatly differs from 20-73% (216-225). Prevalence in part differs by the way adrenal lesions are defined (i.e. also including hyperplasia) and the manner of diagnosis, with prevalence being the highest (73%) in an EUS study (n=49) including all adrenal lesions from ‘plump’ adrenals to adenomas (221). In the series (n=27) with the second-highest prevalence (63%) all CT scans were re-read with the purpose of classifying adrenal lesions and every adrenocortical lesion >5 mm was considered a nodule (225). The largest series to date from the French GTE (n=715) has the lowest prevalence of 20.4% (217). Adrenal lesions are rarely the reason for an MEN1 diagnosis or the first manifestation of the disease, and are most frequently diagnosed asymptomatically by screening/surveillance imaging during follow-up or at the time of initial comprehensive imaging after the diagnosis of MEN1 is made (217, 223). Mean age of diagnosis is usually in the fifth decade, but ranges vary widely (217, 223, 224).

The French GTE series compared MEN1-related adrenal lesions to a cohort of sporadic incidentalomas (n=144) and found that adrenal lesions in patients with MEN1 were diagnosed at a younger age and were similar in size and in prevalence of bilateral lesions (217).

Benign Adrenocortical Tumors

Most MEN1-related adrenal lesions are benign adrenocortical lesions and include hyperplasia, (macro)nodular hyperplasia, and adenomas. Bilateral lesions are frequently seen, but again prevalence reported varies widely from 12.5% to >50% in different series( 216-219, 221-224). Most adrenocortical lesions in MEN1 are non-functioning and generally stable over the course of follow-up (216-224). In a minority of the cases ACTH-independent hypercortisolism, autonomous cortisol secretion, or primary hyperaldosteronism are seen (216-219, 221-224). Interestingly, in the French series, when comparing MEN1-related adrenal lesions with adrenal incidentalomas, functional tumors were more common (15% vs 6.9%), especially primary hyperaldosteronism and ACTH-independent hypercortisolism (217). Pheochromocytomas on the other hand were more common among sporadic incidentalomas.

Adrenocortical Carcinoma

Adrenocortical carcinoma (ACC) is a rare occurrence among patients with MEN1, with a 2019 review of literature identifying 19 published cases (226). In the Swedish cohort, one patient had an ACC and in the tumor LOH at the MEN1 locus was seen (216, 222). Three papers reporting from the same German institutions reported two, four and one case of ACC respectively, but some of these might represent the same patients (220, 224, 227). In the large French series, eight patients with 10 ACCs were reported, which was 5% of patients with an adrenal lesion, but 13.8% of those with an adrenal tumor (>10mm) (217). ACC prevalence was also significantly higher than in the sporadic adrenal incidentaloma cohort (217). There are also several case reports describing patients with MEN1-related ACC (226, 228-233). It is important to mention that there are several cases reported were the ACC developed from an initially observed relatively small adrenocortical tumor, although when reported these lesions did not have Houndsfield Units (HU) ≤ 10. In the French cohort one patient had two nodules (8 and 13 mm, 38 HU) in one adrenal, which grew to 29 and 30 mm after 5 years, which turned out to be ACCs. Another patient had a 25 mm calcified lesion (40 HU) which grew to 40 mm in 4 years(217). One case report describes a female patient with a 2 cm left adrenal tumor which grew to 3 cm in 7 years (advised to undergo surgery but refused) and then to 4 cm in 4 years, after which the tumor was removed and turned out to be an ACC (233). In other cases more rapidly progressing tumors are described (220, 231). When ACCs are functioning, they are mostly cortisol producing or sex-steroid producing. In sporadic ACC,MEN1 is considered one of the driver genes (234).

Pheochromocytoma

Pheochromocytoma is one of the hallmark conditions of Multiple Endocrine Neoplasia type 2 (MEN2), caused by germline mutations in the RET oncogene. In MEN1, the occurrence of pheochromocytoma is rare, with a 2020 case report and review of literature describing 20 published cases (235). The authors identified LOH at the MEN1 locus in the resected pheochromocytoma of the patient they report (235), and in another published series, two resected pheochromocytomas from patients with MEN1 were examined and LOH at the MEN1 locus was found in both, with one having absent menin staining and one weak menin staining (236).

Screening, Treatment and Follow-up

In patients with MEN1, minimal recommended screening for adrenal lesions as per the current guidelines, is abdominal imaging with CT or MRI every 3 years for those without adrenal lesions (2). Since abdominal imaging is also performed to screen for and/or surveille pancreatic lesions this can often be combined. However, care should be taken that the adrenals are properly imaged in the right phase to not only judge their size but also, should a lesion be present, to judge its characteristics. The preponderance to develop adrenal lesions should be mentioned in the clinical information to the radiologist and the images should be read by a radiologist experienced in adrenal imaging.

If an adrenal lesion is identified hormonal screening is recommended if patients are symptomatic or lesions are >1 cm (2). The current guidelines recommend that screening be focused on hyperaldosteronism and hypercortisolism (2), but given that pheochromocytomas do occur in MEN1 as described above and the consequence of missing the diagnosis can be serious, it is prudent to screen for the presence of a pheochromocytoma by either plasma free (nor)metanephrines or urinary fractionated (nor)metanephrines.

Indications for surgical resection parallel those of adrenal incidentalomas, being clinically significant hormone excess and/or concerns about malignancy either due to atypical characteristics on imaging, size (>4 cm), or significant growth over a 6-month period, which in the adrenal incidentaloma guidelines is suggested as increase in 20% (in addition to at least 5mm increase in actual size) (2, 237). Given the reports of ACCs in MEN1 arising from initially small lesions, some authors recommend to use a size cut-off of 3 cm in MEN1 (220).

If there is no indication for surgery, surveillance imaging is indicated, initially after 6 months. In the absence of a surgical indication at this point, frequency of further imaging follow-up should be determined individually and discussed by the multidisciplinary team. Unlike in sporadic adrenal incidentalomas, surveillance cannot be ended, given that multiple adrenal lesions can arise. There may be indications during follow-up to repeat initially negative hormonal screening, such as the development of symptoms or a new adrenal lesion.

CUTANEOUS LESIONS

Facial angiofibromas and collagenomas are the main skin lesions in MEN1 (Figure 1) (238, 239). Frequencies of 64% for angiofibromas and 62% for collagenomas have been described. Multiple angiofibromas and collagenomas are present in 77–81% of the MEN1 patients (238). Primarily angiofibromas are seen in patients with MEN1 (238, 240). An odds ratio of 6.6 (95% CI, 1.09–40.43) for cutaneous lesions in MEN1 in 29 patients with MEN1 in comparison with their non- affected family members is described (240).

These findings are further supported by the allelic loss of the MEN1 gene in six angiofibromas, three collagenomas, and one lipoma, suggesting that loss of function of the wild-type MEN1 gene product plays a role in the development of these skin lesions in patients with MEN1(241). Melanomas and other skin lesions are also described in the MEN1 population, but not with significant prevalences.

Lipomas (Figure 1) are reported in 17-34% of patients with MEN1 (238-240). Loss of heterozygosity of the MEN1 gene is described in MEN1-related lipomas (97, 241, 242) and may also play a role in sporadic lipomas (242). Menin seems to be an important factor for adipogenesis and contributes to lipoma development (243, 244).

A case of a novel MEN1 gene mutation with a recurrent sarcoma addresses the need for cautiousness of (atypical) skin lesions in patients with MEN1 (245).

BREAST CANCER AND MEN1

A higher incidence of breast cancer (Figure 1) was found in four independent MEN1 cohorts in the Netherlands, France, Tasmania and the United States. In the Dutch cohort a relative risk of 2.83 was found, which was significantly higher than in the general Dutch population (3). The median age for breast cancer was 45 years, which is approximately 15 years younger than the general Dutch population. The increased risk for breast cancer for MEN1 carriers was not associated with other breast cancer risk factors or a familial breast cancer risk. Considering the younger age of breast cancer occurrence and an earlier age of breast cancer, surveillance should be considered. Breast cancer surveillance from the age of 40 is initiated in the Dutch MEN1 cohort (4). After the latter publication, several cases of early breast cancer in MEN1 patients were reported (246-249).

These epidemiological findings are supported by basic research. Loss-of-function Men1 mouse models have shown an increased incidence of both in situ and invasive mammary cancer (250). Menin, the tumor suppressor protein encoded by MEN1, is co-localized with the estrogen receptor (ER) alfa in breast cancer cells. In this manner, menin functions as a direct activator of Erα (251). In sporadic ER-positive breast cancer, menin seems to have a proliferative role, which is in contrast with breast cancer in MEN1 carriers, in whom LOH of the MEN1 gene could be found (3, 252). Recent studies showed that reduced menin staining is associated with ER-negative breast cancer and in ER-positive breast cancer with larger tumors, higher grade tumor, and luminal subtypes tumors. Providing further evidence that there is an important role of menin in ERα regulation and the breast cancer formation (253).

PSYCHOSOCIAL ASPECTS

Recently there has been more interest in the psychosocial wellbeing of patients with MEN1 (254-259).The first study was published in 2003, which showed that psychosocial outcomes such as anxiety, depression, intrusion, and avoidance are not altered by the hospital or home setting. A higher burden of disease led to more depression. Compared to the population-based norm values, patients with MEN1 scored lower for General Health and Social Functioning according to the SF-36 (260).

Postoperatively, quality of life (QOL) scores did not differ after pancreaticoduodenal surgery in MEN1 patients in comparison with the general population. Financial difficulties caused by the treatment were significantly worse in MEN1 patients (261). Financial burden seems to be associated with having MEN1. The degree of financial burden has a linear relationship with worse health-related QOL. Patients were three-times more likely to be unemployed in comparison with the US population (256).

The largest QOL-related study showed that employment status was the most consistent predictor for QOL. This is in line with the former studies. The health-related QOL according to the SF-36 was significantly lower for patients with MEN1 on all subscales except for the physical functioning scale. Patients who are aware of their PA and PanNET have worse QOL scores in comparison with patients who are not aware of having these tumors(259). The degree of fear of disease recurrence is high in patients with MEN1. This fear is negatively associated with health-related QOL and is higher in patients who consider themselves at high risk for developing a MEN1-related tumor. More MEN1-related manifestations lead to more fear of disease occurrence (258). In comparison with other chronic diseases MEN1 scores worse regarding anxiety, depression and fatigue (257).

QOL did not overly differ from the general population in the Italian cohort (255) and patients were more optimistic than in the Swedish cohort (260). This could be due to cultural differences, population selection, and awareness of the disease and its implications.

The high response rate in the MEN1 population illustrates the motivation of patients to participate in research and care about their wellbeing (259, 260).

CONCLUSION

In conclusion, in the past decades there have been great advances in the understanding of the natural course of MEN1-related tumors, which has had direct consequences on clinical care. In the coming decade one of the main research objectives will be the identification of individual predictors of disease course which can guide personalized treatment and surveillance. Increasing international collaborations will enable prospective studies. Given the complexity of the disease, it is strongly advised that patients, whenever possible, be followed and treated in centers of expertise. If this is not feasible, consultation with a center of expertise should be considered.

REFERENCES

Lloyd, R.V., et al., WHO Classification of Tumours of Endocrine Organs. 4th ed. 2017, Lyon: IARC.
Thakker, R.V., et al., Clinical practice guidelines for multiple endocrine neoplasia type 1 (MEN1). The Journal of clinical endocrinology & metabolism, 2012. 97(9): p. 2990-3011.
Dreijerink, K.M.a., et al., Breast-Cancer Predisposition in Multiple Endocrine Neoplasia Type 1. New England Journal of Medicine, 2014. 371(6): p. 583-584.
van Leeuwaarde, R.S., et al., MEN1-Dependent Breast Cancer: Indication for Early Screening? Results From the Dutch MEN1 Study Group. J Clin Endocrinol Metab, 2017. 102(6): p. 2083-2090.
Brandi, M.L., et al., Guidelines for diagnosis and therapy of MEN type 1 and type 2. The Journal of clinical endocrinology & metabolism, 2001. 86(12): p. 5658-71.
Chandrasekharappa, S.C., et al., Positional Cloning of the Gene for Multiple Endocrine Neoplasia-Type 1 Science, 1997. 276: p. 404-407.
Lemmens, I., et al., Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. Human molecular genetics, 1997. 6: p. 1177-83.
Lemos, M.C. and R.V. Thakker, Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Human mutation, 2008. 29(1): p. 22-32.
Concolino, P., A. Costella, and E. Capoluongo, Multiple endocrine neoplasia type 1 (MEN1): An update of 208 new germline variants reported in the last nine years. Cancer Genet, 2016. 209(1-2): p. 36-41.
Kooblall, K.G., et al., Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression. J Bone Miner Res, 2021. 36(1): p. 100-109.
Agarwal, S.K., The future: genetics advances in MEN1 therapeutic approaches and management strategies. Endocr Relat Cancer, 2017. 24(10): p. T119-t134.
Romanet, P., et al., Proposition of adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants. Hum Mutat, 2019. 40(6): p. 661-674.
Thevenon, J., et al., Higher risk of death among MEN1 patients with mutations in the JunD interacting domain: a Groupe d'etude des Tumeurs Endocrines (GTE) cohort study. Human molecular genetics, 2013. 22: p. 1940-1948.
Bartsch, D.K., et al., Higher risk of aggressive pancreatic neuroendocrine tumors in MEN1 patients with MEN1 mutations affecting the CHES1 interacting MENIN domain. J Clin Endocrinol Metab, 2014. 99(11): p. E2387-91.
Klein Haneveld, M.J., et al., Initiating pancreatic neuroendocrine tumour (pNET) screening in young MEN1 patients: results from the DutchMEN Study Group. J Clin Endocrinol Metab, 2021.
van den Broek, M.F.M., et al., Clues For Genetic Anticipation In Multiple Endocrine Neoplasia Type 1. J Clin Endocrinol Metab, 2020. 105(7).
Beijers, H., et al., Germline and somatic mosaicism in a family with multiple endocrine neoplasia type 1 (MEN1) syndrome. Eur J Endocrinol, 2019. 180(2): p. K15-k19.
Dreijerink, K.M.A., H.T.M. Timmers, and M. Brown, Twenty years of menin: emerging opportunities for restoration of transcriptional regulation in MEN1. Endocr Relat Cancer, 2017. 24(10): p. T135-t145.
Canaff, L., et al., Impaired transforming growth factor-β (TGF-β) transcriptional activity and cell proliferation control of a menin in-frame deletion mutant associated with multiple endocrine neoplasia type 1 (MEN1). The Journal of biological chemistry, 2012. 287: p. 8584-97.
Iyer, S. and S.K. Agarwal, Epigenetic regulation in the tumorigenesis of MEN1-associated endocrine cell types. J Mol Endocrinol, 2018. 61(1): p. R13-r24.
Walls, G.V., et al., MEN1 gene replacement therapy reduces proliferation rates in a mouse model of pituitary adenomas. Cancer research, 2012. 72: p. 5060-8.
Pieterman, C.R., et al., Care for patients with multiple endocrine neoplasia type 1: the current evidence base. Fam Cancer, 2011. 10(1): p. 157-71.
van Leeuwaarde, R.S., et al., The future: medical advances in MEN1 therapeutic approaches and management strategies. Endocr Relat Cancer, 2017. 24(10): p. T179-t193.
de Laat, J.M., et al., MEN1 redefined, a clinical comparison of mutation-positive and mutation-negative patients. BMC Med, 2016. 14(1): p. 182.
Pieterman, C.R.C., et al., Understanding the clinical course of genotype-negative MEN1 patients can inform management strategies. Surgery, 2020.
Goudet, P., et al., Risk factors and causes of death in MEN1 disease. A GTE (Groupe d'Etude des Tumeurs Endocrines) cohort study among 758 patients. World journal of surgery, 2010. 34(2): p. 249-55.
Geerdink, E.A., R.B. Van der Luijt, and C.J. Lips, Do patients with multiple endocrine neoplasia syndrome type 1 benefit from periodical screening? European journal of endocrinology, 2003. 149(6): p. 577-82.
de Laat, J.M., R.S. van Leeuwaarde, and G.D. Valk, The Importance of an Early and Accurate MEN1 Diagnosis. Front Endocrinol (Lausanne), 2018. 9: p. 533.
de Laat, J.M., et al., Predicting the risk of multiple endocrine neoplasia type 1 for patients with commonly occurring endocrine tumors. European journal of endocrinology, 2012. 167(2): p. 181-7.
van Leeuwaarde, R.S., et al., Impact of delay in diagnosis in outcomes in MEN1: results from the Dutch MEN1 study group. J Clin Endocrinol Metab, 2016: p. jc20153766.
Alrezk, R., F. Hannah-Shmouni, and C.A. Stratakis, MEN4 and CDKN1B mutations: the latest of the MEN syndromes. Endocr Relat Cancer, 2017. 24(10): p. T195-t208.
Frederiksen, A., et al., Clinical Features of Multiple Endocrine Neoplasia Type 4: Novel Pathogenic Variant and Review of Published Cases. J Clin Endocrinol Metab, 2019. 104(9): p. 3637-3646.
Georgitsi, M., et al., Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia. The Journal of clinical endocrinology and metabolism, 2007. 92: p. 3321-3325.
van der Tuin, K., et al., CDC73-Related Disorders: Clinical Manifestations and Case Detection in Primary Hyperparathyroidism. J Clin Endocrinol Metab, 2017. 102(12): p. 4534-4540.
van den Broek, M.F.M., et al., Clinical Relevance of Genetic Analysis in Patients With Pituitary Adenomas: A Systematic Review. Front Endocrinol (Lausanne), 2019. 10: p. 837.
Stratakis, C.A., et al., Pituitary macroadenoma in a 5-year-old: an early expression of multiple endocrine neoplasia type 1. J Clin Endocrinol Metab, 2000. 85(12): p. 4776-80.
Manoharan, J., et al., Is Routine Screening of Young Asymptomatic MEN1 Patients Necessary? World J Surg, 2017. 41(8): p. 2026-2032.
Romanet, P., et al., UMD-MEN1 Database: An Overview of the 370 MEN1 Variants Present in 1676 Patients From the French Population. J Clin Endocrinol Metab, 2019. 104(3): p. 753-764.
Goudet, P., et al., Hyperparathyroidism in multiple endocrine neoplasia type I: surgical trends and results of a 256-patient series from Groupe D'etude des Néoplasies Endocriniennes Multiples Study Group. World J Surg, 2001. 25(7): p. 886-90.
Pieterman, C.R., et al., Multiple endocrine neoplasia type 1 (MEN1): its manifestations and effect of genetic screening on clinical outcome. Clinical endocrinology, 2009. 70(4): p. 575-81.
Marini, F., F. Giusti, and M.L. Brandi, Multiple endocrine neoplasia type 1: extensive analysis of a large database of Florentine patients. Orphanet J Rare Dis, 2018. 13(1): p. 205.
Giusti, F., et al., Multiple endocrine neoplasia syndrome type 1: institution, management, and data analysis of a nationwide multicenter patient database. Endocrine, 2017. 58(2): p. 349-359.
Sakurai, A., et al., Multiple endocrine neoplasia type 1 in Japan: establishment and analysis of a multicentre database. Clinical endocrinology, 2012. 76(4): p. 533-9.
Soczomski, P., et al., Multiple endocrine neoplasia type 1 in Poland: a two-centre experience. Endokrynol Pol, 2019. 70(5): p. 385-391.
Waldmann, J., et al., Screening of patients with multiple endocrine neoplasia type 1 (MEN-1): a critical analysis of its value. World journal of surgery, 2009. 33(6): p. 1208-18.
Herath, M., et al., Paediatric and young adult manifestations and outcomes of multiple endocrine neoplasia type 1. Clin Endocrinol (Oxf), 2019. 91(5): p. 633-638.
Goudet, P., et al., MEN1 disease occurring before 21 years old: a 160-patient cohort study from the Groupe d'etude des Tumeurs Endocrines. J Clin Endocrinol Metab, 2015. 100(4): p. 1568-77.
Shariq, O.A., et al., Multiple endocrine neoplasia type 1 in children and adolescents: Clinical features and treatment outcomes. Surgery, 2021.
Vannucci, L., et al., MEN1 in children and adolescents: Data from patients of a regional referral center for hereditary endocrine tumors. Endocrine, 2018. 59(2): p. 438-448.
Marx, S.J. and D.M. Lourenço, Jr., Questions and Controversies About Parathyroid Pathophysiology in Children With Multiple Endocrine Neoplasia Type 1. Front Endocrinol (Lausanne), 2018. 9: p. 359.
Doherty, G.M., T.C. Lairmore, and M.K. DeBenedetti, Multiple endocrine neoplasia type 1 parathyroid adenoma development over time. World J Surg, 2004. 28(11): p. 1139-42.
d'Alessandro, A.F., et al., Supernumerary parathyroid glands in hyperparathyroidism associated with multiple endocrine neoplasia type 1. Rev Assoc Med Bras (1992), 2012. 58(3): p. 323-7.
Yeh, M.W., et al., Incidence and prevalence of primary hyperparathyroidism in a racially mixed population. J Clin Endocrinol Metab, 2013. 98(3): p. 1122-9.
Press, D.M., et al., The prevalence of undiagnosed and unrecognized primary hyperparathyroidism: a population-based analysis from the electronic medical record. Surgery, 2013. 154(6): p. 1232-7; discussion 1237-8.
Eller-Vainicher, C., et al., Sporadic and MEN1-related primary hyperparathyroidism: differences in clinical expression and severity. J Bone Miner Res, 2009. 24(8): p. 1404-10.
Twigt, B.A., et al., Differences between sporadic and MEN related primary hyperparathyroidism; clinical expression, preoperative workup, operative strategy and follow-up. Orphanet J Rare Dis, 2013. 8: p. 50.
Wilhelm, S.M., et al., The American Association of Endocrine Surgeons Guidelines for Definitive Management of Primary Hyperparathyroidism. JAMA Surg, 2016. 151(10): p. 959-968.
Iacobone, M., et al., Hereditary hyperparathyroidism--a consensus report of the European Society of Endocrine Surgeons (ESES). Langenbecks Arch Surg, 2015. 400(8): p. 867-86.
Silva, A.M., et al., Operative intervention for primary hyperparathyroidism offers greater bone recovery in patients with sporadic disease than in those with multiple endocrine neoplasia type 1-related hyperparathyroidism. Surgery, 2017. 161(1): p. 107-115.
Wang, W., et al., Impaired geometry, volumetric density, and microstructure of cortical and trabecular bone assessed by HR-pQCT in both sporadic and MEN1-related primary hyperparathyroidism. Osteoporos Int, 2020. 31(1): p. 165-173.
Kong, J., et al., Clinical and Genetic Analysis of Multiple Endocrine Neoplasia Type 1-Related Primary Hyperparathyroidism in Chinese. PLoS One, 2016. 11(11): p. e0166634.
Lourenço, D.M., Jr., et al., Early-onset, progressive, frequent, extensive, and severe bone mineral and renal complications in multiple endocrine neoplasia type 1-associated primary hyperparathyroidism. J Bone Miner Res, 2010. 25(11): p. 2382-91.
Burgess, J.R., et al., Osteoporosis in multiple endocrine neoplasia type 1: severity, clinical significance, relationship to primary hyperparathyroidism, and response to parathyroidectomy. Arch Surg, 1999. 134(10): p. 1119-23.
Lourenco, D.M., Jr., et al., Multiple endocrine neoplasia type 1 in Brazil: MEN1 founding mutation, clinical features, and bone mineral density profile. European journal of endocrinology, 2008. 159(3): p. 259-74.
Coutinho, F.L., et al., Bone mineral density analysis in patients with primary hyperparathyroidism associated with multiple endocrine neoplasia type 1 after total parathyroidectomy. Clin Endocrinol (Oxf), 2010. 72(4): p. 462-8.
Kann, P.H., et al., Peripheral bone mineral density in correlation to disease-related predisposing conditions in patients with multiple endocrine neoplasia type 1. J Endocrinol Invest, 2012. 35(6): p. 573-9.
Green, P., et al., High prevalence of chronic kidney disease in patients with multiple endocrine neoplasia type 1 and improved kidney function after parathyroidectomy. Surgery, 2019. 165(1): p. 124-128.
Bilezikian, J.P., et al., Guidelines for the management of asymptomatic primary hyperparathyroidism: summary statement from the Fourth International Workshop. J Clin Endocrinol Metab, 2014. 99(10): p. 3561-9.
Norton, J.A., et al., Prospective study of surgery for primary hyperparathyroidism (HPT) in multiple endocrine neoplasia-type 1 and Zollinger-Ellison syndrome: long-term outcome of a more virulent form of HPT. Ann Surg, 2008. 247(3): p. 501-10.
Hackeng, W.M., et al., A Parathyroid-Gut Axis: Hypercalcemia and the Pathogenesis of Gastrinoma in Multiple Endocrine Neoplasia 1. Mol Cancer Res, 2021. 19(6): p. 946-949.
Nobecourt, P.F., et al., Intraoperative Decision-Making and Technical Aspects of Parathyroidectomy in Young Patients With MEN1 Related Hyperparathyroidism. Front Endocrinol (Lausanne), 2018. 9: p. 618.
Nastos, C., et al., Optimal extent of initial parathyroid resection in patients with multiple endocrine neoplasia syndrome type 1: A meta-analysis. Surgery, 2021. 169(2): p. 302-310.
Schreinemakers, J.M., et al., The optimal surgical treatment for primary hyperparathyroidism in MEN1 patients: a systematic review. World J Surg, 2011. 35(9): p. 1993-2005.
Manoharan, J., et al., Single gland excision for MEN1-associated primary hyperparathyroidism. Clin Endocrinol (Oxf), 2020. 92(1): p. 63-70.
Nilubol, N., et al., Preoperative localizing studies for initial parathyroidectomy in MEN1 syndrome: is there any benefit? World J Surg, 2012. 36(6): p. 1368-74.
Nilubol, N., et al., Utility of intraoperative parathyroid hormone monitoring in patients with multiple endocrine neoplasia type 1-associated primary hyperparathyroidism undergoing initial parathyroidectomy. World J Surg, 2013. 37(8): p. 1966-72.
Kluijfhout, W.P., et al., Unilateral Clearance for Primary Hyperparathyroidism in Selected Patients with Multiple Endocrine Neoplasia Type 1. World J Surg, 2016. 40(12): p. 2964-2969.
Montenegro, F.L.M., et al., Could the Less-Than Subtotal Parathyroidectomy Be an Option for Treating Young Patients With Multiple Endocrine Neoplasia Type 1-Related Hyperparathyroidism? Front Endocrinol (Lausanne), 2019. 10: p. 123.
Choi, H.R., et al., Benefit of diverse surgical approach on short-term outcomes of MEN1-related hyperparathyroidism. Sci Rep, 2020. 10(1): p. 10634.
Nilubol, N., et al., Limited Parathyroidectomy in Multiple Endocrine Neoplasia Type 1-Associated Primary Hyperparathyroidism: A Setup for Failure. Ann Surg Oncol, 2016. 23(2): p. 416-23.
Keutgen, X.M., et al., Reoperative Surgery in Patients with Multiple Endocrine Neoplasia Type 1 Associated Primary Hyperparathyroidism. Ann Surg Oncol, 2016. 23(Suppl 5): p. 701-707.
Bioletto, F., et al., Comparison of the diagnostic accuracy of 18F-Fluorocholine PET and 11C-Methionine PET for parathyroid localization in primary hyperparathyroidism: a systematic review and meta-analysis. Eur J Endocrinol, 2021. 185(1): p. 109-120.
Hindié, E., et al., Primary Hyperparathyroidism: Defining the Appropriate Preoperative Imaging Algorithm. J Nucl Med, 2021. 62(Suppl 2): p. 3s-12s.
Gauthé, M., et al., (18)F-fluorocholine PET/CT in MEN1 Patients with Primary Hyperparathyroidism. World J Surg, 2020. 44(11): p. 3761-3769.
Fyrsten, E., et al., Long-Term Surveillance of Treated Hyperparathyroidism for Multiple Endocrine Neoplasia Type 1: Recurrence or Hypoparathyroidism? World J Surg, 2016. 40(3): p. 615-21.
Pieterman, C.R., et al., Primary hyperparathyroidism in MEN1 patients: a cohort study with longterm follow-up on preferred surgical procedure and the relation with genotype. Ann Surg, 2012. 255(6): p. 1171-8.
Filopanti, M., et al., MEN1-related hyperparathyroidism: response to cinacalcet and its relationship with the calcium-sensing receptor gene variant Arg990Gly. Eur J Endocrinol, 2012. 167(2): p. 157-64.
Giusti, F., et al., Cinacalcet therapy in patients affected by primary hyperparathyroidism associated to Multiple Endocrine Neoplasia Syndrome type 1 (MEN1). Endocrine, 2016. 52(3): p. 495-506.
Moyes, V.J., et al., Clinical Use of Cinacalcet in MEN1 Hyperparathyroidism. Int J Endocrinol, 2010. 2010: p. 906163.
Geslot, A., et al., New therapies for patients with multiple endocrine neoplasia type 1. Ann Endocrinol (Paris), 2021. 82(2): p. 112-120.
Singh Ospina, N., et al., Safety and efficacy of percutaneous parathyroid ethanol ablation in patients with recurrent primary hyperparathyroidism and multiple endocrine neoplasia type 1. J Clin Endocrinol Metab, 2015. 100(1): p. E87-90.
Williams, M.D., et al., Pathology data set for reporting parathyroid carcinoma and atypical parathyroid neoplasm: recommendations from the International Collaboration on Cancer Reporting. Hum Pathol, 2021. 110: p. 73-82.
Song, A., et al., Prevalence of Parathyroid Carcinoma and Atypical Parathyroid Neoplasms in 153 Patients With Multiple Endocrine Neoplasia Type 1: Case Series and Literature Review. Front Endocrinol (Lausanne), 2020. 11: p. 557050.
Christakis, I., et al., Parathyroid carcinoma and atypical parathyroid neoplasms in MEN1 patients; A clinico-pathologic challenge. The MD Anderson case series and review of the literature. Int J Surg, 2016. 31: p. 10-6.
Singh Ospina, N., et al., Prevalence of parathyroid carcinoma in 348 patients with multiple endocrine neoplasia type 1 - case report and review of the literature. Clin Endocrinol (Oxf), 2016. 84(2): p. 244-249.
Erdheim, J., Zur normalen und pathologischen Histologie der Glandula thyreoidea, parathyreoidea, und Hypophysis. Beitr Pathol Anat, 1903. 33: p. 158-236.
Dong, Q., et al., Loss of heterozygosity at 11q13: analysis of pituitary tumors, lung carcinoids, lipomas, and other uncommon tumors in subjects with familial multiple endocrine neoplasia type 1. The Journal of clinical endocrinology & metabolism, 1997. 82(5): p. 1416-20.
Thakker, R.V., The molecular genetics of the multiple endocrine neoplasia syndromes. Clinical Endocrinology, 1993. 38(1): p. 1-14.
Weil, R.J., et al., 11Q13 Allelic Loss in Pituitary Tumors in Patients With Multiple Endocrine Neoplasia Syndrome Type 1. Clinical Cancer Research, 1998. 4(7): p. 1673-1678.
Yoshimoto, K., et al., Allele Loss on Chromosome 11 in a Pituitary Tumor from a Patient with Multiple Endocrine Neoplasia Type 1. 1991. p. 886-889.
Thakker, R.V., et al., Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations. J Clin Invest, 1993. 91(6): p. 2815-21.
Boggild, M.D., et al., Molecular genetic studies of sporadic pituitary tumors. J Clin Endocrinol Metab, 1994. 78(2): p. 387-92.
Asa, S.L., K. Somers, and S. Ezzat, The MEN-1 gene is rarely down-regulated in pituitary adenomas. J Clin Endocrinol Metab, 1998. 83(9): p. 3210-2.
Farrell, W.E., et al., Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours. Br J Cancer, 1999. 80(1-2): p. 44-50.
Prezant, T.R., J. Levine, and S. Melmed, Molecular characterization of the men1 tumor suppressor gene in sporadic pituitary tumors. J Clin Endocrinol Metab, 1998. 83(4): p. 1388-91.
Tanaka, C., et al., Analysis of loss of heterozygosity on chromosome 11 and infrequent inactivation of the MEN1 gene in sporadic pituitary adenomas. J Clin Endocrinol Metab, 1998. 83(8): p. 2631-4.
Zhuang, Z., et al., Mutations of the MEN1 tumor suppressor gene in pituitary tumors. Cancer Res, 1997. 57(24): p. 5446-51.
Ballard, H.S., B. Fame, and R.J. Hartsock, Familial multiple endocrine adenoma-peptic ulcer complex. Medicine (Baltimore), 1964. 43(0025-7974 (Print)): p. 481-516.
Verges, B., et al., Pituitary disease in MEN type 1 (MEN1): Data from the France-Belgium MEN1 multicenter study. Journal of Clinical Endocrinology & Metabolism, 2002. 87(2): p. 457-465.
Trouillas, J., et al., Pituitary tumors and hyperplasia in multiple endocrine neoplasia type 1 syndrome (MEN1): a case-control study in a series of 77 patients versus 2509 non-MEN1 patients. Am J Surg Pathol, 2008. 32(4): p. 534-43.
de Laat, J.M., et al., Long-term natural course of pituitary tumors in patients with MEN1: results from the Dutch MEN1 study group (DMSG). J Clin.Endocrinol.Metab, 2015(1945-7197 (Electronic)): p. JC20152015-JC20152015.
Wu, Y., et al., Pituitary adenomas in patients with multiple endocrine neoplasia type 1: a single-center experience in China. Pituitary, 2019. 22(2): p. 113-123.
Cohen-Cohen, S., et al., Pituitary adenomas in the setting of multiple endocrine neoplasia type 1: A single-institution experience. Journal of Neurosurgery, 2021. 134(4): p. 1132-1138.
Bras, M.L., et al., Pituitary adenoma in patients with multiple endocrine neoplasia type 1 – a cohort study. 2021: p. 1-34.
Makri, A., et al., Children with MEN1 gene mutations may present first (and at a young age) with Cushing disease. Clin Endocrinol (Oxf), 2018. 89(4): p. 437-443.
Melmed, S., et al., Diagnosis and treatment of hyperprolactinemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 2011. 96(2): p. 273-88.
Nieman, L.K., et al., The diagnosis of Cushing's syndrome: An endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism, 2008. 93(5): p. 1526-1540.
Katznelson, L., et al., Acromegaly: An endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism, 2014. 99(11): p. 3933-3951.
Incandela, F., et al., Malignancy course of pituitary adenoma in MEN1 syndrome: Clinical-Neuroradiological signs. Eur J Radiol Open, 2020. 7: p. 100242.
Philippon, M., et al., Long-term control of a MEN1 prolactin secreting pituitary carcinoma after temozolomide treatment. Ann Endocrinol (Paris), 2012. 73(3): p. 225-9.
Benito, M., et al., Gonadotroph tumor associated with multiple endocrine neoplasia type 1. J Clin Endocrinol Metab, 2005. 90(1): p. 570-4.
Scheithauer, B.W., et al., Multiple endocrine neoplasia type 1-associated thyrotropin-producing pituitary carcinoma: report of a probable de novo example. Hum Pathol, 2009. 40(2): p. 270-8.
Morokuma, H., et al., A case of nonfunctioning pituitary carcinoma that responded to temozolomide treatment. Case Rep Endocrinol, 2012. 2012: p. 645914.
Triponez, F., et al., Epidemiology data on 108 MEN 1 patients from the GTE with isolated nonfunctioning tumors of the pancreas. Annals of surgery, 2006. 243(2): p. 265-72.
Vinault, S., et al., Metastatic Potential and Survival of Duodenal and Pancreatic Tumors in Multiple Endocrine Neoplasia Type 1: A GTE and AFCE Cohort Study (Groupe d'etude des Tumeurs Endocrines and Association Francophone de Chirurgie Endocrinienne). Ann Surg, 2018.
Pipeleers-Marichal, M., et al., Gastrinomas in the duodenums of patients with multiple endocrine neoplasia type 1 and the Zollinger-Ellison syndrome. The New England journal of medicine, 1990. 322(11): p. 723-7.
Levy-Bohbot, N., et al., Prevalence, characteristics and prognosis of MEN 1-associated glucagonomas, VIPomas, and somatostatinomas: study from the GTE (Groupe des Tumeurs Endocrines) registry. Gastroenterologie clinique et biologique, 2004. 28(11): p. 1075-81.
Borson-Chazot, F., et al., Acromegaly induced by ectopic secretion of GHRH: a review 30 years after GHRH discovery. Ann Endocrinol (Paris), 2012. 73(6): p. 497-502.
Milanesi, A., et al., Concurrent primary hyperparathyroidism and humoral hypercalcemia of malignancy in a patient with multiple endocrine neoplasia type 1. Pancreas, 2011. 40(4): p. 634-7.
Klöppel, G., et al., Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas. Endocr Pathol, 2014. 25(2): p. 181-5.
Anlauf, M., et al., Allelic deletion of the MEN1 gene in duodenal gastrin and somatostatin cell neoplasms and their precursor lesions. Gut, 2007. 56(5): p. 637-44.
van Beek, D.J., et al., Surgery for multiple endocrine neoplasia type 1-related insulinoma: long-term outcomes in a large international cohort. Br J Surg, 2020. 107(11): p. 1489-1499.
Gibril, F., et al., Multiple endocrine neoplasia type 1 and Zollinger-Ellison syndrome: a prospective study of 107 cases and comparison with 1009 cases from the literature. Medicine, 2004. 83(1): p. 43-83.
Ito, T., et al., Causes of death and prognostic factors in multiple endocrine neoplasia type 1: a prospective study: comparison of 106 MEN1/Zollinger-Ellison syndrome patients with 1613 literature MEN1 patients with or without pancreatic endocrine tumors. Medicine, 2013. 92(3): p. 135-81.
van Beek, D.J., et al., Prognostic factors and survival in MEN1 patients with gastrinomas: Results from the DutchMEN study group (DMSG). J Surg Oncol, 2019. 120(6): p. 966-975.
Jensen, R.T. and T. Ito, Gastrinoma, in Endotext, K.R. Feingold, et al., Editors. 2000, MDText.com, Inc. Copyright © 2000-2021, MDText.com, Inc.: South Dartmouth (MA).
Hampel, H., et al., A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med, 2015. 17(1): p. 70-87.
Gibril, F., et al., Prospective study of the natural history of gastrinoma in patients with MEN1: definition of an aggressive and a nonaggressive form. The Journal of clinical endocrinology & metabolism, 2001. 86(11): p. 5282-93.
Conemans, E.B., et al., Prognostic factors for survival of MEN1 patients with duodenopancreatic tumors metastatic to the liver: results from the DMSG. Endocr Pract, 2017. 23(6): p. 641-648.
Giudici, F., et al., Natural History of MEN1 GEP-NET: Single-Center Experience After a Long Follow-Up. World J Surg, 2017. 41(9): p. 2312-2323.
Christakis, I., et al., Genotype-phenotype pancreatic neuroendocrine tumor relationship in multiple endocrine neoplasia type 1 patients: A 23-year experience at a single institution. Surgery, 2018. 163(1): p. 212-217.
Nagtegaal, I.D., et al., The 2019 WHO classification of tumours of the digestive system. Histopathology, 2020. 76(2): p. 182-188.
van Treijen, M.J.C., et al., Diagnosing Nonfunctional Pancreatic NETs in MEN1: The Evidence Base. J Endocr Soc, 2018. 2(9): p. 1067-1088.
Pieterman, C.R.C., et al., HEREDITARY ENDOCRINE TUMOURS: CURRENT STATE-OF-THE-ART AND RESEARCH OPPORTUNITIES: MEN1-related pancreatic NETs: identification of unmet clinical needs and future directives. Endocr Relat Cancer, 2020. 27(8): p. T9-t25.
Cuthbertson, D.J., et al., The Impact of (68)Gallium DOTA PET/CT in Managing Patients With Sporadic and Familial Pancreatic Neuroendocrine Tumours. Front Endocrinol (Lausanne), 2021. 12: p. 654975.
Sadowski, S.M., et al., Prognostic factors for the outcome of nonfunctioning pancreatic neuroendocrine tumors in MEN1: a systematic review of literature. Endocr Relat Cancer, 2020. 27(6): p. R145-r161.
Partelli, S., et al., Active Surveillance versus Surgery of Nonfunctioning Pancreatic Neuroendocrine Neoplasms </=2 cm in MEN1 Patients. Neuroendocrinology, 2016. 103(6): p. 779-786.
Nell, S., et al., Management of MEN1 Related Nonfunctioning Pancreatic NETs: A Shifting Paradigm: Results From the DutchMEN1 Study Group. Ann Surg, 2018. 267(6): p. 1155-1160.
Triponez, F., et al., Is surgery beneficial for MEN1 patients with small (< or = 2 cm), nonfunctioning pancreaticoduodenal endocrine tumor? An analysis of 65 patients from the GTE. World journal of surgery, 2006. 30(5): p. 654-62; discussion 663-4.
Pieterman, C.R.C., et al., Long-Term Natural Course of Small Nonfunctional Pancreatic Neuroendocrine Tumors in MEN1-Results From the Dutch MEN1 Study Group. J Clin Endocrinol Metab, 2017. 102(10): p. 3795-3805.
Hackeng, W.M., et al., Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size. Gut, 2021.
Cejas, P., et al., Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors. Nat Med, 2019. 25(8): p. 1260-1265.
Chan, C.S., et al., ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup. Nat Commun, 2018. 9(1): p. 4158.
Fahrmann, J.F., et al., A Blood-Based Polyamine Signature Associated With Disease Progression in Patients With Multiple Endocrine Neoplasia Type 1-Related Duodenopancreatic Neuroendocrine Tumors. Journal of the Endocrine Society, 2021. 5(Supplement_1): p. A1009-A1009.
Niederle, B., et al., Multiple Endocrine Neoplasia Type 1 (MEN1) and the Pancreas - Diagnosis and Treatment of Functioning and Non-Functioning Pancreatic and Duodenal Neuroendocrine Neoplasia within the MEN1 Syndrome - An International Consensus Statement. Neuroendocrinology, 2020.
Falconi, M., et al., ENETS Consensus Guidelines Update for the Management of Patients with Functional Pancreatic Neuroendocrine Tumors and Non-Functional Pancreatic Neuroendocrine Tumors. Neuroendocrinology, 2016. 103(2): p. 153-71.
Metz, D.C., et al., Diagnosis of Zollinger-Ellison syndrome in the era of PPIs, faulty gastrin assays, sensitive imaging and limited access to acid secretory testing. Int J Endocr Oncol, 2017. 4(4): p. 167-185.
Singh Ospina, N., et al., Assessing for Multiple Endocrine Neoplasia Type 1 in Patients Evaluated for Zollinger-Ellison Syndrome-Clues to a Safer Diagnostic Process. Am J Med, 2017. 130(5): p. 603-605.
Albers, M.B., J. Manoharan, and D.K. Bartsch, Contemporary surgical management of the Zollinger-Ellison syndrome in multiple endocrine neoplasia type 1. Best Pract Res Clin Endocrinol Metab, 2019. 33(5): p. 101318.
Hackeng, W.M., et al., Metastatic Patterns of Duodenopancreatic Neuroendocrine Tumors in Patients With Multiple Endocrine Neoplasia Type 1. Am J Surg Pathol, 2021.
Norton, J.A., et al., Comparison of surgical results in patients with advanced and limited disease with multiple endocrine neoplasia type 1 and Zollinger-Ellison syndrome. Annals of surgery, 2001. 234(4): p. 495-505; discussion 505-6.
Endall, R., et al., The Relationship of Gastrinoma in MEN 1 to Helicobacter pylori infection. J Clin Endocrinol Metab, 2020. 105(3).
Cryer, P.E., et al., Evaluation and management of adult hypoglycemic disorders: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 2009. 94(3): p. 709-28.
Bartsch, D.K., et al., Enucleation and limited pancreatic resection provide long-term cure for insulinoma in multiple endocrine neoplasia type 1. Neuroendocrinology, 2013. 98(4): p. 290-8.
Vezzosi, D., et al., Long-term results of the surgical management of insulinoma patients with MEN1: a Groupe d'étude des Tumeurs Endocrines (GTE) retrospective study. Eur J Endocrinol, 2015. 172(3): p. 309-19.
Tonelli, F., et al., Operation for insulinomas in multiple endocrine neoplasia type 1: When pancreatoduodenectomy is appropriate. Surgery, 2017. 161(3): p. 727-734.
Antwi, K., et al., 68Ga-Exendin-4 PET/CT Detects Insulinomas in Patients With Endogenous Hyperinsulinemic Hypoglycemia in MEN-1. J Clin Endocrinol Metab, 2019. 104(12): p. 5843-5852.
Shah, R., et al., Exendin-4-based imaging in insulinoma localization: Systematic review and meta-analysis. Clin Endocrinol (Oxf), 2021. 95(2): p. 354-364.
van Beek, D.J., et al., Prognosis after surgery for multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors: Functionality matters. Surgery, 2021. 169(4): p. 963-973.
Dasari, B.V.M., et al., Outcomes of Surgical and Endoscopic Resection of Duodenal Neuroendocrine Tumours (NETs): a Systematic Review of the Literature. J Gastrointest Surg, 2018. 22(9): p. 1652-1658.
Delle Fave, G., et al., ENETS Consensus Guidelines Update for Gastroduodenal Neuroendocrine Neoplasms. Neuroendocrinology, 2016. 103(2): p. 119-24.
Rimbaş, M., et al., Interventional endoscopic ultrasound for pancreatic neuroendocrine neoplasms. Dig Endosc, 2020. 32(7): p. 1031-1041.
Caplin, M.E., et al., Lanreotide in metastatic enteropancreatic neuroendocrine tumors. N Engl J Med, 2014. 371(3): p. 224-33.
Caplin, M.E., et al., Anti-tumour effects of lanreotide for pancreatic and intestinal neuroendocrine tumours: the CLARINET open-label extension study. Endocr Relat Cancer, 2016. 23(3): p. 191-9.
Ramundo, V., et al., Impact of long-acting octreotide in patients with early-stage MEN1-related duodeno-pancreatic neuroendocrine tumours. Clinical endocrinology, 2014. 80(6): p. 850-5.
Cioppi, F., et al., The LARO-MEN1 study: a longitudinal clinical experience with octreotide Long-Acting Release in patients with Multiple Endocrine Neoplasia type 1 Syndrome. Clin Cases Miner Bone Metab, 2017. 14(2): p. 123-130.
Faggiano, A., et al., Lanreotide Therapy vs Active Surveillance in MEN1-Related Pancreatic Neuroendocrine Tumors < 2 Centimeters. J Clin Endocrinol Metab, 2020. 105(1).
Pavel, M., et al., ENETS Consensus Guidelines Update for the Management of Distant Metastatic Disease of Intestinal, Pancreatic, Bronchial Neuroendocrine Neoplasms (NEN) and NEN of Unknown Primary Site. Neuroendocrinology, 2016. 103(2): p. 172-85.
Yao, J.C., et al., Everolimus for advanced pancreatic neuroendocrine tumors. The New England journal of medicine, 2011. 364(6): p. 514-23.
Raymond, E., et al., Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. The New England journal of medicine, 2011. 364(6): p. 501-13.
Strosberg, J., et al., Phase 3 Trial of (177)Lu-Dotatate for Midgut Neuroendocrine Tumors. N Engl J Med, 2017. 376(2): p. 125-135.
Frost, M., K.E. Lines, and R.V. Thakker, Current and emerging therapies for PNETs in patients with or without MEN1. Nat Rev Endocrinol, 2018. 14(4): p. 216-227.
Debelenko, L.V., et al., The multiple endocrine neoplasia type I gene locus is involved in the pathogenesis of type II gastric carcinoids. Gastroenterology, 1997. 113(3): p. 773-81.
Lee, L., et al., Insights into Effects/Risks of Chronic Hypergastrinemia and Lifelong PPI Treatment in Man Based on Studies of Patients with Zollinger-Ellison Syndrome. Int J Mol Sci, 2019. 20(20).
Manoharan, J., et al., Gastric enterochromaffin-like cell changes in multiple endocrine neoplasia type 1. Clin Endocrinol (Oxf), 2021. 95(3): p. 439-446.
Richards, M.L., et al., Regression of type II gastric carcinoids in multiple endocrine neoplasia type 1 patients with Zollinger-Ellison syndrome after surgical excision of all gastrinomas. World J Surg, 2004. 28(7): p. 652-8.
Kloppel, G., M. Anlauf, and A. Perren, Endocrine precursor lesions of gastroenteropancreatic neuroendocrine tumors. Endocrine pathology, 2007. 18: p. 150-155.
Berna, M.J., et al., A prospective study of gastric carcinoids and enterochromaffin-like cell changes in multiple endocrine neoplasia type 1 and Zollinger-Ellison syndrome: identification of risk factors. J Clin Endocrinol Metab, 2008. 93(5): p. 1582-91.
Bordi, C., et al., Aggressive forms of gastric neuroendocrine tumors in multiple endocrine neoplasia type I. Am J Surg Pathol, 1997. 21(9): p. 1075-82.
Norton, J.A., et al., Gastric carcinoid tumors in multiple endocrine neoplasia-1 patients with Zollinger-Ellison syndrome can be symptomatic, demonstrate aggressive growth, and require surgical treatment. Surgery, 2004. 136(6): p. 1267-74.
Mandl, A., et al., Two distinct classes of thymic tumors in patients with MEN1 show LOH at the MEN1 locus. Endocr Relat Cancer, 2021. 28(11): p. L15-l19.
Volante, M., et al., Molecular Pathology of Well-Differentiated Pulmonary and Thymic Neuroendocrine Tumors: What Do Pathologists Need to Know? Endocr Pathol, 2021. 32(1): p. 154-168.
Teh, B.T., et al., Clinicopathologic Studies of Thymic Carcinoids in Multiple Endocrine Neoplasia Type 1. Medicine (Baltimore), 1997. 76(1): p. 21-29.
Ye, L., et al., Clinical features and prognosis of thymic neuroendocrine tumours associated with multiple endocrine neoplasia type 1: A single-centre study, systematic review and meta-analysis. Clinical Endocrinology, 2017. 87(6): p. 706-716.
Singh Ospina, N., et al., Thymic and Bronchial Carcinoid Tumors in Multiple Endocrine Neoplasia Type 1: The Mayo Clinic Experience from 1977 to 2013. Hormones and Cancer, 2015. 6(5-6): p. 247-253.
Sakurai, A., et al., Thymic neuroendocrine tumour in multiple endocrine neoplasia type 1: Female patients are not rare exceptions. Clinical Endocrinology, 2013. 78(2): p. 248-254.
Goudet, P., et al., Thymic neuroendocrine tumors in multiple endocrine neoplasia type 1: A comparative study on 21 cases among a series of 761 MEN1 from the GTE (Groupe des Tumeurs Endocrines). World Journal of Surgery, 2009. 33(6): p. 1197-1207.
Gibril, F., et al., Prospective study of thymic carcinoids in patients with multiple endocrine neoplasia type 1. Journal of Clinical Endocrinology and Metabolism, 2003. 88(3): p. 1066-1081.
Ferolla, P., et al., Thymic neuroendocrine carcinoma (carcinoid) in multiple endocrine neoplasia type 1 syndrome: The Italian series. Journal of Clinical Endocrinology and Metabolism, 2005. 90(5): p. 2603-2609.
de Laat, J.M., et al., Natural course and survival of neuroendocrine tumors of thymus and lung in MEN1 patients. Journal of Clinical Endocrinology and Metabolism, 2014. 99(9): p. 3325-3333.
Christakis, I., et al., Clinical Features, Treatments, and Outcomes of Patients with Thymic Carcinoids and Multiple Endocrine Neoplasia Type 1 Syndrome at MD Anderson Cancer Center. Hormones and Cancer, 2016. 7(4): p. 279-287.
Lim, L.C., et al., Thymic carcinoid in multiple endocrine neoplasia 1: Genotype-phenotype correlation and prevention. Journal of Internal Medicine, 2006. 259(4): p. 428-432.
Sadowski, S.M., et al., The future: surgical advances in MEN1 therapeutic approaches and management strategies. Endocrine-related cancer, 2017. 24(10): p. T243-T260.
van den Broek, M.F.M., et al., The Management of Neuroendocrine Tumors of the Lung in MEN1: Results From the Dutch MEN1 Study Group. The Journal of clinical endocrinology and metabolism, 2021. 106(2): p. e1014-e1027.
Sachithanandan, N., R.A. Harle, and J.R. Burgess, Bronchopulmonary carcinoid in multiple endocrine neoplasia type 1. Cancer, 2005. 103(3): p. 509-515.
Lecomte, P., et al., Histologically Proven Bronchial Neuroendocrine Tumors in MEN1: A GTE 51-Case Cohort Study. World Journal of Surgery, 2018. 42(1): p. 143-152.
Bartsch, D.K., et al., Bronchopulmonary Neuroendocrine Neoplasms and Their Precursor Lesions in Multiple Endocrine Neoplasia Type 1. Neuroendocrinology, 2016. 103(3-4): p. 240-247.
van den Broek, M.F.M., et al., Well-Differentiated Bronchopulmonary Neuroendocrine Tumors: More Than One Entity. J Thorac Oncol, 2021. 16(11): p. 1810-1820.
Sadowski, S.M., et al., Results of 68Gallium-DOTATATE PET/CT Scanning in Patients with Multiple Endocrine Neoplasia Type 1. Journal of the American College of Surgeons, 2015. 221(2): p. 509-517.
Lastoria, S., et al., Role of 68Ga-DOTATATE PET/CT in patients with multiple endocrine neoplasia type 1 (MEN1). Endocrine, 2016. 52(3): p. 488-494.
Froeling, V., et al., Impact of Ga-68 DOTATOC PET/CT on the diagnosis and treatment of patients with multiple endocrine neoplasia. Annals of Nuclear Medicine, 2012. 26(9): p. 738-743.
Albers, M.B., et al., Limited Value of Ga-68-DOTATOC-PET-CT in Routine Screening of Patients with Multiple Endocrine Neoplasia Type 1. World journal of surgery, 2017. 41(6): p. 1521-1527.
Harding, B., et al., Multiple endocrine neoplasia type 1 knockout mice develop parathyroid, pancreatic, pituitary and adrenal tumours with hypercalcaemia, hypophosphataemia and hypercorticosteronaemia. Endocr Relat Cancer, 2009. 16(4): p. 1313-27.
Loffler, K.a., et al., Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1. International journal of cancer. Journal international du cancer, 2007. 120: p. 259-67.
Ludwig, L., et al., Loss of wild-type MEN1 gene expression in multiple endocrine neoplasia type 1-associated parathyroid adenoma. Endocr J, 1999. 46(4): p. 539-44.
Skogseid, B., et al., Clinical and genetic features of adrenocortical lesions in multiple endocrine neoplasia type 1. J Clin Endocrinol Metab, 1992. 75(1): p. 76-81.
Gatta-Cherifi, B., et al., Adrenal involvement in MEN1. Analysis of 715 cases from the Groupe d'etude des Tumeurs Endocrines database. Eur J Endocrinol, 2012. 166(2): p. 269-79.
Barzon, L., et al., Multiple endocrine neoplasia type 1 and adrenal lesions. J Urol, 2001. 166(1): p. 24-7.
Burgess, J.R., et al., Adrenal lesions in a large kindred with multiple endocrine neoplasia type 1. Arch Surg, 1996. 131(7): p. 699-702.
Langer, P., et al., Adrenal involvement in multiple endocrine neoplasia type 1. World J Surg, 2002. 26(8): p. 891-6.
Schaefer, S., et al., Natural course of small adrenal lesions in multiple endocrine neoplasia type 1: an endoscopic ultrasound imaging study. Eur J Endocrinol, 2008. 158(5): p. 699-704.
Skogseid, B., et al., Adrenal lesion in multiple endocrine neoplasia type 1. Surgery, 1995. 118(6): p. 1077-82.
Ventura, M., M. Melo, and F. Carrilho, Outcome and long-term follow-up of adrenal lesions in multiple endocrine neoplasia type 1. Arch Endocrinol Metab, 2019. 63(5): p. 516-523.
Waldmann, J., et al., Adrenal involvement in multiple endocrine neoplasia type 1: results of 7 years prospective screening. Langenbecks Arch Surg, 2007. 392(4): p. 437-43.
Whitley, S.A., et al., The appearance of the adrenal glands on computed tomography in multiple endocrine neoplasia type 1. Eur J Endocrinol, 2008. 159(6): p. 819-24.
Wang, W., et al., Adrenocortical carcinoma in patients with MEN1: a kindred report and review of the literature. Endocr Connect, 2019. 8(3): p. 230-238.
Dotzenrath, C., et al., Malignant endocrine tumors in patients with MEN 1 disease. Surgery, 2001. 129(1): p. 91-5.
Febrero, B., A. Ríos, and J.M. Rodríguez, Aggressive adrenal carcinoma in a young patient with Multiple Endocrine Neoplasia 1 syndrome. Med Clin (Barc), 2017. 149(10): p. 463.
Griniatsos, J.E., et al., Bilateral adrenocortical carcinoma in a patient with multiple endocrine neoplasia type 1 (MEN1) and a novel mutation in the MEN1 gene. World J Surg Oncol, 2011. 9: p. 6.
Haase, M., et al., A new mutation in the menin gene causes the multiple endocrine neoplasia type 1 syndrome with adrenocortical carcinoma. Endocrine, 2011. 39(2): p. 153-9.
Harada, K., et al., A novel case of myxoid variant of adrenocortical carcinoma in a patient with multiple endocrine neoplasia type 1. Endocr J, 2019. 66(8): p. 739-744.
Kharb, S., et al., Hidden diagnosis of multiple endocrine neoplasia-1 unraveled during workup of virilization caused by adrenocortical carcinoma. Indian J Endocrinol Metab, 2013. 17(3): p. 514-8.
Ohara, N., et al., Lung adenocarcinoma and adrenocortical carcinoma in a patient with multiple endocrine neoplasia type 1. Respir Med Case Rep, 2017. 20: p. 77-81.
Assié, G., et al., Integrated genomic characterization of adrenocortical carcinoma. Nat Genet, 2014. 46(6): p. 607-12.
Tepede, A.A., et al., 18F-FDOPA PET/CT accurately identifies MEN1-associated pheochromocytoma. Endocrinol Diabetes Metab Case Rep, 2020. 2020.
Dénes, J., et al., Heterogeneous genetic background of the association of pheochromocytoma/paraganglioma and pituitary adenoma: results from a large patient cohort. J Clin Endocrinol Metab, 2015. 100(3): p. E531-41.
Fassnacht, M., et al., Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol, 2016. 175(2): p. G1-g34.
Asgharian, B., et al., Cutaneous tumors in patients with multiple endocrine neoplasm type 1 (MEN1) and gastrinomas: Prospective study of frequency and development of criteria with high sensitivity and specificity for MEN1. Journal of Clinical Endocrinology and Metabolism, 2004. 89(11): p. 5328-5336.
Darling, T.N., et al., Multiple Facial Angiofibromas and Collagenomas in Patients With Multiple Endocrine Neoplasia Type 1. Arch Dermatol., 1997. 7(133): p. 853-857.
Vidal, A., et al., Cutaneous lesions associated to multiple endocrine neoplasia syndrome type 1. Journal of the European Academy of Dermatology and Venereology, 2008. 22(7): p. 835-838.
Pack, S., et al., Cutaneous tumors in patients with multiple endocrine neoplasia type 1 show allelic deletion of the MEN1 gene. Journal of Investigative Dermatology, 1998. 110(4): p. 438-440.
Vortmeyer, A.O., et al., Multiple endocrine neoplasia 1 gene alterations in MEN1-associated and sporadic lipomas. Journal of the National Cancer Institute, 1998. 90(5): p. 398-399.
Dreijerink, K.M.A., et al., The Multiple Endocrine Neoplasia Type 1 (MEN1) Tumor Suppressor Regulates Peroxisome Proliferator-Activated Receptor γ-Dependent Adipocyte Differentiation. Molecular and Cellular Biology, 2009. 29(18): p. 5060-5069.
Parekh, V.I., et al., Consequence of Menin Deficiency in Mouse Adipocytes Derived by in Vitro Differentiation. International Journal of Endocrinology, 2015. 2015.
Radman, M. and T. Milicevic, A novel mutation of the MEN1 gene in a patient with multiple endocrine neoplasia type 1 and recurrent fibromyxoid sarcoma- A case report. BMC Medical Genetics, 2020. 21(1): p. 1-4.
Cheah, S.K., et al., Breast cancer in multiple endocrine neoplasia type 1 (MEN1). Endocrinology, Diabetes & Metabolism Case Reports, 2021. 2021(May): p. 1-7.
Jeong, Y.J., H.K. Oh, and J.G. Bong, Multiple endocrine neoplasia type 1 associated with breast cancer: A case report and review of the literature. Oncology letters, 2014. 8(1): p. 230-234.
Kim, S.H. and J.H. Park, Adrenal incidentaloma, breast cancer and unrecognized multiple endocrine neoplasia type 1. Acta Endocrinologica, 2019. 15(4): p. 513-517.
Maria, B.C., et al., Incidental finding of MEN-1 syndrome during staging and follow-up of breast carcinoma. BMJ Case Reports, 2020. 13(12): p. 1-6.
Seigne, C., et al., High incidence of mammary intraepithelial neoplasia development in Men1-disrupted murine mammary glands. The Journal of pathology, 2013. 229(4): p. 546-58.
Imachi, H., et al., Menin, a product of the MENI gene, binds to estrogen receptor to enhance its activity in breast cancer cells: possibility of a novel predictive factor for tamoxifen resistance. Breast cancer research and treatment, 2010. 122(2): p. 395-407.
Dreijerink, K.M., et al., Article Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer. Cell Reports, 2017. In Press(1).
Teinturier, R., et al., Reduced menin expression leads to decreased ERα expression and is correlated with the occurrence of human luminal B-like and ER-negative breast cancer subtypes. Breast Cancer Research and Treatment, 2021(0123456789).
Goswami, S., et al., Disease and treatment factors associated with lower quality of life scores in adults with multiple endocrine neoplasia type I. Surgery, 2017: p. 1-8.
Marini, F., et al., Management impact: effects on quality of life and prognosis in MEN1. Endocrine-Related Cancer, 2017. 24(10): p. T227-T242.
Peipert, B.J., et al., Financial burden is associated with worse health-related quality of life in adults with multiple endocrine neoplasia type 1. Surgery, 2017: p. 1-8.
Peipert, B.J., et al., Health-related quality of life in MEN1 patients compared with other chronic conditions and the united states general population. Surgery (United States), 2017. 163: p. 205-211.
van Leeuwaarde, R.S., et al., High fear of disease occurrence is associated with low quality of life in patients with Multiple Endocrine Neoplasia type 1 (MEN1): Results from the Dutch MEN1 Study Group. The Journal of Clinical Endocrinology & Metabolism, 2018. 103(June): p. 2354-2361.
van Leeuwaarde, R.S., et al., Health-related quality of life in patients with Multiple Endocrine Neoplasia type 1. Neuroendocrinology, 2020: p. 1-8.
Berglund, G., et al., Quality of life in patients with multiple endocrine neoplasia type 1 ( MEN1 ). Familial Cancer, 2003. 1(2): p. 27-33.
You, Y.N., et al., Pancreatoduodenal surgery in patients with multiple endocrine neoplasia type 1: Operative outcomes, long-term function, and quality of life. Surgery, 2007. 142(6): p. 829-836.

Snakebite Envenomation and Endocrine Dysfunction

ABSTRACT

Snakebite envenoming (SBE) is a life-threatening medical emergency encountered in tropical parts of Asia, Africa, and Latin America. Toxins in the venom cause local damage and multi-organ dysfunction, predominantly affecting neurological, hematological, and vascular systems. Endocrine anomalies are less frequently reported and often masked by more severe disorders. Anterior pituitary insufficiency is the most common endocrine manifestation and mainly observed after Russell’s viper (Daboia russelii and D. siamensis) bite. SBE-induced hypopituitarism can manifest early or have a delayed presentation. Primary adrenal insufficiency, hyponatremia, hypokalemia, hyperkalemia, and hyperglycemia are also described. These complications are uncommon and under-reported, as SBE occurs in remote areas and medical facilities for endocrine assessment might not be available. Timely identification and management of these problems are critical for optimum medical outcome.

INTRODUCTION

Snakebite envenoming (SBE) occurs predominantly in rural parts of Asia, Africa, and Latin America (1,2). The World Health Organization (WHO) included SBE in the priority list of neglected tropical diseases in 2018. According to the WHO, 4.5–5.4 million people get bitten by snakes annually, but many cases are not reported as people living in remote areas with limited healthcare access are affected. Clinical illness from SBE develops in 1.8–2.7 million, and the annual mortality is around 81,000 to 138,000 (3).

Toxins in snake venom can cause local tissue destruction, neurological damage, hemorrhagic tendency, renal failure, and cardiovascular compromise. Endocrine dysfunctions are uncommon but can have ominous consequences if not recognized. Anterior pituitary insufficiency (API) after Russell’s viper (RV) envenomation (RVE) is the most common endocrine manifestation of SBE. Electrolyte disturbances and hyperglycemia are the other complications described (4). Timely recognition and appropriate management of endocrine derangements like hypocortisolism and electrolyte imbalances can save lives.

SPECIES OF SNAKES AND SNAKE VENOM

The medically relevant poisonous snakes usually belong to the Elapidae and Viperidae families. Rare cases of envenoming from Atractaspididae and Colubridae families are also described. The common Elapidae snakes include cobras, mambas, kraits, coral snakes, death adders, and sea snakes. The Viperidae snakes of significance are vipers, including RV, adders, asps, and pit vipers. The general notion that Elapidae envenomation results in neuroparalytic manifestation and Viperidae bite induce local reaction and vasculotoxicity does not always hold.

Snake venoms contain mixtures of polypeptides, amines, carbohydrates, lipids, phospholipids, nucleosides, and minerals. The principal constituents are proteins belonging to the four families: phospholipase A2, metalloprotease, serine protease, and three-finger peptides. Additional secondary protein families include cysteine-rich secretory proteins, l-amino acid oxidases, kunitz peptides, C-type lectins/snaclecs, disintegrins, and natriuretic peptides (5).

Snake venom toxicity can be classified into three main categories: vasculotoxic, neurotoxic, and cytotoxic. Various proteins with enzymatic properties such as phospholipase A2, hyaluronidases, peptidases, and metalloprotease can cause local tissue destruction. Phospholipase A2, metalloproteases, and other protein components can cause neurotoxicity, damage the coagulation cascade, induce muscle necrosis, and sometimes exert cardiotoxic and nephrotoxic effects (6). Cardiac compromise and acute kidney injury (AKI) can also emerge as secondary complications. Endocrine disorders after SBE are uncommon and pathophysiologic mechanisms are incompletely understood.

ANTERIOR PITUITARY INSUFFICIENCY

Anterior pituitary insufficiency (API) is the most well-recognized endocrine manifestation of SBE. Most cases are from Sri Lanka, India, and Myanmar and occur after RVE (Daboia russelii and D. siamensis).

Etiology

Wolff first narrated SBE-induced hypopituitarism in 1958 after bite from Bothrops jararacussu (7). The first description of API following RVE in the Indian subcontinent came from Eapen et al. (8). Although RV is found in many south Asian countries, most accounts of RVE-induced API are almost exclusively from Myanmar, India, and Sri Lanka (9–16). It could be related to the geographic variation in venom composition among the same snake species. The incidence of API in a study from northern India was 14.6% (6/41) among patients admitted with vasculotoxic snakebites (presumed RVE) (12).

Pathophysiology

The pituitary gland is a highly vascular structure enclosed in a bony cavity called the sella tursica. The low-pressure hypothalamic-pituitary portal system originating from the superior hypophyseal artery provides blood supply to the anterior pituitary. The hypophyseal portal system is susceptible to compressive effects from an enlarged or engorged gland and renders the anterior pituitary vulnerable to vascular insults after stimulation from any cause.

Sheehan’s syndrome and RVE-induced hypopituitarism share similar pathophysiology (16,17). In Sheehan’s syndrome, hemorrhagic infarction of the pregnancy-induced hyperplastic gland occurs during severe postpartum bleeding-related hypovolemic shock (18). Predisposition to vascular damage in RVE could result from gland engorgement due to a generalized increase in capillary permeability as in capillary leak syndrome (19–21). Additionally, the toxins in RV venom can stimulate pituitary cells as suggested by in-vitro studies, further increasing the susceptibility to damage (22).

The vascular supply to an engorged and stimulated gland might be compromised due to microthrombi deposition or hemorrhage from disseminated intravascular coagulation (DIC) (16,23), circulatory or hypovolemic shock (14), thrombotic occlusion of major vessels including cerebral venous thrombosis (24,25), and increased intracranial pressure (14). Autoimmune damage has been postulated to contribute to delayed pituitary injury in Sheehan’s syndrome (26,27). The role of similar immune-mediated damage in the development of delayed hypopituitarism after RVE has not been studied.

Clinical Features

ACUTE HYPOPITUITARISM

Acute onset API has been observed after RVE in several series and can present as early as the first day (11,12,15). In one series of nine patients, API occurred after a median interval of nine days (range 2-14 days) (15). The usual manifestations of RVE include local reaction, coagulopathy, neuromuscular paralysis, and AKI (28,29). Circulatory shock, another feature of RVE, is multifactorial in etiology (14). In the acute phase, adrenal insufficiency (AI) dominates the clinical presentation of API. The symptoms of AI often get masked by other systemic effects of RVE. Clinical clues could be refractory hypotension and the presence of hypoglycemia or hyponatremia.

Central hypothyroidism may coexist with secondary AI and is diagnosed if serum thyroid-stimulating hormone (TSH) is low or normal along with decreased serum thyroxine levels. TSH can also get suppressed due to sick euthyroid syndrome and glucocorticoid administration. The diagnosis of central hypothyroidism is difficult to discern in the acute phase, and a follow-up test after recovery is necessary for confirmation. Reassessment of hypothalamic-pituitary-adrenal (HPA) and additional evaluation of the gonadotrophic and growth hormone (GH) secretion should be performed after 4-6 weeks. Acute hypopituitarism is sometimes transient, but the typical outcome is a permanent disease (12,13).

DELAYED HYPOPITUITARISM

API is often diagnosed years after RVE (30–33). The symptoms depend on the hormone axis involved and the extent of hormone deficiency (34). Delayed hypopituitarism often presents as secondary amenorrhea and infertility in females. In males, hypogonadotropic hypogonadism usually manifests as loss of libido and erectile dysfunction. Loss of secondary sexual characteristics can be present in both genders.

Standard features of secondary hypothyroidism include cold intolerance, weight gain, constipation, dry skin, and hoarseness. Secondary AI presents as fatigue, loss of appetite, and orthostatic dizziness (34). Involvement in early childhood can cause stunted growth and delayed or absent puberty (31). In a case series of delayed API, secondary hypothyroidism and hypogonadotropic hypogonadism were present in all the cases (8/8); and GH and secondary AI were present in 75% (6/8) (30). Table 1 depicts the recent case series describing API.

Predictors of Hypopituitarism

The presence of acute kidney injury (AKI) most consistently correlates with the development of API after RVE (13,30). Bhat et al. found that in patients (n=51) with vasculotoxic snakebite-associated AKI, the risk factors for API were younger age, the number of hemodialysis sessions, and 20-min whole blood clotting time (13). There was a history of AKI in 75% of cases of delayed hypopituitarism in another series (30). In a study describing nine patients with acute API, the predictors were multi-organ dysfunction, lower platelet counts, and more bleeding with a requirement for transfusions (15). However, coagulopathy, AKI, hemodialysis, and clinical severity scores failed to show any association with hypopituitarism in a prospective study (12).

Table 1. Case Series of Hypopituitarism After Snakebite Envenoming
Author, year	Region	Snake species	No. of patients	Onset, Time	Clinical features/ hormone axes involved/ comments
Tun Pe, 1987(10)	Myanmar	Not defined	Snakebite – 220 Acute API – 3 PH (on autopsy) – 4 Delayed API – 11	Acute: 21 hr - 9 d Delayed: 2 wk - 24 yr	Acute - C, GH, PRL Delayed Symptomatic - 7 Asymptomatic – 4
Proby, 1989 (35)	Myanmar	RV	Acute API (probable) – 20 Delayed API – 11/12	Acute, NA Delayed – 8 - 226 wk	C - 10/15 T - 19/20 G -12/17
Golay, 2014 (11)	West Bengal, India	Vasculotoxic snakebite	API - 9/96 cases of snakebite associated AKI	Acute and delayed, 2 wk - 10 yr	C - 6/9 G, GH, T - 9/9 1 empty sella, rest normal
Rajagopala, 2014 (15)	Puducherry, India	Vasculotoxic snakebite	9/989 cases	Acute, 2-14 d	Hypoglycemia (100%), hypotension (67%) C - 9/9 Partial empty sella in 6/9
Naik, 2018 (12)	India	Vasculotoxic snakebite	9/41 cases	Acute (10%), Mean - 32 hr	Primary AI - 2/6, C, GH, G - 6/6 PRL - 2/6
White, 2019 (36)	Myanmar	RV (85.4%), Rest - cobra, krait, green pit viper, others	20/948 cases	Acute (2%)	Coagulopathy - 68.9%, AKI - 72.2%
Gopalkrishnan, 2018 (14)	India	RV, saw-scaled viper	SB - 248 AI – 12 API - 4	Acute	C -19/48 Autopsy - 52. PH or ischemic necrosis - 46% Bilateral adrenal hemorrhage - 26%, Adrenal ischemic necrosis – 6%
Shivaprasad, 2018 (30)	Karnataka, India	RV	Delayed API - 8	Delayed, 5-11 yr	C, GH - 6/8 T, G - 8/8
Bhat, 2019 (13)	West Bengal, India	Vasculotoxic snakebite	API - 11/51 at 7 d and 13/33 at 3 mn after snakebite associated AKI	Acute and delayed, 7 d – 3 mn	C – 12/13 PRL – 9/13 G – 9/13 GH – 5/13 T – 4/13

RV - Russell’s viper, C - cortisol, GH - growth hormone, G - gonadotropin, PRL - prolactin, T - thyroid, API – anterior pituitary insufficiency, AI - adrenal insufficiency, PH – pituitary hemorrhage, AKI – acute kidney injury, SB- snake bite

Diagnosis

ACUTE HYPOPITUITARISM

It is challenging to diagnose API during the acute phase. The indicators associated with API are summarized in table 2. The assessment of the HPA axis is required to decide the necessity for glucocorticoid replacement. Hypocortisolism in the acute phase is diagnosed from random cortisol or with the cosyntropin stimulation test. In remote areas, if a delay is anticipated in obtaining the cortisol report, hydrocortisone replacement should be started in suspected cases. The different criteria that have been used to diagnose hypocortisolism in the acute phase are (a) fasting serum cortisol < 3 μg/dL (83 nmol/L) (13), (b) random serum cortisol < 5 μg/dL (138 nmol/L) in suspected pituitary apoplexy (37), (c) random serum cortisol < 10 μg/dL (275 nmol/L) in a critically ill patient (14), (d) post-cosyntropin peak cortisol < 18 μg/dl (500 nmol/L), and (e) post-cosyntropin delta cortisol < 9 μg/dL (250 nmol/L) (38). Note in very ill patients serum cortisol levels can be artifactually low secondary to a decrease in cortisol binding protein.

The interpretation of the thyroid function test can be problematic in the acute phase. Low luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin might be indicative but not diagnostic of API. The gonadal axis and sex hormone secretion (testosterone in males and estradiol in females) is usually suppressed during any severe illness. The pituitary function should be reassessed after 4-6 weeks of recovery. The MRI findings reveal a normal gland on imaging in acute cases (11,12,15). Pituitary hemorrhage has been demonstrated in autopsy findings but is seldom observed in imaging studies (15).

Table 2. Features Suggestive Of or Associated with Acute Hypopituitarism after Russell’s Viper Bite
Clinical features	Laboratory results	Imaging
Persistent or unexplained hypotension	Hypoglycemia	Pituitary hemorrhage or infarct on MRI
Acute kidney injury	Hyponatremia	Pituitary hemorrhage or infarct on MRI
Capillary leak syndrome
Disseminated intravascular coagulation

DELAYED HYPOPITUITARISM

Acute hypopituitarism may progress to chronic disease or manifest insidiously years later (11,12,30). It may be prudent to perform periodic surveillance to rule out the development of API following RVE. Hypogonadism has been described in 100% of cases, central hypothyroidism in 96.4%, secondary AI in 82%, and GH deficiency in 77% (30). Central diabetes insipidus (CDI) is very rare and discussed next. The clinical presentation depends on the age, the hormone axes involved, and the mode of onset. The tests recommended for the diagnosis of pituitary hormone deficiency are outlined in table 3. However, facilities for the dynamic tests may not be available in remote areas where SBE is prevalent (34). MRI usually reveals a normal pituitary during the initial year, but partial or complete empty sella may be found later on (4).

Table 3. Investigations for Diagnosis of Chronic Anterior Pituitary Insufficiency
Hormone	Tests	Interpretation/comments
ACTH	Cortisol, ACTH between 8- 9 AM	Serum cortisol values < 3 μg/dL (83 nmol/L) at 8–9 AM on 2 occasions strongly suggest AI in an appropriate clinical setting. Intermediate levels (3-18 μg/dl; 83 – 497 nmol/L) require cosyntropin stimulation test. Concomitant normal or low ACTH levels indicate secondary AI.
	Cosyntropin (Synacthen) stimulation test	Cosyntropin injection 250 μg (i.v. or i.m.) followed by serum cortisol at 30 min and 60 min. Peak cortisol < 18 μ/dl (500 nmol/L) is suggestive of AI.
	Insulin tolerance test	Serum cortisol < 20 μg/dL (550 nmol/L) at the time of insulin induced hypoglycemia < 40 mg/dL (2.2 mmol/L). Extreme caution required, not practiced in many centers.

TSH	T4 (total or free), TSH	Low T4 with low or normal TSH suggests the diagnosis of central hypothyroidism

GH	IGF-1	Low IGF-1 suggestive but not diagnostic.
	GH stimulation test	Adults: Insulin tolerance test, arginine, GHRH stimulation test, Macimorelin stimulation test, glucagon stimulation test. Children: Clonidine stimulation test in addition to tests used for adults.

Gonadotrophin (Males)	LH, FSH, testosterone (total), SHBG	Low total testosterone (<300 ng/dl (10.41 nmol/L)) between 8–9 AM, preferably on 2 occasions along with low or normal LH, FSH is suggestive of gonadotrophin deficiency. SHBG and free or bioavailable testosterone measurement should be considered in borderline cases.
Gonadotrophin (Females)	LH, FSH, estradiol	Low estradiol in the setting of low or normal LH and FSH in the appropriate clinical setting (amenorrhea/ oligomeorrhea) suggests gonadotrophin deficiency.

Prolactin	Prolactin	Low levels found in hypopituitarism

ACTH – adrenocorticotrophic hormone, AI – adrenal insufficiency, TSH – thyroid-stimulating hormone, T4 – thyroxine, GH - growth hormone, IGF-1 – insulin-like growth factor 1, LH – luteinizing hormone, FSH – follicle-stimulating hormone, SHBG – sex-hormone binding globulin

Management

Acute hypopituitarism typically occurs in critically ill patients with severe envenomation from RV. Intravenous hydrocortisone is required if AI is suspected or diagnosed. Thyroxine supplementation for hypothyroidism should be started only after correcting AI. There is a risk of precipitating adrenal crisis because of the accelerated metabolic clearance of cortisol, if thyroxine is administered before treatment of AI (39). Monitoring of electrolytes, and slow correction of hyponatremia when present, in order to prevent central pontine myelinolysis, are important adjuncts.

If oral intake is proper and the patient is hemodynamically stable, intravenous hydrocortisone can be substituted by oral glucocorticoids. Hormonal evaluation of the entire pituitary axes should be performed after 4-6 weeks of recovery. Replacement of deficient hormones as per standard practice should be instituted if API persists (34).

Post-mortem Findings

Pituitary hemorrhage and ischemic necrosis have been described in autopsy studies of 43% (36/84) cases of RVE in Myanmar (40). Areas of ischemic necrosis with hemorrhage at the center were observed in studies from India (15). Deposition of fibrin microthrombi in the pituitary and other organs, including the kidney, suggests a possible role of DIC in the pathogenesis of API (23).

DIABETES INSIPIDUS

Involvement of the posterior pituitary gland is exceedingly rare in SBE. There are only a few case reports of central diabetes insipidus (CDI) after RVE (13,31,41,42). The posterior pituitary receives direct arterial supply from the inferior hypophyseal artery and is resistant to vascular damage. On the other hand, the anterior gland is susceptible to vascular compromise as it is supplied by the low-pressure hypophyseal-portal system (43). Moreover, CDI occurs when more than 80% of arginine vasopressin (AVP)-producing hypothalamic magnocellular neurons are lost. The posterior pituitary acts as a storage and secretory organ, and persistent CDI ensues only in the presence of significant damage to the hypothalamus (44). Polyuria, a cardinal feature of CDI, may be obscured due to concomitant hypocortisolism and manifest only after glucocorticoid replacement (45). CDI should be treated with nasal or oral desmopressin.

ADRENAL DISORDERS

Etiopathogenesis

Secondary AI from hypopituitarism is the classically described adrenal disorder resulting from SBE. Primary AI is exceptionally uncommon, though adrenal hemorrhage (AH) has been described in imaging studies and autopsy findings. There are cases of AH occurring after RVE and saw scale viper (Echis carinatus) bite (40,46,47).

The pathophysiology of AH is related to DIC and has been postulated to resemble Waterhouse–Friderichsen syndrome (48). The adrenal gland is a highly vascular structure that derives its arterial supply from three arteries but is drained by only one adrenal vein and has a dense internal network of capillaries (49). The causal factors behind predisposition to AH after RVE are summarized in table 4 (14,50,51).

Table 4. Factors Predisposing to Hemorrhage after Russell’s Viper Envenomation

Intrinsic predisposition of adrenal vascular structure due to arterial supply by 3 vessels but drainage by one vein

Rich subcapsular plexus with limited drainage by venules forming a “dam”

Disseminated intravascular coagulation and hemorrhagic toxins in the venom increase bleeding tendency

Formation of microthrombi in venules impair venous drainage and cause pooling of blood

Pooling of blood due to capillary leak syndrome

Stress-induced trophic effect of adrenocorticotrophic hormone induces adrenal cortical hyperplasia and increase vascularity

Stress-induced catecholamine secretion causes adrenal venous constriction resulting in pooling of blood in the adrenal gland

Diagnosis And Treatment

AH has been described in 36% of cases in an autopsy series, though primary AI is rare (40). Refractory hypotension, hypoglycemia, hyponatremia and hyperkalemia should raise suspicion of primary AI. The presence of associated secondary AI can confound the diagnosis. Bilateral AH with transient AI has been described following RVE. The hemorrhage and adrenal function resolved in weeks (47). Cases depicting chronic AI have been published after vasculotoxic SBE (12). Diagnosis and treatment are similar to secondary AI; the primary differentiating point is elevated plasma ACTH. Mineralocorticoid supplementation may be additionally required in primary AI.

HYPERGLYCEMIA

Etiopathogenesis

Hyperglycemia, an infrequent endocrine complication after SBE, has been described following both elapid (Bungarus multicinctus multicinctus) and viper envenomation (Vipera ammodytes ammodytes, European viper spp) (52–54). In rat models, common krait (Bungarus caeruleus) venom produces hyperglycemia (55). Intraperitoneal injection of saw-scaled viper (Echis carinatus) venom in rats suppressed plasma insulin and depleted liver glycogen stores (56). The hyperglycemic effect of Egyptian cobra (Naja haje) was also associated with concomitant depletion of liver and kidney glycogen stores. The mechanism of hyperglycemia is presumed to be triggered by a massive surge of catecholamines, a phenomenon observed after scorpion envenomation and in pheochromocytoma (4,57). Scorpion toxins stimulate sodium and inhibit potassium channels leading to intense and persistent excitation of the autonomic nervous system and release of neurotransmitters from the adrenal medulla, activating parasympathetic (early hours) and sympathetic nerve endings (4–48 hours). Catecholamine-mediated activation of the alpha receptors inhibits insulin secretion and contributes to hyperglycemia (54,58).

Clinical Features and Management

In a series of 83 children, viper envenomation resulted in hyperglycemia starting 4 hours after the bite, was moderate in severity, and usually transient. Moreover, hyperglycemia at presentation was a marker of high-grade envenomation (54). Severe hyperglycemia up to 480 mg/dl (26.7 mmol/L) occurred in a 45-day baby after two hours of bite from a nose-horned viper (V. a. ammodytes). (53). A retrospective study from Taiwan found hyperglycemia in 15% (7/44) patients of Bungarus multicinctus envenomation. Only one of them had persistent diabetes after recovery (52). Acute pancreatitis can result from the bite of the adder (Vipera berus), but associated hyperglycemia was not observed (59,60). Insulin and other antihyperglycemic drugs should be administered for management of hyperglycemia as and when necessary.

ELECTROLYTE DISTURBANCES

Hyponatremia

ETIOPATHOGENESIS

Envenomation by Malayan krait (Bungarus candidus), banded krait (Bungarus fasciatus)), and vipers can result in hyponatremia (61–68). Hyponatremia sometimes occur secondary to anterior pituitary insufficiency (API) after vasculotoxic envenoming but it has also been reported in the absence of API (4). Initial descriptions suggested that the syndrome of inappropriate antidiuretic hormone secretion (SIADH) could be responsible for hyponatremia (64). However, subsequent accounts revealed that urinary salt loss from natriuretic peptides in venom, rather than SIADH, is the pathogenic mechanism. The urinary salt loss is secondary to venom-derived natriuretic peptides, similar to endogenous natriuretic peptides, and acts on the renal tubules to decrease sodium and water reabsorption (69). Cerebral salt wasting has also been postulated to cause hyponatremia (61). An unusually high prevalence of hyponatremia (89%) was observed in a series of 14 patients with berg adder (Bitis atropos) bite in South Africa (67). Many-banded krait (Bungarus multicinctus) envenomation caused hyponatremia in 42% of cases (63).

Natriuretic peptides are found in the venom of Elapidae species such as Bungarus candidus, Bungarus multicinctus, Dendroaspi sangusticeps, Oxyuranus microlepidotus, Pseudonaja textillis, and Pseudechis australis and a few Viperidae species e.g. Hypnale hypnale, Psudocerastus persicus, and Macrovipera lebetina (66).

CLINICAL FEATURES

The presentation of hyponatremia depends on the severity and acuteness of onset. The clinical profile ranges from asymptomatic hyponatremia to varying alteration in sensorium to frank coma (61,62). Seizures occur in severe cases (62). Usually there are associated systemic features but isolated hyponatremia, hypovolemia, urinary salt loss, and generalized tonic-clonic seizures, following hump-nosed pit viper bite (Hypnale hypnale) has been described (66). In a case series of 42 patients admitted in Vietnam, 31 people (73.8%) had hyponatremia, the lowest values occurring an average of two days after the bite. Approximately 42–50% of patients who did not receive antivenom developed significant hyponatremia (< 130 mmol/L) 2–3 days post-bite. (70). Another series of 78 cases of krait bite from Thailand reported hyponatremia in 17.6%, with severe hyponatremia (< 120 mmol/L) developing in four pediatric patients, two of whom developed seizures (71).

MANAGEMENT

Hyponatremia resulting from natriuretic peptides should be corrected by intravenous saline administration. SIADH is not the cause of hyponatremia, and fluid restriction is not recommended. If chronic hyponatremia is suspected, the correction rate should not exceed 10-12 meq/L in any 24 hours to avoid osmotic demyelination (72). If primary or secondary AI is the cause of hyponatremia, glucocorticoid supplementation is necessary.

Hypokalemia

ETIOPATHOGENESIS

Hypokalemia results from both elapid (73,74) and viper envenomation (75–77). Patients with hypokalemia after RV, common krait (Bungarus caeruleus), and Balkan adder (Vipera berus) bite demonstrated low trans-tubular potassium gradient (TTKG) ruling out renal potassium loss. The intracellular redistribution of potassium has been suggested as the likely pathophysiological mechanism, as gastrointestinal loss was also unlikely. Beta-adrenergic stimulation from toxin-mediated autonomic dysfunction leads to the intracellular shift of potassium and is the likely cause of hypokalemia (74,75). Concomitant hypomagnesemia and high urinary magnesium excretion were also observed in patients with hypokalemia, following Viperidae bite, presumably resulting from the direct toxic action of venom on the renal tubules (77). A high incidence (71%) of hypokalemia (<3.5 mmol/l) was found in a series of 210 patients from Sri Lanka during the first 48 hours. It was accompanied by metabolic acidosis but not respiratory alkalosis (78).

CLINICAL FEATURES AND MANAGEMENT

Hypokalemia manifests as muscular cramps or weakness, constipation, abdominal bloating, polyuria, and sometimes cause life-threatening complications like arrhythmias, rhabdomyolysis, hypokalemic paralysis, diaphragmatic palsy, and respiratory failure (75). The treatment strategy is similar to that of hypokalemic periodic paralysis. Rebound hyperkalemia is a potential complication during recovery. Potassium should be replaced orally or intravenously, along with appropriate monitoring. Magnesium deficit should be corrected if present (79).

Hyperkalemia

ETIOPATHOGENESIS

Hyperkalemia can complicate envenomation by nose-horned viper (Vipera ammodytes ammodytes), European viper (Vipera berus), and hump-nosed viper (Hypnale hypnale) (53,80–83). Severe envenomation from these snakes causes hyperkalemia secondary to rhabdomyolysis and AKI, and can be fatal (53,80). Hyperkalemia was present in 7% of cases of SBE in 258 patients from Thailand (77).

Type 4 renal tubular acidosis (T4RTA) is another possible cause of hyperkalemia. It was described during the recovery phase of bite by hump-nosed viper (81,82). Renal biopsy from these patients showed tubular atrophy and focal segmental glomerulosclerosis pattern (81). The underlying cause could be thrombotic microangiopathy caused by toxins in venom leading to patchy cortical necrosis with delayed or partial recovery of renal functions (83).

CLINICAL FEATURES AND MANAGEMENT

Hyperkalemia associated with rhabdomyolysis and AKI can cause life-threatening arrhythmias. The presence of hyperkalemia along with hyperchloremic metabolic acidosis and low trans-tubular potassium gradient (TTKG) is suggestive of T4RTA and has been described in victims of hump-nosed viper bites during recovery from AKI. Fludrocortisone has been used successfully in such cases. T4RTA, in most cases, was transient (82). Hyperkalemia associated with rhabdomyolysis and AKI will require potassium lowering therapy and, in severe cases, dialysis.

Table 5. Summary of Snakebite Envenoming Induced Endocrine Dysfunctions
Endocrine manifestation	Pathophysiology	Onset of symptoms	Clinical features	Management
Acute hypopituitarism	Hemorrhagic infarction of the anterior pituitary (pathogenesis similar to Sheehan’s syndrome)	Hours to days	Hypotension not responding to standard therapy, hypoglycemia, hyponatremia	Glucocorticoid +/- thyroxine replacement
Delayed hypopituitarism	Sequalae of vascular insult to pituitary during acute phase	Months to years	Amenorrhea, hypogonadism, hypothyroidism, secondary adrenal insufficiency, growth hormone deficiency	Replacement of deficient hormones
Diabetes Insipidus	Very rare, possible vascular insult during acute phase	Immediate or delayed	Polyuria, polydipsia	Desmopressin
Adrenal insufficiency	Hemorrhage with or without infarction in the adrenals secondary to coagulopathy	Hours to days	Hypotension and circulatory collapse	Glucocorticoid +/- mineralocorticoid replacement
Hyperglycemia	Massive catecholamine surge	First 4-6 hours	Children more than adults	Standard treatment of hyperglycemia
Hyponatremia	Venom derived natriuretic peptides – renal salt wasting	First 2-3 days	Asymptomatic to varying alteration in sensorium to coma, seizures	Intravenous saline
Hyponatremia	Pituitary or adrenal insufficiency	First 2-3 days		Glucocorticoid replacement
Hypokalemia	Intracellular redistribution of potassium secondary to autonomic dysfunction.	Within first 24 hours	Muscle cramps, constipation, abdominal bloating, paralysis, respiratory failure, arrhythmia	Replacement of potassium with precaution to avoid rebound hyperkalemia
Hypokalemia	Venom-mediated renal tubular damage	Within first 24 hours
Hyperkalemia	Venom mediated thrombotic microangiopathy in the kidneys leading to type 4 renal tubular acidosis	Weeks	Arrhythmia	Fludrocortisone
Hyperkalemia	Rhabdomyolysis or kidney injury related	Days	Arrhythmia	Supportive, dialysis in severe cases

CONCLUSION

Endocrine dysfunctions associated with SBE are rare. However, missing the diagnosis can have life-threatening consequences. Acute or delayed anterior pituitary insufficiency is the most common manifestation. Establishing the diagnosis of hypocortisolism and timely glucocorticoid initiation in acute hypopituitarism are critical. Reports of adrenal dysfunction are scarce, though adrenal hemorrhage following RVE has been described more often in autopsy series. Electrolyte abnormalities should be anticipated and managed appropriately. Awareness and appropriate treatment of endocrine dysfunctions in resource-limited settings are necessary for optimal outcome.

REFERENCES

Feasey N, Wansbrough-Jones M, Mabey DCW, Solomon AW. Neglected tropical diseases. Br Med Bull. 2010 Mar 1;93(1):179–200.
Williams DJ, Faiz MA, Abela-Ridder B, Ainsworth S, Bulfone TC, Nickerson AD, et al. Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl Trop Dis. 2019 Feb 21;13(2):e0007059.
Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nat Rev Dis Primer. 2017 Sep 14;3:17063.
Bhattacharya S, Krishnamurthy A, Gopalakrishnan M, Kalra S, Kantroo V, Aggarwal S, et al. Endocrine and Metabolic Manifestations of Snakebite Envenoming. Am J Trop Med Hyg. 2020 Oct;103(4):1388–96.
Tasoulis T, Isbister GK. A Review and Database of Snake Venom Proteomes. Toxins. 2017 18;9(9).
Ferraz CR, Arrahman A, Xie C, Casewell NR, Lewis RJ, Kool J, et al. Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis. Front Ecol Evol. 2019 Jun 19;7:218.
Wolff H. Insuficiência hipofisária anterior por picada de ofídio. Arq Bras Endocrinol Metab. 1958;7:25–47.
Animal, Plant, and Microbial Toxins - Volume 1—Biochemistry | Akira Ohsaka | Springer [Internet]. [cited 2021 May 2]. Available from: https://www.springer.com/gp/book/9781468408881
Myint-Lwin, Warrell DA, Phillips RE, Tin-Nu-Swe, Tun-Pe, Maung-Maung-Lay. Bites by Russell’s viper (Vipera russelli siamensis) in Burma: haemostatic, vascular, and renal disturbances and response to treatment. Lancet Lond Engl. 1985 Dec 7;2(8467):1259–64.
Tun-Pe, Phillips RE, Warrell DA, Moore RA, Tin-Nu-Swe, Myint-Lwin, et al. Acute and chronic pituitary failure resembling Sheehan’s syndrome following bites by Russell’s viper in Burma. Lancet Lond Engl. 1987 Oct 3;2(8562):763–7.
Golay V, Roychowdhary A, Dasgupta S, Pandey R. Hypopituitarism in patients with vasculotoxic snake bite envenomation related acute kidney injury: a prospective study on the prevalence and outcomes of this complication. Pituitary. 2014 Apr;17(2):125–31.
Naik BN, Bhalla A, Sharma N, Mokta J, Singh S, Gupta P, et al. Pituitary dysfunction in survivors of Russell’s viper snake bite envenomation: A prospective study. Neurol India. 2018 Oct;66(5):1351–8.
Bhat S, Mukhopadhyay P, Raychaudhury A, Chowdhury S, Ghosh S. Predictors of hypopituitarism due to vasculotoxic snake bite with acute kidney injury. Pituitary. 2019 Dec;22(6):594–600.
Gopalakrishnan M, Vinod KV, Dutta TK, Shaha KK, Sridhar MG, Saurabh S. Exploring circulatory shock and mortality in viper envenomation: a prospective observational study from India. QJM Mon J Assoc Physicians. 2018 Nov 1;111(11):799–806.
Rajagopala S, Thabah MM, Ariga KK, Gopalakrishnan M. Acute hypopituitarism complicating Russell’s viper envenomation: case series and systematic review. QJM Mon J Assoc Physicians. 2015 Sep;108(9):719–28.
Antonypillai CN, Wass J a. H, Warrell DA, Rajaratnam HN. Hypopituitarism following envenoming by Russell’s vipers (Daboia siamensis and D. russelii) resembling Sheehan’s syndrome: first case report from Sri Lanka, a review of the literature and recommendations for endocrine management. QJM Mon J Assoc Physicians. 2011 Feb;104(2):97–108.
Burke CW. The anterior pituitary, snakebite and Sheehan’s syndrome. Q J Med. 1990 Apr;75(276):331–3.
Diri H, Karaca Z, Tanriverdi F, Unluhizarci K, Kelestimur F. Sheehan’s syndrome: new insights into an old disease. Endocrine. 2016 Jan;51(1):22–31.
Kendre PP, Jose MP, Varghese AM, Menon JC, Joseph JK. Capillary leak syndrome in Daboia russelii bite-a complication associated with poor outcome. Trans R Soc Trop Med Hyg. 2018 Feb 1;112(2):88–93.
Lingam TMC, Tan KY, Tan CH. Capillary leak syndrome induced by the venoms of Russell’s Vipers (Daboia russelii and Daboia siamensis) from eight locales and neutralization of the differential toxicity by three snake antivenoms. Comp Biochem Physiol Toxicol Pharmacol CBP. 2021 Sep 9;250:109186.
Rucavado A, Escalante T, Camacho E, Gutiérrez JM, Fox JW. Systemic vascular leakage induced in mice by Russell’s viper venom from Pakistan. Sci Rep. 2018 Oct 31;8(1):16088.
Hart GR, Proby C, Dedhia G, Yeo TH, Joplin GF, Burrin JM. Burmese Russell’s viper venom causes hormone release from rat pituitary cells in vitro. J Endocrinol. 1989 Aug;122(2):489–94.
Than-Thannull, Francis N, Tin-Nu-Swe null, Myint-Lwin null, Tun-Pe null, Soe-Soe null, et al. Contribution of focal haemorrhage and microvascular fibrin deposition to fatal envenoming by Russell’s viper (Vipera russelli siamensis) in Burma. Acta Trop. 1989 Jan;46(1):23–38.
Hung D-Z, Wu M-L, Deng J-F, Yang D-Y, Lin-Shiau S-Y. Multiple thrombotic occlusions of vessels after Russell’s viper envenoming. Pharmacol Toxicol. 2002 Sep;91(3):106–10.
Das SK, Khaskil S, Mukhopadhyay S, Chakrabarti S. A patient of Russell’s viper envenomation presenting with cortical venous thrombosis: an extremely uncommon presentation. J Postgrad Med. 2013 Sep;59(3):235–6.
Goswami R, Kochupillai N, Crock PA, Jaleel A, Gupta N. Pituitary autoimmunity in patients with Sheehan’s syndrome. J Clin Endocrinol Metab. 2002 Sep;87(9):4137–41.
De Bellis A, Kelestimur F, Sinisi AA, Ruocco G, Tirelli G, Battaglia M, et al. Anti-hypothalamus and anti-pituitary antibodies may contribute to perpetuate the hypopituitarism in patients with Sheehan’s syndrome. Eur J Endocrinol. 2008 Feb;158(2):147–52.
Kularatne S a. M. Epidemiology and clinical picture of the Russell’s viper (Daboia russelii russelii) bite in Anuradhapura, Sri Lanka: a prospective study of 336 patients. Southeast Asian J Trop Med Public Health. 2003 Dec;34(4):855–62.
Hung D-Z, Wu M-L, Deng J-F, Lin-Shiau S-Y. Russell’s viper snakebite in Taiwan: differences from other Asian countries. Toxicon Off J Int Soc Toxinology. 2002 Sep;40(9):1291–8.
Shivaprasad C, Aiswarya Y, Sridevi A, Anupam B, Amit G, Rakesh B, et al. Delayed hypopituitarism following Russell’s viper envenomation: a case series and literature review. Pituitary. 2019 Feb;22(1):4–12.
Golay V, Roychowdhary A, Pandey R, Pasari A, Praveen M, Arora P, et al. Growth retardation due to panhypopituitarism and central diabetes insipidus following Russell’s viper bite. Southeast Asian J Trop Med Public Health. 2013 Jul 4;44(4):697–702.
Ratnakaran B, Punnoose VP, Das S, Kartha A. Psychosis in Secondary Empty Sella Syndrome following a Russell’s Viper Bite. Indian J Psychol Med. 2016;38(3):254–6.
Yerawar C, Punde D, Pandit A, Deokar P. Russell’s viper bite and the empty sella syndrome. QJM Mon J Assoc Physicians. 2021 Jul 28;114(4):255–7.
Chung T-T, Koch CA, Monson JP. Hypopituitarism. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000 [cited 2021 Oct 17]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK278989/
Proby C, Tha-Aung, Thet-Win, Hla-Mon, Burrin JM, Joplin GF. Immediate and long-term effects on hormone levels following bites by the Burmese Russell’s viper. Q J Med. 1990 Apr;75(276):399–411.
White J, Alfred S, Bates D, Mahmood MA, Warrell D, Cumming R, et al. Twelve month prospective study of snakebite in a major teaching hospital in Mandalay, Myanmar; Myanmar Snakebite Project (MSP). Toxicon X. 2019 Jan;1:100002.
Hannoush ZC, Weiss RE. Pituitary Apoplexy. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000 [cited 2021 Oct 27]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK279125/
Annane D, Pastores SM, Rochwerg B, Arlt W, Balk RA, Beishuizen A, et al. Guidelines for the Diagnosis and Management of Critical Illness-Related Corticosteroid Insufficiency (CIRCI) in Critically Ill Patients (Part I): Society of Critical Care Medicine (SCCM) and European Society of Intensive Care Medicine (ESICM) 2017. Crit Care Med. 2017 Dec;45(12):2078–88.
Higham CE, Johannsson G, Shalet SM. Hypopituitarism. The Lancet. 2016 Nov 12;388(10058):2403–15.
Thein CM, Byard RW. Characteristics and relative numbers of lethal snake bite cases in medicolegal practice in central Myanmar - A five year study. J Forensic Leg Med. 2019 Apr;63:52–5.
Krishnan MN, Kumar S, Ramamoorthy KP. Severe panhypopituitarism and central diabetes insipidus following snake bite: unusual presentation as torsades de pointes. J Assoc Physicians India. 2001 Sep;49:923–4.
Gupta UC, Garg OP, Kataria ML. Cranial diabetes insipidus due to viper bite. J Assoc Physicians India. 1992 Oct;40(10):686–7.
Anderson JR, Antoun N, Burnet N, Chatterjee K, Edwards O, Pickard JD, et al. Neurology of the pituitary gland. J Neurol Neurosurg Psychiatry. 1999 Jun 1;66(6):703–21.
Christ-Crain M, Bichet DG, Fenske WK, Goldman MB, Rittig S, Verbalis JG, et al. Diabetes insipidus. Nat Rev Dis Primer. 2019 Aug 8;5(1):1–20.
Garrahy A, Moran C, Thompson CJ. Diagnosis and management of central diabetes insipidus in adults. Clin Endocrinol (Oxf). 2019 Jan;90(1):23–30.
Lakhotia M, Pahadiya HR, Singh J, Gandhi R, Bhansali S. Adrenal hematoma and right hemothorax after echis carinatus bite: an unusual manifestation. Toxicol Int. 2014 Dec;21(3):325–7.
Senthilkumaran S, Menezes RG, Hussain SA, Luis SA, Thirumalaikolundusubramanian P. Russell’s Viper Envenomation-Associated Addisonian Crisis. Wilderness Environ Med. 2018 Dec;29(4):504–7.
Fox B. Disseminated intravascular coagulation and the Waterhouse-Friderichsen syndrome. Arch Dis Child. 1971 Oct;46(249):680–5.
Gagnon R. The venous drainage of the human adrenal gland. Rev Can Biol. 1956 Feb;14(4):350–9.
Tan GXV, Sutherland T. Adrenal congestion preceding adrenal hemorrhage on CT imaging: a case series. Abdom Radiol N Y. 2016 Feb;41(2):303–10.
Senthilkumaran S, Menezes RG, Hussain SA, Luis SA, Thirumalaikolundusubramanian P. Russell’s Viper Envenomation-Associated Addisonian Crisis. Wilderness Environ Med. 2018 Dec;29(4):504–7.
Mao Y-C, Liu P-Y, Chiang L-C, Liao S-C, Su H-Y, Hsieh S-Y, et al. Bungarus multicinctus multicinctus Snakebite in Taiwan. Am J Trop Med Hyg. 2017 Jun 7;96(6):1497–504.
Lukšić B, Culić V, Stričević L, Brizić I, Poljak NK, Tadić Z. Infant death after nose-horned viper (Vipera ammodytes ammodytes) bite in Croatia: A case report. Toxicon Off J Int Soc Toxinology. 2010 Dec;56(8):1506–9.
Claudet I, Grouteau E, Cordier L, Franchitto N, Bréhin C. Hyperglycemia is a risk factor for high-grade envenomations after European viper bites (Vipera spp.) in children. Clin Toxicol Phila Pa. 2016;54(1):34–9.
Kiran KM, More SS, Gadag JR. Biochemical and clinicopathological changes induced by Bungarus coeruleus venom in a rat model. J Basic Clin Physiol Pharmacol. 2004;15(3–4):277–87.
Al-Saleh SSM. The effect of Echis carinatus crude venom and purified protein fractions on carbohydrate metabolism in rats. Cell Biochem Funct. 2002 Mar;20(1):1–10.
Bouaziz M, Bahloul M, Kallel H, Samet M, Ksibi H, Dammak H, et al. Epidemiological, clinical characteristics and outcome of severe scorpion envenomation in South Tunisia: multivariate analysis of 951 cases. Toxicon Off J Int Soc Toxinology. 2008 Dec 15;52(8):918–26.
Bahloul M, Turki O, Chaari A, Bouaziz M. Incidence, mechanisms and impact outcome of hyperglycaemia in severe scorpion-envenomed patients. Ther Adv Endocrinol Metab. 2018 Jul;9(7):199–208.
Pande R, Khan H. Acute pancreatitis following adder bite in the UK: a case report. Ann R Coll Surg Engl. 2010 Sep;92(6):e25–6.
Kjellström BT. Acute pancreatitis after snake bite. Case report. Acta Chir Scand. 1989 May;155(4–5):291–2.
Kumar Keyal N, Shrestha R, Thapa S, Adhikari P. Krait Snake Bite Presenting as a Cerebral Salt Wasting. Indian J Crit Care Med Peer-Rev Off Publ Indian Soc Crit Care Med. 2019 Jul;23(7):347–8.
Höjer J, Tran Hung H, Warrell D. Life-threatening hyponatremia after krait bite envenoming – A new syndrome. Clin Toxicol. 2010 Nov 1;48(9):956–7.
Hung HT, Höjer J, Du NT. Clinical features of 60 consecutive ICU-treated patients envenomed by Bungarus multicinctus. Southeast Asian J Trop Med Public Health. 2009 May;40(3):518–24.
Trinh KX, Khac QL, Trinh LX, Warrell DA. Hyponatraemia, rhabdomyolysis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in southern Viet Nam. Toxicon Off J Int Soc Toxinology. 2010 Nov;56(6):1070–5.
Tongpoo A, Sriapha C, Pradoo A, Udomsubpayakul U, Srisuma S, Wananukul W, et al. Krait envenomation in Thailand. Ther Clin Risk Manag. 2018;14:1711–7.
de Silva U, Sarathchandra C, Senanayake H, Pilapitiya S, Siribaddana S, Silva A. Hyponatraemia and seizures in Merrem’s hump-nosed pit viper (Hypnale hypnale) envenoming: a case report. J Med Case Reports. 2018 Aug 2;12(1):213.
van der Walt AJ, Muller GJ. Berg adder (Bitis atropos) envenoming: an analysis of 14 cases. Clin Toxicol Phila Pa. 2019 Feb;57(2):131–6.
Wium CA, Marks CJ, Du Plessis CE, Müller GJ. Berg adder (Bitis atropos): An unusual case of acute poisoning. South Afr Med J Suid-Afr Tydskr Vir Geneeskd. 2017 Nov 27;107(12):1075–7.
Vink S, Jin AH, Poth KJ, Head GA, Alewood PF. Natriuretic peptide drug leads from snake venom. Toxicon Off J Int Soc Toxinology. 2012 Mar 15;59(4):434–45.
Trinh KX, Khac QL, Trinh LX, Warrell DA. Hyponatraemia, rhabdomyolysis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in southern Viet Nam. Toxicon Off J Int Soc Toxinology. 2010 Nov;56(6):1070–5.
Tongpoo A, Sriapha C, Pradoo A, Udomsubpayakul U, Srisuma S, Wananukul W, et al. Krait envenomation in Thailand. Ther Clin Risk Manag. 2018;14:1711–7.
Spasovski G, Vanholder R, Allolio B, Annane D, Ball S, Bichet D, et al. Clinical practice guideline on diagnosis and treatment of hyponatraemia. Eur J Endocrinol. 2014 Mar;170(3):G1-47.
Gawarammana IB, Mudiyanselage Kularatne SA, Kularatne K, Waduge R, Weerasinghe VS, Bowatta S, et al. Deep coma and hypokalaemia of unknown aetiology following Bungarus caeruleus bites: Exploration of pathophysiological mechanisms with two case studies. J Venom Res. 2010 Dec 14;1:71–5.
Kularatne S a. M. Common krait (Bungarus caeruleus) bite in Anuradhapura, Sri Lanka: a prospective clinical study, 1996-98. Postgrad Med J. 2002 May;78(919):276–80.
Jeevagan V, Katulanda P, Gnanathasan CA, Warrell DA. Acute pituitary insufficiency and hypokalaemia following envenoming by Russell’s viper (Daboia russelii) in Sri Lanka: Exploring the pathophysiological mechanisms. Toxicon Off J Int Soc Toxinology. 2013 Mar 1;63:78–82.
Malina T, Krecsák L, Jelić D, Maretić T, Tóth T, Siško M, et al. First clinical experiences about the neurotoxic envenomings inflicted by lowland populations of the Balkan adder, Vipera berus bosniensis. Neurotoxicology. 2011 Jan;32(1):68–74.
Aye K-P, Thanachartwet V, Soe C, Desakorn V, Thwin K-T, Chamnanchanunt S, et al. Clinical and laboratory parameters associated with acute kidney injury in patients with snakebite envenomation: a prospective observational study from Myanmar. BMC Nephrol. 2017 Mar 16;18(1):92.
Sellahewa K. Lessons from four studies on the management of snake bite in Sri Lanka. Ceylon Med J. 1997 Mar;42(1):8–15.
Kardalas E, Paschou SA, Anagnostis P, Muscogiuri G, Siasos G, Vryonidou A. Hypokalemia: a clinical update. Endocr Connect. 2018 Apr;7(4):R135–46.
Denis D, Lamireau T, Llanas B, Bedry R, Fayon M. Rhabdomyolysis in European viper bite. Acta Paediatr Oslo Nor 1992. 1998 Sep;87(9):1013–5.
Weerakkody RM, Lokuliyana PN, Lanerolle RD. Transient distal renal tubular acidosis following hump nosed viper bite: Two cases from Sri Lanka. Saudi J Kidney Dis Transplant. 2016 Sep 1;27(5):1018.
Karunarathne S, Udayakumara Y, Govindapala D, Fernando H. Type IV renal tubular acidosis following resolution of acute kidney injury and disseminated intravascular coagulation due to hump-nosed viper bite. Indian J Nephrol. 2013;23(4):294–6.
Herath N, Wazil A, Kularatne S, Ratnatunga N, Weerakoon K, Badurdeen S, et al. Thrombotic microangiopathy and acute kidney injury in hump-nosed viper (Hypnale species) envenoming: a descriptive study in Sri Lanka. Toxicon Off J Int Soc Toxinology. 2012 Jul;60(1):61–5.

Vitamin D: Production, Metabolism, and Mechanisms of Action

ABSTRACT

Vitamin D production in the skin under the influence of sunlight (UVB) is maximized at levels of sunlight exposure that do not burn the skin. Further metabolism of vitamin D to its major circulating form (25(OH)D) and hormonal form (1,25(OH)2D) takes place in the liver and kidney, respectively, but also in other tissues where the 1,25(OH)2D produced serves a paracrine/autocrine function: examples include the skin, cells of the immune system, parathyroid gland, intestinal epithelium, prostate, and breast. Parathyroid hormone, FGF23, calcium and phosphate are the major regulators of the renal 1-hydroxylase (CYP27B1, the enzyme producing 1,25(OH)2D); regulation of the extra renal 1-hydroxylase differs from that in the kidney and involves cytokines. The major enzyme that catabolizes 25(OH)D and 1,25(OH)2D is the 24-hydroxylase; like the 1-hydroxylase it is tightly controlled in the kidney in a manner opposite to that of the 1-hydroxylase, but like the 1-hydroxylase it is widespread in other tissues where its regulation is different from that of the kidney. Vitamin D and its metabolites are carried in the blood bound to vitamin D binding protein (DBP) and albumin--for most tissues it is the free (i.e., unbound) metabolite that enters the cell; however, DBP bound metabolites can enter some cells such as the kidney and parathyroid gland through a megalin/cubilin mechanism. Most but not all actions of 1,25(OH)2D are mediated by the vitamin D receptor (VDR). VDR is a transcription factor that partners with other transcription factors such as retinoid X receptor that when bound to 1,25(OH)2D regulates gene transcription either positively or negatively depending on other cofactors to which it binds or interacts. The VDR is found in most cells, not just those involved with bone and mineral homeostasis (i.e., bone, gut, kidney) resulting in wide spread actions of 1,25(OH)2D on most physiologic and pathologic processes. Animal studies indicate that vitamin D has beneficial effects on various cancers, blood pressure, heart disease, immunologic disorders, but these non-skeletal effects have been difficult to prove in humans in randomized controlled trials. Analogs of 1,25(OH)2D are being developed to achieve specificity for non-skeletal target tissues such as the parathyroid gland and cancers to avoid the hypercalcemia resulting from 1,25(OH)2D itself. The level of vitamin D intake and achieved serum levels of 25(OH)D that are optimal and safe for skeletal health and the non-skeletal actions remain controversial, but are likely between an intake of 800-2000IU vitamin D in the diet and 20-50ng/ml 25(OH)D in the blood.

OVERVIEW

Rickets became a public health problem with the movement of the population from the farms to the cities during the Industrial Revolution. Various foods such as cod liver oil and irradiation of other foods including plants were found to prevent or cure this disease, leading eventually to the discovery of the active principle—vitamin D. Vitamin D comes in two forms (D₂ and D₃) which differ chemically in their side chains. These structural differences alter their binding to the carrier protein vitamin D binding protein (DBP) and their metabolism, but in general the biologic activity of their active metabolites is comparable. Vitamin D₃ is produced in the skin from 7-dehydrocholesterol by UV irradiation, which breaks the B ring to form pre-D₃. Pre-D₃ isomerizes to D₃ but with continued UV irradiation to tachysterol and lumisterol. D₃ is preferentially removed from the skin, bound to DBP. The liver and other tissues metabolize vitamin D, whether from the skin or oral ingestion, to 25OHD, the principal circulating form of vitamin D. Several enzymes have 25-hydroxylase activity, but CYP2R1 is the most important. 25OHD is then further metabolized to 1,25(OH)₂D principally in the kidney, by the enzyme CYP27B1, although other tissues including various epithelial cells, cells of the immune system, and the parathyroid gland contain this enzyme. 1,25(OH)₂D is the principal hormonal form of vitamin D, responsible for most of its biologic actions. The production of 1,25(OH)₂D in the kidney is tightly controlled, being stimulated by parathyroid hormone (PTH), and inhibited by calcium, phosphate and FGF23. Extrarenal production of 1,25(OH)₂D as in keratinocytes and macrophages is under different control, being stimulated primarily by cytokines such as tumor necrosis factor alfa (TNFα) and interferon gamma (IFNg). 1,25(OH)₂D reduces 1,25(OH)₂D levels in cells primarily by stimulating its catabolism through the induction of CYP24A1, the 24-hydroxylase. 25OHD and 1,25(OH)₂D are hydroxylated in the 24 position by this enzyme to form 24,25(OH)₂D and 1,24,25(OH)₃D, respectively. This 24-hydroxylation is generally the first step in the catabolism of these active metabolites to the final end product of calcitroic acid, although 24,25(OH)₂D and 1,24,25(OH)₃D have their own biologic activities. CYP24A1 also has 23-hydroxylase activity that leads to a different end product. Different species differ in their ratio of 23-hydroxylase/24-hydroxyase activity in their CYP24A1 enzyme, but in humans the 24-hydroxyase activity predominates. Like CYP27B1, CYP24A1 is widely expressed. CYP24A1 is induced by 1,25(OH)₂D in most tissues, which serves as an important feedback mechanism to avoid vitamin D toxicity. In the kidney, PTH inhibits CYP24A1, whereas FGF23, calcium and phosphate stimulates it, just the opposite of the actions of these hormones and minerals on CYP27B1. However, such regulation is not seen in other tissues. In macrophages, CYP24A1 is either missing or defective, so in situations such as granulomatous diseases like sarcoidosis in which macrophage production of 1,25(OH)₂D is increased, hypercalcemia and hypercalciuria due to elevated 1,25(OH)₂D can occur without the counter regulation by CYP24A1.

The vitamin D metabolites are transported in blood bound to DBP and albumin. Very little circulates as the free form. The liver produces DBP and albumin, production that is decreased in liver disease, and these proteins may be lost in protein losing enteropathies or the nephrotic syndrome. Thus, individuals with liver, intestinal or renal diseases which result in low levels of these transport proteins may have low total levels of the vitamin D metabolites without necessarily being vitamin D deficient as their free concentrations may be normal.

The receptor for 1,25(OH)₂D (VDR) is a transcription factor regulating the expression of genes which mediate its biologic activity. VDR is a member of a rather large family of nuclear hormone receptors which includes the receptors for glucocorticoids, mineralocorticoids, sex hormones, thyroid hormone, and vitamin A metabolites or retinoids. The VDR is widely distributed, and is not restricted to those tissues considered the classic target tissues of vitamin D. The VDR upon binding to 1,25(OH)₂D heterodimerizes with other nuclear hormone receptors, in particular the family of retinoid X receptors. This complex then binds to special DNA sequences called vitamin D response elements (VDRE) generally within the genes it regulates, although these VDREs can be thousands of base pairs from the transcription start site. There are thousands of the VDREs in hundreds of genes, and the profile of active VDREs (and regulated genes) varies from cell to cell. A variety of additional proteins called coregulators complex with the VDR to activate (coactivators) or inhibit (corepressors) VDR transcriptional activity. Coactivator factors involved in VDR mediated transcription include factors with histone acetylase activity, including steroid receptor coactivator (SRC) 1, SRC 2 and SRC 3, and CREB-binding protein p300, in addition to the SWI–SNF ATP dependent chromatin remodeling complex, methyltransferases and the Mediator complex (aka DRIP), which functions to recruit RNA polymerases. VDR binding sites are associated with sites for other transcription factors such as p63, C–EBPα, C–EBPβ, Runx2 and PU.1, which can cooperate with VDR and VDR coregulators to influence 1,25(OH)₂D responses in target cells. Among other functions these coregulators reconfigure the chromatin structure to bring the VDR/VDRE to the transcription start site, explaining how such distant VDR/VDREs can regulate gene transcription. In addition to coactivators there are a number of corepressors. One such corepressor of VDR action in the skin is called hairless, in that its loss or mutation, like that of the VDR, leads to altered hair follicle cycling resulting in baldness. Corepressors typically work by recruiting histone deacetylases (HDAC) or methyl transferases (MT) to the gene which reverses the actions of HAT, leading to a reduction in access to the gene by the transcription machinery. These coregulators can be specific for different genes, and different cells differentially express these coregulators, providing some specificity for the actions of 1,25(OH)₂D and VDR.

In addition to regulating gene expression, 1,25(OH)₂D has a number of non-genomic actions including the ability to stimulate calcium transport across the plasma membrane. The mechanisms mediating these non-genomic actions and their physiologic significance remain unclear. Similarly, it is not clear that all actions of the VDR require the ligand 1,25(OH)₂D. The best example of this is the hair loss in animals and subjects with VDR mutations but not in animals and subjects with mutations in CYP27B1, the enzyme producing 1,25(OH)₂D. As mentioned, the VDR is widely distributed, and the actions of 1,25(OH)₂D are quite varied. The classic target tissues—bone, gut, and kidney—are involved with calcium homeostasis. The mechanisms by which 1,25(OH)₂D regulates transcellular calcium transport are best understood in the intestine. Here 1,25(OH)₂D stimulates calcium entry across the brush border membrane into the cell, transport of calcium through the cell, and removal of calcium from the cell at the basolateral membrane. Calcium entry at the brush border membrane occurs down a steep electrochemical gradient. It is controlled in large measure by a specific calcium channel called TRPV6 and in humans also by a homologous calcium channel TRPV5. Transport of calcium through the cell is regulated by a class of calcium binding proteins called calbindins. Much of the transport occurs within vesicles that form in the terminal web. Removal of calcium from the cell at the basolateral membrane requires energy and is mediated by the ATP requiring calcium pump or CaATPase (PMCA1b) as well as the sodium/calcium exchange protein (NCX1). 1,25(OH)₂D induces TRPV6 and TRPV5, the calbindins, and the CaATPase, but not all aspects of transcellular calcium transport are a function of new protein synthesis. Animals null for calbindin 9k (the major calbindin in mammalian intestine) have little impairment of intestinal calcium transport. Animals null for TRPV6, on the other hand, have a reduction in intestinal calcium transport, but the deficit is not profound. Thus, it is likely that compensatory mechanisms for intestinal calcium transport exist that have yet to be discovered. Similar mechanisms mediate 1,25(OH)₂D regulated calcium reabsorption in the distal tubule of the kidney. The proteins involved are homologous but not identical (TRPV5 and Calbindin 28k, for example). The situation in bone, however, is less clear. VDR are found in osteoblasts, the bone forming cells. 1,25(OH)₂D promotes the differentiation of osteoblasts and regulates the production of proteins such as collagen, alkaline phosphatase, and osteocalcin thought to be important in bone formation. 1,25(OH)₂D also induces RANKL, a membrane bound protein in osteoblasts that enables osteoblasts to stimulate the formation and activity of osteoclasts. Thus 1,25(OH)₂D regulates both bone formation and bone resorption. Some evidence suggests that the major effect of 1,25(OH)₂D on bone is to provide adequate levels of calcium and phosphate from the intestine. The rickets of patients with a mutated VDR or of mice in which the VDR has been deleted can be prevented/corrected by normalizing serum calcium and phosphate levels by dietary means. On the other hand, normal bone formation is not restored, and with time the VDR null mice become osteoporotic despite the high calcium/phosphate diet. Moreover, the VDR in osteoblasts/osteocytes appears to control bone resorption especially when dietary calcium is limited. Whether subjects with VDR mutations also develop osteoporosis prematurely or fail to maintain serum calcium in times of calcium deficiency has not been reported.

The non-classic actions of 1,25(OH)₂D include regulation of cellular proliferation and differentiation, regulation of hormone secretion, and regulation of immune function. The ability of 1,25(OH)₂D to inhibit proliferation and stimulate differentiation has led to the development of a number of analogs in the hopes of treating hyperproliferative disorders such as psoriasis and cancer without raising serum calcium. Psoriasis is now successfully treated with several vitamin D analogs. Observational studies are promising with respect to adequate vitamin D nutrition and cancer prevention. However, supplementation with vitamin D of subjects with adequate vitamin D levels to start with has not been shown to decrease cancer incidence but may be beneficial for cancer mortality. 1,25(OH)₂D inhibits parathyroid hormone secretion and stimulates insulin secretion. A number of analogs and 1,25(OH)₂D itself are currently available for use in the treatment of secondary hyperparathyroidism accompanying renal failure. Epidemiologic evidence indicates that vitamin D deficiency is associated with increased risk of both type 1 and type 2 diabetes mellitus, but prospective clinical trials to demonstrate a role for vitamin D supplementation in preventing the conversion of prediabetes to diabetes has not shown benefit in vitamin D replete individuals. However, there may be benefit in vitamin D deficient patients. The ability of 1,25(OH)₂D to regulate immune function is likely part of its efficacy in the treatment of psoriasis. A number of other autoimmune diseases have been found in animal studies to respond favorably to vitamin D and 1,25(OH)₂D or its analogs, and epidemiologic evidence linking vitamin D deficiency to increased incidence of these diseases has been reported. Similarly, epidemiologic evidence linking vitamin D deficiency to a number of respiratory illnesses is substantial, including increased risk of COVID-19 infections.

DISCOVERY

The first clear description of rickets was by Whistler (1) in 1645. However, it was not until the Industrial Revolution with the mass movement of the population from the farms to the smoke- filled cities that rickets became a public health problem, most notably in England where sunlight intensity was already marginal for much of the year. Mellanby (2) in Great Britain and McCollum (3) in the United States developed animal models for rickets and showed that rickets could be cured with cod liver oil. McCollum heated the cod liver oil to destroy its vitamin A content and found that it still had antirachitic properties; he named the antirachitic factor vitamin D. Steenbock and Black (4) then demonstrated that UV irradiation of food, in particular non saponifiable lipids, could treat rickets. Meanwhile, clinical investigations revealed that rickets could be prevented or cured in children with sunlight or artificial UV exposure (5,6) suggesting that what subsequently became known as vitamin D could be produced by irradiation of precursors in vivo. Ultimately, Askew et al. (7) isolated and determined the structure of vitamin D₂ (ergocalciferol) from irradiated plant sterols (ergosterol), and Windaus et al. (8) determined the structures and pathway by which 7-dehydrocholesterol (7-DHC) in the skin is converted to vitamin D₃ (cholecalciferol). The name vitamin D₁ refers to what proved to be an error of an earlier identification, and is not used. The structures and pathways of production of vitamin D₃ are shown in figure 1. The structures of vitamins D₂ and D₃ differ in the side chain where D₂ contains a double bond (C_22-23) and an additional methyl group attached to C₂₄. In this chapter the designation of D will refer to both D₃ and D₂.

Figure 1. The production of vitamin D3 from 7-dehydrocholesterol in the epidermis. Sunlight (the ultraviolet B component) breaks the B ring of the cholesterol structure to form pre- D3. Pre-D3 then undergoes a thermal induced rearrangement to form D3. Continued irradiation of pre- D3 leads to the reversible formation of lumisterol3 and tachysterol3 which can revert back to pre-D3 in the dark.

Figure 2. The metabolism of vitamin D. The liver converts vitamin D to 25OHD. The kidney converts 25OHD to 1,25(OH)2D and 24,25(OH)2D. Other tissues contain these enzymes, but the liver is the main source for 25-hydroxylation, and the kidney is the main source for 1α-hydroxylation. Control of metabolism of vitamin D to its active metabolite, 1,25(OH)2D, is exerted primarily at the renal level where calcium, phosphorus, parathyroid hormone, FGF23, and 1,25(OH)2D regulate the levels of 1,25(OH)2D produced.

METABOLISM

Vitamin D₃ produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes place primarily in the liver, although other tissues have this enzymatic activity as well. As will be discussed below, there are several 25-hydroxylases. 25OHD is the major circulating form of vitamin D. However, in order for vitamin D metabolites to achieve maximum biologic activity they must be further hydroxylated in the 1α position by the enzyme CYP27B1; 1,25(OH)₂D is the most potent metabolite of vitamin D and accounts for most of its biologic actions. The 1α hydroxylation occurs primarily in the kidney, although as for the 25-hydroxylase, other tissues have this enzyme. Vitamin D and its metabolites, 25OHD and 1,25(OH)₂D, can also be hydroxylated in the 24 position. This may serve to activate the metabolite or analog as 1,25(OH)₂D and 1,24(OH)₂D have similar biologic potency, and 1,24,25(OH)₃D has activity approximately 1/10 that of 1,25(OH)₂D. However, 24-hydroxylation of metabolites with an existing 25OH group leads to further catabolism. The details of these reactions are described below.

Cutaneous Production of Vitamin D₃

The precursor of vitamin D, 7-dehydrocholesterol (7-DHC) is on the Kandutsch-Russell cholesterol pathway. The final enzymatic reaction mediated by 7-dehyrocholesterol reductase converting 7-DHC to cholesterol is regulated by a number of factors including vitamin D and cholesterol which enhance its degradation thus enabling increased levels of 7-DHC for conversion to vitamin D (9). Although irradiation of 7-DHC was known to produce pre-D₃ (which subsequently undergoes a temperature rearrangement of the triene structure to form D₃), lumisterol, and tachysterol (figure 1), the physiologic regulation of this pathway was not well understood until the studies of Holick and his colleagues (10-12). They demonstrated that the formation of pre-D₃ under the influence of solar or UV irradiation (maximal effective wavelength between 290-310) is relatively rapid and reaches a maximum within hours. UV irradiation further converts pre-D₃ to lumisterol and tachysterol. Both the degree of epidermal pigmentation and the intensity of exposure correlate with the time required to achieve this maximal concentration of pre-D₃, but do not alter the maximal level achieved. Although pre-D₃ levels reach a maximum level, the biologically inactive lumisterol continues to accumulate with continued UV exposure. Tachysterol is also formed, but like pre-D₃, does not accumulate with extended UV exposure. The formation of lumisterol is reversible and can be converted back to pre-D₃ as pre-D₃ levels fall. At 0^oC, no D₃ is formed; however, at 37^oC pre-D₃ is slowly converted to D₃. Thus, short exposure to sunlight would be expected to lead to a prolonged production of D₃ in the exposed skin because of the slow thermal conversion of pre-D₃ to D₃ and the conversion of lumisterol to pre-D₃. Prolonged exposure to sunlight would not produce toxic amounts of D₃ because of the photoconversion of pre-D₃ to lumisterol and tachysterol as well as the photoconversion of D₃ itself to suprasterols I and II and 5,6 transvitamin D₃ (13).

Melanin in the epidermis, by absorbing UV irradiation, can reduce the effectiveness of sunlight in producing D₃ in the skin. This may be one important reason for the lower 25OHD levels (a well-documented surrogate measure for vitamin D levels in the body) in Blacks and Hispanics living in temperate latitudes (14). Sunlight exposure increases melanin production, and so provides another mechanism by which excess D₃ production can be prevented. The intensity of UV irradiation is also important for effective D₃ production. The seasonal variation of 25OHD levels can be quite pronounced with higher levels during the summer months and lower levels during the winter. The extent of this seasonal variation depends on the latitude, and thus the intensity of the sunlight striking the exposed skin. In Edmonton, Canada (52^oN) very little D₃ is produced in exposed skin from mid-October to mid-April; Boston (42^oN) has a somewhat longer period for effective D₃ production; whereas in Los Angeles (34^oN) and San Juan (18^oN) the skin is able to produce D₃ all year long (15). These findings apply to sea level. At higher elevations there is less atmospheric absorption of UVB, so that skiers can make vitamin D even in winter on sunny days. Peak D₃ production occurs around noon, with a larger portion of the day being capable of producing D₃ in the skin during the summer than other times of the year. Clothing (16) and sunscreens (17) effectively prevent D₃ production in the covered areas. This is one likely explanation for the observation that the Bedouins in the Middle East, who totally cover their bodies with clothing, are more prone to develop rickets and osteomalacia than the Israeli Jews with comparable sunlight exposure.

Hepatic Production of 25OHD

The next step in the bioactivation of D₂ and D₃, hydroxylation to 25OHD, takes place primarily in the liver although a number of other tissues express this enzymatic activity. 25OHD is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. DeLuca and colleagues were the first to identify 25OHD and demonstrate its production in the liver over 30 years ago, but ambiguity remains as to the actual enzyme(s) responsible for this activity. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities appear to differ indicating different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1. However, in mouse knockout studies and in humans with mutations in these enzymes, only CYP2R1 loss is associated with decreased 25OHD levels (18,19). However, deletion or mutation of CYP2R1 does not totally eliminate 25OHD production These are mixed function oxidases, but differ in apparent Kms and substrate specificities.

The mitochondrial 25-hydroxylase is now well accepted as CYP27A1, an enzyme first identified as catalyzing a critical step in the bile acid synthesis pathway. This is a high capacity, low affinity enzyme consistent with the observation that 25-hydroxylation is not generally rate limiting in vitamin D metabolism. Although initial studies suggested that the vitamin D₃-25-hydroxylase and cholestane triol 27-hydroxyase activities in liver mitochondria were due to distinct enzymes with differential regulation, the cloning of CYP27A1 and the demonstration that it contained both activities has put this issue to rest (20-22). CYP27A1 is widely distributed throughout different tissues with highest levels in liver and muscle, but also in kidney, intestine, lung, skin, and bone (20-23). Mutations in CYP27A1 lead to cerebrotendinous xanthomatosis (24,25), and are associated with abnormal vitamin D and/or calcium metabolism in some but not all of these patients (25-27). However, mice in which CYP27A1 is deleted actually have elevated 25OHD levels along with the disruption in bile acid synthesis (28). CYP27A1 can hydroxylate vitamin D and related compounds at the 24, 25, and 27 positions. However, D₂ appears to be preferentially 24-hydroxylated, whereas D₃ is preferentially 25-hydroxylated (29). The 1αOH derivatives of D are more rapidly hydroxylated than the parent compounds (30). These differences between D₂ and D₃ and their 1αOH derivatives may explain the differences in biologic activity between D₂ and D₃ or between 1αOHD₂ and 1αOHD₃.

The major microsomal 25-hydroxylase is CYP2R1, although other enzymes have been shown in in vitro studies to have 25-hydroxylase activity. This enzyme like that of CYP27A1 is widely distributed, although it is most abundantly expressed in liver, skin and testes (30). Unlike CYP27A1, CYP2R1 25-hydroxylates D₂ and D₃ equally (30). Several Nigerian families have been shown to have CYP2R1 mutations in family members with rickets (19,31). These subjects respond to D therapy but suboptimally (19,31). Mice lacking CYP2R1 have reduced 25OHD levels, unlike mice lacking CYP27A1, but even the combined deletion of CYP2R1 and CYP27A1 does not reduce these levels more than about 70% (18). Thus, neither CYP27A1 nor CYP2R1 by themselves account for all 25-hydroxyase activity in the body, suggesting a role of other yet to be described 25-hydroxylases.

Studies of the regulation of 25-hydroxylation have not been completely consistent, most likely because of the initial failure to appreciate that at least two enzymatic activities were involved and because of species differences. In general, 25-hydroxylation in the liver is little affected by vitamin D status. However, CYP27A1 expression in the intestine (32) and kidney (33) is reduced by 1,25(OH)₂D. Not surprisingly bile acids decrease CYP27A1 expression (34) as does insulin (35) through an unknown mechanism. Dexamethasone, on the other hand, increases CYP27A1 expression (36). CYP2R1 appears to be mediated by aspects of metabolism. Roizen et al. (37) found that the serum concentration of 25OHD, but not vitamin D, was decreased in mice fed a high fat diet to induce obesity compared with normal weight mice. Moreover, mRNA and protein levels of CYP2R1 were decreased in these obese mice. The expression of other 25-hydroxylases (CYP27A1, CYP3A) or the catabolizing enzyme CYP24A1 was not altered. Aatsinki et al (38) examined the effect of high fat diet induced obesity, fasting, and type 2 diabetes as well as streptozotocin induced (type 1) diabetes on 25OHD levels in mice. All these metabolic manipulations decreased the hepatic mRNA and protein concentration of CYP2R1. These authors then demonstrated that the decrease in CYP2R1 was mediated by PPARγ-coactivator-1α (PGC1α), a key metabolic regulator increased by fasting or diabetes. They then showed that the control of CYP2R1 gene expression by PGC1α involved another transcriptional regulator, estrogen-related receptor α (ERRα), which also binds to other nuclear receptors such as VDR and the glucocorticoid receptor (GR). Consistent with this is that dexamethasone, a ligand for GR, decreased hepatic CYP2R1 mRNA and protein concentrations by a mechanism mediated by increased PGC1α.

Renal Production of 1,25(OH)₂D

1,25(OH)₂D is the most potent metabolite of vitamin D, and mediates most of its hormonal actions. 1,25(OH)₂D is produced from 25OHD by the enzyme 25OHD-1α hydroxylase (CYP27B1). The cloning of CYP27B1 by four independent groups (40-43) ended a long effort to determine the structure of this critical enzyme in vitamin D metabolism. Mutations in this gene are responsible for the rare autosomal disease of pseudovitamin D deficiency rickets (40,42,44,45). An animal model in which the gene is knocked out by homologous recombination reproduces the clinical features of this disease including retarded growth, rickets, hypocalcemia, hyperparathyroidism, and undetectable 1,25(OH)₂D (46). Unlike Vdr null mice and VDR mutations in humans, alopecia is not part of this phenotype.

CYP27B1 is a mitochondrial mixed function oxidase with significant homology to other mitochondrial steroid hydroxylases including CYP27A1 (39%), CYP24A1 (30%), CYP11A1 (32%), and CYP11β (33%) (40). However, within the heme-binding domain the homology is much greater with 73% and 65% sequence identity with CYP27A1 and CYP24A1 (40). These mitochondrial P450 enzymes are located in the inner membrane of the mitochondrion, and serve as the terminal acceptor for electrons transferred from NADPH through ferrodoxin reductase and ferrodoxin. Expression of CYP27B1 is highest in epidermal keratinocytes (40), cells that previously had been shown to contain high levels of this enzymatic activity (47). However, the kidney also expresses this enzyme in the renal tubules as do the brain, placenta, testes, intestine, lung, breast, macrophages, lymphocytes, parathyroid gland, osteoblasts and chondrocytes (40,48-51). That said, the kidney is generally considered the major source of circulating levels of 1,25(OH)₂D, with the extrarenal CYP27B1 activities providing for local needs under normal circumstances. However, extrarenal sources can lead to increased 1,25(OH)₂D and calcium levels in some pathologic conditions to be discussed subsequently.

The principal regulators of CYP27B1 activity in the kidney are parathyroid hormone (PTH), FGF23, calcium, phosphate, and 1,25(OH)₂D. Extrarenal production tends to be stimulated by cytokines such as IFN-gamma and TNF-α more effectively than PTH (52) and may be less inhibited by calcium, phosphate, and 1,25(OH)₂D depending on the tissue. Administration of PTH in vivo (53) or in vitro (54,55) stimulates renal production of 1,25(OH)₂D. This action of PTH can be mimicked by cAMP (53,55) and forskolin (56,57) indicating that at least part of the effect of PTH is mediated via its activation of adenylate cyclase. However, PTH activation of protein kinase C (PKC) also appears to be involved in that concentrations of PTH sufficient to stimulate PKC activation and 1,25(OH)₂D production are below that required to increase cAMP levels (58). Furthermore, synthetic fragments of PTH lacking the ability to activate adenylate cyclase but which stimulate PKC activity were found to increase 1,25(OH)₂D production (59). Direct activation of PKC with phorbol esters results in increased 1,25(OH)₂D production. Although the promoter of CYP27B1 contains several AP-1 (PKC activated) and cAMP response elements, it is not yet clear how PTH regulates CYP27B1 gene expression (60). However, several mechanisms have been proposed. In one study the nuclear receptor 4A2 acting through a C/EBP consensus element appears to be involved (61). Another mechanism involves VDIR that is proposed to bind to a negative VDRE in the CYP27B1 promoter. When PKA is activated by PTH VDIR is phosphorylated and recruits the p300 complex with HAT activity, inducing gene transcription (62). Calcium modulates the ability of PTH to increase 1,25(OH)₂D production. Calcium by itself can decrease CYP27B1activity (63,64) and block the stimulation by PTH (65). Given in vivo, calcium can exert its effect in part by reducing PTH secretion, but this does not explain its direct actions in vitro or its effects in parathyroidectomized or PTH infused animals. Phosphate deprivation can stimulate CYP27B1 activity in vivo (66,67) and in vitro (68). The in vivo actions of phosphate deprivation can be blocked by hypophysectomy (69,70) and partially restored by growth hormone (GH) (70,71) and insulin-like growth factor (IGF-I) (72). However, like PTH, the exact mechanism by which GH and/or IGF-I mediates the effects of phosphate on CYP27B1 expression remains unclear. More recently FGF23 has been shown to inhibit CYP27B1 activity in vivo and in vitro (73). FGF23 has been implicated as at least one of the factors responsible for impaired phosphate reabsorption and 1,25(OH)₂D production in conditions such as X-linked and autosomal dominant hypophosphatemic rickets and oncogenic osteomalacia (74,75). FGF23 acts through FGF receptors 1 and 3 in conjunction with the coreceptor Klotho, but the mechanism by which FGF23 regulates CYP27B1 remains obscure. High phosphate stimulates FGF23 production from bone, and this is likely the major mechanism by which phosphate leads to decreased CYP27B1 activity (76). 1,25(OH)₂D administration leads to reduction in CYP27B1 activity. In the kidney Meyer et al. (77) identified a region in the Cyp27b1 gene that when deleted blocked 1,25(OH)₂D production. However, in other tissues no vitamin D response element has been identified in the promoter of the 1α-hydroxylase gene (60). In keratinocytes, 1,25(OH)₂D has little or no effect on CYP27B1 mRNA and protein levels when given in vitro. When 24-hydroxylase activity is blocked, 1,25(OH)₂D administration fails to reduce the levels of 1,25(OH)₂D produced (78,79). Thus, the apparent feedback regulation of CYP27B1 activity by 1,25(OH)₂D in most tissues, with the possible exception of the kidney, appears to be due to its stimulation of CYP24A1 and subsequent catabolism, not to a direct effect on CYP27B1 expression or activity. Moreover, 1,25(OH)₂D stimulates FGF23 production and inhibits PTH production. Both actions will decrease, indirectly, the ability of 1,25(OH)₂D to inhibit its own production (76). Thus, renal and extrarenal regulation of CYP27B1 by 1,25(OH)₂D may differ.

Renal Production of 24,25(OH)₂D

The kidney is also the major producer of a second important metabolite of 25OHD, namely 24,25(OH)₂D, and the enzyme responsible is 25OHD-24 hydroxylase (CYP24A1) [75]. CYP24A1 and CYP27B1 are homologous enzymes that coexist in the mitochondria of tissues where both are found, such as the kidney tubule. However, there genes are located on different chromosomes (chromosome 20q13 and chromosome 12q14 for CYP24A1 and CYP27B1, respectively, in humans). They share the same ferrodoxin and ferrodoxin reductase components. While CYP27B1 activates the parent molecule, 25OHD, CYP24A1 initiates a series of catabolic steps that lead to its inactivation. However, in some tissues 24,25(OH)₂D has been shown to have biologic effects different from 1,25(OH)₂D as will be described subsequently. CYP24A1 24-hydroxylates both 25OHD and 1,25(OH)₂D. The 24-hydroxylation is then followed by oxidation of 24OH to a 24-keto group, 23-hydroxylation, cleavage between C23-24, and the eventual production of calcitroic acid, a metabolite with no biologic activity. CYP24A1 also has 23-hydroxylase activity, initiating steps that lead to 23/26 lactone formation. Different species have CYP24A1s that differ in their preference for the 24-hydroxylation vs 23-hydroxylation pathway. The human enzyme follows the 24-hydroxylation pathway. Analogs with differences in their side chain are also likely to differ in the pathway utilized. CYP24A1 catalyzes all the steps in this catabolic pathway (81) (82). Although CYP24A1 is highly expressed in the kidney tubule, its tissue distribution is quite broad. In general, CYP24A1 can be found wherever the VDR is found. The affinity for 1,25(OH)₂D is higher than that for 25OHD, making this enzyme an efficient means for eliminating 1,25(OH)₂D. Thus, CYP24A1 is likely to play the important role of protecting the body against excess 1,25(OH)₂D. Indeed, inactivating mutations in CYP24A1 have been found to underlie the disease idiopathic infantile hypercalcemia (83), manifesting as the name suggests with elevated serum calcium and 1,25(OH)₂D levels. These individuals may present for the first time as adults, often in the context of increased 1,25(OH)₂D production as in pregnancy (84). An animal model in which CYP24A1 has been knocked out likewise showed very high levels of 1,25(OH)₂D when treated with vitamin D and impaired mineralization of intramembranous bone (85). The skeletal abnormalities could be corrected by crossing this mouse to one lacking the VDR suggesting that excess 1,25(OH)₂D (which acts through the VDR) rather than deficient 24,25(OH)₂D (which does not) is to blame (85).

The regulation of CYP24A1 in the kidney is almost the mirror image of that of CYP27B1. PTH and 1,25(OH)₂D are the dominant regulators, but calcium, phosphate, insulin, FGF23, IGF-I, GH, and sex steroids may also play a role. 1,25(OH)₂D induces CYP24A1. The promoter of CYP24A1 has two vitamin D response elements (VDREs) critical for this induction (86-88). Protein kinase C activation as by phorbol esters enhances this induction by 1,25(OH)₂D (89). An AP-1 site is found adjacent to the proximal VDRE, but mutation of this site does not appear to block phorbol ester enhancement of CYP24A1 induction by 1,25(OH)₂D (90). PTH, on the other hand, inhibits the expression of CYP24A1 in the kidney (91). This action can be reproduced with cAMP (92) and forskolin (56) indicating the role of PTH activated adenylate cyclase (93). PTH has no effect on intestinal CYP24A1, most likely because the intestine does not have PTH receptors. Surprisingly, however, PTH is synergistic with 1,25(OH)₂D in stimulating CYP24A1 expression and activity in bone cells which do have PTH receptors, again through a cAMP mediated mechanism (94). This synergism is further potentiated by the addition of insulin (95) (96). FGF23 also induces CYP24A1 expression (97). Surprisingly this requires the VDR (97), since FGF23 also inhibits 1,25(OH)₂D production and so would be expected to reduce CYP24A1 via a 1,25(OH)₂D/VDR mechanism. Restriction in dietary phosphate reduces CYP24A1 expression consistent with a decrease in FGF23, but also in a manner blocked by hypophysectomy (98). GH and IGF-I can reduce CYP24A1 expression in hypophysectomized animals, suggesting that the phosphate effect on CYP24A1 like its opposing effect on CYP27B1, is mediated by GH and IGF-I (98) as well as FGF23. The region(s) of the CYP24A1 promoter mediating these actions of PTH and FGF23 as well as 1,25(OH)2D have recently been mapped (96). Similar to that for CYP27B1 this regulation differs in different cell types. Thus, although different regulators tend to have opposite effects on CYP24A1 and CYP27B1 expression the molecular mechanisms by which the regulation occurs also differ for each enzyme.

TRANSPORT IN BLOOD

The vitamin D metabolites are transported in blood bound primarily to vitamin D binding protein (DBP) (85-88%) and albumin (12-15%) (99-101). DBP concentrations are normally 4-8µM, well above the concentrations of the vitamin D metabolites, such that DBP is only about 2% saturated. DBP has high affinity for the vitamin D metabolites (Ka=5x10⁸M^-1 for 25OHD and 24,25(OH)₂D, 4x10⁷M^-1 for 1,25(OH)₂D and vitamin D), such that under normal circumstances only approximately 0.03% 25OHD and 24,25(OH)₂D and 0.4% 1,25(OH)₂D are free (100-102). Conditions such as liver disease and nephrotic syndrome resulting in reduced DBP and albumin levels will lead to a reduction in total 25OHD and 1,25(OH)₂D levels without necessarily affecting the free concentrations (103) (figure 3). Similarly, DBP levels are reduced during acute illness, potentially obscuring the interpretation of total 25OHD levels (104). Earlier studies with a monoclonal antibody to measure DBP levels suggested a decreased level in African Americans consistent with their lower total 25OHD levels, but these results were not confirmed using polyvalent antibody-based assays (105). Vitamin D intoxication can increase the degree of saturation sufficiently to increase the free concentrations of 1,25(OH)₂D and so cause hypercalcemia without necessarily raising the total concentrations (106).

The vitamin D metabolites bound to DBP are in general not available to most cells. Thus, the free or unbound concentration is that which is critical for cellular uptake as postulated by the free hormone hypothesis. Support for the concept that the role of DBP is to provide a reservoir for the vitamin D metabolites but that it is the free concentration that enters cells and exerts biologic function comes from studies in mice in which DBP has been deleted and in humans in which the gene is mutated. In DBP knockout mice the vitamin D metabolites are presumably all free and/or bioavailable. These mice do not show evidence of vitamin D deficiency unless placed on a vitamin D deficient diet despite having very low levels of serum 25OHD and 1,25(OH)₂D (107). Tissue levels of 1,25(OH)₂D were found to be normal in the DBP knockout mice as were markers of vitamin D action such as expression of intestinal TRPV6, calbindin 9k, PMCA1b, and renal TRPV5 (108). Recently a family in which a large deletion of the coding portion of the DBP gene (and adjacent NPFFR2 gene) has been reported (109). The proband had normal calcium, phosphate and PTH levels with vitamin D supplementation despite very low levels of 25OHD, 24,25(OH)₂D, and 1,25(OH)₂D that were not responsive to massive doses of vitamin D (oral or parenteral). The free 25OHD was nearly normal. The carrier sibling had vitamin D metabolite levels between those of the proband and the normal sibling. Thus, both the studies in DBP null mice and humans support the free hormone hypothesis while also supporting the role of DBP as a circulating reservoir for the vitamin D metabolites. Therefore, there is currently a debate as to whether the free concentration of 25OHD, for example, is a better indicator of vitamin D nutritional status than total 25OHD, given that DBP levels, and hence total 25OHD levels, can be influenced by liver disease, nephrotic syndrome, pregnancy, and inflammatory states (110,111). However, certain tissues such as the kidney, placenta, and parathyroid gland express the megalin/cubilin complex which is able to transport vitamin D metabolites bound to DBP into the cell. This is critical for preventing renal losses of the vitamin metabolites (112) and may be important for vitamin D metabolite transport into the fetus and regulation of PTH secretion. Indeed, mice lacking the megalin/cubilin complex have poor survival with evidence of osteomalacia indicating its role in vitamin D transport into critical cells involved with vitamin D signaling

Figure 3. Correlation of total 25OHD (A) and 1,25(OH)2D (C) levels to DBP; lack of correlation of free 25OHD (B) and 1,25(OH)2D (D) levels to DBP. Data from normal subjects (open triangles), subjects with liver disease (closed triangles, open circles), subjects on oral contraceptives (open triangles*), and pregnant women (open squares) are included. These data demonstrate the dependence of total 25OHD and 1,25(OH)2D concentrations on DBP levels which are reduced by liver disease. However, the free concentrations of 25OHD and 1,25(OH)2D are normal in most patients with liver disease. Reprinted with permission from the American Society for Clinical Investigation.

DBP was originally known as group specific component (Gc-globulin) before its properties as a vitamin D transport protein became known. It has three common polymorphisms which are useful in population genetics. These alleles have somewhat different affinities for the vitamin D metabolites (113), but which do not appear to alter its function. DBP is a 58kDa protein with 458 amino acids that is homologous to albumin and α-fetoprotein (αFP) (40% homology at the nucleotide level, 23% at the amino acid level) (114). These three genes cluster on chromosome 4q11-13 (115). DBP, like albumin and αFP, is made primarily but not exclusively in the liver-other sites include the kidney, testes, and fat. DBP like other steroid hormone binding proteins is increased by oral (not transdermal) estrogens and pregnancy (100). In vitro, glucocorticoids and cytokines such as EGF, IL-6 and TGF-β have been shown to increase (glucocorticoids, EGF, IL-6) or decrease (TGF-β) DBP production (116).

Although transport of the vitamin D metabolites may be the major function for DBP, it has other properties. DBP has high affinity for actin, and may serve as a scavenger for actin released into the blood during cell death (117). DBP has also been shown to activate macrophages (118) and osteoclasts (119). However, in a mouse rendered deficient in DBP by homologous recombination (knock out) no obvious abnormality was observed except for increased turnover in vitamin D and increased susceptibility to osteomalacia on a vitamin D deficient diet (120). Evidence for osteopetrosis (indicating failure of osteoclast function) was not found.

MECHANISM OF ACTION

The hormonal form of vitamin D, 1,25(OH)₂D, is the ligand for a transcription factor, the vitamin D receptor (VDR). Most if not all effects of 1,25(OH)₂D are mediated by VDR acting primarily by regulating the expression of genes whose promoters contain specific DNA sequences known as vitamin D response elements (VDREs). There are thousands of VDREs throughout the gene, often thousands of base pairs away from the coding portion of the gene regulated. However, some actions of 1,25(OH)₂D are more immediate, and may be mediated by a membrane bound vitamin D receptor that has been less well characterized than the nuclear VDR or by the VDR acting outside of the nucleus. On the other hand, some actions of VDR do not require its ligand 1,25(OH)₂D. Our understanding of the mechanism by which VDR regulates gene expression has increased enormously over the past few years.

VDR and Transcriptional Regulation

The VDR was discovered in 1969 (121) (although only as a binding protein for an as yet unknown vitamin D metabolite subsequently identified as 1,25(OH)₂D), and was eventually cloned and sequenced in 1987 (122,123). Inactivating mutations in the VDR result in hereditary vitamin D resistant rickets (HVDRR) (124). Animal models in which the VDR has been knocked out (125) (126) have the full phenotype of severe vitamin D deficiency indicating that the VDR is the major mediator of vitamin D action. The one major difference is the alopecia seen in HVDRR and VDR knockout animals, a feature not associated with vitamin D deficiency, suggesting that the VDR may have functions independent of 1,25(OH)₂D at least in hair follicle cycling. The VDR is a member of a large family of proteins (over 150 members) that includes the receptors for the steroid hormones, thyroid hormone, vitamin A family of metabolites (retinoids), and a variety of cholesterol metabolites, bile acids, isoprenoids, fatty acids and eicosanoids. A large number of family members have no known ligands, and are called orphan receptors. VDR is widely, although not universally, distributed throughout the different tissues of the body (127). Many of these tissues were not originally considered target tissues for 1,25(OH)₂D. The discovery of VDR in these tissues along with the demonstration that 1,25(OH)₂D altered function of these tissues has markedly increased our appreciation of the protean effects of 1,25(OH)₂D.

The VDR is a molecule of approximately 50-60kDa depending on species. The basic structure is shown in figure 4. The VDR is unusual in that it has a very short N-terminal domain before the DNA binding domain when compared to other nuclear hormone receptors. The human VDR has two potential start sites. A common polymorphism (Fok 1) alters the first ATG start site to ACG. Individuals with this polymorphism begin translation three codons downstream such that in these individuals the VDR is three amino acids shorter (424 aas vs 427 aas). This polymorphism has been correlated with reduced bone density suggesting it is of functional importance (128). The most conserved domain in VDR from different species and among the nuclear hormone receptors in general is the DNA binding domain. This domain is comprised of two zinc fingers. The name derives from the cysteines within this stretch of amino acids that form tetrahedral complexes with zinc in a manner which creates a loop or finger of amino acids with the zinc complex at its base. The proximal (N-terminal) zinc finger confers specificity for DNA binding to the VDREs while the second zinc finger and the region following provide at least one of the sites for heterodimerization of the VDR to the retinoid X receptor (RXR). The second half of the molecule is the ligand binding domain, the region responsible for binding 1,25(OH)₂D, but which also contains regions necessary for heterodimerization to RXR. At the C-terminal end is the major activation domain, AF-2, which is critical for the binding to coactivators such as those in the steroid receptor coactivator (SRC) and vitamin D receptor interacting protein (DRIP) or Mediator families (129). In mutation studies of the homologous thyroid receptor, corepressors were found to bind in overlapping regions with coactivators in helices 3 and 5, a region blocked by helix 12 (the terminal portion of the AF2 domain) in the presence of ligand (130). Deletion of helix 12 promoted corepressor binding while preventing that of coactivators (130).

Figure 4. Model of the vitamin D receptor (VDR). The N terminal region is short relative to other steroid hormone receptors. This region is followed by two zinc fingers which constitute the principal DNA binding domain. Nuclear localization signals (NLS) are found within and just C-terminal to the DNA binding domain. The ligand binding domain makes up the bulk of the C-terminal half of the molecule, with the AF2 domain comprising the most C-terminal region. The AF2 domain is largely responsible for binding to co-activators such as the SRC family and DRIP (Mediator) in the presence of ligand. Regions on the second zinc finger and within the ligand binding domain facilitate heterodimerization with RXR. Corepressor binding is less well characterized but appears to overlap that of coactivators in helices 3 and 5, a region blocked by helix 12 in the presence of ligand.

The ligand binding domain (LBD) for VDR has been crystallized and its structure solved (131). More recently the structure of the VDR/RXR heterodimer has been analyzed by high resolution cryoelectron microscopy (132). These studies show that the VDR has a high degree of structural homology to other nuclear hormone receptors. It is comprised of 12 helices joined primarily by beta sheets. The 1,25(OH)₂D is buried deep in the ligand binding pocket and covered by helix 12 (the terminal portion of the AF-2 domain). Assuming analogy with the unliganded LBD of RXRα and the ligand bound LBD of RARγ (133), the binding of 1,25(OH)₂D to the VDR triggers a substantial movement of helix 12 from an open position to a closed position, covering the ligand binding pocket and putting helix 12 in position with critical residues from helices 3, 4, and 5 to bind coactivators. Coactivator complexes bridge the gap from the VDRE to the transcription machinery at the transcription start site (figure 5) (134).

Figure 5. 1,25(OH)2D-initiated gene transcription. 1,25(OH)2D enters the target cell and binds to its receptor, VDR. The VDR then heterodimerizes with the retinoid X receptor (RXR). This increases the affinity of the VDR/RXR complex for the vitamin D response element (VDRE), a specific sequence of nucleotides in the promoter region of the vitamin D responsive gene. Binding of the VDR/RXR complex to the VDRE attracts a complex of proteins termed coactivators to the VDR/RXR complex. The DRIP (Mediator) coactivator complex spans the gap between the VDRE and RNA polymerase II and other proteins in the initiation complex centered at or around the TATA box (or other transcription regulatory elements). SRC coactivators recruit histone acetyl transferases (HAT) to the gene promoting the opening up of its structure to enable the transcription machinery to work. Transcription of the gene is initiated to produce the corresponding mRNA, which leaves the nucleus to be translated to the corresponding protein.

Nuclear hormone receptors including the VDR are further regulated by protein complexes that can be activators or repressors (135). The role of corepressors in VDR function has been demonstrated (136) but is less well studied than the role of coactivators. One such corepressor, hairless, is found in the skin and may regulate 1,25(OH)₂D mediated epidermal proliferation and differentiation as well as ligand independent VDR regulation of hair follicle cycling (137-139). The coactivators, which are essential for VDR function, form two distinct complexes, the interaction of which remains unclear (129). The SRC family has three members, SRC 1-3, all of which can bind to the VDR in the presence of ligand (1,25(OH)₂D) (140). These coactivators recruit additional coactivators such as CBP/p300 and p/CAF that have histone acetyl transferase activity (HAT), an enzyme that by acetylation of lysines within specific histones appears to help unravel the chromatin allowing the transcriptional machinery to do its job. The domain in these molecules critical for binding to the VDR and other nuclear hormone receptors is called the NR box, and has as its central motif LxxLL where L stands for leucine and x for any amino acid. Each SRC family member contains three well conserved NR boxes in the region critical for nuclear hormone receptor binding. The DRIP (Mediator) complex is comprised of 15 or so proteins several of which contain LxxLL motifs (141). However, DRIP205 (Mediator 1) is the protein critical for binding the complex to VDR. It contains 2 NR boxes. Different NR boxes in these coactivators show specificity for different nuclear hormone receptors (142). Unlike the SRC complex, the DRIP complex does not have HAT activity (129). Rather the DRIP complex spans the gene from the VDRE to the transcription start site linking directly with RNA polymerase II and its associated transcription factors. DRIP and SRC appear to compete for binding to the VDR. In keratinocytes DRIP binds preferentially to the VDR in undifferentiated cells, whereas SRC 2 and 3 bind in the more differentiated cells in which DRIP levels have declined (143). Thus in these cells DRIP appears to regulate the early stages of 1,25(OH)₂D induced differentiation, whereas SRC may be more important in the later stages, although overlap in gene specificity is also observed (144,145) (146). These coregulators are not specific for VDR, but interact with a large number of other transcription factors. The DRIP (Mediator) complex can mark regions in the genome containing large numbers of sites for transcription factors including VDREs. These sites are known as super enhancers often regulating genes involved with cell fate determination (147). Recently, SMAD 3, a transcription factor in the TGF-β pathway, has been found to complex with the SRC family members and the VDR, enhancing the coactivation process (148). Phosphorylation of the VDR may also control VDR function (149). Furthermore, VDR has been shown to suppress β-catenin transcriptional activity (150), whereas β-catenin enhances that of VDR (151). Thus, control of VDR activity may involve crosstalk between signaling pathways originating in receptors at the plasma membrane as well as within the nucleus.

VDR acts in concert with other nuclear hormone receptors, in particular RXR (152). Unlike VDR, there are three forms of RXR--α, β, γ--and all three are capable of binding to VDR with no obvious differences in terms of functional effect. RXR and VDR form heterodimers that optimize their affinity for the vitamin D response elements (VDREs) in the genes being regulated. RXR appears to be responsible for keeping VDR in the nucleus in the absence of ligand (153). VDR may also partner with other receptors including the thyroid receptor (TR) and the retinoic acid receptor (RAR) (154,155), but these are the exceptions, whereas RXR is the rule. The VDR/RXR heterodimers bind to VDREs, which typically are comprised of two half sites each with six nucleotides separated by three nucleotides of nonspecific type; this type of VDRE is known as a DR3 (direct repeats with three nucleotide spacing). RXR binds to the upstream half site, while VDR binds to the downstream site (156). However, a wide range of VDRE configurations have been found at nearly any location within a gene (5’, 3’, introns) (157). Moreover, different tissues differ as to which VDREs actively bind VDR (158). 1,25(OH)₂D is required for high affinity binding and activation, but the RXR ligand, 9-cis retinoic acid, may either inhibit (159) or activate (160) 1,25(OH)₂D stimulation of gene transcription. A DR6 has been identified in the phospholipase C-γ1 gene that recognizes VDR/RAR heterodimers (154), and a DR4 has been found in the mouse calbindin 28k gene (161). Inverted palinodromes with 7 to 12 bases between half sites have also been found (151). Furthermore, the half sites of the various known VDREs show remarkable degeneracy (table 1). The G in the second position of each site appears to be the only nearly invariant nucleotide. 1,25(OH)₂D can also inhibit gene transcription through its VDR. This may occur by direct binding of the VDR to negative VDREs that in the PTH and PTHrP genes are remarkably similar in sequence to positive VDREs of other genes (162,163). However, inhibition may also be indirect. For example, 1,25(OH)₂D inhibits IL-2 production by blocking the NFATp/AP-1 complex of transcription factors from activating this gene (164) through a mechanism not yet clear. Similarly, 1,25(OH)₂D inhibits CYP27B1 in at least one renal cell line by an indirect mechanism involving VDR binding to VDIR (62,80). Thus, a variety of factors including the flanking sequences of the genes around the VDREs and tissue specific factors play a large role in dictating the ability of 1,25(OH)₂D to regulate gene expression.

Non-Genomic Actions

A variety of hormones that serve as ligands for nuclear hormone receptors also exert biologic effects that do not appear to require gene regulation and may work through membrane receptors rather their cognate nuclear hormone receptors. Examples include estrogen (165), progesterone (166), testosterone (167), corticosteroids (168), and thyroid hormone (169). 1,25(OH)₂D has also been shown to have rapid effects on selected cells that are not likely to involve gene regulation and that appear to be mediated by a different, probably membrane receptor. A model for such effects is shown in figure 6. Similar to other steroid hormones, 1,25(OH)₂D has been shown to regulate calcium and chloride channel activity, protein kinase C activation and distribution, and phospholipase C activity in a number of cells including osteoblasts (170), liver (171), muscle (172), and intestine (173,174). These rapid effects of 1,25(OH)₂D have been most extensively studied in the intestine. Norman's laboratory coined the term transcaltachia to describe the rapid onset of calcium flux across the intestine of a vitamin D replete chick perfused with 1,25(OH)₂D (175). This increased flux could not be blocked with actinomycin D pretreatment (176), but was blocked by voltage gated L type channel inhibitors (177) and protein kinase C inhibitors (178). These animals had to be vitamin D replete and contain the VDR, indicating that the basic machinery for calcium transport was intact. On the other hand L type channel activators such as BAY K-8644 (179) and protein kinase C activators such as phorbol esters (177) could activate transcaltachia similar to 1,25(OH)₂D.

Figure 6. Model for the non-genomic actions of 1,25(OH)2D. 1,25(OH)2D binds to a putative membrane receptor. This leads to activation of a G protein (GTP displacement of GDP and dissociation of the β and γ subunits from the now active α subunit). Gα -GTP activates phospholipase C (PLC) (β or γ) to hydrolyze phosphatidyl inositol bis phosphate (PIP2) to inositol tris phosphate (IP3) and diacyl glycerol (DG). IP3 releases calcium from intracellular stores via the IP3 receptor in the endoplasmic reticulum; DG activates protein kinase C (PKC). Both calcium and PKC may regulate the influx of calcium across the plasma membrane through various calcium channels including L-type calcium channels.

A putative membrane receptor for 1,25(OH)₂D (1,25(OH)₂D membrane associated rapid response steroid binding protein (1,25D-MARRSBP) also known as ERp57) has been purified from the intestine (180) and subsequently cloned and sequenced (181). Its size is approximately 66kDa. Antibodies have been made against this putative receptor (182). These antibodies block the ability of 1,25(OH)₂D to stimulate calcium uptake by isolated chick intestinal cells (183) and to stimulate protein kinase C activity in resting zone chondrocytes while inhibiting proliferation of both resting zone and proliferating zone chondrocytes (182). Analog studies also support the existence of a separate membrane receptor for 1,25(OH)₂D. Because of the breaking of the B ring during vitamin D₃ production from 7-dehydrocholesterol, the A ring can assume a conformation similar to the parent cholesterol molecule (6-s-cis) (shown as previtamin D₃ in figure 1) or the more commonly depicted 6-s-trans form in which the A ring rotates away from the rest of the molecule (shown as vitamin D₃ in figure 1). Analogs of 1,25(OH)₂D can be produced which favor the 6-s-cis conformation or the 6-s-trans conformation. 1,25(OH)₂-d5-previtamin D₃ is one such analog locked into the 6-s-cis conformation. This analog has only weak activity with respect to VDR binding or transcriptional activation but is fully effective in terms of stimulating transcaltachia and calcium uptake by osteosarcoma cells when compared to 1,25(OH)₂D (184). 6-s-trans analogs are not effective. However, some of these rapid actions of 1,25(OH)₂D are not found in cells from VDR null mice suggesting that the VDR may be required for the expression and/or function of the membrane receptor or be the membrane receptor. In other cells both 1,25D-MARRSBP and VDR appear to be required for these rapid effects of 1,25(OH)₂D (185,186).

The model (figure 6) emerging from these studies is that 1,25(OH)₂D interacts with a membrane receptor to activate phospholipase C possibly through a G protein coupled process. Phospholipase C then hydrolyzes phosphatidyl inositol bis phosphate (PIP₂) in the membrane releasing inositol tris phosphate (IP₃) and diacyl glycerol (DG). These second messengers may then activate both the intracellular release of calcium from intracellular stores via the IP₃ receptor and protein kinase C, either one or both of which could stimulate calcium channel activity leading to a further rise in intracellular calcium levels. In the intestine and kidney, the increased flux of calcium across the brush border membrane is then transported out of the cell at the basolateral membrane, completing transcellular transport. In other cells the increased calcium would need to be removed by other mechanisms after the signal conveyed by the rise in calcium is no longer required. Much work remains to prove this model including the physiologic requirement for a unique membrane receptor.

TARGET TISSUE RESPONSES: CALCIUM REGULATING ORGANS

Intestine

Intestinal calcium absorption, in particular the active component of transcellular calcium absorption, is one of the oldest and best known actions of vitamin D having been first described in vitro by Schachter and Rosen (187) in 1959 and in vivo by Wasserman et al. (188) in 1961. Absorption of calcium from the luminal contents of the intestine involves both transcellular and paracellular pathways. The transcellular pathway dominates in the duodenum and cecum, and this is the pathway primarily regulated by 1,25 dihydroxyvitamin D (1,25(OH)₂D) (189), although elements of the paracellular pathway such as the claudins 2 and 12 are likewise regulated by 1,25(OH)₂D (reviews in (190,191). Figure 7 shows a model of our current understanding of how this process is regulated by 1,25(OH)₂D. Calcium entry across the brush border membrane (BBM) occurs down a steep electrical-chemical gradient and requires no input of energy. Removal of calcium at the basolateral membrane must work against this gradient, and energy is required. This is achieved by the CaATPase (PMCA1b), an enzyme induced by 1,25(OH)₂D in the intestine. Calcium movement through the cell occurs with minimal elevation of the intracellular free calcium concentration (192) by packaging the calcium in calbindin containing vesicles (193-195) that form in the terminal web following 1,25(OH)₂D administration.

Figure 7. Model of intestinal calcium transport. Calcium enters the microvillus of the intestinal epithelial cell through TRPV6 (previously known as CaT1) calcium channel. Within the microvillus calcium is bound to calmodulin (CaM) which is itself bound to brush border myosin I (BBMI). BBMI may facilitate the movement of the calcium/CaM complex into the terminal web where the calcium is picked up by calbindin (CaBP) and transported through the cytoplasm in endocytic vesicles. At the basolateral membrane the calcium is pumped out of the cell by the Ca-ATPase (PMCA1b). 1,25(OH)2D enhances intestinal calcium transport by inducing TRPV6, CaBP, and PMCAb as well as increasing the amount of CaM bound to BBMI in the brush border.

1,25(OH)₂D regulates transcellular calcium transport using a combination of genomic and nongenomic actions. The first step, calcium entry across the BBM, is accompanied by changes in the lipid composition of the membrane including an increase in linoleic and arachidonic acid (196,197) and an increase in the phosphatidylcholine:phosphatidylethanolamine ratio (198). These changes are associated with increased membrane fluidity (197), which we have shown results in increased calcium flux (199). The changes in lipid composition occur within hours after 1,25(OH)₂D administration and are not blocked by pretreatment with cycloheximide (198). In addition, an epithelial specific calcium channel, TRPV6, is expressed in the intestinal epithelium (200). This channel has a high degree of homology to TRPV5, a channel originally identified in the kidney (201,202). The tissue distributions of these channels are overlapping and can be found in other tissues, but TRPV6 appears to be the main form in the intestine (203,204). TRPV6 mRNA levels in the intestine of vitamin D deficient mice are markedly increased by 1,25(OH)₂D, although similar changes are not found in the kidney (205). Mice null for TRPV6 have decreased intestinal calcium transport (206).

Calcium entering the brush border must then be moved into and through the cytoplasm without disrupting the function of the cell. Electron microscopic observations indicate that in the vitamin D deficient animal, calcium accumulates along the inner surface of the plasma membrane of the microvilli (207,208). Following vitamin D or 1,25(OH)₂D administration calcium leaves the microvilli and subsequently can be found in mitochondria and vesicles within the terminal web (193,194,207,208). The vesicles appear to shuttle the calcium to the lateral membrane where it is pumped out of the cell by the basolateral CaATPase, PMCA1b. These morphologic observations have been confirmed by direct measurements of calcium using x-ray microanalysis that demonstrate equivalent amounts of calcium within the microvilli of D deficient and 1,25(OH)₂D treated animals but much higher amounts of calcium in the mitochondria and vesicles of the 1,25(OH)₂D treated animals (194,209). Such data suggest that 1,25(OH)₂D controls calcium entry into the cell primarily by regulating its removal from the microvillus and accumulation by subcellular organelles in the terminal web, although flux through calcium channels in the membrane such as TRPV6 also plays a major role.

The ability of 1,25(OH)₂D to stimulate calcium entry into and transport from the microvillus does not require new protein synthesis (193,198,210). Cycloheximide does not block the ability of 1,25(OH)₂D to increase the capacity of brush border membrane vesicles (BBMV) to accumulate calcium, although it does block the increase in alkaline phosphatase in the same BBMV [193]. Likewise, cycloheximide does not block the increase in mitochondrial calcium following 1,25(OH)₂D administration, although it blocks the rise in calbindin and prevents the normal vesicular transport of calcium through the cytosol (193,211). Thus, nongenomic actions underlie at least some of these first steps in 1,25(OH)₂D stimulated intestinal calcium transport within the microvillus, although the changes take hours, not minutes, to observe. The exact role for these nongenomic effects on calcium influx relative to the role of TRPV6 remains to be elucidated.

Calmodulin is the major calcium binding protein in the microvillus (212). Its concentration in the microvillus is increased by 1,25(OH)₂D; no new calmodulin synthesis is required or observed after 1,25(OH)₂D administration (213). Calmodulin is likely to play a major role in calcium transport within the microvillus, and inhibitors of calmodulin block 1,25(OH)₂D stimulated calcium uptake by BBMV (214). Within the microvillus calmodulin is bound to a 110kD protein, myosin 1A (myo1A)) (previously referred to as brush border myosin 1). 1,25(OH)₂D increases the binding of calmodulin to myo1A in brush border membrane preparations (213), although binding of calmodulin to the myo1A attached to the actin core following detergent extraction of the membrane appears to be reduced (215). The calmodulin/myo1A complex appears late in the development of the brush border, and is found in highest concentration in the same cells of the villus which have the highest capacity for calcium transport (216). Myo1A is located primarily in the microvillus of the mature intestinal epithelial cell, although small amounts have been detected associated with vesicles in the terminal web (217). Thus, the calmodulin/myo1A complex may be responsible for moving calcium out of the microvillus. Its exact role in calcium transport is unclear in that mice null for myo1A do not show reduced intestinal calcium transport(218)). Calbindin is the dominant calcium binding protein in the cytoplasm (212,219), where it appears to play the major role in calcium transport from the terminal web to the basolateral membrane (190). The increase in calbindin levels in the cytosol following 1,25(OH)₂D administration is blocked by protein synthesis inhibitors (210). Indeed, calbindin was the first protein discovered to be induced by vitamin D (219). Glenney and Glenney (212) observed that calbindin has a higher affinity for calcium than does calmodulin. The differential distribution of calmodulin and calbindin between microvillus and cytosol combined with the differences in affinity for calcium led Glenney and Glenney (212) to propose that in the course of calcium transport calcium flowed from calmodulin in the microvillus to calbindin in the cytosol with minimal change in the free calcium concentration in either location. However, the role of calbindin in intestinal calcium transport does not appear to be critical in that mice null for calbindin9k grow normally, have normal intestinal calcium transport, and their serum calcium levels and bone mineral content are equivalent to wildtype mice regardless of the calcium content of the diet (220). The CaATPase (PMCA1b) at the basolateral membrane and the sodium/calcium exchanger (NCX1) are responsible for removing calcium from the cell against the same steep electrochemical gradient as favored calcium entry at the brush border membrane (221). Related proteins are found in the renal distal tubule. As its name implies, the extrusion of calcium from the cell by the calcium pump requires ATP. This pump is a member of the PMCA family, and in the intestine the isoform PMCA1b is the major isoform found. This pump is induced by 1,25(OH)₂D (222). Calmodulin activates the pump, but calbindin may do likewise (223). Deletion of Pmca1b reduces calcium absorption and blocks 1,25(OH)₂D stimulation of such resulting in reduction in growth and bone mineralization (224)., Moreover, the deletion of protein 4.1R, which regulates PMCA1b expression in the intestine, results in decreased intestinal calcium transport (225). The role of NCX is not considered to be as important as PMCA1b for intestinal calcium transport (226).

The paracellular pathway has received less study, but accounts for the bulk of intestinal calcium transport in that the ileum accounts for around 80% of total calcium absorption essentially all by the paracellular pathway. Paracellular calcium absorption depends to a considerable extent on the gradient between the luminal calcium concentrations and the interstitial calcium concentrations. Thus, it is faster in the duodenum and upper jejunum than the ileum, but because the transit time in the ileum is so much longer than that of the upper GI tract, the ileum is where most of the calcium absorption takes place. Solvent drag plays a large part in moving calcium across the tight junctions between the epithelial cells (227) . Solvent flow follows the osmotic gradient which is maintained distal to the tight junction by the Na/K ATPase and sodium glucose cotransporter of the basolateral membrane which may be stimulated by 1,25(OH)2D (226,227). The tight junction itself provides both charge and size selectivity. The actomyosin ring around the tight junction contributes to the size selectivity (228). The claudins and occludins contribute to charge selectivity. Claudin 2, 12, 15 are negatively charged proteins enabling cations such as sodium and calcium to pass (229,230). 1,25(OH)₂D stimulates the expression of claudins 2 and 12 (231). Prolactin stimulates claudin 15 expression, thought to contribute to the increased calcium absorption during pregnancy (232).

Although less studied, intestinal phosphate transport is also under the control of vitamin D. This was first demonstrated by Harrison and Harrison (233) in 1961. Active phosphate transport is greatest in the jejunum, in contrast to active calcium transport that is greatest in the duodenum. Cycloheximide blocks 1,25(OH)₂D stimulated phosphate transport (234), indicating that protein synthesis is involved. Phosphate transport at both the brush border and basolateral membranes requires sodium. A sodium-phosphate transporter in the small intestine (NaP_i-IIb), homologous to the type IIa sodium phosphate transporter in kidney, has been cloned and sequenced (235). Expression of NaP_i-IIb is increased by 1,25(OH)₂D (236). Transport of phosphate through the cytosol from one membrane to the other is poorly understood. However, cytochalasin B, a disrupter of microfilaments, has been shown to disrupt this process (237) suggesting that as for calcium, intracellular phosphate transport occurs in vesicles.

Bone

Nutritional vitamin D deficiency, altered vitamin D responsiveness such as vitamin D receptor mutations (hereditary vitamin D resistant rickets), and deficient production of 1,25(OH)₂D such as mutations in the CYP27B1 gene (pseudo vitamin D deficiency) all have rickets as their main phenotype. This would suggest that vitamin D, and in particular 1,25(OH)₂D, is of critical importance to bone. Furthermore, VDR are found in bone cells (238,239), and vitamin D metabolites have been shown to regulate many processes in bone. However, the rickets resulting from vitamin D deficiency or VDR mutations (or knockouts) can be corrected by supplying adequate amounts of calcium and phosphate either by infusions or orally [214-217]. Moreover, deletion of VDR from bone cells does not result in rickets (240). This would suggest either that vitamin D metabolites do not directly impact bone, or that substantial redundancy has been built into the system. However, arguing for a physiologically non-redundant direct action of vitamin D on bone is the development of osteoporosis and decreased bone formation in these VDR or CYP27B1 null mice not corrected by the high calcium/phosphate diet (241). A further complicating factor in determining the role of vitamin D metabolites in bone is the multitude of effects these metabolites have on systemic calcium homeostatic mechanisms which themselves impact on bone. The lack of vitamin D results in hypocalcemia and hypophosphatemia that as implied above is sufficient to cause rickets. Moreover, part of the skeletal phenotype in vitamin D deficiency is also due to the hyperparathyroidism that develops in the vitamin D deficient state as PTH has its own actions on bone and cartilage. Furthermore, within bone the vitamin D metabolites can alter the expression and/or secretion of a large number of skeletally derived factors including insulin like growth factor-1 (IGF-I) (242), its receptor (243), and binding proteins (244,245), transforming growth factor β (TGFβ) (246), vascular endothelial growth factor (VEGF) (247), interleukin-6 (IL-6) (248), IL-4 (249), and endothelin receptors (250) all of which can exert effects on bone of their own as well as modulate the actions of the vitamin D metabolites on bone. Understanding the impact of vitamin D metabolites on bone is additionally complicated by species differences, differences in responsiveness of bone and cartilage cells according to their states of differentiation, and differences in responsiveness in terms of the vitamin D metabolite being examined. Thus, the study of vitamin D on bone has had a complex history, and uncertainty remains as to how critical the direct actions of the vitamin D metabolites on bone are for bone formation and resorption.

Bone develops intramembranously (e.g., skull) or from cartilage (endochondral bone formation, e.g., long bones with growth plates). Intramembranous bone formation occurs when osteoprogenitor cells proliferate and produce osteoid, a type I collagen rich matrix. The osteoprogenitor cells differentiate into osteoblasts which then deposit calcium phosphate crystals into the matrix to produce woven bone. This bone is remodeled into mature lamellar bone. Endochondral bone formation is initiated by the differentiation of mesenchymal stem cells into chondroblasts that produce the proteoglycan rich type II collagen matrix. These cells continue to differentiate into hypertrophic chondrocytes that shift from making type II collagen to producing type X collagen. These cells also initiate the degradation and calcification of the matrix by secreting matrix vesicles filled with degradative enzymes such as metalloproteinases and phospholipases, alkaline phosphatase (thought to be critical for the mineralization process), and calcium phosphate crystals. Vascular invasion and osteoclastic resorption are stimulated by the production of VEGF and other chemotactic factors from the degraded matrix. The hypertrophic chondrocytes also begin to produce markers of osteoblasts such as osteocalcin, osteopontin, and type I collagen resulting in the initial deposition of osteoid. Moreover, at least some of these chondrocytes further differentiate (or trans differentiate) into osteoblasts (251). Terminal differentiation of the hypertrophic chondrocytes and the subsequent calcification of the matrix are markedly impaired in vitamin D deficiency leading to the flaring of the ends of the long bones and the rachitic rosary along the costochondral junctions of the ribs, classic features of rickets. Although supply of adequate amounts of calcium and phosphate may correct most of these defects in terminal differentiation and calcification, the vitamin D metabolites, 1,25(OH)₂D and 24,25(OH)₂D, have been shown to exert distinct roles in the process of endochondral bone formation.

The VDR makes its first appearance in the fetal rat at day 13 of gestation in the condensing mesenchyme of the vertebral column then subsequently in osteoblasts and the proliferating and hypertrophic chondrocytes by day 17 (252). However, fetal development is quite normal in vitamin D deficient rats (253) and VDR knockout mice (126) suggesting that vitamin D and the VDR are not critical for skeletal formation. Rickets develops postnatally, becoming most manifest after weaning. The impairment of endochondral bone formation observed in vitamin D deficiency is associated with decreased alkaline phosphatase activity of the hypertrophic chondrocytes (254), alterations in the lipid composition of the matrix (255) perhaps secondary to reduced phospholipase activity (256), and altered proteoglycan degradation (257) due to changes in metalloproteinase activity (257,258). Both 1,25(OH)₂D and 24,25(OH)₂D appear to be required for optimal endochondral bone formation (259). However, in the CYP24A1 knockout mouse, that fails to produce any 24-hydroxylated metabolites of vitamin D, the skeletal lesion is defective mineralization of intramembranous (not endochondral) bone. Furthermore, the skeletal abnormality appears to be due to high circulating 1,25(OH)₂D levels in that crossing this mouse with one lacking the VDR corrects the problem (85). Whether this reflects species differences between mice and other species (most studies demonstrating the role of 24,25(OH)₂D in bone and cartilage have used rats and chicks) remains unknown. Chondrocytes from the resting zone of the growth plate of rats tend to be more responsive to 24,25(OH)₂D than 1,25(OH)₂D, whereas the reverse is true for chondrocytes from the growth zone with respect to stimulation of alkaline phosphatase activity (260), regulation of phospholipase A2 (stimulation by 1,25(OH)₂D, inhibition by 24,25(OH)₂D) (261), changes in membrane fluidity (increased by 1,25(OH)₂D, decreased by 24,25(OH)₂D) (262), and stimulation of protein kinase C activity (263). These actions of 1,25(OH)₂D and 24,25(OH)₂D do not require the VDR and are non-genomic in that they take place with isolated matrix vesicles and membrane preparations from these cells (260). As discussed earlier membrane receptors for these vitamin D metabolites have been found in chondrocytes that may mediate these non-genomic actions (264). Osteoblasts also differ in their response to 1,25(OH)₂D depending on their degree of maturation (265). In the latter stages of differentiation, rat osteoblasts respond to 1,25(OH)₂D with an increase in osteocalcin production (266), but do not respond to 1,25(OH)₂D in the early stages. Mice, however, differ from rats in that 1,25(OH)₂D inhibits osteocalcin expression (266). Similarly, the effects of 1,25(OH)₂D on alkaline phosphatase (267) and type I collagen (268) are inhibitory in the early stages of osteoblast differentiation but stimulatory in the latter stages (265). Osteopontin is better stimulated by 1,25(OH)₂D in the early stages than the late stages of differentiation (265,269). Osteocalcin and osteopontin in human and rat cells have well described VDREs in their promoters (270-272) (the mouse does not) (273). However, alkaline phosphatase and the COL1A1 and COL1A2 genes producing type I collagen do not have clearly defined VDREs, so it remains unclear how these genes are regulated by 1,25(OH)₂D. These maturation dependent effects of 1,25(OH)₂D on bone cell function may explain the surprising ability of excess 1,25(OH)₂D to block mineralization leading to hyperosteoidosis (274,275) as such doses may prevent the normal maturation of osteoblasts.

In addition to its role in promoting bone formation, 1,25(OH)₂D also promotes bone resorption by increasing the number and activity of osteoclasts (276). Whether mature osteoclasts contain the VDR and are regulated directly by 1,25(OH)₂D remains controversial (277,278), but the VDR in osteoclast precursors is not required for osteoclastogenesis. Rather, the stimulation of osteoclastogenesis by 1,25(OH)₂D is mediated by osteoblasts. Rodan and Martin (279) originally proposed the hypothesis that osteoblasts were required for osteoclastogenesis, and the mechanism has now been elucidated (280). Osteoblasts produce a membrane associated protein known as RANKL (receptor activator of nuclear factor (NF)-kB ligand) that activates RANK on osteoclasts and their hematopoietic precursors. This cell-to-cell contact in combination with m-CSF also produced by osteoblasts stimulates the differentiation of precursors to osteoclasts, and promotes their activity. 1,25(OH)₂D regulates this process by inducing RANKL (281) as does PTH, PGE2, and IL-11, all of which stimulate osteoclastogenesis. 1,25(OH)₂D requires the VDR in osteoblasts for this purpose, although the other hormones and cytokines do not. Osteoblasts from Vdr knockout mice fail to support 1,25(OH)₂D induced osteoclastogenesis, whereas osteoclast precursors from Vdr knockout mice can be induced by 1,25(OH)₂D to form osteoclasts in the presence of osteoblasts from wildtype animals (282).

Kidney

The regulation of calcium and phosphate transport by vitamin D metabolites in the kidney has received less study than that in the intestine, but the two tissues have similar although not identical mechanisms. Eight grams of calcium are filtered by the glomerulus each day, and 98% of that is reabsorbed. Most is reabsorbed in the proximal tubule. This is a paracellular, sodium dependent process with little or no regulation by PTH and 1,25(OH)₂D. Approximately 20% of calcium is reabsorbed in the thick ascending limb of the loop of Henle, 10-15% in the distal tubule, and 5% in the collecting duct (283). Regulation by vitamin D takes place in the distal tubule where calcium moves against an electrochemical gradient (presumably transcellular) in a sodium independent fashion (284). Phosphate, on the other hand, is approximately 80% reabsorbed in the proximal tubule, and this process is regulated by PTH (285). In parathyroidectomized (PTX) animals Puschett et al. (286-288)) demonstrated acute effects of 25OHD and 1,25(OH)₂D on calcium and phosphate reabsorption. Subsequent studies indicated that PTH could enhance or was required for the stimulation of calcium and phosphate reabsorption by vitamin D metabolites (289,290).

The molecules critical for calcium reabsorption in the distal tubule appear to be the VDR, calbindin, TRPV5, and the BLM calcium pump (PMCA1b as in the intestine), a situation similar to the mechanism for calcium transport in the intestine. However, the calbindin in the kidney in most species is 28kDa, whereas the 9kDa form is found in the intestine in most species. The kidney has mostly TRPV5, whereas the intestine is primarily TRPV6. The calcium pump is the same isoform in both tissues (PMCA1b) although other forms of PMCA are also present. Calmodulin and a brush border myosin I like protein are also found in the kidney brush border, but their role in renal calcium transport has not been explored. VDR, calbindin, TRPV5, and PMCA1b colocalize in the distal tubule, but not all distal tubules contain this collection of proteins (201,202,291,292) suggesting that not all distal tubules are involved in calcium transport. 1,25(OH)₂D upregulates the VDR (234), an action opposed by PTH (237). Calbindin is also induced by 1,25(OH)₂D in the kidney(293,294). The activity of the calcium pump is increased by 1,25(OH)₂D (295), but it is not clear that the protein itself is induced. The increased activity may be due to the induction of calbindin that increases its activity. The effect of 1,25(OH)₂D on TRPV5 expression is stimulatory (205).

Phosphate reabsorption in the proximal tubule is mediated at the brush border by sodium dependent phosphate transporters (NaPi-2a and NaPi-2c) that rely on the baso-lateral membrane Na,K ATPase to maintain the sodium gradient that drives the transport process (296). It is not clear whether 1,25(OH)₂D regulates the expression or activities of these transporters as it does in the intestine, although PTH clearly does. Like PTH, FGF23 blocks phosphate reabsorption, presumably by blocking NaPi-2a activity. Unlike PTH, FGF23 also blocks the renal production of 1,25(OH)₂D, as discussed earlier. The link between phosphate reabsorption and 1,25(OH)₂D production remains unclear.

TARGET TISSUE RESPONSES: NON-CALCIUM TRANSPORTING TISSUES

In addition to the its effects on tissues directly responsible for calcium homeostasis, 1,25(OH)₂D regulates the function of a wide number of other tissues. These all contain the VDR. Regulation of differentiation and proliferation is one common theme; regulation of hormone secretion is another; regulation of immune function is the third. In most cases 1,25(OH)₂D acts in conjunction with calcium. Selected examples follow.

Regulation of Hormone Secretion

PARATHYROID GLAND (PTH SECRETION)

As previously mentioned, PTH stimulates the production of 1,25(OH)₂D. In turn 1,25(OH)₂D inhibits the production of PTH (297,298). The regulation occurs at the transcriptional level. Within the promoter of the PTH gene is a region that binds the VDR and mediates the suppression of the PTH promoter by 1,25(OH)₂D (162,293,299-303). However, there is substantial controversy about whether this site is a single half site (299) or a more classic DR3 (292), whether one VDRE is involved or two (300), whether only VDR binds (299,303), whether VDR/RXR heterodimers bind (162,300), or whether VDR partners with a different protein (301). Some of the differences may reflect different species, but the nature of PTH gene suppression by 1,25(OH)₂D remains incompletely understood. Calcium alters the ability of 1,25(OH)₂D to regulate PTH gene expression. Calcium is a potent inhibitor of PTH production and secretion, acting through the calcium sensing receptor (CaSR) on the plasma membrane of the parathyroid cell. 1,25(OH)₂D induces the CaSR in the parathyroid gland making it more sensitive to calcium (304). Animals placed on a low calcium diet have an increase in PTH and 1,25(OH)₂D levels indicating that the low calcium overrides the inhibition by 1,25(OH)₂D on PTH secretion (305,306). One possible explanation involves the protein calreticulin that binds to nuclear hormone receptors including VDR at KXGFFKR sequences, and inhibits their activity (307,308). Low dietary calcium has been shown to increase calreticulin levels in the parathyroid gland (309). The ability of 1,25(OH)₂D to inhibit PTH production and secretion has been exploited clinically in that 1,25(OH)₂D and several of its analogs are used to prevent and/or treat secondary hyperparathyroidism associated with renal failure. The parathyroid gland also expresses CYP27B1 and so can produce its own 1,25(OH)₂D that may act in an autocrine or paracrine fashion to regulate PTH production (310). As noted earlier, the parathyroid gland is one of several tissues expressing the megalin/cubilin complex potentially enabling it to take up 25OHD and other D metabolites still bound to DBP.

PANCREATIC BETA CELLS (INSULIN SECRETION)

1,25(OH)₂D stimulates insulin secretion, although the mechanism is not well defined (311,312). VDR, CYP27B1 and calbindin-D_28k are found in pancreatic beta cells (313-315), and studies using calbindin-D_28k null mice have suggested that calbindin-D_28k, by regulating intracellular calcium, can modulate depolarization-stimulated insulin release (316). Furthermore, calbindin-D_28k, by buffering calcium, can protect against cytokine mediated destruction of beta cells (317). A number of mostly case control and observational studies have suggested that vitamin D deficiency contributes to increased risk for type 2 diabetes mellitus (318). Moreover, several randomized clinical trials evaluating the ability of vitamin D supplementation to prevent the progression of prediabetes to diabetes indicate that vitamin D has a modest protective effect especially in vitamin D deficient subjects (319,320).

FIBROBLAST GROWTH FACTOR (FGF23)

FGF23 is produced primarily by bone, and in particular by osteoblasts and osteocytes. 1,25(OH)₂D₃ stimulates this process, but the mechanism is not clear (322). Inasmuch as FGF23 inhibits 1,25(OH)₂D production by the kidney, this feedback loop like that for PTH secretion maintains a balance in the levels of these important hormones. Mutations in the Phosphate regulating gene with Homologies to Endopeptidases on the X chromosome (PHEX) or FGF23 itself (which prevent its proteolysis) or conditions such as McCune-Albright disease and tumor induced osteomalacia in which FGF23 is overexpressed in the involved tissue led to hypophosphatemia and inappropriately low 1,25(OH)₂D accompanied by osteomalacia. The role of PHEX, which was originally thought to cleave FGF23, in regulating FGF23 levels is not clear. In contrast mutations in UDP-N-acetyl-α-D galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNT3), which glycosylates FGF23, or in FGF23 which blocks this glycosylation result in inhibited FGF23 secretion leading to hyperphosphatemia, increased 1,25(OH)₂D_,and tumoral calcinosis (323).

Regulation of Proliferation and Differentiation

CANCER

1,25(OH)₂D has been evaluated for its potential anticancer activity in animal and cell studies for nearly 40 years (324). The list of malignant cells that express VDR is now quite extensive, and the list of those same cells that express CYP27B1 is growing. The accepted basis for the promise of 1,25(OH)₂D in the prevention and treatment of malignancy includes its antiproliferative, pro-differentiating effects on most cell types. The list of mechanisms proposed for these actions is extensive, and to some extent cell specific (325). Among these mechanisms 1,25(OH)₂D has been shown to stimulate the expression of cell cycle inhibitors p21 and p27 (326) and the expression of the cell adhesion molecule E-cadherin (150), while inhibiting the transcriptional activity of β-catenin (150,327,328). In keratinocytes, 1,25(OH)₂D has been shown to promote the repair of DNA damage induced by ultraviolet radiation (UVR) (329) (330), reduce apoptosis while increasing survival after UVR (331), and increase p53 (332). Epidemiologic evidence supporting the importance of adequate vitamin D nutrition (including sunlight exposure) for the prevention of a number of cancers (333-337) is extensive. Although numerous types of cancers show reduction (338), most attention has been paid to cancers of the breast, colon, and prostate. I (339) recently reviewed a number of meta-analyses of epidemiologic studies evaluating the association of vitamin D intake and/or 25OHD levels and the risk of developing these cancers. The data supporting a reduction in risk for developing colorectal cancer and breast cancer in premenopausal females with higher vitamin D intake or higher serum 25OHD levels were considerably stronger than that for the prevention of prostate cancer. Prospective randomized controlled trial data are limited. In a prospective 4 yr. trial with 1100iu vitamin D and 1400-1500 mg calcium originally designed to look at osteoporosis the authors showed a 77% reduction in cancers after excluding the initial year of study (340), including a reduction in both breast and colon cancers. In this study, vitamin D supplementation raised the 25OHD levels from a mean of 28.8ng/ml to 38.4ng/ml with no changes in the placebo or calcium only arms of the study. However, this was a relatively small study in which cancer prevention was not the primary outcome variable. A substantially larger trial involving over 25,000 subjects treated in a two by two design with vitamin D and/or omega 3 fatty acid did not find a benefit of vitamin supplementation with respect to cancer incidence but appears to have shown a beneficial effect on mortality (341). Trials of 1,25(OH)₂D and its analogs for the treatment of cancer have been disappointing. In a small study involving 7 subjects with prostate cancer treated with doses of 1,25(OH)₂D up to 2.5µg for 6-15 months, 6/7 showed a decrease in the rise of prostate specific antigen (PSA), a marker of tumor progression (342), and one patient showed a decline. However, hypercalciuria was common and limiting. A preliminary report of a larger study involving 250 patients with prostate cancer using 45µg 1,25(OH)₂D weekly in combination with docetaxel demonstrated a non-significant decline in PSA, although survival was significantly improved (HR 0.67) (343). A larger follow-up study did not show increased survival (344). The incidence of either hypercalcemia or hypercalciuria was not reported. Most likely until an analog of 1,25(OH)₂D is developed which is both efficacious and truly non hypercalcemic, treatment of cancer with vitamin D metabolites will remain problematic.

SKIN

Epidermal keratinocytes are the only cells in the body with the entire vitamin D metabolic pathway. As described earlier, production of vitamin D₃ from 7-dehydrocholesterol takes place in the epidermis. However, the epidermis also contains CYP27A1 (345), the mitochondrial enzyme that 25-hydroxylates vitamin D, and CYP27B1 (40,47), the enzyme that produces 1,25(OH)₂D from 25OHD. The CYP27B1 in keratinocytes is differently regulated than CYP27B1 in renal cells. Although PTH stimulates CYP27B1 activity in the keratinocyte, the mechanism appears to be independent of cAMP (346). Cytokines such as tumor necrosis factor-α and interferon-γ stimulate CYP27B1 activity (347,348). 1,25(OH)₂D does not exert a direct effect on CYP27B1 expression in keratinocytes, but regulates 1,25(OH)₂D levels by inducing CYP24A1 thus initiating the catabolism of 1,25(OH)₂D (79). CYP27B1 is expressed primarily in the basal cells of the epidermis (50); as the cells differentiate the mRNA and protein levels of CYP27B1and its activity decline (349).

1,25(OH)₂D regulates keratinocyte differentiation in part by modulating the ability of calcium to do likewise (350). Therefore, it is important to understand the actions of calcium on this cell prior to examining the influence of 1,25(OH)₂D (351-356)(357). If keratinocytes are grown at calcium concentrations below 0.07mM, they continue to proliferate but either fail or are slow to develop intercellular contacts, stratify little if at all, and fail or are slow to form cornified envelopes. Acutely increasing the extracellular calcium concentration (Cao) above 0.1mM (calcium switch) leads to the rapid redistribution of desmoplakin, cadherins, integrins, catenins, plakoglobulin, vinculin, and actinin from the cytosol to the membrane where they participate in the formation of intercellular contacts. Calcium also stimulates the redistribution to the membrane of protein kinase Cα (PKCα) (358,359) and the tyrosine-phosphorylated p62 associated protein of ras GAP (360,361) where they further the calcium signaling process. These early events are accompanied by a rearrangement of actin filaments from a perinuclear to a radial pattern which if disrupted blocks the redistribution of these proteins and blocks the differentiation process. Within hours of the calcium switch keratinocytes switch from making the basal keratins K5 and K14 and begin making keratins K1 and K10 (356) followed, subsequently, by increased levels of profilaggrin (the precursor of filaggrin, an intermediate filament associated protein), involucrin and loricrin (precursors for the cornified envelope) (362,363). Loricrin, involucrin and other proteins (364) are cross linked into the insoluble cornified envelope by the calcium sensitive, membrane bound form of transglutaminase (365,366), which like involucrin and loricrin increases within 24 hours after the calcium switch (367). Within 1-2 days of the calcium switch cornified envelope formation is apparent (355,368), paralleling transglutaminase activation (369). The induction of these proteins represents a genomic action (likely indirect) of calcium as indicated by a calcium induced increase in mRNA levels and transcription rates (356,363,369,370). The relevance of calcium induced differentiation in vitro to the in vivo situation is indicated by the steep gradient of calcium within the epidermis, with the highest levels in the uppermost (most differentiated) nucleated layers (371). Current evidence for the importance of calcium in epidermal function is that barrier disruption, which results in increased proliferation, is associated with loss of the calcium gradient, whereas increasing the calcium concentration in the epidermis with sonophoresis stimulates lamellar body secretion (372-376).

The keratinocyte senses calcium via a seven transmembrane domain, G protein coupled receptor (CaSR) (377) originally cloned from the parathyroid cell by Brown et al (378,379). Knocking out the CaSR blocks calcium induced differentiation in vitro (380,381) and in vivo (382). However, keratinocytes also produce an alternatively spliced variant of the CaSR as they differentiate (383). This variant CaSR lacks exon 5 and so would be missing residues 461-537 in the extracellular domain. A mouse model in which the full length CaSR has been knocked out continues to produce the alternatively spliced form of CaSR, but its epidermis contains lower levels of the terminal differentiation markers loricrin and profilaggrin, and keratinocytes from these mice fail to respond normally to calcium (383) consistent with the results when the full length calcium receptor was deleted in vitro (380,381). We have produced a conditional knockout of the CaSR allowing us to delete CaSR in the tissue of choice using cell specific cre recombinases that avoids the problem with the original global knockout (384). When the CaSR is deleted specifically in the keratinocyte, this mouse has a reduction in epidermal differentiation and barrier repair (382), but unlike the global knockout does not have abnormalities in overall calcium homeostasis, and rather than showing an increased calcium gradient in the epidermis has a blunted one. The conditional knockout mouse also lacks the alternatively spliced CaSR.

Inositol 1,4,5 tris phosphate (IP₃) and diacylglycerol levels increase within seconds to minutes after the calcium switch implicating activation of the phospholipase C (PLC) pathway (385,386). Similar to intracellular calcium levels (Cai), the levels of inositol phosphates (IPs) remain elevated for hours after the calcium switch. The prolonged increase in IPs after the calcium switch may contribute to the plateau phase of Cai elevation and a prolonged elevation of diacylglycerol (DG) that would stimulate the protein kinase C (PKC) pathway. This prolonged increase in IPs appears to be due to calcium induction and activation of PLC (154,386,387), especially PLC-γ1. Activation of PLC-γ1 by calcium involves a chain of events involving src kinase activation of phosphatidyl inositol 3 kinase and phosphatidyl inositol 4 phosphate 5 kinase 1α within the context of a membrane complex with E-cadherin leading to the formation of phosphatidyl inositol tris phosphate in the membrane which activates PLC-γ1 via its PH domain (388). Phosphorylation of PLC-γ1 is not part of its activation by calcium unlike its activation by EGF (389). Knocking out Plcg1 blocks the ability of calcium to increase Cai and to induce involucrin and transglutaminase (387). Thus, like CaSR, PLC-γ1 is critical for the ability of calcium to regulate keratinocyte differentiation.

Phorbol esters, which bind to and activate PKC, are well known tumor promoters in skin However, the initial effects of phorbol esters in vitro are to promote differentiation in cells grown in low calcium (358,390,391), effects which are potentiated by calcium (383). Phorbol esters stimulate PKC, and PKC inhibitors block the ability of both calcium and phorbol esters to promote differentiation (391). Phorbol esters as well as calcium stimulate the expression of both keratin 1 and involucrin gene constructs each of which contains an AP-1 site within the calcium response element (CaRE) of the promoter for these genes (392,393). If the AP-1 site within the CaRE is mutated, neither calcium nor phorbol esters are effective (392,393). These CaREs also contain VDREs (DR3), which at least in the involucrin gene has been shown to mediate 1,25(OH)₂D regulation of this gene (394). Phorbol esters do not reproduce all the actions of calcium on the keratinocyte, and vice versa, but cross talk between their signaling pathways is clearly present.

The observation that 1,25(OH)₂D induces keratinocyte differentiation was first made by Hosomi et al. (395) and provided a rationale for the previous and unexpected finding of 1,25(OH)₂D receptors in the epidermis (396). 1,25(OH)₂D increases the mRNA and protein levels for involucrin and transglutaminase, and promotes CE formation at subnanomolar concentrations in preconfluent keratinocytes (370,397-399). Calcium affects the ability of 1,25(OH)₂D to stimulate keratinocyte differentiation, and vice versa. Calcium in the absence of 1,25(OH)₂D and 1,25(OH)₂D at low (0.03mM) calcium raise the mRNA levels for involucrin and transglutaminase in a dose dependent fashion by stimulating gene expression. The stimulation of mRNA levels by calcium and 1,25(OH)₂D is synergistic at early time points; however, longer periods of incubation lead to a paradoxical fall in the mRNA levels for these proteins. This is due to the fact that although transcription is increased by calcium and 1,25(OH)₂D, stability of the mRNA is reduced in cells incubated with calcium and 1,25(OH)₂D.

The transcriptional regulation by 1,25(OH)₂D is both direct and indirect. Several genes contain VDREs (e.g. involucrin), but VDREs have not been found in all genes that are regulated by 1,25(OH)₂D. Inhibition of PKC activity or mutation of the AP-1 site in the CaRE of the involucrin gene also blocks the ability of 1,25(OH)₂D to regulate expression of involucrin (394). The ability of 1,25(OH)₂D to increase intracellular calcium (Cai) (298) accounts for at least part of the ability of 1,25(OH)₂D to induce differentiation. A rapid (presumably nongenomic) effect of 1,25(OH)₂D on Cai has been described (400), although this response is controversial (398). Our studies indicate that the ability of 1,25(OH)₂D to increase Cai requires time and gene transcription. 1,25(OH)₂D increases CaSR mRNA levels and prevents their fall in cells grown in 0.03mM calcium (401). This results in an enhanced Cai response to extracellular calcium (Cao). 1,25(OH)₂D also induces the family of PLCs (402). PLC-γ1 contains a VDRE in its promoter (154), which unlike the usual VDRE is a DR6 which binds VDR/RAR rather than VDR/RXR. Knocking out PLCG1 blocks 1,25(OH)₂D induced differentiation (403) as well as calcium induced differentiation mentioned earlier. The other PLCs have not been studied as extensively, but are likely to show similar means of regulation by 1,25(OH)₂D.

Our current working model for the mechanisms by which calcium and 1,25(OH)₂D regulate keratinocyte differentiation is shown in figure 8. The keratinocyte expresses a CaSR that by coupling to and activating PLC controls the production of two important second messengers, IP₃ and DG. PLC-β is likely to be activated acutely by CaSR via a G protein coupled mechanism, whereas PLC-γ1 is activated acutely by calcium stimulated non receptor tyrosine kinases and subsequently by PIP3 in the membrane. Both PLCs are induced by calcium and 1,25(OH)₂D. IP₃ stimulates the release of calcium from intracellular stores thus raising Cai. The initial release of calcium from these stores activates the Stim1/Orai1 channel in the membrane (404) that may stimulate proliferation of the basal keratinocytes and initiate their movement out of the basal layer. The increase in Cai and DG stimulates the activation of critical PKCs and their translocation to membrane receptors (RACK). PKC-α appears to be the most critical PKC for the subsequent events triggered by calcium in the keratinocyte, although PKCδ has also been implicated. Activated PKC leads to the induction and activation of AP-1 transcription factors which regulate the transcription of a number of genes including keratin 1, transglutaminase, involucrin, loricrin, and profilaggrin required for the differentiation process. Activation of the CaSR also activates the RhoA kinase leading to activation of src kinases which by phosphorylating various catenins leads to the formation of the Ecadherin/catenin complex in the membrane (405). This complex recruits both PI3K and PIP5K1α required to maintain the PIP2 and PIP3 levels in the membrane (357). PIP3 activates PLC-γ, that is in turn activates the TRPC channels in the membrane to enable the prolonged increase in Cai required for differentiation (406). 1,25(OH)₂D, which is produced by the keratinocyte in a highly regulated fashion, modulates calcium regulated differentiation at several steps. First, 1,25(OH)₂D increases CaSR expression, thus making the cell more responsive to calcium. Secondly, 1,25(OH)₂D induces all the PLCs again increasing the responsiveness of the cell to calcium. Finally, 1,25(OH)₂D has a direct effect on the transcription of the genes such as involucrin. The net result is that both calcium and 1,25(OH)₂D promote keratinocyte differentiation through interactive mechanisms.

Figure 8. A model of 1,25(OH)2D and calcium regulated keratinocyte differentiation. The G-protein coupled calcium receptor (CaSR) when activated by extracellular calcium activates Gα as described in the legend to figure 6. Gα stimulates PLC mediated hydrolysis of PIP2 to IP3 and DG. IP3 releases Cai from intracellular stores, and DG activates PKC. Depletion of intracellular calcium stores leads to influx of calcium across store operated calcium channels. PKC stimulation leads to activation of AP-1 transcription factors which along with calcium and 1,25(OH)2D activated transcription factors stimulate the expression of genes essential for the differentiation process. 1,25(OH)2D regulates this process by inducing CaSR and PLC as well as genes essential for cornified envelope formation such as involucrin and transglutaminase.

The VDR is also critical for hair follicle (HF) cycling. Unlike epidermal differentiation, hair follicle cycling is not dependent on 1,25(OH)₂D. Alopecia is a well described characteristic of mice and humans lacking VDR (125,126,407) due to failure to regenerate the cycling lower portion of the HF after the initial developmental cycle is completed. Deletion of CYP27B1 (408) and CaSR (382) do not result in alopecia. Cianferotti et al. (409) attributed the loss of HF cycling in VDR null mice to a gradual loss of the proliferative potential in the stem cells of the HF bulge region. However, this conclusion has been challenged by Palmer et al. (410), who attributed the failure of HF cycling in the VDR null mouse in part to a failure of the progeny of these stem cells to migrate out of the bulge rather than their loss of proliferative potential suggesting a loss of activation. The role of VDR in the stem cells that regulate both HF cycling and epidermal regeneration is also important in the skin wound healing process. When the skin is wounded the progeny of stem cells from all regions of the HF and interfollicular epidermis (IFE) contribute at least initially (411,412), although the stem cells in the IFE make the most lasting contribution. Tian et al. (413) observed that topical 1,25(OH)₂D enhanced wound healing, suggesting that unlike HF cycling, the wound repair required this VDR ligand. Luderer et al. (414) observed that in the global VDRKO, there was a reduction in TGFβ signaling in the dermis, and subsequently demonstrated that the VDR in macrophages but not in keratinocytes was responsible for macrophage recruitment during the inflammatory phase of cutaneous wound healing (415). Our studies have focused on the VDR in epidermal keratinocytes. We have observed that re-epithelialization by the keratinocytes over the wound is impaired when the deletion of VDR from keratinocytes is accompanied by either a low calcium diet or a deletion of the CaSR (416). Thus like the role of calcium and CaSR in vitamin D regulated keratinocyte differentiation so a similar synergism is seen in wound healing. These results are consistent with the loss of E-cadherin/catenin complex formation in the VDRKO keratinocyte, a complex that maintains stem cells in their niches (417), regulates when stem cell division is symmetric (to maintain stem cell numbers) or asymmetric (initiating differentiation) (418), and is essential for the ability of keratinocytes to migrate as a sheet to re-epithelialize the wound (419). As noted previously calcium and the CaSR along with 1,25(OH)₂D and VDR are required for E-cadherin/catenin complex formation during the differentiation process and so are involved in enabling its role in wound healing (420).

Immune System

The potential role for vitamin D and its active metabolite 1,25(OH)₂D₃ in modulating the immune response has long been recognized since the discovery of vitamin D receptors (VDR) in macrophages, dendritic cells (DC), and activated T and B lymphocytes, the ability of macrophages and DC as well as activated T and B cells to express CYP27B1, and the ability of 1,25(OH)₂D₃ to regulate the proliferation and function of these cells. While these are the key cells mediating the adaptive immune response, 1,25(OH)₂D, VDR, and CYP27B1 are also expressed in a large number of epithelial cells which along with the aforementioned members of the adaptive immune response contribute to host defense by their innate immune response. The totality of the immune response involves both types of responses in complex interactions involving numerous cytokines. The regulation of these different responses and their interactions by 1,25(OH)₂D₃ is nuanced. In general, 1,25(OH)₂D₃ enhances the innate immune response primarily via its ability to stimulate cathelicidin, an antimicrobial peptide important in defense against invading organisms, whereas it inhibits the adaptive immune response primarily by inhibiting the maturation of dendritic cells (DC) important for antigen presentation, reducing T cell proliferation, and shifting the balance of T cell differentiation from the Th1 and Th17 pathways to Th2 and Treg pathways. Inflammatory autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis involve Th17 activation, a cell that expresses RANKL, and so can drive osteoclastogenesis leading to bone loss.

ADAPTIVE IMMUNE RESPONSE

The adaptive immune response is initiated by cells specialized in antigen presentation, DC and macrophages in particular, activating the cells responsible for subsequent antigen recognition, T and B lymphocytes. These cells are capable of a wide repertoire of responses that ultimately determine the nature and duration of the immune response. Activation of T and B cells occurs after a priming period in tissues of the body, e.g., lymph nodes, distant from the site of the initial exposure to the antigenic substance, and is marked by proliferation of the activated T and B cells accompanied by post translational modifications of immunoglobulin production that enable the cellular response to adapt specifically to the antigen presented. Importantly, the type of T cell activated, CD4 or CD8, or within the helper T cell class Th1, Th2, Th17, Treg, and subtle variations of those, is dependent on the context of the antigen presented by which cell and in what environment. Systemic factors such as vitamin D influence this process. Vitamin D in general exerts an inhibitory action on the adaptive immune system. 1,25(OH)₂D₃ decreases the maturation of DC as marked by inhibited expression of the costimulatory molecules HLA-DR, CD40, CD80, and CD86, decreasing their ability to present antigen and so activate T cells (421). Furthermore, by suppressing IL-12 production, important for Th1 development, and IL-23 and IL-6 production important for Th17 development and function, 1,25(OH)₂D₃ inhibits the development of Th1 cells capable of producing IFN- and IL-2, and Th17 cells producing IL-17 (422). These actions prevent further antigen presentation to and recruitment of T lymphocytes (role of IFN-γ), and T lymphocyte proliferation (role of IL-2). Suppression of IL-12 increases the development of Th2 cells leading to increased IL-4, IL-5, and IL-13 production, which further suppresses Th1 development shifting the balance to a Th2 cell phenotype. Treatment of DCs with 1,25(OH)₂D₃ can also induce CD4+/CD25+ regulatory T cells (Treg) cells (423) as shown by increased FoxP3 expression, critical for Treg development. These cells produce IL-10, which suppresses the development of the other Th subclasses. Treg are critical for the induction of immune tolerance (424). In addition, 1,25(OH)₂D₃ alters the homing of properties of T cells for example by inducing expression of CCR10, the receptor for CCL27, a keratinocyte specific cytokine, while suppressing that of CCR9, a gut homing receptor (425). The actions of 1,25(OH)₂D₃ on B cells have received less attention, but recent studies have demonstrated a reduction in proliferation, maturation to plasma cells and immunoglobulin production (426).

1,25(OH)₂D₃ has both direct and indirect effects on regulation of a number of cytokines involved with the immune response (review in (427)). TNF has a VDRE in its promoter to which the VDR/RXR complex binds. 1,25(OH)₂D₃ both blocks the activation of NFκB via an increase in IκBα expression and impedes its binding to its response elements in the genes such as IL-8 and IL-12 that it regulates. 1,25(OH)₂D₃ has also been shown to bring an inhibitor complex containing histone deacetylase 3 (HDAC3) to the promoter of rel B, one of the members of the NFκB family, thus suppressing gene expression. Thus, TNF/NFkB activity is markedly impaired by 1,25(OH)₂D₃ at multiple levels. In VDR null fibroblasts, NFκB activity is enhanced. Furthermore, 1,25(OH)₂D₃ suppresses IFNγ, and a negative VDRE has been found in the IFNγ promoter. GM-CSF is regulated by VDR monomers binding to a repressive complex in the promoter of this gene, competing with nuclear factor of T cells 1(NFAT1) for binding to the promoter.

The ability of 1,25(OH)₂D₃ to suppress the adaptive immune system appears to be beneficial for a number of conditions in which the immune system is directed at self—i.e. autoimmunity (review in (428)). In a number of experimental models including inflammatory arthritis, psoriasis, autoimmune diabetes (e.g., NOD mice), systemic lupus erythematosis (SLE), experimental allergic encephalitis (EAE) (a model for multiple sclerosis), inflammatory bowel disease (IBD), prostatitis, and thyroiditis VDR agonist administration has prevented and/or treated the disease process. As will be discussed later, a number of these conditions are associated with bone loss either directly (e.g., inflammatory arthritis) or indirectly presumably via increased serum levels of inflammatory cytokines. These actions of 1,25(OH)₂D₃ were originally ascribed to inhibition of Th1 function, but Th17 cells have also been shown to play important roles in a number of these conditions including psoriasis (321), experimental colitis (422), and rheumatoid arthritis (429), conditions that respond to 1,25(OH)₂D₃ and its analogs. Although few prospective, randomized, placebo-controlled trials in humans have been performed, epidemiologic and case control studies indicate that a number of these diseases in humans are favorably impacted by adequate vitamin D levels. For example, the incidence of multiple sclerosis correlates inversely with 25OHD levels and vitamin D intake, and early studies suggested benefit in the treatment of patients with rheumatoid arthritis and multiple sclerosis with VDR agonists (427,428). Similarly, IBD is associated with low vitamin D levels (430). Children who are vitamin D deficient have a higher risk of developing type 1 diabetes mellitus, and supplementation with vitamin D during early childhood reduces the risk of developing type 1 diabetes (review in (421)). In VDR null mice myelopoiesis and the composition of lymphoid organs are normal, although a number of abnormalities in the immune response have been found. Some of the abnormalities in macrophage function and T cell proliferation in response to anti-CD3 stimulation in these animals could be reversed by placing the animals on a high calcium diet to normalize serum calcium (431), indicating the important role of calcium in vitamin D regulated immune function as in skeletal development and maintenance, hormone regulation, and keratinocyte differentiation. Other studies have noted an increased number of mature DCs in the lymph nodes of VDR null mice, which would be expected to promote the adaptive immune response (432). Somewhat surprisingly, RANKL also increases the number and retention of DCs in lymph nodes (433) suggesting that at least this mechanism is not mediated via the RANKL/RANK system in VDR null mice, which I will discuss at length subsequently. In contrast to these inhibitory actions of 1,25(OH)₂D₃, Th2 function as indicated by increased IgE stimulated histamine from mast cells is increased in VDR null mice (434). The IL-10 null mouse model of IBD shows an accelerated disease profile when bred with the VDR null mouse with increased expression of Th1 cytokines (435). Surprisingly, despite a reduction in natural killer T cells and Treg cells and a decreased number of mature DCs, VDR null mice bred with NOD mice do not show accelerated development of diabetes (436). Part of the difference in tissue response in VDR null mice may relate to differences in the ability of 1,25(OH)₂D₃ to alter the homing of T cells to the different tissues (425). In allergic airway disease (asthma) Th2 cells, not Th1 cells, dominate the inflammatory response. 1,25(OH)₂D₃ administration to normal mice protected these mice from experimentally induced asthma in one study, blocking eosinophil infiltration, IL-4 production, and limiting histologic evidence of inflammation (437). However, a study with VDR null mice using a comparable method of inducing asthma showed that lack of VDR also protected the mice from an inflammatory response in their lungs (438). In an extension of this study the investigators showed that wildtype (WT) splenocytes were only minimally successful at restoring experimental airway inflammation to VDR null mice, whereas splenocytes from these mice were able to transfer experimental airway inflammation to the unprimed WT host (439). Thus, the impact of vitamin D signaling on adaptive immunity depends on the specifics of the immune response being evaluated.

Inhibition of the adaptive immune response may also have benefit in transplantation procedures (440). In experimental allograft models of the aorta, bone, bone marrow, heart, kidney, liver, pancreatic islets, skin, and small bowel VDR agonists have shown benefit generally in combination with other immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and glucocorticoids (440). Much of the effect could be attributed to a reduction in infiltration of Th1 cells, macrophages and DC into the grafted tissue associated with a reduction in chemokines such as CXCL10, CXCL9, CCL2, and CCL5. CXCL10, the ligand for CXCR3, may be of particular importance for acute rejection in a number of tissues, whereas CXCL9 as well as CXCL10 (both CXCR3 ligands) may be more important for chronic rejection at least in the heart and kidney, respectively. Although there are no prospective trials of the use of VDR agonists in transplant patients, several retrospective studies in patients with renal transplants treated with 1,25(OH)₂D₃ have suggested benefit with respect to prolonged graft survival and reduced numbers of acute rejection episodes.

Suppression of the adaptive immune system may not be without a price. Several publications have demonstrated that for some infections including Leishmania major (441) and toxoplasmosis (442), 1,25(OH)₂D₃ promotes the infection (442), while the mouse null for VDR is protected (441). This may be due at least in part to loss of IFNγ stimulation of ROS and NO production required for macrophage antimicrobial activity (441). Furthermore, atopic dermatitis, a disease associated with increased Th2 activity (443), and allergic airway disease, likewise associated with increased Th2 activity, (437-439), may be aggravated by 1,25(OH)₂D₃ and less severe in animals null for VDR.

THE INNATE IMMUNE RESPONSE

The innate immune response involves the activation of toll-like receptors (TLRs) in polymorphonuclear cells (PMNs), monocytes and macrophages as well as in a number of epithelial cells including those of the epidermis, gingiva, intestine, vagina, bladder and lungs (review in (444)). There are 10 functional TLRs in human cells (of 11 known mammalian TLRs). TLRs are an extended family of host noncatalytic transmembrane pathogen-recognition receptors that interact with specific membrane patterns (PAMP) shed by infectious agents that trigger the innate immune response in the host. A number of these TLRs signal through adapter molecules such as myeloid differentiation factor-88 (MyD88) and the TIR-domain containing adapter inducing IFN-β (TRIF). MyD88 signaling includes translocation of NFkB to the nucleus, leading to the production and secretion of a number of inflammatory cytokines. TRIF signaling leads to the activation of interferon regulatory factor-3 (IRF-3) and the induction of type 1 interferons such as IFNβ. MyD88 mediates signaling from TLRs 2, 4, 5, 7 and 9, whereas TRIF mediates signaling from TLR 3 and 4. TLR1/2, TLR4, TLR5, TLR2/6 respond to bacterial ligands, whereas, TLR3, TLR7, and TLR 8 respond to viral ligands. The TLR response to fungi is less well defined. CD14 serves as a coreceptor for a number of these TLRs. Activation of TLRs leads to the induction of antimicrobial peptides (AMPs) and reactive oxygen species, which kill the organism. Among these AMPs is cathelicidin. Cathelicidin plays a number of roles in the innate immune response. The precursor protein, hCAP18, must be cleaved to its major peptide LL-37 to be active. In addition to its antimicrobial properties, LL-37 can stimulate the release of cytokines such as IL-6 and IL-10 through G protein coupled receptors, and IL-18 through ERK/P38 pathways, stimulate the EGF receptor leading to activation of STAT1 and 3, induce the chemotaxis of neutrophils, monocytes, macrophages, and T cells into the skin, and promote keratinocyte proliferation and migration (445). The expression of this antimicrobial peptide is induced by 1,25(OH)₂D₃ in both myeloid and epithelial cells (446,447). In addition, 1,25(OH)₂D₃ induces the coreceptor CD14 in keratinocytes(448). Stimulation of TLR2 by infectious organisms like tuberculosis in macrophages (449) or stimulation of TLR2 in keratinocytes by wounding the epidermis (448) results in increased expression of CYP27B1, which in the presence of adequate substrate (25OHD) stimulates the expression of cathelicidin. Lack of substrate (25OHD) or lack of CYP27B1 blunts the ability of these cells to respond to a challenge with respect to cathelicidin and/or CD14 production (447-449). In diseases such as atopic dermatitis, the production of cathelicidin and other antimicrobial peptides (AMPs) is reduced, predisposing these patients to microbial superinfections (450). Th2 cytokines such as IL-4 and 13 suppress the induction of AMPs(451). Since 1,25(OH)₂D₃ stimulates the differentiation of Th2 cells, in this disease 1,25(OH)₂D₃ administration may be harmful. An important role of these AMPs besides their antimicrobial properties is to help link the innate and adaptive immune response. This interplay is well demonstrated in SARS-CoV-19 infections in which a dysfunctional and/or delayed innate immune response can lead to an unchecked adaptive immune response resulting in a massive release of proinflammatory cytokines, the “cytokine storm”, leading to destruction of the lungs and death (452). Patients with vitamin D deficiency appear to be more vulnerable to this infection (453).

Although many cells are capable of the innate immune response including bone cells, most studies have focused on the macrophage and the keratinocyte. Vitamin D regulation of the innate immune response in these two cell types is comparable, but differences exist.

Macrophages

The importance of adequate vitamin D nutrition for resistance to infection has long been appreciated but poorly understood. This has been especially true for tuberculosis. Indeed, prior to the development of specific drugs for the treatment of tuberculosis, getting out of the city into fresh air and sunlight was the treatment of choice. In a recent survey of patients with tuberculosis in London (454) 56% had undetectable 25OHD levels, and an additional 20% had detectable levels but below 9 ng/ml (22 nM). In 1986 Rook et al. (455) demonstrated that 1,25(OH)₂D₃ could inhibit the growth of Mycobacterium tuberculosis. The mechanism for this remained unclear until the publication by Liu et al. (449) of their results in macrophages. They observed that activation of the Toll-like receptor TLR2/1 by a lipoprotein extracted from M. tuberculosis reduced the viability of intracellular M. tuberculosis in human monocytes and macrophages concomitant with increased expression of the VDR and of CYP27B1 in these cells. Killing of M. tuberculosis occurred only when the serum in which the cells were cultured contained adequate levels of 25OHD, the substrate for CYP27B1. This provided clear evidence for the importance of vitamin D nutrition (as manifested by adequate serum levels of 25OHD) in preventing and treating this disease, and demonstrated the critical role for endogenous production of 1,25(OH)₂D₃ by the macrophage to enable its antimycobacterial capacity. Activation of TLR2/1 or directly treating these cells with 1,25(OH)₂D₃ induced the antimicrobial peptide cathelicidin, which is toxic for M. tuberculosis. If induction of cathelicidin is blocked as with siRNA, the ability of 1,25(OH)₂D₃ to enhance the killing of M. tuberculosis is prevented (456). Furthermore, 1,25(OH)₂D₃ also induces the production of reactive oxygen species which if blocked likewise prevents the anti-mycobacterial activity of 1,25(OH)₂D₃ treated macrophages (457). The murine cathelicidin gene lacks a known VDR response element in its promoter, and so might not be expected to be induced by 1,25(OH)₂D₃ in mouse cells, yet 1,25(OH)₂D₃ stimulates antimycobacterial activity in murine macrophages. Murine macrophages, unlike human macrophages, utilize inducible nitric oxide synthase (iNOS) for their TLR and 1,25(OH)₂D₃ mediated killing of M. tuberculosis (457,458). Clinical trials attempting to treat tuberculosis patients with high levels of vitamin D have shown mixed results (459)(460).

Keratinocytes

Cathelicidiin and CD14 expression in epidermal keratinocytes is induced by 1,25(OH)₂D₃(445,448). In these cells butyrate, which by itself has little effect, potentiates the ability of 1,25(OH)₂D₃ to induce cathelicidin (461). Keratinocytes treated with 1,25(OH)₂D₃ are substantially more effective in killing Staphyococcus aureus than are untreated keratinocytes. Wounding the epidermis induces the expression of TLR2 and that of its co-receptor CD14 and cathelicidin (448). This does not occur in mice lacking CYP27B1 (448). Unlike macrophages, 1,25(OH)₂D₃ stimulates TLR2 expression in keratinocytes as well as in the epidermis when applied topically (448) providing a feed forward loop to amplify the innate immune response. Wounding also increases the expression of CYP27B1. This may occur as a result of increased levels of cytokines such as TNF-α and IFN-γ, both of which we have shown stimulate 1,25(OH)₂D₃ production, as well as by TGF-β and the TLR2 ligand Malp-2 (448). When the levels of VDR or one of its principal coactivators, SRC3, are reduced using siRNA technology, the ability of 1,25(OH)₂D₃ to induce cathelicidin and CD14 expression in human keratinocytes is markedly blunted (461).

Other Tissues

The VDR is widespread (127,462) (reviews). In some of these tissues the functional significance of the VDR and/or the effect of 1,25(OH)₂D are unclear. Since several of the functions regulated by 1,25(OH)₂D in some of these tissues may have clinical relevance, this section will focus on a select number of these tissues.

HEART

A reduction in contractility has been observed in vitamin D deficient animals (463). This may be due to lack of vitamin D or the accompanying hypocalcemia and hypophosphatemia. However, in vitro 1,25(OH)₂D stimulates calcium uptake by cardiac muscle cells (464,465). In addition, 1,25(OH)₂D inhibits the expression of atrial naturetic factor, one of the few genes with a negative VDRE in its promoter (466). Deletion of the VDR specifically in cardiac muscle leads to hypertrophy and fibrosis (467). Low circulating levels of 25OHD are associated with increased risk of myocardial infarction in men [436]. However, a large randomized clinical trial failed to show a protective effective of vitamin D supplementation to individuals with normal levels of 25OHD with respect to cardiovascular disease (341)

SKELETAL MUSCLE

Proximal muscle weakness is a hallmark of vitamin D deficiency, and reduced high energy substrates (ATP, creatinine phosphate) have been observed in that condition (468). Myoblasts contain VDR, although the expression of VDR in mature muscle cells is controversial. Muscle weakness may reflect the lower levels of calcium and phosphate rather than a reduction in 1,25(OH)₂D. However, evidence for a direct role of 1,25(OH)2D and VDR in muscle function is increasing (469). Moreover, 1,25(OH)₂D may have actions on muscle that do not require the VDR, at least the genomic functions of VDR. The Boland laboratory (470) has demonstrated acute effects of 1,25(OH)₂D on calcium uptake, PLC, PLA₂, PLD, PKC, and adenylate cyclase activities, all of which may alter muscle function.

PITUITARY

VDR have been found primarily in thyrotropes in vivo and in GH and prolactin secreting cell lines in vitro (471,472). 1,25(OH)₂D increases TRH stimulated TSH secretion by a mechanism involving increased Cai and IP₃ production (473,474), suggesting that induction of PLC by 1,25(OH)₂D may be involved.

BREAST

The breast contains VDR (475), and vitamin D plays a role in normal breast development (476). Moreover, breast cancer cells also contain VDR (477), and 1,25(OH)₂D and its analogs reduce their proliferation in vivo and in vitro (478,479). This has obvious clinical implications for the treatment of breast cancer.

LIVER

Low levels of VDR have been found in the liver, particularly in stellate cells (480,481). Hepatic regeneration is impaired in vitamin D deficient animals, even when the serum calcium is normalized by a high calcium diet (482), suggesting a role for 1,25(OH)₂D in hepatic cell growth and in the prevention of hepatic fibrosis (481).

LUNG

VDR have been found in type II epithelial pneumocytes (483). 1,25(OH)₂D stimulates their maturation including increased phospholipid production and surfactant release [437].These results are consistent with the abnormal alveolar development observed in pups born to vitamin D deficient mothers (484). In addition 1,25(OH)₂D stimulates the innate immune response in bronchial epithelial cells and may provide protection in patients with cystic fibrosis with recurrent lung infections as well as in patient with Covid-19 infections (452,485) as discussed previously.

REFERENCES

Whistler D. De morbo puerli anglorum, quem patrio ideiomate

indigenae vocant "the rickets". Journal of History of Medicine. 1645;5:397-415.

Mellanby E. An experimental investigation on rickets. Lancet. 1919;1:407-412.
McCollum EV, Simmonds N, Becker JE, Shipley PG. An experimental demonstration of the existence of a vitamin which promotes calcium deposition. Journal of Biological Chemistry. 1922;53:293-298.
Steenbock H, Black A. Fat-soluble vitamins. XVII. The induction of growth-promoting and calcifying properties in a ration by exposure to ultraviolet light. Journal of Biological Chemistry. 1924;61:405-422.
Huldshinsky K. Heilung von rachitis durch kunstalich hohensonne. Deut Med Wochenschr. 1919;45:712-713.
Hess AF, Unger LF. Cure of infantile rickets by sunlight. Journal of The American Medical Association. 1921;77:39.
Askew FA, Bourdillon RB, Bruce HM, Jenkins RGC, Webster TA. The distillation of vitamin D. Proceedings of the Royal Society. 1931;B107:76-90.
Windaus A, Schenck F, von Werder F. Uber das antirachitisch wirksame bestrahlungs-produkt aus 7-dehydro-cholesterin. Hoppe-Seylers Z Physiological Chemistry. 1936;241:100-103.
Prabhu AV, Luu W, Sharpe LJ, Brown AJ. Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis. The Journal of biological chemistry. 2016;291(16):8363-8373.
Holick MF, McLaughlin JA, Clark MB, Doppelt SH. Factors that influence the cutaneous photosynthesis of previtamin D3. Science. 1981;211:590-593.
Holick MF, McLaughlin JA, Clark MB, Holick SA, J.T. PJ, Anderson RR, Blank IH, Parrish JA. Photosynthesis of previtamin D3 in human and the physiologic consequences. Science. 1980;210:203-205.
Holick MF, Richtand NM, McNeill SC, Holick SA, Frommer JE, Henley JW, Potts JT, Jr. Isolation and identification of previtamin D3 from the skin of rats exposed to ultraviolet irradiation. Biochemistry. 1979;18(6):1003-1008.
Webb AR, DeCosta BR, Holick MF. Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation. Journal of Clinical Endocrinology and Metabolism. 1989;68(5):882-887.
Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J. Evidence for alteration of the vitamin D-endocrine system in blacks. Journal of Clinical Investigation. 1985;76(2):470-473.
Webb AR, Kline L, Holick MF. Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. Journal of Clinical Endocrinology and Metabolism. 1988;67(2):373-378.
Matsuoka LY, Wortsman J, Dannenberg MJ, Hollis BW, Lu Z, Holick MF. Clothing prevents ultraviolet-B radiation-dependent photosynthesis of vitamin D3. The Journal of clinical endocrinology and metabolism. 1992;75(4):1099-1103.
Matsuoka LY, Ide L, Wortsman J, MacLaughlin JA, Holick MF. Sunscreens suppress cutaneous vitamin D3 synthesis. Journal of Clinical Endocrinology and Metabolism. 1987;64(6):1165-1168.
Zhu JG, Ochalek JT, Kaufmann M, Jones G, Deluca HF. CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(39):15650-15655.
Thacher TD, Fischer PR, Singh RJ, Roizen J, Levine MA. CYP2R1 Mutations Impair Generation of 25-hydroxyvitamin D and Cause an Atypical Form of Vitamin D Deficiency. The Journal of clinical endocrinology and metabolism. 2015;100(7):E1005-1013.
Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russell DW. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. Journal of Biological Chemistry. 1989;264(14):8222-8229.
Usui E, Noshiro M, Okuda K. Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria. Febs Letters. 1990;262(1):135-138.
Cali JJ, Russell DW. Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis. Journal of Biological Chemistry. 1991;266(12):7774-7778.
Ichikawa F, Sato K, Nanjo M, Nishii Y, Shinki T, Takahashi N, Suda T. Mouse primary osteoblasts express vitamin D3 25-hydroxylase mRNA and convert 1 alpha-hydroxyvitamin D3 into 1 alpha,25-dihydroxyvitamin D3. Bone. 1995;16(1):129-135.
Cali JJ, Hsieh CL, Francke U, Russell DW. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. Journal of Biological Chemistry. 1991;266(12):7779-7783.
Leitersdorf E, Reshef A, Meiner V, Levitzki R, Schwartz SP, Dann EJ, Berkman N, Cali JJ, Klapholz L, Berginer VM. Frameshift and splice-junction mutations in the sterol 27-hydroxylase gene cause cerebrotendinous xanthomatosis in Jews or Moroccan origin. Journal of Clinical Investigation. 1993;91(6):2488-2496.
Berginer VM, Shany S, Alkalay D, Berginer J, Dekel S, Salen G, Tint GS, Gazit D. Osteoporosis and increased bone fractures in cerebrotendinous xanthomatosis [see comments]. Metabolism: Clinical and Experimental. 1993;42(1):69-74.
Leitersdorf E, Safadi R, Meiner V, Reshef A, Björkhem I, Friedlander Y, Morkos S, Berginer VM. Cerebrotendinous xanthomatosis in the Israeli Druze: molecular genetics and phenotypic characteristics. American Journal of Human Genetics. 1994;55(5):907-915.
Guo YD, Strugnell S, Jones G. Identification of a human liver mitochondrial cytochrome P-450 cDNA corresponding to the vitamin D3-25-hydroxylase. Journal of Bone and Mineral Research. 1991;6:S120.
Guo YD, Strugnell S, Back DW, Jones G. Transfected human liver cytochrome P-450 hydroxylates vitamin D analogs at different side-chain positions. Proc Natl Acad Sci U S A. 1993;90(18):8668-8672.
Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW. De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase. The Journal of biological chemistry. 2003;278(39):38084-38093.
Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(20):7711-7715.
Theodoropoulos C, Demers C, Mirshahi A, Gascon-Barre M. 1,25-Dihydroxyvitamin D(3) downregulates the rat intestinal vitamin D(3)-25-hydroxylase CYP27A. Am J Physiol Endocrinol Metab. 2001;281(2):E315-325.
Axen E, Postlind H, Wikvall K. Effects on CYP27 mRNA expression in rat kidney and liver by 1 alpha, 25- dihydroxyvitamin D3, a suppressor of renal 25-hydroxyvitamin D3 1 alpha- hydroxylase activity. Biochem Biophys Res Commun. 1995;215(1):136-141.
Vlahcevic ZR, Jairath SK, Heuman DM, Stravitz RT, Hylemon PB, Avadhani NG, Pandak WM. Transcriptional regulation of hepatic sterol 27-hydroxylase by bile acids. Am J Physiol. 1996;270(4 Pt 1):G646-652.
Twisk J, Hoekman MF, Lehmann EM, Meijer P, Mager WH, Princen HM. Insulin suppresses bile acid synthesis in cultured rat hepatocytes by down-regulation of cholesterol 7 alpha-hydroxylase and sterol 27- hydroxylase gene transcription. Hepatology. 1995;21(2):501-510.
Stravitz RT, Vlahcevic ZR, Russell TL, Heizer ML, Avadhani NG, Hylemon PB. Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes. J Steroid Biochem Mol Biol. 1996;57(5-6):337-347.
Roizen JD, Long C, Casella A, O'Lear L, Caplan I, Lai M, Sasson I, Singh R, Makowski AJ, Simmons R, Levine MA. Obesity Decreases Hepatic 25-Hydroxylase Activity Causing Low Serum 25-Hydroxyvitamin D. J Bone Miner Res. 2019:e3686.
Aatsinki SM, Elkhwanky MS, Kummu O, Karpale M, Buler M, Viitala P, Rinne V, Mutikainen M, Tavi P, Franko A, Wiesner RJ, Chambers KT, Finck BN, Hakkola J. Fasting-Induced Transcription Factors Repress Vitamin D Bioactivation, a Mechanism for Vitamin D Deficiency in Diabetes. Diabetes. 2019;68(5):918-931.
Saarem K, Pedersen JI. Sex differences in the hydroxylation of cholecalciferol and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in rat liver. Biochemical Journal. 1987;247(1):73-78.
Fu GK, Lin D, Zhang MY, Bikle DD, Shackleton CH, Miller WL, Portale AA. Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol. 1997;11(13):1961-1970.
Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis. Science. 1997;277(5333):1827-1830.
St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1997;12(10):1552-1559.
Shinki T, Shimada H, Wakino S, Anazawa H, Hayashi M, Saruta T, DeLuca HF, Suda T. Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA. Proc Natl Acad Sci U S A. 1997;94(24):12920-12925.
Kitanaka S, Takeyama K, Murayama A, Sato T, Okumura K, Nogami M, Hasegawa Y, Niimi H, Yanagisawa J, Tanaka T, Kato S. Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med. 1998;338(10):653-661.
Wang JT, Lin CJ, Burridge SM, Fu GK, Labuda M, Portale AA, Miller WL. Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families. Am J Hum Genet. 1998;63(6):1694-1702.
Dardenne O, Prud'homme J, Arabian A, Glorieux FH, St-Arnaud R. Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)- hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D- deficiency rickets. Endocrinology. 2001;142(7):3135-3141.
Bikle DD, Nemanic MK, Whitney JO, Elias PW. Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry. 1986;25(7):1545-1548.
Murayama A, Takeyama K, Kitanaka S, Kodera Y, Kawaguchi Y, Hosoya T, Kato S. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha-hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals. Endocrinology. 1999;140(5):2224-2231.
Panda DK, Al Kawas S, Seldin MF, Hendy GN, Goltzman D. 25-hydroxyvitamin D 1alpha-hydroxylase: structure of the mouse gene, chromosomal assignment, and developmental expression. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2001;16(1):46-56.
Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, Hewison M. Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. The Journal of clinical endocrinology and metabolism. 2001;86(2):888-894.
Bikle DD. Extra renal synthesis of 1,25 dihydroxyvitamin D and its Health Implications. Clin Rev in Bone and Min Metab. 2009;7:114-125.
Bikle DD, Pillai S. Vitamin D, calcium, and epidermal differentiation. Endocrine Review. 1993;14:3-19.
Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E. In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of 1alpha,25-dihydroxyvitamin D3 synthesis in rats. Endocrinology. 1977;101(3):969-974.
Rasmussen H, Wong M, Bikle D, Goodman DB. Hormonal control of the renal conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol. The Journal of clinical investigation. 1972;51(9):2502-2504.
Rost CR, Bikle DD, Kaplan RA. In vitro stimulation of 25-hydroxycholecalciferol 1 alpha-hydroxylation by parathyroid hormone in chick kidney slices: evidence for a role for adenosine 3',5'-monophosphate. Endocrinology. 1981;108(3):1002-1006.
Henry HL. Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology. 1985;116(2):503-510.
Armbrecht HJ, Forte LR, Wongsurawat N, Zenser TV, Davis BB. Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro. Endocrinology. 1984;114(2):644-649.
Janulis M, Tembe V, Favus MJ. Role of protein kinase C in parathyroid hormone stimulation of renal 1,25-dihydroxyvitamin D3 secretion. The Journal of clinical investigation. 1992;90(6):2278-2283.
Janulis M, Wong MS, Favus MJ. Structure-function requirements of parathyroid hormone for stimulation of 1,25-dihydroxyvitamin D3 production by rat renal proximal tubules. Endocrinology. 1993;133(2):713-719.
Brenza HL, Kimmel-Jehan C, Jehan F, Shinki T, Wakino S, Anazawa H, Suda T, DeLuca HF. Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha- hydroxylase gene promoter. Proc Natl Acad Sci U S A. 1998;95(4):1387-1391.
Zierold C, Nehring JA, DeLuca HF. Nuclear receptor 4A2 and C/EBPbeta regulate the parathyroid hormone-mediated transcriptional regulation of the 25-hydroxyvitamin D3-1alpha-hydroxylase. Archives of biochemistry and biophysics. 2007;460(2):233-239.
Takeyama K, Kato S. The vitamin D3 1alpha-hydroxylase gene and its regulation by active vitamin D3. Biosci Biotechnol Biochem. 2011;75(2):208-213.
Bikle DD, Murphy EW, Rasmussen H. The ionic control of 1,25-dihydroxyvitamin D3 synthesis in isolated chick renal mitochondria. The role of calcium as influenced by inorganic phosphate and hydrogen-ion. The Journal of clinical investigation. 1975;55(2):299-304.
Bushinsky DA, Riera GS, Favus MJ, Coe FL. Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat. The Journal of clinical investigation. 1985;76(4):1599-1604.
Hulter HN, Halloran BP, Toto RD, Peterson JC. Long-term control of plasma calcitriol concentration in dogs and humans. Dominant role of plasma calcium concentration in experimental hyperparathyroidism. The Journal of clinical investigation. 1985;76(2):695-702.
Hughes MR, Brumbaugh PF, Hussler MR, Wergedal JE, Baylink DJ. Regulation of serum 1alpha,25-dihydroxyvitamin D3 by calcium and phosphate in the rat. Science. 1975;190(4214):578-580.
Portale AA, Halloran BP, Morris RC, Jr. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D. The Journal of clinical investigation. 1987;80(4):1147-1154.
Condamine L, Menaa C, Vrtovsnik F, Vztovsnik F, Friedlander G, Garabedian M. Local action of phosphate depletion and insulin-like growth factor 1 on in vitro production of 1,25-dihydroxyvitamin D by cultured mammalian kidney cells. The Journal of clinical investigation. 1994;94(4):1673-1679.
Gray RW. Control of plasma 1,25-(OH)2-vitamin D concentrations by calcium and phosphorus in the rat: effects of hypophysectomy. Calcif Tissue Int. 1981;33(5):485-488.
Yoshida T, Yoshida N, Monkawa T, Hayashi M, Saruta T. Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha- hydroxylase gene expression. Endocrinology. 2001;142(5):1720-1726.
Gray RW, Garthwaite TL, Phillips LS. Growth hormone and triiodothyronine permit an increase in plasma 1,25(OH)2D concentrations in response to dietary phosphate deprivation in hypophysectomized rats. Calcif Tissue Int. 1983;35(1):100-106.
Halloran BP, Spencer EM. Dietary phosphorus and 1,25-dihydroxyvitamin D metabolism: influence of insulin-like growth factor I. Endocrinology. 1988;123(3):1225-1229.
Saito H, Kusano K, Kinosaki M, Ito H, Hirata M, Segawa H, Miyamoto K, Fukushima N. Human fibroblast growth factor-23 mutants suppress Na+-dependent phosphate co-transport activity and 1alpha,25-dihydroxyvitamin D3 production. The Journal of biological chemistry. 2003;278(4):2206-2211.
White KE, Evans WE, O'Riordan JLH, Speer MC, Econs MJ, Lorenz-Depiereux B, Grabowski M, Meitinger T, Strom TM. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nature Genetics. 2000;26:345.
Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S, Yamashita T. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(11):6500-6505.
Blau JE, Collins MT. The PTH-Vitamin D-FGF23 axis. Reviews in endocrine & metabolic disorders. 2015;16(2):165-174.
Meyer MB, Benkusky NA, Kaufmann M, Lee SM, Redfield RR, Jones G, Pike JW. Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse. The Journal of biological chemistry. 2019;294(24):9518-9535.
Schuster I, Egger H, Astecker N, Herzig G, Schussler M, Vorisek G. Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes. Steroids. 2001;66(3-5):451-462.
Xie Z, Munson S, Huang N, Schuster I, Portale AA, Miller WL, Bikle DD. The mechanism of 1,25-Dihydroxyvitamin D3 auto-regulation in keratinocytes. j bone min Res (Program & abstracts). 2001;16 suppl 1:S556.
Kato S, Fujiki R, Kim MS, Kitagawa H. Ligand-induced transrepressive function of VDR requires a chromatin remodeling complex, WINAC. The Journal of steroid biochemistry and molecular biology. 2007;103(3-5):372-380.
Akiyoshi-Shibata M, Sakaki T, Ohyama Y, Noshiro M, Okuda K, Yabusaki Y. Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli. Eur J Biochem. 1994;224(2):335-343.
Jones G, Prosser DE, Kaufmann M. 25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D. Arch Biochem Biophys. 2012;523(1):9-18.
Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Broking E, Fehrenbach H, Wingen AM, Guran T, Hoenderop JG, Bindels RJ, Prosser DE, Jones G, Konrad M. Mutations in CYP24A1 and idiopathic infantile hypercalcemia. N Engl J Med. 2011;365(5):410-421.
Shah AD, Hsiao EC, O'Donnell B, Salmeen K, Nussbaum R, Krebs M, Baumgartner-Parzer S, Kaufmann M, Jones G, Bikle DD, Wang Y, Mathew AS, Shoback D, Block-Kurbisch I. Maternal Hypercalcemia Due to Failure of 1,25-Dihydroxyvitamin-D3 Catabolism in a Patient With CYP24A1 Mutations. The Journal of clinical endocrinology and metabolism. 2015;100(8):2832-2836.
St-Arnaud R, Arabian A, Travers R, Barletta F, Raval-Pandya M, Chapin K, Depovere J, Mathieu C, Christakos S, Demay MB, Glorieux FH. Deficient mineralization of intramembranous bone in vitamin D-24- hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D. Endocrinology. 2000;141(7):2658-2666.
Hahn CN, Kerry DM, Omdahl JL, May BK. Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene. Nucleic Acids Res. 1994;22(12):2410-2416.
Chen KS, DeLuca HF. Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim Biophys Acta. 1995;1263(1):1-9.
Ohyama Y, Ozono K, Uchida M, Shinki T, Kato S, Suda T, Yamamoto O, Noshiro M, Kato Y. Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene. The Journal of biological chemistry. 1994;269(14):10545-10550.
Chen ML, Boltz MA, Armbrecht HJ. Effects of 1,25-dihydroxyvitamin D3 and phorbol ester on 25- hydroxyvitamin D3 24-hydroxylase cytochrome P450 messenger ribonucleic acid levels in primary cultures of rat renal cells. Endocrinology. 1993;132(4):1782-1788.
Pike JW, Kerner SA, Jin CH, Allegretto EA, Elgort M. Direct activation of the human 25-(OH)2D3 and PMA: Identification of cis elements and transactivators. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1995;10:S144.
Garabedian M, Holick MF, Deluca HF, Boyle IT. Control of 25-hydroxycholecalciferol metabolism by parathyroid glands. Proc Natl Acad Sci U S A. 1972;69(7):1673-1676.
Shigematsu T, Horiuchi N, Ogura Y, Miyahara T, Suda T. Human parathyroid hormone inhibits renal 24-hydroxylase activity of 25- hydroxyvitamin D3 by a mechanism involving adenosine 3',5'- monophosphate in rats. Endocrinology. 1986;118(4):1583-1589.
Armbrecht HJ, Wongsurawat N, Zenser TV, Davis BB. Effect of PTH and 1,25(OH)2D3 on renal 25(OH)D3 metabolism, adenylate cyclase, and protein kinase. Am J Physiol. 1984;246(1 Pt 1):E102-107.
Armbrecht HJ, Hodam TL, Boltz MA, Partridge NC, Brown AJ, Kumar VB. Induction of the vitamin D 24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 is regulated by parathyroid hormone in UMR106 osteoblastic cells. Endocrinology. 1998;139(8):3375-3381.
Armbrecht HJ, Wongsurawat VJ, Hodam TL, Wongsurawat N. Insulin markedly potentiates the capacity of parathyroid hormone to increase expression of 25-hydroxyvitamin D3-24-hydroxylase in rat osteoblastic cells in the presence of 1,25-dihydroxyvitamin D3. FEBS Lett. 1996;393(1):77-80.
Meyer MB, Lee SM, Carlson AH, Benkusky NA, Kaufmann M, Jones G, Pike JW. A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues. The Journal of biological chemistry. 2019;294(39):14467-14481.
Inoue Y, Segawa H, Kaneko I, Yamanaka S, Kusano K, Kawakami E, Furutani J, Ito M, Kuwahata M, Saito H, Fukushima N, Kato S, Kanayama HO, Miyamoto K. Role of the vitamin D receptor in FGF23 action on phosphate metabolism. Biochem J. 2005;390(Pt 1):325-331.
Wu S, Grieff M, Brown AJ. Regulation of renal vitamin D-24-hydroxylase by phosphate: effects of hypophysectomy, growth hormone and insulin-like growth factor I. Biochem Biophys Res Commun. 1997;233(3):813-817.
Cooke NE, Haddad JG. Vitamin D binding protein (Gc-globulin). Endocrine reviews. 1989;10(3):294-307.
Bikle DD, Gee E, Halloran B, Haddad JG. Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease. The Journal of clinical investigation. 1984;74(6):1966-1971.
Bikle DD, Siiteri PK, Ryzen E, Haddad JG. Serum protein binding of 1,25-dihydroxyvitamin D: a reevaluation by direct measurement of free metabolite levels. The Journal of clinical endocrinology and metabolism. 1985;61(5):969-975.
Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E, Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. The Journal of clinical endocrinology and metabolism. 1986;63(4):954-959.
Bikle DD, Halloran BP, Gee E, Ryzen E, Haddad JG. Free 25-hydroxyvitamin D levels are normal in subjects with liver disease and reduced total 25-hydroxyvitamin D levels. The Journal of clinical investigation. 1986;78(3):748-752.
Madden K, Feldman HA, Chun RF, Smith EM, Sullivan RM, Agan AA, Keisling SM, Panoskaltsis-Mortari A, Randolph AG. Critically Ill Children Have Low Vitamin D-Binding Protein, Influencing Bioavailability of Vitamin D. Ann Am Thorac Soc. 2015;12(11):1654-1661.
Nielson CM, Jones KS, Chun RF, Jacobs JM, Wang Y, Hewison M, Adams JS, Swanson CM, Lee CG, Vanderschueren D, Pauwels S, Prentice A, Smith RD, Shi T, Gao Y, Schepmoes AA, Zmuda JM, Lapidus J, Cauley JA, Bouillon R, Schoenmakers I, Orwoll ES, Osteoporotic Fractures in Men Research G. Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations. The Journal of clinical endocrinology and metabolism. 2016;101(5):2226-2234.
Pettifor JM, Bikle DD, Cavaleros M, Zachen D, Kamdar MC, Ross FP. Serum levels of free 1,25-dihydroxyvitamin D in vitamin D toxicity. Ann Intern Med. 1995;122(7):511-513.
Safadi FF, Thornton P, Magiera H, Hollis BW, Gentile M, Haddad JG, Liebhaber SA, Cooke NE. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. The Journal of clinical investigation. 1999;103(2):239-251.
Zella LA, Shevde NK, Hollis BW, Cooke NE, Pike JW. Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo. Endocrinology. 2008;149(7):3656-3667.
Henderson CM, Fink SL, Bassyouni H, Argiropoulos B, Brown L, Laha TJ, Jackson KJ, Lewkonia R, Ferreira P, Hoofnagle AN, Marcadier JL. Vitamin D-Binding Protein Deficiency and Homozygous Deletion of the GC Gene. The New England journal of medicine. 2019;380(12):1150-1157.
Bikle D, Bouillon R, Thadhani R, Schoenmakers I. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? The Journal of steroid biochemistry and molecular biology 2017;173:105-116.
Malmstroem S, Rejnmark L, Imboden JB, Shoback DM, Bikle DD. Current Assays to Determine Free 25-Hydroxyvitamin D in Serum. J AOAC Int 2017;100:1323-1327.
Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, Melsen F, Christensen EI, Willnow TE. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell. 1999;96(4):507-515.
Arnaud J, Constans J. Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP). Hum Genet. 1993;92(2):183-188.
Cooke NE, David EV. Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family. The Journal of clinical investigation. 1985;76(6):2420-2424.
Cooke NE, Levan G, Szpirer J. The rat vitamin D binding protein (Gc-globulin) gene is syntenic with the rat albumin and alpha-fetoprotein genes on chromosome 14. Cytogenet Cell Genet. 1987;44(2-3):98-100.
Guha C, Osawa M, Werner PA, Galbraith RM, Paddock GV. Regulation of human Gc (vitamin D--binding) protein levels: hormonal and cytokine control of gene expression in vitro. Hepatology. 1995;21(6):1675-1681.
Lees A, Haddad JG, Lin S. Brevin and DBP comparison of the effects of two serum protein on actin assembly and disassembly. Biochemistry. 1984;23:3038-3047.
Yamamoto N, Homma S, Haddad JG, Kowalski MA. Vitamin D3 binding protein required for in vitro activation of macrophages after alkylglycerol treatment of mouse peritoneal cells. Immunology. 1991;74(3):420-424.
Schneider GB, Benis KA, Flay NW, Ireland RA, Popoff SN. Effects of vitamin D binding protein-macrophage activating factor (DBP- MAF) infusion on bone resorption in two osteopetrotic mutations. Bone. 1995;16(6):657-662.
Safadi FF, Thornton P, Magiera H, Hollis BW, Gentile M, Haddad JG, Liebhaber SA, Cooke NE. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. The Journal of clinical investigation. 1999;103(2):239-251.
Haussler MR, Norman AW. Chromosomal receptor for a vitamin D metabolite. Proc Natl Acad Sci U S A. 1969;62(1):155-162.
McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, O'Malley BW. Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science. 1987;235(4793):1214-1217.
Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, O'Malley BW. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci U S A. 1988;85(10):3294-3298.
Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, O'Malley BW. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 1988;242(4886):1702-1705.
Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet. 1997;16(4):391-396.
Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proceedings of the National Academy of Sciences of the United States of America. 1997;94(18):9831-9835.
Walters MR. Newly identified actions of the vitamin D endocrine system. Endocr Rev. 1992;13(4):719-764.
Harris SS, Eccleshall TR, Gross C, Dawson-Hughes B, Feldman D. The vitamin D receptor start codon polymorphism (FokI) and bone mineral density in premenopausal American black and white women. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1997;12(7):1043-1048.
Rachez C, Gamble M, Chang CP, Atkins GB, Lazar MA, Freedman LP. The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. Mol Cell Biol. 2000;20(8):2718-2726.
Makowski A, Brzostek S, Cohen RN, Hollenberg AN. Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor. Molecular endocrinology (Baltimore, Md). 2003;17(2):273-286.
Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell. 2000;5(1):173-179.
Orlov I, Rochel N, Moras D, Klaholz BP. Structure of the full human RXR/VDR nuclear receptor heterodimer complex with its DR3 target DNA. The EMBO journal. 2012;31(2):291-300.
Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H. A canonical structure for the ligand-binding domain of nuclear receptors. Nat Struct Biol. 1996;3(1):87-94.
Chen S, Cui J, Nakamura K, Ribeiro RC, West BL, Gardner DG. Coactivator-vitamin D receptor interactions mediate inhibition of the atrial natriuretic peptide promoter. The Journal of biological chemistry.275(20):15039-15048.
McKenna NJ, Lanz RB, O'Malley BW. Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev. 1999;20(3):321-344.
Polly P, Herdick M, Moehren U, Baniahmad A, Heinzel T, Carlberg C. VDR-Alien: a novel, DNA-selective vitamin D(3) receptor-corepressor partnership. Faseb J. 2000;14(10):1455-1463.
Xie Z, Chang S, Oda Y, Bikle DD. Hairless suppresses vitamin D receptor transactivation in human keratinocytes. Endocrinology. 2006;147(1):314-323.
Hsieh JC, Sisk JM, Jurutka PW, Haussler CA, Slater SA, Haussler MR, Thompson CC. Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling. The Journal of biological chemistry. 2003;278(40):38665-38674.
Bikle DD, Elalieh H, Chang S, Xie Z, Sundberg JP. Development and progression of alopecia in the vitamin D receptor null mouse. J Cell Physiol. 2006;207:340-353.
Leo C, Chen JD. The SRC family of nuclear receptor coactivators. Genes. 2000;245:1-11.
Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP. Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature. 1999;398(6730):824-828.
Teichert A, Arnold LA, Otieno S, Oda Y, Augustinaite I, Geistlinger TR, Kriwacki RW, Guy RK, Bikle DD. Quantification of the vitamin D receptor-coregulator interaction. Biochemistry. 2009;48(7):1454-1461.
Oda Y. Abstract 809. Journal of Investigative Dermatology. 2001;116.
Hawker NP, Pennypacker SD, Chang SM, Bikle DD. Regulation of Human Epidermal Keratinocyte Differentiation by the Vitamin D Receptor and its Coactivators DRIP205, SRC2, and SRC3. The Journal of investigative dermatology. 2007;127:874.
Schauber J, Oda Y, Buchau AS, Yun QC, Steinmeyer A, Zugel U, Bikle DD, Gallo RL. Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3. J Invest Dermatol. 2008;128(4):816-824.
Oda Y, Uchida Y, Moradian S, Crumrine D, Elias P, Bikle D. Vitamin D receptor and coactivators SRC 2 and 3 regulate epidermis-specific sphingolipid production and permeability barrier formation. J Invest Dermatol. 2009;129(6):1367-1378.
Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, Rahl PB, Lee TI, Young RA. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 2013;153(2):307-319.
Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, Toriyabe T, Kawabata M, Miyazono K, Kato S. Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science. 1999;283(5406):1317-1321.
Hsieh JC, Jurutka PW, Nakajima S, Galligan MA, Haussler CA, Shimizu Y, Shimizu N, Whitfield GK, Haussler MR. Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. The Journal of biological chemistry. 1993;268(20):15118-15126.
Palmer HG, Gonzalez-Sancho JM, Espada J, Berciano MT, Puig I, Baulida J, Quintanilla M, Cano A, de Herreros AG, Lafarga M, Munoz A. Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. The Journal of cell biology. 2001;154(2):369-387.
Palmer HG, Anjos-Afonso F, Carmeliet G, Takeda H, Watt FM. The Vitamin D Receptor Is a Wnt Effector that Controls Hair Follicle Differentiation and Specifies Tumor Type in Adult Epidermis. PloS one. 2008;3(1):e1483.
Kurokawa R, Yu VC, Naar A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG, Glass CK. Differential orientations of the DNA-binding domain and carboxy- terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev. 1993;7(7B):1423-1435.
Prufer K, Racz A, Lin GC, Barsony J. Dimerization with retinoid X receptors promotes nuclear localization and subnuclear targeting of vitamin D receptors. The Journal of biological chemistry. 2000;275(52):41114-41123.
Xie Z, Bikle DD. Cloning of the human phospholipase C-gamma1 promoter and identification of a DR6-type vitamin D-responsive element. The Journal of biological chemistry. 1997;272(10):6573-6577.
Schrader M, Muller KM, Nayeri S, Kahlen JP, Carlberg C. Vitamin D3-thyroid hormone receptor heterodimer polarity directs ligand sensitivity of transactivation. Nature. 1994;370(6488):382-386.
Lemon BD, Freedman LP. Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo. Mol Cell Biol. 1996;16(3):1006-1016.
Pike JW, Meyer MB. The vitamin D receptor: new paradigms for the regulation of gene expression by 1,25-dihydroxyvitamin D3. Rheumatic diseases clinics of North America. 2012;38(1):13-27.
Carlberg C, Seuter S, Heikkinen S. The first genome-wide view of vitamin D receptor locations and their mechanistic implications. Anticancer research. 2012;32(1):271-282.
MacDonald PN, Dowd DR, Nakajima S, Galligan MA, Reeder MC, Haussler CA, Ozato K, Haussler MR. Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25- dihydroxyvitamin D3-activated expression of the rat osteocalcin gene. Mol Cell Biol. 1993;13(9):5907-5917.
Kang S, Li XY, Duell EA, Voorhees JJ. The retinoid X receptor agonist 9-cis-retinoic acid and the 24- hydroxylase inhibitor ketoconazole increase activity of 1,25- dihydroxyvitamin D3 in human skin in vivo. The Journal of investigative dermatology. 1997;108(4):513-518.
Gill RK, Christakos S. Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses. Proc Natl Acad Sci U S A. 1993;90(7):2984-2988.
Liu SM, Koszewski N, Lupez M, Malluche HH, Olivera A, Russell J. Characterization of a response element in the 5'-flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor. Mol Endocrinol. 1996;10(2):206-215.
Kremer R, Sebag M, Champigny C, Meerovitch K, Hendy GN, White J, Goltzman D. Identification and characterization of 1,25-dihydroxyvitamin D3- responsive repressor sequences in the rat parathyroid hormone-related peptide gene. The Journal of biological chemistry. 1996;271(27):16310-16316.
Alroy I, Towers TL, Freedman LP. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol Cell Biol. 1995;15(10):5789-5799.
Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz JL. A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology. 1992;131(3):1305-1312.
Wistrom CA, Meizel S. Evidence suggesting involvement of a unique human sperm steroid receptor/Cl- channel complex in the progesterone-initiated acrosome reaction. Dev Biol. 1993;159(2):679-690.
Koenig H, Fan CC, Goldstone AD, Lu CY, Trout JJ. Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes. Circ Res. 1989;64(3):415-426.
Orchinik M, Murray TF, Moore FL. A corticosteroid receptor in neuronal membranes. Science. 1991;252(5014):1848-1851.
Segal J. Thyroid hormone action at the level of the plasma membrane. Thyroid. 1990;1(1):83-87.
Caffrey JM, Farach-Carson MC. Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells. The Journal of biological chemistry. 1989;264(34):20265-20274.
Baran DT, Sorensen AM, Honeyman TW, Ray R, Holick MF. 1 alpha,25-dihydroxyvitamin D3-induced increments in hepatocyte cytosolic calcium and lysophosphatidylinositol: inhibition by pertussis toxin and 1 beta,25-dihydroxyvitamin D3. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1990;5(5):517-524.
Morelli S, de Boland AR, Boland RL. Generation of inositol phosphates, diacylglycerol and calcium fluxes in myoblasts treated with 1,25-dihydroxyvitamin D3. Biochem J. 1993;289(Pt 3):675-679.
Wali RK, Baum CL, Sitrin MD, Brasitus TA. 1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium. The Journal of clinical investigation. 1990;85(4):1296-1303.
Khare S, Bolt MJ, Wali RK, Skarosi SF, Roy HK, Niedziela S, Scaglione-Sewell B, Aquino B, Abraham C, Sitrin MD, Brasitus TA, Bissonnette M. 1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation. The Journal of clinical investigation. 1997;99(8):1831-1841.
Nemere I, Yoshimoto Y, Norman AW. Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3. Endocrinology. 1984;115(4):1476-1483.
Nemere I, Norman AW. Rapid action of 1,25-dihydroxyvitamin D3 on calcium transport in perfused chick duodenum: effect of inhibitors. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1987;2(2):99-107.
de Boland AR, Norman AW. Influx of extracellular calcium mediates 1,25-dihydroxyvitamin D3- dependent transcaltachia (the rapid stimulation of duodenal Ca2+ transport). Endocrinology. 1990;127(5):2475-2480.
de Boland AR, Norman A. Evidence for involvement of protein kinase C and cyclic adenosine 3',5' monophosphate-dependent protein kinase in the 1,25-dihydroxy-vitamin D3- mediated rapid stimulation of intestinal calcium transport, (transcaltachia). Endocrinology. 1990;127(1):39-45.
de Boland AR, Nemere I, Norman AW. Ca2(+)-channel agonist BAY K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2+ transport in chick perfused duodenum. Biochem Biophys Res Commun. 1990;166(1):217-222.
Nemere I, Dormanen MC, Hammond MW, Okamura WH, Norman AW. Identification of a specific binding protein for 1 alpha,25- dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia. The Journal of biological chemistry. 1994;269(38):23750-23756.
Nemere I, Safford SE, Rohe B, DeSouza MM, Farach-Carson MC. Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS) binding protein. The Journal of steroid biochemistry and molecular biology. 2004;89-90(1-5):281-285.
Pedrozo HA, Schwartz Z, Rimes S, Sylvia VL, Nemere I, Posner GH, Dean DD, Boyan BD. Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1999;14(6):856-867.
Khanal RC, Peters TM, Smith NM, Nemere I. Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells. Journal of cellular biochemistry. 2008;105(4):1109-1116.
Norman AW, Okamura WH, Farach-Carson MC, Allewaert K, Branisteanu D, Nemere I, Muralidharan KR, Bouillon R. Structure-function studies of 1,25-dihydroxyvitamin D3 and the vitamin D endocrine system. 1,25-dihydroxy-pentadeuterio-previtamin D3 (as a 6- s-cis analog) stimulates nongenomic but not genomic biological responses. The Journal of biological chemistry. 1993;268(19):13811-13819.
Sequeira VB, Rybchyn MS, Tongkao-On W, Gordon-Thomson C, Malloy PJ, Nemere I, Norman AW, Reeve VE, Halliday GM, Feldman D, Mason RS. The role of the vitamin D receptor and ERp57 in photoprotection by 1alpha,25-dihydroxyvitamin D3. Molecular endocrinology (Baltimore, Md). 2012;26(4):574-582.
Mizwicki MT, Keidel D, Bula CM, Bishop JE, Zanello LP, Wurtz JM, Moras D, Norman AW. Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1alpha,25(OH)2-vitamin D3 signaling. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(35):12876-12881.
Schachter D, Rosen S. Active transport of Ca45 by the small intestine and its dependence on vitamin D. Am J Physiol. 1959;196:357-362.
Wasserman RH, Kallfelz FA, Comar CL. Active transport of calcium by rat duodenum in vivo. Science. 1961;133:883-884.
Bikle DD. Regulation of intestinal calcium transport by vitamin D [1,25(OH)2]:role of membrane structure. In: Membrane transport and information storage. New York: Wiley-Liss. 1990, pp 191-219
Wasserman RH, Fullmer CS. Vitamin D and intestinal calcium transport: facts, speculations and hypotheses. J Nutr. 1995;125(7 Suppl):1971S-1979S.
Wongdee K, Chanpaisaeng K, Teerapornpuntakit J, Charoenphandhu N. Intestinal Calcium Absorption. Compr Physiol. 2021;11(3):2047-2073.
Bikle DD, Shoback DM, Munson S. 1,25-dihydroxyvitamin D increases the intracellular free calcium concentration of duodenal epithelial cells. In: Vitamin D: Chemial, biochemical and clinical update. New York: Walter de Gruyter, 1985, p 416
Morrissey RL, Zolock DT, Mellick PW, Bikle DD. Influence of cycloheximide and 1,25-dihydroxyvitamin D3 on mitochondrial and vesicle mineralization in the intestine. Cell Calcium. 1980;1:69-79.
Davis WL, Hagler HK, Jones RG, Farmer GR, Cooper OJ, Martin JH, Bridges GE, Goodman DB. Cryofixation, ultracryomicrotomy, and X-ray microanalysis of enterocytes from chick duodenum: vitamin-D-induced formation of an apical tubulovesicular system. Anat Rec. 1991;229(2):227-239.
Nemere I, Leathers V, Norman AW. 1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium- binding protein (calbindin-D28K). The Journal of biological chemistry. 1986;261(34):16106-16114.
Max EE, Goodman DB, Rasmussen H. Purification and characterization of chick intestine brush border membrane. Effects of 1alpha(OH) vitamin D3 treatment. Biochimica et biophysica acta. 1978;511(2):224-239.
Brasitus TA, Dudeja PK, Eby B, Lau K. Correction by 1,25(OH)2D3 of the abnormal fluidity and lipid composition of enterocyte brush border membranes in vitamin D-deprived rats. Journal of Biological Chemistry. 1981;256:3354-3360.
Matsumoto T, Fontaine O, Rasmussen H. Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action. The Journal of biological chemistry. 1981;256(7):3354-3360.
Bikle DD, Whitney J, Munson S. The relationship of membrane fluidity to calcium flux in chick intestinal brush border membranes. Endocrinology. 1984;114(1):260-267.
Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, Hediger MA. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. The Journal of biological chemistry. 1999;274(32):22739-22746.
Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ. Molecular identification of the apical Ca2+ channel in 1, 25- dihydroxyvitamin D3-responsive epithelia. The Journal of biological chemistry. 1999;274(13):8375-8378.
Peng JB, Chen XZ, Berger UV, Vassilev PM, Brown EM, Hediger MA. A rat kidney-specific calcium transporter in the distal nephron. The Journal of biological chemistry. 2000;275(36):28186-28194.
Muller D, Hoenderop JG, Meij IC, van den Heuvel LP, Knoers NV, den Hollander AI, Eggert P, Garcia-Nieto V, Claverie-Martin F, Bindels RJ. Molecular cloning, tissue distribution, and chromosomal mapping of the human epithelial Ca2+ channel (ECAC1). Genomics. 2000;67(1):48-53.
Peng JB, Brown EM, Hediger MA. Structural conservation of the genes encoding cat1, cat2, and related cation channels. Genomics. 2001;76(1-3):99-109.
Song Y, Peng X, Porta A, Takanaga H, Peng JB, Hediger MA, Fleet JC, Christakos S. Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice. Endocrinology. 2003;144(9):3885-3894.
Bianco SD, Peng JB, Takanaga H, Suzuki Y, Crescenzi A, Kos CH, Zhuang L, Freeman MR, Gouveia CH, Wu J, Luo H, Mauro T, Brown EM, Hediger MA. Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2007;22(2):274-285.
Sampson HW, Matthews JL, Martin JH, Kunin AS. An electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin-D-treated rats. Calcif Tissue Res. 1970;5(4):305-316.
Schaefer HJ. Ultrastructure and ion distribution of the intestinal cell during experimental vitamin D deficiency rickets in rats. Virchows Archiv. 1973;359:111-123.
Chandra S, Fullmer CS, Smith CA, Wasserman RH, Morrison GH. Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: a 44Ca stable isotope study. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(15):5715-5719.
Bikle DD, Zolock DT, Morrissey RL, Herman RH. Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis. The Journal of biological chemistry. 1978;253(2):484-488.
Bikle DD, Morrissey RL, Zolock DT. The mechanism of action of vitamin D in the intestine. Am J Clin Nutr. 1979;32(11):2322-2328.
Glenney JR, Jr., Glenney P. Comparison of Ca++-regulated events in the intestinal brush border. J Cell Biol. 1985;100(3):754-763.
Bikle DD, Gee E. Free, and not total, 1,25-dihydroxyvitamin D regulates 25- hydroxyvitamin D metabolism by keratinocytes. Endocrinology. 1989;124(2):649-654.
Bikle DD, Munson S, Chafouleas J. Calmodulin may mediate 1,25-dihydroxyvitamin D-stimulated intestinal calcium transport. FEBS Lett. 1984;174(1):30-33.
Howe CL, Keller TC, 3rd, Mooseker MS, Wasserman RH. Analysis of cytoskeletal proteins and Ca2+-dependent regulation of structure in intestinal brush borders from rachitic chicks. Proc Natl Acad Sci U S A. 1982;79(4):1134-1138.
Bikle DD, Munson S. The villus gradient of brush border membrane calmodulin and the calcium-independent calmodulin-binding protein parallels that of calcium-accumulating ability. Endocrinology. 1986;118(2):727-732.
Drenckhahn D, Dermietzel R. Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. J Cell Biol. 1988;107(3):1037-1048.
Munson S, Wang Y, Chang W, Bikle DD. Myosin 1a Regulates Osteoblast Differentiation Independent of Intestinal Calcium Transport. J Endocr Soc. 2019;3(11):1993-2011.
Wasserman RH, Taylor AN. Vitamin D-dependent calcium-binding protein. Response to some physiological and nutritional variables. The Journal of biological chemistry. 1968;243(14):3987-3993.
Lee GS, Lee KY, Choi KC, Ryu YH, Paik SG, Oh GT, Jeung EB. Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2007;22(12):1968-1978.
Ghijsen WE, De Jong MD, Van Os CH. ATP-dependent calcium transport and its correlation with Ca2+ -ATPase activity in basolateral plasma membranes of rat duodenum. Biochim Biophys Acta. 1982;689(2):327-336.
Cai Q, Chandler JS, Wasserman RH, Kumar R, Penniston JT. Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression. Proc Natl Acad Sci U S A. 1993;90(4):1345-1349.
Wasserman RH, Chandler JS, Meyer SA, Smith CA, Brindak ME, Fullmer CS, Penniston JT, Kumar R. Intestinal calcium transport and calcium extrusion processes at the basolateral membrane. J Nutr. 1992;122(3 Suppl):662-671.
Ryan ZC, Craig TA, Filoteo AG, Westendorf JJ, Cartwright EJ, Neyses L, Strehler EE, Kumar R. Deletion of the intestinal plasma membrane calcium pump, isoform 1, Atp2b1, in mice is associated with decreased bone mineral density and impaired responsiveness to 1, 25-dihydroxyvitamin D3. Biochem Biophys Res Commun. 2015;467(1):152-156.
Liu C, Weng H, Chen L, Yang S, Wang H, Debnath G, Guo X, Wu L, Mohandas N, An X. Impaired intestinal calcium absorption in protein 4.1R-deficient mice due to altered expression of plasma membrane calcium ATPase 1b (PMCA1b). The Journal of biological chemistry. 2013;288(16):11407-11415.
Hoenderop JG, Nilius B, Bindels RJ. Calcium absorption across epithelia. Physiol Rev. 2005;85(1):373-422.
Charoenphandhu N, Krishnamra N. Prolactin is an important regulator of intestinal calcium transport. Can J Physiol Pharmacol. 2007;85(6):569-581.
Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R, Mrsny RJ, Madara JL. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol. 1997;273(4):C1378-1385.
Fujita H, Chiba H, Yokozaki H, Sakai N, Sugimoto K, Wada T, Kojima T, Yamashita T, Sawada N. Differential expression and subcellular localization of claudin-7, -8, -12, -13, and -15 along the mouse intestine. J Histochem Cytochem. 2006;54(8):933-944.
Van Itallie CM, Fanning AS, Anderson JM. Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudins. Am J Physiol Renal Physiol. 2003;285(6):F1078-1084.
Fujita H, Sugimoto K, Inatomi S, Maeda T, Osanai M, Uchiyama Y, Yamamoto Y, Wada T, Kojima T, Yokozaki H, Yamashita T, Kato S, Sawada N, Chiba H. Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes. Mol Biol Cell. 2008;19(5):1912-1921.
Charoenphandhu N, Nakkrasae LI, Kraidith K, Teerapornpuntakit J, Thongchote K, Thongon N, Krishnamra N. Two-step stimulation of intestinal Ca(2+) absorption during lactation by long-term prolactin exposure and suckling-induced prolactin surge. Am J Physiol Endocrinol Metab. 2009;297(3):E609-619.
Harrison HE, Harrison HC. Intestinal transport of phosphate: Action of vitamin D, calcium, and potassium. American Journal of Physiology. 1961;201:1007-1012.
Peterlik M, Wasserman RH. Regulation by vitamin D of intestinal phosphate absorption. Horm Metab Res. 1980;12(5):216-219.
Xu H, Bai L, Collins JF, Ghishan FK. Molecular cloning, functional characterization, tissue distribution, and chromosomal localization of a human, small intestinal sodium-phosphate (Na+-Pi) transporter (SLC34A2). Genomics. 1999;62(2):281-284.
Xu H, Bai L, Collins JF, Ghishan FK. Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)(2) vitamin D(3). American journal of physiology Cell physiology. 2002;282(3):C487-493.
Fuchs R, Peterlik M. Vitamin D-induced transepithelial phosphate and calcium transport by chick jejunum. Effect of microfilamentous and microtubular inhibitors. FEBS Lett. 1979;100(2):357-359.
Narbaitz R, Stumpf WE, Sar M, Huang S, DeLuca HF. Autoradiographic localization of target cells for 1 alpha, 25- dihydroxyvitamin D3 in bones from fetal rats. Calcif Tissue Int. 1983;35(2):177-182.
Boivin G, Mesguich P, Pike JW, Bouillon R, Meunier PJ, Haussler MR, Dubois PM, Morel G. Ultrastructural immunocytochemical localization of endogenous 1,25- dihydroxyvitamin D3 and its receptors in osteoblasts and osteocytes from neonatal mouse and rat calvaria. Bone Miner. 1987;3(2):125-136.
Nakamichi Y, Udagawa N, Horibe K, Mizoguchi T, Yamamoto Y, Nakamura T, Hosoya A, Kato S, Suda T, Takahashi N. VDR in Osteoblast-Lineage Cells Primarily Mediates Vitamin D Treatment-Induced Increase in Bone Mass by Suppressing Bone Resorption. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2017;32(6):1297-1308.
Panda DK, Miao D, Bolivar I, Li J, Huo R, Hendy GN, Goltzman D. Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis. The Journal of biological chemistry. 2004;279(16):16754-16766.
Chenu C, Valentin-Opran A, Chavassieux P, Saez S, Meunier PJ, Delmas PD. Insulin like growth factor I hormonal regulation by growth hormone and by 1,25(OH)2D3 and activity on human osteoblast-like cells in short- term cultures. Bone. 1990;11(2):81-86.
Kurose H, Yamaoka K, Okada S, Nakajima S, Seino Y. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increases insulin-like growth factor I (IGF-I) receptors in clonal osteoblastic cells. Study on interaction of IGF-I and 1,25-(OH)2D3. Endocrinology. 1990;126(4):2088-2094.
Scharla SH, Strong DD, Mohan S, Baylink DJ, Linkhart TA. 1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts. Endocrinology. 1991;129(6):3139-3146.
Moriwake T, Tanaka H, Kanzaki S, Higuchi J, Seino Y. 1,25-Dihydroxyvitamin D3 stimulates the secretion of insulin-like growth factor binding protein 3 (IGFBP-3) by cultured human osteosarcoma cells. Endocrinology. 1992;130(2):1071-1073.
Sato T, Ono T, Tuan RS. 1,25-Dihydroxy vitamin D3 stimulation of TGF-beta expression in chick embryonic calvarial bone. Differentiation. 1993;52(2):139-150.
Wang DS, Yamazaki K, Nohtomi K, Shizume K, Ohsumi K, Shibuya M, Demura H, Sato K. Increase of vascular endothelial growth factor mRNA expression by 1,25- dihydroxyvitamin D3 in human osteoblast-like cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1996;11(4):472-479.
Lacey DL, Grosso LE, Moser SA, Erdmann J, Tan HL, Pacifici R, Villareal DT. IL-1-induced murine osteoblast IL-6 production is mediated by the type 1 IL-1 receptor and is increased by 1,25 dihydroxyvitamin D3. The Journal of clinical investigation. 1993;91(4):1731-1742.
Lacey DL, Erdmann JM, Tan HL, Ohara J. Murine osteoblast interleukin 4 receptor expression: upregulation by 1,25 dihydroxyvitamin D3. Journal of cellular biochemistry. 1993;53(2):122-134.
Nambi P, Wu HL, Lipshutz D, Prabhakar U. Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: down-regulation by 1,25-dihydroxy- vitamin D3. Mol Pharmacol. 1995;47(2):266-271.
Zhou X, von der Mark K, Henry S, Norton W, Adams H, de Crombrugghe B. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLoS Genet. 2014;10(12):e1004820.
Johnson JA, Grande JP, Roche PC, Kumar R. Ontogeny of the 1,25-dihydroxyvitamin D3 receptor in fetal rat bone. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1996;11(1):56-61.
Miller SC, Halloran BP, DeLuca HF, Lee WSS. Studies on the role of vitamin D in early skeletal development, mineralization, and growth in rats. Calcif Tissue Int. 1983;35:455-460.
Kyeyune-Nyombi E, Lau KH, Baylink DJ, Strong DD. 1,25-Dihydroxyvitamin D3 stimulates both alkaline phosphatase gene transcription and mRNA stability in human bone cells. Arch Biochem Biophys. 1991;291(2):316-325.
Irving JT, Wuthier RE. Histochemistry and biochemistry of calcification with special reference to the role of lipids. Clin Orthop. 1968;56:237-260.
Howell DS, Marquez JF, Pita JC. The nature of phospholipids in normal and rachitic costochondral plates. Arthritis Rheum. 1965;8(6):1039-1046.
Dean DD, Boyan BD, Muniz OE, Howell DS, Schwartz Z. Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner. Calcif Tissue Int. 1996;59(2):109-116.
Roughley PJ, Dickson IR. A comparison of proteoglycan from chick cartilage of different types and a study of the effect of vitamin D on proteoglycan structure. Connect Tissue Res. 1986;14(3):187-197.
Plachot JJ, Du Bois MB, Halpern S, Cournot-Witmer G, Garabedian M, Balsan S. In vitro action of 1,25-dihydroxycholecalciferol and 24,25- dihydroxycholecalciferol on matrix organization and mineral distribution in rabbit growth plate. Metab Bone Dis Relat Res. 1982;4(2):135-142.
Boyan BD, Schwartz Z, Carnes DL, Jr., Ramirez V. The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro. Endocrinology. 1988;122(6):2851-2860.
Schwartz Z, Boyan B. The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro. Endocrinology. 1988;122(5):2191-2198.
Swain LD, Schwartz Z, Caulfield K, Brooks BP, Boyan BD. Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation. Bone. 1993;14(4):609-617.
Sylvia VL, Schwartz Z, Schuman L, Morgan RT, Mackey S, Gomez R, Boyan BD. Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures. J Cell Physiol. 1993;157(2):271-278.
Boyan BD, Chen J, Schwartz Z. Mechanism of Pdia3-dependent 1alpha,25-dihydroxy vitamin D3 signaling in musculoskeletal cells. Steroids. 2012;77(10):892-896.
Owen TA, Aronow MS, Barone LM, Bettencourt B, Stein GS, Lian JB. Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. Endocrinology. 1991;128(3):1496-1504.
Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, Collart D, Zambetti G, Stein G. Structure of the rat osteocalcin gene and regulation of vitamin D- dependent expression. Proc Natl Acad Sci U S A. 1989;86(4):1143-1147.
Manolagas SC, Burton DW, Deftos LJ. 1,25-Dihydroxyvitamin D3 stimulates the alkaline phosphatase activity of osteoblast-like cells. The Journal of biological chemistry. 1981;256(14):7115-7117.
Rowe DW, Kream BE. Regulation of collagen synthesis in fetal rat calvaria by 1,25- dihydroxyvitamin D3. The Journal of biological chemistry. 1982;257(14):8009-8015.
Prince CW, Butler WT. 1,25-Dihydroxyvitamin D3 regulates the biosynthesis of osteopontin, a bone-derived cell attachment protein, in clonal osteoblast-like osteosarcoma cells. Coll Relat Res. 1987;7(4):305-313.
Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM. DNA sequences in the rat osteocalcin gene that bind the 1,25- dihydroxyvitamin D3 receptor and confer responsiveness to 1,25- dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1990;87(1):369-373.
Kerner SA, Scott RA, Pike JW. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(12):4455-4459.
Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT. Identification of a DNA sequence responsible for binding of the 1,25- dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression. Proc Natl Acad Sci U S A. 1990;87(24):9995-9999.
Zhang R, Ducy P, Karsenty G. 1,25-dihydroxyvitamin D3 inhibits Osteocalcin expression in mouse through an indirect mechanism. The Journal of biological chemistry. 1997;272(1):110-116.
Wronski TJ, Halloran BP, Bikle DD, Globus RK, Morey-Holton ER. Chronic administration of 1,25-dihydroxyvitamin D3: increased bone but impaired mineralization. Endocrinology. 1986;119(6):2580-2585.
Hock JM, Gunness-Hey M, Poser J, Olson H, Bell NH, Raisz LG. Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats. Calcif Tissue Int. 1986;38(2):79-86.
Suda T, Takahashi N, Abe E. Role of vitamin D in bone resorption. Journal of cellular biochemistry. 1992;49(1):53-58.
Merke J, Klaus G, Hugel U, Waldherr R, Ritz E. No 1,25-dihydroxyvitamin D3 receptors on osteoclasts of calcium- deficient chicken despite demonstrable receptors on circulating monocytes. The Journal of clinical investigation. 1986;77(1):312-314.
Mee AP, Hoyland JA, Braidman IP, Freemont AJ, Davies M, Mawer EB. Demonstration of vitamin D receptor transcripts in actively resorbing osteoclasts in bone sections. Bone. 1996;18(4):295-299.
Rodan GA, Martin TJ. Role of osteoblasts in hormonal control of bone resorption--a hypothesis. Calcif Tissue Int. 1981;33(4):349-351.
Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev. 1999;20(3):345-357.
Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A. 1998;95(7):3597-3602.
Takeda S, Yoshizawa T, Nagai Y, Yamato H, Fukumoto S, Sekine K, Kato S, Matsumoto T, Fujita T. Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice. Endocrinology. 1999;140(2):1005-1008.
Friedman PA, Gesek FA. Cellular calcium transport in renal epithelia: measurement, mechanisms, and regulation. Physiol Rev. 1995;75(3):429-471.
Winaver J, Sylk DB, Robertson JS, Chen TC, Puschett JB. Micropuncture study of the acute renal tubular transport effects of 25-hydroxyvitamin D3 in the dog. Mineral and electrolyte metabolism. 1980;4:178-188.
Tenenhouse HS. Cellular and molecular mechanisms of renal phosphate transport. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1997;12(2):159-164.
Puschett JB, Beck WS, Jr., Jelonek A, Fernandez PC. Study of the renal tubular interactions of thyrocalcitonin, cyclic adenosine 3',5'-monophosphate, 25-hydroxycholecalciferol, and calcium ion. The Journal of clinical investigation. 1974;53(3):756-767.
Puschett JB, Fernandez PC, Boyle IT, Gray RW, Omdahl JL, DeLuca HF. The acute renal tubular effects of 1,25-dihydroxycholecalciferol. Proc Soc Exp Biol Med. 1972;141(1):379-384.
Puschett JB, Moranz J, Kurnick WS. Evidence for a direct action of cholecalciferol and 25- hydroxycholecalciferol on the renal transport of phosphate, sodium, and calcium. The Journal of clinical investigation. 1972;51(2):373-385.
Popovtzer MM, Robinette JB, DeLuca HF, Holick MF. The acute effect of 25-hydroxycholecalciferol on renal handling of phosphorus. Evidence for a parathyroid hormone-dependent mechanism. The Journal of clinical investigation. 1974;53(3):913-921.
Yamamoto M, Kawanobe Y, Takahashi H, Shimazawa E, Kimura S, Ogata E. Vitamin D deficiency and renal calcium transport in the rat. The Journal of clinical investigation. 1984;74(2):507-513.
Kumar R, Schaefer J, Grande JP, Roche PC. Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P- 450, and calbindin D28k in human kidney. Am J Physiol. 1994;266(3 Pt 2):F477-485.
Borke JL, Minami J, Verma AK, Penniston JT, Kumar R. Co-localization of erythrocyte Ca++-Mg++ ATPase and vitamin D-dependent 28-kDa-calcium binding protein. Kidney Int. 1988;34(2):262-267.
Christakos S, Brunette MG, Norman AW. Localization of immunoreactive vitamin D-dependent calcium binding protein in chick nephron. Endocrinology. 1981;109(1):322-324.
Roth J, Thorens B, Hunziker W, Norman AW, Orci L. Vitamin D--dependent calcium binding protein: immunocytochemical localization in chick kidney. Science. 1981;214(4517):197-200.
Bouhtiauy I, Lajeunesse D, Christakos S, Brunette MG. Two vitamin D3-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. I. Effect of CaBP 28K. Kidney Int. 1994;45(2):461-468.
Biber J, Hernando N, Forster I. Phosphate transporters and their function. Annu Rev Physiol. 2013;75:535-550.
Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci U S A. 1985;82(12):4270-4273.
Cantley LK, Russell J, Lettieri D, Sherwood LM. 1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture. Endocrinology. 1985;117(5):2114-2119.
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25- dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1992;89(17):8097-8101.
Russell J, Ashok S, Koszewski NJ. Vitamin D receptor interactions with the rat parathyroid hormone gene: synergistic effects between two negative vitamin D response elements. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1999;14(11):1828-1837.
Nishishita T, Okazaki T, Ishikawa T, Igarashi T, Hata K, Ogata E, Fujita T. A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D. The Journal of biological chemistry. 1998;273(18):10901-10907.
Hawa NS, O'Riordan JL, Farrow SM. Functional analysis of vitamin D response elements in the parathyroid hormone gene and a comparison with the osteocalcin gene. Biochem Biophys Res Commun. 1996;228(2):352-357.
Mackey SL, Heymont JL, Kronenberg HM, Demay MB. Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor. Mol Endocrinol. 1996;10(3):298-305.
Canaff L, Hendy GN. Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. The Journal of biological chemistry. 2002;277(33):30337-30350.
Naveh-Many T, Marx R, Keshet E, Pike JW, Silver J. Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by 1,25- dihydroxyvitamin D3 in the parathyroid in vivo. The Journal of clinical investigation. 1990;86(6):1968-1975.
Russell J, Bar A, Sherwood LM, Hurwitz S. Interaction between calcium and 1,25-dihydroxyvitamin D3 in the regulation of preproparathyroid hormone and vitamin D receptor messenger ribonucleic acid in avian parathyroids. Endocrinology. 1993;132(6):2639-2644.
Dedhar S, Rennie PS, Shago M, Hagesteijn CY, Yang H, Filmus J, Hawley RG, Bruchovsky N, Cheng H, Matusik RJ, et al. Inhibition of nuclear hormone receptor activity by calreticulin. Nature. 1994;367(6462):480-483.
Wheeler DG, Horsford J, Michalak M, White JH, Hendy GN. Calreticulin inhibits vitamin D3 signal transduction. Nucleic Acids Res. 1995;23(16):3268-3274.
Sela A, Silver J, Naveh-Many T. Chronic hypocalcemia increases PTH mRNA despite high 1,25(OH)2D levels:Roles of calretivulin and the vitamin D receptor. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1996;11: (abstract).
Ritter CS, Haughey BH, Armbrecht HJ, Brown AJ. Distribution and regulation of the 25-hydroxyvitamin D3 1alpha-hydroxylase in human parathyroid glands. J Steroid Biochem Mol Biol. 2012;130(1-2):73-80.
Kadowaki S, Norman AW. Demonstration that the vitamin D metabolite 1,25(OH)2-vitamin D3 and not 24R,25(OH)2-vitamin D3 is essential for normal insulin secretion in the perfused rat pancreas. Diabetes. 1985;34(4):315-320.
Lee S, Clark SA, Gill RK, Christakos S. 1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion. Endocrinology. 1994;134(4):1602-1610.
Clark SA, Stumpf WE, Sar M, DeLuca HF, Tanaka Y. Target cells for 1,25 dihydroxyvitamin D3 in the pancreas. Cell and tissue research. 1980;209(3):515-520.
Morrissey RL, Bucci TJ, Richard B, Empson N, Lufkin EG. Calcium-binding protein: its cellular localization in jejunum, kidney and pancreas. Proc Soc Exp Biol Med. 1975;149(1):56-60.
Bland R, Markovic D, Hills CE, Hughes SV, Chan SL, Squires PE, Hewison M. Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in pancreatic islets. The Journal of steroid biochemistry and molecular biology. 2004;89-90(1-5):121-125.
Sooy K, Schermerhorn T, Noda M, Surana M, Rhoten WB, Meyer M, Fleischer N, Sharp GW, Christakos S. Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines. The Journal of biological chemistry. 1999;274(48):34343-34349.
Rabinovitch A, Suarez-Pinzon WL, Sooy K, Strynadka K, Christakos S. Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis. Endocrinology. 2001;142(8):3649-3655.
Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. The Journal of clinical endocrinology and metabolism. 2007;92(6):2017-2029.
Pittas AG, Jorde R, Kawahara T, Dawson-Hughes B. Vitamin D Supplementation for Prevention of Type 2 Diabetes Mellitus: To D or Not to D? The Journal of clinical endocrinology and metabolism. 2020;105(12).
Pittas A, Dawson-Hughes B, Staten M. Vitamin D Supplementation and Prevention of Type 2 Diabetes. Reply. N Engl J Med. 2019;381(18):1785-1786.
Harris SS, Pittas AG, Palermo NJ. A randomized, placebo-controlled trial of vitamin D supplementation to improve glycaemia in overweight and obese African Americans. Diabetes Obes Metab. 2012;14(9):789-794.
Kolek OI, Hines ER, Jones MD, LeSueur LK, Lipko MA, Kiela PR, Collins JF, Haussler MR, Ghishan FK. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. American journal of physiology Gastrointestinal and liver physiology. 2005;289(6):G1036-1042.
Fukumoto S, Yamashita T. FGF23 is a hormone-regulating phosphate metabolism--unique biological characteristics of FGF23. Bone. 2007;40(5):1190-1195.
Eisman JA, Martin TJ, MacIntyre I, Moseley JM. 1,25-dihydroxyvitamin-D-receptor in breast cancer cells. Lancet. 1979;2(8156-8157):1335-1336.
Fleet JC, DeSmet M, Johnson R, Li Y. Vitamin D and cancer: a review of molecular mechanisms. Biochem J. 2012;441(1):61-76.
Ingraham BA, Bragdon B, Nohe A. Molecular basis of the potential of vitamin D to prevent cancer. Current medical research and opinion. 2008;24(1):139-149.
Shah S, Hecht A, Pestell R, Byers SW. Trans-repression of beta-catenin activity by nuclear receptors. The Journal of biological chemistry. 2003;278(48):48137-48145.
Shah S, Islam MN, Dakshanamurthy S, Rizvi I, Rao M, Herrell R, Zinser G, Valrance M, Aranda A, Moras D, Norman A, Welsh J, Byers SW. The molecular basis of vitamin D receptor and beta-catenin crossregulation. Molecular cell. 2006;21(6):799-809.
Dixon KM, Deo SS, Wong G, Slater M, Norman AW, Bishop JE, Posner GH, Ishizuka S, Halliday GM, Reeve VE, Mason RS. Skin cancer prevention: a possible role of 1,25dihydroxyvitamin D3 and its analogs. The Journal of steroid biochemistry and molecular biology. 2005;97(1-2):137-143.
Demetriou SK, Ona-Vu K, Teichert AE, Cleaver JE, Bikle DD, Oh DH. Vitamin D receptor mediates DNA repair and is UV inducible in intact epidermis but not in cultured keratinocytes. The Journal of investigative dermatology. 2012;132(8):2097-2100.
De Haes P, Garmyn M, Degreef H, Vantieghem K, Bouillon R, Segaert S. 1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes. J Cell Biochem. 2003;89(4):663-673.
Gupta R, Dixon KM, Deo SS, Holliday CJ, Slater M, Halliday GM, Reeve VE, Mason RS. Photoprotection by 1,25 dihydroxyvitamin D3 is associated with an increase in p53 and a decrease in nitric oxide products. The Journal of investigative dermatology. 2007;127(3):707-715.
Garland C, Shekelle RB, Barrett-Connor E, Criqui MH, Rossof AH, Paul O. Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men. Lancet. 1985;1(8424):307-309.
Bostick RM, Potter JD, Sellers TA, McKenzie DR, Kushi LH, Folsom AR. Relation of calcium, vitamin D, and dairy food intake to incidence of colon cancer among older women. The Iowa Women's Health Study. Am J Epidemiol. 1993;137(12):1302-1317.
Kearney J, Giovannucci E, Rimm EB, Ascherio A, Stampfer MJ, Colditz GA, Wing A, Kampman E, Willett WC. Calcium, vitamin D, and dairy foods and the occurrence of colon cancer in men. Am J Epidemiol. 1996;143(9):907-917.
Garland FC, Garland CF, Gorham ED, Young JF. Geographic variation in breast cancer mortality in the United States: a hypothesis involving exposure to solar radiation. Prev Med. 1990;19(6):614-622.
Hanchette CL, Schwartz GG. Geographic patterns of prostate cancer mortality. Evidence for a protective effect of ultraviolet radiation. Cancer. 1992;70(12):2861-2869.
Boscoe FP, Schymura MJ. Solar ultraviolet-B exposure and cancer incidence and mortality in the United States, 1993-2002. BMC Cancer. 2006;6:264.
Diercke K, Kohl A, Lux CJ, Erber R. Strain-dependent up-regulation of ephrin-B2 protein in periodontal ligament fibroblasts contributes to osteogenesis during tooth movement. The Journal of biological chemistry. 2011;286(43):37651-37664.
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. The American journal of clinical nutrition. 2007;85(6):1586-1591.
Manson JE, Cook NR, Lee IM, Christen W, Bassuk SS, Mora S, Gibson H, Gordon D, Copeland T, D'Agostino D, Friedenberg G, Ridge C, Bubes V, Giovannucci EL, Willett WC, Buring JE, Group VR. Vitamin D Supplements and Prevention of Cancer and Cardiovascular Disease. N Engl J Med. 2019;380(1):33-44.
Gross C, Stamey T, Hancock S, Feldman D. Treatment of early recurrent prostate cancer with 1,25-dihydroxyvitamin D3 (calcitriol). J Urol. 1998;159(6):2035-2039; discussion 2039-2040.
Beer TM, Ryan CW, Venner PM, Petrylak DP, Chatta GS, Ruether JD, Redfern CH, Fehrenbacher L, Saleh MN, Waterhouse DM, Carducci MA, Vicario D, Dreicer R, Higano CS, Ahmann FR, Chi KN, Henner WD, Arroyo A, Clow FW. Double-blinded randomized study of high-dose calcitriol plus docetaxel compared with placebo plus docetaxel in androgen-independent prostate cancer: a report from the ASCENT Investigators. J Clin Oncol. 2007;25(6):669-674.
Barnett CM, Beer TM. Prostate cancer and vitamin D: what does the evidence really suggest? Urol Clin North Am. 2011;38(3):333-342.
Lehmann B, Tiebel O, Meurer M. Expression of vitamin D3 25-hydroxylase (CYP27) mRNA after induction by vitamin D3 or UVB radiation in keratinocytes of human skin equivalents-- a preliminary study. Arch Dermatol Res. 1999;291(9):507-510.
Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation. The Journal of clinical investigation. 1986;78(2):557-566.
Bikle DD, Pillai S, Gee E, Hincenbergs M. Tumor necrosis factor-alpha regulation of 1,25-dihydroxyvitamin D production by human keratinocytes. Endocrinology. 1991;129(1):33-38.
Bikle DD, Pillai S, Gee E, Hincenbergs M. Regulation of 1,25-dihydroxyvitamin D production in human keratinocytes by interferon-gamma. Endocrinology. 1989;124(2):655-660.
Pillai S, Bikle DD, Elias PM. 1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation. The Journal of biological chemistry. 1988;263(11):5390-5395.
Bikle DD. Vitamin D and the skin: Physiology and pathophysiology. Reviews in endocrine & metabolic disorders. 2012;13(1):3-19.
Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell. 1980;19(1):245-254.
Hennings H, Holbrook KA. Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study. Experimental cell research. 1983;143(1):127-142.
Hennings H, Holbrook KA, Yuspa SH. Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells. Journal of cellular physiology. 1983;116(3):265-281.
Praeger FC, Stanulis-Praeger BM, Gilchrest BA. Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes. J Cell Physiol. 1987;132(1):81-89.
Pillai S, Bikle DD, Mancianti ML, Cline P, Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium. Journal of Cellular Physiology. 1990;143(2):294-302.
Yuspa SH, Kilkenny AE, Steinert PM, Roop DR. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. The Journal of cell biology. 1989;109(3):1207-1217.
Bikle DD, Xie Z, Tu CL. Calcium regulation of keratinocyte differentiation. Expert review of endocrinology & metabolism. 2012;7(4):461-472.
Sheu HM, Kitajima Y, Yaoita H. Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration. Exp Cell Res. 1989;185(1):176-190.
Denning MF, Dlugosz AA, Williams EK, Szallasi Z, Blumberg PM, Yuspa SH. Specific protein kinase C isozymes mediate the induction of keratinocyte differentiation markers by calcium. Cell Growth Differ. 1995;6(2):149-157.
Filvaroff E, Calautti E, McCormick F, Dotto GP. Specific changes of Ras GTPase-activating protein (GAP) and a GAP- associated p62 protein during calcium-induced keratinocyte differentiation. Mol Cell Biol. 1992;12(12):5319-5328.
Filvaroff E, Calautti E, Reiss M, Dotto GP. Functional evidence for an extracellular calcium receptor mechanism triggering tyrosine kinase activation associated with mouse keratinocyte differentiation. The Journal of biological chemistry. 1994;269(34):21735-21740.
Rice RH, Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell. 1979;18(3):681-694.
Hohl D, Lichti U, Breitkreutz D, Steinert PM, Roop DR. Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. The Journal of investigative dermatology. 1991;96(4):414-418.
Simon M, Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell. 1984;36(4):827-834.
Hohl D. Cornified cell envelope. Dermatologica. 1990;180(4):201-211.
Thacher SM, Rice RH. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell. 1985;40(3):685-695.
Hennings H, Steinert P, Buxman MM. Calcium induction of transglutaminase and the formation of epsilon(gamma-glutamyl) lysine cross-links in cultured mouse epidermal cells. Biochemical and biophysical research communications. 1981;102(2):739-745.
Hennings H, Kruszewski FH, Yuspa SH, Tucker RW. Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes. Carcinogenesis. 1989;10(4):777-780.
Gibson DF, Ratnam AV, Bikle DD. Evidence for separate control mechanisms at the message, protein, and enzyme activation levels for transglutaminase during calcium-induced differentiation of normal and transformed human keratinocytes. Journal of Investigative Dermatology. 1996;106(1):154-161.
Su MJ, Bikle DD, Mancianti ML, Pillai S. 1,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium. The Journal of biological chemistry. 1994;269(20):14723-14729.
Menon GK, Grayson S, Elias PM. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. Journal of Investigative Dermatology. 1985;84(6):508-512.
Mauro T, Bench G, Sidderas-Haddad E, Feingold K, Elias P, Cullander C. Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE. Journal of Investigative Dermatology. 1998;111(6):1198-1201.
Lee SH, Elias PM, Feingold KR, Mauro T. A role for ions in barrier recovery after acute perturbation. The Journal of investigative dermatology. 1994;102(6):976-979.
Menon GK, Price LF, Bommannan B, Elias PM, Feingold KR. Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion. The Journal of investigative dermatology. 1994;102(5):789-795.
Lee SH, Elias PM, Proksch E, Menon GK, Mao-Quiang M, Feingold KR. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis. The Journal of clinical investigation. 1992;89(2):530-538.
Menon GK, Elias PM, Lee SH, Feingold KR. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier. Cell and Tissue Research. 1992;270(3):503-512.
Bikle DD, Ratnam A, Mauro T, Harris J, Pillai S. Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor. J Clin Invest. 1996;97(4):1085-1093.
Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca-2+-sensing receptor from bovine parathyroid. Nature. 1993;366(6455):575-580.
Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, Nemeth EF, Fuller F. Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. Journal of Biological Chemistry. 1995;270(21):12919-12925.
Tu CL, Chang W, Bikle DD. The extracellular calcium-sensing receptor Is Required for calcium- induced differentiation in human keratinocytes. The Journal of biological chemistry. 2001;276(44):41079-41085.
Tu C, Chang W, Xie Z, Bikle D. Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes. J Biol Chem. 2008;283:3519-3528.
Tu CL, Crumrine DA, Man MQ, Chang W, Elalieh H, You M, Elias PM, Bikle DD. Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function. The Journal of investigative dermatology. 2012;132(10):2350-2359.
Oda Y, Tu CL, Pillai S, Bikle DD. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation. Journal of Biological Chemistry. 1998;273(36):23344-23352.
Chang W, Tu C, Chen TH, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal. 2008;1(35):ra1.
Jaken S, Yuspa SH. Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells. Carcinogenesis. 1988;9(6):1033-1038.
Punnonen K, Denning M, Lee E, Li L, Rhee SG, Yuspa SH. Keratinocyte differentiation is associated with changes in the expression and regulation of phospholipase C isoenzymes. The Journal of investigative dermatology. 1993;101(5):719-726.
Xie Z, Bikle DD. Phospholipase C-gamma1 is required for calcium-induced keratinocyte differentiation. The Journal of biological chemistry. 1999;274(29):20421-20424.
Xie Z, Bikle DD. The recruitment of phosphatidylinositol 3-kinase to the E-cadherin-catenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and human keratinocyte differentiation. The Journal of biological chemistry. 2007;282(12):8695-8703.
Xie Z, Singleton PA, Bourguignon LY, Bikle DD. Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1. Molecular biology of the cell. 2005;16(7):3236-3246.
Yuspa SH, Ben T, Hennings H, Lichti U. Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1982;42(6):2344-2349.
Dlugosz AA, Yuspa SH. Protein kinase C regulates keratinocyte transglutaminase (TG-K) gene expression in cultured primary mouse epidermal keratinocytes induced to terminally differentiate by calcium. Journal of Investigative Dermatology. 1994;102(4):409-414.
Lu B, Rothnagel JA, Longley MA, Tsai SY, Roop DR. Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element. The Journal of biological chemistry. 1994;269(10):7443-7449.
Ng DC, Shafaee S, Lee D, Bikle DD. Requirement of an AP-1 site in the calcium response region of the involucrin promoter. The Journal of biological chemistry. 2000;275(31):24080-24088.
Bikle DD, Ng D, Oda Y, Hanley K, Feingold K, Xie Z. The vitamin D response element of the involucrin gene mediates its regulation by 1,25-dihydroxyvitamin D3. The Journal of investigative dermatology. 2002;119(5):1109-1113.
Hosomi J, Hosoi J, Abe E, Suda T, Kuroki T. Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3. Endocrinology. 1983;113(6):1950-1957.
Stumpf WE, Sar M, Reid FA, Tanaka Y, DeLuca HF. Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid. Science. 1979;206(4423):1188-1190.
Smith EL, Walworth NC, Holick MF. Effect of 1 alpha,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions. The Journal of investigative dermatology. 1986;86(6):709-714.
Pillai S, Bikle DD. Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: differences in the mode of action of extracellular calcium and 1,25 dihydroxyvitamin D3. Journal of cellular physiology. 1991;146(1):94-100.
McLane JA, Katz M, Abdelkader N. Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions. In Vitro Cell Dev Biol. 1990;26(4):379-387.
McLaughlin JA, Cantley LC, Holick MF. 1,25(OH)2D3 increased calcium and phosphatidylinositol metabolism in differentiating cultured human keratinocytes. J Nutr Biochem. 1990;1:81-87.
Ratnam AV, Bikle DD, Cho JK. 1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes. Journal of Cellular Physiology. 1999;178(2):188-196.
Pillai S, Bikle DD, Su MJ, Ratnam A, Abe J. 1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels. The Journal of clinical investigation. 1995;96(1):602-609.
Xie Z, Bikle DD. Inhibition of 1,25-dihydroxyvitamin-D-induced keratinocyte differentiation by blocking the expression of phospholipase C-gamma1. The Journal of investigative dermatology. 2001;117(5):1250-1254.
Vandenberghe M, Raphael M, Lehen'kyi V, Gordienko D, Hastie R, Oddos T, Rao A, Hogan PG, Skryma R, Prevarskaya N. ORAI1 calcium channel orchestrates skin homeostasis. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(50):E4839-4848.
Tu CL, Chang W, Bikle DD. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A. The Journal of investigative dermatology. 2011;131(5):1119-1128.
Tu CL, Chang W, Bikle DD. Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes. The Journal of investigative dermatology. 2005;124(1):187-197.
Malloy PJ, Pike JW, Feldman D. The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocrine reviews. 1999;20(2):156-188.
Bikle DD, Chang S, Crumrine D, Elalieh H, Man MQ, Choi EH, Dardenne O, Xie Z, Arnaud RS, Feingold K, Elias PM. 25 Hydroxyvitamin D 1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasis. The Journal of investigative dermatology. 2004;122(4):984-992.
Cianferotti L, Cox M, Skorija K, Demay MB. Vitamin D receptor is essential for normal keratinocyte stem cell function. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(22):9428-9433.
Palmer HG, Martinez D, Carmeliet G, Watt FM. The vitamin D receptor is required for mouse hair cycle progression but not for maintenance of the epidermal stem cell compartment. The Journal of investigative dermatology. 2008;128(8):2113-2117.
Plikus MV, Gay DL, Treffeisen E, Wang A, Supapannachart RJ, Cotsarelis G. Epithelial stem cells and implications for wound repair. Seminars in cell & developmental biology. 2012;23(9):946-953.
Mascre G, Dekoninck S, Drogat B, Youssef KK, Brohee S, Sotiropoulou PA, Simons BD, Blanpain C. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature. 2012;489(7415):257-262.
Tian XQ, Chen TC, Holick MF. 1,25-dihydroxyvitamin D3: a novel agent for enhancing wound healing. Journal of cellular biochemistry. 1995;59(1):53-56.
Luderer HF, Nazarian RM, Zhu ED, Demay MB. Ligand-dependent actions of the vitamin D receptor are required for activation of TGF-beta signaling during the inflammatory response to cutaneous injury. Endocrinology. 2013;154(1):16-24.
Song L, Papaioannou G, Zhao H, Luderer HF, Miller C, Dall'Osso C, Nazarian RM, Wagers AJ, Demay MB. The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury. Endocrinology. 2016;157(10):4066-4075.
Oda Y, Tu CL, Menendez A, Nguyen T, Bikle DD. Vitamin D and calcium regulation of epidermal wound healing. The Journal of steroid biochemistry and molecular biology. 2015.
Chen S, Lewallen M, Xie T. Adhesion in the stem cell niche: biological roles and regulation. Development. 2013;140(2):255-265.
Lechler T, Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature. 2005;437(7056):275-280.
Li L, Hartley R, Reiss B, Sun Y, Pu J, Wu D, Lin F, Hoang T, Yamada S, Jiang J, Zhao M. E-cadherin plays an essential role in collective directional migration of large epithelial sheets. Cellular and molecular life sciences : CMLS. 2012;69(16):2779-2789.
Bikle DD, Ng D, Tu CL, Oda Y, Xie Z. Calcium- and vitamin D-regulated keratinocyte differentiation. Mol Cell Endocrinol. 2001;177(1-2):161-171.
van Etten E, Mathieu C. Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts. The Journal of steroid biochemistry and molecular biology. 2005;97(1-2):93-101.
Daniel C, Sartory NA, Zahn N, Radeke HH, Stein JM. Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. The Journal of pharmacology and experimental therapeutics. 2008;324(1):23-33.
Gregori S, Casorati M, Amuchastegui S, Smiroldo S, Davalli AM, Adorini L. Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. Journal of immunology. 2001;167(4):1945-1953.
Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133(5):775-787.
Sigmundsdottir H, Pan J, Debes GF, Alt C, Habtezion A, Soler D, Butcher EC. DCs metabolize sunlight-induced vitamin D3 to 'program' T cell attraction to the epidermal chemokine CCL27. Nat Immunol. 2007;8(3):285-293.
Chen S, Sims GP, Chen XX, Gu YY, Chen S, Lipsky PE. Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation. Journal of immunology. 2007;179(3):1634-1647.
Bouillon R, Carmeliet G, Verlinden L, van Etten E, Verstuyf A, Luderer HF, Lieben L, Mathieu C, Demay M. Vitamin D and human health: lessons from vitamin D receptor null mice. Endocrine reviews. 2008;29(6):726-776.
Adorini L, Penna G. Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol. 2008;4(8):404-412.
Adamopoulos IE, Bowman EP. Immune regulation of bone loss by Th17 cells. Arthritis Res Ther. 2008;10(5):225.
Limketkai BN, Mullin GE, Limsui D, Parian AM. Role of Vitamin D in Inflammatory Bowel Disease. Nutr Clin Pract. 2017;32(3):337-345.
Mathieu C, Van Etten E, Gysemans C, Decallonne B, Kato S, Laureys J, Depovere J, Valckx D, Verstuyf A, Bouillon R. In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2001;16(11):2057-2065.
O'Kelly J, Hisatake J, Hisatake Y, Bishop J, Norman A, Koeffler HP. Normal myelopoiesis but abnormal T lymphocyte responses in vitamin D receptor knockout mice. The Journal of clinical investigation. 2002;109(8):1091-1099.
Josien R, Li HL, Ingulli E, Sarma S, Wong BR, Vologodskaia M, Steinman RM, Choi Y. TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo. The Journal of experimental medicine. 2000;191(3):495-502.
Baroni E, Biffi M, Benigni F, Monno A, Carlucci D, Carmeliet G, Bouillon R, D'Ambrosio D. VDR-dependent regulation of mast cell maturation mediated by 1,25-dihydroxyvitamin D3. J Leukoc Biol. 2007;81(1):250-262.
Froicu M, Weaver V, Wynn TA, McDowell MA, Welsh JE, Cantorna MT. A crucial role for the vitamin D receptor in experimental inflammatory bowel diseases. Molecular endocrinology (Baltimore, Md). 2003;17(12):2386-2392.
Gysemans C, van Etten E, Overbergh L, Giulietti A, Eelen G, Waer M, Verstuyf A, Bouillon R, Mathieu C. Unaltered diabetes presentation in NOD mice lacking the vitamin D receptor. Diabetes. 2008;57(1):269-275.
Topilski I, Flaishon L, Naveh Y, Harmelin A, Levo Y, Shachar I. The anti-inflammatory effects of 1,25-dihydroxyvitamin D3 on Th2 cells in vivo are due in part to the control of integrin-mediated T lymphocyte homing. European journal of immunology. 2004;34(4):1068-1076.
Wittke A, Weaver V, Mahon BD, August A, Cantorna MT. Vitamin D receptor-deficient mice fail to develop experimental allergic asthma. Journal of immunology. 2004;173(5):3432-3436.
Wittke A, Chang A, Froicu M, Harandi OF, Weaver V, August A, Paulson RF, Cantorna MT. Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation. Archives of biochemistry and biophysics. 2007;460(2):306-313.
Adorini L. Intervention in autoimmunity: the potential of vitamin D receptor agonists. Cellular immunology. 2005;233(2):115-124.
Ehrchen J, Helming L, Varga G, Pasche B, Loser K, Gunzer M, Sunderkotter C, Sorg C, Roth J, Lengeling A. Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2007;21(12):3208-3218.
Rajapakse R, Mousli M, Pfaff AW, Uring-Lambert B, Marcellin L, Bronner C, Jeanblanc M, Villard O, Letscher-Bru V, Klein JP, Candolfi E. 1,25-Dihydroxyvitamin D3 induces splenocyte apoptosis and enhances BALB/c mice sensitivity to toxoplasmosis. The Journal of steroid biochemistry and molecular biology. 2005;96(2):179-185.
Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, Gilliet M, Ho S, Antonenko S, Lauerma A, Smith K, Gorman D, Zurawski S, Abrams J, Menon S, McClanahan T, de Waal-Malefyt Rd R, Bazan F, Kastelein RA, Liu YJ. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol. 2002;3(7):673-680.
Liu PT, Krutzik SR, Modlin RL. Therapeutic implications of the TLR and VDR partnership. Trends Mol Med. 2007;13(3):117-124.
Schauber J, Gallo RL. The vitamin D pathway: a new target for control of the skin's immune response? Experimental dermatology. 2008;17(8):633-639.
Gombart AF, Borregaard N, Koeffler HP. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2005;19(9):1067-1077.
Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, Tavera-Mendoza L, Lin R, Hanrahan JW, Mader S, White JH. Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. Journal of immunology. 2004;173(5):2909-2912.
Schauber J, Dorschner RA, Coda AB, Buchau AS, Liu PT, Kiken D, Helfrich YR, Kang S, Elalieh HZ, Steinmeyer A, Zugel U, Bikle DD, Modlin RL, Gallo RL. Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism. The Journal of clinical investigation. 2007;117(3):803-811.
Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006;311(5768):1770-1773.
Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL, Leung DY. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. The New England journal of medicine. 2002;347(15):1151-1160.
Howell MD, Gallo RL, Boguniewicz M, Jones JF, Wong C, Streib JE, Leung DY. Cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus. Immunity. 2006;24(3):341-348.
Bilezikian JP, Bikle D, Hewison M, Lazaretti-Castro M, Formenti AM, Gupta A, Madhavan MV, Nair N, Babalyan V, Hutchings N, Napoli N, Accili D, Binkley N, Landry DW, Giustina A. MECHANISMS IN ENDOCRINOLOGY: Vitamin D and COVID-19. Eur J Endocrinol. 2020;183(5):R133-R147.
Nogues X, Ovejero D, Pineda-Moncusi M, Bouillon R, Arenas D, Pascual J, Ribes A, Guerri-Fernandez R, Villar-Garcia J, Rial A, Gimenez-Argente C, Cos ML, Rodriguez-Morera J, Campodarve I, Quesada-Gomez JM, Garcia-Giralt N. Calcifediol Treatment and COVID-19-Related Outcomes. The Journal of clinical endocrinology and metabolism. 2021;106(10):e4017-e4027.
Ustianowski A, Shaffer R, Collin S, Wilkinson RJ, Davidson RN. Prevalence and associations of vitamin D deficiency in foreign-born persons with tuberculosis in London. J Infect. 2005;50(5):432-437.
Rook GA, Steele J, Fraher L, Barker S, Karmali R, O'Riordan J, Stanford J. Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology. 1986;57(1):159-163.
Liu PT, Stenger S, Tang DH, Modlin RL. Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin. Journal of immunology. 2007;179(4):2060-2063.
Sly LM, Lopez M, Nauseef WM, Reiner NE. 1alpha,25-Dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase. The Journal of biological chemistry. 2001;276(38):35482-35493.
Brightbill HD, Libraty DH, Krutzik SR, Yang RB, Belisle JT, Bleharski JR, Maitland M, Norgard MV, Plevy SE, Smale ST, Brennan PJ, Bloom BR, Godowski PJ, Modlin RL. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science. 1999;285(5428):732-736.
Salahuddin N, Ali F, Hasan Z, Rao N, Aqeel M, Mahmood F. Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebo-controlled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis'. BMC Infect Dis. 2013;13:22.
Martineau AR, Timms PM, Bothamley GH, Hanifa Y, Islam K, Claxton AP, Packe GE, Moore-Gillon JC, Darmalingam M, Davidson RN, Milburn HJ, Baker LV, Barker RD, Woodward NJ, Venton TR, Barnes KE, Mullett CJ, Coussens AK, Rutterford CM, Mein CA, Davies GR, Wilkinson RJ, Nikolayevskyy V, Drobniewski FA, Eldridge SM, Griffiths CJ. High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lancet. 2011;377(9761):242-250.
Schauber J, Oda Y, Buchau AS, Steinmeyer A, Zugel U, Bikle DD, Gallo RL. Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3. The Journal of investigative dermatology. 2008;128(4):816-824.
Norman AW, Roth J, Orci L. The vitamin D endocrine system: steroid metabolism, hormone receptors, and biological response (calcium binding proteins). Endocr Rev. 1982;3(4):331-366.
Weishaar RE, Kim SN, Saunders DE, Simpson RU. Involvement of vitamin D3 with cardiovascular function. III. Effects on physical and morphological properties. Am J Physiol. 1990;258(1 Pt 1):E134-142.
Walters MR, Ilenchuk TT, Claycomb WC. 1,25-Dihydroxyvitamin D3 stimulates 45Ca2+ uptake by cultured adult rat ventricular cardiac muscle cells. The Journal of biological chemistry. 1987;262(6):2536-2541.
Selles J, Boland R. Rapid stimulation of calcium uptake and protein phosphorylation in isolated cardiac muscle by 1,25-dihydroxyvitamin D3. Mol Cell Endocrinol. 1991;77(1-3):67-73.
Wu J, Garami M, Cao L, Li Q, Gardner DG. 1,25(OH)2D3 suppresses expression and secretion of atrial natriuretic peptide from cardiac myocytes. Am J Physiol. 1995;268(6 Pt 1):E1108-1113.
Gardner DG, Chen S, Glenn DJ. Vitamin D and the heart. Am J Physiol Regul Integr Comp Physiol. 2013;305(9):R969-977.
Boland R. Role of vitamin D in skeletal muscle function. Endocr Rev. 1986;7(4):434-448.
Girgis CM, Clifton-Bligh RJ, Hamrick MW, Holick MF, Gunton JE. The roles of vitamin D in skeletal muscle: form, function, and metabolism. Endocrine reviews. 2013;34(1):33-83.
Boland RL. VDR activation of intracellular signaling pathways in skeletal muscle. Mol Cell Endocrinol. 2011;347(1-2):11-16.
Gelbard HA, Stern PH, U'Prichard DC. 1 alpha, 25-Dihydroxyvitamin D3 nuclear receptors in pituitary. Science. 1980;209(4462):1247-1249.
Haug E, Gautvik KM. Demonstration and characterization of a 1 alpha,25-(OH)2D3 receptor- like macromolecule in cultured rat pituitary cells. J Steroid Biochem. 1985;23(5A):625-635.
Tornquist K. Pretreatment with 1,25-dihydroxycholecalciferol enhances thyrotropin- releasing hormone- and inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ in permeabilized GH4C1 pituitary cells. Endocrinology. 1992;131(4):1677-1681.
Tornquist K, Lamberg-Allardt C. The effect of 1,25-dihydroxy-cholecalciferol on the TRH induced TSH release in rats. Acta Endocrinol (Copenh). 1987;114(1):55-59.
Narbaitz R, Sar M, Stumpf WE, Huang S, DeLuca HF. 1,25-Dihydroxyvitamin D3 target cells in rat mammary gland. Horm Res. 1981;15(4):263-269.
Lopes N, Paredes J, Costa JL, Ylstra B, Schmitt F. Vitamin D and the mammary gland: a review on its role in normal development and breast cancer. Breast Cancer Res. 2012;14(3):211.
Eisman JA, Suva LJ, Martin TJ. Significance of 1,25-dihydroxyvitamin D3 receptor in primary breast cancers. Cancer Res. 1986;46(10):5406-5408.
Eisman JA, Sutherland RL, McMenemy ML, Fragonas JC, Musgrove EA, Pang GY. Effects of 1,25-dihydroxyvitamin D3 on cell-cycle kinetics of T 47D human breast cancer cells. J Cell Physiol. 1989;138(3):611-616.
Colston KW, Berger U, Coombes RC. Possible role for vitamin D in controlling breast cancer cell proliferation. Lancet. 1989;1(8631):188-191.
Duncan WE, Whitehead D, Wray HL. A 1,25-dihydroxyvitamin D3 receptor-like protein in mammalian and avian liver nuclei. Endocrinology. 1988;122(6):2584-2589.
Cao Y, Shu XB, Yao Z, Ji G, Zhang L. Is vitamin D receptor a druggable target for non-alcoholic steatohepatitis? World J Gastroenterol. 2020;26(38):5812-5821.
Rixon RH, MacManus JP, Whitfield JF. The control of liver regeneration by calcitonin, parathyroid hormone and 1 alpha,25-dihydroxycholecalciferol. Mol Cell Endocrinol. 1979;15(2):79-89.
Nguyen TM, Guillozo H, Marin L, Dufour ME, Tordet C, Pike JW, Garabedian M. 1,25-dihydroxyvitamin D3 receptors in rat lung during the perinatal period: regulation and immunohistochemical localization. Endocrinology. 1990;127(4):1755-1762.
Gaultier C, Harf A, Balmain N, Cuisinier-Gleizes P, Mathieu H. Lung mechanics in rachitic rats. Am Rev Respir Dis. 1984;130(6):1108-1110.
Herscovitch K, Dauletbaev N, Lands LC. Vitamin D as an anti-microbial and anti-inflammatory therapy for Cystic Fibrosis. Paediatr Respir Rev. 2014;15(2):154-162.

Diabetes Mellitus in People with Cancer

ABSTRACT

There is increasing evidence of an association between cancer and diabetes mellitus. Patients with type II diabetes are at increased risk of malignancy due to shared risk factors between the two conditions, and people with a diagnosis of cancer may develop new onset diabetes or impaired glycemic control, partly as a result of the systemic anti-cancer treatments (SACT) they receive. Many newer targeted anti-cancer treatments can have off-target metabolic toxicities not seen with conventional chemotherapy agents. Early recognition of diabetes or hyperglycemia in people with cancer can improve outcomes. This chapter aims to summarize these associations, provide an overview of how different SACT modalities can impact on glycemic control, and highlight key recommendations for the management of this complex patient group.

INTRODUCTION

Diabetes mellitus (DM) is a rising global public health emergency, with recent estimates suggesting that over 780 million people globally will be affected by 2045 (1). DM is typically classified into broad categories including type 1 (T1DM), type 2 (T2DM), gestational, monogenic, pharmacologically-induced, endocrinopathy-driven and DM due to pancreatic disease/deficiency (sometimes referred to as type 3c) (2, 3). T2DM is regarded as the most common subtype and is reported to account for over 85% of cases (1). All types of DM can lead to multisystem microvascular (nephropathy, retinopathy, neuropathy) and macrovascular (ischemic heart disease, stroke and peripheral vascular disease) complications, with management of these complications placing a strain upon many health services.

People with a diagnosis of DM are also at higher risk for developing several cancers (4), with reasons for this in part due to shared risk factors between the two, including age, obesity, sedentary lifestyle, and diet (5,6). A recent umbrella review of meta-analyses found that risks of developing most cancers were higher in people with DM compared to those without, with the most convincing evidence seen in breast cancer, intrahepatic cholangiocarcinoma, colorectal cancer, and endometrial cancer. One exception in this study was prostate cancer, where the risk appeared lower in individuals with DM (4). In view of this increased cancer risk in people with DM, some groups even advocate that regular screening for underlying cancer should be part of routine DM assessments (7).

It is estimated that approximately 20% of people with cancer have concurrent diabetes (8). Individuals with cancer are also at an increased risk of developing new onset DM or hyperglycemia, independent of an underlying diagnosis of diabetes, whilst cancer patients with concurrent DM often experience worsening glycemic control (9). Reasons for poor glycemic control in these individuals include complications from systemic anticancer treatments (SACT) along with supportive medications to treat treatment side effects, and symptoms of the underlying malignancy. This chapter aims to summarize the complex relationship between malignancy and DM, particularly the effects of SACT on glycemic control and risk of DM, as well as outlining management guidelines for DM in people with cancer.

DIABETES/HYPERGLYCEMIA AND CANCER OUTCOMES

A number of observational studies have demonstrated that hyperglycemia is associated with poorer overall survival (OS) and increased risk of disease recurrence in a number of malignancies, solid and hematological (10-17), with a number of individual studies, and larger meta-analyses supporting this. One meta-analysis reviewed 12 studies comprising 9,872 people with a diagnosis of cancer without known diabetes. Individuals with hyperglycemia were found to have significantly worse disease-free survival (DFS) (hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.20-3.27) compared to those without, as well as worse OS (HR 2.05, 95% CI 1.67-2.551) (18). A further meta-analysis of 4,241 patients with pancreatic cancer suggested that those individuals with concurrent DM (1,034) have poorer OS (HR 1.16, 95% CI 1.08-1.25) and a higher risk of on-treatment death than those without concurrent DM (19). Furthermore, in a meta-analysis of 8 studies in breast cancer, concurrent DM was found to confer a greater risk of death, and a later stage at presentation, as well as impact on the treatment given (20). People with DM also have a higher prevalence of oral cancers, as well as a higher mortality from these cancers (21).

In addition to this, a number of preclinical studies have suggested that hyperglycemia may specifically attenuate the efficacy of chemotherapy in people with cancer with or without diabetes, which could in part account for these observations (22). For example, hyperglycemia may attenuate chemotherapy-induced reactive oxygen species (ROS) production, which in-turn can diminish the efficacy of treatment (23). In vivo, there are some small series that have demonstrated an association between hyperglycemia and resistance to chemotherapy. A clinical study of 88 people with estrogen-receptor positive breast cancer demonstrated impaired glucose tolerance significantly correlated with disease progression in those patients receiving chemotherapy (24). Furthermore, high blood glucose levels irrespective of an underlying DM diagnosis, were shown to significantly enhance oxaliplatin resistance in individuals with stage III colorectal cancer receiving adjuvant chemotherapy (22). Studies such as these highlight the importance of adequate glycemic control during treatment for cancer to potentially improve outcomes, although these data are mainly from observational studies, with interventional studies lacking.

EFFECT OF DIABETES OR HYPERGLYCEMIA ON QUALITY OF LIFE IN PEOPLE WITH CANCER

Cancer-related symptoms and SACT side effects, such as fatigue, nausea, anorexia and pain can be debilitating to patients. When confounded by symptoms of hyperglycemia, the impact upon an individuals’ quality of life can be significant (25). Furthermore, the impact of a cancer diagnosis, as well as treatment and cancer-related symptoms can have major negative impacts on diabetes self-care (26), with data suggesting that adherence to glucose lowering drugs often decreases in individuals following a cancer diagnosis (27). A cancer diagnosis can also have financial and social impacts upon individuals, affecting access to healthy food and outpatient diabetes services, resulting in lower quality of life and a higher symptom burden (28). A systematic review of 10 studies, demonstrated poorer patient reported outcomes (PROs) in those diagnosed with both cancer and DM compared to having either one of these diseases alone (29).

People with DM are known to be at higher risk from infections, and undergoing SACT can exacerbate this, resulting in higher rates of infection and hospitalization observed in those with cancer and DM (30, 31). This in turn leads to higher rates of chemotherapy dose reductions and early treatment cessation (28, 32-34). A meta-analysis of 10 observational studies involving 8,688 cases found that the likelihood of developing chemotherapy-induced neutropenia was higher amongst individuals with DM/hyperglycemia than those without (odds ratio (OR) 1.32, 95% CI 1.06-1.64) (31). Chemotherapy-induced neutropenia poses a significant risk for infection and hospitalization in all people with cancer, with an associated rate of morbidity and mortality which is higher in those with raised blood glucose levels (30). In addition to severe hematological toxicity, more severe rates of non-hematological toxicity have also been associated with hyperglycemia during chemotherapy in people with prostate cancer and lymphoma (35). A single-center retrospective analysis found that individuals with cancer and DM who had good glycemic control had no increased risk of treatment-related complications compared with individuals without DM (36), suggesting that optimal glycemic control during SACT could improve tolerability, thereby reducing rates of admission and dose-limiting toxicity.

Conceivably, people with DM may be more prone to neuro- and nephrotoxic agents due to their underlying predisposition conferred by the DM. Indeed, a previous report suggested that taxane-based chemotherapy regimens resulted in a significantly higher rates of peripheral neuropathy in those with DM compared to those without (74.4% vs. 58.5%) (37). There are no convincing data to suggest that a concurrent cancer diagnosis accelerates the risk of diabetic nephropathy or retinopathy.

EFFECTS OF SYSTEMIC ANTICANCER THERAPIES ON GLYCEMIA

Systemic anti-cancer therapies (SACT) encompass a wide range of treatments including cytotoxic chemotherapy, hormone therapy, targeted therapy, and immunotherapy, many of which can impact upon glycemic control directly or as a result of toxicity management or supportive medications which are given alongside treatment. Several anti-cancer agents have been demonstrated to increase the risk of hyperglycemia as summarized in Table 1, and many can do this even in those without a known diagnosis of DM. People receiving SACT are also at risk of developing a new diagnosis of diabetes. One study demonstrated that 11% of people (15/134) undergoing routine chemotherapy met the criteria for a new diagnosis of diabetes (using the diagnostic criteria as per guidelines from the UK National Institute for Clinical and Healthcare Excellence (NICE) and without a previous known diagnosis) based upon HbA_1cmeasurements). The majority of these individuals (73%) had been receiving short course steroids with chemotherapy, and 40% were being treated in the curative/adjuvant setting (38). A second prospective cohort study in 90 people taking glucocorticoids as part of therapy protocols for primary brain tumor or metastases, lymphoma, or for bone marrow transplant, found non-DM range hyperglycemia in 58% and DM-range hyperglycemia in 18.9% (39). These individuals with hyperglycemia are also more likely to present with an emergency admission during cancer therapy than those with normoglycemia (40).

Table 1. SACT used in the Treatment of Cancer Demonstrated to be Associated with Worsening Glycemic Control
Type of SACT	Drug Examples	Risk of Diabetes/Hyperglycemia (Range of any grade)	Type of diabetes most likely to develop
Targeted therapy
mTOR inhibitors	Everolimus (41, 42)	12-50%	T2DM
mTOR inhibitors	Temsirolimus (42)	26%	T2DM
PI3K inhibitors	Alpelisib (43)	37%	T2DM
PI3K inhibitors	Idelialisib (44)	28/30%	T2DM
EGFR inhibitor	Osimertinib (45)	2%	T2DM
EGFR inhibitor	Panitumumab (46, 47)	1-10%	T2DM
Multikinase inhibitor	Sunitinib (48-50)	0-8% Risk of hypoglycemia	Reverses T1/T2DM, but also causes hyperglycemia
Multikinase inhibitor	Pazopanib (50)	0-8% Risk of hypoglycemia	Reverses T1/T2DM, but also causes hyperglycemia
Tyrosine kinase inhibitor (TKI)	Nilotinib (51)	6%	T2DM
Tyrosine kinase inhibitor (TKI)	Ponatinib (52)	3%	T2DM
ALK Inhibitor	Ceritinib (53)	49%	T2DM
FLT3 inhibitor	Midostaurin (54, 55)	7-20%	T2DM
FLT3 inhibitor	Gilteritinib (56)	13%	T2DM
Monoclonal antibody	Gemtuzumab (anti-CD33) *inpatient use (57)	10%	T2DM
Somatostatin Analogues	Octreotide, Lanreotide (58)	Up to 30%	T2DM, but risk of hypoglycemia
Chemotherapy
Anti-metabolite	5-fluorouracil (59, 60)	Up to 10%	T2DM
	Pemetrexed (61, 62)	4%
	Decitadine/Azacitidine (63)	6-33%
Alkylating agents	Busulfan (64)	66-67%
Platinum based	Oxaliplatin (65, 66)	4%
Anthracyclines	Doxorubicin (60, 67)	Up to 10%
Other	Arsenic trioxide (ATO) (68)	45%
Immune Checkpoint Inhibitors
PD-1	Nivolumab (69)	<1%	T1DM
PD-1	Pembrolizumab (70)	1-2.2%
CTLA-4	Ipilumumab (69)	<1%
	Combination ICP (71)	4%
Hormone Therapy
Hormone Treatment	ADT (44, 72)	Risk ratio 1.39 (95% CI 1.27-1.53) n=65,595 cases	T2DM
Hormone Treatment	Tamoxifen (73)	Diabetes risk adj. odds ratio 1.24 (95% CI 1.08-1.42)	T2DM

Abbreviations: ADT = androgen deprivation therapy; ALK – anaplastic lymphoma kinase; ATO – arsenic trioxide; CTLA-4 – cytotoxic T-lymphocyte protein-4; EGFR – epidermal growth factor receptor; FLT3 – FMS-like tyrosine kinase-3; ICP – immune checkpoint inhibitor; TKI – tyrosine kinase inhibitor; mTOR – mechanistic target of rapamycin; PI3K – phosphoinositide-3 kinase; PD-1 – programmed cell death protein-1; T1DM – type 1 diabetes mellitus; T2DM – type 2 diabetes mellitus

Cytotoxic Chemotherapy

Hyperglycemia occurs in between 10 and 30% of people undergoing cytotoxic chemotherapy for malignancy (74), and although often transient during treatment, can persist, or even lead to DM in some people. Poor glycemic control can increase the risk of infections and hospitalization (28, 34), as previously discussed, leading to treatment interruptions and dose reductions, as well as significant morbidity, and even mortality (33). A number of cytotoxic chemotherapy regimens are reported to cause hyperglycemia in people without diabetes, including commonly used drugs such as 5-fluorouracil (5-FU), platinum-based drugs (oxaliplatin, carboplatin, cisplatin) and anthracyclines (doxorubicin, epirubicin) (75). In one cohort study of 422 people receiving 5FU-based chemotherapy regimens for the treatment of early or advanced colorectal cancer, 11.6% (42 people) developed diabetes and a further 11.3% developed impaired fasting blood glucose (FBG) levels. Of the 42 people who developed diabetes, 7 required no treatment, 13 received diet control and physiotherapy only, and 22 received antidiabetic medication (75). In a second cohort of 185 people with head and neck cancer treated with platinum-containing regimens, 3.8% developed type 2 DM, with 3 presenting with hyperglycemic crises (DKA, HHS) (65). One possible contributing factor for developing impaired FBG levels and/or type 2 DM is the concurrent use of corticosteroids in highly emetogenic chemotherapy regimens, but an analysis of type 2 DM following anthracycline use in 3,147 lymphoma patients suggested that the use of these drugs independently increases the risk of T2DM, when data was adjusted for corticosteroid use, comorbidities, age, and gender. A threshold doxorubicin dose of 253mg was identified, below which there was no increased risk of developing T2DM (76). Risk of diabetes from cytotoxic chemotherapy may also increase with age, with one pediatric study suggesting that the risk was higher in acute lymphoblastic leukemia (ALL) patients aged > 10, compared with those < 10 years old (77).

Exact mechanisms of how and why some cytotoxic chemotherapies can lead to hyperglycemia or T2DM remain unclear. Proposed mechanisms include the induction of an inflammatory state which predisposes to hyperglycemia (78) or direct metabolic effects on tissues vital to glucose homeostasis such as skeletal muscles (79).

Oral Targeted Anticancer Agents

Many new targeted cancer therapies inhibit various points in the insulin receptor signaling pathway including the commonly used class of tyrosine kinase inhibitors (TKIs) (80). Reported effects of targeted TKIs on blood glucose metabolism range from the development of metabolic syndrome and diabetes via the blocking of insulin signaling (80), as well as erratic glycemic control and even hypoglycemia in those with pre-existing type 1 or type 2 DM49, (81, 82). In contrast some TKIs may improve glycemic control suggesting that management of these individuals needs to be individualized with no one-size-fits-all management algorithm. Reversibility of these effects is also unclear, with reported improvements in glycemic control and HbA1c levels following dose reductions or treatment termination (83).

Inhibitors of mTOR (everolimus, temsirolimus or ridaforolimus) have also been shown to impact glycemic control since mTOR is a protein kinase that plays a key role in regulating cell growth as well as lipid and glucose metabolism (84, 85). Meta-analyses looking into these effects have demonstrated significantly higher rates of hyperglycemia, hypercholesterolemia, and hypertriglyceridemia compared with controls (86, 87) In isolated cases, the effects have been severe enough to precipitate DKA (88). To date, studies have not demonstrated either positive or negative associations between treatment response rates and incidence of metabolic complications (89).

As novel targeted agents continue to be introduced to manage a range of cancers, it is expected that metabolic toxicities continue to be reported given the homeostatic function of many of these druggable targets. Whilst some of these agents will provide meaningful benefit in terms of survival for people with advanced cancers, such as the PI3Kainhibitor alpelisib for PI3KA-mutated metastatic breast cancer (43), glycemic control needs to be at the forefront of the prescriber’s mind at initiation, to ensure adequate management of toxicities.

Hormone Therapy

ANDROGEN DEPRIVATION THERAPY

Androgen deprivation therapy (ADT) is recognized as a risk factor for development of diabetes, metabolic syndrome, and cardiovascular disease (72, 90, 91). In a large observational study of over 35,000 men treated for prostate cancer, ADT in the form of gonadotropin-releasing hormone (GnRH) agonists, oral antiandrogens, a combination of the two, or orchiectomy was associated with a significantly increased risk of diabetes, coronary heart disease, myocardial infarction, and sudden cardiac death (90). These findings are supported by other studies, including a meta-analysis of over 150,000 men with prostate cancer receiving ADT (72), with association observed with all forms of ADT, with the weakest association with anti-androgen therapy alone.

ESTROGEN TARGETED THERAPY

Studies examining the effect of estrogen-targeted therapies on the development of diabetes in women with breast cancer are less clear cut. Whilst one retrospective cohort analysis failed to demonstrate a link between tamoxifen use and the development of DM (92), two large population-based studies demonstrated a significant association between tamoxifen use and the development of diabetes in women diagnosed with breast cancer (73, 93); The first of these studies included almost 15,000 Canadian women aged 65 years or older diagnosed with early breast cancer, whilst the second included over 22,000 women in Taiwan aged 20 years and over. Whilst tamoxifen appears to increase the risk of developing DM, aromatase inhibitor therapy does not, with no link found in any of these three studies.

Immune Checkpoint Inhibitors

Immune checkpoint inhibitors (ICPi), including cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PDL-1) inhibitors are a sub-class of monoclonal antibody treatments that have revolutionized cancer treatment over the last decade. First approved for use in the treatment of melanoma, ICPi are now recognized as providing a survival benefit across a number of cancers, and are increasingly used in early-stage cancers in the adjuvant setting and also in combination with chemotherapy (94). Whilst clinically effective, ICPi can lead to a spectrum of immune-related adverse events (IRAEs). Endocrine IRAEs include hypophysitis, thyroiditis, adrenalitis and de novo diabetes. The risk of developing de novo diabetes is low, occurring in 0.2-4% of ICPi treated individuals depending on the immunotherapy given (69). The immune checkpoint PD-1 and its ligand PD-L1 have been shown to have an important immune homeostatic function in the pancreas by promoting beta cell maturation and preventing immune-mediated beta cell destruction (95). To date, there is no convincing evidence for a physiological role for CTLA-4 within the pancreas. PD-1 inhibitors, PD-L1 inhibitors, and combination CTLA-4/PD-1 therapy have been demonstrated to precipitate diabetes more commonly than CTLA-4 inhibitors alone. The underlying clinical presentation is akin to type 1 diabetes (70) and believed to be precipitated by inappropriate activation of self-reactive T-cells and destruction of insulin-producing pancreatic islet β-cells. ICP-induced insulin deficiency may lead to new-onset insulin-dependent diabetes or worsening pre-existing type 2 diabetes. Up to 75% of people who develop ICP-induced hyperglycemia/diabetes present with diabetic ketoacidosis (DKA) (96-98). Presentations are frequently acute with a precipitous increase in blood glucose (99). Therefore ICP-induced diabetes can be discriminated from ‘standard’ type 1 diabetes mellitus, by its tendency towards a faster onset, apparently fulminant course, and high degree of antibody negativity (99). The nomenclature of the condition in the published literature varies mainly between ‘type 1 like’ to ‘fulminant’ with there being differences between the presentation of ICPi-induced diabetes and type 1 and fulminant diabetes. Kyriacou and colleagues compared the characteristics of 75 published cases and concluded that there is some overlap with type 1 DM and fulminant DM. However, this was felt to be insufficient overlap for ICPi diabetes to be wholly classified as either type 1 like or fulminant (100). Nevertheless, the recognition that these agents can precipitate rapid beta cell destruction which results in an unusually high number of emergency presentations is key. Treatment of non-endocrine IRAEs is typically with high dose steroids, often for prolonged periods of time. At present, steroids are used in up to a third of people receiving ICPs, further increasing the risk of hyperglycemia, and steroid induced T2DM.

An analysis of the World Health Organizations (WHO) pharmacovigilance database over a 4-year period detected 283 cases of ICP-induced diabetes mellitus, 50.2% of which presented with DKA, and 6% of whom were on concurrent steroids at diagnosis (101). There was a wide variability in duration of ICP treatment, and timing of DM onset, occurring even up to 8 months after cessation of ICP treatment. A systematic review of 90 cases, demonstrated a diagnosis of DM on average after 4.5 cycles of ICP (102). C-peptide levels were usually low or undetectable at diagnosis, islet autoantibodies were positive in 53%, with a predominance of glutamic acid decarboxylase antibodies, and susceptible HLA genotypes present in 65% (102). HbA1c levels were relatively low, consistent with the observed rapid onset of beta cell inflammation. Importantly, an elegant albeit small single-center study, used radiological and biochemical phenotyping to demonstrate that ICPi DM is irreversible (103). This has important clinical implications such that any individual diagnosed with ICPi-induced DM should be counselled around an expected life-long requirement of insulin.

Glucocorticoid (Steroid) Treatment

Glucocorticoids (GC) increase insulin resistance and glucose production and inhibit the production and secretion of insulin by pancreatic beta cells, as well as acting centrally to counteract the appetite-reducing effects of insulin (104). As such they are commonly associated with the development of hyperglycemia and diabetes. GCs have a direct hyperglycemic effect which starts very early after ingestion (105, 106). They typically cause an increase in blood glucose levels 4-8 hours after ingestion leading to a peak blood glucose level between midday meal and evening meal (106, 107). One in ten people not known to have diabetes develop GC-induced diabetes (108) an effect which is dose dependent (109). The incidence of glucocorticoid-induced hyperglycemia has been shown to occur in up to 30% of individuals without diabetes (110), but could be as high as 50%. The consequences of missing it can lead to significant harm, including the development of Hyperosmolar Hyperglycemic State (HHS), hospitalization, and in extreme circumstances, death. In a single center UK prevalence study 12.8% (120/940) of inpatients were found to be on glucocorticoids, however only 20.5% of these individuals (25/120) had their blood glucose levels measured during admission, demonstrating how infrequently glucose is measured in hospital (111). It is important to ensure that if glucocorticoid (steroid) induced hyperglycemia does occur, it is picked up early.

The use of GCs, is common in advanced cancer, to reduce peri-lesional edema, relieve pain, control nausea, combat fatigue, or boost appetite. For oncological emergencies such as cerebral metastases, superior vena-cava obstruction (SVCO), or metastatic spinal cord compression (MSCC), high dose GC treatment is integral to patient management. Furthermore, GC treatments are the backbone of many hematological cancer treatment regimens, and are often used as supportive anti-emetic medications, or to prevent allergic reactions, in many solid tumor regimens (105), and, as discussed above, the main first-line treatment for the management of ICP toxicity. In one study, the incidence of glucocorticoid-induced diabetes was 20% in those with newly diagnosed gastrointestinal cancer following at least 3 cycles of highly or moderately emetogenic chemotherapy, including dexamethasone as a supportive medication. Furthermore, almost 60% of people in the study exhibited signs of insulin resistance and multivariate analysis showed a significant association between the cumulative dose of dexamethasone and the incidence of corticosteroid-induced diabetes (112). In a separate smaller study of 16 women without diabetes with ovarian or endometrial cancer receiving carboplatin/paclitaxel chemotherapy with dexamethasone as supportive care, almost all experienced elevated interstitial glucose levels with diurnal variation during the first five days of treatment (113). For those who receive prednisolone as part of a treatment regimen for hematological malignancies, rates of steroid-induced diabetes and hyperglycemia have been reported to be as high as 32.5% and 47% respectively, highlighting the scale of this issue (114, 115).

Supra-physiological doses of glucocorticoids approximate to a dose of prednisolone greater than 5mg per day – or an equivalent dose of the alternative synthetic GC (Table 2). With increasing dose of GC, the risk of potential hyperglycemia increases, and in people without pre-existing diabetes, a glucocorticoid dose equivalent of >12mg dexamethasone and longer acting steroids are associated with a greater degree of hyperglycemia (116). As duration of GC treatment increases, it becomes increasingly likely that hyperglycemia may not resolve once the GCs are withdrawn, with those groups at particular risk of developing glucocorticoid induced diabetes, shown in Table 3.

Table 2. Glucocorticoid Dose Equivalent
Glucocorticoid (steroid)	Potency (equivalent doses)	Duration of action (half-life, in hours)
Hydrocortisone	20 mg	8
Prednisolone	5 mg	16-36
Methylprednisolone	4 mg	18-40
Dexamethasone	0.8 mg	36-54
Betamethasone	0.8 mg	26-54

Table 3. Risk Factors for Glucocorticoid-Inducted Diabetes

Pre-existing type 1 or type 2 diabetes

Family history of diabetes

Increasing age

Obesity

Ethnic minorities

Impaired fasting glucose or impaired glucose tolerance

Polycystic ovarian syndrome

Previous gestational diabetes

Previous development of hyperglycemia on glucocorticoid therapy

Concurrent cytotoxic therapy known to cause hyperglycemia

HYPOGYCEMIA IN PEOPLE ON SACT

Although anti-cancer therapies and glucocorticoid use lead predominantly to hyperglycemia, there are risks of hypoglycemia that require consideration. People at risk of hypoglycemia should be counselled on the signs and symptoms to be aware of, and of the requirement to inform the driver and vehicle licensing agency should they experience any episodes of hypoglycemia requiring third party assistance.

Poor oral intake and nausea/vomiting from the underlying cancer or treatments put individuals at increased risk of hypoglycemia. Poor glycemic control can cause weight loss and precipitate nutrition impact symptoms (NIS) such as nausea, poor appetite, and altered bowel movements, further increasing the risks of hypoglycemia, particularly when dietary intake has been poor for some time. People with diabetes on an insulin secretagogue (sulfonylureas or meglitinides), or those on insulin, are also at higher risk of hypoglycemia.

In patients with end-stage metastatic disease, and shortened life expectancy, tight glucose control is not indicated, potentially placing individuals at unnecessary risk for hypoglycemia, particularly in those with a poor performance status >2. Individual risk for hypoglycemia and prognosis should be considered and recommended glycemic measurement targets are between 6.0 mmol/L – 15 mmol/L (108 – 225 mg/dl) (117).

People with new onset ICPi-induced insulin deficiency often have labile glucose control (99). More relaxed glucose targets may be required to avoid hypoglycemia wherever possible. Immune checkpoint inhibitors can also induce hypopituitarism leading to secondary adrenal insufficiency. This may lead to hypoglycemia (together with any of the following - hyponatremia, hyperkalemia and hypotension). Adrenalitis leading to primary adrenal insufficiency is very rare. Presentation of adrenal insufficiency ranges from asymptomatic laboratory alterations to the acutely unwell, with management depending on the severity (118). Other causes of adrenal or pituitary deficiency leading to hypoglycemia include metastases at these sites, surgery, irradiation, azole class of anti-fungal medication, and inappropriate abrupt cessation of glucocorticoid medication.

In oncology patients being weaned from long-term steroids, glucose monitoring will need to be continued after glucocorticoid cessation, with doses of anti-diabetic treatments adjusted accordingly, and individuals advised on risks of hypoglycemia. Caution is also required whilst using certain hematological anti-cancer therapies, including lenalidomide (119) and bortezomib (120), which can precipitate hypoglycemia, particularly in people with an underlying diagnosis of diabetes.

All cancer patients at risk from hypoglycemia should receive advice regarding appropriate treatment with 15–20 g of fast-acting carbohydrate, taken immediately (121). Comprehensive guidelines from the Joint British Diabetes Societies for Inpatient Care on the management of hypoglycemia can be found at this reference (122).

MANAGEMENT RECOMMENDATIONS

Despite the effects of hyper- and hypoglycemia in people with diabetes (PWD) and those without known diabetes in cancer, there is a sparsity of guidance on the specific management considerations of these individuals. To address this, collaborative guidelines have recently been produced by the UK Chemotherapy Board (UKCB) and Joint British Diabetes Society for Inpatient Care (JBDS) (123, 124). The scope of these guidelines are to provide advice for the oncology/hemato-oncology and diabetes multidisciplinary teams to manage people with diabetes, commencing anti-cancer/ steroid therapy, as well as identifying individuals without a known diagnosis of diabetes who are at risk of developing hyperglycemia and new onset diabetes. These guidelines are intended for the outpatient management of people with cancer, particularly in the setting of the oncology/hemato-oncology clinic, and provision of advice for individuals at home, but where necessary, may be applied to inpatients as well. Whilst covering these guidelines in detail is beyond the scope of this chapter, key management considerations are summarized in tables 4-9.

Table 4. At Baseline

· HbA_1c and venous plasma glucose should be checked in all people with cancer at baseline clinic visit

· Provide high risk individuals with capillary blood glucose (CBG) meter and glucose testing strips, or if baseline plasma glucose is ≥12 mmol/L (216 mg/dl)

· Individuals with raised baseline HbA_1c (>47 mmol/mol [6.5%]) should be referred to primary care for management of hyperglycemia prior to next follow up visit

· When initiating SACT/glucocorticoids individuals must be informed of the risk of developing hyperglycemia/diabetes and potential symptoms to expect

· The recommended glucose target level is 6.0-10.0 mmol/L (108 – 180 mg/dl), allowing a range of 6.0-12.0 mmol/L (108 – 216 mg/dl)

· There are differences in opinion at where the threshold for intervention should be drawn - 12.0 mmol/L (216 mg/dl) is a pragmatic threshold

Table 5. Commencing Glucocorticoids (GC) /Systemic Anti-Cancer Therapy

· Check baseline HbA_1c and random venous plasma glucose before starting therapy

· Monitor random plasma glucose at each treatment visit

· Educate patients in symptoms of hyperglycemia

· Consider commencing gliclazide 40mg if raised blood glucose ≥12mmol/L (216 mg/dl) on two occasions

· Gliclazide may require frequent and significant increases in dose to reduce glucose levels, particularly on high dose steroids

· Inform diabetes care provider if persistently raised blood glucose

· If blood glucose is ≥20mmol/L (360 mg/dl), rule out DKA/HHS

Table 6. Commencing Immune Checkpoint Inhibitors (ICP)

· Educate patients to be aware of symptoms of hyperglycemia

· Rule out DKA or HHS which often occurs precipitously

· Withhold ICP if evidence of ICP-induced diabetes emergency. Once patient has been regulated with insulin substitution, consider restarting ICP

· Almost all patients require insulin therapy – refer urgently to diabetes team

Table 7. Managing Nausea and Vomiting

· People with diabetes should be made aware of likely exacerbation of hyperglycemia whilst on anti-emetic therapy

· People with diabetes receiving emetogenic chemotherapy should be offered an NK1 antagonist (e.g., aprepitant) with a long acting 5HT3 inhibitor (e.g., ondansetron)

· Consider the use of a GC in the first cycle and reduce doses or withdraw completely based on the PWD’s emetic control and on blood glucose management

Table 8. For Non-Insulin-Treated Individuals with Type 2 Diabetes

· Check baseline HbA_1c and random venous plasma glucose before starting therapy

· Monitor random plasma glucose at each treatment visit

· Educate patients in symptoms of hyperglycemia

· If plasma glucose is ≥12 mmol/L (216 mg/dl) on two occasions, screen for symptoms of hyperglycemia and ketonuria/ketonemia

· In individuals already on a sulphonyurea such as gliclazide or meglitinides, up-titrate morning dose of gliclazide to a maximum doses of 240 mg. Evening dose of gliclazide may be initiated to achieve a maximum daily dose of 320 mg

· Insulin therapy may be required

· In individuals on a diet-controlled regimen, or on other non-sulfonylurea treatments (e.g., metformin, DPP4 inhibitors, pioglitazone, SGLT2 inhibitors) commence gliclazide 40 mg, and up-titrate

Table 9. For Insulin-Treated Individuals with Type 2 Diabetes

· Check baseline HbA_1c and random venous plasma glucose before starting therapy

· Monitor random plasma glucose at each treatment visit

· If plasma glucose is ≥12 mmol/L (216 mg/dl) on two occasions, screen for symptoms of hyperglycemia and ketonuria/ketonemia

· Contact usual diabetes team for support in titrating insulin

· Consider titrating insulin by 10-20% of the original dose daily

· Individuals should be made aware of ‘sick day rules’ with insulin administration

Full management guidelines can be found at the UK Chemotherapy Board (UKCB) and Joint British Diabetes Society for Inpatient Care (JBDS) websites.

ADDITIONAL MANAGEMENT CONSIDERATIONS: CHOICE OF DIABETES THERAPEUTIC AGENT

Special consideration should also be given to the non-glycemic effects of hypoglycemic agents, including specific side effects and the impact on weight. Although weight reduction is associated with improvement in glycemic and metabolic profile in people with type 2 diabetes and is a key consideration in the choice of therapy, significant weight loss would usually be an unwanted effect in the oncology population. Indeed, weight gain is often used as a metric of improving nutritional state, especially in cancer related cachexia. This also has implications when counselling people with cancer about dietary choices when there is an additional cancer diagnosis. It is imperative that personalized advice is offered by healthcare professionals considering the global impact on the individual of any dietary or even lifestyle advice. SGLT2 inhibitors and GLP-1 agonists, for their potential weight reduction effects, are therefore less attractive options in the oncology setting. Insulin and sulfonylureas, on the other hand, offer an anabolic effect and therefore may be more desirable. Gastrointestinal side effects are common among hypoglycemic agents including metformin, DPP4 inhibitors, and GLP-1 agonists, and have the potential to complicate issues with nausea, vomiting, and oral intake from the underlying cancer and its treatment. Similarly, poor oral intake and nephrotoxic effects of certain SACT, added to a potential osmotic diuretic effect of SGLT2

inhibitors, could also increase the risk of acute kidney injury. The associated risk of genital tract infections with SGLT2 inhibitors would also be an additional consideration especially within an immunocompromised population (125). The impact and significance of these non-glycemic effects in the oncology population clearly differ to that of the general population, therefore highlighting the importance of a personalized approach with regular review of patients’ diabetes treatment through their oncology journey.

CONCLUSIONS

It is common practice in oncology to initiate systemic anti-cancer therapy (including chemotherapy, targeted treatment, immunotherapy and steroids) in people with pre-existing diabetes. Diabetes, or risk of developing diabetes are by no means a contraindication to treatment but treating clinicians should be aware of the risks to patients, and counsel them appropriately. As more sophisticated anti-cancer treatments become licensed for use, the metabolic effects of these treatments will become better understood, and oncology teams should utilize and collaborate with endocrinology and primary care services to minimize the risks to individuals from poor glycemic control and diabetes. The recent publication of specific guidelines should act as a reference aid for clinicians and wider healthcare professionals to aid in risk recognition, diagnostic and screening for treatment induced diabetes, and provide the tools to appropriately manage these individuals and reduce the risks of complications.

REFERENCES

International Diabetes Federation. IDF Diabetes Atlas 2021. Available at https://diabetesatlas.org/atlas/tenth-edition/. Accessed Dec 2021.
American Diabetes Association. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2021. Diabetes Care [Internet]. 2021; 44(Supplement 1): S15-S33.https://doi.org/10.2337/dc21-S002
Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010;33 Suppl 1(Suppl 1):S62-9. doi:10.2337/dc10-S062
Tsilidis KK, Kasimis JC, Lopez DS, Ntzani EE, Ioannidis JPA. Type 2 diabetes and cancer: umbrella review of meta-analyses of observational studies. BMJ : British Medical Journal. 2015;350:g7607. doi:10.1136/bmj.g7607
Giovannucci E, Harlan DM, Archer MC, et al. Diabetes and cancer: a consensus report. CA Cancer J Clin. 2010;60(4):207-21. doi:10.3322/caac.20078
Yan P, Wang Y, Yu X, Liu Y, Zhang ZJ. Type 2 diabetes mellitus and risk of head and neck cancer subtypes: a systematic review and meta-analysis of observational studies. Acta Diabetol. 2021;58(5):549-565. doi:10.1007/s00592-020-01643-0
Suh S, Kim KW. Diabetes and Cancer: Cancer should be screened in routine diabetes assessment. Diabetes Metab J. 2019;43(6):733-743. doi:10.4093/dmj.2019.0177
Diabetes UK. Diabetes and Cancer. https://wwwdiabetesorguk/diabetes-the-basics/related-conditions/diabetes-and-cancer. Accessed 28th February 2020
Barone BB, Yeh HC, Snyder CF, et al. Long-term all-cause mortality in cancer patients with preexisting diabetes mellitus: a systematic review and meta-analysis. JAMA. 2008;300(23):2754-64. doi:10.1001/jama.2008.824
Calderillo-Ruiz G, Lopez H, Herrera M, et al. P-149 - Obesity and hyperglycemia as a bad prognosis factor for recurrence and survival in colon cancer. Annals of Oncology. 2019;30:iv40-iv41. doi:https://doi.org/10.1093/annonc/mdz155.148
Villarreal-Garza C, Shaw-Dulin R, Lara-Medina F, et al. Impact of diabetes and hyperglycemia on survival in advanced breast cancer patients. Exp Diabetes Res. 2012;2012:732027. doi:10.1155/2012/732027
Hosokawa T, Kurosaki M, Tsuchiya K, et al. Hyperglycemia is a significant prognostic factor of hepatocellular carcinoma after curative therapy. World journal of gastroenterology. 2013;19(2):249-57. doi:10.3748/wjg.v19.i2.249
Wright JL, Plymate SR, Porter MP, et al. Hyperglycemia and prostate cancer recurrence in men treated for localized prostate cancer. Prostate cancer and prostatic diseases. 2013;16(2):204-8. doi:10.1038/pcan.2013.5
Barba M, Sperati F, Stranges S, et al. Fasting glucose and treatment outcome in breast and colorectal cancer patients treated with targeted agents: results from a historic cohort. Ann Oncol. 2012;23(7):1838-45. doi:10.1093/annonc/mdr540
Chen S, Tao M, Zhao L, Zhang X. The association between diabetes/hyperglycemia and the prognosis of cervical cancer patients: A systematic review and meta-analysis. Medicine (Baltimore). 2017;96(40):e7981. doi:10.1097/md.0000000000007981
Liu H, Liu Z, Jiang B, et al. Prognostic significance of hyperglycemia in patients with brain tumors: a meta-analysis. Mol Neurobiol. 2016;53(3):1654-1660. doi:10.1007/s12035-015-9115-4
Ali NA, O'Brien Jr JM, Blum W, et al. Hyperglycemia in patients with acute myeloid leukemia is associated with increased hospital mortality. https://doi.org/10.1002/cncr.22777. Cancer. 2007/07/01 2007;110(1):96-102. doi:https://doi.org/10.1002/cncr.22777
Barua R, Templeton AJ, Seruga B, Ocana A, Amir E, Ethier JL. Hyperglycaemia and survival in solid tumours: A systematic review and meta-analysis. Clin Oncol (R Coll Radiol). 2018;30(4):215-224. doi:10.1016/j.clon.2018.01.003
Ma J, Wang J, Ge L, Long B, Zhang J. The impact of diabetes mellitus on clinical outcomes following chemotherapy for the patients with pancreatic cancer: a meta-analysis. Acta Diabetol. 2019;56(10):1103-1111. doi:10.1007/s00592-019-01337-2
Peairs KS, Barone BB, Snyder CF, et al. Diabetes mellitus and breast cancer outcomes: a systematic review and meta-analysis. J Clin Oncol. 2011;29(1):40-6. doi:10.1200/jco.2009.27.3011
Ramos-Garcia P, Roca-Rodriguez MDM, Aguilar-Diosdado M, Gonzalez-Moles MA. Diabetes mellitus and oral cancer/oral potentially malignant disorders: A systematic review and meta-analysis. Oral Dis. 2021;27(3):404-421. doi:10.1111/odi.13289
Gerards MC, van der Velden DL, Baars JW, et al. Impact of hyperglycemia on the efficacy of chemotherapy—A systematic review of preclinical studies. Critical reviews in oncology/hematology. 2017;113:235-241. doi:https://doi.org/10.1016/j.critrevonc.2017.03.007
Garufi A, Traversi G, Gilardini Montani MS, et al. Reduced chemotherapeutic sensitivity in high glucose condition: implication of antioxidant response. Oncotarget. 2019;10(45):4691-4702. doi:10.18632/oncotarget.27087
Stebbing J, Sharma A, North B, et al. A metabolic phenotyping approach to understanding relationships between metabolic syndrome and breast tumour responses to chemotherapy. Ann Oncol. 2012;23(4):860-6. doi:10.1093/annonc/mdr347
Hershey DS, Given B, Given C, Von Eye A, You M. Diabetes and cancer: impact on health-related quality of life. Oncol Nurs Forum. 2012;39(5):449-57. doi:10.1188/12.Onf.449-457
Hershey DS, Tipton J, Given B, Davis E. Perceived impact of cancer treatment on diabetes self-management. Diabetes Educ. 2012;38(6):779-90. doi:10.1177/0145721712458835
Pettit S, Cresta E, Winkley K, Purssell E, Armes J. Glycaemic control in people with type 2 diabetes mellitus during and after cancer treatment: A systematic review and meta-analysis. PLoS One. 2017;12(5):e0176941. doi:10.1371/journal.pone.0176941
Hershey DS. Importance of glycemic control in cancer patients with diabetes: treatment through end of life. Asia-Pacific journal of oncology nursing. 2017;4(4):313-318. doi:10.4103/apjon.apjon_40_17
Vissers PAJ, Falzon L, van de Poll-Franse LV, Pouwer F, Thong MSY. The impact of having both cancer and diabetes on patient-reported outcomes: a systematic review and directions for future research. J Cancer Surviv. 2016;10(2):406-415. doi:10.1007/s11764-015-0486-3
Alenzi EO, Kelley GA. The association of hyperglycemia and diabetes mellitus and the risk of chemotherapy-induced neutropenia among cancer patients: A systematic review with meta-analysis. J Diabetes Complications. Jan 2017;31(1):267-272. doi:10.1016/j.jdiacomp.2016.09.006
Chao C, Page JH, Yang SJ, Rodriguez R, Huynh J, Chia VM. History of chronic comorbidity and risk of chemotherapy-induced febrile neutropenia in cancer patients not receiving G-CSF prophylaxis. Annals of Oncology. 2014;25(9):1821-1829. doi:10.1093/annonc/mdu203
Yang IP, Miao ZF, Huang CW, et al. High blood sugar levels but not diabetes mellitus significantly enhance oxaliplatin chemoresistance in patients with stage III colorectal cancer receiving adjuvant FOLFOX6 chemotherapy. Ther Adv Med Oncol. 2019;11:1758835919866964. doi:10.1177/1758835919866964
Liu X, Ji J, Sundquist K, Sundquist J, Hemminki K. The impact of type 2 diabetes mellitus on cancer-specific survival: a follow-up study in Sweden. Cancer. 2012;118(5):1353-61. doi:10.1002/cncr.26420
Park JH, Kim H-Y, Lee H, Yun EK. A retrospective analysis to identify the factors affecting infection in patients undergoing chemotherapy. European Journal of Oncology Nursing. 2015;19(6):597-603. doi:https://doi.org/10.1016/j.ejon.2015.03.006
Brunello A, Kapoor R, Extermann M. Hyperglycemia during chemotherapy for hematologic and solid tumors is correlated with increased toxicity. Am J Clin Oncol. 2011;34(3):292-6. doi:10.1097/COC.0b013e3181e1d0c0
Attili V, Bapsy P, Dadhich HK, Batra U, Lokanatha D, Babu KG. Impact of diabetes on cancer chemotherapy outcome: A retrospective analysis. International Journal of Diabetes in Developing Countries. 2007;27(4)122-128
de la Morena Barrio P, Conesa M, González-Billalabeitia E, et al. Delayed recovery and increased severity of Paclitaxel-induced peripheral neuropathy in patients with diabetes. J Natl Compr Canc Netw. 2015;13(4):417-23. doi:10.6004/jnccn.2015.0057
Kim S, Garg A, Raja F. Cancer patients with undiagnosed and poorly managed diabetes mellitus. Journal of Clinical Oncology. 2017;35(15_suppl):e18232-e18232. doi:10.1200/JCO.2017.35.15_suppl.e18232
Hamilton A, Todd A, Wallace H, et al. Improving identification and management of steroid-induced hyperglycaemia in day-case chemotherapy patients: Results from a quality improvement project in a regional oncology centre. 2018. (Abstract) Diabetic Medicine 2018;35 (Suppl 1)
Zylla D, Gilmore G, Eklund J, Richter S, Carlson A. Impact of diabetes and hyperglycemia on health care utilization, infection risk, and survival in patients with cancer receiving glucocorticoids with chemotherapy. J Diabetes Complications. 2019;33(4):335-339. doi:10.1016/j.jdiacomp.2018.12.012
Xu KY, Shameem R, Wu S. Risk of hyperglycemia attributable to everolimus in cancer patients: A meta-analysis. Acta Oncol. 2016;55(9-10):1196-1203. doi:10.3109/0284186x.2016.1168939
Verges B, Walter T, Cariou B. Endocrine side effects of anti-cancer drugs: effects of anti-cancer targeted therapies on lipid and glucose metabolism. Eur J Endocrinol. 2014;170(2):R43-55. doi:10.1530/EJE-13-0586
André F, Ciruelos E, Rubovszky G, et al. Alpelisib for PIK3CA-mutated, hormone receptor–positive advanced breast cancer. New England Journal of Medicine. 2019;380(20):1929-1940. doi:10.1056/NEJMoa1813904
Shariff AI, Syed S, Shelby RA, et al. Novel cancer therapies and their association with diabetes. 2019;62(2):R187. doi:10.1530/jme-18-0002
Sullivan I, Planchard D. Osimertinib in the treatment of patients with epidermal growth factor receptor T790M mutation-positive metastatic non-small cell lung cancer: clinical trial evidence and experience. Therapeutic Advances in Respiratory Disease. 2016;10(6):549-565. doi:10.1177/1753465816670498
Wadlow RC, Hezel AF, Abrams TA, et al. Panitumumab in patients with KRAS wild-type colorectal cancer after progression on cetuximab. Oncologist. 2012;17(1):14. doi:10.1634/theoncologist.2011-0452
Joint Formulary Committee. British National Formulary. Available at: https://bnf.nice.org.uk/drug/panitumumabhtml. 2020;Accessed 3rd March 2020
Armstrong AJ, Halabi S, Eisen T, et al. Everolimus versus sunitinib for patients with metastatic non-clear cell renal cell carcinoma (ASPEN): a multicentre, open-label, randomised phase 2 trial. The Lancet Oncology. 2016;17(3):378-388. doi:10.1016/S1470-2045(15)00515-X
Agostino NM, Chinchilli VM, Lynch CJ, et al. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract. 2011;17(3):197-202. doi:10.1177/1078155210378913
Chang L, An Y, Yang S, Zhang X. Meta-analysis of therapeutic effects and the risks of hypertension and hyperglycemia in patients with renal cell carcinoma who were receiving antiangiogenic drugs. J Cancer Res Ther. 2016;12(Supplement):96-103. doi:10.4103/0973-1482.191614
Saglio G, Larson RA, Hughes TP, et al. Efficacy and Safety of Nilotinib In Chronic Phase (CP) Chronic Myeloid Leukemia (CML) Patients (Pts) with Type 2 Diabetes In the ENESTnd Trial. Blood. 2010;116(21):3430-3430. doi:10.1182/blood.V116.21.3430.3430
Chan O, Talati C, Isenalumhe L, et al. Side-effects profile and outcomes of ponatinib in the treatment of chronic myeloid leukemia. Blood advances. 2020;4(3):530-538. doi:10.1182/bloodadvances.2019000268
Villadolid J, Ersek JL, Fong MK, Sirianno L, Story ES. Management of hyperglycemia from epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) targeting T790M-mediated resistance. Transl Lung Cancer Res. 2015;4(5):576-583. doi:10.3978/j.issn.2218-6751.2015.10.01
Schlenk RF, Weber D, Fiedler W, et al. Midostaurin added to chemotherapy and continued single-agent maintenance therapy in acute myeloid leukemia with FLT3-ITD. Blood. 2019;133(8):840-851. doi:10.1182/blood-2018-08-869453
Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. New England Journal of Medicine. 2017;377(5):454-464. doi:10.1056/NEJMoa1614359
Perl AE, Martinelli G, Cortes JE, et al. Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML. New England Journal of Medicine. 2019;381(18):1728-1740. doi:10.1056/NEJMoa1902688
US FDA. Mylotarg (gemtuzumab ozogamicin for injection). Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/021174s020lblpdf. Accessed January 2021;
Wolin EM. The expanding role of somatostatin analogs in the management of neuroendocrine tumors. Gastrointest Cancer Res. 2012;5(5):161-168.
Yang J, Jia B, Qiao Y, Chen W, Qi X. Variations of blood glucose in cancer patients during chemotherapy. Original Article. Nigerian Journal of Clinical Practice. 2016 2016;19(6):704-708. doi:10.4103/1119-3077.187323
Yang J, Jia B, Yan J, He J. Glycaemic adverse drug reactions from anti-neoplastics used in treating pancreatic cancer. Niger J Clin Pract. 2017;20(11):1422-1427. doi:10.4103/njcp.njcp_444_16
Paz-Ares L, de Marinis F, Dediu M, et al. Maintenance therapy with pemetrexed plus best supportive care versus placebo plus best supportive care after induction therapy with pemetrexed plus cisplatin for advanced non-squamous non-small-cell lung cancer (PARAMOUNT): a double-blind, phase 3, randomised controlled trial. Lancet Oncol. 2012;13(3):247-55. doi:10.1016/s1470-2045(12)70063-3
Clinical Trials. Pemetrexed and best supportive care versus placebo and best supportive care in non-small cell lung cancer (NSCLC). Available at: https://clinicaltrialsgov/ct2/show/results/NCT00102804. 2014;Accessed 3rd March 2020
Ma YY, Zhao M, Liu Y, et al. Use of decitabine for patients with refractory or relapsed acute myeloid leukemia: a systematic review and meta-analysis. Hematology. 2019;24(1):507-515. doi:10.1080/16078454.2019.1632407
Electronic Medicines Compendium (emc). Busulfan concentrate for infusion. Available at : https://www.medicines.org.uk/emc/product/2160/smpc#gref. Accessed January 2021;
Huang CY, Lin YS, Liu YH, Lin SC, Kang BH. Hyperglycemia crisis in head and neck cancer patients with platinum-based chemotherapy. J Chin Med Assoc. 2018;81(12):1060-1064. doi:10.1016/j.jcma.2018.05.008
Nan DN, Fernandez-Ayala M, Vega Villegas ME, et al. Diabetes mellitus following cisplatin treatment. Acta Oncol. 2003;42(1):75-8. doi:10.1080/0891060310002276
Yang J, Wang Y, Liu K, Yang W, Zhang J. Risk factors for doxorubicin-induced serious hyperglycaemia-related adverse drug reactions. Diabetes Ther. 2019;10(5):1949-1957. doi:10.1007/s13300-019-00677-0
Kritharis A, Bradley TP, Budman DR. Association of diabetes mellitus with arsenic trioxide (ATO) evaluated in the treatment of acute promyelocytic leukemia (APL). Journal of Clinical Oncology. 2011;29(15_suppl):e19724-e19724. doi:10.1200/jco.2011.29.15_suppl.e19724
Haanen JBAG, Carbonnel F, Robert C, et al. Management of toxicities from immunotherapy: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Annals of Oncology. 2017;28:iv119-iv142. doi:https://doi.org/10.1093/annonc/mdx225
Kotwal A, Haddox C, Block M, Kudva YC. Immune checkpoint inhibitors: an emerging cause of insulin-dependent diabetes. BMJ Open Diabetes Research & Care. 2019;7(1):e000591. doi:10.1136/bmjdrc-2018-000591
Lu J, Yang J, Liang Y, Meng H, Zhao J, Zhang X. Incidence of immune checkpoint inhibitor-associated diabetes: A meta-analysis of randomized controlled studies. Systematic Review. Frontiers in Pharmacology. 2019;10(1453)doi:10.3389/fphar.2019.01453
Wang H, Sun X, Zhao L, Chen X, Zhao J. Androgen deprivation therapy is associated with diabetes: Evidence from meta-analysis. J Diabetes Investig. 2016;7(4):629-636. doi:10.1111/jdi.12472
Lipscombe LL, Fischer HD, Yun L, et al. Association between tamoxifen treatment and diabetes: a population-based study. Cancer. 2012;118(10):2615-22. doi:10.1002/cncr.26559
Hwangbo Y, Lee EK. Acute hyperglycemia associated with anti-cancer medication. Endocrinol Metab (Seoul). 2017;32(1):23-29. doi:10.3803/EnM.2017.32.1.23
Feng JP, Yuan XL, Li M, et al. Secondary diabetes associated with 5-fluorouracil-based chemotherapy regimens in non-diabetic patients with colorectal cancer: results from a single-centre cohort study. Colorectal Dis. 2013;15(1):27-33. doi:10.1111/j.1463-1318.2012.03097.x
Teng CJ, Chang KH, Tsai IJ, Hwang WL, Hsu CY, Sheu WH. Impact of anthracyclines on diabetes mellitus development in B-cell lymphoma patients: A nationwide population-based study. Clin Drug Investig. 2018;38(7):603-610. doi:10.1007/s40261-018-0645-1
Koltin D, Sung L, Naqvi A, Urbach SL. Medication induced diabetes during induction in pediatric acute lymphoblastic leukemia: prevalence, risk factors and characteristics. Support Care Cancer. 2012;20(9):2009-15. doi:10.1007/s00520-011-1307-5
van Niekerk G, Engelbrecht AM. Inflammation-induced metabolic derangements or adaptation: An immunometabolic perspective. Cytokine Growth Factor Rev. 2018;43:47-53. doi:10.1016/j.cytogfr.2018.06.003
de Lima Junior EA, Yamashita AS, Pimentel GD, et al. Doxorubicin caused severe hyperglycaemia and insulin resistance, mediated by inhibition in AMPk signalling in skeletal muscle. J Cachexia Sarcopenia Muscle. 2016;7(5):615-625. doi:10.1002/jcsm.12104
Giovannucci E. The critical need for guidance in managing glycaemic control in patients with cancer. https://doi.org/10.1111/dme.14624. Diabetic Medicine. 2021;e14624. doi:https://doi.org/10.1111/dme.14624
Buffier P, Bouillet B, Smati S, Archambeaud F, Cariou B, Verges B. Expert opinion on the metabolic complications of new anticancer therapies: Tyrosine kinase inhibitors. Ann Endocrinol (Paris). 2018;79(5):574-582. doi:10.1016/j.ando.2018.07.011
Romero-Ventosa EY, Otero-Millán L, González-Costas S, et al. Effect of tyrosine kinase inhibitors on the glucose levels in diabetic and nondiabetic patients. Indian journal of cancer. 2017;54(1):136-143. doi:10.4103/ijc.IJC_190_17
Sakuma I, Nagano H, Yoshino I, Yokote K, Tanaka T. Ceritinib aggravates glycemic control in insulin-treated patients with diabetes and metastatic ALK-positive lung cancer. Intern Med. 2019;58(6):817-820. doi:10.2169/internalmedicine.1870-18
Bouillet B, Buffier P, Smati S, Archambeaud F, Cariou B, Vergès B. Expert opinion on the metabolic complications of mTOR inhibitors. Ann Endocrinol (Paris). 2018;79(5):583-590. doi:10.1016/j.ando.2018.07.010
Vergès B. mTOR and cardiovascular diseases: diabetes mellitus. Transplantation. 2018;102(2S Suppl 1):S47-s49. doi:10.1097/tp.0000000000001722
Martel S, Bruzzone M, Ceppi M, et al. Risk of adverse events with the addition of targeted agents to endocrine therapy in patients with hormone receptor-positive metastatic breast cancer: A systematic review and meta-analysis. Cancer treatment reviews. 2018;62:123-132. doi:10.1016/j.ctrv.2017.09.009
Sivendran S, Agarwal N, Gartrell B, et al. Metabolic complications with the use of mTOR inhibitors for cancer therapy. Cancer treatment reviews. 2014;40(1):190-6. doi:10.1016/j.ctrv.2013.04.005
Acharya GK, Hita AG, Yeung SJ, Yeung SJ. Diabetic ketoacidosis and acute pancreatitis: serious adverse effects of everolimus. Ann Emerg Med. 2017;69(5):666-667. doi:10.1016/j.annemergmed.2017.01.002
Bono P, Oudard S, Bodrogi I, et al. Outcomes in patients with metastatic renal cell carcinoma who develop everolimus-related hyperglycemia and hypercholesterolemia: combined subgroup analyses of the RECORD-1 and REACT Trials. Clin Genitourin Cancer. 2016;14(5):406-414. doi:10.1016/j.clgc.2016.04.011
Keating NL, O'Malley AJ, Freedland SJ, Smith MR. Diabetes and cardiovascular disease during androgen deprivation therapy: observational study of veterans with prostate cancer. J Natl Cancer Inst. 2010;102(1):39-46. doi:10.1093/jnci/djp404
Ng HS, Koczwara B, Roder D, Vitry A. Development of comorbidities in men with prostate cancer treated with androgen deprivation therapy: an Australian population-based cohort study. Prostate cancer and prostatic diseases. 2018;21(3):403-410. doi:10.1038/s41391-018-0036-y
Santorelli ML, Hirshfield KM, Steinberg MB, Rhoads GG, Lin Y, Demissie K. Hormonal therapy for breast cancer and diabetes incidence among postmenopausal women. Ann Epidemiol. 2016;26(6):436-40. doi:10.1016/j.annepidem.2016.04.004
Sun LM, Chen HJ, Liang JA, Li TC, Kao CH. Association of tamoxifen use and increased diabetes among Asian women diagnosed with breast cancer. Br J Cancer. 2014;111(9):1836-42. doi:10.1038/bjc.2014.488
Quandt Z, Young A, Anderson M. Immune checkpoint inhibitor diabetes mellitus: a novel form of autoimmune diabetes. https://doi.org/10.1111/cei.13424. Clinical & Experimental Immunology. 2020;200(2):131-140. doi:https://doi.org/10.1111/cei.13424
Falcone M, Fousteri G. Role of the PD-1/PD-L1 dyad in the maintenance of pancreatic immune tolerance for prevention of type 1 diabetes. Review. Frontiers in Endocrinology. 2020;11(569)doi:10.3389/fendo.2020.00569
Clotman K, Janssens K, Specenier P, Weets I, De Block CEM. Programmed cell death-1 inhibitor–induced type 1 diabetes mellitus. The Journal of Clinical Endocrinology & Metabolism. 2018;103(9):3144-3154. doi:10.1210/jc.2018-00728
Stamatouli AM, Quandt Z, Perdigoto AL, et al. Collateral damage: insulin-dependent diabetes induced with checkpoint inhibitors. Diabetes. 2018;67(8):1471-1480. doi:10.2337/dbi18-0002
Akturk HK, Kahramangil D, Sarwal A, Hoffecker L, Murad MH, Michels AW. Immune checkpoint inhibitor-induced Type 1 diabetes: a systematic review and meta-analysis. https://doi.org/10.1111/dme.14050. Diabetic Medicine. 2019;36(9):1075-1081. doi:https://doi.org/10.1111/dme.14050
Perdigoto AL, Quandt Z, Anderson M, Herold KC. Checkpoint inhibitor-induced insulin-dependent diabetes: an emerging syndrome. The Lancet Diabetes & Endocrinology. 2019;7(6):421-423. doi:10.1016/S2213-8587(19)30072-5
Kyriacou A, Melson E, Chen W, Kempegowda P. Is immune checkpoint inhibitor-associated diabetes the same as fulminant type 1 diabetes mellitus? Clinical Medicine. 2020;20(4):417-423. doi:10.7861/clinmed.2020-0054
Wright JJ, Salem J-E, Johnson DB, et al. Increased reporting of immune checkpoint inhibitor–associated diabetes. Diabetes Care. 2018;41(12):e150. doi:10.2337/dc18-1465
de Filette JMK, Pen JJ, Decoster L, et al. Immune checkpoint inhibitors and type 1 diabetes mellitus: a case report and systematic review. Eur J Endocrinol. Sep 2019;181(3):363-374. doi:10.1530/eje-19-0291
Byun DJ, Braunstein R, Flynn J, et al. Immune checkpoint inhibitor-associated diabetes: A single-institution experience. Diabetes Care. 2020;43(12):3106-3109. doi:10.2337/dc20-0609
Perez A, Jansen-Chaparro S, Saigi I, Bernal-Lopez MR, Miñambres I, Gomez-Huelgas R. Glucocorticoid-induced hyperglycemia. J Diabetes. 2014;6(1):9-20. doi:10.1111/1753-0407.12090
Dunning T, Martin P. Palliative and end of life care of people with diabetes: Issues, challenges and strategies. Diabetes Res Clin Pract. 2018;143:454-463. doi:10.1016/j.diabres.2017.09.018
Joint British Diabetes Society (JBDS) for Inpatient Care Group. Steroid use for inpatients with diabetes. Available at: https://abcdcare/resource/steroid-use-inpatients-diabetes. May 202
Tamez-Pérez HE, Quintanilla-Flores DL, Rodríguez-Gutiérrez R, González-González JG, Tamez-Peña AL. Steroid hyperglycemia: Prevalence, early detection and therapeutic recommendations: A narrative review. World J Diabetes. 2015;6(8):1073-1081. doi:10.4239/wjd.v6.i8.1073
Pilkey J, Streeter L, Beel A, Hiebert T, Li X. Corticosteroid-induced diabetes in palliative care. J Palliat Med. 2012;15(6):681-9. doi:10.1089/jpm.2011.0513
Gannon C, Dando N. Dose-sensitive steroid-induced hyperglycaemia. Palliat Med. 2010;24(7):737-9. doi:10.1177/0269216310377816
Liu XX, Zhu XM, Miao Q, Ye HY, Zhang ZY, Li YM. Hyperglycemia induced by glucocorticoids in nondiabetic patients: a meta-analysis. Ann Nutr Metab. 2014;65(4):324-32. doi:10.1159/000365892
Narwani V, Swafe L, Stavraka C, Dhatariya K. How frequently are bedside glucose levels measured in hospital inpatients on glucocorticoid treatment? Clin Med (Lond). 2014;14(3):327-328. doi:10.7861/clinmedicine.14-3-326a
Jeong Y, Han HS, Lee HD, et al. A pilot study evaluating steroid-induced diabetes after antiemetic dexamethasone therapy in chemotherapy-treated cancer patients. Cancer Res Treat. 2016;48(4):1429-1437. doi:10.4143/crt.2015.464
Lyall MJ, Thethy I, Steven L, et al. Diurnal profile of interstitial glucose following dexamethasone prophylaxis for chemotherapy treatment of gynaecological cancer. Diabet Med. 2018;35(11):1508-1514. doi:10.1111/dme.13770
Lamar ZS, Dothard A, Kennedy L, et al. Hyperglycemia during first-line R-CHOP or dose adjusted R-EPOCH chemotherapy for non-Hodgkin lymphoma is prevalent and associated with chemotherapy alteration - a retrospective study. Leuk Lymphoma. 2018;59(8):1871-1877. doi:10.1080/10428194.2017.1410889
Lee S-y, Kurita N, Yokoyama Y, et al. Glucocorticoid-induced diabetes mellitus in patients with lymphoma treated with CHOP chemotherapy. Supportive Care in Cancer. 2014;22(5):1385-1390. doi:10.1007/s00520-013-2097-8
Healy SJ, Nagaraja HN, Alwan D, Dungan KM. Prevalence, predictors, and outcomes of steroid-induced hyperglycemia in hospitalized patients with hematologic malignancies. Endocrine. 2017;56(1):90-97. doi:10.1007/s12020-016-1220-2
Roberts A, James J, Dhatariya K. Management of hyperglycaemia and steroid (glucocorticoid) therapy: a guideline from the Joint British Diabetes Societies (JBDS) for Inpatient Care group. Diabet Med. 2018;35(8):1011-1017. doi:10.1111/dme.13675
Higham CE, Olsson-Brown A, Carroll P, et al. Society for Endocrinology emergency guidance: Acute management of the endocrine complications of checkpoint inhibitor therapy. Endocrine connections. 2018;7(7):G1-G7. doi:10.1530/EC-18-0068
Przybylski DJ, Birhiray R, Reeves DJ. Severe Hypoglycemia Caused by Lenalidomide. Pharmacotherapy. 2018;38(1):e1-e6. doi:10.1002/phar.2061
Electronic Medicines Compendium (emc). Bortezomib. Available from: https://wwwmedicinesorguk/emc/product/10201/smpc#gref. Accessed September 2021;
Joint British Diabetes Society (JBDS) for Inpatient Care Group. The Hospital Management of Hypoglycaemia in Adults with Diabetes Mellitus 3rd edition.

Available from: https://abcdcare/joint-british-diabetes-societies-jbds-inpatient-care-group. 2018;

Joint British Diabetes Society for Inpatient Care. The Hospital Management of Hypoglycaemia in Adults with Diabetes Mellitus 4th Edition. Available at: https://abcdcare/sites/abcdcare/files/site_uploads/JBDS_HypoGuideline_4th_edition_FINALpdf. 2020;
Joharatnam-Hogan N, Chambers P, Dhatariya K, Board R. A guideline for the outpatient management of glycaemic control in people with cancer. Diabet Med. Jul 9 2021:e14636. doi:10.1111/dme.14636
The UK Chemotherapy Board (UKCB) and Joint British Diabetes Society (JBDS). Management of Glycaemic Control in Patients with Cancer. Available from: https://wwwukchemotherapyboardorg/publications. Accessed September 2021;
Liu J, Li L, Li S, et al. Effects of SGLT2 inhibitors on UTIs and genital infections in type 2 diabetes mellitus: a systematic review and meta-analysis. Sci Rep. Jun 6 2017;7(1):2824. doi:10.1038/s41598-017-02733-w

Monogenic Disorders Causing Hypobetalipoproteinemia – copy

ABSTRACT

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia, abetalipoproteinemia, chylomicron retention disease, loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familiar Combined Hypolipidemia). This chapter describes the etiology, pathogenesis, clinical and laboratory findings, and the treatment of these rare monogenic disorders.

INTRODUCTION

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia (FHBL), abetalipoproteinemia (ABL), chylomicron retention disease (CMRD), loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familial Combined Hypolipidemia, FCH). Increased understanding of the genetic and the molecular underpinnings of these disorders has allowed a focused prioritization of therapeutic targets for drug development. Table 1 summarizes genetic, lipid, and clinical features of the major hypobetalipoproteinemia syndromes. Of note the parental lipid profile is normal in abetalipoproteinemia and chylomicron retention disease.

It should be recognized that secondary, non-familial, forms of hypobetalipoproteinemia occur and include strict vegan diet, malnutrition, hyperthyroidism, malignancy, and chronic liver disease. In addition, hypobetalipoproteinemia can also be due to polymorphisms in multiple genes that together result in hypobetalipoproteinemia (polygenic etiology) (1-3). In a study of 111 patients with LDL-C levels below the fifth percentile 36% had monogenic hypobetalipoproteinemia, 34% had polygenic hypobetalipoproteinemia, and 30% had hypobetalipoproteinemia from an unknown cause (1). In a study of women with an LDL-C ≤1st percentile (≤50 mg/dL) 15.7% carried mutations causing monogenic hypocholesterolemia and 49.6% were genetically predisposed to a low LDL-C on the basis of an extremely low weighted polygenetic risk score (3).

Table 1. Characteristics of the Hypobetalipoproteinemia Syndromes
	Inheritance	Effected gene	Prevalence	Lipids	Clinical features
FHBL	ACD	Truncation mutations in Apo B	1:1000 – 1:3000	Apo B <5th percentile, LDL-C 20- 50 mg/dL	Hepatic steatosis Mild elevation of transaminases. Lower prevalence of ASCVD
ABL	AR	MTTP	<1:1,000,000	Triglycerides < 30 mg/dl, Cholesterol < 30 mg/dl), LDL and Apo B undetectable	Hepatic steatosis Malabsorption, steatorrhea, diarrhea, and failure to thrive. Deficiency of fat-soluble vitamins.
PCSK9	ACD	Loss of function mutations in PCSK9		Heterozygous – mild to moderate reduction in LDL-C Homozygous – LDL-C ~15 mg/dl	Normal health; significantly lower prevalence of ASCVD
FCH	ACD	Loss of function mutations in ANGPTL3	Very rare	Panhypolipidemia	Normal health; significantly lower prevalence of ASCVD
CMRD	AR	SAR1B	Very rare	LDL-C and HDL-C -decreased by 50%, Triglycerides - normal	hypocholesterolemia associated with failure to thrive, diarrhea, steatorrhea, and abdominal distension

ACD- autosomal co-dominant; AR- autosomal recessive; FHBL- familial hypobetalipoproteinemia; ABL- abetalipoproteinemia; FCH- Familiar Combined Hypolipidemia; CMRD- chylomicron retention disease, MTTP- microsomal triglyceride transfer protein; ANGPTL3- angiopoietin-like protein 3; ASCVD- atherosclerotic cardiovascular disease

FAMILIAL HYPOBETALIPOPROTEINEMIA

Familial Hypobetalipoproteinemia (FHBL) is most commonly due to truncation mutations in the gene coding for Apo B (4-6). Variants that lead to truncated proteins that are 30% in length or shorter have more severe signs and symptoms than those with longer truncated proteins (4,5). The truncated forms of Apo B found in FHBL are generally non-functional (truncation decreases lipidation and secretion) and are catabolized quickly, resulting in markedly reduced levels in the plasma (Apo B <5th percentile and LDL-C typically between 20- 50 mg/dL) (5,6). Although there is one normal allele in heterozygous FHBL, plasma Apo B levels are approximately 25% of normal rather than the predicted 50% (6). These lower than expected levels result from a lower secretion rate of VLDL Apo B from the liver, decreased production of LDL Apo B, increased catabolism of VLDL, and extremely low secretion of the truncated Apo B (4-6). Given the reduced substrate (Apo B) for lipid (predominantly triglyceride) loading, fatty liver develops in these patients (4,7). Hepatic steatosis and mild elevation of liver enzymes are common in heterozygous FHBL (4,7). Interestingly, individuals with monogenic hypobetalipoproteinemia had a much greater prevalence of hepatic dysfunction than individuals with polygenic hypobetalipoproteinemia (1). In contrast to non-alcoholic fatty liver disease, FHBL is not associated with hepatic or peripheral insulin resistance (7). This observation, however, does not imply that hepatic steatosis associated with FHBL is benign. There are several reports of steatohepatitis, cirrhosis, and hepatocellular carcinoma in patients with FHBL and it is estimated that 5-10% of individuals with FHBL develop relatively more severe nonalcoholic steatohepatitis (4). Because of the risk of developing liver disease liver function tests should be checked every 1-2 years and a hepatic ultrasound in those with elevated liver transaminases (4). While hepatic fat accumulation is the rule, there is generally sufficient chylomicron production to handle dietary fat. However, oral fat intolerance and intestinal fat malabsorption have been reported (4). On the positive side the decrease in proatherogenic lipoproteins has been associated with a reduced risk of cardiovascular disease (8).

Given the association of FHBL and low LDL-C, Apo B has been an attractive target for drug development. Indeed, unraveling the genetic and molecular mechanisms of FHBL provided the motivation to pharmacologically antagonize Apo B synthesis for therapeutic gains. This culminated in the production of mipomersen, a synthetic single strand anti-sense oligonucleotide to Apo B (9,10). Essentially, anti-sense oligonucleotides contain approximately ~20 deoxyribonucleic acid (DNA) base pairs complementary to a unique messenger ribonucleic acid (mRNA) sequence. The hybridization of the anti-sense oligonucleotide to the mRNA of interest leads to its catabolism via RNase H1, with markedly reduced mRNA levels and ultimately reduced target protein levels. In this case, mipomersen binds to Apo B mRNA leading to reduced production of the protein, and mimicking (albeit to a lesser extent) FHBL. Mipomersen is the first anti-sense oligonucleotide approved by the United States Food and Drug Administration (FDA) and was commercialized in 2013 with a limited indication for adjunctive LDL-C lowering in patients with homozygous familial hypercholesterolemia (HoFH) (10). It is an injectable agent administered subcutaneously once a week. In the clinical trials, mipomersen was associated with a reduction of LDL-C by 21% in subjects with HoFH and 33% in subjects with heterozygous familial hypercholesterolemia (HeFH) (10). Interestingly, it was also found to lower Lp(a) by 21- 23% (10). While it is highly efficacious in LDL-C lowering, it has side effects, many of which can be predicted based on the experience with FHBL (e.g., hepatic steatosis, elevated liver enzymes) (10). It is also associated with injection site reactions in a considerable number of subjects (10). In May 2018 sales were discontinued due to safety concerns related to increased liver transaminases and fatty liver.

Homozygous hypobetalipoproteinemia (HHBL) is extremely rare (4). These patients are homozygous or compound heterozygous for mutations in the Apo B gene. The clinical manifestations mimic ABL (see below) (4).

ABETALIPOPROTEINEMIA

Abetalipoproteinemia (ABL) is a rare disorder characterized by very low plasma concentrations of triglyceride and cholesterol (under 30 mg/dl) and undetectable levels of LDL and Apo B (5,11,12). HDL-C levels are usually normal or modestly reduced. It is due to mutations in the gene that codes for microsomal triglyceride transfer protein (MTTP) (5,11-13). MTTP lipidates nascent Apo B in the endoplasmic reticulum to produce VLDL and chylomicrons in the liver and small intestine, respectively (13,14). Unlipidated Apo B is targeted for proteasomal degradation leading to the absence of Apo B containing lipoproteins in the plasma (and thus markedly reduced levels of LDL-C and triglycerides) (13,14). Similar to FHBL, VLDL production is inhibited (12). The reduced triglyceride export from the liver leads to hepatic steatosis, which rarely may progress to steatohepatitis, fibrosis, and cirrhosis (7,11). Additionally, lack of MTTP facilitated lipidation of chylomicrons in the small intestine results in lipid accumulation in enterocytes with associated malabsorption, steatorrhea, and diarrhea (5,11). The malabsorption and diarrhea lead to failure to thrive during infancy (5,11). Acanthocytosis may encompass 50% of circulating red blood cells (red blood cells with spiked cell membranes, due to thorny projections) (11,12). An additional issue of importance related to ABL is deficiency of fat-soluble vitamins (11). Early diagnosis of ABL and homozygous hypobetalipoproteinemia is extremely important as vitamin E deficiency culminates in atypical retinitis pigmentosa, spinocerebellar degeneration with ataxia, and vitamin K deficiency can lead to a significant bleeding diathesis (11). High dose supplementation with fat soluble vitamins early in life can prevent these devastating complications (5,11). Additional treatment measures include a low-fat diet and supplementation with essential fatty acids (5,11).

Given the very low level of atherogenic lipoproteins and lipids associated with ABL, there was interest in inhibiting MTTP therapeutically. Lomitapide is an oral MTP inhibitor that has been developed over the course of many years (10,15). In early trials, it was tested at a relatively high dose and the side effect profile was prohibitive (nausea, flatulence, and diarrhea). The more recent clinical trial program tested lower doses with drug titration in subjects with HoFH (10,15). On an intention to treat basis, LDL-C was decreased by 40% and apolipoprotein B by 39% (10). In patients who were actually taking lomitapide, LDL-C levels were reduced by 50% (10). In addition to decreasing LDL-C levels, non-HDL-C levels were decreased by 50%, Lp(a) by 15%, and triglycerides by 45% (10). Lomitapide received the same limited indication as mipomersen for adjunctive treatment of patients with HoFH (10). Besides the gastrointestinal issues already alluded to, its side effect profile includes hepatic steatosis (10). Its long-term safety has not been established.

PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 (PCSK9)

Proprotein convertase subtilisin/ kexin type 9 (PCSK9) belongs to the proprotein convertase class of serine proteases (16-18). After synthesis, PCSK9 undergoes autocatalytic cleavage. This step is required for secretion, most likely because the prodomain functions as a chaperone and facilitates folding (16,17). PCSK9 is associated with LDL particles and the LDL-receptor (LDLR) (18). In 2003, Abifadel reported the seminal work that mapped PCSK9 as the third locus for autosomal dominant hypercholesterolemia (Familial Hypercholesterolemia- FH) (19). This finding revealed a previously unknown actor involved in cholesterol homeostasis and served to launch a series of investigations into PCSK9 biology. As it turns out, PCSK9 functions as a central regulator of plasma LDL-C concentration (16-18). It binds to the LDLR and targets it for destruction in the lysosome (16-18). Overactivity of PCSK9 results in a decrease in LDLR and an increase in LDL-C levels while decreased activity of PCSK9 results in an increase in LDLR and a decrease in LDL-C.

Since the discovery of gain-of-function mutations in PCSK9 as a cause of FH, investigators have also uncovered loss of function mutations of PCSK9. Loss-of-function mutations in PCSK9 are associated with low LDL-C levels and markedly reduced ASCVD (16,17). In African Americans 2.6 percent had nonsense mutations in PCSK9 that resulted in a 28 percent reduction in LDL-C and an 88 percent reduction in the risk of coronary heart disease (20). The hypolipidemia is not associated with liver abnormalities or other disorders. Interestingly, rare individuals homozygous or compound heterozygotes for loss of function mutations in PCSK9 have been reported with extremely low levels of LDL-C (~15 mg/dl), normal health and reproductive capacity, and no evidence of neurologic or cognitive dysfunction (18,21,22). Collectively, these observations served as further motivation to pursue antagonism of PCSK9 as a therapeutic target. Antagonizing PCSK9 would prolong the lifespan of LDLR, leading to significant reductions in plasma LDL-C levels.

There are numerous approaches to inhibiting PCSK9 including humanized monoclonal antibodies (mAbs), gene silencing, and use of small inhibitory peptides (18). Thus far, approaches utilizing mAbs are FDA approved (10). The two fully human monoclonal antibodies (alirocumab and evolocumab) targeting PCSK9 became commercially available in 2015. Clinical trials of mAbs targeted to PCSK9 have demonstrated remarkable efficacy in LDL-C reduction (~50% reduction in LDL-C as monotherapy and ~65% reduction in LDL-C in combination with a statin) with an excellent short-term safety and tolerability profile (10). Moreover, a large randomized controlled trial (FOURIER) demonstrated incremental improvement with a 15% reduction in the composite primary endpoint of major adverse cardiovascular outcome with addition of evolocumab on top of standard of care in patients with stable vascular disease (23). Additionally, the ODYSSEY OUTCOMES trial also demonstrated a similar reduction in major adverse cardiovascular events with alirocumab vs. placebo in patients with recent acute coronary syndromes (24). Finally, inclisiran, a small interfering RNA that inhibits translation of PCSK9, is approved in Europe but not yet in the US (10).

FAMILIAL COMBINED HYPOLIPIDEMIA

Familial combined hypolipidemia (FCH) is due to loss of function mutations in the gene encoding angiopoietin-like protein 3 (ANGPTL3) (25,26). ANGPTL3 inhibits various lipases, such as lipoprotein lipase and endothelial lipase (25,26). Therefore, loss of function mutations in ANGPTL3 relinquishes this inhibition resulting in more efficient metabolism of VLDL and HDL particles (25,26). In addition, to increasing VLDL clearance the secretion of VLDL is also decreased due to a decrease in free fatty acid flux to the liver (25). LDL clearance is increased but the mechanism remains to be fully elucidated (25). Studies have suggested that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production due to ANGPTL3 inhibition and increased endothelial lipase activity reducing VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation rather than being further metabolized to LDL (27). Clinically, FCH manifests as panhypolipidemia (decreased triglycerides, LDL-C, and HDL-C) (25,26). Interestingly, heterozygotes for certain nonsense mutations in the first exon of ANGPTL3 have moderately reduced LDL-C and triglyceride levels while compound heterozygotes have significant reductions in HDL-C as well (25,26). Homozygosity or compound heterozygosity for other loss-of-function mutations in exon 1 of ANGPTL3 have no detectable ANGPTL3 in plasma and striking reductions of atherogenic lipoproteins with HDL particles containing only apo A-I and preß-HDL. Individuals who are heterozygous for the loss of function mutations in ANGPTL3 have normal HDL-C levels and significantly reduced LDL-C (<25^th percentile) (25,26).

A pooled analysis of cases of familial combined hypolipidemia was published 2013 (28). One hundred fifteen individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were evaluated. Homozygotes and compound heterozygotes (two mutant alleles) had no measurable ANGPTL3 protein. In heterozygotes, ANGPTL3 was reduced by 34-88%, according to genotype. All cases (homozygotes and heterozygotes) demonstrated significantly lower concentrations of all plasma lipoproteins (except for Lp(a)) as compared to controls. Familial combined hypolipidemia is not associated with any comorbidity. In fact, the prevalence of fatty liver was the same as controls. However, ANGPTL3 deficiency is associated with a reduced risk of cardiovascular disease (25,29).

Recently, evinacumab, a human monoclonal antibody against ANGPTL3, was approved for the treatment of Homozygous Familial Hypercholesterolemia (10). Evinacumab decreases LDL-C levels by mechanisms independent of LDL receptor activity (10).

CHYLOMICRON RETENTION DISEASE

Chylomicron retention disease (CMRD), known also as Anderson’s disease for the individual who first described the condition in 1961, is a rare inherited lipid malabsorption syndrome (30,31). It is due to mutations in the SAR1B gene which codes for the protein SAR1b, a small GTPase, involved in intracellular protein trafficking (30). Mutations in SAR1b result in the failure of pre-chylomicrons to move from the endoplasmic reticulum to the golgi (30). This disorder usually presents in young infants with diarrhea, steatorrhea, abdominal distention, and failure to thrive (30,31). Patients with CMRD demonstrate a specific autosomal recessive hypocholesterolemia that differs from other familial hypocholesterolemias. CMRD is associated with a 50% reduction in both plasma LDL-C and HDL-C with normal fasting triglyceride levels (30,31). Mutations in SAR1B do not affect VLDL secretion by the liver. The decrease in HDL-C is postulated to be due to a decrease in Apo A-I secretion and cholesterol efflux by the small intestine (30). The mechanism accounting for the decrease in LDL-C is not clear. The usual increase in triglycerides and chylomicron levels following a fat meal is blocked (30). The duodenal mucosa is white on endoscopy and intestinal biopsy reveals cytosolic lipid droplets and lipoprotein-sized particles in enterocytes (30). As one would expect the absorption of fat-soluble vitamins (A, D, K, and E) and essential fatty acids is impaired (30,31).

Treatment for individuals with CMRD is similar to that described above for individuals with ABL (31).

REFERENCES

Rimbert A, Vanhoye X, Coulibaly D, Marrec M, Pichelin M, Charriere S, Peretti N, Valero R, Wargny M, Carrie A, Lindenbaum P, Deleuze JF, Genin E, Redon R, Rollat-Farnier PA, Goxe D, Degraef G, Marmontel O, Divry E, Bigot-Corbel E, Moulin P, Cariou B, Di Filippo M. Phenotypic Differences Between Polygenic and Monogenic Hypobetalipoproteinemia. Arterioscler Thromb Vasc Biol 2021; 41:e63-e71
Blanco-Vaca F, Martin-Campos JM, Beteta-Vicente A, Canyelles M, Martinez S, Roig R, Farre N, Julve J, Tondo M. Molecular analysis of APOB, SAR1B, ANGPTL3, and MTTP in patients with primary hypocholesterolemia in a clinical laboratory setting: Evidence supporting polygenicity in mutation-negative patients. Atherosclerosis2019; 283:52-60
Balder JW, Rimbert A, Zhang X, Viel M, Kanninga R, van Dijk F, Lansberg P, Sinke R, Kuivenhoven JA. Genetics, Lifestyle, and Low-Density Lipoprotein Cholesterol in Young and Apparently Healthy Women. Circulation 2018; 137:820-831
Burnett JR, Hooper AJ, Hegele RA. APOB-Related Familial Hypobetalipoproteinemia. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, eds. GeneReviews((R)). Seattle (WA) 2021.
Hooper AJ, Burnett JR. Update on primary hypobetalipoproteinemia. Curr Atheroscler Rep 2014; 16:423
Hooper AJ, van Bockxmeer FM, Burnett JR. Monogenic hypocholesterolaemic lipid disorders and apolipoprotein B metabolism. Crit Rev Clin Lab Sci 2005; 42:515-545
Welty FK. Hypobetalipoproteinemia and abetalipoproteinemia: liver disease and cardiovascular disease. Curr Opin Lipidol 2020; 31:49-55
Peloso GM, Nomura A, Khera AV, Chaffin M, Won HH, Ardissino D, Danesh J, Schunkert H, Wilson JG, Samani N, Erdmann J, McPherson R, Watkins H, Saleheen D, McCarthy S, Teslovich TM, Leader JB, Lester Kirchner H, Marrugat J, Nohara A, Kawashiri MA, Tada H, Dewey FE, Carey DJ, Baras A, Kathiresan S. Rare Protein-Truncating Variants in APOB, Lower Low-Density Lipoprotein Cholesterol, and Protection Against Coronary Heart Disease. Circ Genom Precis Med 2019; 12:e002376
Crooke ST, Geary RS. Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B. Br J Clin Pharmacol 2013; 76:269-276
Feingold KR. Cholesterol Lowering Drugs. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, Dungan K, Grossman A, Hershman JM, Hofland J, Kalra S, Kaltsas G, Koch C, Kopp P, Korbonits M, Kovacs CS, Kuohung W, Laferrere B, McGee EA, McLachlan R, Morley JE, New M, Purnell J, Sahay R, Singer F, Stratakis CA, Trence DL, Wilson DP, eds. Endotext. South Dartmouth (MA) 2021.
Burnett JR, Hooper AJ, Hegele RA. Abetalipoproteinemia. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, eds. GeneReviews((R)). Seattle (WA) 2018.
Lee J, Hegele RA. Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management. J Inherit Metab Dis 2014; 37:333-339
Wetterau JR, Lin MC, Jamil H. Microsomal triglyceride transfer protein. Biochim Biophys Acta 1997; 1345:136-150
Hussain MM, Shi J, Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly. J Lipid Res 2003; 44:22-32
Cuchel M, Rader DJ. Microsomal transfer protein inhibition in humans. Curr Opin Lipidol 2013; 24:246-250
Debose-Boyd RA, Horton JD. Opening up new fronts in the fight against cholesterol. Elife 2013; 2:e00663
Seidah NG, Awan Z, Chretien M, Mbikay M. PCSK9: a key modulator of cardiovascular health. Circ Res 2014; 114:1022-1036
Shapiro MD, Tavori H, Fazio S. PCSK9: From Basic Science Discoveries to Clinical Trials. Circ Res 2018; 122:1420-1438
Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, Derre A, Villeger L, Farnier M, Beucler I, Bruckert E, Chambaz J, Chanu B, Lecerf JM, Luc G, Moulin P, Weissenbach J, Prat A, Krempf M, Junien C, Seidah NG, Boileau C. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003; 34:154-156
Cohen JC, Boerwinkle E, Mosley TH, Jr., Hobbs HH. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006; 354:1264-1272
Zhao Z, Tuakli-Wosornu Y, Lagace TA, Kinch L, Grishin NV, Horton JD, Cohen JC, Hobbs HH. Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote. Am J Hum Genet 2006; 79:514-523
Cariou B, Ouguerram K, Zair Y, Guerois R, Langhi C, Kourimate S, Benoit I, Le May C, Gayet C, Belabbas K, Dufernez F, Chetiveaux M, Tarugi P, Krempf M, Benlian P, Costet P. PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia. Arterioscler Thromb Vasc Biol 2009; 29:2191-2197
Sabatine MS, Giugliano RP, Keech AC, Honarpour N, Wiviott SD, Murphy SA, Kuder JF, Wang H, Liu T, Wasserman SM, Sever PS, Pedersen TR, FOURIER Steering Committee and Investigators. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. N Engl J Med 2017; 376:1713-1722
Schwartz GG, Steg PG, Szarek M, Bhatt DL, Bittner VA, Diaz R, Edelberg JM, Goodman SG, Hanotin C, Harrington RA, Jukema JW, Lecorps G, Mahaffey KW, Moryusef A, Pordy R, Quintero K, Roe MT, Sasiela WJ, Tamby JF, Tricoci P, White HD, Zeiher AM, ODYSSEY OUTCOMES Committees and Investigators. Alirocumab and Cardiovascular Outcomes after Acute Coronary Syndrome. N Engl J Med 2018; 379:2097-2107
Arca M, D'Erasmo L, Minicocci I. Familial combined hypolipidemia: angiopoietin-like protein-3 deficiency. Curr Opin Lipidol 2020; 31:41-48
Kersten S. Angiopoietin-like 3 in lipoprotein metabolism. Nat Rev Endocrinol 2017; 13:731-739
Adam RC, Mintah IJ, Alexa-Braun CA, Shihanian LM, Lee JS, Banerjee P, Hamon SC, Kim HI, Cohen JC, Hobbs HH, Van Hout C, Gromada J, Murphy AJ, Yancopoulos GD, Sleeman MW, Gusarova V. Angiopoietin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance. J Lipid Res2020; 61:1271-1286
Minicocci I, Santini S, Cantisani V, Stitziel N, Kathiresan S, Arroyo JA, Marti G, Pisciotta L, Noto D, Cefalu AB, Maranghi M, Labbadia G, Pigna G, Pannozzo F, Ceci F, Ciociola E, Bertolini S, Calandra S, Tarugi P, Averna M, Arca M. Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis. J Lipid Res 2013; 54:3481-3490
Dewey FE, Gusarova V, Dunbar RL, O'Dushlaine C, Schurmann C, Gottesman O, McCarthy S, Van Hout CV, Bruse S, Dansky HM, Leader JB, Murray MF, Ritchie MD, Kirchner HL, Habegger L, Lopez A, Penn J, Zhao A, Shao W, Stahl N, Murphy AJ, Hamon S, Bouzelmat A, Zhang R, Shumel B, Pordy R, Gipe D, Herman GA, Sheu WHH, Lee IT, Liang KW, Guo X, Rotter JI, Chen YI, Kraus WE, Shah SH, Damrauer S, Small A, Rader DJ, Wulff AB, Nordestgaard BG, Tybjaerg-Hansen A, van den Hoek AM, Princen HMG, Ledbetter DH, Carey DJ, Overton JD, Reid JG, Sasiela WJ, Banerjee P, Shuldiner AR, Borecki IB, Teslovich TM, Yancopoulos GD, Mellis SJ, Gromada J, Baras A. Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease. N Engl J Med 2017; 377:211-221
Levy E, Poinsot P, Spahis S. Chylomicron retention disease: genetics, biochemistry, and clinical spectrum. Curr Opin Lipidol 2019; 30:134-139
Sissaoui S, Cochet M, Poinsot P, Bordat C, Collardeau-Frachon S, Lachaux A, Lacaille F, Peretti N. Lipids Responsible for Intestinal or Hepatic Disorder: When to Suspect a Familial Intestinal Hypocholesterolemia? J Pediatr Gastroenterol Nutr 2021; 73:4-8

OBESITY

Hyperparathyroidism in Chronic Kidney Disease

ABSTRACT

Chronic kidney disease (CKD) is associated with a mineral and bone disorder (CKD-MBD) which starts early in the course of the disease and worsens with its progression. The main initial serum biochemistry abnormalities are increases in fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) and decreases in 1,25 dihydroxy vitamin D (calcitriol) and soluble α-Klotho (Klotho), allowing serum calcium and phosphate to stay normal. Subsequently, serum 25 hydroxy vitamin D (calcidiol) decreases and in late CKD stages hyperphosphatemia develops in the majority of patients. Serum calcium may stay normal, decrease, or increase. More recent reports showed that sclerostin, Dickkopf-1, and activin A also play an important role in the pathogenesis of CKD-MBD. Both the synthesis and the secretion of PTH are continuously stimulated in the course of CKD, resulting in secondary hyperparathyroidism. In addition to the above systemic disturbances, downregulation of vitamin D receptor, calcium-sensing receptor, and Klotho expression in parathyroid tissue further enhances PTH overproduction. Last but not least, miRNAs have also been shown to be involved in the hyperparathyroidism of CKD. The chronic stimulation of parathyroid secretory function is not only characterized by a progressive rise in serum PTH but also by parathyroid gland hyperplasia. It results from an increase in parathyroid cell proliferation which is not fully compensated by a concomitant increase in parathyroid cell apoptosis. Parathyroid hyperplasia is initially of the diffuse, polyclonal type. In late CKD stages it often evolves towards a nodular, monoclonal or multiclonal type of growth. Enhanced parathyroid expression of transforming growth factor-α and its receptor, the epidermal growth factor receptor, is involved in polyclonal hyperplasia. Chromosomal changes have been found to be associated with clonal outgrowth in some, but not the majority of benign parathyroid tumors removed from patients with end-stage kidney disease. In initial CKD stages skeletal resistance to the action of PTH may explain why low bone turnover predominates in a significant proportion of patients, together with other conditions inhibiting bone turnover such as reduced calcitriol levels, sex hormone deficiency, diabetes, Wnt inhibitors, and uremic toxins. High turnover bone disease (osteitis fibrosa) occurs only later on, when increased serum PTH levels are able to overcome skeletal PTH resistance. The diagnosis of secondary uremic hyperparathyroidism and osteitis fibrosa relies mainly on serum biochemistry. X-ray and other imaging methods of the skeleton provide diagnostically relevant information only in severe forms. From a therapeutic point of view, it is important to prevent the development of secondary hyperparathyroidism as early as possible in the course of CKD. A variety of prophylactic and therapeutic approaches are available, as outlined in the final part of the chapter.

INTRODUCTION

Chronic kidney disease (CKD) is almost constantly associated with a systemic disorder of mineral and bone metabolism, at present named CKD-MBD (1). According to this definition, the disorder is manifested by either one or a combination of biochemical abnormalities (abnormal calcium, phosphate, PTH, or vitamin D metabolism), bone abnormalities (abnormal bone turnover, mineralization, volume, linear growth, or strength) and vascular or other soft tissue calcification. More recently, the underlying pathophysiology has become more complex, with the progressive awareness that fibroblast growth factor 23 (FGF23), a-Klotho (subsequently called "Klotho") as well as the Wnt-b-catenin signaling pathway also play an important role (see below). CKD-MBD generally becomes apparent in CKD stage G3, i.e., at a glomerular filtration rate between 60 and 30 ml/min x 1.73 m². Initially, it is characterized by a tendency towards hypocalcemia, fasting normo- or hypophosphatemia, and diminished plasma 25OH vitamin D (calcidiol) and 1,25diOH vitamin D (calcitriol) concentrations, together with a progressive increase in plasma FGF23 and intact parathyroid hormone (iPTH), a decrease in plasma soluble Klotho (2–5) and the development of renal osteodystrophy. Renal osteodystrophy often presents initially as adynamic bone disease and subsequently transforms into osteitis fibrosa or mixed bone disease (6). Pure osteomalacia is seen only infrequently. The low bone turnover observed in a significant proportion of patients in early stages of CKD could be due to the initial predominance of bone turnover inhibitory conditions such as resistance to the action of PTH, reduced serum calcitriol levels, sex hormone deficiency, diabetes, inflammation and malnutrition, and uremic toxins leading to the repression of osteocyte Wnt-β-catenin signaling and increased expression of Wnt antagonists such as sclerostin, Dickkopf-1, and secreted frizzled-related protein 4 (7,8). According to this scenario, high turnover bone disease occurs only later on, when sufficiently elevated serum PTH levels are able to overcome the skeletal resistance to its action. Even at that stage, over suppression of PTH by the administration of excessive calcium and/or vitamin D supplements can again induce adynamic bone disease (9). Nephrologists became progressively aware of the fact that the abnormally high serum phosphorus levels in late CKD stages, associated with either hyperparathyroidism or (mostly iatrogenically induced) hypoparathyroidism, may be detrimental to the patients not only in terms of abnormal bone structure and strength, but also in terms of the relative risk of soft-tissue calcifications and cardiovascular as well as all-cause mortality (10–13). As regards serum PTH levels, observational studies have consistently reported an increased relative risk of death in patients with CKD stage G5 and PTH values at the extremes, that is less than two or greater than nine times the upper normal limit of the assay (14,15). For PTH values within the range of two to nine times the upper normal limitreports of associations with relative risk of cardiovascular events or death in patients with CKD have been inconsistent. Of note, however, a report in elderly men in the community showed a strong association between plasma iPTH in the normal range and cardiovascular mortality (16).

SECONDARY HYPERPARATHYROIDISM IN CKD – SEQUENCE OF PLASMA BIOCHEMISTRY CHANGES IN EARLY CKD STAGES (Figure 1)

Figure 1. Schematic view of the time profile of disturbances in mineral hormones and bone turnover with progression of chronic kidney disease (CKD). From Drueke & Massy (6).

Phosphate Retention

The precise sequence of metabolic anomalies in incipient CKD leading to secondary hyperparathyroidism remains a matter of debate. Many years ago, it was postulated that a retention of phosphate in the extracellular space due to the decrease in glomerular filtration rate and the accompanying reduction in plasma ionized calcium concentration was the primary event in the pathogenesis of secondary hyperparathyroidism. These anomalies would only be transient and a new steady state would rapidly be reached, with normalization of plasma calcium and phosphate in response to increased PTH secretion and the well-known inhibitory effect of this hormone on the tubular reabsorption of phosphate (“trade-off hypothesis” of Bricker and Slatopolsky) (17). However, this hypothesis has become less attractive since it was demonstrated that plasma phosphate is only rarely elevated in early CKD, and phosphate balance was found to be not positive but negative, at least in rats with moderate-degree CKD (18). Most often, plasma phosphate remains normal until CKD stages G4-G5 (2,19). It may even be moderately diminished in CKD (20). Oral phosphate absorption remains normal in early stages of experimental CKD (18), and urinary phosphate excretion after an oral overload in patients with mild CKD was actually found to be accelerated (20). Nonetheless, one could argue that in early kidney failure normal or even subnormal concentrations of plasma phosphate might be observed after a slight, initial plasma phosphate increase following phosphate ingestion and stimulation of the secretion of FGF23 and PTH, which in turn could overcorrect plasma phosphate rapidly, due to a more potent inhibition of tubular phosphate reabsorption. However, a more recent study identified slight increases of plasma phosphate in a large US population sample (NHANES III) with CKD stage G3 as compared to a healthy control population without evidence of kidney disease (21). Probably both the time of plasma phosphate determinations during the day as well as subtle changes in circulating and local factors involved in the control of phosphate balance determine the actual plasma level of phosphate in patients with CKD.

Fibroblast Growth Factor 23 (FGF23) and Klotho

FGF23 is recognized at present as a major, if not the most important player in the control of phosphate metabolism. It is mainly produced by osteocytes and osteoblasts. It decreases plasma phosphate by reducing tubular phosphate reabsorption similar to, but independent of PTH. Moreover, in contrast to PTH it decreases the renal synthesis of calcitriol. To activate its receptors FGFR-1 and FGFR-3 on tubular epithelial cells it requires the presence of Klotho (or more precisely α-Klotho), which in its function as a co-receptor confers FGF receptor specificity for FGF23 (22). Although initially Klotho expression was found only in the distal tubule, it has subsequently been demonstrated to occur in the proximal tubule as well. In line with this finding, ablation of Klotho specifically from the distal tubules certainly resulted in a hyperphosphatemic phenotype, but to a lesser degree than in systemic or whole nephron Klothoknockout models (23). The regulation of FGF23 production and its interrelations with PTH, calcitriol, calcium, phosphate, and Klotho are complex, being only progressively unraveled. Isakova et al. provided evidence that serum FGF23 increased earlier than serum iPTH in patients with CKD (4). This observation is also supported by experiments in an animal model of CKD and the use of anti-FGF23 antibodies (24). However, the authors of a subsequent large-scale population study took issue with the claim that the increase in circulating FGF23 preceded that of PTH (25). Klotho expression in kidney, Klotho plasma levels, and Klotho urinary excretion decrease with progressive CKD (26,27). The presence of Klotho is required to allow FGF23 to exert its action in the kidney. In addition, Klotho also exerts FGF23 independent effects. It acts from the tubular luminal side as an autocrine or paracrine enzyme to regulate transporters and ion channels. By modifying the Na-phosphate cotransporter NaPi2a it can enhance phosphaturia directly (28). However, its purported glycosidase activity has been put into question recently (29). The issue then arises which comes first in CKD, an increase in FGF23 or a decrease in Klotho? The answer remains a matter of debate (30). Some studies showed that secreted soluble Klotho levels decrease before FGF23 levels increase (31,32) but the sequence of events may differ depending on experimental models and diverse clinical conditions (33). CKD is probably the most common cause of chronically elevated serum FGF23 levels (34). FGF23 production in bone is increased by phosphate, calcitriol, calcium, PTH, Klotho, and iron. Not all of these effects are necessarily direct. The effect of PTH clearly is both direct, via stimulation of PTH receptor-1 (PTH-R1) (35) and the orphan nuclear receptor Nurr1 (36), and indirect, via an increase in calcitriol synthesis (37). On the other hand, FGF23 inhibits PTH synthesis and secretion although in CKD this effect is mitigated by reduced Klotho and FGFR-1 expression in parathyroid tissue (38–40).

The increase in circulating FGF23 with the progression of CKD is independently associated with serum phosphate, calcium, iPTH, and calcitriol (41,42). Despite its direct inhibitory action on the parathyroid tissue FGF23 contributes to the progression of secondary hyperparathyroidism by reducing renal calcitriol synthesis and subsequently decreasing active intestinal calcium transport. Figure 2 shows the complex interrelations between serum FGF23, Klotho, phosphate, calcium, calcitriol, and parathyroid function in CKD.

Figure 2. Chronic kidney disease-associated mineral and bone disorder (CKD-MBD). Complex interactions between phosphate, FGF23, FGF receptor-1c (FGFR1c), Klotho, 1,25diOH vitamin D (calcitriol), renal 1α 25OH vitamin D hydroxylase (1α hydroxylase), vitamin D receptor (VDR), calcium, Ca-sensing receptor (CaSR), and parathyroid hormone (PTH). From Komaba & Fukagawa (43), modified.

Calcium Deficiency

In early CKD stages, disturbances of calcium metabolism may already be present. They include a calcium deficiency state due to a negative calcium balance resulting from low oral calcium intakes and impaired active intestinal calcium absorption (although a positive calcium balance can be induced by the ingestion of high amounts of calcium-containing phosphate binders) (44,45), a tendency towards hypocalcemia due to skeletal resistance to the action of PTH (46), and reduced calcium-sensing receptor (CaSR) expression in the parathyroid cell. All these factors contribute to the development of parathyroid over function (46,47). Their relative importance increases with the progression of CKD. It also depends on individual patient characteristics such as the underlying type of nephropathy, comorbidities, dietary habits, and amount of food intake.

Inhibition of Calcitriol Synthesis

The progressive loss of functioning nephrons and increased production of FGF23 are mainly responsible for the reduction in renal calcitriol synthesis, favoring the development of parathyroid over function. Although PTH in turn stimulates renal tubular 1α-OH vitamin D hydroxylase activity resistance to its action probably attenuates this counter-regulatory mechanism. Whether the direct inhibition of 1α-OH vitamin D hydroxylase activity by FGF23 is more powerful than its stimulation by PTH depends on several other additional factors such as the presence of hyperphosphatemia, metabolic acidosis, and uremic toxins. The marked disturbances of the calcitriol synthesis pathway probably explain the long reported direct relation in CKD patients between plasma calcidiol and calcitriol, and between plasma calcitriol and glomerular filtration rate (48). Such relations are not observed in people with normal kidney function.

Yet another hypothesis is based on the observation that calcidiol does not penetrate into proximal tubular epithelium from the basolateral side, but only from the luminal side. The complex formed by calcidiol and its binding protein (DBP) is ultrafiltered by the glomerulus, subsequently enters the tubular epithelium from the apical side via the multifunctional brush border membrane receptor megalin, and then serves as substrate for the renal enzyme, 1α-OH vitamin D hydroxylase for calcitriol synthesis (Figure 3) (49). Reduced glomerular filtration leads to a decrease in calcidiol-DBP complex transfer into the proximal tubular fluid and hence reduced availability of calcidiol substrate for luminal reabsorption and calcitriol formation. However, the validity for the human situation of this mechanism established in the mouse has subsequently been questioned since 1α-OH vitamin D hydroxylase expression was found not only in proximal, but also in distal tubular epithelium of human kidney, that is in tubular areas in which megalin apparently is not expressed (50).

Figure 3. Schematic representation of the role of megalin in renal tubular 25 OH vitamin D reabsorption. Megalin is a multifunctional brush border membrane receptor expressed in the proximal renal tubule. It enables endocytic reabsorption of 25 OH vitamin D (calcidiol) filtered by the glomerulus and the subsequent synthesis of 1,25 diOH vitamin D (calcitriol) by mitochondrial 1-a 25 OH vitamin D hydroxylase. After Nykjaer et al (49).

Finally, the concentration of plasma calcidiol is diminished in the majority of patients with CKD (51,52). The reasons for this vitamin D deficiency state include insufficient hours of sunshine or sun exposure especially in the elderly, skin pigmentation, intake of antiepileptic drugs (like in general population), and in addition enhanced urinary excretion of calcidiol complexed to vitamin D binding protein (DBP) in the presence of proteinuria, and loss into the peritoneal cavity in those on peritoneal dialysis treatment. All these factors may also contribute to the reduction in calcitriol synthesis (53). However, low plasma calcidiol has also been postulated to be a risk factor per se for secondary hyperparathyroidism, as suggested by an observational study in Algerian hemodialysis patients with insufficient exposure to sunshine (54) and the observation that calcidiol is able to directly suppress PTH synthesis and secretion in bovine parathyroid cells in vitro, although with much less potency than calcitriol (55).

SECONDARY HYPERPARATHYROIDISM IN CKD – PLASMA BIOCHEMISTRY CHANGES IN ADVANCED CKD STAGES (Figure 1)

The above-mentioned roles of relative or absolute deficiency states of calcium and vitamin D are steadily gaining importance with the progression of CKD, and phosphate becomes a major player.

Role of Hyperphosphatemia

In CKD stages G4-G5 hyperphosphatemia becomes an increasingly frequent feature (19), due to phosphate retention caused by the progressive loss of functioning nephrons and the increasing difficulty in augmenting glomerular phosphate ultrafiltration and to further reduce its tubular reabsorption when it is already maximally inhibited by high serum FGF23 and PTH levels.

FGF23 Excess and Klotho Deficiency

Circulating FGF23 may reach extremely high, maladaptive concentrations in patients with end-stage kidney disease (ESKD) (56). In parallel, a reduction of Klotho expression is observed in kidney and parathyroid tissue, as well as of soluble Klotho in the plasma and urine of patients and animals with CKD (26,27,30). The reduction is particularly marked in advanced stages of CKD. The resulting resistance to the action of FGF23 in kidney and parathyroid tissue favors hyperparathyroidism (see below).

The uremic syndrome itself could also play a role. In addition to phosphate many other so-called uremic toxins, that is substances which accumulate in the uremic state, are known to interfere with vitamin D metabolism and action (57,58). Indoxyl sulfate has been shown to participate in the pathogenesis of skeletal resistance to the action of PTH (59), in addition to direct inhibitory effects on bone turnover (60).

MECHANISMS INVOLVED IN THE PATHOGENESIS OF SECONDARY HYPERPARA-THYROIDISM

Generally speaking, there are at least two major different mechanisms which determine the magnitude of secondary hyperparathyroidism in CKD. The first is an increase in PTH synthesis and secretion, and the second an increase in parathyroid gland mass, mostly due to enhanced cell proliferation (hyperplasia), and to a lesser degree also an increase in cell size (hypertrophy) (see schematic representation in Figure 4). Whereas acute stimulation of PTH synthesis and/or release generally occurs in the absence of cell growth stimulation, these two processes appear to be tightly linked whenever there is chronic stimulation. The main factors involved in the control of the two processes are again calcitriol, calcium, and phosphate whereas the direct effects of FGF23 appear to be essentially limited to the control of PTH synthesis and secretion. In the following, the disturbances of the mechanisms controlling parathyroid function will be discussed subsequently for each of these three factors, although there are numerous interactions between them. Subsequently, the influence of other factors and comorbid conditions related to CKD will be presented.

Figure 4. Pathogenesis of secondary hyperparathyroidism. Schematic representation of parathyroid hormone (PTH) synthesis and secretion (upper part) and parathyroid cell proliferation and apoptosis (lower part), as regulated by a number of hormones and growth factors.

Calcitriol

The above-mentioned decrease in plasma calcitriol aggravates hyperparathyroidism via several mechanisms. The first is direct and results from an insufficient inhibition of PTH synthesis due to low circulating calcitriol levels and a disturbed action of calcitriol at the level of the preproPTH gene. It is well established that calcitriol, after forming a complex with its receptor, vitamin D receptor (VDR) and heterodimerizing with the retinoic acid receptor (RXR), directly inhibits preproPTH gene transcription by binding to a specific DNA response element (VDRE) located in the 5’-flanking region of the gene. In CKD, in addition to low extracellular concentrations of calcitriol, at least two other factors interfere with calcitriol’s action on the preproPTH gene (61). The first factor is a reduced expression of the VDR gene in hyperplastic parathyroid tissue of CKD patients (62). This reduction is particularly marked in nodular, as compared to diffusely hyperplastic parathyroid tissue. The second factor is reduced binding of calcitriol to VDR, slowed nuclear migration of the calcitriol–VDR complex, and less efficient inhibitory action on the preproPTH gene, in association with the uremic state (58,63). Of note, extracellular Ca²⁺ concentration [Ca²⁺_e] appears to play a role in the regulation of VDR expression. In rat parathyroid glands, low [Ca²⁺_e] reduced VDR expression independently of calcitriol, whereas high [Ca²⁺_e] increased it (64). Hypocalcemia may attenuate by this mechanism the feedback of increased plasma calcitriol concentrations on the parathyroids.

The second level at which calcitriol regulates PTH gene expression involves calreticulin. Calreticulin is a calcium binding protein which is present in the endoplasmic reticulum of the cell, and also may have a nuclear function. It regulates gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors of sterol hormones. Sela-Brown et al. proposed that calreticulin might inhibit vitamin D's action on the PTHgene, based on in vitro and in vivo experiments (65). They fed rats either a control diet or a low calcium diet, which led to increased PTH mRNA levels despite high serum calcitriol levels that would be expected to inhibit PTH gene transcription. Their postulate that high calreticulin levels in the nuclear fraction might prevent the effect of calcitriol on the PTH gene was strongly supported by the observation that hypocalcemic rats had increased levels of calreticulin protein in parathyroid nuclear fraction. This could explain why hypocalcemia leads to increased PTH gene expression despite high serum calcitriol levels, and might also be relevant for the refractoriness of secondary hyperparathyroidism to calcitriol treatment observed in many patients with CKD.

The third mechanism of calcitriol’s action could be indirect, via a stimulatory effect on parathyroid CaSR expression, as shown by Brown et al (66) and subsequently confirmed by Mendoza et al (67).

The fourth mechanism is again a direct one. It concerns the well-known inhibitory effect of vitamin D on cell proliferation and the induction of differentiation towards mature, slowly growing cells. A decrease in plasma calcitriol and a perturbed action at molecular targets favors abnormal cell growth. This is the case with parathyroid tissue as well, and parathyroid hyperplasia ensues (68). The importance of vitamin D in the pathogenesis of parathyroid hyperplasia of experimental uremia has first been shown by Szabo et al (69). These authors administered increasing doses of calcitriol to rats either at the time of inducing chronic kidney failure or at a later time point, when uremia was already well established. They were able to prevent parathyroid cell proliferation entirely when calcitriol was given in initial CKD stages, but not when given later on. Fukagawa et al showed that pharmacologic doses of calcitriol repressed c-myc expression in the parathyroid tissue of uremic rats and suggested that the hormone might suppress parathyroid hyperplasia by this pathway (70). In contrast, Naveh-Many et al. (71) failed to observe such an antiproliferative effect of calcitriol in parathyroid cells of uremic rats but they administered the hormone for only three days. Such short-term administration may not have been sufficient for an efficacious suppression of cell turnover.

To answer the question of a possible direct calcitriol action on parathyroid cells, several studies were performed in experimental models in vitro. Nygren et al. (72) showed in primary cultures of bovine parathyroid cells, maintained in short-term culture, that these cells underwent significant increases both in number and size in response to fetal calf serum, and that the addition of 10-100 ng/mL calcitriol almost completely inhibited cell proliferation whereas cell hypertrophy was unaffected. Kremer et al (73) subsequently confirmed in the same parathyroid cell model that calcitriol exerted an anti-proliferative action. They further suggested that this inhibition occurred via a reduction of c-myc mRNA expression. One report showed an inhibitory action under long-term culture conditions (up to 5 passages) of the effect of calcitriol on bovine parathyroid cell proliferation (74). Our group subsequently confirmed such a direct antiproliferative effect of calcitriol in a human parathyroid cell culture system derived from hyperplastic parathyroid tissue of patients with severe secondary uremic hyperparathyroidism (75) (Figure 5).

Figure 5. Antiproliferative effect of 1,25 diOH vitamin D on parathyroid cells. Reduction of parathyroid cell proliferation in response to increasing medium 1,25diOH vitamin D (calcitriol) concentrations in the incubation milieu of a human parathyroid cell culture system, with parathyroid cells derived from hyperplastic parathyroid tissue of patients with severe secondary uremic hyperparathyroidism. From Roussanne et al (75).

A fifth mechanism is the potential association between parathyroid function and vitamin D receptor (VDR) polymorphism. Fernandez et al (76) separated hemodialysis patients with same serum calcium and time on dialysis treatment into two groups, according to their serum iPTH levels, namely low PTH (<12 pmol/L) or high PTH (>60 pmol/L). They found that the BB genotype and the B allele were significantly more frequent in the low PTH than in the high PTH group (32.3 % vs 12.5 %, and 58.8% vs 39.1%, respectively). This information suggests that VDR gene polymorphism influences parathyroid function in CKD. Similar results have been reported by an Italian group (77) and in a large sample of Japanese hemodialysis patients (78). In this latter study, after excluding patients with diabetes and patients with a dialysis vintage of less than ten years, the authors observed lower plasma iPTH levels in ESKD patients with BB than with Bb or bb alleles. A relationship between Apa I polymorphism (A/a alleles) and the severity of hyperparathyroidism has also been sought in Japanese hemodialysis patients (79). Plasma PTH levels in AA and Aa groups were approximately half that of the aa group. However, other groups found no difference in PTH levels for various VDR polymorphisms (80–82). Moreover, although in some clinical conditions VDR polymorphism may be associated with variations of the half-life of the VDR gene transcript (83) or of VDR function (84), there has been no report showing that in uremic patients with secondary hyperparathyroidism the density of parathyroid cell VDR varies with different VDR genotypes. In addition, although VDR genotypes may have some influence on the degree of parathyroid cell proliferation, the mechanism by which this could occur remains unknown.

Finally, Egstrand et al recently provided experimental evidence for the role of a circadian clock operating in parathyroid glands. This clock and downstream cell cycle regulators were shown to be disturbed in uremic rats, potentially contributing to dysregulated parathyroid proliferation in secondary hyperparathyroidism (85).

Calcium

It has long been known that [Ca²⁺_e] is the primary regulator of PTH secretion. Small changes in serum Ca²⁺concentration result in immediate changes of PTH release which are short-lived or long-lived, depending on the velocity of the restoration of serum Ca²⁺towards normal. Thus, postprandial urinary calcium excretion was increased in patients with CKD as it was in healthy volunteers, but only in the patients was this accompanied by significantly reduced serum Ca²⁺ and increased PTH levels (86). The inverse relation between Ca²⁺ and PTH in the circulation obeys a sigmoidal curve (87). While the majority of in vitro studies have reported a decreased responsiveness of hyperplastic parathyroid cells to changes in [Ca²⁺_e] in vivo studies have not always confirmed this. Such discrepant findings are likely due to different methods used to assess the dynamics of PTH secretion (88).

Several in vitro studies have shown that the set point of calcium for PTH secretion (that is the Ca²⁺ concentration required to produce half maximal PTH secretion) is greater in parathyroid cells from primary adenomas and secondary (uremic) hyperplastic parathyroid glands than in normal parathyroid cells (89). Such a relatively poor response to [Ca²⁺_e] should contribute to the increased PTH levels observed in uremic patients with secondary hyperparathyroidism.

We and others have demonstrated that both primary parathyroid adenoma and secondary uremic, hyperplastic parathyroid gland tissue exhibit a decrease in the expression of CaSR protein (90,91). In secondary uremic hyperparathyroidism, there is a significant decrease of CaSR in diffusely growing hyperplastic tissue, with the decrease being even more marked in nodular areas (characteristic of advanced hyperparathyroidism with autonomously growing cells) (90) (Figure 6). Since changes in intracellular Ca²⁺elicited by hyper or hypocalcemia depend on the expression and activity of the CaSR, any decrease explains, at least in part, an impaired intracellular calcium response to [Ca²⁺_e] and hence a reduced inhibitory effect of the cation on PTH secretion. Several factors contribute to the downregulation of CaSR expression and/or activity in CKD including reduced calcitriol levels (66,67), low magnesium levels (92), dietary phosphate (probably indirect action) (93), and metabolic acidosis (94). However, raising extracellular phosphate has been recently shown to also exert a direct inhibitory action on parathyroid cell CaSR activity of isolated human parathyroid cells resulting in an increase in PTH secretion (95). Almaden et al studied calcium-regulated PTH response in vitro, using respectively primary parathyroid adenoma and uremic hyperplastic tissue, the latter either of the nodular or the diffuse type (96). They found that in primary adenoma tissue PTH secretion was less responsive to an increase in [Ca²⁺_e] than in uremic hyperplastic parathyroid tissue; among the latter, nodular tissue was less responsive than diffusely hyperplastic tissue. The decreased secretory response to Ca²⁺observed in nodular uremic hyperplasia may be explained by the markedly reduced CaSR expression in CKD, as demonstrated by Gogusev et al (90). This decrease can be overcome, at least partially, by PTHrp, as shown by Lewin et al (97), who observed that the administration of PTHrp significantly stimulated the impaired secretory capacity of the parathyroid glands of uremic rats in response to hypocalcemia. Of note, this observation also implies that the PTH/PTHrp receptor is expressed on the parathyroid cell.

Figure 6. Calcium-sensing receptor (CaSR) expression in normal and hyperplastic parathyroid glands. Normal parathyroid tissue (in blue), secondary (2°) hyperparathyroidism from dialysis patients (glands with diffuse hyperplasia in yellow; glands with nodular hyperplasia in green), and primary (1°) adenomatous hyperparathyroidism from patients with conserved kidney function (in orange). Decreased expression of both CaSR protein and mRNA in the majority of hyperplastic glands, with a particularly marked decrease in nodular type secondary uremic hyperparathyroidism. After Gogusev et al (90).

The shift of the calcium set point to the right in dialysis patients in vivo has been a much less constant finding than the right shift observed in the above-mentioned studies in uremic parathyroid tissue in vitro. While in CKD patients with a mild to moderate degree of hyperparathyroidism the set point was most often found to be normal, an altered set point was observed in presence of severe parathyroid over function with hypercalcemia (98). This anomaly could at least in part be due to CaSR down-regulation. As regards CKD patients with less severe parathyroid over function, a considerable controversy took place regarding the results of in vivo assessments of parathyroid gland function (99,100). In part, disparities among study results reflected technical differences in experimental methods and/or variations in the mathematical modeling of PTH secretion in vivo (101). Another difficulty in interpreting the results of in vivo dynamic tests of parathyroid gland function relates to the issue of parathyroid gland size. Because there is a basal, or non-suppressible, component of PTH release from the parathyroid cell even at high [Ca²⁺_e], excessive PTH secretion may result solely from increases in parathyroid gland mass (98). This can theoretically occur in the absence of any defect in calcium sensing at the level of the parathyroid cell. Since parathyroid gland hyperplasia is present to some extent in nearly all patients with CKD stages G3-G5, alterations in PTH secretion due to increases in parathyroid gland mass cannot readily be distinguished from those attributable to changes in calcium-sensing by the parathyroid cell using the four-parameter model for in vivo studies (100).

The role of calcium in parathyroid cell proliferation is less clear than is generally assumed. Calcium deficiency, in the presence or absence of hypocalcemia, together with vitamin D deficiency or reduced production of calcitriol, probably is a major stimulus of parathyroid hyperplasia. Naveh-Many et al showed that calcium deprivation, together with vitamin D deficiency, greatly enhanced the rate of parathyroid cell proliferation in normal rats and also in rats with CKD, using the cell cycle-linked antigen, PCNA (71). The concomitant decrease in CaSR expression in CKD, as observed in parathyroid glands of both dialysis patients and uremic rats (90,102), should theoretically enhance parathyroid tissue hyperplasia further. Indirect support for this contention came from the observation that the administration of the calcimimetic compound NPS R-568, a CaSR agonist, led to the suppression of parathyroid cell proliferation in rats with CKD (103). However, in the study by Naveh-Many et al the dietary regimen was poor in both calcium and vitamin D. In contrast, when feeding normal rats on a calcium-deficient diet alone, in the absence of concomitant vitamin D deficiency, Wernerson et al observed parathyroid cell hypertrophy, not hyperplasia (104).

The question whether the effect of calcium is direct or indirect remains therefore unsolved at present. It can only be answered by in vitro studies. For a long time, available culture systems using normal parathyroid cells did not allow the maintenance of functionally active cells for prolonged time periods. They were all characterized by a rapid and significant loss of PTH secretion within 3 to 4 days (105–107). One culture model has been described, using bovine parathyroid cell organoids, which maintained the ability to modulate PTH secretion in response to [Ca²⁺_e] and tissue-like morphology for 2 weeks (108). However, only one long-term study using bovine parathyroid cells demonstrated a release of bioactive bovine PTH but with reduced sensitivity to [Ca²⁺_e] (109). Other reports showed that the rapid decrease in PTH responsiveness of cultured bovine parathyroid cells to changes in [Ca²⁺_e] was associated with a marked reduction in CaSR expression (110,111). Yet other parathyroid cell-derived culture models proposed in the literature were in fact devoid of any PTH secretory capacity (112,113).

To study direct effects of [Ca²⁺_e] on the parathyroid cell in vitro, we developed a functional human parathyroid cell culture system capable of maintaining regulation of its secretory activity and the expression of extracellular CaSR mRNA and protein for several weeks. For this purpose, we used parathyroid cells derived from hyperplastic parathyroid tissue of hemodialysis patients with severe secondary hyperparathyroidism (114). In a subsequent study with this experimental model, we surprisingly obtained evidence that parathyroid cell proliferation index, as estimated by [³H]-thymidine incorporation into an acid-precipitable fraction as a measure of DNA synthesis, could be directly stimulated by high [Ca²⁺_e] in the incubation medium, compared with low [Ca²⁺_e] (75) (Figure 7).

Figure 7. Effect of medium calcium concentration on parathyroid cell proliferation. Stimulatory effect on parathyroid cell proliferation (measured by KI-67 staining method) of high medium calcium concentrations in the incubation milieu of a human parathyroid cell culture system derived from hyperplastic parathyroid tissue of patients with severe secondary uremic hyperparathyroidism. From Roussanne et al (75).

We confirmed this finding in independent experiments using the cell cycle-linked antigen Ki-67 to determine parathyroid cell proliferation. However, the addition of the calcimimetic NPS R-467 to the incubation medium led to a decrease in cell proliferation (Figure 8).

Figure 8. Inhibitory effect of calcimimetic on parathyroid cell proliferation. Human parathyroid cells derived from hyperplastic parathyroid tissue of patients with severe secondary uremic hyperparathyroidism were maintained in high medium calcium incubation milieu, and exposed to increasing concentrations of calcimimetic NPS R-467. Determination of cell proliferation by [3H]-thymidine incorporation method. After Roussanne et al (75).

Of interest, calcimimetics have subsequently been shown to upregulate the expression of both CaSR (67,115) and VDR (67) in parathyroid glands of uremic rats. In an attempt to unify our apparently contradictory in-vitro observations with respect to findings made in vivo, we proposed the following hypothesis. The effect of calcium on parathyroid cell proliferation could occur along two different pathways, via two distinct mechanisms. Inhibition of proliferation would occur via the well-known parathyroid CaSR-dependent pathway, whereas stimulation of proliferation would occur via an alternative pathway (Figure 9). Note that the parathyroid tissue samples used in our study stemmed from uremic patients with long-term ESKD and severe secondary hyperparathyroidism. Since such parathyroid tissue generally exhibits decreased CaSR expression, it is possible that the number of CaSR expressed in the parathyroid cell membranes of our culture model was insufficient to inhibit cell proliferation. Of note, the human CaSR gene has two promoters and two 5’ untranslated exons; therefore, the alternative usage of these exons leads to production of multiple CaSR mRNAs in parathyroid cells (116). The expression of CaSR mRNA produced by one of the two promoters of CaSR gene is specifically reduced in parathyroid adenomas, suggesting a role in PTH hypersecretion and proliferation. Moreover, the membrane-bound 550-kD Ca²⁺-binding glycoprotein megalin, belonging to the low-density lipoprotein receptor superfamily, has been identified in parathyroid chief cells as another putative calcium-sensing molecule which could be involved in calcium-regulated cellular signaling processes as well (117). Based on these observations, one can postulate that parathyroid cells express multiple CaSR-like molecules. Consequently, if the well-known parathyroid CaSR is downregulated, parathyroid cell proliferation induced by increases in [Ca²⁺_e] may occur via a different type of CaSR. Another possibility is an alteration in post-receptor signal transduction that could occur in hyperparathyroid states or under cell culture conditions. Our observations are in line with findings by Ishimi et al. which were incompatible with a direct effect of low [Ca²⁺_e] in the pathogenesis of parathyroid hyperplasia (74). However, any extrapolation from such in vitro observations to the in vivo setting should be done with caution, and further work is needed to define the precise pathway(s) by which calcium regulates parathyroid tissue growth.

Figure 9. Hypothesis of the regulation of parathyroid cell proliferation by extracellular [Ca2+]. 1) Inhibitory pathway via the calcium-sensing receptor (CaR). 2) Stimulatory pathway via an unknown transmembrane transduction mechanism. Physiologically, pathway 1 predominates over pathway 2. In presence of parathyroid hyperplasia with calcium-sensing receptor down-regulation pathway 2 could become dominant and favor parathyroid cell proliferation over suppression. After Roussanne et al (75).

Phosphate

Hyperphosphatemia is associated with increased PTH secretion. The stimulation of PTH release occurs via direct and indirect mechanisms. The initially proposed indirect mechanism, which remains true according to present knowledge, is via a decrease in plasma Ca²⁺ concentration (see above). Hyperphosphatemia also leads to an inhibition of the renal synthesis of calcitriol, probably mostly via stimulation of FGF23 production.

A direct action of phosphate on PTH secretion by the parathyroid cell has long been suspected. However, it has been formally demonstrated in vitro only in 1996 (118–120). This demonstration required the use of either intact parathyroid glands (from rats) (Figure 10) or parathyroid tissue slices (from cows) whereas it had not been possible to obtain such direct stimulation using the classic model of isolated bovine parathyroid cells. Elevating plasma phosphate concentration in the incubation milieu of experimental models using intact (or partially intact) parathyroid tissue leads to a stimulation of PTH secretion within some hours, in the absence of any change in [Ca²⁺_e]. It can however be abrogated by an increase in cytosolic Ca²⁺concentration (121).

Figure 10. Direct inhibition of parathyroid hormone (PTH) secretion by phosphate. Intact parathyroid glands obtained from normal rats were maintained in culture and exposed to increasing in phosphate concentrations in the incubation medium. After Almaden et al (121).

Silver’s group reported subsequently that phosphate, like calcium, regulates pre-pro-PTH gene expression post-transcriptionally by changes in protein-PTH mRNA interactions at the 3'-UTR which determine PTH mRNA stability. They identified the minimal sequence required for protein binding in the PTH mRNA 3'-UTR and determined its functionality. They found that the conserved PTH RNA protein-binding region conferred responsiveness to calcium and phosphate and determined PTH mRNA stability and levels (122). Thus, a low calcium diet increased stability, whereas a low phosphate diet decreased stability of PTH mRNA (123) (Figure 11). The PTH mRNA 3’-untranslated region-binding protein was subsequently identified by this research group as adenylate-uridylate-rich element RNA binding protein 1 (AUF1) (124).

Figure 11. Post-transcriptional regulation of PTH mRNA stability by calcium, phosphate, and kidney failure. Pre-pro-PTH gene expression is modulated via changes in protein-PTH mRNA interactions at the 3'-UTR region which determine PTH mRNA stability. Low calcium diet increases stability, whereas low phosphate diet decreases stability of PTH mRNA. PTH mRNA protective factor AUF1 in yellow, PTH mRNA degrading endonuclease in orange. After Yalcindag et al (123).

In addition to its stimulatory effect on PTH secretion a high phosphate diet also rapidly induces parathyroid over function and hyperplasia, as shown in experimental animal models (125). Subsequent studies showed that hyperphosphatemia induced by phosphate-rich diets in animals with CKD induced parathyroid hyperplasia even when changes in plasma Ca²⁺and calcitriol concentration were carefully avoided, pointing to a direct effect of phosphate on cell proliferation (71,120). Conversely, early dietary phosphate restriction in the course of CKD was capable of preventing both PTH over secretion and parathyroid hyperplasia (71,120,126). Interestingly, dietary phosphate restriction following phosphate overload in rats led to an immediate decrease in PTH secretion despite no regression of parathyroid gland size (127).

Our group wished to know whether the stimulatory effect of phosphate on parathyroid cell proliferation was direct or indirect. To answer this question, we used the above described in vitro model of human parathyroid cells maintained in long-term culture (114). We could show that cell proliferation index was directly stimulated by high phosphate concentrations in the incubation medium, compared with low phosphate concentration (75) (Figure 12). These experiments demonstrated that phosphate is capable of stimulating not only PTH secretion, but also of inducing parathyroid tissue hyperplasia by a direct mode of action.

Figure 12. Direct stimulatory effect of extracellular phosphate on parathyroid cell proliferation. Response of parathyroid cell growth to increasing phosphate concentrations in the incubation milieu of a human parathyroid cell culture system derived from hyperplastic parathyroid tissue of patients with severe secondary uremic hyperparathyroidism. Determination of cell proliferation by [3H]-thymidine incorporation method. After Roussanne et al (128).

FGF23 plays an important role in the control of plasma phosphate. Elevated FGF23 in CKD allows efficient inhibition of proximal tubular phosphate reabsorption and maintenance of plasma phosphorus in the normal range. However, since hyperphosphatemia directly stimulates PTH secretion, its correction by FGF23 indirectly leads to a reduction of PTH release, in addition to the direct inhibitory action of FGF23 on parathyroid secretory activity (see above).

FGF23 and Klotho

As mentioned before FGF23 directly inhibits PTH synthesis and secretion via its action on parathyroid FGFR-1 (129). FGF23 also increases parathyroid CaSR and VDR expression, further contributing to the suppression of PTH by this hormone (Canalejo 2010). In advanced stages of CKD FGF23’s effect is partially or even completely abolished owing to downregulation of the expression of its receptor and co-receptor Klotho (38–40). Of interest, in early stages of CKD there could be an initial upregulation of FGFR-1 and Klotho, with enhanced PTH secretion in response to FGF23 via an Na+/K+ -ATPase driven pathway (130). Subsequent findings suggested a function for Klotho in suppressing PTH biosynthesis and parathyroid gland growth, even in the absence of CaSR (131). Moreover, they pointed to a physical interaction between Klotho and CaSR. Specific deletion of CaSR in parathyroid tissue led to elevated serum PTH levels and parathyroid gland hyperplasia, and additional deletion of Klotho in parathyroid glands exacerbated this condition. However, a recent review concluded that role of parathyroid Klotho remains controversial (132).

MicroRNAs

More recently, Shilo et al provided evidence for the important role of microRNAs (miRNAs) in the physiological regulation of parathyroid function, and its dysregulation in the secondary hyperparathyroidism of CKD (133,134). The authors found an abnormal regulation of many miRNAs in experimental uremic hyperparathyroidism supporting a key role for miRNAs in this condition. Specifically, their studies showed that inhibition of the abundant let-7 family increased PTH secretion in normal and uremic rats, as well as in mouse parathyroid organ cultures. Conversely, inhibition of the upregulated miRNA-148 family prevented the increase in serum PTH of uremic rats, and inhibition of let-7 family also reduced PTH secretion in parathyroid cultures. Thus, miRNA dysregulation represents yet another crucial step in the pathogenesis of secondary hyperparathyroidism.

Other Factors and Conditions

As already pointed out above the uremic state with its accumulation of numerous uremic toxins is another long suspected, albeit yet ill-defined factor in the pathogenesis of secondary hyperparathyroidism. Recently, several pieces of evidence have been provided in favor of a role of the uremic state which interferes with the binding of calcitriol to VDR (58) and with the nuclear uptake of the hormone-receptor complex (63). This should have consequences not only for PTH synthesis and secretion, but also for parathyroid cell proliferation. Another mechanism of excessive proliferation involves the mTOR pathway, which has been shown to be activated in secondary hyperparathyroidism (135). Inhibition of mTOR complex 1 by rapamycin decreased parathyroid cell proliferation in vivo and in vitro.

Patients with diabetes receiving dialysis therapy have relatively low plasma PTH levels, as compared to those without diabetes. The high incidence of low bone turnover in uremic patients with diabetes (136–139) has been attributed to low levels of biologically active PTH, possibly via an inhibition of PTH secretion or a modification of the PTH peptide by the accumulation of advanced glycation end-products such as pentosidine (140) or else oxidation of PTH (141,142). However, experimental studies have demonstrated that the metabolic abnormalities associated with diabetes can also directly decrease bone turnover, independent of PTH (143). In general, patients with low bone turnover tend to develop hypercalcemia when on a normal or high dietary calcium intake, probably due to the diminished skeletal capacity of calcium uptake. This in turn tends to reduce plasma PTH. Thus, not only does hypoparathyroidism promote adynamic bone disease but adynamic bone disease also favors hypoparathyroidism. Another issue is whether in patients with diabetes abnormalities such as hyperglycemia and insulin deficiency or resistance may directly affect parathyroid function. In an in vitro study using dispersed bovine parathyroid cells, high glucose and low insulin concentrations suppressed the PTH response to low Ca²⁺ concentration (144). These results are compatible with the view that diabetes directly inhibits parathyroid function. However, when uremic rats were fed on a high phosphate diet to induce secondary hyperparathyroidism, the presence of diabetes did not affect the development of parathyroid over function (143).

Aluminum bone disease is generally associated with low serum PTH levels (145,146) and a decreased PTH response to stimulation by hypocalcemia (147,148). In aluminum intoxicated patients, high amounts of aluminum are also found in parathyroid tissue (149). The relatively low PTH levels may reflect either an inhibition of PTH secretion by the hypercalcemia commonly observed in this condition (150) or a direct inhibitory effect of aluminum on parathyroid cell function (151). Direct toxic effects of the trace element have also been demonstrated in studies in vitro (152,153).Observations made in experimental animals and results of clinical studies have been less clear. Whereas some experiments indicated that aluminum overload did not decrease plasma PTH levels in vivo (152,153), other experiments reported a decrease (154,155). Whatever the mechanisms involved, subsequent clinical data clearly showed that the introduction of an aluminum-free dialysis fluid and the discontinuation of aluminum contamination of the dialysate or aluminum removal with deferoxamine resulted in an increase in plasma PTH levels and in PTH response to hypocalcemia (156). Thus, although there appears to be an association between aluminum toxicity and parathyroid gland function, the interaction is complex.

Post-Receptor Mechanisms Involved in Polyclonal Parathyroid Tissue Growth

As pointed out above, calcitriol reduces parathyroid cell proliferation by decreasing the expression of the early gene, c-myc. This gene modulates cell cycle progression from G1 to S phase. A decrease in plasma calcitriol and/or a disturbance of its action at the level of the parathyroid cell, which are both frequently observed in uremic patients, may cause disinhibition of c-myc expression and progression into the cell cycle. Another mode of action involves the cyclin kinase inhibitor p21^WAF1. Calcitriol has long been shown to induce the differential expression of p21^WAF1 in the myelo-monocytic cell line U937 and to activate the p21 gene transcriptionally in a VDR-dependent, but p53-independent, manner, thereby arresting parathyroid growth (157). Slatopolsky’s group further showed that the administration of calcitriol to moderately uremic rats enhanced parathyroid p21 expression and prevented high phosphate-induced increase in parathyroid TGF-α content (157). In addition, they found that calcitriol altered membrane trafficking of the epithelial growth factor receptor (EGFR), which binds both EGF and TGF-α, and down-regulated EGFR mediated growth signaling (158). Induction of p21 and reduction of TGF-α content in the parathyroid glands also occurred when uremia-induced parathyroid hyperplasia was suppressed by high dietary Ca intake. The mechanisms by which a phosphate-rich diet and hyperphosphatemia induce parathyroid hyperplasia, and conversely a phosphate-poor diet and hypophosphatemia inhibit parathyroid tissue growth have also been examined by this group in a detailed fashion. Thus, Dusso et al showed that feeding a low phosphate diet to uremic rats increased parathyroid p21 gene expression through a vitamin D-independent mechanism (159). When administering a high phosphate diet, p21 expression was not suppressed. In this condition, they observed an increase in parathyroid tissue TGF-α expression and a direct correlation between this expression and parathyroid cell proliferation rate. This finding is in line with the previous observation by our group of de novo TGF-α expression in severely hyperplastic parathyroid tissue of patients with ESKD (160). The inducer of TGF-α gene transcription could be activator protein 2α (AP2), whose expression and transcriptional activity at the TGF-α promoter is increased in the secondary hyperparathyroidism of CKD (161).

Although these findings provide more insight into the pathways by which changes in phosphate intake, and ultimately variations in extracellular phosphate concentration, control parathyroid tissue growth the exciting question of the transmembrane signal transduction mechanism and subsequent nuclear events triggered by phosphate remains yet to be answered.

In addition to p21 and TGF-α, a variety of other growth factors and inhibitors are probably involved in polyclonal parathyroid hyperplasia. Thus, PTHrp has been proposed as a possible growth suppressor in the human parathyroid (162). PTHrp, and probably PTH itself, also exert an inhibitory effect on PTH secretion by acting via a negative feedback loop on PTH-R1 which appears to be expressed in the parathyroid cell membrane as well (97). Table 1summarizes various changes in gene and growth factor expression, which are potentially involved in the parathyroid tissue hyperplasia of secondary uremic hyperparathyroidism. Gcm2 has been identified as a master regulatory gene of parathyroid gland development, since Gcm2 knockout mice lack parathyroid glands (163). Correa et al. found high Gcm2 mRNA expression in human parathyroid glands in comparison with other non-neural tissues and under expression in parathyroid adenomas but not in lesions of HPT secondary to uremia (164). Gcm2 expression itself is regulated by Gata3, and Gata3, in cooperation with Gcm2 and MafB, stimulates PTH gene expression, by interacting with the ubiquitous transcription factor SP1 (165). MafB probably plays a role in uremic hyperparathyroidism as well. Thus, stimulation of the parathyroid by CKD in MafB+/-mice resulted in an impaired increase in serum PTH, PTH mRNA, and parathyroid cell proliferation (166,167).

Table 1. Changes in Gene and Growth Factor Expression Potentially Involved in Parathyroid Tissue Hyperplasia of Secondary Uremic Hyperparathyroidism

Early immediate genes and receptor/coreceptor genes

-Enhanced c-myc gene expression (Fukagawa et al, Kidney Int 1991; 39: 874-81)

-Decreased calcium-sensing receptor (CaSR) gene expression (Kifor et al, J Clin Endocrinol Metab 1996; 81: 1598-1606. Gogusev et al, Kidney Int 1997; 51: 328-36)

-Decreased vitamin D receptor (VDR) gene expression (Fukuda et al, J Clin Invest 1993; 92: 1436-42)

-Decrease in parathyroid Klotho and FGFR1c gene expression (Galitzer et al, Kidney Int 2010; 77: 211-8. Canalejo et al, JASN 2010; 21: 1125-35. Komaba et al, Kidney Int 2010; 77: 232-8)

Gene polymorphisms

-Vitamin-D receptor (VDR) gene polymorphism (Olmos et al, Methods Find Exp Clin Pharmacol 1998; 20: 699-707. Fernandez et al, J Am Soc Nephrol 1997; 8: 1546-52. Tagliabue et al, Am J Clin Pathol 1999; 112: 366-70)

Growth factors and cell cycle inhibitors

=Increased acidic growth factor (aFGF) gene expression (Sakaguchi, J Biol Chem 1992; 267: 24554-62)

-Decreased parathyroid hormone-related peptide (PTHrp) gene expression (Matsushita et al, Kidney Int 1999; 55: 130-8)

-De novo transforming growth factor-α (TGF-α) gene expression (Gogusev et al, Nephrol Dial Transplant 1996; 11: 2155-62)

-Induction of TGF-α by high phosphate diet (Dusso et al, Kidney Int 2001; 59: 855-865)

-Insufficient inhibition of cyclin kinase inhibitor p21WAF1 (Dusso et al, Kidney Int 2001; 59: 855-65); p21WAF1can be induced by calcitriol (Cozzolino et al, Kidney Int 2001; 60: 2109-2117)

-mTOR activation and rpS6 phosphorylation (Volovelsky et al, JASN 2016; 27: 1091–1101)

Gene mutations: association with monoclonal or multiclonal growth

-Mutation of menin gene (Falchetti et al, J Clin Endocrinol Metab 1993; 76: 139-44. Tahara et al, J Clin Endocrinol Metab 2000; 85: 4113-7. Imanishi et al, J Am Soc Nephrol 2002;13:1490-8)

-Mutation of Ha-ras gene (Inagaki et al, Nephrol Dial Transplant 1998; 13: 350-7)

-No involvement of VDR or CaSR gene mutations (Degenhardt et al, Kidney Int 1998; 53: 556-61. Brown et al, J Clin Endocrinol Metab 2000; 85: 868-72)

SECONDARY HYPERPARATHYROIDISM IN CKD – MECHANISMS INVOLVED IN THE TRANSFORMATION OF POLYCLONAL TO MONOCLONAL PARATHYROID GROWTH

In severe forms of secondary hyperparathyroidism nodular formations within diffusely hyperplastic tissue are a frequent finding (168). This observation probably corresponds to the occurrence of a monoclonal type of cell proliferation within a given tissue, which initially exhibits polyclonal growth. Clonal, benign tumoral growth was initially shown by Arnold et al using chromosome X-inactivation analysis method (169), and subsequently confirmed by other groups (170,171). After the initially diffuse, polyclonal hyperplasia, with the progression of CKD towards ESKD foci of nodular, monoclonal growth may arise within one or several parathyroid glands which eventually may transform to diffuse monoclonal neoplasia leading to an aspect comparable to that of primary parathyroid adenoma. Several different clones often coexist in same patient, and sometimes even in a single parathyroid gland. Figure 13 shows the progression from polyclonal to monoclonal and/or multiclonal parathyroid hyperplasia (172). It also shows corresponding changes in ultrasonographic features.

Figure 13. Schematic representation of the transformation of parathyroid hyperplasia from polyclonal to nodular, monoclonal/multiclonal growth with the progression of CKD towards ESKD. After Tominaga et al (172).

Acquired mutations of tumor enhancer or tumor suppressor genes are almost certainly involved in the development of such cell clones but precise knowledge about acquired genetic abnormalities remains limited (170). To identify new locations of parathyroid oncogenes or tumor suppressor genes important in this disease, Imanishi et al performed both comparative genomic hybridization (CGH) and genome-wide molecular allelotyping on a large number of uremia-associated parathyroid tumors (173). One or more chromosomal changes were present in 24% of tumors, markedly different from the values in common sporadic adenomas (28%), whereas no gains or losses were found in 76% of tumors. Two recurrent abnormalities were found, namely gain of chromosome 7 (9% of tumors) and gain of chromosome 12 (11% of tumors). Losses on chromosome 11, the location of the MEN1 tumor suppressor gene, occurred in only one uremia-associated tumor (2%), as compared to 34% in adenomas. The additional search for allelic losses with polymorphic microsatellite markers led to the observation of recurrent allelic loss on 18q (13% of informative tumors). Lower frequency loss was detected on 7p, 21q, and 22q. Interestingly, the cyclin D1 oncogene, activated and overexpressed by clonal gene rearrangement or other mechanisms in 20-40% of parathyroid adenomas (174,175) has not been found to be overexpressed in uremia-associated tumors (175).

Another interesting question was if somatic genes played a major role in the normal regulation of parathyroid function, such as the CaSR and VDR genes. The expression of these two genes was found to be decreased in the hyperplastic parathyroid tissue of uremic patients (62,90,91). The decrease was particularly marked in nodular areas, as compared to diffuse areas of parathyroid gland hyperplasia. Moreover, in uremic rats the decrease in CaSR expression was inversely related to the degree of parathyroid cell proliferation (93). However, the search for mutations or deletions of the VDR gene or the CaSR gene in uremic hyperparathyroidism has remained unsuccessful (170,176,177). The question remains unsolved whether the downregulation of CaSR and VDR expression is a primary event or whether it is secondary to hyperplasia.

Whether benign parathyroid tumors may evolve towards malignant forms is still subject to debate. Since in patients on dialysis therapy parathyroid carcinoma is a rare event (178–180), malignant transformation of clonal parathyroid neoplasms is probably exceptional.

Genome-wide allelotyping and CGH have directly confirmed the presence of monoclonal parathyroid neoplasms in uremic patients with refractory secondary hyperparathyroidism whereas the candidate gene approach has led to only modest results. Somatic inactivation of the MEN1 gene does contribute to the pathogenesis of uremia-associated parathyroid tumors, but its role in this disease appears to be limited, and there is probably no role for DNA changes of the CaSR and VDR genes. Recurrent DNA abnormalities suggest the existence of new oncogenes on chromosomes 7 and 12, and tumor suppressor genes on 18q and 21q, involved in uremic hyperparathyroidism. Finally, patterns of somatic DNA alterations indicate that markedly different molecular pathogenetic pathways exist for clonal outgrowth in severe uremic hyperparathyroidism, as compared to common sporadic parathyroid adenomas. Our group did not find a correlation between the presence of microscopically evident nodules and the clonal character of resected parathyroid tissue, and appearances of several glands with histologic patterns of diffuse hyperplasia also were unequivocally monoclonal in the absence of detectable nodular formations, suggesting that the current criteria for pathological diagnosis do not reflect the genetic differences among these two histopathological types (169).

Parathyroid Cell Apopotosis

It remains uncertain whether reduced apoptosis rates can also contribute to parathyroid tissue hyperplasia (68,181,182). One research group examined this issue in rats with short-term kidney failure (5 days). They were unable to detect apoptosis in hyperplastic parathyroid glands (183). However, this failure could be due to lack of sensitivity of the employed methods.

Negative findings in rats, with no identifiable apoptotic figures at all in parathyroid glands (68,182,183), contrast with subsequent positive observations in rats by others (184,185) and with personal observations of significant apoptotic figures in hyperplastic parathyroid glands removed from uremic, severely hyperparathyroid patients during surgery (186). In our study of human parathyroid glands from patients with ESKD approximately ten times higher apoptotic cell numbers were observed than in normal parathyroid tissue, using Tunel method (Figure 14) (186).

Figure 14. Increased proportion of apoptotic (TUNEL positive) cells in parathyroid glands from patients with primary or secondary uremic hyperparathyroidism, as compared to normal parathyroid tissue. After Zhang et al (186).

Of note, the uremic state appears to stimulate apoptosis in other cell types as well such as circulating monocytes (187), possibly via the well-known increase of cytosolic Ca²⁺ which has been observed in a variety of cell types in kidney failure (188), and also possibly via the noxious effect of bioincompatible dialysis membranes used for renal replacement therapy (189). The observed enhancement of parathyroid tissue apoptosis could compensate, at least in part, for the increase in parathyroid cell proliferation observed in secondary uremic hyperparathyroidism.

SECONDARY HYPERPARATHYROIDISM IN CKD – REGRESSION OF PARATHYROID HYPERPLASIA?

Whether regression of parathyroid hyperplasia occurs in animals or patients with advanced stages of CKD remains a matter of debate. According to some authors regression must be an extremely slow process, if it occurs at all (71,182). This is in sharp contrast to the rapid reversibility of excessive PTH secretion in uremic rats which was observed after normalization of renal function by kidney transplantation (190), although parathyroid mass probably did not rapidly decrease in this acute experimental model.

The issue of regression is of clinical importance. As an example, if a patient on dialysis therapy has a dramatic increase in total parathyroid mass there is practically no chance to obtain gland mass regression after successful kidney transplantation. In this condition it would seem appropriate to perform a surgical parathyroidectomy prior to transplantation. If, however significant regression of hyperplasia can occur as an active or passive process, namely by enhanced apoptosis or reduced proliferation, prophylactic surgery could be avoided. That regression of parathyroid hyperplasia secondary to vitamin D deficiency can occur has been convincingly demonstrated many years ago in experiments done in chicks (191). The administration of cholecalciferol to these birds that had developed an increase in parathyroid gland mass when fed a rachitogenic, vitamin D-free diet for 8-10 weeks led to a significant (50%) reduction in gland weight. Calcitriol failed to achieve same effect at a low, albeit hypercalcemic, dose but was capable of reducing gland mass at a higher dose. However, in an experimental dog model no parathyroid mass regression was found when the animals were first treated with a low-calcium, low-sodium, and vitamin D deficient diet for two years and subsequently a normal diet for another 17 months (192). In uremic animals, evidence for or against the possibility of regression of increased parathyroid tissue mass remains sparse and inconclusive.

The calcimimetic drug NPS R-568 was shown to decrease parathyroid cell proliferation and to prevent parathyroid hyperplasia in 5/6th nephrectomized rats; however, it was unable to entirely revert established hyperplasia (183,193). In apparent contrast, Miller et al showed that in rats with established secondary hyperparathyroidism, cinacalcet administration led to complete regression of parathyroid hyperplasia (194). The cinacalcet-mediated decrease in parathyroid gland size was accompanied by increased expression of the cyclin-dependent kinase inhibitor p21. However, these were short-term experiments over an 11-week time period. Interestingly, the prevention of cellular proliferation with cinacalcet occurred despite increased serum phosphorus and decreased serum calcium levels.

In patients with primary hyperparathyroidism spontaneous remission of parathyroid over function has been observed in rare instances, caused by parathyroid “apoplexy” due to tissue necrosis (195). The diagnosis of parathyroid tissue necrosis is more difficult to ascertain in secondary than in primary forms of hyperparathyroidism because the hyperplasia of the former is not limited to a single gland.

Regression of parathyroid hyperplasia in hemodialysis patients in response to intravenous calcitriol pulse therapy for 12 weeks has been reported by Fukagawa et al using ultrasonography (196). These authors observed a significant decrease in mean gland volume from 0.87 to 0.51 cm³ of this time period, together with a reduction in serum iPTH of more than 50%. In contrast, Quarles et al who also examined parathyroid gland morphology in hemodialysis patients in vivo in response to intermittent intravenous or oral calcitriol treatment for 36 weeks failed to observe a decrease in parathyroid gland size as assessed by high resolution ultrasound and/or magnetic resonance imaging (197). Mean gland size was 1.9 and 2.1 cm³ before and 3.3 and 2.3 cm³ after oral and intravenous calcitriol therapy, respectively. The authors achieved an overall maximum average serum PTH reduction of 43% over this time period. There were marked differences between these two studies which may explain the apparently diverging results. Hyperparathyroidism probably was more severe in the latter than in the former. Although initial mean serum iPTH levels were similar, serum phosphorus was higher and the decrease in serum PTH achieved in response to calcitriol was less marked in the latter. Moreover, parathyroid mass was more than double. In another study, Fukagawa et al examined the possible relation between parathyroid size and the long-term outcome after calcitriol pulse therapy, by subdividing patients into different groups according to initial parathyroid gland volume assessments (198). In two hemodialysis patients with detectable gland(s), in whom the size of all parathyroid glands as well as PTH hypersecretion regressed to normal, secondary hyperparathyroidism remained controllable for at least 12 months after switching to conventional oral active vitamin D therapy. In contrast, in seven hemodialysis patients, in whom the size of all parathyroid glands did not regress to normal by calcitriol pulse therapy, secondary hyperparathyroidism relapsed after switching to conventional therapy although PTH hypersecretion could be controlled temporarily. Similarly, Okuno et al. showed in a study in hemodialysis patients that plasma PTH levels and the number of detectable parathyroid glands decreased in response to the active vitamin D derivative maxacalcitol (22-oxacalcitriol) given for 24 weeks only when the mean value of the maximum diameter of one of the parathyroid glands was less than 11.0 mm, but not when it was above that value (199).

Taken together, these findings suggest that the degree of parathyroid hyperplasia, as detected by ultrasonography, is an important determinant for regression in response to calcitriol therapy. It is probable, although not proven, that the type of hyperplasia, namely monoclonal/multiclonal vs polyclonal growth, is even more important as regards the potential of regression than the mere size of each gland.

Figure 2 (see above) summarizes in a schematic view the main mechanisms involved in the abnormal PTH synthesis and secretion and in parathyroid tissue hyperplasia. Figure 2 further points to the possible counterregulatory role of apoptosis.

ALTERED PTH METABOLISM AND RESISTANCE TO PTH ACTION

PTH metabolism is greatly disturbed in CKD. Normally, most of full-length PTH1-84 is transformed in the liver to the biologically active N-terminal PTH1-34 fragment and several other, inactive C-terminal fragments. The latter are mainly catabolized in the kidney and the degradation process involves solely glomerular filtration and tubular reabsorption, whereas the N-terminal PTH1-34 fragment undergoes both tubular reabsorption and peritubular uptake, as does the full-length PTH1-84 molecule (200). Tubular reabsorption involves the multifunctional receptor megalin (201).

With the progression of CKD, both pathways of renal PTH degradation are progressively impaired. This leads to a marked prolongation of the half-life of C-terminal PTH fragments in the circulation (202–204) and their accumulation in the extracellular space. Moreover, there is no peritubular metabolism of PTH1-84 in uremic non-filtering kidneys, in contrast to peritubular uptake by normal, filtering kidneys (205). Hepatic PTH catabolism appears however to be unchanged in CKD since uremic livers and control livers released equal amounts of immunoreactive C-terminal PTH fragments (205).

A decreased response to the action of PTH may be another factor involved in the stimulation of the parathyroid glands in CKD. A diminished calcemic response to the infusion of PTH has long been reported, suggesting that PTH over secretion was necessary to maintain eucalcemia. The skeletal resistance to PTH has been attributed to variousmechanisms, including impaired vitamin D action in association with hyperphosphatemia, overestimation of true PTH(1-84) by assays measuring iPTH (see below), accumulation of inhibitory PTH fragments, oxidative modification of PTH, increase in circulating osteoprotegerin and sclerostin levels, administration of active vitamin D derivatives and calcimimetics, and altered PTH-R1 expression (7,141,206,207). Concerning the latter mechanism, studies have suggested the presence of PTH receptor isoforms in various organs of normal rats. Downregulation of PTH-R1 mRNA has been observed in various tissues in uremic rats (208–211) and also in osteoblasts of patients with end-stage renal disease (212). However, the issue of PTH-R1 expression in bone tissue remains a matter of controversy since onegroup found it to be upregulated in patients with moderate to severe renal hyperparathyroid bone disease (213). A recent study claimed that inhibition of PTH binding to PTH-R1 by soluble Klotho could represent yet another mechanism of PTH resistance (214). This observation would be compatible with the presence of upregulated, yet biologically inactive PTH-R1.

Other mechanisms involved in the control of the normal balance between bone formation and resorption and their response to PTH are the Wnt-β-catenin signaling pathway and its inhibition by sclerostin and Dickkopf-related protein 1 (Dkk1) (7,56,215), and the activin A pathway with its inhibition by a decoy receptor (216). Wnt-β-catenin inhibitors are expressed predominantly in osteocytes. Whereas reduced activity of sclerostin and Dkk1 leads to increased bone mass and strength, the opposite occurs in animal models with overexpression of both sclerostin and Dkk1. In CKD, circulating levels of both Wnt—β-catenin inhibitors have generally been found to be increased (56), and serum sclerostin was found to correlate negatively with serum PTH (217,218), and PTH has been shown to blunt osteocytic production of this Wnt inhibitor (219). Since high PTH and sclerostin levels coexist in CKD this raises the suspicion that sclerostin contributes to PTH resistance in CKD (7). Calcitonin and bone morphogenetic proteins stimulate, whereas PTH and estrogens suppress the expression of sclerostin and/or Dkk1 (220,221). Bone formation induced by intermittent PTH administration to patients with osteoporosis could be explained, at least in part, by the ability of PTH to downregulate sclerostin expression in osteocytes, permitting the anabolic Wnt signaling pathway to proceed (222).In patients with ESKD sclerostin is a strong predictor of bone turnover and osteoblast number (223). Serum levels of sclerostin correlate negatively with serum iPTH in such patients. Sclerostin was superior to iPTH for the positive prediction of high bone turnover and number of osteoblasts. In contrast, iPTH was superior to sclerostin for the negative prediction of high bone turnover and had similar predictive values as sclerostin for the number of osteoblasts. Serum sclerostin levels increase after parathyroidectomy (7). As regards activin A, a member of the transforming growth factor-b superfamily, Hruska’s group has demonstrated increased serum levels and systemic activation of its receptors in mouse models of CKD (216). In humans, serum activin A levels increase already at early stages of CKD, before elevations in intact PTH and FGF23, equally pointing to a role in CKD-MBD and PTH resistance (224).

Interesting new pathways have recently been identified by Pacifici’s group. First, they used various mouse models to demonstrate a permissive activity of butyrate produced by the gut microbiota, required to allow stimulation of bone formation by PTH (225). Butyrate’s effect was mediated by short-chain fatty acid receptor GPR43 signaling in dendritic cells and by GPR43-independent signaling in T cells. Second, the group showed that intestinal segmented filamentous bacteria (SFB) enabled PTH to expand intestinal TNF+ T and Th17 cells and thereby increase their egress from the intestine and recruitment to the bone marrow to cause bone loss (226). Figure 15 shows these recently detected pathways involving the gut microbiota.

Figure 15. Importance of intestinal microbiota for PTH action in bone. The stimulation of bone anabolism by PTH requires butyrate formation by short chain fatty acid (SCFA) producing gut bacteria. CKD probably reduces its production. Butyrate increases the frequency of regulatory T (Treg) cells in the intestine and in the bone marrow and potentiates the capacity of intermittently administered PTH to induce the differentiation of naïve CD4+ T cells into Tregs, a population of T cells which induces conventional CD8+ T cells to release Wnt10b. This osteogenic Wnt ligand activates Wnt signaling in osteoblastic cells and stimulates bone formation. Butyrate enables intermittent PTH dosing to expand Tregs via GPR43 signaling in dendritic cells (DCs) and GPR43 independent targeting of T cells. Butyrate may also affect bone remodeling by modulating osteoclast genes. The stimulation of bone resorption by PTH requires the presence of segmented filamentous bacteria (SFB) within gut microbiota for the production of Th17 cells in intestinal Peyers' plaques. Continuously elevated PTH levels lead to TNF+ T cell expansion in the gut and the bone marrow via a microbiota-dependent, but SFB independent mechanism. Furthermore, intestinal TNF producing T cells are required for PTH to increase the number of intestinal Th17 cells, and TNF mediates the migration of intestinal Th17 cells to the bone marrow. This migration depends on upregulation of chemokine receptor CXCR3 and chemokine CCL20. Bone marrow Th17 cells then induce osteoclastogenesis by secreting IL-17A, RANKL, TNF, IL-1, and IL-6. From Massy & Drueke (227).

It will be interesting to examine the hypotheses that the excessive bone resorption associated with secondary hyperparathyroidism in CKD is at least partially due to either insufficient intestinal butyrate availability, excessive intestinal SFB activity, or both (227).

SECONDARY HYPERPARATHYROIDISM IN CKD – CLINICAL FEATURES

In most patients with ESKD, even advanced secondary hyperparathyroidism remains a clinically silent disease. Clinical manifestations are generally related to severe osteitis fibrosa and to the consequences of hypercalcemia and/or hyperphosphatemia.

Osteoarticular pain may be present. When patients become symptomatic, they usually complain of pain on exertion in skeletal sites that are subjected to biomechanical stress. Pain at rest and localized pain are rather unusual and suggest other underlying causes. Severe proximal myopathy is seen in some patients, even in the absence of vitamin D deficiency. These symptoms and signs are more frequent in patients who suffer from mixed renal osteodystrophy, resulting from a combination of parathyroid over function and vitamin D deficiency. Skeletal fractures may occur after only minor injury. They may also develop on the ground of cystic bone lesions, the so-called “brown tumors”, which occur for still unknown reasons in a small number of uremic patients with secondary hyperparathyroidism. Rupture of the patella or avulsion of tendons may be seen in advanced cases.

Uremic pruritus is most often associated with an elevated Ca x P product although other factors may also be involved. Related symptoms and signs are the red eye syndrome due to the deposition of calcium in the conjunctiva, cutaneous calcification, and pseudogout. The latter is a form of painful arthralgia of acute or subacute onset caused by intra-articular deposition of radio-opaque crystals of calcium pyrophosphate dehydrate.

The syndrome of “calciphylaxis” is an infrequent manifestation of cutaneous and vascular calcification in uremic patients which may occur in association with secondary hyperparathyroidism, although this association is by no means constant. It is characterized by a rapidly progressive skin necrosis involving buttocks and the legs, particularly the thighs. It can produce gangrene and may be fatal. It occurs as the result of arteriolar calcification and has also been termed “calcific uremic arteriolopathy” to reflect more accurately the nature of the lesion (228). Of interest, a post-hoc analysis of the EVOLVE trial in chronic hemodialysis patients recently showed that cinacalcet administration, which allows improved PTH control, resulted in a significant decrease in the incidence of calcific uremic arteriolopathy as compared to placebo (229).

SECONDARY HYPERPARATHYROIDISM IN CKD -- DIAGNOSIS

The biochemical diagnosis relies on the determination of plasma iPTH. This is also true for primary hyperparathyroidism. In patients with CKD, it has however become apparent in recent years that there are several limitations to the measurement of iPTH, in addition to the usual day-to-day variations in healthy people (230). Physiological iPTH plasma values are not normal for uremic patients since values in the normal range are often associated with low bone turnover (adynamic bone disease) whereas normal bone turnover may be observed in presence of elevated plasma intact PTH levels (231–234). It is currently unclear to what extent this is due to imperfections in the PTH assays used (see below), PTH receptor status, post-receptor events, non-PTH-mediated changes in bone metabolism (e.g., supply of vitamin D or its metabolites, supply of estrogens or androgens), or a combination of these factors.

The accumulation of a large non (1-84) molecular form of PTH, which is detected by iPTH (so-called "intact" PTH) assays, has been described in patients with CKD (235). The large PTH fragment was tentatively identified as hPTH(7-84) (236). This finding is of importance in the interpretation of PTH values, since true hPTH(1-84) represents only about 50-60% of the levels detected by the currently used intact PTH assays, and since PTH(7-84) antagonizes PTH(1-84) effects on serum calcium and on osteoblasts (237). Moreover, the secretory responses of hPTH(1-84) and non-hPTH(1-84) to changes in [Ca²⁺_e] are not proportional for these two PTH moieties (87). Moreover, a large variability has been found between different assay methods used for plasma PTH measurement in patients with CKD, recognizing PTH(7-84) with various cross-reactivities (238). Varying plasma sampling and storage conditions may further complicate the interpretation of PTH results provided by clinical laboratories (239). The development of assays which detect full-length (whole) human PTH, but not amino-terminally truncated fragments (240), was initially considered as a major progress in this field. To further improve the assessment of uremic hyperparathyroidism and the associated increase in bone turnover Monier-Faugere et al proposed to calculate the ratio of PTH-(1-84) to large C-PTH fragments (241). The usefulness in the clinical setting of the whole PTH assay and of the ratio of whole PTH to PTH fragments has however not been convincingly established for the diagnosis of parathyroid over function in adult (242,243) or pediatric (244) dialysis patients. From a practical point of view, it must be pointed out that at present measurement of PTH with third-generation assays is not widely available. Another potential issue is the presence of oxidized, inactive PTH in the circulation of patients with CKD, with concentrations much higher than those of iPTH (141), although there were large interindividual variations (141). Whereas one study showed a U-shaped association of non-oxidized, but not oxidized, PTH with survival in patients on hemodialysis therapy (245), a subsequent study done in CKD stage 2-4 patients found iPTH, but not non-oxidized PTH, to be associated with all-cause death in multivariable analysis (246). The reasons for these apparently opposite findings are unclear. The assertion that PTH oxidation is a vitro artifact has been disproven recently (247). Based on personal findings, Hocher and Zeng postulated that oxidized and non-oxidized PTH should be measured separately to correctly evaluate the degree of severity and clinical relevance of parathyroid over function in CKD (142). However, in a very recent study Ursem et al observed a strong correlation between serum non-oxidized PTH and total PTH in patients with ESKD (248). Most importantly, they found that both histomorphometric and circulating bone turnover markers exhibited similar correlations with non-oxidized PTH and total PTH. The authors therefore concluded that non-oxidized PTH is not superior to total PTH as a biomarker of bone turnover in ESKD. However, presently available methods do not enable a precise distinction between biologically active and inactive PTH forms, be it through oxidative or other post-translational modifications of the hormone (249). Most importantly, we will hopefully be able in the future to rely not only on serum PTH but also on appropriate direct markers of bone structure and function for the assessment of renal osteodystrophy and on markers of cardiovascular disease related to secondary hyperparathyroidism (250).

Bone x-ray diagnosis is impossible in mild to moderate forms of secondary hyperparathyroidism, but relatively easy in severe forms. Nevertheless, to date x-ray diagnosis is rarely used in routine clinical praxis. Typical lesions include resorptive defects on the external and internal surfaces of cortical bone, with the resorption particularly pronounced on the subperiosteal surface. Resorption within cortical bone enlarges the Haversian channels, resulting in longitudinal striation; resorption at the endosteal surface causes cortical thinning. These lesions can be generally detected first in the hand skeleton, most characteristically at the periosteal surface of the middle phalanges (Figure 16).

Accelerated bone deposition at this site (periosteal neostosis) can also be seen. Another characteristic feature is resorptive loss of acral bone (acro-osteolysis), in particular at the terminal phalanges, at the distal end of the clavicles, and in the skull (‘pepper-pot’ aspect) (Figure 17). Whereas cortical bone is progressively thinning, the mass of spongy bone tends to increase, particularly in the metaphyses. The latter phenomenon results in a characteristic sclerotic aspect of the upper and lower thirds of the vertebrae, contrasting with rarefaction of the center (‘rugger jersey spine’). Osteosclerosis is also commonly seen in radiographs of the metaphyses of the radius and tibia.

Figure 16. Periosteal resorption and small vessel calcification in severe secondary uremic hyperparathyroidism. (a) X-ray aspect of periosteal resorption within cortical bone of middle phalanges of the hand, indicative of osteitis fibrosa, and extensive finger artery calcification in a CKD stage 5 patient with severe secondary hyperparathyroidism. (b) One year after surgical parathyroidectomy: complete bone lesion healing and disappearance of arterial calcification.

Figure 17. X-ray pepper-and-salt aspect of the skull in in a chronic hemodialysis patient with severe secondary hyperparathyroidism.

In addition to the skeletal lesions, radiographs often reveal various types of soft tissue calcification. These comprise vascular calcifications, i.e., calcification of intimal plaques (aorta, iliac arteries) (Figure 18a), as well as diffuse calcification (Mönckeberg type) of the media of peripheral muscular arteries (Figure 18b) (251).

Figure 18. Massive intima (a) and media (b) calcification of hypogastric artery in a chronic hemodialysis patient.

Of interest, media calcification of digital arteries can entirely regress after surgical parathyroidectomy (Figure 16). Calcium deposits may also be seen in periarticular tissue or bursas and may exhibit tumor-like features (Figure 19).

Figure 19. X-ray feature of a tumor-like periarticular calcification in the shoulder of a chronic hemodialysis patient with adynamic bone disease due to aluminum intoxication.

Since the development of electron-beam computed tomography (EBCT) and multiple slice computed tomography (MSCT) more reliable means have become available to assess quantitatively vascular calcification and its progression in uremic patients (252). However, these techniques are not universally available and they are costly. Moreover, they do not allow a distinction between arterial intima and media calcifications. Such a distinction can be obtained by radiograms of the pelvis and the thigh, combined with ultrasonography of the common carotid artery. Using these simple methods, London et al could show that hemodialysis patients with arterial media calcification had a longer survival than hemodialysis patients with arterial intima calcification, but in turn their survival was significantly shorter than that of hemodialysis patients without calcifications (253). Of note, both severe hyperparathyroidism and marked hypoparathyroidism favor the occurrence of the two types of calcifications in patients with ESKD (254–256). In contrast to permanent elevations in serum PTH, the intermittent administration of PTH1-34 has been shown to decrease arterial calcification in uremic rats (257) and in diabetic mice with LDL receptor deletion (258). This observation tends to demonstrate that normal parathyroid function is required not only for the maintenance of optimal bone structure and function, but also as an efficacious defense against soft tissue calcification, and that intermittent PTH administration may not only improve osteoporosis (259), but also reduce vascular calcification, at least in experimental animals.

SECONDARY HYPERPARATHYROIDISM IN CKD – TREATMENT

Medical Management

Presently available options of medical treatment should take into account the levels of plasma biochemistry and x-ray findings, and as a more recently recognized parameter also the dimensions of the largest parathyroid glands, as assessed by ultrasonography. A gland diameter of 5-10 mm or more is considered as being indicative of autonomous growth, which often is resistant to medical treatment (198).

Schematically, there are five major medical treatment options which can be combined in some cases, but not in others, namely the restriction of phosphate intake and/or the administration of calcium supplements, oral phosphate binders, vitamin D derivatives, and calcimimetics (260,261). In dialysis patients the weekly dose of renal replacement therapy is an additional important factor. An optimal dialysis technique allows controlling hyperphosphatemia, and providing enough calcium to avoid PTH stimulation by hypocalcemia during dialysis sessions.

When trying to control hyperparathyroidism it is important to avoid both hypocalcemia and hypercalcemia and to reduce or correct hyperphosphatemia as well. In patients with controlled plasma phosphate, this can be achieved by giving either calcitriol or one of its synthetic analogs, or by administering oral calcium supplements. For a long time, calcitriol or alfacalcidol was the preferred therapy in uremic patients with high to very high plasma intact PTH values and normal to moderately elevated plasma calcium levels, when plasma phosphate did not exceed recommended levels, namely 1.5 mmol/L for CKD stages 3-4 and 1.8 mmol/L for CKD stage 5, according to the K/DOQI guidelines of 2003 (262). However, the administration of active vitamin D derivatives often induces hypercalcemia and/or hyperphosphatemia. The KDIGO CKD-MBD guideline of 2009 (263) and its -update in 2017/2018 (264,265) suggest "maintaining iPTH levels in CKD stage 5D patients (i.e., patients receiving dialysis therapy) in the range of approximately two to nine times the upper normal limit for the assay, to keep serum calcium normal, and to decrease serum phosphorus towards the normal range." Thus, the recommended iPTH target range has become larger than with the prior K/DOQI guidelines. The KDIGO guideline further suggests that marked changes in iPTH levels in either direction within the newly defined, broadened range should "prompt initiation or change in therapy to avoid progression to levels outside of this range." The updated guideline recommends that patients with CKD stages G3a-G5 not on dialysis whose levels of intact PTH are progressively rising or persistently above the upper normal limit for the assay be evaluated for modifiable factors, including hyperphosphatemia, hypocalcemia, high phosphate intake, and vitamin D deficiency (grade 2C recommendation).

Vitamin D and Active Vitamin D Derivatives

A satisfactory degree of vitamin D repletion should probably be aimed at in case of vitamin D deficiency since the majority of patients with CKD have at least some degree of vitamin D deficiency (51,266). Relative vitamin D depletion has been shown to be an independent risk factor for secondary hyperparathyroidism in hemodialysis patients (54). Repletion with native vitamin D may lead to improved control of secondary hyperparathyroidism in patients with CKD not yet on dialysis (267) and in those treated by dialysis (268) but a beneficial effect has not been observed in a subsequent meta-analysis (269). Vitamin D repletion may allow optimal bone formation, help to avoid osteomalacia, and exert numerous other positive effects due to the pleiotropic actions of vitamin D, but most of these presumably positive actions remain a matter of debate (269,270). Most importantly, randomized controlled trials with native vitamin D or calcidiol have not been performed so far to evaluate hard clinical outcomes of patients with CKD.

As regards the administration of active vitamin D sterols during the course of CKD, the updated KDIGO guideline suggests that calcitriol and vitamin D analogues not be routinely used in patients with CKD G3a-G5 (Grade 2C recommendation). It further states that it is reasonable to reserve the use of these agents for patients with CKD G4-G5 with severe and progressive hyperparathyroidism (264,265).

To correct secondary hyperparathyroidism of moderate to severe degree the oral administration of active vitamin D derivatives is generally more efficient than that of native vitamin D. In hemodialysis patients, calcitriol or its analogs can be given either orally or intravenously. The oral administration can be on a daily basis (for instance 0.125 to 0.5 µg of calcitriol) or as intermittent bolus ingestions (for instance 0.5 to 2.0 µg of calcitriol for each dose) whereas the intravenous administration is always intermittent (also 0.5 to 2.0 µg of calcitriol or more per injection). The route and mode of administration of calcitriol or alfacalcidol probably play only a minor role. Since the highly active 1α-hydroxylated vitamin D derivatives can easily induce hypercalcemia, intensive research has focused on the development of various non-hypercalcemic analogs, including the natural vitamin D compound 24,25(OH)2 vitamin D3, 22-oxa-calcitriol (maxacalcitol), 19-nor-1,25(OH)2 vitamin D3 (paricalcitol), and 1α-(OH) vitamin D2 (hectorol). Despite numerous studies done in many patients, none of them has been shown to be entirely devoid of inducing increases in plasma calcium or phosphate, and none has been demonstrated thus far to be superior to calcitriol or alfacalcidol in the long run in controlling secondary hyperparathyroidism (271,272). An observational study by Teng et al. showed that paricalcitol administration to a large cohort of hemodialysis patients conferred a remarkable (16%) survival advantage over the administration of calcitriol (273). Numerous subsequent observational studies reported a survival benefit, either comparing treatment with active vitamin D derivatives to no treatment, or novel active vitamin D derivatives to calcitriol in CKD patients not yet on dialysis (274) or those receiving dialysis treatment (275–277). Another observational study conducted in hemodialysis patients, however, did not find a survival advantage with paricalcitol, as compared to calcitriol (278). In the absence of randomized controlled trials, it is impossible to conclude that paricalcitol treatment is superior to calcitriol or alfacalcidol in terms of patient survival. Findings of observational studies can only be considered as hypothesis-generating. They need to be confirmed by a properly designed prospective investigation (279).

Calcimimetics

The introduction of the calcimimetic cinacalcet into clinical practice led to a change in the above treatment strategy since it enables parathyroid over function control without increasing plasma calcium or phosphorus. Calcimimetics modify the configuration of the CaSR, a receptor cloned by Brown et al in 1993 (280) They make the CaSR more sensitive to [Ca²⁺_e] in contrast to the so-called calcilytics which decrease its sensitivity, as schematically shown in Figure 20.

Figure 20. Schematic representation of the modulation of the calcium-sensing receptor (CaSR) by calcimimetics and calcilytics. CaSR is expressed on cell membrane. Calcimimetics increase its sensitivity to calcium ions whereas calcilytics decrease it.

Initial acute studies in chronic hemodialysis patients showed that the calcimimetic cinacalcet was capable of reducing plasma PTH within hours, immediately followed by a rapid decrease in plasma calcium and a minor decrease in plasma phosphate (281–283). In addition, calcimimetics can also reduce parathyroid cell proliferation. Both short-term and long-term studies performed in rats and mice with CKD showed that the administration of the calcimimetic NPS R-568, starting at the time of CKD induction, allowed the prevention of parathyroid hyperplasia (183,193,284). This effect is probably due to a direct inhibitory action on the parathyroid cell, as shown by our group in an experimental study in which we exposed human uremic parathyroid cells to the calcimimetic NPS-R467 (75). An interesting finding of a yet unexplained mechanism and significance is the observation that calcimimetic treatment led to an approximately 5-fold increase in the proportion of oxyphil cells, as compared to chief cells, in parathyroid glands removed from CKD patients with refractory hyperparathyroidism (285–287). Of note, oxyphil cells also exhibited higher CaSR expression than chief cells in such glands (288).

Perhaps more important from a clinical point of view, the administration of calcimimetics enabled an improvement of osteitis fibrosa (103), halted the progression of vascular calcification both in uremic animals (284,289) and probably also in dialysis patients (290), prevented vascular remodeling (291), improved cardiac structure and function (292), and prolonged survival (293) in uremic animals with secondary hyperparathyroidism.

The long-term administration of cinacalcet to chronic hemodialysis patients proved to be superior to optimal standard therapy in controlling secondary uremic hyperparathyroidism, in that it was able to induce not only a decrease in plasma PTH but also in plasma calcium and phosphate (294–297). Figure 21 shows the superior control of severe secondary hyperparathyroidism by cinacalcet as compared to placebo treatment with standard of care (298). The initial daily dose is 30 mg orally, which can be increased up to 180 mg if necessary. Cinacalcet is generally well tolerated, with the exception of gastrointestinal side effects, which however cease in the majority of patients with time. Since its administration generally leads to a decrease in serum calcium, a close follow-up is required, at least initially, to avoid hypocalcemia with possible adverse clinical consequences. Cinacalcet can be associated with calcium-containing and non-calcium containing phosphate binders and also with vitamin D derivatives. For PTH lowering a combination therapy may lead to more complete correction than single drug treatment because of less side-effects and greater efficacy in the control of parathyroid over function (299,300).

Figure 21. Effect of cinacalcet on need of parathyroidectomy in patients on hemodialysis therapy. In the EVOLVE trial, parathyroidectomy was performed in 140 (7%) cinacalcet-treated and 278 (14%) placebo-treated patients. Key independent predictors of parathyroidectomy included younger age, female sex, geographic region, and absence of history of peripheral vascular disease. One hundred and forty-three (7%) cinacalcet-treated and 304 (16%) placebo-treated patients met the biochemical definition of severe, unremitting (tertiary) hyperparathyroidism. Considering the pre-specified biochemical composite or surgical parathyroidectomy as an endpoint, 240 (12%) cinacalcet-treated and 470 (24%) placebo-treated patients developed severe, unremitting hyperparathyroidism (298).

The subsequent development of an intravenously active calcimetic led to another series of clinical studies aimed at controlling secondary hyperparathyroidism in patients on hemodialysis with an easy access to parenteral drug administration, thereby reducing oral pill overload. Two randomized controlled trials were conducted in such patients with moderate to severe secondary hyperparathyroidism, evaluating the efficacy and safety of the intravenous calcimimetic, etelcalcetide as compared to placebo (301). Thrice weekly administration of active drug after hemodialysis led to a greater than 30% reduction in serum PTH compared with less than 8.9% of patients receiving placebo. The reduction in PTH was rapid and sustained over 26 weeks. Treatment with etelcalcetide lowered serum calcium in the majority of patients, with overt symptomatic hypocalcemia reported in 7%. Adverse events occurred in 92% of etelcalcetide-treated and 80% of placebo-treated patients. Nausea, vomiting, and diarrhea were more common in etelcalcetide-treated patients, as were symptoms potentially related to hypocalcemia. A subsequent double-blind, double-dummy randomized controlled trial compared intravenous etelcalcetide to oral cinacalcet in patients on hemodialysis with moderate to severe secondary hyperparathyroidism (302). It showed that the use of etelcalcetide was not inferior to cinacalcet in reducing serum PTH concentrations over 26 weeks. In addition, etelcalcetide met several superiority criteria, including a greater reduction in serum PTH concentrations from baseline, and more potent reductions in serum concentrations of FGF23 and two markers of high-turnover bone disease.

How about hard patient outcomes? The randomized controlled trial EVOLVE examined the question whether better control of secondary uremic hyperparathyroidism by cinacalcet, as compared to placebo treatment with standard of care, reduced the incidence of cardiovascular events and mortality (298). The study enrolled 3803 patients receiving long-term hemodialysis therapy. Using intention-to-treat analysis the study outcome was negative (Figure 22, upper part). However, after adjustment for age and other confounders, and also when using lag-censoring analysis (Figure 22, lower part), there was a nominally significant reduction in the primary cardiovascular endpoint including mortality in the cinacalcet treatment group in whom serum PTH, calcium, and phosphate were better controlled than in the placebo treatment group. Moreover, a post-hoc lag-censoring analysis of EVOLVE further showed that the incidence of clinically ascertained fractures was lower in the cinacalcet than the placebo arm (303).

Figure 22. Effect of cinacalcet on cardiovascular outcomes of patients on hemodialysis therapy. The randomized controlled trial EVOLVE examined the question whether a better control of secondary uremic hyperparathyroidism by cinacalcet, as compared to placebo treatment with standard of care, reduced the incidence of cardiovascular events and mortality. The study enrolled 3803 patients receiving long-term hemodialysis therapy. Using intention-to-treat analysis the study outcome was negative (upper part of Figure). However, with lag-censoring analysis there was a nominally significant reduction in the primary composite cardiovascular endpoint in the cinacalcet treatment group in whom serum PTH, calcium, and phosphorus were better controlled than in the placebo treatment group (lower part of Figure). From Chertow et al (298).

Phosphate Binders, Inhibitors of Intestinal Phosphate Absorption, Oral Phosphate Restriction, and Phosphate Removal by Dialysis

Calcium-containing phosphate binders should be given, preferentially during or at the end of phosphate-rich meals, to patients with CKD and uncontrolled hyperphosphatemia who have no hypercalcemia or radiological evidence of marked soft tissue calcifications. In these latter cases non-calcium-containing phosphate binders should be preferred (see below). The administration of calcium salts alone such as calcium carbonate or calcium acetate may be sufficient for the control of hyperphosphatemia in many instances, particularly in patients with CKD stages G3-G5 not yet on dialysis. At the same time these calcium salts will prevent serum iPTH from rising in the majority of patients (304). They may however lead to calcium overload (44,45) and excessive PTH over suppression, resulting eventually in adynamic bone disease (305). In hemodialysis patients, the efficacy and tolerance of this treatment may be enhanced by the concomitant use of low-calcium dialysate, for instance a calcium concentration of 1.25 mmol/L, especially if plasma intact PTH levels are not very high. However, long-term studies have shown that the continuous use of a dialysate calcium of only 1.25 mmol/L requires close monitoring of plasma calcium and PTH because of the risk of inducing excessive PTH secretion (306,307). A dialysate calcium concentration between 1.25 and 1.5 mmol/L is more appropriate in terms of optimal calcium balance and control of secondary hyperparathyroidism (308). The use of a low calcium dialysate also may require higher doses of active vitamin D derivatives (309) or cinacalcet (310) for the control of secondary hyperparathyroidism. Of note, the use of a low calcium bath favors hemodynamic instability during the hemodialysis session (311) and the occurrence of sudden cardiac arrest (312,313). In CAPD patients, the use of calcium carbonate, in the absence of vitamin D, together with a reduction of the dialysate calcium concentration from 1.75 to 1.45 mmol/L prevents the occurrence of hypercalcemia in most patients (314). However, the addition of daily low-dose alfacalcidol may lead to hypercalcemia, despite a further reduction of dialysate calcium to 1.0 mmol/L.

The development of calcium-free, aluminum-free oral phosphate binders such as sevelamer-HCl (315–317), sevelamer carbonate (318,319), lanthanum carbonate (320–322), sucroferric oxyhydroxide (323) and ferric citrate (324) allows controlling hyperphosphatemia without the potential danger of calcium overload. Their phosphate binding capacity is roughly equivalent to that of Ca carbonate or calcium acetate. Sevelamer offers in addition the advantage to lower serum total cholesterol and LDL-cholesterol and to increase serum HDL-cholesterol, to slow the progression of arterial calcification in dialysis patients (316), and possibly to improve survival in such patients (325). The administration of sevelamer is probably more efficient in halting the progression of vascular calcification than calcium carbonate or calcium acetate but this remains a matter of debate (14,326,327). The administration of lanthanum carbonate to uremic animals has been shown to also reduce progression of vascular calcification (328,329), but studies in patients with CKD have led to variable results (330–332). The effects of calcium-free, aluminum-free phosphate binders on serum iPTH are variable, depending on baseline iPTH and concomitant therapies. In general, iPTH levels are higher in response to these binders than to calcium-containing phosphate binders (Figure 23) (333,334).

Figure 23. Effect of oral calcium vs. sevelamer on serum intact PTH (iPTH) in CKD. In this 54-week, randomized, open-label study the effects of sevelamer hydrochloride on bone structure and various biochemical parameters were compared to that of calcium carbonate in 119 patients on long-term hemodialysis therapy. Serum iPTH was consistently lower with calcium carbonate than with sevelamer treatment. From Ferreira et al (333).

The administration of aluminum-containing phosphate binders should be avoided because of their potential toxicity. They may be given in some treatment resistant cases, but only for short periods of time (263).

Another approach chosen to control hyperphosphatemia and therefore to prevent or delay the development of secondary hyperparathyroidism is pharmacologic interference with active intestinal phosphate transport by oral inhibitors of the phosphate/sodium cotransporter NaPi2b, using either already available drugs such as niacin or nicotinamide (335–337), or recently developed novel inhibitors such as tenapanor (338,339). The rather disappointing results of available studies have not let so far to their introduction into clinical practice (340).

Dietary phosphate intake should be assessed and diminished, if possible. Special attention should be given to the avoidance of foods containing phosphate additives (341). The spontaneous reduction of protein intake with age probably explains the often better control of serum phosphate in elderly as compared to younger patients with ESKD, and this may contribute to the relatively lower PTH levels of the former and their propensity to develop adynamic bone disease (342). However, when reducing dietary phosphate intake and concomitantly protein intake, one has to take care to avoid the induction of a protein malnutrition state. Restricting dietary protein intake excessively may lead to greater mortality (343). In dialysis patients, an attempt should always be made as well to improve the efficiency of the dialysis procedure.

A better correction of metabolic acidosis by bicarbonate-buffered dialysate, as compared to acetate-buffered dialysate, probably helps to delay the progression of osteitis fibrosa in hemodialysis patients (344). One possible mechanism for the beneficial role of acidosis correction is an increase in the sensitivity of the parathyroid gland to plasma ionized calcium (345).

Current recommendations for the medical treatment and prevention of patients with CKD-MBD, including secondary hyperparathyroidism, can be found in the 2009 KDIGO CKD-MBD guideline (263) and its recent update (264,265). It must be pointed out though that there is no definitive proof of a beneficial effect of phosphate lowering on patient-level outcome (247).

Local Injection of Alcohol and Active Vitamin D Derivatives

Since in advanced forms of secondary hyperparathyroidism the hyperplasia of parathyroid glands is asymmetrical, with some glands being grossly enlarged and others remaining relatively small, local injection of ethanol (346,347) or active vitamin D derivatives (348,349) has been proposed as an alternative therapy in patients who become resistant to medical treatment. However, the direct injection technique has not reached widespread use in clinical practice outside of Japan. Other research groups have been unable to obtain convincing results (350,351).

Despite major advances in the medical treatment of CKD-MBD the achievement of the targets for plasma calcium, phosphate, Ca x P product, and PTH, as recommended by the K/DOQI guidelines (262), was found to be far from being optimal in the DOPPS patient population for the years 2002-2004 (352). It was actually rare in the hemodialysis patients of this international cohort to fall within recommended ranges for all four indicators of mineral metabolism, although consistent control of all three main CKD-MBD parameters calcium, phosphate, and PTH was found to be a strong predictor of survival in hemodialysis patients in an observational study (353). A recent report on chronic hemodialysis patients in France confirmed that a satisfactory control of serum calcium, phosphate, or PTH was achieved in less than 20% among them (354).

Surgical Treatment

Surgical correction remains the final, symptomatic therapy of the most severe forms of secondary hyperparathyroidism, which cannot be controlled by medical treatment (355). The most important goal remains to prevent or correct the development of major clinical complications associated with this disease. The presence of severe parathyroid over function must be ascertained by clinical, biochemical and radiological evidence. In general, neck surgery should only be done when plasma iPTH values are greatly elevated (> 600-800 pg/mL), together with an increase in plasma total alkaline phosphatases (or better bone-specific alkaline phosphatase), and only after one or several medical treatment attempts have remained unsuccessful in decreasing plasma iPTH with cinacalcet (in dialysis patients only) and/or active vitamin D derivatives or if their use is relatively or absolutely contraindicated, namely in presence of persistent hypercalcemia, marked hyperphosphatemia, or severe vascular calcifications. Bone histomorphometry examination is rarely needed. Clinical symptoms and signs such as pruritus and osteoarticular pain are non-specific and therefore not good criteria for operation by their own. Similarly, an isolated increase in plasma calcium and/or phosphate, even in case of coexistent soft tissue calcifications, is not a sufficient criterion alone for surgical parathyroidectomy. However, in the presence of a persistently high plasma PTH the latter disturbances may facilitate the decision to proceed to surgery. The results can be spectacular, including in rare instances the complete disappearance of soft tissue calcifications from small peripheral arteries (see Figure 15b). A concomitant aluminum overload should be excluded or treated, if present, before performing surgery.

Two main surgical procedures are generally used, either subtotal parathyroidectomy or total parathyroidectomy with immediate auto-transplantation. There is no substantial difference of operative difficulties and treatment results between the two procedures. We found that the long-term frequency of recurrent hyperparathyroidism was similar (356). One group of authors claimed superiority of total parathyroidectomy without reimplantation of parathyroid tissue in terms of long-term control of parathyroid over function, tolerance, and safety (357), but this claim has been questioned by us and others (358–360). We do not recommend the performance of total parathyroidectomy without auto-transplantation in uremic patients since permanent hypoparathyroidism and adynamic bone disease may ensue, with possible harmful consequences especially for those patients who subsequently undergo kidney transplantation.

As regards the prevalence of parathyroidectomy it was very high before the turn of the century and did not change significantly between 1983 and 1996. According to a survey from Northern Italy in 7371 dialysis patients (361) it was 5.5% in the all patients together but increased with duration of RRT, from 9.2% after 10-15 years to 20.8% after 16-20 years of dialysis therapy. A recent survey from the US showed that parathyroidectomy rates were much lower in the first decade of the 21st century. It decreased from 7.9% in 2003 to a nadir of 3.3% in 2005 - most likely due to the commercial introduction of cinacalcet, then increased again to 5.5% through 2006, and subsequently remained stable until 2011 (362). The authors concluded that despite the use of multiple medical therapies rates of parathyroidectomy in patients with secondary hyperparathyroidism did not decline in recent years. These findings are in contrast with a Canadian study (363) and the international Dialysis Outcomes and Practice Patterns Study (DOPPS) (364). The Canadian study, although restricted to a single province (Quebec), showed a sustained reduction in parathyroidectomy rates after 2006. The DOPPS reported that prescriptions of active vitamin D analogs and cinacalcet increased and that parathyroidectomy rates decreased. Difference in medical treatment modalities between geographic regions and different modes of data analysis may at least partially account for these apparent discrepancies. This is illustrated by the observation that parathyroidectomy rates in Japan fell abruptly after the advent of cinacalcet to approximately 2%, with median serum iPTH around 150 pg/mL between 1996 and 2011 (364).

Parathyroidectomy was associated with higher short-term mortality, but lower long-term mortality among chronic dialysis patients in the US (365). Whether presently available therapeutic and prophylactic measures taken to attenuate secondary hyperparathyroidism play an important role in reducing cardiovascular morbidity and mortality among patients with ESKD remains a matter of debate. The EVOLVE trial points to better clinical outcomes with a more efficient control of parathyroid over function by cinacalcet than by optimal standard treatment but the results, although suggestive, must still be considered as not definitively conclusive (298,303).

ACKNOWLEDGEMENTS

The author wishes to thank Ms Martine Netter, Paris for expert assistance in Figure design.

REFERENCES

Moe S, Drueke T, Cunningham J et al. Definition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int 2006; 69: 1945–1953.
Levin A, Bakris GL, Molitch M et al. Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: Results of the study to evaluate early kidney disease. Kidney Int 2007; 71: 31–38.
Fliser D, Kollerits B, Neyer U et al. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study. J Am Soc Nephrol 2007; 18: 2600–2608.
Isakova T, Wahl P, Vargas GS et al. Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease. Kidney Int 2011; 79: 1370–1378.
Hu MC, Kuro-o M, Moe OW. The emerging role of Klotho in clinical nephrology. Nephrol Dial Transplant 2012; 27: 2650–2657.
Drüeke TB, Massy ZA. Changing bone patterns with progression of chronic kidney disease. Kidney International 2016; 89: 289–302.
Evenepoel P, D’Haese P, Brandenburg V. Sclerostin and DKK1: new players in renal bone and vascular disease. Kidney Int 2015; 88: 235–240.
Haarhaus M, Evenepoel P, European Renal Osteodystrophy (EUROD) workgroup, Chronic Kidney Disease Mineral and Bone Disorder (CKD-MBD) working group of the European Renal Association–European Dialysis and Transplant Association (ERA-EDTA). Differentiating the causes of adynamic bone in advanced chronic kidney disease informs osteoporosis treatment. Kidney International 2021; 100: 546–558.
Malluche HH, Mawad HW, MonierFaugere MC. Renal Osteodystrophy in the First Decade of the New Millennium: Analysis of 630 Bone Biopsies in Black and White Patients. J Bone Miner Res 2011; 26: 1368–1376.
Block GA, Klassen PS, Lazarus JM, Ofsthun N, Lowrie EG, Chertow GM. Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Amer Soc Nephrol 2004; 15: 2208–2218.
Moe SM, Drueke TB. Management of secondary hyperparathyroidism: the importance and the challenge of controlling parathyroid hormone levels without elevating calcium, phosphorus, and calcium-phosphorus product. Am J Nephrol 2003; 23: 369–379.
Tentori F, Blayney MJ, Albert JM et al. Mortality risk for dialysis patients with different levels of serum calcium, phosphorus, and PTH: the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis 2008; 52: 519–530.
Floege J. Calcium-containing phosphate binders in dialysis patients with cardiovascular calcifications: should we CARE-2 avoid them? Nephrol Dial Transplant 2008; 23: 3050–3052.
Floege J, Kim J, Ireland E et al. Serum iPTH, calcium and phosphate, and the risk of mortality in a European haemodialysis population. Nephrol Dial Transplant 2011; 26: 1948–1955.
Fernandez-Martin JL, Martinez-Camblor P, Dionisi MP et al. Improvement of mineral and bone metabolism markers is associated with better survival in haemodialysis patients: the COSMOS study. Nephrol Dial Transplant 2015; 30: 1542–1551.
Hagstrom E, Hellman P, Larsson TE et al. Plasma Parathyroid Hormone and the Risk of Cardiovascular Mortality in the Community. Circulation 2009; 119: 2765-U34.
E Slatopolsky, S Caglar, J P Pannell et al. On the pathogenesis of hyperparathyroidism in chronic experimental renal insufficiency in the dog. J. Clin. Invest. 1971; 50: 492–499.
Vorland CJ, Biruete A, Lachcik PJ et al. Kidney Disease Progression Does Not Decrease Intestinal Phosphorus Absorption in a Rat Model of Chronic Kidney Disease-Mineral Bone Disorder. Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research 2020; 35: 333–342.
Moranne O, Froissart M, Rossert J et al. Timing of onset of CKD-related metabolic complications. J Am Soc Nephrol 2009; 20: 164–171.
F Llach, S G Massry, A Koffler. Secondary hyperparathyroidism in early renal failure: role of phosphate retention. Kidney Int. 1977; 12: 459–463.
Hsu CY, Chertow GM. Elevations of serum phosphorus and potassium in mild to moderate chronic renal insufficiency. Nephrol Dial Transplant 2002; 17: 1419-25.
Kurosu H, Kuro OM. The Klotho gene family as a regulator of endocrine fibroblast growth factors. Mol Cell Endocrinol 2009; 299: 72–78.
Olauson, HannesVervloet, Marc GCozzolino, MarioMassy, Ziad AUrena Torres, PabloLarsson, Tobias EengReview2014/12/17 06:0.
Hasegawa H, Nagano N, Urakawa I et al. Direct evidence for a causative role of FGF23 in the abnormal renal phosphate handling and vitamin D metabolism in rats with early-stage chronic kidney disease. Kidney Int 2010; 78: 975–980.
Dhayat NA, Ackermann D, Pruijm M et al. Fibroblast growth factor 23 and markers of mineral metabolism in individuals with preserved renal function. Kidney International 2016; 90: 648–657.
Hu MC, Shi M, Zhang J et al. Klotho deficiency causes vascular calcification in chronic kidney disease. J Am Soc Nephrol 2011; 22: 124–136.
Lim K, Lu TS, Molostvov G et al. Vascular klotho deficiency potentiates the development of human artery calcification and mediates resistance to fibroblast growth factor 23. Circulation 2012; 125: 2243–2255.
Hu MC, Shi M, Zhang J et al. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J 2010; 24: 3438–3450.
Chen G, Liu Y, Goetz R et al. α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling. Nature 2018; 553: 461–466.
Olauson H, Vervloet MG, Cozzolino M, Massy ZA, Urena Torres P, Larsson TE. New insights into the FGF23-Klotho axis. Semin Nephrol 2014; 34: 586–597.
Pavik I, Jaeger P, Ebner L et al. Secreted Klotho and FGF23 in chronic kidney disease Stage 1 to 5: a sequence suggested from a cross-sectional study. Nephrol Dial Transplant 2013; 28: 352–359.
Shimamura Y, Hamada K, Inoue K et al. Serum levels of soluble secreted alpha-Klotho are decreased in the early stages of chronic kidney disease, making it a probable novel biomarker for early diagnosis. Clin Exp Nephrol 2012; 16: 722–729.
Smith ER, Holt SG, Hewitson TD. αKlotho-FGF23 interactions and their role in kidney disease: a molecular insight. Cellular and molecular life sciences: CMLS 2019; 76: 4705–4724.
Wolf M. Update on fibroblast growth factor 23 in chronic kidney disease. Kidney Int 2012;
Fan Y, Bi R, Densmore MJ et al. Parathyroid hormone 1 receptor is essential to induce FGF23 production and maintain systemic mineral ion homeostasis. FASEB J 2015;
Meir T, Durlacher K, Pan Z et al. Parathyroid hormone activates the orphan nuclear receptor Nurr1 to induce FGF23 transcription. Kidney Int 2014; 86: 1106–1115.
Lopez I, RodriguezOrtiz ME, Almaden Y et al. Direct and indirect effects of parathyroid hormone on circulating levels of fibroblast growth factor 23 in vivo. Kidney Int 2011; 80: 475–482.
Galitzer H, BenDov IZ, Silver J, NavehMany T. Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease. Kidney Int 2010; 77: 211–218.
Canalejo R, Canalejo A, MartinezMoreno JM et al. FGF23 Fails to Inhibit Uremic Parathyroid Glands. J Amer Soc Nephrol 2010; 21: 1125–1135.
Komaba H, Goto S, Fujii H et al. Depressed expression of Klotho and FGF receptor 1 in hyperplastic parathyroid glands from uremic patients. Kidney Int 2010; 77: 232–238.
Larsson T, Nisbeth U, Ljunggren O, Juppner H, Jonsson KB. Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers. Kidney Int 2003; 64: 2272–2279.
Shigematsu T, Kazama JJ, Yamashita T et al. Possible involvement of circulating fibroblast growth factor 23 in the development of secondary hyperparathyroidism associated with renal insufficiency. Am J Kidney Dis 2004; 44: 250–256.
Komaba H, Fukagawa M. FGF23-parathyroid interaction: implications in chronic kidney disease. Kidney Int 2010; 77: 292–298.
Spiegel DM, Brady K. Calcium balance in normal individuals and in patients with chronic kidney disease on low- and high-calcium diets. Kidney Int 2012; 81: 1116–1122.
Hill KM, Martin BR, Wastney ME et al. Oral calcium carbonate affects calcium but not phosphorus balance in stage 3-4 chronic kidney disease. Kidney Int 2013; 83: 959–966.
Koizumi M, Komaba H, Fukagawa M. Parathyroid function in chronic kidney disease: role of FGF23-Klotho axis. Contrib Nephrol 2013; 180: 110–123.
Goodman WG, Quarles LD. Development and progression of secondary hyperparathyroidism in chronic kidney disease: lessons from molecular genetics. Kidney Int 2008; 74: 276–288.
P A Lucas, R C Brown, J S Woodhead, G A Colesi. 1,25 dihydroxycholecalciferol and parathyroid hormone in advanced chronic renal failure : effect of simultaneous protein and phosphorus restriction. Clin. Nephrol. 1986; 25: 7–10.
Nykjaer A, Dragun D, Walther D et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D-3. Cell 1999; 96: 507–515.
Zehnder D, Bland R, Walker EA et al. Expression of 25-hydroxyvitamin D-3-1 alpha-hydroxylase in the human kidney. J Amer Soc Nephrol 1999; 10: 2465–2473.
Cuppari L, Garcia-Lopes MG. Hypovitaminosis D in chronic kidney disease patients: prevalence and treatment. J Ren Nutr 2009; 19: 38–43.
Mehrotra R, Kermah D, Budoff M et al. Hypovitaminosis D in chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1144–1151.
J Cunningham, H Makin. How important is vitamin D deficiency in uraemia ? (Editorial Comment). Nephrol. Dial. Transplant. 1997; 12: 16–18.
Ghazali A, Fardellone P, Pruna A et al. Is low plasma 25-(OH)vitamin D a major risk factor for hyperparathyroidism and Looser’s zones independent of calcitriol? Kidney Int 1999; 55: 2169–2177.
Ritter CS, Armbrecht HJ, Slatopolsky E, Brown AJ. 25-Hydroxyvitamin D(3) suppresses PTH synthesis and secretion by bovine parathyroid cells. Kidney Int 2006; 70: 654–659.
Vervloet MG, Massy ZA, Brandenburg VM et al. Bone: a new endocrine organ at the heart of chronic kidney disease and mineral and bone disorders. Lancet Diabetes Endocrinol 2014; 2: 427–436.
Glorieux G, Hsu CH, de Smet R et al. Inhibition of calcitriol-induced monocyte CD14 expression by uremic toxins: role of purines. J Am Soc Nephrol 1998; 9: 1826–1831.
S R Patel, H Q Ke, R Vanholder, R J Koenig, C H Hsu. Inhibition of calcitriol receptor binding to vitamin D response elements by uremic toxins. J. Clin. Invest. 1995; 96: 50–59.
Goto S, Fujii H, Hamada Y, Yoshiya K, Fukagawa M. Association between indoxyl sulfate and skeletal resistance in hemodialysis patients. Ther Apher Dial 2012; 417–423.
Watanabe K, Tominari T, Hirata M et al. Indoxyl sulfate, a uremic toxin in chronic kidney disease, suppresses both bone formation and bone resorption. FEBS open bio 2017; 7: 1178–1185.
Dusso AS. Vitamin D receptor: Mechanisms for vitamin D resistance in renal failure. Kidney Int Suppl 2003; : 6–9.
N Fukuda, H Tanaka, Y Tominaga, M Fukagawa, K Kurokawa, Y Seino. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J. Clin. Invest. 1993; 92: 1436–1442.
S Patel, H Q Ke, R Vanholder, C H Hsu. Inhibition of nuclear uptake of calcitriol receptor by uremic ultrafiltrate. Kidney Int. 1994; 46: 129–133.
Garfia B, Canadillas S, Canalejo A et al. Regulation of parathyroid vitamin D receptor expression by extracellular calcium. J Amer Soc Nephrol 2002; 13: 2945–2952.
SelaBrown A, Russell J, Koszewski NJ, Michalak M, NavehMany T, Silver J. Calreticulin inhibits vitamin D’s action on the PTH gene in vitro and may prevent vitamin D’s effect in vivo in hypocalcemic rats. Mol Endocrinol 1998; 12: 1193–1200.
A J Brown, M Zhong, J Finch et al. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am. J. Physiol. 1996; 270: F454–F460.
Mendoza FJ, Lopez I, Canalejo R et al. Direct upregulation of parathyroid calcium-sensing receptor and vitamin D receptor by calcimimetics in uremic rats. Amer J Physiol Renal Physiol 2009; 296: F605–F613.
J Silver, S B Sela, T Naveh-Many. Regulation of parathyroid cell proliferation. Curr. Opin. Nephrol. Hypertens. 1997; 6: 321–326.
A Szabo, J Merke, E Beier, G Mall, E Ritz. 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int. 1989; 35: 1045–1056.
M Fukagawa, S-Y Kaname, T Igarashi, E Ogata, K Kurokawa. Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int. 1991; 39: 874–881.
T Naveh-Many, R Rahamimov, N Livni, J Silver. Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J. Clin. Invest. 1995; 96: 1786–1793.
P Nygren, R Larsson, H Johannson, S Ljunghall, J Rastad, G Akerstrom. 1,25 (OH)2 D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells. Calcif. Tissue Int. 1988; 43: 213–218.
R Kremer, I Bolivar, D Goltzman, G N Hendy. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989; 125: 935–941.
Y Ishimi, J Russel, L M Sherwood. Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture. J. Bone Min. Res. 1990; 5: 755–760.
Roussanne MC, Lieberherr M, Souberbielle JC, Sarfati E, Drueke T, Bourdeau A. Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate. Eur J Clin Invest 2001; 31: 610-6.
A Fernandez, J Fibla, A Betriu, J M Piulats, J Almirall, J Montoliu. Association between vitamin D receptor gene polymorphism and relative hypoparathyroidism in patients with chronic renal failure. J. Am. Soc. Nephrol. 1997; 8: 1546–1552.
S Aterini, M Salvadori, E Ippolito et al. The role of vitamin D receptor alleles in the secondary hyperparathyroidism of hemodialysis patients. J Nephrol 1996; 9: 201–206.
Y Nagaba, M Heishi, H Tazawa, Y Tsukamoto, Y Kobayashi. Vitamin D receptor gene polymorphisms affect secondary hyperparathyroidism in hemodialyzed patients. Am J Kidney Dis 1998; 32: 464–469.
Yokoyama K, Shigematsu T, Tsukada T et al. Apa I polymorphism in the vitamin D receptor gene may affect the parathyroid response in Japanese with end-stage renal disease. Kidney Int. 1998; 53: 454–458.
Carling T, Rastad J, Akerstrom G, Westin G. Vitamin D receptor (VDR) and parathyroid hormone messenger ribonucleic acid levels correspond to polymorphic VDR alleles in human parathyroid tumors. J Clin Endocrinol Metab 1998; 83: 2255–2259.
S Schmidt, J Chudek, H Karkoska et al. The BsmI vitamin D-recptor polymorphism and secondary hyperparathyroidism (Letter). Nephrol Dial Transplant 1997; 12: 1771–1772.
A Torres, M Machado, M TConcepcion et al. Influence of vitamin D receptor genotype on bone mass changes after renal transplantation. Kidney Int 1996; 50: 1726–1733.
N S Hawa, F J Cockerill, S Vadher et al. Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping. Clin Endocrinol 1996; 45: 85–92.
N A Morrison, J C Qi, A Tokita et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994; 367: 284–287.
Egstrand S, Nordholm A, Morevati M et al. A molecular circadian clock operates in the parathyroid gland and is disturbed in chronic kidney disease associated bone and mineral disorder. Kidney International 2020; 98: 1461–1475.
Isakova T, Gutierrez O, Shah A et al. Postprandial mineral metabolism and secondary hyperparathyroidism in early CKD. J Am Soc Nephrol 2008; 19: 615–623.
Santamaria R, Almaden Y, Felsenfeld A et al. Dynamics of PTH secretion in hemodialysis patients as determined by the intact and whole PTH assays. Kidney Int 2003; 64: 1867–1873.
Moyses RMA, Pereira RC, dosReis LM, Sabbaga E, Jorgetti V. Dynamic tests of parathyroid hormone secretion using hemodialysis and calcium infusion cannot be compared. Kidney Int 1999; 56: 659–665.
E M Brown. Four-parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J. Clin. Endocrinol. Metab. 1983; 56: 572–581.
J Gogusev, P Duchambon, B Hory, M Giovannini, E Sarfati, T B Drüeke. Depressed expression of calcium receptor in parathyroid gland tissue of patients with primary or secondary uremic hyperparathyroidism. Kidney Int. 1997; 51: 328–336.
O Kifor, F D Moore, P Wang et al. Reduced immunostaining for the extracellular Ca2+ - sensing receptor in primary and uremic secondary hyperparathyroidism. J. Clin. Endocrinol. Metab. 1996; 81: 1598–1606.
Rodríguez-Ortiz ME, Canalejo A, Herencia C et al. Magnesium modulates parathyroid hormone secretion and upregulates parathyroid receptor expression at moderately low calcium concentration. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2014; 29: 282–289.
Brown AJ, Ritter CS, Finch JL, Slatopolsky EA. Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: Role of dietary phosphate. Kidney Int 1999; 55: 1284–1292.
Campion KL, McCormick WD, Warwicker J et al. Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism. Journal of the American Society of Nephrology: JASN 2015; 26: 2163–2171.
Centeno PP, Herberger A, Mun H-C et al. Phosphate acts directly on the calcium-sensing receptor to stimulate parathyroid hormone secretion. Nature Communications 2019; 10: 4693.
Almadén Y, Hernandez A, Torregrosa V et al. High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro. J Amer Soc Nephrol 1998; 9: 1845–1852.
Lewin E, Garfia B, Almaden Y, Rodriguez M, Olgaard K. Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats. Kidney Int 2003; 64: 63–70.
Goodman WG, Veldhuis JD, Belin TR, VanHerle AJ, Juppner H, Salusky IB. Calcium-sensing by parathyroid glands in secondary hyperparathyroidism. J Clin Endocrinol Metab 1998; 83: 2765–2772.
A J Felsenfeld, M Rodriguez. Parathyroid gland function in hemodialysis patients. Semin. Dial. 1996; 9: 303–309.
W G Goodman, T R Belin, I B Salusky. In vivo assessments of calcium-regulated parathyroid hormone release in secondary hyperparathyroidism. Kidney Int. 1996; 50: 1834–1844.
R Ouseph, J D Leiser, S M Moe. Calcitriol and the parathyroid hormone-ionized calcium curve: a comparison of methodologic approaches. J. Am. Soc. Nephrol 1996; 7: 497–505.
Mathias RS, Nguyen HT, Zhang MYH, Portale AA. Reduced expression of the renal calcium-sensing receptor in rats with experimental chronic renal insufficiency. J Amer Soc Nephrol 1998; 9: 2067–2074.
Wada M, Furuya Y, Sakiyama J et al. NPS R-568 halts or reverses osteitis fibrosa in uremic rats. Kidney Int. 1997; 53: 448–453.
A Wernerson, S M Windholm, O Svensson, F P Reinholt. Parathyroid cell number and size in hypocalcemic young rats. APMIS 1991; 99: 1096–1102.
M S LeBoff, H G Rennke, E M Brown. Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells. Endocrinology 1983; 113: 277–284.
M S LeBoff, D Shoback, E M Brown et al. Regulation of parathyroid hormone release and cytosolic calcium by extracellular calcium in dispersed and cultured bovine and pathological human parathyroid cells. J. Clin. Invest. 1985; 75: 49–57.
R R McGregor, M P Sarras, H Houle, D V Cohn. Primary monolayer culture of bovine parathyroids: effect of calcium, isoproterenol, and growth factors. Mol. Cell. Endocrinol. 1983; 30: 313–328.
R D Ridgeway, J W Hamilton, R R M Gregor. Characteristics of bovine parathyroid cell organoid in culture. In Vitro Cell Dev. 1998; 22: 91–99.
M L Brandi, L A Fitzpatrick, H G Coon, G D Aurbach. Bovine parathyroid cell: cultures maintained for more than 140 population doublings. Proc. Natl. Acad. Sci. USA 1986; 83: 1709–1713.
A J Brown, M Zhong, C S Ritter, E M Brown, E Slatopolsky. Loss of calcium responsiveness in cultured parathyroid cells is associated with decreased calcium receptor expression. Biochem. Biophys. Res. Commun. 1995; 212: 861–867.
A Mithal, O Kifor, I Kifor et al. The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca²⁺ is associated with marked reduction in the expression of extracellular Ca²⁺-sensing receptor messenger ribonucleic acid and protein. Endocrinology 1995; 136: 3087–3092.
M L Brandi, R L Ornberg, K Sakaguchi et al. Establishment and characterization of a clonal line of parathyroid endothelial cells. FASEB J 1990; 4: 3152–3158.
K Sakaguchi. Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth. J. Biol. Chem. 1992; 267: 24554–24562.
M-C Roussanne, J Gogusev, B Hory et al. Persistence of Ca²⁺-sensing receptor expression in functionally active, long-term human parathyroid cell cultures. J. Bone Min. Res. 1998; 13: 1–9.
Mizobuchi M, Hatamura I, Ogata H et al. Calcimimetic compound upregulates decreased calcium-sensing receptor expression level in parathyroid glands of rats with chronic renal insufficiency. J Am Soc Nephrol 2004; 15: 2579–2587.
Chikatsu N, Fukumoto S, Takeuchi Y et al. Cloning and characterization of two promoters for the human calcium-sensing receptor (CaSR) and changes of CaSR expression in parathyroid adenomas. J Biol Chem 2000; 275: 7553–7557.
S Lundgren, T Carling, G Hjälm et al. Tissue distribution of human gp330/megalin, a putative Ca²⁺-sensing protein. J. Histochem. Cytochem. 1997; 45: 383–392.
Y Almadén, A Canalejo, A Hernandez et al. Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro. J Bone Min Res 1996; 11: 970–976.
P K Nielsen, U Feldt-Rasmussen, K Olgaard. A direct effect of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol. Dial. Transplant. 1996; 11: 1762–1768.
E Slatopolsky, J Finch, M Denda et al. Phosphorus restriction prevents parathyroid gland growth: high phosphorus directly stimulates PTH secretion in vitro. J. Clin. Invest. 1996; 97: 2534–2540.
Almaden Y, Canalejo A, Ballesteros E, Anon G, Canadillas S, Rodriguez M. Regulation of arachidonic acid production by intracellular calcium in parathyroid cells: Effect of extracellular phosphate. J Amer Soc Nephrol 2002; 13: 693–698.
Moallem E, Kilav R, Silver J, NavehMany T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998; 273: 5253–5259.
Yalcindag C, Silver J, Naveh-Many T. Mechanism of increased parathyroid hormone mRNA in experimental uremia: roles of protein RNA binding and RNA degradation. Journal of the American Society of Nephrology: JASN 1999; 10: 2562–2568.
SelaBrown A, Silver J, Brewer G, NavehMany T. Identification of AUF1 as a parathyroid hormone mRNA 3’-untranslated region-binding protein that determines parathyroid hormone mRNA stability. J Biol Chem 2000; 275: 7424–7429.
E Slatopolsky, E Delmez. Pathogenesis of secondary hyperparathyroidism. Miner. Electrolyte Metab. 1995; 21: 91–96.
H Yi, M Fukagawa, H Yamato, M Kumagai, T Watanabe, K Kurokawa. Prevention of enhanced parathyroid hormone secretion, synthesis and hyperplasia by mild dietary phosphorus restriction in early chronic renal failure in rats: possible direct role of phosphorus. Nephron 1995; 70: 242–248.
Takahashi F, Denda M, Finch JL, Brown AJ, Slatopolsky E. Hyperplasia of the parathyroid gland without secondary hyperparathyroidism. Kidney Int 2002; 61: 1332–1338.
M-C Roussanne, J Gogusev, E Sarfati, T Drüeke, A Bourdeau. Effect of phosphate on PTH secretion and proliferation in human parathyroid cells in culture (Abstract). J. Bone Min. Res. (suppl. 1) 1997; 12: S387.
Silver J, Naveh-Many T. FGF23 and the parathyroid. Advances in Experimental Medicine and Biology 2012; 728: 92–99.
HofmanBang J, Martuseviciene G, Santini MA, Olgaard K, Lewin E. Increased parathyroid expression of klotho in uremic rats. Kidney Int 2010; 78: 1119–1127.
Fan Y, Liu W, Bi R et al. Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia. Proceedings of the National Academy of Sciences of the United States of America 2018; 115: E3749–E3758.
Mace ML, Olgaard K, Lewin E. New Aspects of the Kidney in the Regulation of Fibroblast Growth Factor 23 (FGF23) and Mineral Homeostasis. International Journal of Molecular Sciences 2020; 21: E8810.
Shilo V, Ben-Dov IZ, Nechama M, Silver J, Naveh-Many T. Parathyroid-specific deletion of dicer-dependent microRNAs abrogates the response of the parathyroid to acute and chronic hypocalcemia and uremia. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2015; 29: 3964–3976.
Shilo V, Mor-Yosef Levi I, Abel R et al. Let-7 and MicroRNA-148 Regulate Parathyroid Hormone Levels in Secondary Hyperparathyroidism. Journal of the American Society of Nephrology: JASN 2017; 28: 2353–2363.
Volovelsky O, Cohen G, Kenig A et al. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism. Journal of the American Society of Nephrology: JASN 2016; 27: 1091–1101.
J Aubia, S Serrano, L Mariosso et al. Osteodystrophy of diabetics in chronic dialysis: a histomorphometric study. Calcif Tissue Int 1988; 42: 297–301.
D Hernandez, M T Concepcion, V Lorenzo et al. Adynamic bone disease with negative aluminum staining in predialysis patients: prevalence and evolution after maintenance dialysis. Nephrol Dial Transplant 1994; 9: 517–523.
Y Pei, G Hercz, C Greenwood et al. Renal osteodystrophy in diabetic patients. Kidney Int 1993; 44: 159–164.
F Vicenti, S B Arnaud, R Recker et al. Parathyroid and bone response of the diabetic patient to uremia. Kidney Int 1984; 25: 677–682.
Panuccio V, Mallamaci F, Tripepi G et al. Low parathyroid hormone and pentosidine in hemodialysis patients. Am J Kidney Dis 2002; 40: 810-5.
Hocher B, Armbruster FP, Stoeva S et al. Measuring parathyroid hormone (PTH) in patients with oxidative stress--do we need a fourth generation parathyroid hormone assay? PLoS One 2012; 7: e40242.
Hocher B, Zeng S. Clear the Fog around Parathyroid Hormone Assays: What Do iPTH Assays Really Measure? Clinical journal of the American Society of Nephrology: CJASN 2018; 13: 524–526.
Jara A, Gonzalez S, Felsenfeld AJ et al. Failure of high doses of calcitriol and hypercalcaemia to induce apoptosis in hyperplastic parathyroid glands of azotaemic rats. Nephrol Dial Transplant 2001; 16: 506-12.
T Sugimoto, C Ritter, J Morrisey, C Hayes, E Slatopolsky. Effects of high concentrations of glucose on PTH secretion in parathyroid cells. Kidney Int 1990; 37: 1522–1527.
J B Cannata, J D Briggs, B J R Junor, J S Fell. The influence of aluminium on parathyroid hormone levels in hemodialysis patients. Proc Eur Dial Transplant Assoc 1982; 19: 244–247.
F Llach, A J Felsenfeld, M D Coleman, J J Keveney Jr, J A Pederson, T R Medlock. The natural course of dialysis osteomalacia. Kidney Int 1986; 29 (suppl 18): S74–S79.
D Andress, A J Felsenfeld, A Voigts, F Llach. Parathyroid hormone response to hypocalcemia in hemodilaysis patients with osteomalacia. Kidney Int 1983; 24: 363–370.
J A Kraut, J H Shinaberger, F R Singer et al. Parathyroid hormone response to acute hypocalcemia in dialysis osteomalacia. Kidney Int 1983; 23: 725–730.
C E Cann, S G Prussin, G S Gordan. Aluminum uptake by the parathyroid glands. J Clin Endocrinol Metab 1979; 49: 543–545.
M Rodriguez, A J Felsenfeld, F Llach. The role of aluminum in the development of hypercalcemia in the rat. Kidney Int 1987; 31: 766–771.
J Morrissey, S Slatopolsky. Effect of aluminum on parathyroid hormone secretion. Kidney Int 1986; 29 (suppl 18): S41–S44.
A C Alfrey, A Sedman, Y Chan. The compartmentalization and metabolism of aluminum in uremic rats. J Lab Clin Med 1985; 105: 227–233.
D A Henry, W G Goodman, R K Nudelman et al. Parenteral aluminum administration in the dog: I. Plasma kinetics, tissue levels, calcium metabolism, and parathyroid hormone. Kidney Int 1984; 25: 362–369.
J B DiazLopez, P C D’Haese, E J Noueven, L V Lamberts, J B Cannata, M E De Broe. Estudio del contenido de aluminio en paratiroides de ratas con insuficiencia renal e intoxicacion aluminica cronica. Nefrologia 1988; 8: 35–41.
J L Finch, M Bergfeld, K J Martin, Y L Chan, S Teitelbaum, E Slatopolsky. The effects of discontinuation of aluminum exposure on aluminum-induced osteomalacia. Kidney Int 1986; 30: 318–324.
A J Felsenfeld, M Rofdriguez, M Coleman, D Ross, F Llach. Desferrioxamine therapy in hemodialysis patients with aluminum-associated bone disease. Kidney Int. 1989; 35: 1371–1378.
Cozzolino M, Lu Y, Finch J, Slatopolsky E, Dusso AS. p21(WAF1) and TGF-alpha mediate parathyroid growth arrest by vitamin D and high calcium. Kidney Int 2001; 60: 2109–2117.
Cordero JB, Cozzolino M, Lu Y et al. 1,25-Dihydroxyvitamin D down-regulates cell membrane growth- and nuclear growth-promoting signals by the epidermal growth factor receptor. J Biol Chem 2002; 277: 38965–38971.
Dusso AS, Pavlopoulos T, Naumovich L et al. P21(WAF1) and transforming growth factor-alpha mediate dietary phosphate regulation of parathyroid cell growth. Kidney Int 2001; 59: 855–865.
J Gogusev, P Duchambon, C Stoermann-Chopard, M Giovannini, E Sarfati, T B Drüeke. De novo expression of transforming growth factor-alpha in parathyroid gland tissue of patients with primary or secondary uraemic hyperparathyroidism. Nephrol. Dial. Transplant. 1996; 11: 2155–2162.
Arcidiacono MV, Cozzolino M, Spiegel N et al. Activator protein 2alpha mediates parathyroid TGF-alpha self-induction in secondary hyperparathyroidism. J Am Soc Nephrol 2008; 19: 1919–1928.
Matsushita H, Hara M, Endo Y et al. Proliferation of parathyroid cells negatively correlates with expression of parathyroid hormone-related protein in secondary parathyroid hyperplasia. Kidney Int 1999; 55: 130–138.
Gunther T, Chen ZF, Kim J et al. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 2000; 406: 199–203.
Correa P, Akerstrom G, Westin G. Underexpression of Gcm2, a master regulatory gene of parathyroid gland development, in adenomas of primary hyperparathyroidism. Clin Endocrinol (Oxf) 2002; 57: 501–505.
Han SI, Tsunekage Y, Kataoka K. Gata3 cooperates with Gcm2 and MafB to activate parathyroid hormone gene expression by interacting with SP1. Mol Cell Endocrinol 2015; 411: 113–120.
Morito N, Yoh K, Usui T et al. Transcription factor MafB may play an important role in secondary hyperparathyroidism. Kidney International 2018; 93: 54–68.
Naveh-Many T, Silver J. Transcription factors that determine parathyroid development power PTH expression. Kidney International 2018; 93: 7–9.
V Mendes, V Jorgetti, J Nemeth et al. Secondary hyperparathyroidism in chronic haemodialysis patients: a clinico-pathologic study. Proc. Europ. Dial. Transplant. Assoc. 1983; 20: 731–738.
A Arnold, M F Brown, P Ureña, R D Gaz, E Sarfati, T B Drüeke. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995; 95: 2047–2054.
Chudek J, Ritz E, Kovacs G. Genetic abnormalities in parathyroid nodules of uremic patients. Clin. Cancer Res. 1998; 4: 211–214.
Y Tominaga, S Kohara, Y Namii et al. Clonal analysis of nodular parathyroid hyperplasia in renal hyperparathyroidism. World J. Surg. 1996; 20: 744–752.
Tominaga Y, Takagi H. Molecular genetics of hyperparathyroid disease. Current Opinion in Nephrology and Hypertension 1996; 5: 336–341.
Imanishi Y, Tahara H, Palanisamy N et al. Clonal chromosomal defects in the molecular pathogenesis of refractory hyperparathyroidism of uremia. J Amer Soc Nephrol 2002; 13: 1490–1498.
E D Hsi, L R Zukerberg, W-I Yang, A Arnold. CyclinD1/PRAD1 expression in parathyroid adenomas: an immunohistochemical study. J. Clin. Endocrinol. Metab. 1996; 81: 1736–1739.
Tominaga Y, Tsuzuki T, Uchida K et al. Expression of PRAD1 cyclin D1, retinoblastoma gene products, and Ki67 in parathyroid hyperplasia caused by chronic renal failure versus primary adenoma. Kidney Int 1999; 55: 1375–1383.
Brown SB, Brierley TT, Palanisamy N et al. Vitamin D receptor as a candidate tumor-suppressor gene in severe hyperparathyroidism of uremia. J Clin Endocrinol Metab 2000; 85: 868–872.
Degenhardt S, Toell A, Weideman W, Dotzenrath C, Spindler K-D, Grabensee B. Point mutations of the human parathyroid calcium receptor gene are not responsible for non-suppresible renal hyperparathyroidism. Kidney Intern. 1998; 53: 556–561.
Djema AI, Mahmoud MD, Collin P, Heyman MF. Hyperparathyroïdie tertiaire: cancer parathyroïdien avec métastases hépatiques chez un hémodialysé. Néphrologie 1998; 19: 121–123.
Miki H, Sumitomo M, Inoue H, Kita S, Monden Y. Parathyroid carcinoma in patients with chronic renal failure on maintenance hemodialysis. [Review] [10 refs]. Surgery 1996; 120: 897–901.
Takami H, Kameyama K, Nagakubo I. Parathyroid carcinoma in a patient receiving long-term hemodialysis. Surgery 1999; 125: 239–240.
T B Drüeke, P Zhang, J Gogusev. Apoptosis: background and possible role in secondary hyperparathyroidism (Invited Comment). Nephrol. Dial. Transplant. 1997; 12: 2228–2233.
A M Parfitt. The hyperparathyroidism of chronic renal failure: a disorder of growth. Kidney Int. 1997; 52: 3–9.
Wada M, Furuya Y, Sakiyama J et al. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J. Clin. Invest. 1997; 100: 2977–2983.
Canalejo A, Almaden Y, Torregrosa V et al. The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Amer Soc Nephrol 2000; 11: 1865–1872.
A Jara, J Bover, A J Felsenfeld. Development of secondary hyperparathyroidism and bone disease in diabetic rats with renal failure. Kidney Int. 1995; 47: 1746–1751.
P Zhang, P Duchambon, J Gogusev et al. Apoptosis in parathyroid hyperplasia of patients with primary or secondary uremic hyperparathyroidism. Kidney Int 2000; 57: 437–445.
S Heidenreich, M Schmidt, J Bachmann, B Harrach. Apoptosis of monocytes cultured from long-term hemodialysis patients. Kidney Int. 1996; 49: 792–799.
S G Fadda, S G Massry. Chronic renal failure is a state of cellular calcium toxicity (Review). Am. J. Kidney Dis. 1993; 21: 81–86.
J Carracedo, R Ramirez, A Martin-Malo, M Rodriguez, P Aljama. Nonbiocompatible hemodialysis membranes induce apoptosis in mononuclear cells: the role of G-proteins. J. Am. Soc. Nephrol. 1998; 9: 46–53.
E Lewin, W Wang, K Olgaard. Reversibility of experimental secondary hyperparathyroidism. Kidney Int. 1997; 52: 1232–1241.
H L Henry, A N Taylor, A W Norman. Response of chick parathyroid glands to the vitamin D metabolites, 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol. J. Nutr. 1977; 107: 1918–1926.
Cloutier M, Brossard JH, Gascon-Barre M, D’Amour P. Lack of involution of hyperplastic parathyroid glands in dogs: adaptation via a decrease in the calcium stimulation set point and a change in secretion profile. J Bone Miner Res 1994; 9: 621–629.
Colloton M, Shatzen E, Miller G et al. Cinacalcet HCl attenuates parathyroid hyperplasia in a rat model of secondary hyperparathyroidism. Kidney Int 2005; 67: 467–476.
Miller G, Davis J, Shatzen E, Colloton M, Martin D, Henley CM. Cinacalcet HCl prevents development of parathyroid gland hyperplasia and reverses established parathyroid gland hyperplasia in a rodent model of CKD. Nephrol Dial Transplant 2012; 27: 2198–2205.
E Nylen, A Shah, J Hall. Spontaneous remission of primary hyperparathroidism from parathroid apoplexy. J. Clin. Endocrinol. Metab. 1996; 81: 1326–1328.
M Fukagawa, R Okazaki, K Takano et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N. Engl. J. Med. 1990; 323: 421–422.
L D Quarles, D A Yohay, B A Carroll et al. Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int. 1994; 45: 1710–1721.
M Fukagawa, M Kitaoka, H Yi, N Fukuda, T Matsumoto, E Ogata. Serial evaluation of parathyroid size by ultrasonography is another useful marker for the long-term prognosis of calcitriol pulse therapy in chronic dialysis patients. Nephron 1994; 68: 221–228.
Okuno S, Ishimura E, Kitatani K et al. Relationship between parathyroid gland size and responsiveness to maxacalcitol therapy in patients with secondary hyperparathyroidism. Nephrol Dial Transplant 2003; 18: 2613–2621.
K J Martin, K A Hruska, J Lewis, C Anderson, E Slatopolsky. The renal handling of parathyroid hormone: role of peritubular uptake and glomerular filtration. J. Clin. Invest. 1977; 60: 808–814.
Hilpert J, Nykjaer A, Jacobsen C et al. Megalin antagonizes activation of the parathyroid hormone receptor. J Biol Chem 1999; 274: 5620–5625.
Freitag JJ, Martin KJ, Hruska KA, Slatopolsky E. Impaired parathyroid hormone metabolism in patients with chronic renal failure. N. Engl. J. Med. 1978; 298: 29–34.
K A Hruska, R Kopelman, W E Rutherford, S Klahr, E Slatopolsky. Metabolism of immunoreactive parathyroid hormone in the dog: the role of the kidney and the effects of chronic renal disease. J. Clin. Invest. 1975; 56: 39–46.
Martin KJ, Hruska KA, Freitag JJ, Klahr S, Slatopolsky E. The peripheral metabolism of parathyroid hormone in patients with chronic renal failure. N. Engl. J. Med. 1979; 301: 1092–1098.
Daugaard H, Egfjord M, Lewin E, Olgaard K. Metabolism of intact PTH by isolated perfused kidney and liver from uremic rats. Exp. Nephrol. 1994; 2: 240–248.
Fukagawa M, Kazama JJ, Shigematsu T. Skeletal resistance to PTH as a basic abnormality underlying uremic bone diseases. Am J Kidney Dis 2001; 38: S152-5.
Kazama JJ, Shigematsu T, Yano K et al. Increased circulating levels of osteoclastogenesis inhibitory factor (osteoprotegerin) in patients with chronic renal failure. Am J Kidney Dis 2002; 39: 525–532.
M Smogorzewski, J Tian, S G Massry. Down-regulation of PTH-PTHrP receptor of heart in CRF: role of [Ca²⁺]i. Kidney Int. 1995; 47: 1182–1186.
P Ureña, A Ferreira, C Morieux, T B Drüeke, M C deVernejoul. PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilage growth plate of uraemic rats. Nephrol. Dial. Transplant. 1996; 11: 2008–2016.
P Ureña, M Kubrusly, M Mannstadt et al. The renal PTH/PTHrP receptor is down-regulated in rats with chronic renal failure. Kidney Int. 1994; 45: 605–611.
P Ureña, M Mannstadt, M Hruby et al. Parathyroidectomy does not prevent the renal PTH/PTHrP receptor down-regulation in uremic rats. Kidney Int. 1995; 47: 1797–1805.
Picton ML, Moore PR, Mawer EB et al. Down-regulation of human osteoblast PTH/PTHrP receptor mRNA in end-stage renal failure. Kidney Int 2000; 58: 1440–1449.
Langub MC, MonierFaugere MC, Qi QL, Geng Z, Koszewski NJ, Malluche HH. Parathyroid hormone/parathyroid hormone-related peptide type 1 receptor in human bone. J Bone Miner Res 2001; 16: 448–456.
Takenaka T, Inoue T, Miyazaki T, Hayashi M, Suzuki H. Xeno-Klotho Inhibits Parathyroid Hormone Signaling. J Bone Miner Res 2015; 31: 455–462.
Drueke TB, Lafage-Proust MH. Sclerostin: just one more player in renal bone disease? Clin J Am Soc Nephrol 2011; 6: 700–703.
Sugatani T. Systemic Activation of Activin A Signaling Causes Chronic Kidney Disease-Mineral Bone Disorder. International Journal of Molecular Sciences 2018; 19: E2490.
Viaene L, Behets GJ, Claes K et al. Sclerostin: another bone-related protein related to all-cause mortality in haemodialysis? Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2013; 28: 3024–3030.
Drechsler C, Evenepoel P, Vervloet MG et al. High levels of circulating sclerostin are associated with better cardiovascular survival in incident dialysis patients: results from the NECOSAD study. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2015; 30: 288–293.
Bellido T, Ali AA, Gubrij I et al. Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis. Endocrinology 2005; 146: 4577–4583.
Kramer I, Loots GG, Studer A, Keller H, Kneissel M. Parathyroid hormone (PTH)-induced bone gain is blunted in SOST overexpressing and deficient mice. J Bone Miner Res 2010; 25: 178–189.
Fujita K, Roforth MM, Demaray S et al. Effects of estrogen on bone mRNA levels of sclerostin and other genes relevant to bone metabolism in postmenopausal women. J Clin Endocrinol Metab 2014; 99: E81-8.
Silva BC, Bilezikian JP. Parathyroid hormone: anabolic and catabolic actions on the skeleton. Curr Opin Pharmacol 2015; 22: 41–50.
Cejka D, Herberth J, Branscum AJ et al. Sclerostin and Dickkopf-1 in renal osteodystrophy. Clin J Am Soc Nephrol 2011; 6: 877–882.
Lima F, Mawad H, El-Husseini AA, Davenport DL, Malluche HH. Serum bone markers in ROD patients across the spectrum of decreases in GFR: Activin A increases before all other markers. Clinical Nephrology 2019; 91: 222–230.
Li J-Y, Yu M, Pal S et al. Parathyroid hormone-dependent bone formation requires butyrate production by intestinal microbiota. The Journal of Clinical Investigation 2020; 130: 1767–1781.
Yu M, Malik Tyagi A, Li J-Y et al. PTH induces bone loss via microbial-dependent expansion of intestinal TNF+ T cells and Th17 cells. Nature Communications 2020; 11: 468.
Massy ZA, Drueke TB. Gut microbiota orchestrates PTH action in bone: role of butyrate and T cells. Kidney International 2020; 98: 269–272.
F Llach. Calcific uremic arteriolopathy (calciphylaxis): an evolving entity ? Am J Kidney Dis 1998; 32: 513–518.
Floege J, Kubo Y, Floege A, Chertow GM, Parfrey PS. The Effect of Cinacalcet on Calcific Uremic Arteriolopathy Events in Patients Receiving Hemodialysis: The EVOLVE Trial. Clin J Am Soc Nephrol 2015; 10: 800–807.
Viljoen A, Singh DK, Twomey PJ, Farrington K. Analytical quality goals for parathyroid hormone based on biological variation. Clin Chem Lab Med 2008; 46: 1438–1442.
Barreto FC, Barreto DV, Moyses RM et al. K/DOQI-recommended intact PTH levels do not prevent low-turnover bone disease in hemodialysis patients. Kidney Int 2008; 73: 771–777.
M E Cohen-Solal, B Boudailliez, J L Sebert, P F Westeel, R Bouillon, A Fournier. Comparison of intact, mid region and carboxyterminal assays of parathyroid hormone for the diagnosis of bone disease in hemodialyzed patients. J. Clin. Endocrinol. Metab. 1991; 73: 516–524.
L D Quarles, B Lobaugh, G Murphy. Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J. Clin. Endocrinol. Metab. 1992; 75: 145–150.
M Wang, G Hercz, D J Sherrard, N A Maloney, G V Segre, Y Pei. Relationship between intact 1-84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminium toxicity. Am. J. Kidney Dis. 1995; 26: 836–844.
J H Brossard, M Cloutier, L Roy, R Lepage, M Gascon-Barré, P D’Amour. Accumulation of a non-(1-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values. J. Clin. Endocrinol. Metab. 1996; 81: 3923–3929.
R Lepage, L Roy, J H Brossard et al. A non-(1-84) circulating parathyroid hormone (PTH fragment interferes significantly with intact commercial PTH assay measurements in uremic samples. Clin. Chem. 1998; 44: 805–809.
Langub MC, Monier-Faugere MC, Wang G, Williams JP, Koszewski NJ, Malluche HH. Administration of PTH-(7-84) antagonizes the effects of PTH-(1-84) on bone in rats with moderate renal failure. Endocrinology 2003; 144: 1135–1138.
Souberbielle JC, Boutten A, Carlier MC et al. Inter-method variability in PTH measurement: Implication for the care of CKD patients. Kidney Int 2006; 70: 345–350.
Joly D, Drueke TB, Alberti C et al. Variation in serum and plasma PTH levels in second-generation assays in hemodialysis patients: a cross-sectional study. Am J Kidney Dis 2008; 51: 987–995.
John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: Implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999; 84: 4287–4290.
MonierFaugere MC, Geng ZP, Mawad H et al. Improved assessment of bone turnover by the PTH-(1-84) large C-PTH fragments ratio in ESRD patients. Kidney Int 2001; 60: 1460–1468.
Coen G, Bonucci E, Ballanti P et al. PTH 1-84 and PTH “7-84” in the noninvasive diagnosis of renal bone disease. Am J Kidney Dis 2002; 40: 348–354.
Reichel H, Esser A, Roth HJ, Schmidt-Gayk H. Influence of PTH assay methodology on differential diagnosis of renal bone disease. Nephrol Dial Transplant 2003; 18: 759–768.
Salusky IB, Goodman WG, Kuizon BD et al. Similar predictive value of bone turnover using first- and second-generation immunometric PTH assays in pediatric patients treated with peritoneal dialysis. Kidney Int 2003; 63: 1801–1808.
Tepel M, Armbruster FP, Grön HJ et al. Nonoxidized, biologically active parathyroid hormone determines mortality in hemodialysis patients. The Journal of Clinical Endocrinology and Metabolism 2013; 98: 4744–4751.
Seiler-Mussler S, Limbach AS, Emrich IE et al. Association of Nonoxidized Parathyroid Hormone with Cardiovascular and Kidney Disease Outcomes in Chronic Kidney Disease. Clinical journal of the American Society of Nephrology: CJASN 2018; 13: 569–576.
Vervloet MG, van Ballegooijen AJ. Prevention and treatment of hyperphosphatemia in chronic kidney disease. Kidney International 2018; 93: 1060–1072.
Ursem SR, Heijboer AC, D’Haese PC et al. Non-oxidized parathyroid hormone (PTH) measured by current method is not superior to total PTH in assessing bone turnover in chronic kidney disease. Kidney International 2021; 99: 1173–1178.
Drüeke TB, Floege J. Parathyroid hormone oxidation in chronic kidney disease: clinical relevance? Kidney International 2021; 99: 1070–1072.
Drueke TB, Fukagawa M. Whole or fragmentary information on parathyroid hormone? Clin J Am Soc Nephrol 2007; 2: 1106–1107.
Amann K. Media calcification and intima calcification are distinct entities in chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1599–1605.
Bellasi A, Raggi P. Techniques and technologies to assess vascular calcification. Semin Dial 2007; 20: 129–133.
London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 2003; 18: 1731–1740.
Rostand SG, Drueke TB. Parathyroid hormone, vitamin D, and cardiovascular disease in chronic renal failure. Kidney Int 1999; 56: 383–392.
Tsuchihashi K, Takizawa H, Torii T et al. Hypoparathyroidism potentiates cardiovascular complications through disturbed calcium metabolism: Possible risk of vitamin D-3 analog administration in dialysis patients with end-stage renal disease. Nephron 2000; 84: 13–20.
Galassi A, Spiegel DM, Bellasi A, Block GA, Raggi P. Accelerated vascular calcification and relative hypoparathyroidism in incident haemodialysis diabetic patients receiving calcium binders. Nephrol Dial Transplant 2006; 21: 3215–3222.
Sebastian EM, Suva LJ, Friedman PA. Differential effects of intermittent PTH(1-34) and PTH(7- 34) on bone microarchitecture and aortic calcification in experimental renal failure. Bone 2008; 43: 1022–1030.
Shao JS, Cheng SL, CharltonKachigian N, Loewy AP, Towler DA. Teriparatide (Human parathyroid hormone (1-34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice. J Biol Chem 2003; 278: 50195–50202.
Canalis E, Giustina A, Bilezikian JP. Mechanisms of anabolic therapies for osteoporosis. N Engl J Med 2007; 357: 905–916.
Drueke TB, Ritz E. Treatment of secondary hyperparathyroidism in CKD patients with cinacalcet and/or vitamin D derivatives. Clin J Am Soc Nephrol 2009; 4: 234–241.
Malluche HH, Mawad H, Monier-Faugere MC. Effects of treatment of renal osteodystrophy on bone histology. Clin J Am Soc Nephrol 2008; 3 Suppl 3: S157-63.
Eknoyan G, Levin A, Levin NW. Bone metabolism and disease in chronic kidney disease. Am J Kidney Dis 2003; 42: 1–201.
KidneyDisease-ImprovingGlobalOutcomes(KDIGO)CKD-MBDWorkGroup. KDIGO clinical practice guideline for the diagnosis, evaluation, prevention, and treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD). Kidney Int Suppl 2009; : S1-130.
Ketteler M, Block GA, Evenepoel P et al. Executive summary of the 2017 KDIGO Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD) Guideline Update: what’s changed and why it matters. Kidney International 2017; 92: 26–36.
Ketteler M, Block GA, Evenepoel P et al. Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease-Mineral and Bone Disorder: Synopsis of the Kidney Disease: Improving Global Outcomes 2017 Clinical Practice Guideline Update. Annals of Internal Medicine 2018; 168: 422–430.
Bhan I, Thadhani R. Vitamin D therapy for chronic kidney disease. Semin Nephrol 2009; 29: 85–93.
Kooienga L, Fried L, Scragg R, Kendrick J, Smits G, Chonchol M. The effect of combined calcium and vitamin D3 supplementation on serum intact parathyroid hormone in moderate CKD. Am J Kidney Dis 2009; 53: 408–416.
Jean G, Souberbielle JC, Chazot C. Monthly cholecalciferol administration in haemodialysis patients: a simple and efficient strategy for vitamin D supplementation. Nephrol Dial Transplant 2009; 24: 3799–3805.
Agarwal R, Georgianos PI. Con: Nutritional vitamin D replacement in chronic kidney disease and end-stage renal disease. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2016; 31: 706–713.
Goldsmith DJA. Pro: Should we correct vitamin D deficiency/insufficiency in chronic kidney disease patients with inactive forms of vitamin D or just treat them with active vitamin D forms? Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2016; 31: 698–705.
Drueke TB. Calcimimetics versus vitamin D: what are their relative roles? Blood Purif 2004; 22: 38–43.
Hansen D, Rasmussen K, Danielsen H et al. No difference between alfacalcidol and paricalcitol in the treatment of secondary hyperparathyroidism in hemodialysis patients: a randomized crossover trial. Kidney Int 2011; 80: 841–850.
Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R. Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 2003; 349: 446–456.
Shoben AB, Rudser KD, deBoer IH, Young B, Kestenbaum B. Association of Oral Calcitriol with Improved Survival in Nondialyzed CKD. J Amer Soc Nephrol 2008; 19: 1613–1619.
Shoji T, Shinohara K, Kimoto E et al. Lower risk for cardiovascular mortality in oral 1alpha-hydroxy vitamin D3 users in a haemodialysis population. Nephrol Dial Transplant 2004; 19: 179–184.
Teng M, Wolf M, Ofsthun MN et al. Activated injectable vitamin D and hemodialysis survival: A historical cohort study. J Amer Soc Nephrol 2005; 16: 1115–1125.
Tentori F, Hunt WC, Stidley CA et al. Mortality risk among hemodialysis patients receiving different vitamin D analogs. Kidney Int 2006; 70: 1858–1865.
Tentori F, Albert JM, Young EW et al. The survival advantage for haemodialysis patients taking vitamin D is questioned: findings from the Dialysis Outcomes and Practice Patterns Study. Nephrol Dial Transplant 2009; 24: 963–972.
Drueke TB, McCarron DA. Paricalcitol as compared with calcitriol in patients undergoing hemodialysis. N Engl J Med 2003; 349: 496–499.
E M Brown, G Gamba, D Riccardi et al. Cloning and characterization of an extracellular Ca²⁺-sensing receptor from bovine parathyroid. Nature 1993; 366: 575–580.
Antonsen JE, Sherrard DJ, Andress DL. A calcimimetic agent acutely suppresses parathyroid hormone levels in patients with chronic renal failure - rapid communication. Kidney Int. 1998; 53: 223–227.
Goodman WG, Frazao JM, Goodkin DA, Turner SA, Liu W, Coburn JW. A calcimimetic agent lowers plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. Kidney Int 2000; 58: 436-45.
Goodman WG, Hladik GA, Turner SA et al. The calcimimetic agent AMG 073 lowers plasma parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism. J Am Soc Nephrol 2002; 13: 1017-24.
Ivanovski O, Nikolov IG, Joki N et al. The calcimimetic R-568 retards uremia-enhanced vascular calcification and atherosclerosis in apolipoprotein E deficient (apoE-/-) mice. Atherosclerosis 2009; 205: 55–62.
Basile C, Lomonte C. The function of the parathyroid oxyphil cells in uremia: still a mystery? Kidney International 2017; 92: 1046–1048.
Lomonte C, Vernaglione L, Chimienti D et al. Does vitamin D receptor and calcium receptor activation therapy play a role in the histopathologic alterations of parathyroid glands in refractory uremic hyperparathyroidism? Clinical journal of the American Society of Nephrology: CJASN 2008; 3: 794–799.
Ritter C, Miller B, Coyne DW et al. Paricalcitol and cinacalcet have disparate actions on parathyroid oxyphil cell content in patients with chronic kidney disease. Kidney International 2017; 92: 1217–1222.
Ritter CS, Haughey BH, Miller B, Brown AJ. Differential gene expression by oxyphil and chief cells of human parathyroid glands. The Journal of Clinical Endocrinology and Metabolism 2012; 97: E1499-1505.
Lopez I, Aguilera-Tejero E, Mendoza FJ et al. Calcimimetic R-568 decreases extraosseous calcifications in uremic rats treated with calcitriol. J Am Soc Nephrol 2006; 17: 795–804.
Raggi P, Chertow GM, Torres PU et al. The ADVANCE study: a randomized study to evaluate the effects of cinacalcet plus low-dose vitamin D on vascular calcification in patients on hemodialysis. Nephrol Dial Transplant 2011; 26: 1327–1339.
Koleganova N, Piecha G, Ritz E, Schmitt CP, Gross ML. A calcimimetic (R-568), but not calcitriol, prevents vascular remodeling in uremia. Kidney Int 2009; 75: 60–71.
Koleganova N, Piecha G, Ritz E et al. Interstitial fibrosis and microvascular disease of the heart in uremia: amelioration by a calcimimetic. Lab Invest 2009; 89: 520–530.
Lopez I, Mendoza FJ, AguileraTejero E et al. The effect of calcitriol, paricalcitol, and a calcimimetic on extraosseous calcifications in uremic rats. Kidney Int 2008; 73: 300–307.
Block GA, Martin KJ, de Francisco AL et al. Cinacalcet for secondary hyperparathyroidism in patients receiving hemodialysis. N Engl J Med 2004; 350: 1516–1525.
Lindberg JS, Moe SM, Goodman WG et al. The calcimimetic AMG 073 reduces parathyroid hormone and calcium x phosphorus in secondary hyperparathyroidism. Kidney Int 2003; 63: 248-254.
Quarles LD, Sherrard DJ, Adler S et al. The calcimimetic AMG 073 as a potential treatment for secondary hyperparathyroidism of end-stage renal disease. J Am Soc Nephrol 2003; 14: 575–583.
Parfrey PS, Chertow GM, Block GA et al. The clinical course of treated hyperparathyroidism among patients receiving hemodialysis and the effect of cinacalcet: the EVOLVE trial. J Clin Endocrinol Metab 2013; 98: 4834–4844.
Chertow GM, Block GA, Correa-Rotter R et al. Effect of cinacalcet on cardiovascular disease in patients undergoing dialysis. N Engl J Med 2012; 367: 2482–2494.
Block GA, Zeig S, Sugihara J et al. Combined therapy with cinacalcet and low doses of vitamin D sterols in patients with moderate to severe secondary hyperparathyroidism. Nephrol Dial Transplant 2008; 23: 2311–2318.
Wilkie M, Pontoriero G, Macario F et al. Impact of Vitamin D Dose on Biochemical Parameters in Patients with Secondary Hyperparathyroidism Receiving Cinacalcet. Nephron Clin Pract 2009; 112: c41–c50.
Block GA, Bushinsky DA, Cunningham J et al. Effect of Etelcalcetide vs Placebo on Serum Parathyroid Hormone in Patients Receiving Hemodialysis With Secondary Hyperparathyroidism: Two Randomized Clinical Trials. JAMA 2017; 317: 146–155.
Block GA, Bushinsky DA, Cheng S et al. Effect of Etelcalcetide vs Cinacalcet on Serum Parathyroid Hormone in Patients Receiving Hemodialysis With Secondary Hyperparathyroidism: A Randomized Clinical Trial. JAMA 2017; 317: 156–164.
Moe SM, Abdalla S, Chertow GM et al. Effects of Cinacalcet on Fracture Events in Patients Receiving Hemodialysis: The EVOLVE Trial. J Am Soc Nephrol 2015; 26: 1466–1475.
Shahapuni I, Mansour J, Harbouche L et al. How do calcimimetics fit into the management of parathyroid hormone, calcium, and phosphate disturbances in dialysis patients? Semin Dial 2005; 18: 226–238.
Cannata Andia JB. Adynamic bone and chronic renal failure: an overview. Am J Med Sci 2000; 320: 81–84.
Argilés A, P G Kerr, B Canaud, J L Flavier, C Mion. Calcium kinetics and the long-term effects of lowering dialysate calcium concentration. Kidney Int. 1993; 43: 630–640.
Arenas MD, Alvarez-Ude F, Gil MT et al. Application of NKF-K/DOQI Clinical Practice Guidelines for Bone Metabolism and Disease: changes of clinical practices and their effects on outcomes and quality standards in three haemodialysis units. Nephrol Dial Transplant 2006; 21: 1663–1668.
Basile C, Libutti P, Di Turo AL et al. Effect of dialysate calcium concentrations on parathyroid hormone and calcium balance during a single dialysis session using bicarbonate hemodialysis: a crossover clinical trial. Am J Kidney Dis 2012; 59: 92–101.
Sonikian M, Metaxaki P, Karatzas I, Vlassopoulos D. Paricalcitol treatment of secondary hyperparathyroidism in hemodialysis patients on sevelamer hydrochloride: which dialysate calcium concentration to use? Blood Purif 2009; 27: 182–186.
Touam M, Menoyo V, Attaf D, Thebaud HE, Drueke TB. High dialysate calcium may improve the efficacy of calcimimetic treatment in hemodialysis patients with severe secondary hyperparathyroidism. Kidney Int 2005; 67: 2065; author reply 2065-6.
Drüeke TB, Touam M. Calcium balance in haemodialysis--do not lower the dialysate calcium concentration too much (con part). Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2009; 24: 2990–2993.
Pun PH, Horton JR, Middleton JP. Dialysate calcium concentration and the risk of sudden cardiac arrest in hemodialysis patients. Clin J Am Soc Nephrol 2013; 8: 797–803.
Pun PH, Lehrich RW, Honeycutt EF, Herzog CA, Middleton JP. Modifiable risk factors associated with sudden cardiac arrest within hemodialysis clinics. Kidney Int 2011; 79: 218–227.
J Cunningham, J Beer, R D Coldwell, K Noonan, N Sawyer, H L J Makin. Dialysate calcium reduction in CAPD patients treated with calcium carbonate and alfacalcidol. Nephrol. Dial. Transplant. 1992; 7: 63–68.
Chertow GM, Burke SK, Lazarus JM et al. Poly[allylamine hydrochloride] (RenaGel): a noncalcemic phosphate binder for the treatment of hyperphosphatemia in chronic renal failure. Am J Kidney Dis 1997; 29: 66–71.
Chertow GM, Burke SK, Raggi P. Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int 2002; 62: 245-52.
Slatopolsky EA, Burke SK, Dillon MA. RenaGel (R), a nonabsorbed calcium- and aluminum-free phosphate binder, lowers serum phosphorus and parathyroid hormone. Kidney Int 1999; 55: 299–307.
Delmez J, Block G, Robertson J et al. A randomized, double-blind, crossover design study of sevelamer hydrochloride and sevelamer carbonate in patients on hemodialysis. Clin Nephrol 2007; 68: 386–391.
Ketteler M, Rix M, Fan S et al. Efficacy and tolerability of sevelamer carbonate in hyperphosphatemic patients who have chronic kidney disease and are not on dialysis. Clin J Am Soc Nephrol 2008; 3: 1125–1130.
D’Haese PC, Spasovski GB, Sikole A et al. A multicenter study on the effects of lanthanum carbonate (Fosrenol(TM)) and calcium carbonate on renal bone disease in dialysis patients. Kidney Int 2003; 63: S73–S78.
Hutchison AJ. Oral phosphate binders. Kidney Int 2009; 75: 906–914.
Hutchison AJ, Barnett ME, Krause R, Kwan JT, Siami GA. Long-term efficacy and safety profile of lanthanum carbonate: results for up to 6 years of treatment. Nephron Clin Pract 2008; 110: c15–c23.
Sprague SM, Ketteler M. A safety evaluation of sucroferric oxyhydroxide for the treatment of hyperphosphatemia. Expert Opinion on Drug Safety 2021;
Choi YJ, Noh Y, Shin S. Ferric citrate in the management of hyperphosphataemia and iron deficiency anaemia: A meta-analysis in patients with chronic kidney disease. British Journal of Clinical Pharmacology 2021; 87: 414–426.
Block GA, Raggi P, Bellasi A, Kooienga L, Spiegel DM. Mortality effect of coronary calcification and phosphate binder choice in incident hemodialysis patients. Kidney Int 2007; 71: 438–441.
Block GA, Wheeler DC, Persky MS et al. Effects of phosphate binders in moderate CKD. J Am Soc Nephrol 2012; 23: 1407–1415.
Drueke TB, Massy ZA. Phosphate binders in CKD: bad news or good news? J Am Soc Nephrol 2012; 23: 1277–1280.
Neven E, Dams G, Postnov A et al. Adequate phosphate binding with lanthanum carbonate attenuates arterial calcification in chronic renal failure rats. Nephrol Dial Transplant 2009; 24: 1790–1799.
Nikolov IG, Joki N, Nguyen-Khoa T et al. Lanthanum carbonate, like sevelamer-HCl, retards the progression of vascular calcification and atherosclerosis in uremic apolipoprotein E-deficient mice. Nephrol Dial Transplant 2012; 27: 505–513.
Toussaint ND, Lau KK, Polkinghorne KR, Kerr PG. Attenuation of aortic calcification with lanthanum carbonate versus calcium-based phosphate binders in haemodialysis: A pilot randomized controlled trial. Nephrology (Carlton) 2011; 16: 290–298.
Ohtake T, Kobayashi S, Oka M et al. Lanthanum carbonate delays progression of coronary artery calcification compared with calcium-based phosphate binders in patients on hemodialysis: a pilot study. J Cardiovasc Pharmacol Ther 2013; 18: 439–446.
Seifert ME, de las Fuentes L, Rothstein M et al. Effects of phosphate binder therapy on vascular stiffness in early-stage chronic kidney disease. Am J Nephrol 2013; 38: 158–167.
Ferreira A, Frazao JM, Monier-Faugere MC et al. Effects of sevelamer hydrochloride and calcium carbonate on renal osteodystrophy in hemodialysis patients. J Am Soc Nephrol 2008; 19: 405–412.
Barreto DV, Barreto FD, deCarvalho AB et al. Phosphate Binder Impact on Bone Remodeling and Coronary Calcification - Results from the BRiC Study. Nephron Clin Pract 2008; 110: C273–C283.
Lenglet A, Liabeuf S, El Esper N et al. Efficacy and safety of nicotinamide in haemodialysis patients: the NICOREN study. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association 2017; 32: 870–879.
Lenglet A, Liabeuf S, Guffroy P, Fournier A, Brazier M, Massy ZA. Use of nicotinamide to treat hyperphosphatemia in dialysis patients. Drugs in R&D 2013; 13: 165–173.
Malhotra R, Katz R, Hoofnagle A et al. The Effect of Extended Release Niacin on Markers of Mineral Metabolism in CKD. Clinical journal of the American Society of Nephrology: CJASN 2018; 13: 36–44.
Pergola PE, Rosenbaum DP, Yang Y, Chertow GM. A Randomized Trial of Tenapanor and Phosphate Binders as a Dual-Mechanism Treatment for Hyperphosphatemia in Patients on Maintenance Dialysis (AMPLIFY). Journal of the American Society of Nephrology: JASN 2021; 32: 1465–1473.
Block GA, Rosenbaum DP, Leonsson-Zachrisson M et al. Effect of Tenapanor on Serum Phosphate in Patients Receiving Hemodialysis. Journal of the American Society of Nephrology: JASN 2017; 28: 1933–1942.
Drüeke TB, Massy ZA. Lowering Expectations with Niacin Treatment for CKD-MBD. Clinical journal of the American Society of Nephrology: CJASN 2018; 13: 6–8.
D’Alessandro C, Piccoli GB, Cupisti A. The “phosphorus pyramid”: a visual tool for dietary phosphate management in dialysis and CKD patients. BMC Nephrol 2015; 16: 9.
Lorenzo V, Martin M, Rufino M et al. Protein intake, control of serum phosphorus, and relatively low levels of parathyroid hormone in elderly hemodialysis patients. Am J Kidney Dis 2001; 37: 1260–1266.
Shinaberger CS, Greenland S, Kopple JD et al. Is controlling phosphorus by decreasing dietary protein intake beneficial or harmful in persons with chronic kidney disease? Amer J Clin Nutr 2008; 88: 1511–1518.
A Lefebvre, M C De Vernejoul, J Gueris, B Goldfarb, A M Graulet, C Morieux. Optimal correction of acidosis changes progression of dialysis osteodystrophy. Kidney Int. 1989; 36: 1112–1118.
K A Graham, N A Hoenich, M Tarbit, M K Ward, T H J Goodship. Correction of acidosis in hemodialysis patients increases the sensitivity of the parathyroid glands to calcium. J. Am. Soc. Nephrol. 1997; 8: 627–631.
A Giangrande, A Castiglioni, L Solbiati, P Allaria. US-guided percutaneous fine-needle ethanol injection into parathyroid glands in secondary hyperparathyroidism. Nephrol. Dial. Transplant. 1992; 7: 412–420.
M Kitakoa, M Fukagawa, E Ogata, K Kurokawa. Reduction of functioning parathyroid cell mass by ethanol injection in chronic dialysis patients. Kidney Int. 1994; 46: 1110–1117.
Shiizaki K, Hatamura I, Negi S et al. Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia. Kidney Int 2003; 64: 992–1003.
Shiizaki K, Negi S, Hatamura I et al. Biochemical and cellular effects of direct maxacalcitol injection into parathyroid gland in uremic rats. J Am Soc Nephrol 2005; 16: 97–108.
Fletcher S, Kanagasundaram NS, Rayner HC et al. Assessment of ultrasound guided percutaneous ethanol injection and parathyroidectomy in patients with tertiary hyperparathyroidism. Nephrol Dial Transplant 1998; 13: 3111–3117.
de Barros Gueiros JE, Chammas MC, Gerhard R et al. Percutaneous ethanol (PEIT) and calcitrol (PCIT) injection therapy are ineffective in treating severe secondary hyperparathyroidism. Nephrol Dial Transplant 2004; 19: 657–663.
Young EW, Akiba T, Albert JM et al. Magnitude and impact of abnormal mineral metabolism in hemodialysis patients in the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis 2004; 44: 34–38.
Danese MD, Belozeroff V, Smirnakis K, Rothman KJ. Consistent control of mineral and bone disorder in incident hemodialysis patients. Clin J Am Soc Nephrol 2008; 3: 1423–1429.
Fouque D, Roth H, Darné B et al. Achievement of Kidney Disease: Improving Global Outcomes mineral and bone targets between 2010 and 2014 in incident dialysis patients in France: the Photo-Graphe3 study. Clinical Kidney Journal 2018; 11: 73–79.
Lau WL, Obi Y, Kalantar-Zadeh K. Parathyroidectomy in the Management of Secondary Hyperparathyroidism. Clinical journal of the American Society of Nephrology: CJASN 2018; 13: 952–961.
E R Gagné, P Ureña, S Leite-Silva et al. Short and long-term efficacy of total parathyroidectomy with immediate autografting compared with subtotal parathyroidectomy in hemodialysis patients. J. Am. Soc. Nephrol. 1992; 3: 1008–1017.
Stracke S, Keller F, Steinbach G, HenneBruns D, Wuerl P. Long-Term Outcome after Total Parathyroidectomy for the Management of Secondary Hyperparathyroidism. Nephron Clin Pract 2009; 111: C102–C109.
T Drüeke, J Zingraff. The dilemma of parathyroidectomy in chronic renal failure. Curr. Opin. Nephrol. Hypertens. 1994; 3: 386–395.
Locatelli F, Cannata-Andia JB, Drueke TB et al. Management of disturbances of calcium and phosphate metabolism in chronic renal insufficiency, with emphasis on the control of hyperphosphataemia. Nephrol Dial Transplant 2002; 17: 723–731.
Stratton J, Simcock M, Thompson H, Farrington K. Predictors of recurrent hyperparathyroidism after total parathyroidectomy in chronic renal failure. Nephron Clin Pract 2003; 95: c15-22.
Malberti F, Marcelli D, Conte F, Limido A, Spotti D, Locatelli F. Parathyroidectomy in patients on renal replacement therapy: An epidemiologic study. J Amer Soc Nephrol 2001; 12: 1242–1248.
Kim SM, Long J, Montez-Rath ME, Leonard MB, Norton JA, Chertow GM. Rates and Outcomes of Parathyroidectomy for Secondary Hyperparathyroidism in the United States. Clinical journal of the American Society of Nephrology: CJASN 2016; 11: 1260–1267.
Lafrance J-P, Cardinal H, Leblanc M et al. Effect of cinacalcet availability and formulary listing on parathyroidectomy rate trends. BMC nephrology 2013; 14: 100.
Tentori F, Wang M, Bieber BA et al. Recent changes in therapeutic approaches and association with outcomes among patients with secondary hyperparathyroidism on chronic hemodialysis: the DOPPS study. Clinical journal of the American Society of Nephrology: CJASN 2015; 10: 98–109.
Kestenbaum B, Seliger SL, Gillen DL et al. Parathyroidectomy rates among United States dialysis patients: 1990-1999. Kidney Int 2004; 65: 282–288.

Hypophysitis

ABSTRACT

Hypophysitis is an inflammation of the pituitary gland and is a rare cause of hypopituitarism. It can be primary (idiopathic) or secondary to sella and parasellar lesions, systemic diseases, or drugs (mainly immune checkpoint inhibitors). Primary hypophysitis has five histologic variants: lymphocytic, granulomatous, xanthomatous, IgG4-related, and necrotizing. Lymphocytic hypophysitis is the most common form; it is likely an autoimmune disease and is more frequently observed in females during pregnancy or postpartum. Granulomatous hypophysitis is the second most common variant and possible secondary causes of granulomatous infiltration of the pituitary should be excluded before concluding that a case of granulomatous hypophysitis is idiopathic. Xanthomatous, necrotizing, and IgG4-related hypophysitis are very rare and the latter is often the manifestation of a systemic disease with multi-organ involvement (IgG4-related disease). Immune checkpoint inhibitors are monoclonal antibodies increasingly used for the treatment of solid and hematological malignancies. They cause a T-lymphocyte activation and proliferation that lead to the anti-tumor response, and may cause autoimmune manifestations known as part of what is called “immune-related adverse events”. A significant number of patients treated with immune checkpoint inhibitors develop immune-related hypophysitis and require prompt diagnosis and treatment. Regardless of the etiology, patients with hypophysitis present with various signs and symptoms caused by the pituitary inflammation that can lead to hypopituitarism and compression of sella and parasellar structures. Contrary to other causes of hypopituitarism, adrenocorticotropic hormone and thyroid-stimulating hormone deficiencies are very frequent in the early stages of hypophysitis and must be identified immediately. The diagnosis of hypophysitis is based on clinical, laboratory, and radiological data; while pituitary biopsy is the gold standard test for diagnosing primary hypophysitis, it should be reserved only for selected cases. Magnetic resonance imaging is the technique of choice for suspected hypophysitis, and the main differential diagnoses are pituitary adenomas in adults, germinomas, and Langerhans cell histiocytosis in adolescents, and metastases in those receiving immune checkpoint inhibitors. The mainstay of treatment of patients with hypophysitis is pituitary hormone replacement. Those with severe signs and symptoms of sella compression should be treated with high-dose glucocorticoids, which usually cause an excellent initial response, although relapse of the pituitary inflammation is common. Pituitary surgery should be considered in patients who do not respond to glucocorticoids and have progressive and debilitating symptoms. Pituitary fibrosis and atrophy often develop in the late stage of the disease, with persistent hypopituitarism.

INTRODUCTION

Hypophysitis is a generic term that includes a variety of conditions that cause inflammation of the pituitary gland. It is an infiltrative cause of hypopituitarism and can cause symptoms related to sella compression and pituitary hormone deficiencies.

Hypophysitis can be classified according to the anatomic location of pituitary involvement (adenohypophysitis, infundibulo-neurohypophysitis, or panhypophysitis) and the cause (primary or secondary forms) (Table 1) (1-4). The primary forms are characterized by an idiopathic inflammatory process confined to the pituitary gland, while the secondary forms are triggered by a definite etiology (drugs and intracranial or systemic diseases). Five histologic variants of primary hypophysitis have been described: lymphocytic, granulomatous, xanthomatous, IgG4-related, and necrotizing (Table 1). Lymphocytic hypophysitis is the most common form of hypophysitis and occurs most commonly in women during late pregnancy and the postpartum period. However, thanks to the increasing use over the last two decades of monoclonal antibodies inhibiting immune checkpoints for the treatment of several solid and hematological malignancies, new immune-related adverse events have emerged, with hypophysitis being a relatively common occurrence.

Table 1. Classification of Hypophysitis

CAUSE: PRIMARY AND SECONDARY HYPOPHYSITIS

· Primary hypophysitis:

o Isolated

o Associated with autoimmune diseases:

• Polyglandular autoimmune syndromes

• Autoimmune thyroiditis (Hashimoto thyroiditis)

• Autoimmune adrenalitis

• Type 1 diabetes mellitus

• Lymphocytic parathyroiditis

• Idiopathic inflammatory myopathy

• Systemic lupus erythematosus

• Sjogren’s syndrome

• Rheumatoid arthritis

• Primary biliary cirrhosis

• Atrophic gastritis

• Optic neuritis

• Myocarditis

• Temporal arteritis

• Bechet’s disease

• Retroperitoneal fibrosis

• Erythema nodosum

• Idiopathic thrombocytopenic purpura

• Dacryoadenitis

• Autoimmune thrombocytopenia

• Autoimmune encephalitis

· Secondary hypophysitis:

o Drugs:

• Immune checkpoint inhibitors

• Interferon-α

• Ribavirin

• Ustekinumab

o Sella and parasellar diseases*:

• Germinoma

• Rathke’s cleft cyst

• Craniopharyngioma

• Pituitary adenoma

• Primary pituitary lymphoma

o Systemic diseases:

• IgG4-related disease**

• Sarcoidosis

• Granulomatosis with polyangiitis (Wegener’s granulomatosis)

• Langerhans cell histiocytosis

• Erdheim-Chester’s disease

• Rosai-Dorfman disease

• Inflammatory pseudotumor

• Tolosa-Hunt syndrome

• Takayasu’s arteritis

• Cogan’s syndrome

• Crohn’s disease

o Thymoma and other malignancies (anti-Pit-1 antibody syndrome)

o Infections:

• Bacteria (Mycobacterium tuberculosis; Treponema pallidum; Tropheryma whipplei; Borrelia; Brucella)

• Viruses (Cytomegalovirus; Herpes simplex; Varicella-zoster virus; Influenza viruses; Coronavirus; Enterovirus; Coxsackie; Tick-Borne encephalitis virus; Hantavirus)

• Mycoses (Aspergillus; Nocardia; Candida albicans; Pneumocystis jirovecii)

• Parasites (Toxoplasma gondii)

ANATOMIC LOCATION OF PITUITARY INVOLVEMENT

· Adenohypophysitis: the inflammation involves the anterior pituitary. It accounts for ~65% of cases of primary hypophysitis

· Infundibulo-neurohypophysitis: the inflammation involves the posterior pituitary and the stalk. It accounts for ~10% of cases of primary hypophysitis

· Panhypophysitis: the inflammation involves the entire gland. It accounts for ~25% of cases of primary hypophysitis

HISTOPATHOLOGY FORMS OF PRIMARY HYPOPHYSITIS

· Lymphocytic hypophysitis (68%)

· Granulomatous hypophysitis (19%)

· IgG4-related (plasmocytic) hypophysitis (8%)**

· Xanthomatous hypophysitis (4%)

· Necrotizing hypophysitis (<1%)

· Mixed forms (lymphogranulomatous; xanthogranulomatous)

* The infiltrate focuses around the lesion rather than diffuse in the entire gland. This secondary form of pituitary infiltration is generally a histopathological finding and patient’s signs and symptoms are otherwise related to the primary sella and parasellar mass.

** IgG4-related hypophysitis can be isolated, but is often a manifestation of systemic disease with the involvement of multiple organs.

PRIMARY HYPOPHYSITIS

Primary hypophysitis is a rare disease, with just over 1300 published cases so far (5). The incidence is estimated to be ~1 in 9 million/year (4,6), and hypophysitis accounts for ~0.4% of pituitary surgery cases (2). Five histologic variants of primary hypophysitis have been described, and there are mixed forms as well. Table 2 summarizes the epidemiological and histopathological features of these variants (2,5,7-9). Primary hypophysitis, apart from the rare IgG4-related and necrotizing variants, occurs more frequently in young females. The clinical manifestations of all forms of primary hypophysitis are similar and are linked to the degree of pituitary involvement and the associated hormonal deficiencies.

Table 2. Characteristics of the Various Forms of Primary Hypophysitis
	Lymphocytic	Granulomatous	IgG4-related	Xanthomatous	Necrotizing
Prevalence	The most common subtype (68%*).	The second most common subtype (19%*).	Rare (8%*). Higher prevalence in Japan and Korea.	Very rare (4%*).	Extremely rare (<1%).
Gender predominance	Female, ~3:1	Female, ~3:1	Male, ~2:1	Female, ~3:1	Male, ~2:1
Association with pregnancy	Yes. ~70% of patients present during pregnancy or postpartum.	No	No	No	No
Mean age at presentation	4th decade (women). 5th decade (men).	5th decade	7th decade (men). 2nd-3rd decade (women).	4th decade	Six cases reported (aged 12, 20, 33, 39, 40, and 52).
Histopathology	Diffuse lymphocyte infiltration (primarily T cells) of the pituitary gland. Lymphoid follicles can be observed and occasional plasma cells, eosinophils, and fibroblasts may also be present. Pituitary fibrosis and atrophy may occur in later stages of the disease.	Large numbers of multinucleated giant cells and histiocytes with granuloma formation.	Extensive gland infiltration by plasma cells with a high degree of IgG4 positivity. Storiform fibrosis is observed**. Pituitary fibrosis and atrophy occur in later stages of the disease, if not treated.	Foamy histiocytes (lipid-rich macrophages) without the presence of granulomas. Plasma cells and small round mature lymphocytes are also observed. Pituitary fibrosis may be seen in later stages of the disease.	Diffuse non-hemorrhagic necrosis with surrounding lymphocytes, plasma cells and eosinophils.

* Prevalence derived by published cases after excluding those where the pathologic variant is unknown. Forty-one cases with mixed histology findings have been published.

** Storiform fibrosis: dense, wire-like strands of fibrotic collagen deposition radiating outward from a central point.

Lymphocytic Hypophysitis

Lymphocytic hypophysitis is the most common histologic variant of primary hypophysitis (4,5,8). It shows a striking temporal association with pregnancy, with ~70% of cases in women presenting during pregnancy or postpartum. Most patients present in the last month of pregnancy or in the first 2 months after delivery (4). Lymphocytic hypophysitis is believed to have an autoimmune etiology. This is supported by the lymphocytic infiltration of the pituitary, the link with pregnancy, the frequent association with other autoimmune diseases (Table 1), the frequent finding of pituitary antibodies in these patients (see below), the association with particular human leukocyte antigen alleles (1), the improvement of symptoms in response to immunosuppressive drugs, and animal models of primary hypophysitis (10).

Granulomatous Hypophysitis

Granulomatous hypophysitis is the second most common subtype of primary hypophysitis and its etiology is unknown. Before concluding that a case of granulomatous hypophysitis is “primary” (i.e., idiopathic), known possible causes of granulomatous infiltration of the pituitary should be excluded. Possible secondary causes of granulomatous hypophysitis include tuberculosis, sarcoidosis, syphilis, Langerhans’ histiocytosis, granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis), and Rathke’s cleft cyst rupture (see “Hypophysitis secondary to sella and parasellar disease” and “Hypophysitis secondary to systemic disease” below). (11).

IgG4-related hypophysitis can be isolated (primary hypophysitis) but is often a manifestation of systemic disease with involvement of multiple organs (14,15). Some authors include IgG4-related hypophysitis among the histologic variants of primary hypophysitis, while others report this among the secondary forms of hypophysitis. Considering that the diagnosis and management does not change according to the classification used, we will discuss the features of IgG4-related hypophysitis in this section.

The etiology of this disease is poorly understood and may involve autoimmunity and/or an abnormal tolerance to unspecified allergens and infectious agents (16,17). IgG4-related disease is diagnosed more frequently in older males and is characterized by a dense lymphoplasmacytic infiltration with a predominance of IgG4-positive plasma cells in the affected tissue and storiform fibrosis in the more advanced stages of the disease (Table 2). One or (more frequently) multiple organs can be affected including lymph nodes, pancreas, liver, salivary and lacrimal glands, retroperitoneum, aorta, pericardium, thyroid, lungs, kidneys, skin, stomach, prostate, ovaries, and the pituitary gland (17-19). Overall, the prevalence of pituitary involvement in IgG4-related disease is believed to be low (2-8%) (20). Nonetheless, a recent cohort study from Japan screened 27 patients with IgG4-related pancreatitis via pituitary MRI and found 1 case of hypophysitis with hypopituitarism and 4 cases of empty sella (21). Patients with pituitary abnormalities were more likely to have multi-organ disease. If confirmed by large-scale studies, these findings would advocate for screening for hypophysitis especially in patients with multiple IgG4-related organ involvement.

IgG4-related disease is considered a rare cause of hypophysitis, although a Japanese group reported a strikingly high prevalence of IgG4-related hypophysitis in 170 consecutive patients with hypopituitarism/central diabetes insipidus and a clinical diagnosis of hypophysitis (4% and 30% respectively) (22). Moreover, Bernreuther et al. reviewed retrospectively 29 cases of biopsy-proven primary hypophysitis previously diagnosed as “lymphocytic” or “not otherwise specified, non-granulomatous” and found that 41.4% of cases fulfilled the criteria for IgG4-related hypophysitis, suggesting that this entity might be more frequent than previously thought (23). Two recent reviews of the literature found that the epidemiology of IgG4-related hypophysitis may differ according to sex: affected men were older, more likely to have systemic disease and higher IgG4 serum levels; women were younger and often presenting with isolated pituitary disease, lower IgG4 serum levels, and a concomitant diagnosis of other autoimmune diseases (24,25).

The diagnosis of IgG4-related hypophysitis is confirmed by characteristic histopathologic findings at pituitary biopsy. However, pituitary biopsy is an invasive procedure and other criteria can be used to establish the diagnosis (Table 3)(26).

Table 3. Diagnostic Criteria for IgG4-related Hypophysitis
Criteria		Established diagnosis
Criterion 1	PITUITARY HISTOPATHOLOGY: Mononuclear infiltration of the pituitary gland, rich in lymphocytes and plasma cells, with >10 IgG4-positive cells/high-power field. *	CRITERION 1 or CRITERIA 2 + 3 or CRITERIA 2 + 4 + 5
Criterion 2	PITUITARY MRI: Sella mass or thickened pituitary stalk.
Criterion 3	OTHER INVOLVEMENT: Biopsy-proven involvement in other organs.
Criterion 4	SEROLOGY: Serum IgG4 level >140 mg/dL (1.4 g/L).
Criterion 5	RESPONSE TO TREATMENT: Shrinkage of the pituitary mass and symptom improvement with corticosteroids.

* Low level of infiltration may be seen if the patient is receiving treatment with glucocorticoids (27)

It should be considered that patients with IgG4-related hypophysitis have multi-organ involvement in 60-90% of cases. Therefore, they should receive an extensive evaluation for establishing the extent of the disease after the initial diagnosis. The diagnostic work-up should include physical examination, laboratory evaluation, and whole-body imaging (19).

Xanthomatous Hypophysitis

The pituitary shows cystic-like areas of liquefaction infiltrated by lipid-rich macrophages. It has been suggested that many cases of xanthomatous hypophysitis may represent an inflammatory response to components of a ruptured Rathke’s cleft cyst (see “Hypophysitis secondary to sella and parasellar disease” below) (12,13).

Necrotizing Hypophysitis

Necrotizing hypophysitis has been reported in six patients (of which only five histology-proven) (28-30). Five patients presented with diabetes insipidus and some degree of anterior pituitary dysfunction was described in all reported cases. Frontal headache at presentation was reported in three patients (28,29). One patient presented with photophobia (29). Five patients were treated surgically and all but one had persistent postoperative panhypopituitarism and central diabetes insipidus (28-31).

Clinical Presentation of Primary Hypophysitis

The signs and symptoms at diagnosis, as well as the pituitary hormone abnormalities depend on the degree of pituitary involvement (Table 4) (4,5,8).

Primary hypophysitis more frequently involves the anterior pituitary and patients typically present with severe headaches, visual disturbances due to chiasmal compression, and symptoms of adrenal insufficiency. Contrary to other causes of hypopituitarism, impaired adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) secretion is very frequent in the early stages of primary hypophysitis, putting these patients at increased risk of life-threatening adrenal insufficiency. A large case series from Germany has highlighted that secretion of gonadotropins is also impaired very frequently in these patients (32). Growth hormone (GH) deficiency and hyperprolactinemia can also occur.

Less frequently, the inflammation can involve primarily the posterior pituitary and the stalk. Patients with infundibulo-neurohypophysitis typically present with diabetes insipidus and other pituitary hormone deficiencies are less common. As expected, signs of both anterior and posterior pituitary involvement coexist in panhypophysitis (that is, inflammation of the entire gland).

Table 4. Clinical Presentation and Prevalence of Pituitary Hormone Abnormalities at Diagnosis in Patients with Primary Hypophysitis According to the Degree of Pituitary Involvement

SIGNS AND SYMPTOMS AT DIAGNOSIS

Adenohypophysitis

(~65% of cases)

Infundibulo-neurohypophysitis

(~10% of cases)

Panhypophysitis

(~25% of cases)

All forms *

· Headache: 53%

· Visual disturbances: 43%

· Adrenal insufficiency: 42%

· Hyperprolactinemia: 23%

· Hypothyroidism: 18%

· Hypogonadism: 12%

· Lactation failure: 11%

· Polydipsia/polyuria: 1%

· Polydipsia/polyuria: 98%

· Headache: 13%

· Adrenal insufficiency: 8%

· Hyperprolactinemia: 5%

· Hypogonadism: 3%

· Visual disturbances: 3%

· Hypothyroidism: 0%

· Lactation failure: 0%

· Polydipsia/polyuria: 83%

· Headache: 41%

· Adrenal insufficiency: 19%

· Visual disturbances: 18%

· Hypothyroidism: 17%

· Hyperprolactinemia: 17%

· Hypogonadism: 14%

· Lactation failure: 5%

· Headache: 48%

· Adrenal insufficiency: 38%

· Polydipsia/polyuria: 34%

· Visual disturbances: 32%

· Hypogonadism: 21%

· Hyperprolactinemia: 20%

· Hypothyroidism: 16%

· Lactation failure: 8%

PITUITARY HORMONE ABNORMALITIES AT DIAGNOSIS

Adenohypophysitis

(~65% of cases)

Infundibulo-neurohypophysitis

(~10% of cases)

Panhypophysitis

(~25% of cases)

All forms

· ACTH deficiency: 56%

· TSH deficiency: 44%

· FSH/LH deficiency: 42%

· GH decreased: 26%

· Hyperprolactinemia: 25%

· Hyperprolactinemia: 23% ***

· ADH deficiency: 0%

· ADH deficiency: 98%

· FSH/LH deficiency: 8% **

· Hyperprolactinemia: 5% ***

· Hyperprolactinemia: 0%

· ACTH deficiency: 0%

· TSH deficiency: 0%

· GH decreased: 0% **

· ADH deficiency: 95%

· GH decreased: 51%

· FSH/LH deficiency: 47%

· ACTH deficiency: 46%

· Hyperprolactinemia: 40% ***

· TSH deficiency: 39%

· Hyperprolactinemia: 16%

· ADH deficiency: 63%

· ACTH deficiency: 60%

· FSH/LH deficiency: 55%

· TSH deficiency: 50%

· Hyperprolactinemia: 39%

· GH decreased: 37%

Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone.

* Other possible symptoms at diagnosis include weight gain (~20%) and temperature dysregulation (rare) (32,33).

** Some case series have reported a high prevalence of GH and FSH/LH deficiency in patients with infundibulo-neurohypophysitis (34).

*** Hyperprolactinemia may be related to stalk compression (disconnection hyperprolactinemia) or to the immune-mediated destruction of prolactin-secreting cells.

Granulomatous hypophysitis can be associated with more severe symptoms than lymphocytic hypophysitis, with two case series documenting more frequent occurrence of headache, chiasmal compression, and hypopituitarism (32,35). A review of the literature found that the most common symptoms of granulomatous hypophysitis at presentation were headache (61%), visual changes (40%), polyuria/polydipsia (27%) and cranial nerve palsies (27%); panhypopituitarism and diabetes insipidus were found in 49% and 27% of cases, respectively (11). Cases of compression of the cavernous part of the internal carotid artery have also been described (36).

Clinical data regarding xanthomatous and IgG4-related hypophysitis are less robust due to the rarity of these variants. Gutenberg et al. found that xanthomatous hypophysitis did not cause chiasmal compression and was associated with a low risk of diabetes insipidus and a less severe anterior pituitary hormone impairment than lymphocytic or granulomatous hypophysitis (FSH/LH and GH deficiencies are more common than TSH and ACTH deficiencies) (35). IgG4-related hypophysitis involves frequently both the pituitary and the stalk (~65%) and causes panhypopituitarism, anterior hypopituitarism and central diabetes insipidus in ~50%, ~25% and ~18% of cases, respectively (37). Cases of intrachiasmal abscess and spreading to the cavernous sinus have also been reported (38,39).

Primary hypophysitis is rare in children, with less than 100 cases reported in the literature of which only a few were biopsy-proven (40-42). The clinical presentation, however, seems to differ from adults. A review of the literature showed that the most common presenting symptoms in children are caused by antidiuretic hormone (ADH) deficiency (85%) (42). GH deficiency is found in 76% of cases, while FSH/LH, TSH and ACTH deficiencies were less common than in adults (32%, 29% and 20%, respectively). Headaches and visual disturbances were also rarely reported (17% and 8% of cases, respectively) (42). As central diabetes insipidus and growth retardation are the most common presenting symptoms in children with primary hypophysitis, the more frequent intracranial germinomas and Langerhans cell histiocytosis, as well as craniopharyngiomas, have to be considered in the differential diagnosis (43). Moreover, children with a presumptive diagnosis of hypophysitis are at risk of developing germinomas later in life (up to 3 years after the initial diagnosis) and require extended follow-up (42,44). Germinomas are also a documented cause of secondary hypophysitis (see “Hypophysitis secondary to sella and parasellar disease” below).

Imaging and Differential Diagnosis of Primary Hypophysitis

Magnetic resonance imaging (MRI) of the sella region typically shows an enlarged pituitary. In order to avoid unnecessary surgery, primary hypophysitis needs to be differentiated from other sella and parasellar masses (Table 5)(45), with pituitary adenomas being the most frequent differential diagnosis in adults.

Table 5. Differential Diagnosis of Hypophysitis

SELLA AND PARASELLAR MASSES

· Pituitary adenomas (including pituitary apoplexy);

· Pituitary metastases: the differential diagnosis is particularly important in patients with suspected hypophysitis and malignant tumors receiving immune checkpoint inhibitors;

· Other sella and parasellar tumors (e.g., craniopharyngiomas, germinomas, gliomas, lymphomas, meningiomas, pituicytomas, chordomas, teratomas, dermoids and epidermoids);

· Rathke’s cleft cyst;

· Abscesses.

OTHER

· Physiological hypertrophy of the pituitary in children and adolescents (especially pubertal females) and perimenopausal women;

· Pituitary hyperplasia associated with pregnancy;

· Sheehan’s syndrome at onset;

· Thyrotropic hyperplasia associated with severe, untreated primary hypothyroidism.

Primary hypophysitis typically presents as a homogeneous pituitary enlargement with intense and homogeneous enhancement post-gadolinium and no deviation of the stalk (Figure 1); these and other features can help differentiate between primary hypophysitis and pituitary adenomas at MRI (Table 6) (1,4,46,47). Gutenberg et al. developed a score using variables such as age, association with pregnancy, and MRI findings to distinguish hypophysitis from pituitary adenomas with high accuracy (47). Further differential diagnoses, especially for lymphocytic hypophysitis, are the physiologic pituitary enlargement associated with pregnancy and Sheehan’s syndrome, although these patients have no history of obstetric hemorrhage (48,49). A cautious balance between radiological, clinical, and laboratory findings is necessary to reach the correct diagnosis and avoid inappropriate treatment (50).

Table 6. Differential Imaging Characteristics of Primary Hypophysitis and Pituitary Adenomas
MRI	Primary hypophysitis	Pituitary adenoma
Pre-gadolinium	ACUTE / SUB-ACUTE PHASE: · Homogeneous pituitary enlargement with symmetrical suprasellar expansion; · Suprasellar extension with compression and displacement of chiasm; · Stalk thickened but not deviated; * · Loss of bright spot of the neurohypophysis in case of involvement of the posterior pituitary. ** CHRONIC PHASE: · Pituitary atrophy; · Empty sella.	· Microadenoma (<1cm): unilateral, asymmetric endosellar mass; · Macroadenoma (>1cm): expanding, not homogeneous pituitary mass with asymmetrical suprasellar expansion; · Compression and displacement of chiasm (macroadenoma); · Contralateral deviation of the stalk; · The bright spot of the neurohypophysis can be usually seen. **
Post-gadolinium	· Intense and homogeneous enhancement of the pituitary mass. Cystic areas have been described, especially in the xanthomatous variant; · Dural tail sign can be present (thickening of the enhanced dura that resembles a tail extending from a mass). ***	· Slight, delayed and not homogeneous enhancement. Cystic and necrotic areas are frequently observed in macroadenomas; · Dural tail usually absent. ***

Abbreviations: MRI, magnetic resonance imaging.

* An enlarged pituitary stalk can also be found in other intracranial pathologies (e.g., sarcoidosis, metastases, Langerhans cell histiocytosis, germinoma, craniopharyngioma, astrocytoma, pituitary adenoma, lymphoma, tuberculosis, Erdheim-Chester’s disease) (51).

** The bright spot may be absent in up to 20% of healthy subjects (especially the elderly).

*** The dural tail sign is not specific to hypophysitis. It can be observed in meningioma (most frequently) and other intracranial pathologies (e.g. lymphoma, chloroma, metastasis, multiple myeloma, glioblastoma multiforme, aspergillosis, chordoma, schwannoma, pleomorphic xanthoastrocytoma, hemangiopericytoma, granulomatosis with polyangiitis, sarcoidosis, medulloblastoma, eosinophilic granuloma, pituitary adenoma, pituitary apoplexy, Erdheim-Chester’s disease) (52)

Figure 1. Magnetic resonance imaging findings in a case of primary hypophysitis. Panel A) T1-weighted image, sagittal section. Panel B) T1-weighted image, coronal section. Panel C) T1-weighted image post-gadolinium, sagittal section. Panel D) T1-weighted image post-gadolinium, coronal section. A homogeneous enlargement of the pituitary with thickening of the stalk can be seen. The mass shows intense and homogeneous enhancement post-gadolinium.

Autoantibodies in Primary Hypophysitis

Several authors have assessed the presence and utility of serum autoantibodies (pituitary and/or hypothalamic antibodies) in patients with primary hypophysitis:

An autoimmune etiology for lymphocytic hypophysitis was suggested by the presence of pituitary antibodies that may recognize α-enolase, GH, the pituitary gland-specific factors 1a and 2 (PGSF1a and PGSF2), regulatory prohormone-processing enzymes commonly produced in the pituitary gland (PC1/3, PC2, CPE and 7B2), secretogranin II, chromosome 14 open reading frame 166 (C14orf166), the corticotroph-specific transcription factor TPIT, and chorionic somatomammotrophin (HCS) (53-61). Several techniques have been used to detect pituitary antibodies in primary hypophysitis (ELISA, radioligand assay, immunoblotting, and immunofluorescence) and the prevalence of antibody-positive hypophysitis is 11-73% depending from the antigen(s) tested and the technique used (7,62). However, the pathogenic role of these autoantibodies is unclear and they are not specific to hypophysitis. For example, pituitary antibodies were identified by indirect immunofluorescence in ~45% of patients with biopsy-proven hypophysitis, but were also found in the serum of patients with isolated central diabetes insipidus (35%), germinomas (33%), isolated anterior hormone deficiencies (29%), prolactinomas (27%), Rathke’s cleft cysts (25%), craniopharyngiomas (17%), non-functioning pituitary tumors (13%), GH-secreting pituitary tumors (12%), and healthy subjects (5%) (62-65). They can also be found in patients with autoimmune endocrine disorders, especially Hashimoto thyroiditis (63). However, indirect immunofluorescence using human pituitary gland as a substrate and showing a granular cytosolic staining pattern was most commonly found in patients with hypophysitis and isolated hormone deficiencies (62); therefore, the finding of this staining pattern can be useful to clinicians in establishing a diagnosis of hypophysitis;

The detection of hypothalamic antibodies targeting corticotropin-releasing hormone (CRH)-secreting cells in some patients with GH/ACTH deficiency but with pituitary antibodies targeting only GH-secreting cells indicates that an autoimmune aggression to the hypothalamus can be responsible for some cases of lymphocytic hypophysitis (66). Consequently, not only pituitary but also hypothalamic autoimmunity may contribute to anterior pituitary dysfunction in a subset of patients with primary hypophysitis;

A search for ADH antibodies in patients with primary hypophysitis may help identifying patients who are prone to developing autoimmune central diabetes insipidus (67). These antibodies alone are not a good diagnostic marker for posterior pituitary involvement, but may serve as a predictive marker of gestational or post-partum diabetes insipidus (68,69);

Anti-Rabphilin antibodies have been proposed to be a biomarker for lymphocytic infundibulo-neurohypophysitis (70). Rabphilin is involved in the release of hormones or neurotransmitters and is expressed mainly in the brain, including the posterior pituitary and hypothalamus where ADH is present. Whether anti-Rabphilin antibodies are a cause of central diabetes insipidus or a result of infundibulo-neurohypophysitis is unknown. However, anti-Rabphilin antibodies are detected in 76% of patients with infundibulo-neurohypophysitis and 11% of patients with lymphocytic hypophysitis. In contrast, these antibodies are absent in patients with sella/suprasellar masses without lymphocytic hypophysitis, suggesting that this antibody may serve as a biomarker for the diagnosis of infundibulo-neurohypophysitis and may be useful for the differential diagnosis in patients with central diabetes insipidus (45);

Primary hypophysitis can eventually evolve in pituitary fibrosis and atrophy, documented at imaging as an “empty sella”. Lupi et al. have found pituitary antibodies in 6% of patients with an empty sella not linked to previous head trauma. In this cohort, the presence of pituitary antibodies also correlated with the presence of hypopituitarism (71);

Antibodies recognizing GH and one peptide from proopiomelanocortin (POMC) have been described in a patient with IgG4-related hypophysitis (72).

Natural History of Primary Hypophysitis

Primary hypophysitis can be self-limiting and spontaneous remission may occur (Figure 2). Considering the low prevalence of the disease, however, robust data regarding the natural history of primary hypophysitis are lacking (54). Moreover, most of the literature regards lymphocytic hypophysitis, while data from other histology subtypes are less robust. A review of 76 cases of primary hypophysitis from Germany has shown that patients not receiving any active treatment had improvement, stability or progression of the pituitary involvement at MRI in 46%, 27% and 27% of cases, respectively; pituitary deficiencies improved, remained stable or worsened in 27%, 55% and 18% of patients, respectively (73). A previous study by Khare et al. showed that spontaneous resolution of sella compression symptoms occurred in all patients managed conservatively and that 33% had complete or partial recovery of pituitary function (74). Park et al. also reported similar findings (75).

Primary hypophysitis frequently evolves to fibrosis and pituitary atrophy in the chronic stages of the disease, which often impair pituitary function (Figure 2). The evolution to empty sella has also been shown in a mouse model of autoimmune hypophysitis (76). Caturegli et al. reported that only 4% of patients had spontaneous remission with recovery of pituitary function, while most patients will require long-term replacement of one or more pituitary axes (4,54). Whether medical treatment with glucocorticoids has a positive impact on the natural history of primary hypophysitis is still a matter of debate.

Figure 2. Natural History of Primary Hypophysitis.

Most of the published case series mainly focus on the more frequent lymphocytic hypophysitis. Granulomatous hypophysitis can cause more severe signs and symptoms (headache, chiasmal compression and anterior/posterior hypopituitarism). Xanthomatous hypophysitis seems to cause sella compression and pituitary dysfunction less frequently. IgG4-related hypophysitis can cause various degree of involvement of the anterior pituitary, posterior pituitary and the stalk. Necrotizing hypophysitis is extremely rare and is associated with a high risk of panhypopituitarism and diabetes insipidus. The chronic stage of the disease is most likely related to the extent of damage of the pituitary. Some authors have suggested that some cases of lymphocytic hypophysitis may evolve to the granulomatous form, as mixed forms can rarely be observed. A death rate of 7% has been described in large case series of patients with primary hypophysitis and is probably related to unrecognized acute adrenal insufficiency.

Diagnosis and Treatment of Primary Hypophysitis

Pituitary biopsy is the gold standard to confirm the diagnosis of primary hypophysitis. This procedure, however, should be considered only in equivocal cases and when the outcome of the biopsy is expected to change the therapeutic management, and should be performed by a neurosurgeon with extensive expertise in pituitary surgery.

Due to the rarity of the disease, the management of hypophysitis is controversial. An algorithm in line with the more recent literature is reported in Figure 3. Initial evaluation of patients with suspected hypophysitis involves clinical and laboratory assessment. Patients with a suspicion of hypophysitis based on biochemical results should undergo a pituitary MRI, as well as visual assessment to check visual fields and acuity. Secondary causes of hypophysitis and other sella/parasellar masses should be considered in the differential diagnosis.

The mainstay of treatment of primary hypophysitis is pituitary hormone replacement (77,78). As outlined above, ACTH production is frequently impaired at presentation, and most patients will require glucocorticoid replacement. This should be started before thyroxine replacement (if TSH deficiency is present as well) to avoid precipitating acute adrenal insufficiency.

Conservative management is recommended for primary hypophysitis unless symptoms are severe and progressive. The only exception to this rule is IgG4-related hypophysitis that – like other manifestations of the disease – should be promptly treated to revert symptoms and prevent irreversible fibrosis (79,80). The mainstay of treatment are glucocorticoids, which often cause remission of symptoms within a few weeks. A typical starting dose is prednisone 30-40 mg/day (or equivalent), which should be continued for 2-4 weeks, and then tapered gradually over 2-6 months (19). However, some patients may benefit from long-term maintenance glucocorticoid therapy (with or without a steroid-sparing agent), especially in case of extensive multi-organ involvement. Relapse is possible and multiple courses of high-dose glucocorticoids are often necessary. Rituximab has also been used in patients with poor response to glucocorticoids (19,81,82). A case of IgG4-related hypophysitis successfully treated with azathioprine has also been reported (83).

High-dose glucocorticoids are the first-line treatment to improve the swelling of the pituitary and improve the symptoms related to significant sella compression. Anterior pituitary function can recover after pulse corticosteroid therapy, although >70% of patients will require long-term replacement with one or more hormones (4); central diabetes insipidus rarely recovers. Honegger et al. documented excellent initial responses to high-dose glucocorticoids, with radiological improvement, stability and progression in 65%, 31% and 4% of cases, respectively (73). However, these patients carried a higher risk of side effects (weight gain, psychiatric symptoms, peripheral edema, diabetes mellitus and glaucoma) and relapse of the pituitary inflammation was documented in 38% of cases. Relapses occurred 2-17 months after starting pulse steroids and the risk or relapse did not correlate with either initial glucocorticoid dose or treatment duration (73). Hormone deficiencies improved with glucocorticoids only in 15% of patients, while they remained stable or worsened in 70% and 15% of cases, respectively (73). Lupi et al. performed a review of the literature and found somewhat better outcomes with medical therapy, reporting pituitary mass reduction in 84% of cases, improving anterior pituitary function in 45%, and restored posterior pituitary function in 41% after high-dose glucocorticoids and/or azathioprine, with a relatively low risk of relapse (14%) (84). Recently, Chiloiro et al. found in a small prospective double-arm study that high-dose glucocorticoid treatment – compared with simple observation – was associated with higher rates of hypophysitis resolution and pituitary function recovery (85). The authors also showed that positive pituitary antibodies, a diagnosis of diabetes insipidus and secondary hypogonadism at the time of presentation, and specific MRI findings (a thicker pituitary stalk, a smaller pituitary volume, and the evidence of posterior pituitary involvement at MRI including absent bright spot) predicted better clinical outcomes following glucocorticoid therapy. These findings should be confirmed in a larger prospective cohort.

Whether central diabetes insipidus is an unfavorable prognostic factor for response to glucocorticoids is unclear. The abovementioned study by Chiloiro et al. suggests better outcomes in patients with central diabetes insipidus at the time of hypophysitis diagnosis (85); however, Lupi et al. found that patients with concomitant anterior and posterior pituitary dysfunction responded poorly to glucocorticoids, which were unable to revert the hypopituitarism (86). Glucocorticoid therapy was also found to be less effective in granulomatous or xanthomatous hypophysitis (35). In glucocorticoid-resistant cases and when high-dose glucocorticoids cause unacceptable side effects, immunosuppressive drugs such as azathioprine, methotrexate, and cyclosporin A have been used successfully. However, the long-term effects are unclear (1). Rituximab has also been employed to treat steroid-refractory hypophysitis (36,87-89).

Surgery should be considered only in cases with serious and progressive deficits of the visual field, visual acuity, or nerve paralysis not responsive to medical treatment. Surgery generally improves sella compression in the short term; however, Honegger et al. observed progression/relapse of the disease in 25% of patients after a mean follow-up of 3 years (73). Pituitary function improved only in 8% of patients after surgery, and the rates of resolution of chiasmal compression were also low (73). Further supporting the limited role of surgery in the management of hypophysitis, two small observational studies found that surgery did not impact significantly on the resolution of neurological symptoms or hormonal deficits during follow-up (90,91).

Stereotactic radiotherapy (radiosurgery) has been effectively employed in selected patients who have failed medical treatment or suffer from repeated recurrence of lymphocytic hypophysitis (92,93).

Figure 3. Diagnosis and management of primary hypophysitis. 1 Check random ACTH and cortisol if acute adrenal insufficiency is suspected. Consider confirmatory testing (e.g., Synacthen) if equivocal or borderline results. The Synacthen test can give false-positive results in the early stages of central adrenal insufficiency. During pregnancy and in patients receiving oral estrogens, the rise of corticosteroid-binding globulin (CBG) leads to falsely elevated cortisol levels and the normal reference ranges and stimulated cortisol cut-offs do not apply. 2 Pituitary surgery can also provide histology for definitive diagnosis.

DRUG-INDUCED HYPOPHYSITIS: IMMUNE CHECKPOINT INHIBITORS

Immune checkpoint inhibitors are monoclonal antibodies increasingly used for solid and hematological malignancies (94). They block several regulators of the immune activation (immune checkpoints), enhancing the host’s immune response to tumor cells (Figure 4). These drugs have shown a favorable toxicity profile and significant anti-tumor activity but, because of their mechanism of action, new typical side-effects have emerged (immune-related adverse events, irAEs) (Figure 4) (95,96).

Figure 4. Mechanism of Action of Immune Checkpoint Inhibitors. Tumor antigens are presented to T-cells by antigen-presenting-cells (APCs) via the interaction of the major histocompatibility complex (MHC) and the T-cell receptors, representing the primary signal for activating T-cells. Another costimulatory signal involving interaction between B7 on APCs and CD28 on T-cells is needed to complete T-cell activation and expansion (Panel A). Several co-receptors act as negative modulators of immune response at different molecular checkpoints. The CTLA-4 is induced in T-cells at the time of their initial response to antigen. CTLA-4 is transported to the cell surface proportionally to the antigen stimulation; it binds to B7 with greater affinity than CD28, resulting in specific T-cell inactivation (Panel B). The PD-1/PD1-L1 pathway is not involved in initial T-cell activation: it regulates inflammatory responses in peripheral tissues sustained by already activated effector T-cells. Activated T-cells up-regulate PD-1 and inflammatory signals in the tissue induce the expression of PD1-L1s, which downregulate the activity of T-cells, protecting normal tissues from collateral destruction; this mechanism is also exploited by tumor cells to evade the immune system response (Panel B). Monoclonal antibodies that block either CTLA-4 or PD1/PD1-L1 increase cytotoxic T-cell activity by expanding T-cell activation and proliferation (Panel C). The eventual T-cell reactivation is responsible for the both anti-tumor response and the immune-related adverse events associated with these drugs.

irAES mirror the immune response reactivation induced by immune checkpoint inhibitors and may predict better survival and response to the treatment of the underlying malignancy (97-100). irAEs can affect multiple organs and systems, including the pituitary and other endocrine glands (Table 7) (101).

Table 7. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors

ENDOCRINOPATHIES

OTHER SYSTEMS AND ORGANS

PITUITARY: Hypophysitis.*

THYROID: Thyroiditis (both hypo- and hyperthyroidism); Euthyroid Graves’ ophthalmopathy.

ADRENAL GLANDS: Adrenalitis.*

PANCREAS: Insulinopenic diabetes mellitus.

SKIN: Rash/inflammatory dermatitis; Bullous dermatoses; Stevens-Johnson syndrome; Toxic epidermal necrolysis; Drug rash with eosinophilia and systemic symptoms syndrome; Drug-induced hypersensitivity syndrome; Acute generalized exanthematous pustulosis; Alopecia areata; Vitiligo; Psoriasis.

GASTROINTESTINAL SYSTEM: Colitis; Hepatitis; Pancreatitis.

LUNGS: Pneumonitis.

MUSCOSKELETAL SYSTEM: Arthritis; Polymyalgia-like syndrome; Myositis; Vasculitis.

KIDNEY: Nephritis.

CARDIOVASCULAR SYSTEM: Myocarditis; Pericarditis; Arrhythmias; Heart failure; Vasculitis; Venous thromboembolism.

NERVOUS SYSTEM: Guillain-Barré syndrome; Myasthenia gravis; Peripheral neuropathy; Autonomic neuropathy; Aseptic meningitis; Encephalitis; Transverse myelitis.

HEMATOLOGY: Autoimmune hemolytic anemia; Acquired thrombotic thrombocytopenic purpura; Hemolytic uremic syndrome; Aplastic anemia; Lymphopenia; Immune thrombocytopenia; Acquired hemophilia.

EYE: Uveitis; Iritis; Episcleritis; Blepharitis.

* Immune checkpoint inhibitors can cause both primary adrenal insufficiency (rarer) and secondary adrenal insufficiency (more frequent).

Epidemiology

Hypophysitis may occur as a complication during treatment with immune checkpoint inhibitors. Ipilimumab, a monoclonal antibody against the cytotoxic T lymphocyte antigen-4 (CTLA-4) is the drug that has been more strongly associated with this immune-related adverse event (Table 8) (5,102-112). The overall incidence of hypophysitis is 12% in patients treated with anti-CTLA-4 antibodies and 0.5% in patients treated with anti-programmed death 1 (PD1) antibodies (113,114).

Table 8. Immune Checkpoint Inhibitors and the Risk of Hypophysitis
Category	Drug	Approved and off-label indications	Incidence of reported hypophysitis in clinical studies
Anti-CTLA-4 (70% of published hypophysitis cases)	Ipilimumab	Unresectable or metastatic melanoma; Adjuvant treatment in melanoma; Relapsed hematologic cancer.	Up to 17.4% (G3-G4: 0.3-17.4%)
Anti-CTLA-4 (70% of published hypophysitis cases)	Tremelimumab	Malignant mesothelioma; Hepatocellular carcinoma. This drug is not FDA approved.	0-2.6% (G3-G4: 1%)
Anti-PD1 (24% of published hypophysitis cases)	Nivolumab	Metastatic colorectal cancer; Recurrent or metastatic squamous cell head and neck cancer; Hepatocellular carcinoma; Classical Hodgkin’s lymphoma; Unresectable or metastatic melanoma; Adjuvant treatment in melanoma; Progressive, metastatic non-small cell lung cancer; Progressive small cell lung cancer; Advanced renal cell cancer; Urothelial carcinoma; Platinum-resistant ovarian cancer.	0-3% (G3-G4: 0.5%)
	Pembrolizumab	Metastatic or recurrent locally advanced gastric cancer; Recurrent or metastatic squamous cell head and neck cancer; Relapsed or refractory classical Hodgkin’s lymphoma; Relapsed chronic lymphocytic leukemia; Unresectable or metastatic melanoma; Unresectable or metastatic microsatellite instability-high cancer; Metastatic non-small cell lung cancer; Metastatic, non-squamous Non-small cell lung cancer (in combination with Pemetrexed and Carboplatin); Locally advanced or metastatic urothelial carcinoma; Advanced Merkel cell carcinoma.	0-4.8% (G3-G4: 0-2.4%)
	Dostarlimab	Mismatch repair deficient recurrent or advanced endometrial cancer; Mismatch repair deficient recurrent or advanced solid tumors.	No cases published. Hypophysitis is listed as a possible adverse reaction in <10% of treated patients in the product information.
	Cemiplimab	Cutaneous squamous cell carcinoma.	1 case reported
	Toripalimab	Melanoma; several solid malignancies (development stage).	1 case reported
	Geptanolimab (still in development stage)	Peripheral T-cell lymphoma; Alveolar soft part sarcoma; Cervical cancer; Non-Hodgkin's lymphoma; Liver cancer; Colorectal cancer; Non-small cell lung cancer.	1 case reported
Anti-PD1-L1 (2% of published hypophysitis cases)	Atezolizumab	Metastatic non-small cell lung cancer; Locally advanced or metastatic urothelial carcinoma.	1% (G3-G4: 1%)
	Avelumab	Metastatic Merkel cell carcinoma; Locally advanced or metastatic urothelial carcinoma; Advanced non-small cell lung cancer.	1 case reported
	Durvalumab	Advanced non-small cell lung cancer; Locally advanced or metastatic urothelial carcinoma.	1 case reported
Combination therapy (4% of published hypophysitis cases)	Ipilimumab + Nivolumab	Unresectable or metastatic melanoma; Progressive small cell lung cancer; Non-small cell lung cancer; Advanced renal cell cancer; Malignant mesothelioma; Recurrent glioblastoma.	Up to 12.8% (G3-G4: 1.5-8.7%)
	Ipilimumab + Pembrolizumab	Advanced melanoma; Advanced renal cell carcinoma.	0-9.1% (G3-G4: 0-6%)
	Durvalumab + Tremelimumab	Advanced non-small cell lung cancer.	0%

Abbreviations: CTLA-4, cytotoxic T lymphocyte antigen-4; FDA, Food and Drug Administration; G3, grade 3 immune checkpoint inhibitor-induced hypophysitis (see Table 11); G4, grade 4 immune checkpoint inhibitor-induced hypophysitis (see Table 11); PD1, programmed death 1; PD1-L1, programmed death 1 Ligand 1.

Pathogenesis

The pathogenesis of anti-CTLA-4 antibody-induced hypophysitis involves type II and IV hypersensitivity, as well as the humoral immune response (Figure 5). This has been suggested by histopathological findings of patients with hypophysitis following treatment with Ipilimumab (alone or in combination with Nivolumab or Pembrolizumab), evidence of pituitary antibodies in the serum of these patients, association with specific human leucocyte antigens, and animal models of anti-CTLA-4-induced hypophysitis (8,15,115-118).

Evidence regarding the pathophysiology of anti-PD1/PD1-L1 antibody-induced hypophysitis is scant, but immune response reactivation most likely targets ACTH-secreting cells because of the very frequent isolated ACTH deficiency (5). Kanie et al. recently postulated that ectopic expression of ACTH in the tumor may contribute to some cases of anti-PD1/PD1-L1 antibody-induced hypophysitis, as a form of paraneoplastic syndrome (119). Furthermore, Bellastella et al. identified a higher prevalence of anti-pituitary and anti-hypothalamus antibodies in patients with cancer treated with anti-PD1/PD1-L1 agents (120). In a small longitudinal study, the same group also found that more than half of patients who start anti-PD1/PD1-L1 treatment developed anti-pituitary or anti-hypothalamus antibodies after 9 weeks of treatment, with a concomitant increase prolactin and a reduction in ACTH and IGF-1 levels compared to baseline (120). These preliminary results need to be validated in a larger cohort, but the presence of anti-hypothalamus antibodies would suggest that – at least in some patients – hypothalamic autoimmunity might contribute to the development of anti-PD1/PD1-L1 antibody-induced pituitary dysfunction.

Figure 5. Proposed pathogenesis of anti-CTLA-4 antibody-induced hypophysitis. Anti-CTLA-4 antibody-induced hypophysitis accounts for ~70% of immune-checkpoint induced hypophysitis cases. The CTLA-4 antibody binds to pituitary CTLA-4 antigen, inducing complement activation and infiltration with macrophages and other inflammatory cells, leading to phagocytosis and enhanced antigen presentation. Subsequently, autoimmune type IV hypersensitivity reactions start, with infiltration of the anterior pituitary by autoreactive T lymphocytes that eventually leads to pituitary cytotoxicity and inflammation. Moreover, patients with anti-CTLA-4 antibody-induced hypophysitis develop pituitary antibodies that predominantly recognize TSH- FSH- and ACTH-secreting cells. Pituitary cytotoxicity in anti-PD1/PD1-L1 antibody-induced hypophysitis presumably affects mostly ACTH-secreting cells, as isolated ACTH deficiency is the most common occurrence in these patients.

Clinical Characteristics

There are important differences between primary hypophysitis and immune checkpoint-induced hypophysitis (Table 9)(5,8,103). The latter does not have a female predominance (8,121) and seems to present more frequently with hypopituitarism at diagnosis. Both forms of hypophysitis are more commonly associated with an initial deficit of ACTH, FSH/LH and TSH, but symptoms of adrenal insufficiency and confirmed ACTH deficiency are much more common in patients with immune checkpoint-induced hypophysitis (8,113,114). Central diabetes insipidus can occur in a substantial share of primary hypophysitis cases (i.e., the infundibulo-neurohypophysitis and panhypophysitis variants), while it is extremely rare in immune-checkpoint induced hypophysitis. Pituitary enlargement and visual impairment are much more common in primary hypophysitis, while the size of the pituitary may appear normal in immune checkpoint inhibitor-induced hypophysitis (in absence of a baseline pituitary MRI) and optic chiasm involvement is rare (5,8).

Table 9. Comparison Between Primary and Immune Checkpoint Inhibitor-Induced Hypophysitis
Characteristics	Primary hypophysitis	Immune checkpoint inhibitor-induced hypophysitis
Etiology	Autoimmune.	Immune response reactivation.
Epidemiology	· More prevalent in young females (female:male ratio ~3:1), apart from the rare IgG4-related form that is more common in older males. · The onset of the lymphocytic subtype is strongly associated with late pregnancy and the post-partum period.	· The epidemiology is most likely influenced by the underlying malignancy. · 0.5-12% of treated patients develop hypophysitis, depending on the drug used. · The female:male ratio is ~1:4 and the mean age at onset is ~60 years (older male patients appear to be the group carrying the higher risk). · No prior cancer therapy is associated with higher risk of developing hypophysitis.
Time after the initiating event	Unknown. The median duration of symptoms before clinical presentation is varies according to the anatomic location of the pituitary involvement: · Adenohypophysitis (during pregnancy): 4 months; · Adenohypophysitis (outside of pregnancy): 12 months; · Infundibulo-neurohypophysitis: 3 months; · Panhypophysitis: 4 months.	· Ipilimumab: median time to onset 9-11 weeks (range 1-35); * · Pembrolizumab: median time to onset 16 weeks (range 1-52); * · Nivolumab: median time to onset 21-22 weeks (range 6-48); * · Ipilimumab + Nivolumab: median time to onset 11-12 weeks (range 3-32). *
Symptoms at presentation	· Headache: 48% · Adrenal insufficiency: 38% · Polydipsia/polyuria: 34% · Visual disturbances: 32% · Hypogonadism: 21% · Hypothyroidism: 16%	·Adrenal insufficiency: 81% ** ·Headache: 45% ·Hypothyroidism: 18% ·Hypogonadism: 11% ·Visual disturbances: 6% ·Polydipsia/polyuria: 2%
Pituitary hormone abnormalities	· ADH deficiency: 63% · ACTH deficiency: 60% · FSH/LH deficiency: 55% · TSH deficiency: 50% · GH decreased: 37% · Hyperprolactinemia: 39%	· ACTH deficiency: 96% ** · TSH deficiency: 63% · FSH/LH deficiency: 59% · GH decreased: 19% · Hyperprolactinemia: 11% · ADH deficiency: 4%
Abnormal MRI at presentation	97% of cases	64% of cases ***
Histopathology	Marked infiltration of lymphocytes of the pituitary gland, typically accompanied by scattered plasma cells, eosinophils and fibroblasts, and in later disease stages by fibrosis.	T-cell infiltration and IgG-dependent complement fixation and phagocytosis.
Treatment	Usually good response to glucocorticoids.	Good response to glucocorticoids of the symptoms related to sella compression.
Outcome	Variable: from complete recovery to persistent hypopituitarism.	Pituitary enlargement (if present) eventually resolves. TSH and FSH/LH deficiencies often recover, while central adrenal insufficiency persists almost invariably.

Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; CTLA-4, cytotoxic T lymphocyte antigen-4; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GH, growth hormone; MRI, magnetic resonance imaging; TSH, thyroid-stimulating hormone.

* Data from the prescribing information of Ipilimumab, Pembrolizumab and Nivolumab.

** Anti-PD-1/PD1-L1 antibody-induced hypophysitis typically presents with isolated ACTH deficiency, while CTLA-4 antibody-induced hypophysitis more frequently leads to multiple hormone deficiencies (Table 10).

*** MRI abnormalities are transient, can be subtle and precede clinical symptoms in ~50% of cases. Anti-PD-1/PD1-L1 antibody-induced hypophysitis typically lacks MRI changes and causes no mass effect symptoms (Table 10).

The onset of immune checkpoint-induced hypophysitis varies according to the drug used (Table 9); early onset has been reported and it can appear also several months after the initiation of the immunotherapy (122,123). The risk of hypophysitis with Ipilimumab appears to be dose-dependent, with a higher prevalence in those receiving 10 mg/kg vs. 3 mg/kg (124-126). Conversely, patients receiving concomitant cytotoxic chemotherapy or with brain radiotherapy-pretreated metastases might be protected from the risk of developing hypophysitis, presumably through immune cell depletion (95,127).

Patients with immune checkpoint-induced hypophysitis typically present with nonspecific symptoms of adrenal insufficiency like fatigue, headache, myalgia, nausea, vomiting, reduced appetite, light-headedness, and dizziness, whilst symptoms of other anterior pituitary hormone deficiencies are less common at the time of diagnosis (Table 9) (113,114). Manifestations of adrenal insufficiency often overlap with those of the underlying malignancy but must not be overlooked because of the risk of developing life-threatening adrenal crisis. Visual disturbances are very rare (the pituitary enlargement, if present, is often minor and transient) and central diabetes insipidus is extremely uncommon (95,113,114,128,129). Other less frequent symptoms include confusion, hallucinations, memory loss, labile moods and depression (including suicidal ideation), insomnia, temperature intolerance, and chills (130,131). Importantly, up to 45% of patients can be asymptomatic and are diagnosed only at laboratory evaluation, highlighting the importance of regular monitoring (123,132).

Associated irAEs have been reported in about half of patients with immune checkpoint inhibitor-induced hypophysitis (133). By far, the most common associated irAE was thyroiditis (~30%), followed by colitis (~20%), skin reactions (~15%), pneumonitis (~5%), and hepatitis (~5%) (133).

Patients with anti-CTLA-4 antibody-induced hypophysitis tend to have a more diverse clinical presentation than those with anti-PD-1/PD1-L1 antibody-induced hypophysitis. The latter typically presenting later during treatment, with severe isolated ACTH deficiency (which frequently leads to hyponatremia at the time of diagnosis), and no significant pituitary enlargement both clinically and radiologically. Also, treatment discontinuation is less frequently required in patients with anti-PD-1/PD1-L1 antibody-induced hypophysitis (Table 10) (5,81,114,134-136).

Table 10. Comparison Between Anti-CTLA-4 and Anti-PD1/PD1-L1 Antibody-Induced Hypophysitis
Characteristics	Anti-CTLA-4 antibody-induced hypophysitis	Anti-PD1/PD1-L1 antibody-induced hypophysitis
Number of cases reported	192 (74% males)	69 (72% males)
Mean time to onset (95% CI)	10.5 weeks (9.8-11.2)	Anti-PD1: 27.0 weeks (20.9-33.1) Anti-PD1-L1: 27.8 weeks (0-58.0)
Mean doses to onset	3.4 doses	10.3 doses
Symptoms at presentation	· Adrenal insufficiency: 75% · Headache: 60% · Hypothyroidism: 21% · Hypogonadism: 16% · Visual disturbances: 8% · Polydipsia/polyuria: <1%	·Adrenal insufficiency: 91% ·Hypothyroidism: 7% ·Headache: 4% ·Polydipsia/polyuria: 3% ·Hypogonadism: 0% ·Visual disturbances: 0%
Pituitary hormone abnormalities at presentation *	· ACTH deficiency: 95% · TSH deficiency: 85% · FSH/LH deficiency: 75% · GH decreased: 27% · Hyperprolactinemia: 7% · ADH deficiency: 2%	· ACTH deficiency: 97% · Hyperprolactinemia: 20% · FSH/LH deficiency: 13% · TSH deficiency: 4% · GH decreased: 3% · ADH deficiency: 3%
Prevalence of hyponatremia at presentation **	39% of cases	62% of cases
Abnormal MRI at presentation	81% of cases	18% of cases
Discontinuation of the immune checkpoint inhibitor	· No: 56% · Yes, temporarily: 3% · Yes, permanently: 41%	· No: 70% · Yes, temporarily: 20% · Yes, permanently: 10%
Outcome	· Long-term hypopituitarism: 89% · Pituitary function recovery after treatment: 5% · Spontaneous resolution: 1% · Death: 5% · Recurrence after treatment: 0%	· Long-term hypopituitarism: 90% · Pituitary function recovery after treatment: 6% · Spontaneous resolution: 0% · Death: 4% · Recurrence after treatment: 0%

* Pituitary hormone deficiencies can be isolated or combined (especially in the case of anti-CTLA-4 antibody-induced hypophysitis). ACTH + TSH deficiency is the most frequent combination observed in these patients.

** Most likely related to cortisol deficiency. It can be a clue to the diagnosis.

According to the degree of symptoms and of the severity of the disease, immune checkpoint-induced hypophysitis is graded 1 to 4 (Table 11) (78). Grade 3 toxicity or worse (including death) has been described in 2-10% of reported hypophysitis cases (137,138).

Abbreviations: ADL, activities of daily living.

Table 11. Grading of Immune Checkpoint Inhibitor-Induced Hypophysitis
Grade	Description
Grade 1	Asymptomatic or mild symptoms.
Grade 2	Moderate symptoms, able to perform ADL.
Grade 3	Severe symptoms, medically significant consequences, unable to perform ADL.
Grade 4	Severe symptoms, life-threatening consequences, unable to perform ADL.
Grade 5	Death.

Diagnosis and Management

An algorithm for diagnosis and management of immune checkpoint-induced hypophysitis in line with the more recent literature is shown in Figure 6. Patients should be regularly monitored with clinical assessment and hormonal tests during treatment with immune checkpoint inhibitors. Almost invariably, patients who develop hypophysitis have ACTH deficiency and cases of fatal acute adrenal insufficiency have been reported (139); this highlights the importance of pituitary function assessment at baseline and during treatment, also in asymptomatic patients (140). If there is a strong suspicion of adrenal insufficiency on clinical grounds (e.g. G3-G4 symptoms), glucocorticoid replacement should be started without delay (141). TSH deficiency is also very common (>60% of patients). Indeed, a fall in serum TSH and free T4 have been suggested to be early signs of immune checkpoint inhibitor-induced hypophysitis and can be a clue to the diagnosis (141-144). A recent paper has identified antibodies against two autoantigens (anti-GNAL and anti-ITM2B) that may aid in the diagnosis of and predict the risk of developing immune checkpoint-induced hypophysitis (145). Moreover, Kobayashi et al. evaluated the usefulness of pituitary antibodies and human leukocyte antigen alleles in predicting immune checkpoint-induced pituitary dysfunction. The authors showed distinct and overlapped patterns of pituitary antibodies and human leukocyte antigen alleles between patients who developed hypophysitis (n=5) or isolated ACTH deficiency (n=17) (117). The usefulness of anti-GNAL and anti-ITM2B antibodies, pituitary antibodies, and human leukocyte antigen alleles as biomarkers in the clinical setting needs to be validated in larger cohorts of patients.

Patients with suspected drug-induce hypophysitis should undergo a pituitary MRI and visual assessment (141,146). The importance of obtaining pituitary imaging was recently highlighted in a retrospective study by Nguyen et al., where 33% of hypophysitis cases would have been missed if no MRI were carried out (143). Also, pituitary MRI is important for the differential diagnosis of other pituitary lesions, in particular metastases (Table 12). Faje et al. reported that ~50% of patients with immune checkpoint-induced hypophysitis presented with diffuse pituitary enlargement at MRI before the onset of clinical symptoms (98). ¹⁸F-FDG PET performed as part of the staging of the underlying malignancy can show intense radiotracer uptake and may precede clinical symptoms and biochemical abnormalities (147,148); however, its routine use for the diagnosis of hypophysitis is not recommended.

Current guidelines on the management of immune-checkpoint induced hypophysitis suggest clinicians to consider with holding treatment in G1-G2 hypophysitis until the patient is stabilized on hormone replacement (101). We believe that patients with immune checkpoint inhibitor-induced hypophysitis should not stop treatment unless they develop severe and progressive symptoms (G3-G4 hypophysitis). In fact, this type of hypophysitis if often self-limiting and most of patients do not show progression of sella compression. Therefore, the decision whether to withhold a treatment that can have a significant impact on the progression-free survival of the underlying malignancy should be balanced carefully. When G3-G4 hypophysitis is suspected, a course of high-dose corticosteroids given during the acute phase may result in inflammation reversal and ameliorate the compression of sella and parasellar structures. Whether high-dose glucocorticoids have an impact on the anti-tumor effect of immune checkpoint inhibitors is uncertain. Earlier evidence suggested a neutral effect on survival (99,149,150); however, a study from Faje et al. questioned this, showing reduced survival among patients with melanoma treated with high-doses glucocorticoids for Ipilimumab-induced hypophysitis (100,126). Nonetheless, treatment should not be delayed in patients with severe symptoms of sella compression.

The resolution of the neuroradiological abnormalities is usually observed within 2 months (128,130). Treatment with high-dose glucocorticoids, however, does not restore ACTH deficiency and most patients will require long-term replacement (Table 10) (5,123). On the other hand, thyroid and gonadal deficiencies often recover and the need for hormone replacement needs to be reassessed in the long term (123,124,143,151,152). In addition, patients developing irAEs can be severely ill and can present with a “euthyroid sick syndrome” and/or a “sick eugonadal syndrome” that can affect the interpretation of the laboratory results (130).

Figure 6. Diagnosis and Management of Immune Checkpoint Inhibitor-Induced Hypophysitis. 1 Some authors suggest laboratory evaluation before the first infusion, then at 8 weeks for patients receiving Ipilimumab (i.e., prior to cycle 3) and then at week 16 if there are no interim signs/symptoms suggestive of hypophysitis. Other authors recommend laboratory evaluation for hypophysitis prior to each infusion of immune checkpoint inhibitors in the first 12-16 weeks of treatment, in order to pick up early or late onset of the disease. 2 Check random ACTH and cortisol if acute adrenal insufficiency is suspected. Exclude recent glucocorticoid use and concomitant treatment that may alter serum cortisol measurement (e.g., oral estrogens). As a guide, in patients that are unwell serum cortisol >450 nmol/L makes the diagnosis of adrenal insufficiency unlikely. Adrenal insufficiency is possible if morning cortisol 200-450 nmol/L or random cortisol 100-450 nmol/L; consider confirmatory testing with Synacthen, although this can give false-positive results in the early stages of central adrenal insufficiency. Adrenal insufficiency is likely if morning cortisol <200 nmol/L or random cortisol <100 nmol/L and patients should be started on hormone replacement. These cut-offs should be seen only as a guide and need to be adapted to local laboratory assays and reference ranges. Patients receiving immune checkpoint inhibitors can also develop adrenalitis and primary adrenal insufficiency. These patients have high ACTH and renin/aldosterone should be measured to investigate mineralocorticoid deficiency. 3 IGF-1 is valuable to confirm changes from baseline that may suggest new-onset hypophysitis. However, further tests to prove GH deficiency are not required because these patients would not be treated (active malignancy). 4 Pituitary MRI is normal in ~20% and ~80% of hypophysitis cases associated with anti-CTLA-4 and anti-PD1/PD1-L1 antibodies, respectively. Therefore, normal imaging does not exclude hypophysitis. MRI changes can be very subtle (Table 11). 5 We believe that patients with immune checkpoint inhibitor-induced hypophysitis should not stop treatment unless they develop severe and progressive symptoms (G3-G4 hypophysitis). Once the acute symptoms of hypophysitis have resolved, restarting treatment with immune checkpoint inhibitors is not contraindicated. Adequately treated, long-term hypopituitarism is not a contraindication to restarting immune checkpoint inhibitors.

An important differential diagnosis in patients with suspected drug-induced hypophysitis and a sella mass are pituitary metastases (Table 12) (95,141,153-155). The early studies on immune checkpoint inhibitors mainly assessed their efficacy in patients with advanced melanoma. Pituitary metastases are rare in melanoma (~2.5% of pituitary metastasis cases reported in the literature); however, these drugs are increasingly used for other malignancies including lung cancer, which accounts for ~25% of pituitary metastases (153). Central diabetes insipidus in immune checkpoint inhibitor-induced hypophysitis is extremely rare; therefore, a sella mass associated with diabetes insipidus is strongly suggestive of a metastasis.

Table 12. Differential Diagnosis of Immune Checkpoint Inhibitor-Induced Hypophysitis and Pituitary Metastases
Characteristics	Immune checkpoint inhibitor-induced hypophysitis	Pituitary Metastases
Clinical presentation	· Central diabetes insipidus is extremely rare; · Anterior pituitary insufficiency is very common (chiefly ACTH, FSH/LH and TSH deficiency). · Headache is a frequent presenting symptom.	· Central diabetes insipidus is the most common hormonal abnormality (~45%); · Cranial nerve deficits due to involvement of the chiasm and the cavernous sinus are common (22-28%); · Anterior pituitary insufficiency has been described in ~24% of patients; · Headache and retro-orbital pain have been reported in ~16% of patients;
Imaging	MRI: · Mild-to-moderate diffuse enlargement of the pituitary (up to 60-100% of the baseline size). Pituitary height typically does not exceed 2 cm. Pituitary enlargement resolves in most cases over the course of weeks/months. Empty sella can develop in the long term. · Extension into the cavernous sinus or above the sellar diaphragm is uncommon. · Homogeneous (more frequent) or heterogeneous enhancement (less frequent) post-gadolinium; · Suprasellar extension with compression and displacement of chiasm is uncommon; · The pituitary stalk may be thickened but not deviated; · The posterior pituitary is preserved in most of cases.	MRI: · Sella or suprasellar mass; · Isointense or hypointense mass on T1WI, with a usually high-intensity sign on T2WI; · Homogeneous enhancement post-gadolinium, although hemorrhage, necrosis and areas of cystic degeneration can be observed; · Thickened and enhancing pituitary stalk is possible, but it is typically less common than immune checkpoint inhibitor-induced hypophysitis; · Presence of other brain metastases (~15%); · Invasion of the cavernous sinus, chiasm, or hypothalamus (~14%) · Loss of bright spot of the neurohypophysis (~13%); · Dumbbell-shaped mass (~11%); · Sphenoid sinus invasion (~9%); CT: may show bony destruction.

Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging; T1WI, T1 weighted images; T2WI, T2 weighted images.

DRUG-INDUCED HYPOPHYSITIS: OTHER DRUGS

Reversible or irreversible hypopituitarism may be a rare side effect following treatment with interferon-α, and interferon-α/ribavirin combination therapy has been associated with cases of granulomatous hypophysitis with anterior pituitary dysfunction (156-160). The anti-interleukin-12 and -23 monoclonal antibody ustekinumab (used in the treatment of psoriasis) has been associated with a case of hypophysitis with panhypopituitarism (161).

HYPOPHYSITIS SECONDARY TO SELLA AND PARASELLAR DISEASE

Pituitary inflammation can be triggered by sella and parasellar disease. The infiltrate is mainly lymphocytic or xanthogranulomatous and focuses around the lesion rather than diffusing to the entire gland (4).

Germinoma

Germinomas are rare brain tumors predominantly affecting prepubertal children. They are highly immunogenic tumors and can induce a strong immune response that can involve the pituitary leading to secondary hypophysitis (162-169). Histologically, lymphocytic or granulomatous hypophysitis is seen in ~80% and ~20% of cases linked to germinomas, respectively (169).

Germinomas arising in the sella and parasellar region are difficult to differentiate from hypophysitis in children because of similar clinical features (diabetes insipidus + GH deficiency + visual disturbances). This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. Biopsy-proven cases of primary hypophysitis are extremely rare in children and adolescents (41); therefore, in children below 10 years a germinoma should be considered the most likely diagnosis.

Tumor markers such as α-fetoprotein, β-human chorionic gonadotropin, or placental alkaline phosphatase in the cerebrospinal fluid may be useful for diagnosing germinoma. However, a pituitary biopsy is the gold standard for differentiating the two conditions, although germinomas can have a marked lymphocytic infiltrate that can outnumber the neoplastic cells making differential diagnosis difficult (168). If germinoma is part of the histologic differential diagnosis, markers for germinomas such as Oct3/4, PLAP and NANOG may be useful.

Finally, it should be noted that the hypopituitarism caused by sella germinomas can precede for years a visible pituitary mass, so that prolonged symptomatic periods prior to diagnosis are common (168).

Rathke’s Cleft Cyst

The rupture of Rathke’s cleft cyst can cause hypophysitis associated with visual disturbances, headache and hypopituitarism including – very frequently – central diabetes insipidus (170-175). Histopathology can show lymphocytic, granulomatous, xanthomatous or mixed forms of hypophysitis (174). Some authors have suggested that many cases of xanthomatous hypophysitis may actually be related to rupture of Rathke’s cleft cysts (12,13).

Other Sella and Parasellar Masses

Cases of secondary hypophysitis have been described in association with craniopharyngiomas (176), pituitary adenomas (177-182) and primary pituitary lymphomas (177,183).

HYPOPHYSITIS SECONDARY TO SYSTEMIC DISEASE

Sarcoidosis

Sarcoidosis is a multisystem inflammatory disease of unknown origin characterized by the formation of non-caseating granulomas that can involve all organ systems. The central nervous system can be affected in 5-15% of patients (neurosarcoidosis) and may be the presenting feature of the disease (184). Granulomas can involve the pituitary, hypothalamus and the stalk in ~0.5% of patients with sarcoidosis, resulting in varying grade of hypopituitarism (185,186). ~60% of the cases reported in the literature are males presenting in the 3^rd and 4^th decade. Central diabetes insipidus, FSH/LH deficiency and hyperprolactinemia are among the most frequent hormone abnormalities (187). Patients with sarcoidosis and hypothalamic-pituitary involvement tend to have more frequent multi-organ involvement, as well as neurosarcoidosis and sinonasal involvement (187).

Granulomatosis with Polyangiitis

Granulomatosis with polyangiitis (previously known as Wegener’s Granulomatosis) is an antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis of unknown etiology with multisystem involvement and formation of necrotizing granulomas and vasculitis in small- and medium-sized blood vessels. Pituitary involvement is a rare and usually late manifestation of the disease (186,188,189), but it can also be the presenting complaint (190,191). Secondary hypogonadism and central diabetes insipidus are the most common endocrine abnormalities; diabetes insipidus can recover after adequate treatment of the underlying vasculitis, while anterior pituitary dysfunction is permanent in the majority of patients (192).

Langerhans Cell Histiocytosis

Langerhans cell histiocytosis is a rare disease mainly occurring in childhood, involving clonal proliferation of myeloid Langerhans cells that can infiltrate multiple organs (bones, skin, lymph nodes, lungs, thymus, liver, spleen, bone marrow, and central nervous system including the pituitary). Patients often carry the BRAF V600E mutation in the clonal myeloid cells (193).

The most common endocrine abnormality in patients with Langerhans cell histiocytosis is hypothalamic-pituitary infiltration causing central diabetes insipidus. These patients usually have multi-organ and cranio-facial involvement, although localized disease of the hypothalamic-pituitary region has been reported (194,195). Up to 40% of patients develop symptoms consistent with diabetes insipidus within the first four years, particularly if there is multisystem involvement and proptosis (196-198). Anterior pituitary hormone deficiency is also possible at diagnosis and during follow up (194,199).

Langerhans cell histiocytosis and germinoma are the most common cause of central diabetes insipidus in children and adolescents; therefore, germinoma should always been considered in the differential diagnosis (200).

The definitive diagnosis of Langerhans cell histiocytosis is the biopsy-proven infiltration of the pituitary with Langerhans cells with eosinophils, neutrophils, small lymphocytes, and histiocytes. However, pituitary biopsy is invasive and the diagnosis can be suggested by the presence of the characteristic histopathologic features in other tissues when a multisystem disease is present. For patients with suspected disease isolated to the pituitary, identification of BRAF-V600E in the peripheral blood or cerebrospinal fluid can support the diagnosis and rule out germinoma, although it does not distinguish Langerhans cell histiocytosis from Erdheim-Chester disease (see below) (201).

When hypophysitis secondary to Langerhans cell histiocytosis is suspected but pituitary biopsy is not available, it is reasonable to initiate therapy empirically with a plan to follow disease response with MRI. Treatment options include prednisone, alone or in combination with vinblastine, cladribine and vemurafenib, alongside desmopressin and other pituitary hormone replacements to treat hypopituitarism.

Erdheim-Chester Disease

Erdheim-Chester’s disease is a rare multisystem histiocytic disorder, most often seen in adults, which may be confused with Langerhans cell histiocytosis. Histiocytic infiltration leads to xanthogranulomatous infiltrates of multiple tissues (bones, skin, lungs, facial, orbital and retro-orbital tissue, retroperitoneum, cardiovascular system and cerebral nervous system including the pituitary). Long bone pain and symmetric osteosclerotic lesions suggest this diagnosis, which is confirmed by tissue biopsies showing histiocytes with non-Langerhans features. Patients often carry the BRAF V600E mutation in the clonal myeloid progenitor cells (193).

Pituitary involvement may manifest as central diabetes insipidus and anterior hypopituitarism, which typically persist even with radiographic regression of the disease. As for Langerhans cell histiocytosis, the definitive diagnosis of Erdheim-Chester’s disease is the finding of the typical histologic features at pituitary biopsy, which can be supported by the finding of the BRAF V600E mutation. Treatment options include vemurafenib, interferon-α, dabrafenib, trametinib, cobimetinib, cladribine, cyclophosphamide and glucocorticoids.

Rosai-Dorfman Disease

Pituitary involvement has been described in Rosai-Dorfman disease, a rare histiocytic disorder. Patients may have both anterior pituitary dysfunction, central diabetes insipidus and visual disturbances (202,203).

Inflammatory Pseudotumor

The inflammatory pseudotumor is a rare inflammatory disorder commonly involving the lung and orbit. It can be isolated or associated with the IgG4-related disease (204). Pituitary infiltration is a rare manifestation and patients can present with anterior and posterior hypopituitarism. The inflammatory pseudotumor can also spread to the sphenoid sinus, the cavernous sinus and the optic chiasm (205-207).

Tolosa-Hunt Syndrome

Tolosa-Hunt syndrome is a painful ophthalmoplegia caused by idiopathic retro-orbital inflammation involving the cavernous sinus or the superior orbital fissure. Histology shows nonspecific granulomatous or nongranulomatous inflammation. Patients with pituitary involvement present with anterior and posterior hypopituitarism, diplopia and retro-orbital pain (often unilateral) (208-212).

Other Systemic Diseases

Cases of secondary hypophysitis have been described in association with Takayasu’s arteritis (granulomatous hypophysitis) (213), Cogan’s syndrome (214) and Crohn’s disease (215,216). A case of isolated ACTH deficiency in a patient with Crohn’s disease has also been published (217).

OTHER CAUSES OF SECONDARY HYPOPHYSITIS

Thymoma and Other Malignancies (Anti-Pit-1 Antibody Syndrome)

Pit-1 is essential for the differentiation, proliferation, and maintenance of somatotrophs, lactotrophs, and thyrotrophs in the pituitary (218). Yamamoto et al. described three cases of acquired combined TSH, GH, and PRL deficiency, with circulating anti-Pit-1 antibodies (219). Cytotoxic T-cells that react against Pit-1 are likely the cause of anti-Pit-1 antibody syndrome (220-222). All these patients later developed thymomas that express Pit-1. Removal of the thymoma resulted in a decline in antibody titer, suggesting that aberrant expression of Pit-1 in the thymoma plays a causal role in the development of this syndrome (223). A handful of cases of anti-Pit-1 antibody syndrome not associated with thymoma have since been published. The malignancies causing this paraneoplastic syndrome included diffuse large B-cell lymphoma of the bladder and a metastatic cancer of unknown origin (222,224). Based on these cases, Yamamoto et al. have proposed diagnostic criteria for anti-PIT-1 hypophysitis (Table 13).

Table 13. Diagnostic Criteria for Anti-PIT-1 Hypophysitis
Criteria		Probable diagnosis	Established diagnosis
Criterion 1	Acquired specific GH, PRL, and TSH deficiency. *	CRITERION 1	CRITERION 1 and CRITERION 2
Criterion 2	Presence of anti-PIT-1 antibody or PIT-1-reactive T cells in the circulation.
Criterion 3	Coexistence of thymoma or malignant neoplasm. **

* The secretion of other pituitary hormones is not impaired. The MRI of the pituitary is typically normal, but a slight atrophy of the anterior pituitary can be observed.

** Criterion 3 may help the diagnosis and clarify pathogenesis but may not be necessarily obvious at the time of diagnosis.

Infections

Infections of the pituitary are a rare cause of hypophysitis and hypopituitarism (225). They can affect either exclusively the pituitary area or as a part of disseminated infections. Risk factors are diabetes mellitus, organ transplantation, human immunodeficiency virus infection, non-Hodgkin lymphoma, chemotherapy, and Cushing’s syndrome. They can occur by (186):

Hematogenous spread in immuno-compromised hosts;
Contiguous extension from adjacent anatomical sites (meninges, sphenoid sinus, cavernous sinus and skull base);
Previous infectious diseases of the CNS of different etiologies;
Iatrogenic inoculation during trans-sphenoidal surgery.

However, in the majority of cases of pituitary abscess an obvious cause cannot be identified.

Tuberculosis can cause granulomatous involvement of the hypothalamus, the pituitary or the stalk. Tubercular meningitis and hypothalamic-pituitary involvement seem to affect mostly anterior pituitary function (226).

Several viruses can cause meningitis, meningoencephalitis and encephalitis that can involve the hypothalamic-pituitary region. Partial or complete hypopituitarism may develop as a result (186). A study by Leow et al. has shown that ~40% of patients with severe acute respiratory syndrome (SARS)-associated with Coronavirus infection can develop reversible central adrenal insufficiency, suggesting a possible inflammation of the pituitary in these patients (227). Hantavirus can also cause viral hypophysitis with pituitary ischemia and hemorrhage as part of the hemorrhagic fever with renal syndrome (HFRS), leading to partial or complete hypopituitarism, including diabetes insipidus (186,228,229).

Mycoses with hypothalamic-pituitary involvement are extremely rare. Patients frequently present with central diabetes insipidus and anterior pituitary dysfunction (mainly FSH/LH deficiency and hyperprolactinemia) (186).

REFERENCES

Bellastella G, Maiorino MI, Bizzarro A, et al. Revisitation of autoimmune hypophysitis: knowledge and uncertainties on pathophysiological and clinical aspects. Pituitary. 2016;19(6):625-642.
Faje A. Hypophysitis: Evaluation and Management. Clin Diabetes Endocrinol. 2016;2:15.
Carmichael JD. Update on the diagnosis and management of hypophysitis. Curr Opin Endocrinol Diabetes Obes. 2012;19(4):314-321.
Caturegli P, Newschaffer C, Olivi A, Pomper MG, Burger PC, Rose NR. Autoimmune hypophysitis. Endocr Rev. 2005;26(5):599-614.
Di Dalmazi G, Ippolito S, Lupi I, Caturegli P. Hypophysitis induced by immune checkpoint inhibitors: a 10-year assessment. Expert Rev Endocrinol Metab. 2019;14(6):381-398.
Buxton N, Robertson I. Lymphocytic and granulocytic hypophysitis: a single centre experience. Br J Neurosurg. 2001;15(3):242-245, discussion 245-246.
Guaraldi F, Giordano R, Grottoli S, Ghizzoni L, Arvat E, Ghigo E. Pituitary Autoimmunity. Front Horm Res. 2017;48:48-68.
Caturegli P, Di Dalmazi G, Lombardi M, et al. Hypophysitis Secondary to Cytotoxic T-Lymphocyte-Associated Protein 4 Blockade: Insights into Pathogenesis from an Autopsy Series. Am J Pathol. 2016;186(12):3225-3235.
Larkin S, Ansorge O. Pathology And Pathogenesis Of Pituitary Adenomas And Other Sellar Lesions. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA) 2000.
Chalan P, Thomas N, Caturegli P. Th17 Cells Contribute to the Pathology of Autoimmune Hypophysitis. J Immunol. 2021;206(11):2536-2543.
Hunn BH, Martin WG, Simpson S, Jr., McLean CA. Idiopathic granulomatous hypophysitis: a systematic review of 82 cases in the literature. Pituitary. 2014;17(4):357-365.
Kleinschmidt-DeMasters BK, Lillehei KO, Hankinson TC. Review of xanthomatous lesions of the sella. Brain Pathol. 2017;27(3):377-395.
Duan K, Asa SL, Winer D, Gelareh Z, Gentili F, Mete O. Xanthomatous Hypophysitis Is Associated with Ruptured Rathke's Cleft Cyst. Endocr Pathol. 2017;28(1):83-90.
Amirbaigloo A, Esfahanian F, Mouodi M, Rakhshani N, Zeinalizadeh M. IgG4-related hypophysitis. Endocrine. 2021;73(2):270-291.
Takahashi Y. MECHANISMS IN ENDOCRINOLOGY: Autoimmune hypopituitarism: novel mechanistic insights. Eur J Endocrinol. 2020;182(4):R59-R66.
Kuruma S, Kamisawa T, Tabata T, et al. Allergen-specific IgE antibody serologic assays in patients with autoimmune pancreatitis. Intern Med. 2014;53(6):541-543.
Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med. 2012;366(6):539-551.
Weindorf SC, Frederiksen JK. IgG4-Related Disease: A Reminder for Practicing Pathologists. Arch Pathol Lab Med. 2017;141(11):1476-1483.
Khosroshahi A, Wallace ZS, Crowe JL, et al. International Consensus Guidance Statement on the Management and Treatment of IgG4-Related Disease. Arthritis Rheumatol. 2015;67(7):1688-1699.
Lin W, Lu S, Chen H, et al. Clinical characteristics of immunoglobulin G4-related disease: a prospective study of 118 Chinese patients. Rheumatology (Oxford). 2015;54(11):1982-1990.
Kanie K, Bando H, Iguchi G, et al. IgG4-related hypophysitis in patients with autoimmune pancreatitis. Pituitary. 2019;22(1):54-61.
Bando H, Iguchi G, Fukuoka H, et al. The prevalence of IgG4-related hypophysitis in 170 consecutive patients with hypopituitarism and/or central diabetes insipidus and review of the literature. Eur J Endocrinol. 2014;170(2):161-172.
Bernreuther C, Illies C, Flitsch J, et al. IgG4-related hypophysitis is highly prevalent among cases of histologically confirmed hypophysitis. Brain Pathol. 2017;27(6):839-845.
Uccella S, Amaglio C, Brouland JP, et al. Disease heterogeneity in IgG4-related hypophysitis: report of two histopathologically proven cases and review of the literature. Virchows Arch. 2019;475(3):373-381.
Li Y, Gao H, Li Z, Zhang X, Ding Y, Li F. Clinical Characteristics of 76 Patients with IgG4-Related Hypophysitis: A Systematic Literature Review. Int J Endocrinol. 2019;2019:5382640.
Leporati P, Landek-Salgado MA, Lupi I, Chiovato L, Caturegli P. IgG4-related hypophysitis: a new addition to the hypophysitis spectrum. J Clin Endocrinol Metab. 2011;96(7):1971-1980.
Ohkubo Y, Sekido T, Takeshige K, et al. Occurrence of IgG4-related hypophysitis lacking IgG4-bearing plasma cell infiltration during steroid therapy. Intern Med. 2014;53(7):753-757.
Gutenberg A, Caturegli P, Metz I, et al. Necrotizing infundibulo-hypophysitis: an entity too rare to be true? Pituitary. 2012;15(2):202-208.
Cacic M, Marinkovic J, Kruljac I, et al. Ischemic Pituitary Apoplexy, Hypopituitarism and Diabetes Insipidus: A Triad Unique to Necrotizing Hypophysitis. Acta Clin Croat. 2018;57(4):768-771.
Magalhaes-Ribeiro C, Furtado A, Baggen Santos R, et al. Necrotizing infundibulo-hypophysitis: case-report and literature review. Br J Neurosurg. 2021:1-4.
Ahmed SR, Aiello DP, Page R, Hopper K, Towfighi J, Santen RJ. Necrotizing infundibulo-hypophysitis: a unique syndrome of diabetes insipidus and hypopituitarism. J Clin Endocrinol Metab. 1993;76(6):1499-1504.
Honegger J, Schlaffer S, Menzel C, et al. Diagnosis of Primary Hypophysitis in Germany. J Clin Endocrinol Metab. 2015;100(10):3841-3849.
Jain A, Dhanwal DK. A rare case of autoimmune hypophysitis presenting as temperature dysregulation. J Clin Diagn Res. 2015;9(2):OD09-10.
Chiloiro S, Tartaglione T, Angelini F, et al. An Overview of Diagnosis of Primary Autoimmune Hypophysitis in a Prospective Single-Center Experience. Neuroendocrinology. 2017;104(3):280-290.
Gutenberg A, Hans V, Puchner MJ, et al. Primary hypophysitis: clinical-pathological correlations. Eur J Endocrinol. 2006;155(1):101-107.
Gendreitzig P, Honegger J, Quinkler M. Granulomatous hypophysitis causing compression of the internal carotid arteries reversible with azathioprine and rituximab treatment. Pituitary. 2020;23(2):103-112.
Shikuma J, Kan K, Ito R, et al. Critical review of IgG4-related hypophysitis. Pituitary. 2017;20(2):282-291.
Kanoke A, Ogawa Y, Watanabe M, Kumabe T, Tominaga T. Autoimmune hypophysitis presenting with intracranial multi-organ involvement: three case reports and review of the literature. BMC Res Notes. 2013;6:560.
Hadjigeorgiou GF, Lund EL, Poulsgaard L, et al. Intrachiasmatic abscess caused by IgG4-related hypophysitis. Acta Neurochir (Wien). 2017;159(11):2229-2233.
Murray PG, Clayton PE. Disorders of Growth Hormone in Childhood. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Gellner V, Kurschel S, Scarpatetti M, Mokry M. Lymphocytic hypophysitis in the pediatric population. Childs Nerv Syst. 2008;24(7):785-792.
Kalra AA, Riel-Romero RM, Gonzalez-Toledo E. Lymphocytic hypophysitis in children: a novel presentation and literature review. J Child Neurol. 2011;26(1):87-94.
Gan HW, Bulwer C, Spoudeas H. Pituitary and Hypothalamic Tumor Syndromes in Childhood. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Di Iorgi N, Morana G, Maghnie M. Pituitary stalk thickening on MRI: when is the best time to re-scan and how long should we continue re-scanning for? Clin Endocrinol (Oxf). 2015;83(4):449-455.
Allix I, Rohmer V. Hypophysitis in 2014. Ann Endocrinol (Paris). 2015;76(5):585-594.
Evanson J. Radiology of the Pituitary. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Gutenberg A, Larsen J, Lupi I, Rohde V, Caturegli P. A radiologic score to distinguish autoimmune hypophysitis from nonsecreting pituitary adenoma preoperatively. AJNR Am J Neuroradiol. 2009;30(9):1766-1772.
Gonzalez JG, Elizondo G, Saldivar D, Nanez H, Todd LE, Villarreal JZ. Pituitary gland growth during normal pregnancy: an in vivo study using magnetic resonance imaging. Am J Med. 1988;85(2):217-220.
Molitch ME. Pituitary and Adrenal Disorders of Pregnancy. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA) 2000.
Biswas SN, Barman H, Sarkar TS, Chakraborty PP. Physiological pituitary hyperplasia misinterpreted and treated as lymphocytic hypophysitis. BMJ Case Rep. 2018;2018.
Catford S, Wang YY, Wong R. Pituitary stalk lesions: systematic review and clinical guidance. Clin Endocrinol (Oxf). 2016;85(4):507-521.
Sotoudeh H, Yazdi HR. A review on dural tail sign. World J Radiol. 2010;2(5):188-192.
O'Dwyer DT, Smith AI, Matthew ML, et al. Identification of the 49-kDa autoantigen associated with lymphocytic hypophysitis as alpha-enolase. J Clin Endocrinol Metab. 2002;87(2):752-757.
Caturegli P, Lupi I, Landek-Salgado M, Kimura H, Rose NR. Pituitary autoimmunity: 30 years later. Autoimmun Rev. 2008;7(8):631-637.
Crock PA. Cytosolic autoantigens in lymphocytic hypophysitis. J Clin Endocrinol Metab. 1998;83(2):609-618.
Takao T, Nanamiya W, Matsumoto R, Asaba K, Okabayashi T, Hashimoto K. Antipituitary antibodies in patients with lymphocytic hypophysitis. Horm Res. 2001;55(6):288-292.
Tanaka S, Tatsumi KI, Kimura M, et al. Detection of autoantibodies against the pituitary-specific proteins in patients with lymphocytic hypophysitis. Eur J Endocrinol. 2002;147(6):767-775.
Scherbaum WA, Schrell U, Gluck M, Fahlbusch R, Pfeiffer EF. Autoantibodies to pituitary corticotropin-producing cells: possible marker for unfavourable outcome after pituitary microsurgery for Cushing's disease. Lancet. 1987;1(8547):1394-1398.
Bensing S, Hulting AL, Hoog A, Ericson K, Kampe O. Lymphocytic hypophysitis: report of two biopsy-proven cases and one suspected case with pituitary autoantibodies. J Endocrinol Invest. 2007;30(2):153-162.
Lupi I, Broman KW, Tzou SC, Gutenberg A, Martino E, Caturegli P. Novel autoantigens in autoimmune hypophysitis. Clin Endocrinol (Oxf). 2008;69(2):269-278.
Smith CJ, Bensing S, Burns C, et al. Identification of TPIT and other novel autoantigens in lymphocytic hypophysitis: immunoscreening of a pituitary cDNA library and development of immunoprecipitation assays. Eur J Endocrinol. 2012;166(3):391-398.
Ricciuti A, De Remigis A, Landek-Salgado MA, et al. Detection of pituitary antibodies by immunofluorescence: approach and results in patients with pituitary diseases. J Clin Endocrinol Metab. 2014;99(5):1758-1766.
Guaraldi F, Caturegli P, Salvatori R. Prevalence of antipituitary antibodies in acromegaly. Pituitary. 2012;15(4):490-494.
Chiloiro S, Giampietro A, Angelini F, et al. Markers of humoral and cell-mediated immune response in primary autoimmune hypophysitis: a pilot study. Endocrine. 2021;73(2):308-315.
Patti G, Calandra E, De Bellis A, et al. Antibodies Against Hypothalamus and Pituitary Gland in Childhood-Onset Brain Tumors and Pituitary Dysfunction. Front Endocrinol (Lausanne). 2020;11:16.
De Bellis A, Sinisi AA, Pane E, et al. Involvement of hypothalamus autoimmunity in patients with autoimmune hypopituitarism: role of antibodies to hypothalamic cells. J Clin Endocrinol Metab. 2012;97(10):3684-3690.
De Bellis A, Colao A, Di Salle F, et al. A longitudinal study of vasopressin cell antibodies, posterior pituitary function, and magnetic resonance imaging evaluations in subclinical autoimmune central diabetes insipidus. J Clin Endocrinol Metab. 1999;84(9):3047-3051.
De Bellis A, Colao A, Bizzarro A, et al. Longitudinal study of vasopressin-cell antibodies and of hypothalamic-pituitary region on magnetic resonance imaging in patients with autoimmune and idiopathic complete central diabetes insipidus. J Clin Endocrinol Metab. 2002;87(8):3825-3829.
Bellastella G, Bizzarro A, Aitella E, et al. Pregnancy may favour the development of severe autoimmune central diabetes insipidus in women with vasopressin cell antibodies: description of two cases. Eur J Endocrinol. 2015;172(3):K11-17.
Iwama S, Sugimura Y, Kiyota A, et al. Rabphilin-3A as a Targeted Autoantigen in Lymphocytic Infundibulo-neurohypophysitis. J Clin Endocrinol Metab. 2015;100(7):E946-954.
Lupi I, Manetti L, Raffaelli V, et al. Pituitary autoimmunity is associated with hypopituitarism in patients with primary empty sella. J Endocrinol Invest. 2011;34(8):e240-244.
Landek-Salgado MA, Leporati P, Lupi I, Geis A, Caturegli P. Growth hormone and proopiomelanocortin are targeted by autoantibodies in a patient with biopsy-proven IgG4-related hypophysitis. Pituitary. 2012;15(3):412-419.
Honegger J, Buchfelder M, Schlaffer S, et al. Treatment of Primary Hypophysitis in Germany. J Clin Endocrinol Metab. 2015;100(9):3460-3469.
Khare S, Jagtap VS, Budyal SR, et al. Primary (autoimmune) hypophysitis: a single centre experience. Pituitary. 2015;18(1):16-22.
Park SM, Bae JC, Joung JY, et al. Clinical characteristics, management, and outcome of 22 cases of primary hypophysitis. Endocrinol Metab (Seoul). 2014;29(4):470-478.
Lupi I, Zhang J, Gutenberg A, et al. From pituitary expansion to empty sella: disease progression in a mouse model of autoimmune hypophysitis. Endocrinology. 2011;152(11):4190-4198.
Nicolaides NC, Chrousos GP, Charmandari E. Adrenal Insufficiency. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Weiss RE. Hypopituitarism: Emergencies. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Huguet I, Clayton R. Pituitary-Hypothalamic Tumor Syndromes: Adults. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Joshi H, Hikmat M, Devadass AP, Oyibo SO, Sagi SV. Anterior hypopituitarism secondary to biopsy-proven IgG4-related hypophysitis in a young man. Endocrinol Diabetes Metab Case Rep. 2019;2019.
Waytz J, Jan R. IgG-4-Related Hypophysitis Treated With Rituximab: Case Report. J Clin Rheumatol. 2020.
Boharoon H, Tomlinson J, Limback-Stanic C, et al. A Case Series of Patients with Isolated IgG4-related Hypophysitis Treated with Rituximab. J Endocr Soc. 2020;4(6):bvaa048.
Caputo C, Bazargan A, McKelvie PA, Sutherland T, Su CS, Inder WJ. Hypophysitis due to IgG4-related disease responding to treatment with azathioprine: an alternative to corticosteroid therapy. Pituitary. 2014;17(3):251-256.
Lupi I, Manetti L, Raffaelli V, et al. Diagnosis and treatment of autoimmune hypophysitis: a short review. J Endocrinol Invest. 2011;34(8):e245-252.
Chiloiro S, Tartaglione T, Capoluongo ED, et al. Hypophysitis Outcome and Factors Predicting Responsiveness to Glucocorticoid Therapy: A Prospective and Double-Arm Study. J Clin Endocrinol Metab. 2018;103(10):3877-3889.
Lupi I, Cosottini M, Caturegli P, et al. Diabetes insipidus is an unfavorable prognostic factor for response to glucocorticoids in patients with autoimmune hypophysitis. Eur J Endocrinol. 2017;177(2):127-135.
Schreckinger M, Francis T, Rajah G, Jagannathan J, Guthikonda M, Mittal S. Novel strategy to treat a case of recurrent lymphocytic hypophysitis using rituximab. J Neurosurg. 2012;116(6):1318-1323.
Vauchot F, Bourdon A, Hay B, Mariano-Goulart D, Ben Bouallegue F. Therapeutic Response to Rituximab in IgG4-Related Hypophysitis Evidenced on 18F-FDG PET and MRI. Clin Nucl Med. 2019;44(5):e362-e363.
Bullock DR, Miller BS, Clark HB, Hobday PM. Rituximab treatment for isolated IgG4-related hypophysitis in a teenage female. Endocrinol Diabetes Metab Case Rep. 2018;2018.
Kyriacou A, Gnanalingham K, Kearney T. Lymphocytic hypophysitis: modern day management with limited role for surgery. Pituitary. 2017;20(2):241-250.
Zhu Q, Qian K, Jia G, et al. Clinical Features, Magnetic Resonance Imaging, and Treatment Experience of 20 Patients with Lymphocytic Hypophysitis in a Single Center. World Neurosurg. 2019;127:e22-e29.
Ray DK, Yen CP, Vance ML, Laws ER, Lopes B, Sheehan JP. Gamma knife surgery for lymphocytic hypophysitis. J Neurosurg. 2010;112(1):118-121.
Selch MT, DeSalles AA, Kelly DF, et al. Stereotactic radiotherapy for the treatment of lymphocytic hypophysitis. Report of two cases. J Neurosurg. 2003;99(3):591-596.
Torino F, Barnabei A, Paragliola RM, Marchetti P, Salvatori R, Corsello SM. Endocrine side-effects of anti-cancer drugs: mAbs and pituitary dysfunction: clinical evidence and pathogenic hypotheses. Eur J Endocrinol. 2013;169(6):R153-164.
Corsello SM, Barnabei A, Marchetti P, De Vecchis L, Salvatori R, Torino F. Endocrine side effects induced by immune checkpoint inhibitors. J Clin Endocrinol Metab. 2013;98(4):1361-1375.
Ouyang T, Cao Y, Kan X, et al. Treatment-Related Serious Adverse Events of Immune Checkpoint Inhibitors in Clinical Trials: A Systematic Review. Front Oncol. 2021;11:621639.
Hiniker SM, Reddy SA, Maecker HT, et al. A Prospective Clinical Trial Combining Radiation Therapy With Systemic Immunotherapy in Metastatic Melanoma. Int J Radiat Oncol Biol Phys. 2016;96(3):578-588.
Faje AT, Sullivan R, Lawrence D, et al. Ipilimumab-induced hypophysitis: a detailed longitudinal analysis in a large cohort of patients with metastatic melanoma. J Clin Endocrinol Metab. 2014;99(11):4078-4085.
Downey SG, Klapper JA, Smith FO, et al. Prognostic factors related to clinical response in patients with metastatic melanoma treated by CTL-associated antigen-4 blockade. Clin Cancer Res. 2007;13(22 Pt 1):6681-6688.
Faje AT, Lawrence D, Flaherty K, et al. High-dose glucocorticoids for the treatment of ipilimumab-induced hypophysitis is associated with reduced survival in patients with melanoma. Cancer. 2018;124(18):3706-3714.
Brahmer JR, Lacchetti C, Thompson JA. Management of Immune-Related Adverse Events in Patients Treated With Immune Checkpoint Inhibitor Therapy: American Society of Clinical Oncology Clinical Practice Guideline Summary. J Oncol Pract. 2018;14(4):247-249.
Konda B, Nabhan F, Shah MH. Endocrine dysfunction following immune checkpoint inhibitor therapy. Curr Opin Endocrinol Diabetes Obes. 2017;24(5):337-347.
Gonzalez-Rodriguez E, Rodriguez-Abreu D, Spanish Group for Cancer I-B. Immune Checkpoint Inhibitors: Review and Management of Endocrine Adverse Events. Oncologist. 2016;21(7):804-816.
Freeman-Keller M, Kim Y, Cronin H, Richards A, Gibney G, Weber JS. Nivolumab in Resected and Unresectable Metastatic Melanoma: Characteristics of Immune-Related Adverse Events and Association with Outcomes. Clin Cancer Res. 2016;22(4):886-894.
Hui R, Garon EB, Goldman JW, et al. Pembrolizumab as first-line therapy for patients with PD-L1-positive advanced non-small cell lung cancer: a phase 1 trial. Ann Oncol. 2017;28(4):874-881.
Long GV, Atkinson V, Cebon JS, et al. Standard-dose pembrolizumab in combination with reduced-dose ipilimumab for patients with advanced melanoma (KEYNOTE-029): an open-label, phase 1b trial. Lancet Oncol. 2017;18(9):1202-1210.
Postow MA, Chesney J, Pavlick AC, et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med. 2015;372(21):2006-2017.
Weber J, Mandala M, Del Vecchio M, et al. Adjuvant Nivolumab versus Ipilimumab in Resected Stage III or IV Melanoma. N Engl J Med. 2017;377(19):1824-1835.
Wolchok JD, Chiarion-Sileni V, Gonzalez R, et al. Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med. 2017;377(14):1345-1356.
Yamazaki N, Takenouchi T, Fujimoto M, et al. Phase 1b study of pembrolizumab (MK-3475; anti-PD-1 monoclonal antibody) in Japanese patients with advanced melanoma (KEYNOTE-041). Cancer Chemother Pharmacol. 2017;79(4):651-660.
Zhai Y, Ye X, Hu F, et al. Endocrine toxicity of immune checkpoint inhibitors: a real-world study leveraging US Food and Drug Administration adverse events reporting system. J Immunother Cancer. 2019;7(1):286.
Sato K, Mano T, Iwata A, Toda T. Neurological and related adverse events in immune checkpoint inhibitors: a pharmacovigilance study from the Japanese Adverse Drug Event Report database. J Neurooncol. 2019;145(1):1-9.
Barroso-Sousa R, Barry WT, Garrido-Castro AC, et al. Incidence of Endocrine Dysfunction Following the Use of Different Immune Checkpoint Inhibitor Regimens: A Systematic Review and Meta-analysis. JAMA Oncol. 2018;4(2):173-182.
Faje A, Reynolds K, Zubiri L, et al. Hypophysitis secondary to nivolumab and pembrolizumab is a clinical entity distinct from ipilimumab-associated hypophysitis. Eur J Endocrinol. 2019;181(3):211-219.
Iwama S, De Remigis A, Callahan MK, Slovin SF, Wolchok JD, Caturegli P. Pituitary expression of CTLA-4 mediates hypophysitis secondary to administration of CTLA-4 blocking antibody. Sci Transl Med. 2014;6(230):230ra245.
Mihic-Probst D, Reinehr M, Dettwiler S, et al. The role of macrophages type 2 and T-regs in immune checkpoint inhibitor related adverse events. Immunobiology. 2020;225(5):152009.
Kobayashi T, Iwama S, Sugiyama D, et al. Anti-pituitary antibodies and susceptible human leukocyte antigen alleles as predictive biomarkers for pituitary dysfunction induced by immune checkpoint inhibitors. J Immunother Cancer. 2021;9(5).
Yano S, Ashida K, Sakamoto R, et al. Human leucocyte antigen DR15, a possible predictive marker for immune checkpoint inhibitor-induced secondary adrenal insufficiency. Eur J Cancer. 2020;130:198-203.
Kanie K, Iguchi G, Bando H, et al. Mechanistic insights into immune checkpoint inhibitor-related hypophysitis: a form of paraneoplastic syndrome. Cancer Immunol Immunother. 2021.
Bellastella G, Carbone C, Scappaticcio L, et al. Hypothalamic-Pituitary Autoimmunity in Patients Treated with Anti-PD-1 and Anti-PD-L1 Antibodies. Cancers (Basel). 2021;13(16).
de Filette J, Andreescu CE, Cools F, Bravenboer B, Velkeniers B. A Systematic Review and Meta-Analysis of Endocrine-Related Adverse Events Associated with Immune Checkpoint Inhibitors. Horm Metab Res. 2019;51(3):145-156.
Yamazaki N, Kiyohara Y, Uhara H, et al. Phase II study of ipilimumab monotherapy in Japanese patients with advanced melanoma. Cancer Chemother Pharmacol. 2015;76(5):997-1004.
Scott ES, Long GV, Guminski A, Clifton-Bligh RJ, Menzies AM, Tsang VH. The spectrum, incidence, kinetics and management of endocrinopathies with immune checkpoint inhibitors for metastatic melanoma. Eur J Endocrinol. 2018;178(2):175-182.
Albarel F, Gaudy C, Castinetti F, et al. Long-term follow-up of ipilimumab-induced hypophysitis, a common adverse event of the anti-CTLA-4 antibody in melanoma. Eur J Endocrinol. 2015;172(2):195-204.
Maker AV, Yang JC, Sherry RM, et al. Intrapatient dose escalation of anti-CTLA-4 antibody in patients with metastatic melanoma. J Immunother. 2006;29(4):455-463.
Ascierto PA, Del Vecchio M, Robert C, et al. Ipilimumab 10 mg/kg versus ipilimumab 3 mg/kg in patients with unresectable or metastatic melanoma: a randomised, double-blind, multicentre, phase 3 trial. Lancet Oncol. 2017;18(5):611-622.
Snyders T, Chakos D, Swami U, et al. Ipilimumab-induced hypophysitis, a single academic center experience. Pituitary. 2019;22(5):488-496.
Faje A. Immunotherapy and hypophysitis: clinical presentation, treatment, and biologic insights. Pituitary. 2016;19(1):82-92.
Zhao C, Tella SH, Del Rivero J, et al. Anti-PD-L1 Treatment Induced Central Diabetes Insipidus. J Clin Endocrinol Metab. 2018;103(2):365-369.
Torino F, Corsello SM, Salvatori R. Endocrinological side-effects of immune checkpoint inhibitors. Curr Opin Oncol. 2016;28(4):278-287.
Rogiers A, Leys C, Lauwyck J, et al. Neurocognitive Function, Psychosocial Outcome, and Health-Related Quality of Life of the First-Generation Metastatic Melanoma Survivors Treated with Ipilimumab. J Immunol Res. 2020;2020:2192480.
Ducloux R, Tavernier JY, Wojewoda P, Toullet F, Romanet S, Averous V. Corticotropic insufficiency in a monocentric prospective cohort of patients with lung cancer treated with nivolumab: Prevalence and etiology. Ann Endocrinol (Paris). 2021;82(1):8-14.
Guerrero E, Johnson DB, Bachelot A, Lebrun-Vignes B, Moslehi JJ, Salem JE. Immune checkpoint inhibitor-associated hypophysitis-World Health Organisation VigiBase report analysis. Eur J Cancer. 2019;113:10-13.
Lupi I, Brancatella A, Cosottini M, et al. Clinical heterogeneity of hypophysitis secondary to PD-1/PD-L1 blockade: insights from four cases. Endocrinol Diabetes Metab Case Rep. 2019;2019.
Iglesias P, Sanchez JC, Diez JJ. Isolated ACTH deficiency induced by cancer immunotherapy: a systematic review. Pituitary. 2021;24(4):630-643.
Levy M, Abeillon J, Dalle S, et al. Anti-PD1 and Anti-PDL1-Induced Hypophysitis: A Cohort Study of 17 Patients with Longitudinal Follow-Up. J Clin Med. 2020;9(10).
Mikami T, Liaw B, Asada M, et al. Neuroimmunological adverse events associated with immune checkpoint inhibitor: a retrospective, pharmacovigilance study using FAERS database. J Neurooncol. 2021;152(1):135-144.
Da L, Teng Y, Wang N, et al. Organ-Specific Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitor Monotherapy Versus Combination Therapy in Cancer: A Meta-Analysis of Randomized Controlled Trials. Front Pharmacol. 2019;10:1671.
Hodi FS, Chesney J, Pavlick AC, et al. Combined nivolumab and ipilimumab versus ipilimumab alone in patients with advanced melanoma: 2-year overall survival outcomes in a multicentre, randomised, controlled, phase 2 trial. Lancet Oncol. 2016;17(11):1558-1568.
Higham CE, Olsson-Brown A, Carroll P, et al. SOCIETY FOR ENDOCRINOLOGY ENDOCRINE EMERGENCY GUIDANCE: Acute management of the endocrine complications of checkpoint inhibitor therapy. Endocr Connect. 2018;7(7):G1-G7.
Albarel F, Castinetti F, Brue T. MANAGEMENT OF ENDOCRINE DISEASE: Immune check point inhibitors-induced hypophysitis. Eur J Endocrinol. 2019;181(3):R107-R118.
De Sousa SMC, Sheriff N, Tran CH, et al. Fall in thyroid stimulating hormone (TSH) may be an early marker of ipilimumab-induced hypophysitis. Pituitary. 2018;21(3):274-282.
Nguyen H, Shah K, Waguespack SG, et al. Immune checkpoint inhibitor related hypophysitis: diagnostic criteria and recovery patterns. Endocr Relat Cancer. 2021;28(7):419-431.
Siddiqui MS, Lai ZM, Spain L, et al. Predicting development of ipilimumab-induced hypophysitis: utility of T4 and TSH index but not TSH. J Endocrinol Invest. 2021;44(1):195-203.
Tahir SA, Gao J, Miura Y, et al. Autoimmune antibodies correlate with immune checkpoint therapy-induced toxicities. Proc Natl Acad Sci U S A. 2019;116(44):22246-22251.
Crowne E, Gleeson H, Benghiat H, Sanghera P, Toogood A. Effect of cancer treatment on hypothalamic-pituitary function. Lancet Diabetes Endocrinol. 2015;3(7):568-576.
Mekki A, Dercle L, Lichtenstein P, et al. Detection of immune-related adverse events by medical imaging in patients treated with anti-programmed cell death 1. Eur J Cancer. 2018;96:91-104.
van der Hiel B, Blank CU, Haanen JB, Stokkel MP. Detection of early onset of hypophysitis by (18)F-FDG PET-CT in a patient with advanced stage melanoma treated with ipilimumab. Clin Nucl Med. 2013;38(4):e182-184.
Weber J. Review: anti-CTLA-4 antibody ipilimumab: case studies of clinical response and immune-related adverse events. Oncologist. 2007;12(7):864-872.
Araujo PB, Coelho MC, Arruda M, Gadelha MR, Neto LV. Ipilimumab-induced hypophysitis: review of the literature. J Endocrinol Invest. 2015;38(11):1159-1166.
Dillard T, Yedinak CG, Alumkal J, Fleseriu M. Anti-CTLA-4 antibody therapy associated autoimmune hypophysitis: serious immune related adverse events across a spectrum of cancer subtypes. Pituitary. 2010;13(1):29-38.
Min L, Vaidya A, Becker C. Association of ipilimumab therapy for advanced melanoma with secondary adrenal insufficiency: a case series. Endocr Pract. 2012;18(3):351-355.
Komninos J, Vlassopoulou V, Protopapa D, et al. Tumors metastatic to the pituitary gland: case report and literature review. J Clin Endocrinol Metab. 2004;89(2):574-580.
Mekki A, Dercle L, Lichtenstein P, et al. Machine learning defined diagnostic criteria for differentiating pituitary metastasis from autoimmune hypophysitis in patients undergoing immune checkpoint blockade therapy. Eur J Cancer. 2019;119:44-56.
Lasocki A, Iravani A, Galligan A. The imaging of immunotherapy-related hypophysitis and other pituitary lesions in oncology patients. Clin Radiol. 2021;76(5):325-332.
Chan WB, Cockram CS. Panhypopituitarism in association with interferon-alpha treatment. Singapore Med J. 2004;45(2):93-94.
Ridruejo E, Christensen AF, Mando OG. Central hypothyroidism and hypophysitis during treatment of chronic hepatitis C with pegylated interferon alpha and ribavirin. Eur J Gastroenterol Hepatol. 2006;18(6):693-694.
Tebben PJ, Atkinson JL, Scheithauer BW, Erickson D. Granulomatous adenohypophysitis after interferon and ribavirin therapy. Endocr Pract. 2007;13(2):169-175.
Concha LB, Carlson HE, Heimann A, Lake-Bakaar GV, Paal AF. Interferon-induced hypopituitarism. Am J Med. 2003;114(2):161-163.
Sakane N, Yoshida T, Yoshioka K, Umekawa T, Kondo M, Shimatsu A. Reversible hypopituitarism after interferonalfa therapy. Lancet. 1995;345(8960):1305.
Ramos-Levi AM, Gargallo M, Serrano-Somavilla A, Sampedro-Nunez MA, Fraga J, Marazuela M. Hypophysitis following Treatment with Ustekinumab: Radiological and Pathological Findings. Front Endocrinol (Lausanne). 2018;9:83.
Bettendorf M, Fehn M, Grulich-Henn J, et al. Lymphocytic hypophysitis with central diabetes insipidus and consequent panhypopituitarism preceding a multifocal, intracranial germinoma in a prepubertal girl. Eur J Pediatr. 1999;158(4):288-292.
Nishiuchi T, Imachi H, Murao K, et al. Suprasellar germinoma masquerading as lymphocytic hypophysitis associated with central diabetes insipidus, delayed sexual development, and subsequent hypopituitarism. Am J Med Sci. 2010;339(2):195-199.
Ozbey N, Sencer A, Tanyolac S, et al. An intrasellar germinoma with normal cerebrospinal fluid beta-HCG concentrations misdiagnosed as hypophysitis. Hormones (Athens). 2006;5(1):67-71.
Mikami-Terao Y, Akiyama M, Yanagisawa T, et al. Lymphocytic hypophysitis with central diabetes insipidus and subsequent hypopituitarism masking a suprasellar germinoma in a 13-year-old girl. Childs Nerv Syst. 2006;22(10):1338-1343.
Endo T, Kumabe T, Ikeda H, Shirane R, Yoshimoto T. Neurohypophyseal germinoma histologically misidentified as granulomatous hypophysitis. Acta Neurochir (Wien). 2002;144(11):1233-1237.
Fehn M, Bettendorf M, Ludecke DK, Sommer C, Saeger W. Lymphocytic hypophysitis masking a suprasellar germinoma in a 12-year-old girl--a case report. Pituitary. 1999;1(3-4):303-307.
Gutenberg A, Bell JJ, Lupi I, et al. Pituitary and systemic autoimmunity in a case of intrasellar germinoma. Pituitary. 2011;14(4):388-394.
Pal R, Rai A, Vaiphei K, et al. Intracranial Germinoma Masquerading as Secondary Granulomatous Hypophysitis: A Case Report and Review of Literature. Neuroendocrinology. 2020;110(5):422-429.
McLaughlin N, Lavigne F, Kilty S, Berthelet F, Bojanowski MW. Hypophysitis secondary to a ruptured Rathke cleft cyst. Can J Neurol Sci. 2010;37(3):402-405.
Yang C, Wu H, Bao X, Wang R. Lymphocytic Hypophysitis Secondary to Ruptured Rathke Cleft Cyst: Case Report and Literature Review. World Neurosurg. 2018;114:172-177.
Hayashi Y, Oishi M, Kita D, Watanabe T, Tachibana O, Hamada J. Pure Lymphocytic Infundibuloneurohypophysitis Caused by the Rupture of Rathke's Cleft Cyst: Report of 2 Cases and Review of the Literature. Turk Neurosurg. 2015;25(2):332-336.
Nishikawa T, Takahashi JA, Shimatsu A, Hashimoto N. Hypophysitis caused by Rathke's cleft cyst. Case report. Neurol Med Chir (Tokyo). 2007;47(3):136-139.
Schittenhelm J, Beschorner R, Psaras T, et al. Rathke's cleft cyst rupture as potential initial event of a secondary perifocal lymphocytic hypophysitis: proposal of an unusual pathogenetic event and review of the literature. Neurosurg Rev. 2008;31(2):157-163.
Roncaroli F, Bacci A, Frank G, Calbucci F. Granulomatous hypophysitis caused by a ruptured intrasellar Rathke's cleft cyst: report of a case and review of the literature. Neurosurgery. 1998;43(1):146-149.
Puchner MJ, Ludecke DK, Saeger W. The anterior pituitary lobe in patients with cystic craniopharyngiomas: three cases of associated lymphocytic hypophysitis. Acta Neurochir (Wien). 1994;126(1):38-43.
Martinez JH, Davila Martinez M, Mercado de Gorgola M, Montalvo LF, Tome JE. The coexistence of an intrasellar adenoma, lymphocytic hypophysitis, and primary pituitary lymphoma in a patient with acromegaly. Case Rep Endocrinol. 2011;2011:941738.
Ballian N, Chrisoulidou A, Nomikos P, Samara C, Kontogeorgos G, Kaltsas GA. Hypophysitis superimposed on a non-functioning pituitary adenoma: diagnostic clinical, endocrine, and radiologic features. J Endocrinol Invest. 2007;30(8):677-683.
Cuthbertson DJ, Ritchie D, Crooks D, et al. Lymphocytic hypophysitis occurring simultaneously with a functioning pituitary adenoma. Endocr J. 2008;55(4):729-735.
Moskowitz SI, Hamrahian A, Prayson RA, Pineyro M, Lorenz RR, Weil RJ. Concurrent lymphocytic hypophysitis and pituitary adenoma. Case report and review of the literature. J Neurosurg. 2006;105(2):309-314.
McConnon JK, Smyth HS, Horvath E. A case of sparsely granulated growth hormone cell adenoma associated with lymphocytic hypophysitis. J Endocrinol Invest. 1991;14(8):691-696.
Sivakoti S, Nandeesh BN, Bhatt AS, Chandramouli BA. Pituitary Adenoma with Granulomatous Hypophysitis: A Rare Coexistence. Indian J Endocrinol Metab. 2019;23(4):498-500.
Huang YY, Lin SF, Dunn P, Wai YY, Hsueh C, Tsai JS. Primary pituitary lymphoma presenting as hypophysitis. Endocr J. 2005;52(5):543-549.
Baughman RP, Teirstein AS, Judson MA, et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med. 2001;164(10 Pt 1):1885-1889.
Stern BJ, Krumholz A, Johns C, Scott P, Nissim J. Sarcoidosis and its neurological manifestations. Arch Neurol. 1985;42(9):909-917.
Pekic S, Popovic V. DIAGNOSIS OF ENDOCRINE DISEASE: Expanding the cause of hypopituitarism. Eur J Endocrinol. 2017;176(6):R269-R282.
Langrand C, Bihan H, Raverot G, et al. Hypothalamo-pituitary sarcoidosis: a multicenter study of 24 patients. QJM. 2012;105(10):981-995.
Goyal M, Kucharczyk W, Keystone E. Granulomatous hypophysitis due to Wegener's granulomatosis. AJNR Am J Neuroradiol. 2000;21(8):1466-1469.
Bando H, Iguchi G, Fukuoka H, et al. A diagnostic pitfall in IgG4-related hypophysitis: infiltration of IgG4-positive cells in the pituitary of granulomatosis with polyangiitis. Pituitary. 2015;18(5):722-730.
Chu JN, Goglin S, Patzek S, Brondfield S. Granulomatosis with Polyangiitis Presenting as Hypophysitis. Am J Med. 2019;132(1):e21-e22.
How Hong E, Shalid A, Gatt D, Deepak S, Bahl A. Primary pituitary granulomatosis with polyangiitis and the role of pituitary biopsy, case report and literature review. Br J Neurosurg. 2021:1-7.
Kapoor E, Cartin-Ceba R, Specks U, Leavitt J, Erickson B, Erickson D. Pituitary dysfunction in granulomatosis with polyangiitis: the Mayo Clinic experience. J Clin Endocrinol Metab. 2014;99(11):3988-3994.
Milne P, Bigley V, Bacon CM, et al. Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults. Blood. 2017;130(2):167-175.
Radojkovic D, Pesic M, Dimic D, et al. Localised Langerhans cell histiocytosis of the hypothalamic-pituitary region: case report and literature review. Hormones (Athens). 2018;17(1):119-125.
Zhou W, Rao J, Li C. Isolated Langerhans cell histiocytosis in the hypothalamic-pituitary region: a case report. BMC Endocr Disord. 2019;19(1):143.
Dunger DB, Broadbent V, Yeoman E, et al. The frequency and natural history of diabetes insipidus in children with Langerhans-cell histiocytosis. N Engl J Med. 1989;321(17):1157-1162.
Grois N, Fahrner B, Arceci RJ, et al. Central nervous system disease in Langerhans cell histiocytosis. J Pediatr. 2010;156(6):873-881, 881 e871.
Grois N, Potschger U, Prosch H, et al. Risk factors for diabetes insipidus in langerhans cell histiocytosis. Pediatr Blood Cancer. 2006;46(2):228-233.
Prosch H, Grois N, Prayer D, et al. Central diabetes insipidus as presenting symptom of Langerhans cell histiocytosis. Pediatr Blood Cancer. 2004;43(5):594-599.
Prosch H, Grois N, Bokkerink J, et al. Central diabetes insipidus: Is it Langerhans cell histiocytosis of the pituitary stalk? A diagnostic pitfall. Pediatr Blood Cancer. 2006;46(3):363-366.
Allen CE, Ladisch S, McClain KL. How I treat Langerhans cell histiocytosis. Blood. 2015;126(1):26-35.
Kelly WF, Bradey N, Scoones D. Rosai-Dorfman disease presenting as a pituitary tumour. Clin Endocrinol (Oxf). 1999;50(1):133-137.
Rotondo F, Munoz DG, Hegele RG, et al. Rosai-Dorfman disease involving the neurohypophysis. Pituitary. 2010;13(3):256-259.
Patnana M, Sevrukov AB, Elsayes KM, Viswanathan C, Lubner M, Menias CO. Inflammatory pseudotumor: the great mimicker. AJR Am J Roentgenol. 2012;198(3):W217-227.
Soares D, Crandon I, Char G, Webster D, Carpenter R. Orbital psuedotumour with intracranial extension. A case report. West Indian Med J. 1998;47(2):68-71.
Hansen I, Petrossians P, Thiry A, et al. Extensive inflammatory pseudotumor of the pituitary. J Clin Endocrinol Metab. 2001;86(10):4603-4610.
Al-Shraim M, Syro LV, Kovacs K, Estrada H, Uribe H, Al-Gahtany M. Inflammatory pseudotumor of the pituitary: case report. Surg Neurol. 2004;62(3):264-267; discussion 267.
Hida C, Yamamoto T, Endo K, Tanno Y, Saito T, Tsukamoto T. Inflammatory involvement of the hypophysis in Tolosa-Hunt syndrome. Intern Med. 1995;34(11):1093-1096.
Hama S, Arita K, Kurisu K, Sumida M, Kurihara K. Parasellar chronic inflammatory disease presenting Tolosa-Hunt syndrome, hypopituitarism and diabetes insipidus: a case report. Endocr J. 1996;43(5):503-510.
Yamakita N, Hanamoto T, Muraoka N, et al. Hypopituitarism and diabetes insipidus with localized hypertrophic pachymeningitis (Tolosa-Hunt syndrome) associated with Hashimoto thyroiditis. Am J Med Sci. 2004;327(1):38-43.
Kambe A, Tanaka Y, Numata H, et al. A case of Tolosa-Hunt syndrome affecting both the cavernous sinuses and the hypophysis, and associated with C3 and C4 aneurysms. Surg Neurol. 2006;65(3):304-307; discussion 307.
Kita D, Tachibana O, Nagai Y, Sano H, Yamashita J. Granulomatous pachymeningitis around the sella turcica (Tolosa-Hunt syndrome) involving the hypophysis--case report. Neurol Med Chir (Tokyo). 2007;47(2):85-88.
Toth M, Szabo P, Racz K, et al. Granulomatous hypophysitis associated with Takayasu's disease. Clin Endocrinol (Oxf). 1996;45(4):499-503.
Kanatani M, Nakamura R, Kurokawa K, et al. Hypopituitarism associated with Cogan's syndrome; high-dose glucocorticoid therapy reverses pituitary swelling. Jpn J Med. 1991;30(2):164-169.
de Bruin WI, van 't Verlaat JW, Graamans K, de Bruin TW. Sellar granulomatous mass in a pregnant woman with active Crohn's disease. Neth J Med. 1991;39(3-4):136-141.
Freeman HJ, Maguire J. Sellar inflammatory mass with inflammatory bowel disease. Can J Gastroenterol. 2010;24(1):58-60.
Kalambokis G, Vassiliou V, Vergos T, Christou L, Tsatsoulis A, Tsianos EV. Isolated ACTH deficiency associated with Crohn's disease. J Endocrinol Invest. 2004;27(10):961-964.
Larkin S, Ansorge O. Development And Microscopic Anatomy Of The Pituitary Gland. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Yamamoto M, Iguchi G, Takeno R, et al. Adult combined GH, prolactin, and TSH deficiency associated with circulating PIT-1 antibody in humans. J Clin Invest. 2011;121(1):113-119.
Bando H, Iguchi G, Fukuoka H, et al. Involvement of PIT-1-reactive cytotoxic T lymphocytes in anti-PIT-1 antibody syndrome. J Clin Endocrinol Metab. 2014;99(9):E1744-1749.
Kanie K, Bando H, Iguchi G, et al. Pathogenesis of Anti-PIT-1 Antibody Syndrome: PIT-1 Presentation by HLA Class I on Anterior Pituitary Cells. J Endocr Soc. 2019;3(11):1969-1978.
Yamamoto M, Iguchi G, Bando H, et al. Autoimmune Pituitary Disease: New Concepts With Clinical Implications. Endocr Rev. 2020;41(2).
Bando H, Iguchi G, Okimura Y, et al. A novel thymoma-associated autoimmune disease: Anti-PIT-1 antibody syndrome. Sci Rep. 2017;7:43060.
Kanie K, Iguchi G, Inuzuka M, et al. Two Cases of anti-PIT-1 Hypophysitis Exhibited as a Form of Paraneoplastic Syndrome not Associated With Thymoma. J Endocr Soc. 2021;5(3):bvaa194.
Chung TT, Monson JP. Hypopituitarism. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA)2000.
Dhanwal DK, Vyas A, Sharma A, Saxena A. Hypothalamic pituitary abnormalities in tubercular meningitis at the time of diagnosis. Pituitary. 2010;13(4):304-310.
Leow MK, Kwek DS, Ng AW, Ong KC, Kaw GJ, Lee LS. Hypocortisolism in survivors of severe acute respiratory syndrome (SARS). Clin Endocrinol (Oxf). 2005;63(2):197-202.
Tarvainen M, Makela S, Mustonen J, Jaatinen P. Autoimmune polyendocrinopathy and hypophysitis after Puumala hantavirus infection. Endocrinol Diabetes Metab Case Rep. 2016;2016.
Schwab S, Lissmann S, Schafer N, et al. When polyuria does not stop: a case report on an unusual complication of hantavirus infection. BMC Infect Dis. 2020;20(1):713.

Benign Prostate Disorders

ABSTRACT

Benign prostatic hyperplasia (BPH) is among the commonest urological abnormalities affecting the aging male. The cause of the increase in prostatic volume is multifactorial, but current research has implicated hormonal aberrations. Clinical assessment of the patient is integral to determining the optimal treatment strategy. Exclusion of prostatic cancer and complications of BPH are critical prior to the commencement of conservative and non-invasive strategies. Recently, the introduction of pharmaceutical agents has changed the landscape of management of BPH. Alpha-blockers, 5-alpha reductase inhibitors, and phosphodiesterase-5 inhibitors provide significant symptomatic improvement for BPH, particularly when used in combination. Invasive surgical therapies remain the gold standard for refractory and complicated BPH disease. Advances in technology have provided new methods to perform prostatectomy including: bipolar resection, laser resection, ablation, enucleation or vaporization. Newer, minimally invasive measures have been introduced in an attempt to limit patient morbidity, specifically operative complications, sexual and urinary function. While results are promising, these emerging therapies have limited long-term data. The purpose of the current chapter is to provide an overview of the current knowledge of benign prostatic hyperplasia.

INTRODUCTION

The prostate is an organ linked inextricably to the endocrine system. During the development of the prostate, the epithelium and mesenchyme are under the control of testicular androgens, and interact to form an organized secretory organ. Furthermore, the endocrine system plays a key mechanistic role in many prostate diseases, and many therapies for prostatic diseases are aimed at the manipulation of the endocrine system. The gland resides in the true anatomical pelvis and forms the most proximal aspect of the urethra. It has been stated that the prostate gland is the male organ most commonly afflicted with either benign or malignant neoplasms (1). Therefore, it is an organ with which every physician and surgeon needs to be familiar. We will focus on BPH, the most prevalent of benign disorders affecting the prostate.

EMBRYOLOGY

The development, growth and cytodifferentiation of the prostate are androgen-dependent and occur via embryonic cell-to-cell interactions between the mesenchyme (undifferentiated connective tissue) that induce epithelial development while the epithelium induces mesenchymal differentiation (2).

In the developing prostate, urogenital sinus mesenchyme acting under the influence of testicular androgens induces ductal morphogenesis, the expression of epithelial androgen receptors, regulates epithelial proliferation and specifies the expression of prostatic-lobe specific secretory proteins. The developing prostatic epithelium reciprocally induces the differentiation and morphological patterning of smooth muscle in the urogenital sinus mesenchyme (2). In the prostate, it is traditional to consider androgens as promoters of growth, while activin and tumor growth factor-beta1 (TFG-β1) are regarded as potent growth inhibitors. These factors do not act independently, however, and cross-talk occurs between the signaling pathways at a sub-cellular level (3).

The first step in development of the prostate begins with the urogenital sinus mesenchyme signaling to the epithelium, causing it to form epithelial buds. Androgens then induce bud elongation, branching and epithelial differentiation (3). Prenatally, the androgen receptor (AR) is expressed only in the mesenchyme, not in the epithelium. Initial epithelial development is thus controlled via paracrine interactions where activation of stromal androgen receptors stimulates growth factors and induces growth in adjacent prostatic epithelial cells (4).

At the 5^th week, the mesonephric (Wolffian) duct opens onto the lateral surface of the urogenital sinus and gives rise to the ureteric bud (Figure 1). By the 7^th week, the growth of the urogenital sinus involves the progressive incorporation of the terminal part of the mesonephric duct into the wall of the urogenital sinus. They eventually open into the Mullerian tubercle that is the future verumontanum of the prostate. At their termination the paramesonephric (Mullerian) ducts fuse and are surrounded by the mesonephric ducts. At 10 weeks, prostatic epithelial buds begin to arise from the circumference of the urethra, around the orifice of the paramesonephric ducts. They develop predominantly on the posterior surface of the junction of the mesonephric ducts, forming two concentrations, above and below them (5).

Figure 1. The embryological origin and development of the prostatic urethra and the prostate, adapted from Delmas (5).

During the fetal period at about 6 months, multiple outgrowths arise from the prostatic portion of the urethra, particularly the posterior surface of the urethra, and grow into the surrounding mesenchyme. Glandular epithelium of the prostate differentiates from the endodermal cells of the urethra, and outgrowths of glandular epithelium protrude into the associated mesenchyme differentiate into the dense stroma and smooth muscle fibers of the prostate. In contrast, the prostatic glandular epithelium outgrowths situated on the anterior surface regress and are replaced by fibromuscular tissue. This region becomes the future anterior commissure of the prostate 5,6).

ANATOMY

According McNeal’s model of the prostate (7), four different anatomical zones may be distinguished that have anatomo-clinical correlation (Figure 2):

The peripheral zone: is the area forming the postero-inferior aspect of the gland and represents 70% of the prostatic volume. It is the zone where the majority (60-70%) of prostate cancers form.
The central zone: represents 25% of the prostate volume and contains the ejaculatory ducts. It is the zone which usually gives rise to inflammatory processes (e.g., prostatitis).
The transitional zone: this represents only 5% of the total prostatic volume. This is the zone where benign prostatic hypertrophy occurs and consists of two lateral lobes together with periurethral glands. Approximately 25% of prostatic adenocarcinomas also occur it this zone.
The anterior zone: predominantly fibromuscular with no glandular structures.

The prostate weighs approximately 20g by the age of 20 and has the shape of an inverted cone, with the base at the bladder neck and the apex at the urogenital diaphragm (8). The prostatic urethra does not follow a straight line as it runs through the center of the prostate gland but it is actually bent anteriorly approximately 35 degrees at the verumontanum (where the ejaculatory ducts join the prostate) (9).

Figure 2. 1= Peripheral Zone, 2= Central Zone, 3= Transitional Zone, 4= Anterior Fibromuscular Zone. B= Bladder, U= Urethra, SV= Seminal Vesicle (adapted from Algaba (10)).

HISTOLOGY

The prostate consists of stromal and epithelial elements. Smooth muscle cells, fibroblasts and endothelial cells are in the stroma and the epithelial cells are secretory cells, basal cells and neuroendocrine cells (Figure 3).

Figure 3. Histology of a prostate gland affected by benign prostatic hyperplasia.

The columnar secretory cells are tall with pale to clear cytoplasm. These cells stain positively with prostate-specific antigen (PSA) (11). Basal cells are less differentiated than secretory cells and are devoid of secretory products such as PSA (12). Finally, neuroendocrine cells are irregularly distributed throughout ducts and acini, with a greater proportion in the ducts. The prostate has the greatest number of neuroendocrine cells of any of the genitourinary organs (13). Glands are structured with open and closed cell types with the open type facing the inside of the duct having a monitoring role over its contents. Most cells contain serotonin, but other peptides that are present include somatostatin, calcitonin, gene-related peptides and katacalcin (11). The cells co-express PSA and prostatic acid phosphatase. Their function is unclear, but it is speculated that these cells are involved with local regulation by paracrine release of peptides (11). Prostatic ducts and acini are distinguished by architectural pattern at low power magnification. The prostate becomes more complex with ducts and branching glands arranged in lobules and surrounded by stroma with advancing age.

Figure 4. Diagram outlining the structure of the prostate gland with regard to ducts, glandular cells and their relationship to blood vessels.

PHYSIOLOGY

At present, there is only limited knowledge of all of the secretory products of the prostate and how these relate to reproduction and infertility. However, the main role of the prostate as a male reproductive organ is to produce prostatic fluid that accounts for up to 30 per cent of the semen volume. Prostatic fluid promotes sperm motility, and it is a milky, alkaline fluid containing PSA, citric acid, calcium, zinc, acid phosphatase and fibrinolysin among its many constituents (Table 1) (14). During ejaculation, alpha-adrenergic stimulation of prostatic smooth muscle expresses seminal fluid containing sperm from the ampulla of the vas deferens into the posterior urethra (15). Interestingly, abnormal growth of the prostate is only experienced by humans and dogs, and why other mammals are spared is a mystery (16).

Table 1. The Composition of Human Semen (adapted from Ganong (17))
Color	White, opalescent
Specific Gravity	1.028
pH	7.35-7.50
Volume	3ml
SPECIFIC COMPONENTS OF SEMEN
Gland/Site	Volume in ejaculate	Features
Testis/Epididymis	0.15ml (5%)	Average approximately spermatozoa 80 million/ml
Seminal Vesicle	1.5-2ml (50-65%)	Fructose (1.5-6.5 mg/ml) phosphorylcholine ergothioneine, ascorbic acid, flavins prostaglandins, bicarbonate
Prostate	0.6-0.9ml (20-30%)	Spermine, citric acid, cholesterol, phospholipids, fibrinolysin, fibrinogenase, zinc, acid phosphatase, prostate-specific antigen
Bulbourethral Glands	< 0.15ml (<5%)	Clear mucus

ENDOCRINE CONTROL OF PROSTATIC GROWTH

Intraprostatic signaling systems are important for the regulation of cell proliferation and extracellular matrix production in the prostatic stroma. Central to this premise is the balance between factors such as TGF-β1, that induces extracellular matrix production, suppresses collagen breakdown and cell proliferation and factors such as fibroblast growth factor 2 and insulin-like growth factors that are mitogenic in the stromal compartment (18). Other endocrine pathways are being investigated, and there is a growing body of evidence suggesting an abnormality in the insulin-like growth factor axis is playing a role in the pathogenesis of BPH (19).

Testosterone

Prostatic epithelial cells express the androgen receptor (20). From the beginning of embryonic differentiation to pubertal maturation and beyond, androgens are a prerequisite for the normal development and physiological control of the prostate (21). Androgens help maintain the normal metabolic and secretory functions of the prostate, and they are also implicated in the development of BPH and prostate cancer. Androgens do not act in isolation, and other hormones and growth factors are being investigated (22).

Androgens also interact with prostate stromal cells which release soluble paracrine factors that induce growth and development of the prostatatic epithelium (4). These paracrine pathways might be critical in regulating the balance between proliferation and apoptosis of prostate epithelial cells in the adult (22).

The appropriate balance between testosterone and its 5-alpha reduced metabolites is key to normal prostate physiology (note the metabolic pathways for androgen metabolism are described in Endotext, Endocrinology of Male Reproduction, Androgen Physiology, Pharmacology and Abuse, D Handelsman). The metabolism of testosterone to dihydrotestosterone (DHT) and its aromatization to estradiol are recognized as the key events in prostatic steroid response.

Figure 5. Conversion of testosterone to dihydrotestosterone (DHT) by 5alpha reductase

Testosterone, to be maximally active in the prostate, must be converted to DHT by the enzyme 5-alpha reductase (Figure 5) (23). DHT has a much greater affinity for the androgen receptor than does testosterone, and DHT accumulates in the prostate even when circulating concentrations of testosterone are low (24,25). Based on rat studies, DHT is about twice as potent as testosterone at equivalent androgen concentrations (26). Therefore, prostatic DHT concentrations may remain similar to those in young and elderly men, despite the fact that serum testosterone concentrations generally decline with age (23). In the prostate, the total level of testosterone is 0.4 ng/g and the total of DHT is 4.5 ng/g (27). The total serum concentration of testosterone in the blood is approximately 10 times higher than DHT (17). Circulating DHT, by virtue of its low serum plasma concentration and tight binding to plasma proteins, is of diminished importance as a circulating androgen affecting prostate growth (16). Intra-prostatic androgens are remarkably independent of serum concentrations (28), and circulating androgen concentrations often do not correlate with intraprostatic concentrations (29).

Estrogen

A role for estrogens in the prostate pathology of the ageing male appears likely with accumulating evidence that estrogens, alone or in combination with androgens, are involved in inducing aberrant growth and/or malignant change. Animal models have supported this hypothesis in the canine model, where estrogens “sensitize” the ageing dog prostate to the effects of androgen (30). The evidence is less clear in humans. Estrogens in the male are predominantly the products of peripheral aromatization of testicular and adrenal androgens (31). While the testicular and adrenal production of androgens declines with ageing, concentrations of total plasma estradiol do not decline. This has been ascribed to the increase in fat mass with ageing (the primary site of peripheral aromatization) and to an increased aromatase activity with ageing. However, free or bioavailable estrogens may decline due to an increase in sex hormone binding globulin, which could translate to lower intraprostatic concentrations of the hormone. The potentially adverse effects of estrogens on the prostate might be due to a shift in the intra-prostatic estrogen: androgen ratio with ageing.

Estrogen, which acts through estrogen receptors (ER) alpha and beta, has been implicated in the pathogenesis of benign and malignant human prostatic tumors (32-34). As stated above, BPH is thought to originate in the transitional zone (TZ) and prostate cancer the peripheral zone (PZ) of the prostate. Receptor studies have found ER-alpha and ER-beta types distributed in human normal and hyperplastic prostate tissues, using in situ hybridization and immunohistochemistry. ER-alpha expression was restricted to stromal cells of the PZ. In contrast, ER-beta was expressed in the stromal and epithelial cells of PZ as well as TZ. These findings suggest that estrogen might play a crucial role in the pathogenesis of BPH through ER-beta (33). Investigations are ongoing and could result in a new range of therapies directed against BPH and prostate cancer. Dietary phytoestrogens (in soy and other vegetables) or selective estrogen receptor modulators are currently being investigated with regard to their role in the development of BPH and prostate cancer (31). Such ER modifiers might oppose some of the effects of natural estrogen by modulating ER receptors, thus reducing the local impact of androgens that need active ER receptors, effectively making them anti-androgenic compounds, but this hypothesis requires more investigation (35).

BENIGN PROSTATIC HYPERPLASIA (BPH)

BPH is an age-related and progressive neoplastic condition of the prostate gland (36). BPH can only be diagnosed definitively by histology. BPH in the clinical setting is characterized by lower urinary tract symptoms (LUTS, see below and Table 2). There is no causal relationship between BPH and prostate cancer (37). Clinically apparent BPH has a significant effect on quality of life, particularly its effects on nocturia and bladder dysfunction. The overall prevalence of BPH is 10.3%, with an overall annual incidence rate of 15 per 1000 man-years, increasing with age (3 per 1000 at age 45-49 years, to 38 per 1000 at 75-79 years). For a symptom-free man at age 46, the 30-year risk of clinical BPH is 45% (38). The true prevalence and incidence of clinical BPH will vary according to the criteria used to describe the condition; however, it has been estimated that the prevalence of BPH is rising due to increases in modifiable risk factors such as obesity (39). It is crucial to acknowledge that LUTS can exist without signs of BPH – as the symptoms can be caused by variations in the sympathetic nervous stimulation of prostatic smooth muscle, variability of prostatic anatomy (viz., enlarged median lobe of the prostate), and the variable effects of bladder physiology from the obstruction and aging.

There have been several studies demonstrating the fact that clinical BPH is a progressive disease. The Olmsted county study (40) showed that with each year there were deteriorations in symptom scores, peak flow rates, and increases in prostate volumes based on transrectal ultrasound scanning (TRUS). The risk of acute urinary retention (AUR) increased with flow rates below 12 ml/sec and with glands greater than 30ml. Studies have also demonstrated that those with larger prostates (>40 ml) and with serum PSA greater than 1.4 ng/ml were more likely to develop acute urinary retention (41). Treatment, however, has changed with the advent of effective non-surgical therapies. Between 1992-1998 there has been a significant lengthening of the period between first diagnosis of LUTS secondary to clinical BPH and surgery, associated with the earlier and increased use of specific medical treatments (42). From the patients perspective, the goals of therapy are to improve quality of life, reduce symptoms, and avoid surgery while ensuring safety from the complications of BPH (43).

Risk Factors for BPH

The only clearly defined risk factors for BPH are age and the presence of circulating androgens. BPH does not develop in men castrated before the age of forty (44), but other factors may influence the prevalence of clinical disease. These include the following:

GENETICS

Clinical BPH appears to run in families. If one or more first degree relatives are affected, an individual is at greater risk of being afflicted by the disorder (45). In a study by Sanda et al (46), the hazard-function ratio for surgically treated BPH amongst first degree relatives of the BPH patients as compared to controls was 4.2 (95% CI, 1.7 to 10.2). The incidence of BPH is highest and starts earliest in blacks than Caucasians and is lowest in Asians (37). Interestingly, despite having larger prostate glands, the age-adjusted risk of BPH was the same for blacks as for whites (RR = 1.0, 95% Cl 0.8-1.2) (47). Furthermore, in an Asian population, men presenting with BPH are likely to have higher symptom scores than blacks or Caucasians (48).

DIET

Diet has been reported as a risk factor for the development of BPH. Large amounts of vegetables and soy products in the diet may explain the lower rate of BPH in Asia when compared to countries with Western, non-Asian diets. In particular, certain vegetables and soy are said to be high in phytoestrogens, such as genestin, that have anti-androgenic effects by an undetermined mechanism on the prostate in vitro (49).

Studying migrant populations with their heterogeneous environmental exposures increases the probabilities of identifying potential risk factors for BPH. Therefore, the association of alcohol, diet, and other lifestyle factors with obstructive uropathy was investigated in a cohort of 6581 Japanese-American men, examined and interviewed from 1971 to 1975 in Hawaii. After 17 years of follow-up, 846 incident cases of surgically treated obstructive uropathy were diagnosed with BPH. Total alcohol intake was inversely associated with obstructive uropathy (p < 0.0001). The relative risk was 0.64 (95% confidence interval: 0.52-0.78) for men drinking at least 25 grams of alcohol per month compared with nondrinkers. Among the 4 sources of alcohol, a significant inverse association was present for beer, wine, and sake, but not for spirits. No association was found with education, number of marriages, or cigarette smoking. Increased beef intake was weakly related to an increased risk (p = 0.047), while no association was found with the consumption of 32 other food items in the study (50).

METABOLIC SYNDROME

There is a growing body of evidence supporting the association between obesity or metabolic syndrome and the development of BPH. The risk of BPH appears to be independently associated with the individual components of BPH including central obesity, hyperinsulinemia, insulin resistance and dyslipidemia. Despite this, the precise causation of this association has not been clearly identified. Recent studies have suggested that in this setting, BPH is a consequence of the metabolic syndrome-associated metabolic derangements, altered sex hormone concentrations and lowered sex-hormone binding globulin concentrations (51). One study found in a cohort of 415 men, that indicators of metabolic syndrome (abnormal concentrations of insulin resistance, subclinical inflammatory state, and sex hormone globulin changes) were significantly associated with increased risk of BPH (52). Mechanisms associated with metabolic syndrome have been discussed as possible targets for future therapies for BPH (53).

CHRONIC INFLAMMATION

There is strong evidence to suggest that inflammation and inflammatory markers are involved in the pathogenesis of BPH. Inflammation within the prostate can be caused by several factors including bacteria, virus, autoimmune disease, diet, metabolic syndrome, and hormone imbalances (53). This leads to the activation of inflammatory cells, release of cytokines, expression of growth factors, and ultimately abnormal proliferation of epithelial and stromal cells of the prostate. Proliferation induces a cycle of hypoxia and recruitment of more growth factors resulting in increased prostate volume and BPH (54). The REDUCE study of 8224 prostate biopsy samples of men with BPH found that 77.6% had cells of chronic inflammation on histology (55).

Anti-inflammatory medications have been studied in combination with BPH medications. A meta-analysis of three randomized controlled trials (n=183) in this area found that non-steroidal anti-inflammatory drugs improved IPSS scores by a mean of 2.89 points and increase peak urine flow by a mean of 0.89m/s (56).

OTHER RISK FACTORS

It has not been possible to delineate any other risk factors for BPH such as coronary artery disease, liver cirrhosis, or diabetes mellitus. Traditionally it has been believed that there is no causal relationship between malignant and benign prostatic hypertrophy (37) and recent data from large trials continue to support this premise (57). Alternative theories have emerged but more data directly linking association with causality are required (58).

PATHOPHYSIOLOGY OF BPH

Natural History

BPH is a histological diagnosis, but its clinical manifestations occur after growth has occurred to such a degree and in such a strategic location within the gland, namely the transitional zone, that it impairs bladder emptying and results in LUTS. One can consider the natural history of BPH as involving two phases:

(i) The pathological or first phase of BPH is asymptomatic and involves a progression from microscopic to macroscopic BPH. Microscopic BPH will develop in almost all men if they live long enough but in only about half will progress to macroscopic BPH. This would suggest that additional factors are necessary to cause microscopic to progress to macroscopic BPH (59). The pathological phase involves development of hyperplastic changes in the transitional zone of the prostate (60). While there is wide variability in prostate growth rates at an individual level, prostate volume appears to increase steadily at about 1.6% per year in randomly selected community men (61).

(ii) The clinical or second phase of BPH involves the progression from pathological to ‘clinical BPH’ that is synonymous with the development of LUTS. Only about one half of patients with macroscopic BPH progress to develop clinical BPH (59). BPH consists of mechanical and dynamic components and it is these components that are responsible for the progression from pathological to clinical BPH (62). In clinical BPH, the ratio of stroma to epithelium is 5:1 whereas in the case of asymptomatic hyperplasia the ratio is 2.7:1. A significant contribution is therefore made by stroma to the infravesical obstruction of BPH (63).

DISEASE MANIFESTATIONS OF BPH

Lower Urinary Tract Symptoms (LUTS)

Lower urinary tract symptoms (LUTS) are highly prevalent and the majority of LUTS in men is produced by BPH, but may be contributed to by a variety of conditions (Figure 6). LUTS are traditionally divided into voiding or obstructive and storage or irritative symptoms (Table 2). Voiding symptoms are more common, however it is storage symptoms that are most bothersome and have a greater impact on a patient's life (64,65). The prevalence of clinical BPH rises with age and approximately 25% of men age 40 or over will suffer from LUTS (66).

Figure 6. Interaction of the many factors involved in the pathogenesis of LUTS. Other causes of LUTS (top right) include all of the differential diagnoses included in Table 5 (see below).

Hesitancy

Poor stream

Intermittent stream

Straining to pass urine
Prolonged micturition
Sense of incomplete bladder emptying
Terminal dribbling

Urinary frequency

Urgency

Urge incontinence

Nocturia

In the past, LUTS suggestive of bladder outflow obstruction (BOO) secondary to BPH were referred to as ‘prostatism’, once other causes such as a urinary tract infection or prostate cancer were excluded. The pathology behind the symptoms was thought to be obstruction due to prostatic gland enlargement alone. However, it is now recognized that voiding/obstructive symptoms result from direct urinary flow obstruction whilst storage/irritative symptoms appear to be due to secondary bladder dysfunction (67). Thus, LUTs occurs after the prostate enlargement causes obstruction, and the bladder voiding is secondarily affected leading irritable symptoms (Figure 7).

Figure 7. Diagrammatic representation of BPH with the enlarged prostate transition zone causing obstruction of the prostatic urethra and the secondary changes in the bladder leading to hypertrophy of the detrusor muscle (copyright Nathan Lawrentschuk 2012).

This concept has been further refined in that obstructive symptoms are thought to result not only from mechanical obstruction due to glandular enlargement, but also dynamic obstruction secondary to contraction of the smooth muscle of the prostate, urethra and bladder neck. This dynamic obstruction is a result of sympathetic nervous system mediated stimulation of alpha-1 adrenoceptors. Storage symptoms appear to be caused by detrusor instability related to detrusor muscle changes in response to obstruction, such as bladder wall hypertrophy and collagen deposition in the bladder (68,69). Adrenoceptors may be further sub-divided into alpha1A and alpha1D subtypes, with alpha1A predominant in the prostate and alpha 1D in the bladder. Thus, blockade of alpha1A may be necessary for reduction of obstruction whereas the blockade of alpha1D may be required to relieve storage symptoms (70) (see below).

It has been suggested that the etiology of LUTS related to BPH is even more complex than outlined above, with extra-prostatic mechanisms such as bladder wall ischemia and changes in the central nervous system being implicated (71). Normal lower urinary tract function is complex, and theoretically any disruption of the pathway for micturition (Figure 8 below) may lead to LUTS (72).

It is worth noting the relationship between LUTS and sexual dysfunction, with sexual dysfunction being highly prevalent in men with LUTS

(73).

By sexual dysfunction, we refer to decreased libido, erectile dysfunction, decreased ejaculation and other ejaculation disorders. Kassabian

(74)

expands on the relationship and agrees with Leilefeld et al

(75)

in suggesting that the relationship is coincidental and both are common in the ageing male.

Figure 8- Normal micturition pathways (reproduced with permission from Physiology and pathophysiology of lower urinary tract symptoms, Drugs of Today, Vol 37, p. 7, Michel MC(72)).

COMPLICATIONS OF BPH

The complications of BPH are summarized in Table 3.

Table 3. Common Complications of BPH

· Urinary retention

· Recurrent Urinary Tract Infections

· Bladder Calculi

· Hematuria

· Secondary bladder instability

· Renal Impairment

Urinary Retention (Acute and Chronic)

As the prostate volume increases with age, the likelihood of acute urinary retention (AUR) and symptom severity both increase while urinary flow rates fall. In one study of more than 2000 men, those with a maximum urinary flow rate (Qmax) <12 ml/s had a 4 times greater risk for AUR than did men with a Qmax >12ml/s (76). AUR is usually painful and necessitates the insertion of a per urethral indwelling or suprapubic urinary catheter.

If the urinary retention is not dealt with in a timely fashion, the detrusor muscle becomes distended and damaged, contributing to poor detrusor function and an inability to adequately empty the bladder. The retention of urine becomes painless over time, and the sequelae of retained urine such as recurrent UTI, calculi, and renal impairment may develop.

Furthermore, a situation of overflow incontinence may develop whereby the bladder automatically empties once the volume reached exceeds its new, larger capacity. The passage of urine is typically uncontrolled, and this may often be the first presentation for someone with advanced BPH. The bladder remains full despite the emptying, which is only partial.

In situations of chronic urinary retention, relieving the bladder outflow obstruction might not restore normal detrusor function. These patients often need to use intermittent self-catheterization or have permanent drainage to keep their bladder empty and to reduce damage to the upper urinary tract.

Recurrent Lower Urinary Tract Infection (UTI)

The best host defense against infection in the lower urinary tract might be normal flow of urine and bladder emptying. In BPH, bladder outflow obstruction results in disruption of this mechanism with retention and pooling of urine in the bladder, giving organisms the opportunity to multiply rather than be flushed out. Despite this logical assumption, there is little evidence in the literature to support this theory. Nevertheless, men with significant clinical BPH are probably at risk of UTI, and men with UTI should be assessed for signs of BPH.

Bladder Calculi

In developed countries, the most prevalent cause of bladder calculi is bladder outlet obstruction owing to BPH (77). Of those who undergo prostate surgery for BPH, approximately 2% of all patients are found to have bladder stones (78). Stones occur in this situation due to urinary stasis combined with high urinary solute concentrations, which leads to crystal precipitation (79). Chronic infection with urease-producing organisms may predispose to the development of stones and rarely stones pass from the upper tract to act as a nidus in the bladder (79) . Bladder calculi associated with BPH remains an absolute indication for transurethral resection of the prostate (TURP) (80,81) because of the risk or recurrence of stone formation. However, the necessity of surgery is being challenged by the expanding use of medical management in treating BPH (81).

Hematuria

The incidence of hematuria with BPH is uncertain, however; in a retrospective review of almost 4000 patients undergoing TURP, Mebust et al (80) noted that hematuria was an indication for surgery in 12% of patients. It is hypothesized that BPH, with its increased acinar and stromal cell proliferation, stimulates increased vascularity via angiogenesis. These new and prolific vessels may be easily disrupted leading to recurrent bleeding (82). This is supported by Foley et al (83) who found the microvessel density to be higher in those patients with BPH having hematuria after histological studies. It is also hypothesized that 5-alpha reductase inhibitors might reduce angiogenesis and theoretically reduce the risk prostate bleeding. Finasteride has been suggested as an option in treating the problem of hematuria (84-86).

Detrusor (Bladder) Instability

The definition of detrusor instability is the development of a detrusor contraction which exceeds 15cm H₂O at a bladder volume of less than 300ml (87). Detrusor instability is not a specific term related to BPH, but implies LUTS secondary to detrusor pathology. These symptoms are normally storage related and consist of urgency, frequency, urge incontinence, and nocturia. In BPH, the normal dynamics of the bladder are altered due to detrusor muscle stretching due in turn to retention of urine and contraction against an obstructed outlet. Although not completely understood, some of the detrusor instability may be related to changes at the adrenoceptors level, rather than just from obstruction and its consequences alone. In normal bladder physiology, beta-adrenoceptors are believed to be involved in the relaxation of the bladder during storage of urine (71). In some patients, however, the administration of noradrenaline leads to contraction of the detrusor muscle which may be blocked by an alpha-1 adrenoceptor antagonist (88). This implies the presence of alpha-adrenoceptors in the detrusor muscle in at least some patients. Furthermore, alpha-adrenoceptor antagonists have been shown to relieve storage and voiding symptoms in men without obstruction and storage symptoms in women (71,89-92). Alpha adrenoceptor subtypes in the human bladder are predominantly of the alpha1D and alpha1A type. In animal models, the alpha1D receptors become more abundant with bladder obstruction (93), and it may be speculated that this is the case in humans and that these receptors, once up-regulated, play a role in storage symptoms (71).

Renal Insufficiency

Renal insufficiency results from obstructive uropathy secondary to the bladder outlet obstruction of BPH. In an analysis of patients receiving treatment for BPH, 13.6% (range 0.3-30%) had renal insufficiency (78). Certainly, an abnormal creatinine is an indication to further investigate the upper urinary tract with imaging. Obviously, other concurrent causes of renal insufficiency need to be excluded. Those patients with renal insufficiency undergoing surgery are at increased risk (25%) of postoperative complications such as acute renal failure and urosepsis compared to patients without (17%) insufficiency (80).

HISTORY

A comprehensive medical history must be evaluated and should include the use of a voiding diary, the International Prostate Symptom Score (IPSS) and a discussion of the role of PSA testing (94). An outline of the evaluation and treatment options for LUTS is shown in Table 4 and is discussed in greater depth below (95,96). Previous urological disease should be documented including previous urological surgery, UTI, bladder or renal calculi, renal disease and penoscrotal pathology. Any risk factors for surgery such as diabetes mellitus, immunosuppression, ischemic heart disease, respiratory problems, smoking as well as a comprehensive list of medications should be noted. Medications with anti-cholinergic properties should be noted, as these may contribute to the patient’s symptoms. The use of antihypertensives must be noted as any alpha-blocker treatment initiated could potentially cause severe hypotension.

As discussed in the section on differential diagnosis, consideration needs to be given to neurologic causes of voiding dysfunction such as stroke or Parkinson’s disease.

Table 4. A Summary of Diagnosis and Treatment Options in BPH

EVALUATION of LUTS

ESSENTIAL

1. History

2. Digital Rectal Exam (DRE)

3. Urinalysis
4. Serum creatinine
5. PSA, if > 10-year life expectancy
6. International Prostate Symptom Score (IPSS) or AUA symptom index

SELECTED
1. Uroflowmetry
2. Imaging - especially if hematuria, UTI, urolithiasis
3. Post Void Residual (PVR) estimation
4. +/-Pressure flow studies
5. +/-Cystoscopy

TREATMENT OPTIONS

MEDICAL THERAPY

1. Phytotherapy

2. Monotherapy:

a. Alpha blockers

b. 5-alpha reductase inhibitors

c. PDE5 Inhibitors

3. Combination therapy:

a. Alpha blocker + 5-alpha reductase inhibitor

b. PDE5 inhibitor + alpha blocker (experimental)

SURGERY
1. Invasive surgery

a. Transurethral resection of the prostate (TURP)
b. Laser prostatectomy/treatment

c. Open prostatectomy
2. Minimally invasive measures

a. Transurethral Incision of the prostate (TUIP)

b. Thermo ablative strategies (TUMT, TUNA)

c. Chemical ablative (PRX-302, NX-1207, TEAP)

d. Mechanical (Urolift, prostatic stent)

e. Others (prostatic artery embolization, histotripsy, Rezum, aquablation)

International Prostate Symptom Score (IPSS)

The American Urologic Association (AUA) Symptom Index was developed as a standardized instrument to assess the degree of bladder outlet obstruction in men (89). It is widely used and consists of seven questions that assess emptying, frequency, intermittency, urgency, weak stream and straining with each graded with a score of 0-5. Total score ranges 0-35. The index categorizes patients as:

Mild (score £7)
Moderate (score 8-19)
Severe (score 20-35).

The International Prostate Symptom Score (IPSS) is a modification of the AUA Symptom Index adding a single question assessing the quality of life or bother score based on the patient’s perception of the problem (Figure 9) (97). Both the AUA and IPSS questionnaires, although not specific for BPH, prostate volume, urinary flow rate, post-void residual volume, or bladder outlet obstruction, have been validated and are sensitive enough to be to be used in the evaluation of symptoms and selection of treatment (98-100). Many would argue that the score is the primary determinant of whether or not a patient proceeds to further treatment. Further, these questionnaires are a valuable objective measure when determining the response to treatments for BPH.

Figure 9. International Prostate Symptom Score (IPSS) Sheet (101,102)

EXAMINATION

General appearance is of importance, especially in identifying those with neurological disease (e.g., past stroke, Parkinson’s disease) or other major co-morbidities (obesity, severe osteoarthritis, diabetes) that may impact on treatment or further investigation. An abdominal examination should identify those in marked urinary retention, any abnormal masses, and previous surgical scars. A careful assessment of the scrotum and its contents as well as the penis is also warranted to exclude any other pathology. The digital rectal examination (DRE) is important in identifying prostatic abnormalities, including clinically apparent prostate carcinoma (103). Prostate size, texture, and tenderness should all be assessed, as should anal tone. Any nodules should be carefully noted. Constipation may also be a contributing factor to urinary retention and anal tone should also be recorded.

DIFFERENTIAL DIAGNOSIS OF BPH

It is important to acknowledge that the diagnosis of BPH often relies on surrogate measures until a histological diagnosis is confirmed. These range from clinical (symptom scores), physiological (uroflowmetry), anatomical (prostatic volume on DRE or TRUS) and biochemical (PSA values) measurement. Although all of these measurements capture some component of BPH, none of them is specific for BPH (104). Surrogate measures are likely to represent a continuum of disease severity without the existence of a threshold. Thus, differential diagnoses need to always be considered and where appropriate, excluded. In table 5 below, some of the more obvious differential diagnoses are listed, but will not be examined in detail.

Table 5. Differential Diagnoses for LUTS

Inflammatory Conditions

1. Urinary Tract Infection

2. Prostatitis

3. Bladder Calculi
4. Interstitial Cystitis
5. Tuberculous Cystitis

Neoplastic Conditions

1. Prostate cancer

2. Bladder transitional cell carcinoma (usually CIS)

3. Urethral cancer

Neurological Conditions

1. Parkinson's disease

2. Stroke

3. Multiple Sclerosis
4. Cerebral Atrophy
5. Shy-Drager Syndrome

Other Causes of Urinary Obstruction

1. Urethral stricture

2. Severe phimosis

3. Bladder neck dyssynergia
4. External sphincter dyssynergia

PROSTATITIS

Prostatitis is a common condition that must be excluded from other causes of LUTS and is a common cause of visits to primary care physicians and urologists. It may present as an acute bacterial infection or may be chronic, occasionally progressing to a debilitating illness. In practice, the clinical diagnosis of prostatitis depends on the history and physical examination, but there is no characteristic physical finding or diagnostic laboratory test. Patients with prostatitis experience considerable morbidity and may remain symptomatic for many years. Unfortunately, there is limited understanding of the pathophysiology and optimal treatment for most patients. Prostatitis has been sub-classified and an abbreviated version is shown in Table 6.

· Table 6. The National Institute of Health (USA) Consensus Classification of Prostatitis Syndromes

· Acute bacterial prostatitis

· Chronic bacterial prostatitis

· Chronic prostatitis/chronic pelvic pain syndrome

· Inflammatory

· Non-inflammatory

· Asymptomatic inflammatory prostatitis

Acute Prostatitis

Clinical features suggestive of acute prostatitis (Type 1, in Table 6 above) include dysuria and urinary frequency as well as perineal pain (Table 7). Systemic symptoms such as fever, rigors, myalgia and sweats are often a feature. On examination, the patient is normally febrile, and may be overtly septic depending on the infection severity. A digital rectal exam finds an extremely tender prostate, which is often intolerable to the patient. An abscess is occasionally palpated.

Table 7. Clinical Symptoms in Prostatitis (adapted from Lobel (105))

Genital symptoms

1. Dribbling

2. Inguinal pain

3. Testicular pain

4. Retropubic pain
5. Perineal pain
6. Urethral Burning

General Symptoms

1. Backache

2. Sweating

3. Tiredness

4. Cold feet

Investigations should include a mid-stream urine sample for microscopy, culture for bacteria, and antibiotic sensitivity. The most common organisms are typical uropathogenic bacteria such as Escherichia coli (E. coli). Blood cultures for bacteria and antibiotic sensitivity should also be considered. Prostatic massage is usually contraindicated in patients with acute prostatitis due to pain and the risk of precipitating sepsis. A treatment regime is highlighted in Table 8. If there is failure to respond to therapy, evaluation for a prostatic abscess using a transrectal ultrasound scan or computed tomography scan may be required. If necessary, perineal or transurethral drainage of an abscess may be undertaken. At least 4 weeks of antibiotic therapy is recommended in all patients to try to prevent chronic bacterial prostatitis. Following resolution of acute prostatitis, the urinary tract should be investigated for any structural problems (106,107).

Table 8. Treatment of Acute Prostatitis

1. Hydration

2. Rest and hospitalization if severe

3. Empirical therapy with antibiotic until urine culture and sensitivities available

4. For patients requiring parenteral therapy antibiotics covering the likely organisms: broad spectrum cephalosporins, for example, cefuroxime, cefotaxime, or ceftriaxone plus gentamicin

5 Oral treatment according to sensitivities.: quinolones, such as ciprofloxacin or norfloxacin. For patients intolerant of, or allergic to, quinolones: trimethoprim or co-trimoxazole;

6. Analgesics, such as non-steroidal anti-inflammatory drugs Suprapubic catheterization if catheterization needed - per urethral catheters may precipitate abscess formation

Chronic Prostatitis

As the presentation may be localized to the genital region or non-specific (see Table 7) a careful history and examination along with specialized diagnostic tests are needed to identify this condition. Investigations may involve prostatic massage to express organisms and/or white blood cells for analysis. Urine sample collection is often done in phases to aid in the localization process: first void urethral urine; mid-stream bladder urine; post-prostatic massage sample. Urine microscopy and quantitative culture is then undertaken. Semen analysis for excessive white blood cell numbers may also be indicative of chronic prostatitis. Serum PSA concentrations are often elevated in acute prostatitis or in an active phase of chronic prostatitis. Trans-rectal ultrasound might be considered but not recommended to differentiate the different forms of chronic prostatitis. Urinary tract localization procedures (culture of first void urethral urine; mid-stream bladder urine; post-prostatic massage samples of urine correlating to urethra, bladder and prostate) although theoretically correct, are often not used in clinical practice (106,107).

The various classifications of chronic prostatitis are listed in Table 6. Patients with chronic bacterial prostatitis (type II prostatitis) experience recurrent episodes of bacterial urinary tract infection caused by the same organism, usually E. coli, another Gram-negative organism, or enterococcus. Between symptomatic episodes of bacteriuria, lower urinary tract cultures can be used to document an infected prostate gland as the focus of these recurrent infections. Acute and chronic bacterial prostatitis represent the best understood, but least common, prostatitis syndromes (106,107).

Unfortunately, more than 90% of symptomatic patients have chronic prostatitis/chronic pelvic pain syndrome (type III). This term recognizes the limited understanding of the causes of this syndrome for most patients and the possibility that organs other than the prostate gland may contribute to this syndrome. Urological pain (normally in the perineum or associated with voiding or intercourse) is now recognized as a primary component of this syndrome. Active urethritis, urogenital cancer, urinary tract disease, functionally significant urethral stricture, or neurological disease affecting the bladder must be excluded. Patients with the inflammatory subtype (type IIIA) of chronic prostatitis/chronic pelvic pain syndrome have leukocytes in their expressed prostatic secretions post prostate massage urine or in semen.

In contrast, patients with the non-inflammatory subtype of chronic prostatitis (type III B) have no evidence of inflammation. In essence, they have no evidence of active infection nor of inflammation on available investigative techniques taken at a particular point in time. Repeat investigations are therefore done to be sure adequate sampling has been undertaken. This condition may be difficult to treat and requires intensive counselling, information and reassurance to the patient to be successfully managed (107).

Finally, asymptomatic inflammatory prostatitis (type IV) is diagnosed in patients who have no history of genitourinary tract pain complaints. It is often an incidental finding on prostatic biopsy done for other reasons (e.g., a raised PSA). Treatment is usually not required.

Treatment of Chronic Prostatitis

All patients should have investigations as outlined above. A summary of treatment options is shown in Table 9. Those patients with chronic prostatitis secondary to bacterial infection (type II) require a prolonged course of antibiotics (often up to three months) and should then be re-cultured to ensure eradication of the organism. Some urologists argue that these patients should also have investigation of their urinary tract by way of cystoscopy and at minimum, an ultrasound to ensure no anatomical abnormality that may be responsible.

Patients with asymptomatic prostatitis (IV) require no treatment but those with the inflammatory (IIIA) and non-inflammatory (IIIB) are more difficult. Patients with type IIIA disease have excessive leukocytosis in their specimens but no bacteria. However, because their symptoms may be due to a pathogen that is difficult to isolate, a further course of antibiotics (6-12 weeks) with coverage of chlamydia and ureaplasma should be given (105). If this antibiotic course is not therapeutic, then a focus should be on anti-inflammatory medications (which may be used in conjunction with the course of antibiotics). If anti-inflammatory treatment fails, then patients should be treated as below, for type IIIB.

Current treatment for Type IIIB patients requires multiple therapies. Triple-therapy involves high dose alpha-blocker (3 month minimum), analgesia, and muscle relaxant (benzodiazepines). Initially, a narcotic analgesic should be changed to a non-steroidal anti-inflammatory (NSAID) if a response occurs after 2 weeks. The NSAID should be continued for at least 6 weeks, but stopped if there is no response at 2 weeks. If the triple treatment fails, other avenues must be explored, including biofeedback, relaxation exercises, psychotherapy, and lifestyle changes (soft cushions, cease bike-riding). The focus is on improving quality of life and minimizing symptoms, not curing the disease (105).

· Table 9. Management and Treatment of Chronic Prostatitis

· Oral and written patient education

· Pharmacological treatment for chronic bacterial prostatitis chosen according to antimicrobial sensitivities include quinolones such as ciprofloxacin; ofloxacin; norfloxacin. For those allergic to quinolones: minocycline; doxycycline; trimethoprim-sulfamethoxazole; co-trimoxazole; in many regions, trimethoprim sulfamethoxazole is first line therapy because of better safety profile than quinolones.

· Other treatments for chronic bacterial prostatitis: radical transurethral prostatectomy or total prostatectomy in carefully selected patients.

· Empirical treatments for chronic abacterial prostatitis

· Treat as for chronic bacterial prostatitis with a quinolone or tetracycline

· Alpha blockers: terazosin, doxazosin, alfuzosin, tamsulosin, silodosin

· Non-steroidal anti-inflammatory drugs

· Stress management. Referral for psychological assessment as appropriate; diazepam. Note: benzodiazepines are considered but not recommended in clinical practice because of dependency

· Adequate follow-up and counselling, often with professional support

· Cernilton (pollen extract)

· Bioflavonoid quercetin

· Transurethral microwave thermotherapy

INVESTIGATIONS OF LUTS

As outlined by Tubarro et al (94), the aim of investigations for LUTS should be threefold: (1) to evaluate the possible relationship between prostatic enlargement, lower urinary tract symptoms and signs of bladder outlet obstruction; (2) to quantify the severity of benign prostatic enlargement-related symptoms and signs and (3) to rule out the presence of a prostate cancer.

Urinalysis

Urinalysis is used to screen for urinary tract infection as a cause of LUTS in order to identify those with microscopic or macroscopic hematuria. A formal urine culture may be undertaken if the analysis was suspicious for infection.

Post-Void Residual Urine Volume (PVRU)

Although there is a high degree of intra-individual variation in the PVRU, it may still provide valuable information with regard to bladder emptying. Although it does not distinguish adequately between bladder outlet obstruction or poor detrusor function, it can identify a bladder emptying problem and be used as a marker for improvement. Due to its inability to differentiate between causes, the United States guidelines on BPH suggest it is an optional investigation (78). Greater than 300ml is considered a potential risk factor for upper urinary tract dilatation and renal impairment (108). The PVRU does have the advantage of being used as a monitoring investigation in those opting for non-surgical therapy for BPH. It is readily and quickly performed in the office or hospital setting using portable ultrasound equipment.

Laboratory Investigations

Serum creatinine is recommended by most guidelines for the investigation of BPH and an elevated serum creatinine would be an indication to evaluate the upper urinary tract (96).

Serum PSA has several implications in the diagnosis and management of BPH, including (1) providing a prediction of the prostate volume (2) providing the prediction of disease course, and (3) providing a risk assessment for prostate cancer. Indeed, in multiple placebo arms of large double-blind clinical trials, the serum PSA is an independent predictor of the risk of acute urinary retention and progression to BPH-related surgery (109). While the PSA provides useful information in the aforementioned domains, in clinical practice the main utility of PSA testing in the setting of LUTS is to exclude prostate malignancy. In patients presenting with isolated LUTS, current guidelines suggest its use only if a diagnosis of prostate cancer will change management or if the PSA can assist in decision-making in patients at high risk of BPH progression.

Upper Urinary Tract Imaging

Urinary tract ultrasound or computerized tomography are appropriate modalities. Most would consider upper tract imaging as mandatory if hematuria is present and recommend it if there was a history of urolithiasis, urinary tract infection, or renal insufficiency. Intravenous pyelography still has a role in certain cases, as other modalities do not outline the anatomy of the collecting system with such definition (94).

Urodynamics

Urodynamics is a general term for a collection of investigations useful in quantifying the activity of the lower urinary tract during micturition (110). Complete pressure-flow urodynamics are complex and usually involve fluoroscopy, video recording, bladder and rectal pressure measurement, as well as an assessment of urine flow. The simplest urodynamics are pressure-flow studies, requiring only voiding into a measuring device to obtain flow rates, and may easily be done in the office setting.

With regard to the investigation and diagnosis of conditions underlying LUTS, when considering inexpensive, safe, and completely reversible treatments, one may opt to avoid urodynamics studies initially. However, when considering irreversible, expensive, or potentially morbid therapy, such studies are considered mandatory. Many patients will not have urodynamics studies based on the first premise above (110). However, in reality, many surgeons and physicians will have simple pressure-flow studies readily available and will perform these as part of an initial consultation. More complex studies require time and are costly, and so should be reserved for particular situations as discussed below.

Urinary Flow Rate (Uroflowmetry)

Uroflowmetry is considered by some as the single most useful urodynamic technique for the assessment of obstructive uropathy. The purpose of the uroflow examination is to record one or more micturitions that are representative of the patient’s usual voiding pattern. Therefore, more than one micturition is often required and it is necessary to confirm with the patient if the flow was better, worse or about the same as their normal pattern, otherwise intra-individual variability may lead to false assumptions (111). The study may be performed in the office or as part of other urodynamic studies in the laboratory or operating suite.

Figure 10 indicates the most common urinary flow parameters measured. Of these, the peak flow rate is the most closely correlated with the extent of outflow obstruction (Table 10). Total voiding time is prolonged in obstruction and has a reduced Qmax. Poor detrusor contractility is impossible to distinguish from bladder outflow obstruction on uroflowmetry so other urodynamics investigations such as a cytometry are indicated.

Figure 10. Uroflowmetry in a normal individual- diagram above and actual reading below (Table 10).

Table 10. Interpretation of Uroflowmetry Results.
Flow rate- Qmax	Interpretation
>15ml/sec	Unlikely to be significant obstruction
<10ml/sec	Likely to be significant obstruction or weak detrusor activity
10-15ml/sec	Equivocal

Urodynamics- Pressure-Flow Studies

Various measurements may be used to define detrusor pressures and urethral sphincter pressures as an aid to diagnosis in specific circumstances. This is relevant in patients with LUTS who have had a stroke (or other neurologic disease) where bladder function may have sensory deficits or unstable detrusor contractions that may need alternate management. Nevertheless, detrusor instability is not considered a negative factor with respect to the outcome of BPH surgery (94), provided it is adequately managed. Some have even suggested that the detection of detrusor instability in patients with LUTS is only of minor diagnostic importance (112).

Urethrocystoscopy

The performance of this investigation depends on patient history and proposed surgical intervention. It is necessary where there is a history of microscopic or macroscopic hematuria to exclude bladder tumors or stones. A history or suspicion of urethral strictures, bladder tumors, or prior lower urinary tract surgery should also prompt this investigation. Surgeons may also use urethroscystoscopy when planning different surgical treatments or invasive therapies.

Transrectal Ultrasound Scanning (TRUS)

Compared to TRUS, methods of determining prostate size such as DRE, urethrocystoscopy, and retrograde urethrography are poor (113). It is often conducted in unison with biopsies of the prostate for suspected carcinoma, but is also a useful tool for assessing the size of an enlarged prostate so that the best mode of management may be undertaken, such as open versus endoscopic surgery.

OVERVIEW OF TREATMENT OF BPH

The primary aim of any treatment for BPH in the vast majority of men is to relieve bothersome obstructive and irritative symptoms (114) (Table 2). Treatment is often undertaken on an elective basis for such patients. Those in whom complications of BPH occur have treatment done urgently as a matter of course. A range of treatment options are available and may be tailored to the needs of every individual, taking into account their disease manifestations, success rates of treatment, possible complications, and patient preference.

WATCH AND WAIT/LIFESTYLE CHANGE

Many men who present with LUTS are often seeking a full assessment of their prostatic health rather than immediate treatment of symptoms that may not be exceptionally bothersome. People with mild symptoms may wish to pursue lifestyle changes as a way of improving their quality of life but with the option of review if such measures fail or symptoms worsen. Furthermore, when an adequate history is taken, hidden agendas such as fear of prostate cancer may even be revealed and fears allayed.

Often drinking habits may be responsible for symptoms such as nocturia, where considerable fluid volumes are consumed in the evening. Reducing fluid intake may diminish nocturia and evening urgency. Furthermore, caffeine and alcohol acting as diuretics can further exacerbate LUTS. Simple shifts in daily fluid intake may fulfil patient expectations and result in satisfactory outcomes. Voiding diaries are useful for making patients aware of drinking habits and may be the catalyst for initiating and monitoring changes. Bladder retraining (by using timed voiding, strengthening pelvic floor exercises, and monitoring oral intake) is also an option in some individuals, once a voiding diary has been examined.

Medications may also play a role with LUTS. Measures such as diuretic restriction in evenings often prevents nocturia and frequency, provided the diuretic can be taken earlier in the afternoon.

It is important to discuss options with the patient and that they he be made aware that the possibility of damage to their upper urinary tract or to the detrusor muscle may result if their symptoms deteriorate and they do not seek medical attention.

PHYTOTHERAPY FOR BPH

Phytotherapy, or the use of plant extracts, is becoming widely used in the management of many medical conditions including BPH (Table 11) (115). Often these agents are promoted to aid “prostatic health” and a significant proportion of men try them. Factors also contributing to their widespread use include the perception that they are supposedly ''natural'' products; the presumption of their safety (although this is not adequately proven); their alleged potential to assist in avoiding surgery, and even the unproven claim that they may prevent prostate cancer. The widespread availability of these products (without prescription) in vitamin shops, supermarkets, pharmacies, and over the internet has contributed to their usage and reflects the demand for these phytotherapeutic agents. The mechanisms of action are poorly understood but have been proposed to be (1) anti-inflammatory, (2) inhibitors of 5-alpha reductase, and more recently (3) through alteration in growth factors (116).

Phytotherapy, although promising, lacks long-term, good quality clinical data (117). Nevertheless, because there is a large placebo effect associated with treatment of voiding symptoms, the use of herbal products that have few or no side effects may be a reasonable first-line approach for many patients (118). However, patients should be counselled that the efficacy, mechanisms of action and long-term effects of these agents are not known and they must be aware of the limitations before proceeding (119).

The most popular phytotherapeutic agents are extracted from the seeds, barks and fruits of plants. Products may contain extracts from one or more plants and different extraction procedures are often used by manufacturers. Thus, the composition and purity of products may differ even if they originated from the same plant. Basic research on one product may not be easily transferred to another making the gathering of data and giving of advice difficult (120).

Table 11. Phytotherapy Used in the Treatment of Benign Prostatic Hyperplasia
Phytotherapeutic plant extract	Proposed Mechanism of action
Saw palmetto- fruit (Serenoa repens)	Antiandrogenic, Anti-inflammatory
African plum- bark (Pygeum africanum)	Antiandrogenic, potential growth factor manipulation, anti-inflammation actions
Pumpkin- seed (Cucurbita pepo)	Phytosterols are thought to be amongst the active compounds
Cernilton- pollen (Secale cereal, Rye)	Inhibition of alpha-adrenergic receptors
South African star grass- root (Hypoxis rooperi)	Antiandrogenic, alteration in detrusor function
Stinging nettle- root	Steroid hormone manipulation reducing prostate growth
Opuntia- flower (Cactus)	Unknown
Pinus- flower (Pine)	Unknown

Saw Palmetto Berry (Serenoa repens)

Extracts from the berries of the American dwarf palm (saw palmetto) are the most popular and widely available plant extracts used to treat symptomatic BPH today (121,122). At least eight possible mechanisms of action for saw palmetto have been advocated including anti-androgenic properties, anti-inflammatory properties, induction of apoptosis to name a few (120). Several studies have found that saw palmetto suppresses growth and induces apoptosis of prostate epithelial cells by inhibition of various signal transduction pathways (123). However, it is most commonly believed that saw palmetto works as a naturally occurring weak 5-alpha reductase inhibitor, blocking the conversion of testosterone to DHT, as demonstrated in several in vitro studies (118, 124-127). Thus, saw palmetto may be expected to reduce prostate size. While demonstrated in animal models (128), this is not the case in several trials using saw palmetto in men with BPH (129,130). The only trial to show in vivo effects of saw palmetto involved needle biopsies of the prostate gland, before and after treatment with saw palmetto or placebo. Although the mechanism is unclear, there was a significant increase in prostatic epithelial contraction in the saw palmetto group (131).

Clinical evidence reporting the use of saw palmetto is conflicting. In a meta-analysis of 18 randomized studies relating to saw palmetto extracts, almost 3000 men with BPH were studied and the authors concluded that “the evidence suggests that saw palmetto improves urologic symptoms and flow rates but that further research is needed using standardized preparations to determine long term effectiveness” (115). When analyzing flow rate and symptom score alone from this meta-analysis, the effect of Seronoa repens (the scientific name of saw palmetto was to increase the flow rate by a further 2.28 ml/sec (standard error, SE, 0.29) over placebo which gave an increase of 1.09 ml/sec (SE 0.45). Serenoa repens also reduced the IPSS by 4.7 (SE 0.41), which is comparable to that found with finasteride and tamsulosin monotherapy (132).

Conversely, a recently published Cochrane review concluded Serenoa repens was no more effective than placebo for treatment of urinary symptoms consistent with BPH (133). This update of a prior review, nine new trials involving 2053 additional men (a 65% increase) were included. The main comparison was again Serenoa repens versus placebo where three trials were added with 419 subjects and three endpoints (IPSS, peak urine flow, prostate size). Overall, 5222 subjects from 30 randomized trials ranging from four to 60 weeks were assessed. The vast majority were double blinded and treatment allocation concealment was adequate in just over half the studies.

In summary, some saw palmetto studies have shown improved symptom scores compared to placebo but generally no change in flow rates (134). However, large reviews cast doubt on its efficacy. In general, there is a real paucity of well performed, adequately powered, and placebo-controlled trials in the use of phytotherapy in clinical BPH. It is generally well tolerated at a dose of 320mg/day, but its efficacy has not been compared with alpha-blockers regarding efficacy, and has not been shown to reduce complications of BPH with long term use. Finally, the product quality and purity cannot always be assured.

African Plum Tree (Pygeum africanum)

Extracts come from the bark of the African plum tree. It is hypothesized, based on in vitro observation, that it acts on the prostate through inhibition of fibroblast growth factors, has anti-estrogenic effects, and inhibits chemotactic leukotrienes. No strong clinical data exists of its efficacy although trials are in progress (116,119).

Pumpkin Seed (Cucurbita pepo)

Dried or fresh seeds have been taken to relieve symptoms. Phytosterols are thought to be amongst the active compounds. Side effects have not been reported but evidence is lacking with no current clinical trials (135).

Rye Pollen (Secale cereale)

This is prepared from rye grass pollen extract. In a systematic review summarizing evidence from randomized and clinically controlled trials (114), rye pollen was found to be well tolerated but only achieved modest improvement in symptom outcomes and did not significantly improve objective measures such as peak and mean urinary flow rates. Again, several mechanisms of action have been proposed including an improvement in detrusor activity, a reduction in prostatic urethral resistance, inhibition of 5-alpha reductase activity, and an influence on androgen metabolism in the prostate (119).

Other Extracts

South African Star Grass (Hypoxis rooperi), Opuntia (Cactus flower), stinging nettle, and Pinus (Pine flower) have also been studied and used, however the data numbers are small and the types of trials do not allow conclusions to be drawn at this stage (116).

MEDICAL THERAPY FOR BPH – MONOTHERAPY AGENTS

In 1986, Caine (62) proposed that infravesical obstruction in men with symptomatic BPH comprised both static and dynamic components. The static component of obstruction is related primarily to the mechanical obstruction caused by the enlarging prostatic adenoma whereas the dynamic component is principally determined by the tone of the prostatic smooth muscle. Two avenues for pharmacotherapy have therefore evolved, namely shrinking the prostate tissue or relaxing the smooth muscle of the prostate. Prostatic smooth muscle tone is under the influence of the autonomic nervous system. Thus, any pharmacologic agent that may interfere with the functioning of this system could alter resistance in smooth muscle tone and resulting symptoms.

Medical therapy is now first-line treatment for most men with symptomatic BPH. They are non-invasive, reversible, cause minimal side effects, and significantly improve symptoms (81,136). With these recommendations, the rates of prescriptions for the medical management for BPH have increased drastically over the past decade (137,138). This increased interest has further led to the development of safer, more efficacious agents.

Alpha-Blockers

There are 3 main components to clinically significant BPH: static, dynamic and detrusor muscle components as outlined above. The dynamic component is associated with an increase in smooth muscle tone of the prostate. These smooth muscle cells contract under the influence of noradrenergic sympathetic nerves, thereby constricting the urethra (139). Prostatic tissue contains high concentrations of both alpha₁ and alpha₂ adrenoceptors – 98% of the alpha₁ adrenoceptors are associated with stromal elements of the prostate (140). Thus alpha₁-receptor blockers relax smooth muscle, resulting in relief of bladder outlet obstruction that enhances urine flow (87). Different subtypes of alpha₁ receptors have been identified, with alpha_1A predominating. Two alpha_1A-adrenoceptors generated by genetic polymorphism have been identified with different ethnic distributions but similar pharmacologic properties (36).

It was demonstrated in 1978 that phenoxybenzamine, a non-selective alpha₁/alpha₂ blocker, was effective in relieving the symptoms of BPH (141). Side effects were significant and included dizziness and palpitations. Many of the side effects of the alpha-blockers were mediated by alpha₂-receptors (142).Thus, alpha₁ selective antagonists such as terazosin, doxazosin, and prazosin and were developed that had fewer side effects than phenoxybenzamine (67). Doxazosin, alfuzosisn, and terazosin have gained favor in clinical practice because they are longer acting than prazosin. Due to side effects, many alpha₁ selective antagonists need to be titrated and are often started at the lowest dose and built up over time to the maximal dose or a dose where clinical effects are satisfactory.

More recently, highly uro-selective alpha_1A selective agents have been introduced including tamsulosin and silodosin. Due to the uro-selective nature, there is significant reduction in risk of systemic side-effects when compared to the less selective agents. However, the increased potency of these agents results in an increased compromise to bladder neck function and as a result, increases the risk of ejaculatory dysfunction.

PRAZOSIN

Prazosin (titrated up to 5mg day) has been shown to significantly increase flow rates by 36-59% compared to placebo 6-28% but 17% of men discontinued the drug due to side effects such as dizziness (21%), headache (14%), syncope (3.4%) and retrograde ejaculation (13%).

ALFUZOSIN

Alfuzosin (5 mg bid or 10 mg daily) has shown symptom score reduction of 31-65% (compared to placebo 18-39%) and flow rate increases of 22-54% (compared to placebo 10-30%). Hence the results were similar to those of prazosin but with only 3-7% discontinuations due to dizziness (3-7%), headache (1-6%) and syncope (<1 %) (143,144).

TERAZOSIN

Terazosin (2-10mg) had a symptom score reduction of 40-70% (compared to placebo 16-58%) and improved flow rates 19-40% (placebo 5-46%). Between 9-15 % of men discontinued the drug, related to dizziness (10-20%), headache (1-7%), asthenia (7-10%), syncope (0.5-1.0%), and postural hypotension (3-9%). Thus, terazosin was effective and superior to placebo in reducing symptoms and increasing the peak urinary flow rate. The effect of terazosin on the peak urinary flow rate was apparent in studies as soon as 8 weeks of therapy. Most importantly, the effect of terazosin on symptoms and peak urinary flow rate was independent of the baseline prostate size for the range of prostate volumes reported (145).

DOXAZOSIN

Doxazosin (4-12mg/day) is a selective alpha₁-adrenoceptor antagonist, and produced a significant increase in maximum urinary flow rate (2.3 to 3.6 ml. per sec) at doses of 4 mg, 8 mg and 12 mg, and in average flow rate compared with placebo. The increase in maximum flow rate was significantly greater than placebo within 1 week of initiating therapy and the drug significantly decreased patient-assessed total, obstructive, and irritative BPH symptoms. Blood pressure was significantly lower with all doxazosin doses compared with placebo. Adverse events, primarily mild to moderate in severity, were reported in 48% of patients on doxazosin compared to 35% on placebo, with only 11% discontinuing treatment (a similar number to placebo). The main side effects were dizziness (15-24%), headache (12%) and hypotension (5-8%), and abnormal ejaculation (0.4%) (146,147).

TAMSULOSIN

Tamsulosin (0.4 mg once or twice daily dose) is a selective alpha blocker for the alpha_1A subtype which predominates in the human prostate, having 12 times more affinity for the receptors in the prostate than in the aorta thereby reducing side effects mediated through blood vessels receptors. Symptom scores were reduced by 20-50% (placebo 18-30%), flow rates improved 20-45% (placebo 5-15%) but only 3-7% of men discontinued drug because of dizziness (3-20%), headache (3-20%), syncope (0.3%), and retrograde ejaculation (5-10%). The rate of retrograde ejaculation was much higher than alfuzosin but the blood pressure lowering side effects are less with tamsulosin(148). There are different formulations including extended release with lower pharmacological peaks and troughs which may offer fewer side effects.

SILODOSIN

Silodosin (8mg daily) is a highly selective blocker for the alpha_1A receptor subtype. It has the highest affinity for alpha_1A receptors of the medications discussed here. Symptoms scores were reduced by 40-50% (placebo 20-30%), flow rates improved by 17-30% (placebo 5-14%). Despite these favorable urinary outcomes, a significant proportion of patients experienced ejaculatory dysfunction (13-23%). These rates are higher compared to tamsulosin, however discontinuation rates secondary to ejaculatory dysfunction remains at 1-2%. Typical side effects include thirst (10%), loose stools (9%) and dizziness (5%) (149,150). Similar results were found in a recent meta-analysis of silodosin. Compared to tamsulosin, the combination of 13 studies found silodosin showed little to no difference in urological symptom scores and quality of life whilst increasing sexual adverse events. The same results were reported when silodosin was compared to naftopidil and alfuzosin (151).

Several meta-analyses have demonstrated that all non-selective alpha1-adrenoceptor antagonists seem to have similar efficacy in improving symptoms and flow rates (152). The difference between non-selective alpha 1-adrenoceptor antagonists is related to their side effect profile. Overall, alfuzosin appear to be better tolerated than doxazosin, terazosin and prazosin(153). More recent analyses suggest that the highly uro-selective alpha_1A blockers are more efficacious compared to non-selective alpha blockers with regards to urinary symptoms and urine flow improvement (154-156). Further, these highly selective agents appear to have a favorable systemic side-effect profile at a cost of ejaculatory function when compared to non-selective alpha blockers.

Table 12. Commonly Used Alpha-Blockers
Group	Drug
Nonselective alpha blockers	· Phenoxybenzamine · Nicergoline · Thymoxamine
Selective alpha1 blockers	· Prazosin · Alfuzosin
Super-selective alpha1_A blockers	· Tamsulosin · Silodosin
Long-acting alpha1 blockers	· Terazosin · Doxazosin

5-Alpha Reductase Inhibitors

The enzyme 5-alpha reductase is crucial in the amplification of androgen action in the prostate by modulating the conversion of testosterone to DHT (Figure 5). Within the prostate, 90% of testosterone is converted to DHT (78,157). There are 2 isoforms of the enzyme 5-alpha reductase which are encoded by separate genes (158). Type 1 isoenzyme is expressed highly in the skin, liver, hair follicles, sebaceous glands, and prostate whereas type 2 is responsible for male virilization of the male fetus, and in adulthood resides in prostate, genital skin, facial and scalp follicles (159,160). Inhibitors of these enzymes potentially decrease serum and intra-prostatic DHT concentrations, thus reducing prostatic tissue growth.

FINASTERIDE

Finasteride was the first of these to be studied in humans and shown to decrease DHT concentrations (161). It acts predominantly on the type 2 isoenzyme of 5-alpha reductase. There is some evidence that patients on finasteride experience fewer serious complications associated with the progression of BPH compared with those prescribed an alpha blocker, such as acute urinary retention or undergoing BPH-related surgery, but more prospective data is needed (162). Finasteride reduces serum DHT concentrations by 65-70% and prostatic concentrations by 85-90%, although the intraprostatic concentrations of testosterone are reciprocally elevated as the testosterone is not being converted to DHT.

Because 5-alpha reductase inhibitors work by reducing prostatic tissue volume, baseline prostate size has a significant impact on its efficacy with larger glands (>50ml) being likely to respond (163,164). After treatment for one year with finasteride, there was a significant decrease (17-30%) in total gland size with the greatest size reduction in the periurethral component of the prostate, which has the greatest impact on obstructive symptoms (78,165,166). There was a 60-70% decrease in serum DHT concentration, a 25% decrease in prostate volume, and a symptom score reduction of 13-30% (vs placebo 4-20%). Urinary flow rate improved 7-20% (vs placebo 3-15%) and was more pronounced with prostates > 40ml. The side effect profile included a decreased libido in 10%, ejaculatory dysfunction in 7.7%, and impotence in 15.8%. But adverse events resulted in only 4% of patients discontinuing treatment (117,167). There was a 50% reduction in the risk of AUR and in the need for surgery (30%). Finasteride has also found a role in the treatment of BPH-related hematuria although its role in reduction of perioperative bleeding is not well defined (84,117).

More recently, as part of the Prostate Cancer Prevention Trial involving over 18,000 men, it was concluded that finasteride delays the appearance of prostate cancer whilst reducing the risk of urinary problems. However, there was a reported increased risk of high-grade prostate cancer leading to the discontinuation of this study. This point remains controversial as some believe due to the gland shrinking that sampling was altered and by virtue of a smaller area the likelihood of finding an aggressive tumor was increased (168). In any case, the benefits in terms of improved LUTS needs to be weighed against the potential sexual side effects and potential small but significant increased risk of high-grade prostate carcinoma (169,170) and compared to the option of using adrenoreceptor blockers. Despite the findings, more evidence is needed before advising patients to cease finasteride. However, they do need to be counselled on the small, but significant risks of developing aggressive prostate cancer (171).

DUTASTERIDE

Dutasteride unlike finasteride blocks both the type I and Type II 5-alpha reductase isomers showing a 60-fold greater inhibition of the type 1 isoenzyme than finasteride plus activity against the type 2 isoform (23,117). In terms of monotherapy, a one year randomized, double-blinded comparison of finasteride and dutasteride in men with BPH (EPICS: Enlarged Prostate International Comparator Study) found a trend for dutasteride improvement over finasteride in IPSS (International Prostate Symptom Score) that did not reach statistical significance (abstract) (172). Another non-randomized comparative trial with 240 patients, published only in abstract form, showed a small improvement in AUASI and Q_max for dutasteride (173). However, dutasteride and finasteride have never been compared in long-term therapy, either as monotherapy or in combination with an alpha-blocker. These medications appear to exert continued effects beyond 1 year so comparison after only 1 year is likely to be premature.

The tolerability of 5-alpha reductase inhibitors in most studies has been excellent with the most relevant adverse effects being related to sexual function. They include reduced libido, erectile dysfunction, and, less frequently, abnormal ejaculation (74,174). Specifically for dutasteride in the Combat study (175), in the monotherapy arm of 1623 patients the side effect were: erectile dysfunction (6.0%) ; retrograde ejaculation (0.6%); altered (decreased) libido (2.8%); ejaculation failure (0.5%); semen volume decreased (0.3%); loss of libido (1.3%); breast enlargement (1.8%); nipple pain (0.6%); breast tenderness (1.0%), and dizziness (0.7%).

As with finasteride, the REduction by DUtasteride of prostate cancer Events (REDUCE) trial now fully reported has demonstrated similar results to the PCPT trial in reducing prostate cancer (176). Again, a higher risk of developing more aggressive cancer was demonstrated- but in this study it was not statistically significant. Indeed, some organizations such as the Canadian Urological Association have been dismissive of this point in recent guidelines (171). Needless to say, careful counselling of men regarding this issue is again required, particularly for younger men who will be on dutasteride for many years.

5-Alpha Reductase Inhibitors to Reduce Hematuria and Intraoperative Hemorrhage for Prostate Surgery

While considered an off-label use, there is some evidence that suggests that 5-alpha reductase inhibitors may be useful in the setting of (177):

Recurrent hematuria secondary to BPH
To reduce gland size and/or impact on angiogenesis to reduce intraoperative bleeding for prostate surgery.

No large randomized trials exist but an extensive summary of the literature is available (177).

Phosphodiesterase 5 Inhibitors

Phosphodiesterase 5 (PDE5) inhibitors (e.g., sildenafil, tadalafil and vardenafil) have been used predominantly to treat erectile dysfunction in men. However, recent data suggest they are effective for the treatment of LUTS secondary to BPH. Specifically, the cyclic nucleotide monophosphate cyclic GMP represents an important mediator in the control of the lower urinary tract outflow region (bladder, urethra). PDE5 inhibitors exert effects by several mechanisms including: calcium-dependent relaxation of endothelial smooth muscle, alteration of the spinal micturition reflex pathways, and increased blood flow to the lower urinary tract. PDE inhibitors are regarded as efficacious, have a rapid onset of action, and favorable effect-to-side-effect ratio (178).

The rationale for using tadalafil for BPH stems from the following three observations: first, the prevalence of LUTS, BPH, and erectile dysfunction (ED) increases with age; second, phosphodiesterase-5 inhibition mediates smooth muscle relaxation in the lower urinary tract; and third, early evidence demonstrates that PDE5 inhibitors such as tadalafil are successful in treating LUTS and ED (179). Results of several randomized controlled trials have demonstrated reproducible reductions in IPSS, symptoms, and improved quality of life compared to placebo. Data suggests tadalafil 5mg improves IPSS by 22-37% and the improvement occurs within one week of commencement, with a duration of 52 weeks (180). The adverse event profile was acceptable and consistent with that previously reported in men with ED (blurred vision, headache, back ache, nausea, etc.), with discontinuation rates of 2%. Not unexpectedly, in the same study tadalafil significantly improved the International Index of Erectile Function-Erectile Function score in sexually active men with erectile dysfunction at twelve weeks. Meta-analytical data confirms these findings suggesting that PDE5 inhibitors improve IPPS and erectile function, with no significant effect on maximal urinary flow rate (181). Other PDE5 inhibitors are being studied including sildenafil and vardenafil (178). The theoretical advantage is treating BPH and erectile dysfunction with one agent (182). To date, tadalafil is the only PDE5 inhibitor that is FDA approved for use for the treatment of BPH. Data on the long-term effects on symptoms and disease progression is not available at present.

Anticholinergic Medications

High-level evidence suggests that for selected patients with bladder outlet obstruction due to BPH and concomitant detrusor overactivity, combination therapy with an alpha-receptor antagonist and anticholinergic can be helpful (183).Such agents help particularly with the irritative urinary symptoms of frequency and urgency. Caution is recommended, however, when considering these agents in men with an elevated residual urine volume or a history of spontaneous urinary retention (171).

Botulinum Toxin A Injection

Injection of botulinum toxin A into the prostate is a novel treatment for LUTS secondary to BPH. First reported in 2003 (184), trans-perineal injection of 100 units of botulinum toxin into each lobe of the prostate under trans-rectal guidance is required. In this randomized controlled trial, thirty patients demonstrated significant improvement in IPSS (65% decrease) and serum PSA (51% decrease) compared to controls, who had injections of saline without botulinum toxin A, at a median follow-up of 20 months. Subsequent long-term follow-up of 77 patients up to 30 months has shown similar results – significant reduction in IPSS (approximately 50% lower), significant improvement in maximum flow rate (approximately 70% higher), and significant reduction in serum PSA values (approximately 50% lower).Importantly, no adverse events were noted (185).

Summary of Monotherapy Medical Treatment

The first line of medical treatment is an alpha-blocker, as the majority of patients treated have a prostate volume of less than 40ml. In men with larger prostates (greater than 40cc), a 5-alpha reductase inhibitor (e.g., finasteride or duasteride) alone or in combination with an alpha-blocker would be appropriate. Patients who are likely to respond to 5-alpha reductase inhibition will do so at the same relative magnitude as an alpha-blocker, but it will take a longer period of time (months as opposed to weeks). There is likely to be a 20-30 reduction in symptoms and a 1-2ml per second increase in urinary flow (167). Side-effect profiles of medical treatments are also important, as discussed above. For example, with regard to sexual function, tamsulosin and silodosin have an increased risk of retrograde ejaculation and finasteride increases sexual dysfunction (74). These may be important factors in choosing therapies. Finally, the emergence of PDE5 inhibitors for the treatment of men with LUTS secondary to BPH alters the landscape with an ability to treat men with BPH and ED with one agent. Multiple randomized trials and associated meta-analyses demonstrate the reproducible benefits of PDE5 inhibitors on urinary and erectile function.

MAJOR STUDIES OF MEDICAL TREATMENT OF BPH

The medical treatment of clinical BPH has come under increasing scrutiny through larger trials that have become imperative for their introduction into clinical practice. Some of these larger trials have been selected and are discussed below.

Veterans Affairs Study

In the Veterans Affairs Cooperative Studies Benign Prostatic Hyperplasia Study Group (186), a total of 1,229 subjects with clinical BPH were randomized to 1 year of placebo, finasteride, terazosin or drug combination. The primary outcome measures were the AUA symptom score and the peak urinary flow rate. The percentage of subjects who rated improvement as marked or moderate with placebo, finasteride, terazosin and combination was 39, 44, 61 and 65%, respectively, only the latter two were superior to placebo. There was no significant relationship between baseline prostate volume and treatment response to finasteride or with the other treatments (terazosin or combination). There was a significant but weak relationship between change in AUA symptom score and peak flow rate in the finasteride and combination groups. The symptom responses with terazosin were not related to peak flow rate or baseline prostate volume. In men with clinical BPH, finasteride and placebo are equally effective, while terazosin and combination are significantly more effective. In men with clinical BPH and large prostates, the advantage of finasteride over placebo in terms of symptom reduction, impact on bother due to symptoms and quality of life was small at best, while the advantage of terazosin (alone or in combination with finasteride) over finasteride alone and placebo was highly significant. The authors concluded that alpha₁blockers, such as terazosin, should be first line medical treatment for BPH(186). Another arm of this study observing surgical treatment versus watchful waiting is discussed below.

PLESS Study

The Proscar Long-term Efficacy and Safety Study (PLESS) was a 4-year, randomized, double-blind, placebo-controlled trial assessing the efficacy and safety of finasteride 5mg (Proscarä) in 3040 men, aged 45 to 78 years, with symptomatic BPH, enlarged prostates on TRUS volume criteria, and no evidence of prostate cancer (187,188). Finasteride use reduced the risk of developing acute urinary retention by 57% and the need for BPH-related surgery by 55% (189) in comparison to placebo. A modified AUA symptom score was used (because trial was undertaken prior to formal AUA being developed) and showed a statistically significant reduction in mean score of 3 for finasteride and 1.2 for placebo, starting at a level of 15 for both groups (188). Compared with placebo, men treated with finasteride experienced an increased incidence of new drug-related sexual adverse events (erectile dysfunction, decreased libido, ejaculation disorder) only during the first year of therapy with 4% of men discontinuing because of such events (187).

PREDICT Trial

The Prospective European Doxazosin and Combination Therapy (PREDICT) Trial was constructed to evaluate the efficacy and tolerability of the selective alpha₁-adrenergic antagonist doxazosin and the 5-alpha reductase inhibitor finasteride, alone and in combination, for the symptomatic treatment of benign prostatic hyperplasia. It was a prospective, double-blind, placebo-controlled trial involving 1,095 men aged 50 to 80 years. The dose of finasteride was 5 mg/day. Doxazosin was initiated at 1 mg/day, and titrated up to a maximum of 8 mg/day over approximately 10 weeks according to the response of the maximal urinary flow rate (Qmax) and IPSS. An intent-to-treat analysis of 1,007 men showed doxazosin and doxazosin plus finasteride combination therapy produced statistically significant improvements in total IPSS and Qmax compared with placebo and finasteride alone. Finasteride alone was not significantly different statistically from placebo with respect to Qmax or total IPSS. The treatments were generally well tolerated. They concluded that doxazosin was effective in improving urinary symptoms and urinary flow rate in men with benign prostatic hyperplasia, and was more effective than finasteride alone or placebo. The addition of finasteride did not provide further benefit to that achieved with doxazosin alone (146).

MTOPS Study and Predictors of Clinical Progression

The Medical Therapy of Prostatic Symptoms (MTOPS) study is a double-masked, placebo-controlled, multi-center, randomized clinical trial with 4 study arms 1) placebo; 2) doxazosin (4 to 8 mg); 3) finasteride (5 mg) and 4) combination of both doxazosin and finasteride. 3,047 men were randomized equally to the 4 groups (190). Baseline parameters analyzed included age at randomization, transrectal ultrasound (TRUS) volume, AUA symptom score, Qmax, PVRU, and PSA. Reduction in the risk of BPH progression was analyzed by one covariate at a time regression models of absolute risk of BPH progression versus baseline covariates. Groups compared were combination versus doxazosin, combination versus finasteride and finasteride versus doxazosin (190).

In the main finding, disease progression, defined as an increase in AUASS (American Urology Association Symptom Score- similar to IPSS to score LUTS) of 4, AUR, renal insufficiency, recurrent UTIs and urinary incontinence, was prevented equally by doxazosin and finasteride with an even greater effect when both medications were combined. In conflict with the Veterans Affairs and PREDICT studies, finasteride alone did improve overall symptoms and peak urine flow compared to placebo at 4 years and with even more so when combined with doxazosin (190). This finding coincides with the long-term open label ARIA dutasteride study described above showing a cumulative symptom benefit of treatment up to 4 years (191).

A small part of the MTOPS study focused on the routinely available measure of serum PSA, in an attempt to predict a patient’s future risk of BPH clinical progression, acute urinary retention and BPH-related invasive therapy, permitting an informed decision concerning the value of medical therapy over watchful waiting (192). In MTOPS, 737 patients were assigned to placebo and followed for an average of 4.5 years. Clinical progression of BPH was pre-defined as either a 4-point increase in AUA symptom score, acute urinary retention, incontinence, renal insufficiency, or recurrent UTI. The need for BPH-related invasive therapy was a secondary outcome. These data are summarized in Table 13, where those having a lower PSA, had a lower rise in symptom score, and a reduced risk of acute urinary retention or invasive treatment compared to those with a higher PSA. The sub-group with the highest baseline PSA was also likely to have larger prostate glands, making the findings intuitive. However, as with many such findings, translating an individual PSA to a population study is difficult, as other factors will determine progression or regression of symptoms, not just PSA.

Table 13. Progression of BPH Symptomatically of Placebo Group only, to AUR or Further Intervention Based on Baseline PSA (adapted from Kaplan et al).
Baseline PSA tertiles (ng/ml)	Progression of symptom score (points)	Acute urinary retention Risk over study period	Invasive Treatment Risk
<1.2	3.10	0.18	0.6
1.2-2.5	3.47	0.35	1.33
>2.5	7.21	1.46	2.13

CombAT Trial

Combination therapy with a 5-alpha reductase (dutasteride) and the alpha blocker, tamsulosin, in men with moderate-to-severe benign prostatic hyperplasia and prostate enlargement was also further studied in the Combination of Avodart™ and Tamsulosin™ (CombAT) trial. The rationale was the same as those outlined for the MTOPS trial. In summary, it is a 4-year, global, multicenter, randomized, double-blind, parallel-group study designed to investigate the benefits of combination therapy with the dual 5-ARI dutasteride and the alpha-blocker tamsulosin compared with each monotherapy in improving symptoms and long-term outcomes in men with moderate-to-severe symptoms of BPH and prostate enlargement. Symptoms and long-term outcomes (AUR and surgery) were assessed as separate primary endpoints at two and four years, respectively. Eligible patients were at least 50 years old with prostate volume ≥30 cm³ and PSA level ≥1.5 ng/mL. Almost 5,000 men were enrolled (193). Perhaps the only criticism is the lack of placebo control arm in the study.

The results at four years (194) demonstrated that combination therapy was superior to tamsulosin monotherapy but not dutasteride monotherapy at reducing the relative risk of AUR or BPH-related surgery. Combination therapy was also significantly superior to both monotherapies at reducing the relative risk of BPH clinical progression. Combination therapy provided significantly greater symptom benefit than either monotherapy. Safety and tolerability were reasonable and in line with expectations for both medications. Certainly, at four years the CombAT data supports the long-term use of dutasteride and tamsulosin combination therapy in men with moderate-to-severe LUTS due to BPH and prostatic enlargement.

EMERGING COMBINATION THERAPY REGIMES

With the increasing body of evidence supporting the use of PDE5 inhibitors in the setting of BPH, a number of trials support its use in combination therapy. To date, there are smalls studies of alfuzosin and tadalafil, tamsulosin and sildenafil, and tamsulosin and vardenafil. These early studies suggest that combination therapy is more effective than monotherapy for urinary and erectile function with a good safety profile (195,196). A meta-analysis of 11 randomized controlled trials (n=855) looked at alpha blockers with or without PDE5 inhibitors. This analysis found men receiving PDE5 inhibitors had a mean improvement of 1.66 points on IPSS, mean increase of 0.94 ml/s maximum urinary flow rate, and improved erectile function (197). Larger series with longer-term follow up is required to definitively define the role of these combination therapies in current practice.

Summary of Combination Therapy for Men with BPH

In the larger studies where the standard endpoint of prostate symptom score was measured, a greater impact of dutasteride over tamsulosin was observed. Considering urinary flow rate (Q_max), combination therapy outperformed dutasteride in those with PSA and prostate volumes above the 75 percentile. Clearly, those with larger prostates and higher PSAs derive a greater benefit with dutasteride coinciding with the size reduction impact of this drug.

In summary, the results of the MTOPS and CombAT trials both suggest combination therapy is better than 5-alpha reductase monotherapy at the 4-year mark. The higher incidence of adverse effects, the increased cost of combination therapy, and the need for prolonged therapy argue for a reductionist medical approach to this condition. One recent small study investigated the discontinuation of 5-alpha reductase inhibitors in patients on combination therapy and found prostate regrowth and worsening of symptoms after 1 year of cessation, emphasizing the importance of 5-alpha reductase inhibitors in prolonged therapy (198). In an opposing design, the SMART trial (Symptom Management After Reducing Therapy) observed the effect of removing the alpha blocker (tamsulosin) after 6 months of combined therapy with dutasteride (199). With I-PSS as the primary outcome, the investigators found that 77% of patients had symptoms that were the same or better after only 3 months of alpha blocker removal. In reference to the CombAT study, the effects of dutasteride continue past two years suggesting that removal of the alpha blocker at later time points may be even less noticeable. However, the CombAT study is quite powerful as it does demonstrate that the natural history of BPH is not altered by taking alpha blocker alone. The rate of AUR and need for surgery was unaltered at about 18%, and thus while tamsulosin helps LUTS, it does not alter disease progression. Combination therapy did lead to reductions in prostate volume, and changed natural history to reduce rates of AUR and surgery. The largest benefit was in the men with the largest glands.

The emergence of newer agents including PDE5 inhibitors gives rise to an increasing number of combination-therapies under investigation. Long-term follow-up is required on these newer combinations. As such, combination treatment will continue to shape the management of BPH for years to come.

SURGICAL TREATMENT OF BPH

Invasive Surgical Therapies

Traditionally prostatectomy by an open approach or TURP has been considered the gold-standard for refractory or complicated BPH (indications listed in Table 14). At present approximately 90% of prostatectomies are done by TURP. Open prostatectomy should be considered when a gland is estimated to weigh more that 75g, where large bladder calculi exist that may not be dealt with endoscopically, where large bladder diverticula requiring repair exist, if complex urethral conditions or when orthopedic abnormalities prevent positioning in lithotomy for TURP. Contraindications to open prostatectomy include a small fibrous gland, prostate adenocarcinoma, previous prostatectomy or other surgery of the pelvis preventing access (200).

Table 14. Indications for Prostatectomy

· Acute Urinary Retention

· Recurrent or persistent urinary tract infections

· Significant bother from LUTS secondary to bladder outflow obstruction not responding to medical therapy

· Recurrent hematuria known to be of prostatic origin

· Bladder Calculi

TRANSURETHRAL RESECTION OF THE PROSTATE (TURP)

TURP remains the most common surgical treatment for BPH (201) and remains the ‘gold standard’ by which other surgical (and even medical) treatments are measured (74). TURP involves either regional or general anesthesia, with most patients spending a minimum of one night in the hospital. TURP involves surgically debulking the periurethral and transitional zones of the prostate to relieve obstruction. Debulking is done by electrocautery in the standard TURP through endoscopic instruments introduced into the urethra and bladder. Tissue is resected in small pieces until the hyperplastic tissue is removed and a new channel for passage in the prostatic urethra created in the capsule left behind, much like fashioning a pumpkin for a Halloween jack o’lantern. Despite using electrocautery, there are mild to severe degrees of hemorrhage, depending on the gland size. However, transfusions are rarely needed and the procedure is relatively free of life-threatening complications and most patients experience satisfactory resolution of their micturition symptoms. Studies on urinary peak flow rates and invasive pressure flow have demonstrated the superiority of TURP over minimally invasive therapies (202). Complications of TURP include failure to void (6%), hemorrhage requiring transfusion (1-4%), clot retention (3%), infection (2%), bladder neck contracture or urethral stricture (6%), transurethral resection syndrome (2%), and rarely incontinence (80,203,204).

TURP is plagued by the potential for morbidity, specifically: retrograde ejaculation, erectile dysfunction, and urinary incontinence. Retrograde ejaculation is reported to occur in almost all patients undergoing TURP as the normal bladder neck mechanism which contracts to allow antegrade ejaculation is surgically resected. Counselling prior to surgery must include a discussion of the impact on sexual performance and also fertility. Erectile dysfunction (ED) may be associated with TURP either via thermal nerve injury or emotional stress and was reported in early studies at a rate of 4-40%. This has now been shown to be an overestimation (74,206). The rate of ED in the AUA Cooperative study was found to be 13% in 1,000 men (80), however this must be compared to increases of around 20% of ED in untreated groups with BPH. Although ED is often quoted as a side effect of TURP, Kassabian concluded that TURP (or even any other surgical therapy) did not appear to have a long-term effect on erectile function or libido (74). Incontinence is infrequent and typically is a result of intra-operative damage to the external urinary sphincter. Large pooled analysis revealed rates of incontinence following TURP of around 1% (207).

There has been one randomized controlled trial (Veterans Affairs Cooperative Study Group, see above) comparing TURP to “watchful waiting” or reassurance (203). This demonstrated that TURP showed greater benefit with 66% of patients having a decrease in symptoms post TURP compared to 28% who were undergoing ‘watchful waiting”.

One significant modification to the standard TURP using monopolar cautery with glycine as an irrigant has been the use of bipolar cautery using normal saline as the irrigant. The latter has been termed bipolar transurethral resection in saline (TURIS). Glycine alters serum osmolality when absorbed through venous channels in the prostate as the system is under pressure which potentially leading to hyponatremia and also glycine directly itself has an impact on the nervous system. This syndrome is termed “TURP syndrome” and dictates that monopolar resections should be abandoned at around the one-hour mark or when significant venous breach occurs. The TURIS or bipolar technologies thus have the advantage of the ability to carry out resections for a longer time due to very few issues with absorption of saline systemically as opposed to glycine. Large series meta-analysis illustrated comparable efficacy and morbidity profiles when compared to monopolar TURP (207,208). A further modification of the bipolar technology is so called plasmakinetic vaporization where some data is emerging (209,210). This vaporizes rather than resects the prostate tissue. Compared to TURP, plasmakinetic energies results in similar improvements to IPSS up to 12-month follow up (211).

LASER THERAPY FOR BPH – ENUCLEATION OR VAPORIZATION OF THE PROSTATE

There are several evolving therapies for BPH involving various lasers including Nd-YAG, Holmium, and now Thulium lasers. These laser energies may be utilized in various methods to resect, enucleate, or vaporize the prostate. Laser as an energy source has an advantage over standard electrocautery by being relatively bloodless and does not carry the risk of hyponatremia, which may rarely occur via absorption of irrigation fluid in a standard TURP (212).

Photoselective Vaporization of the Prostate (PVP)

The characteristic 532-nm wavelength laser is selectively absorbed by hemoglobin within prostatic tissue (213,214). Introducing this energy to the prostate results in selective vaporization of prostatic tissue, with effective hemostasis and relatively little tissue coagulation (1.5 – 0.3mm margin). Initially launched as a 60W prototype, the laser was ultimately introduced to the urology community as an 80W system that has been the predominant device used in clinical trials. This first generation used an Nd:YAG laser beam passed through a potassium-titanyl-phosphate (KTP) crystal, halving the wavelength (to 532nm), doubling the laser's frequency, and resulting in a green light. In 2006, the 120W lithium triborate laser (LBO) laser was introduced using a diode pumped Nd:YAG laser light that is emitted through an LBO instead of aKTP crystal, resulting in a higher-powered 532 nm wavelength while still using the same 70-degree deflecting, sidefiring, silica fiber delivery system. More recently, a 180W version has been released (215). This increase in energy corresponds with reduced lasering and operating time (216). Two-year data from the GOLIATH trial illustrates that the 180W version provides durable symptom improvement that is comparable to traditional TURP (217).

Compared to TURP, PVP has been shown to have an improved side-effect profile, time of catheterization, hospital stay, and improvement in urinary flow rate (218,219). Clinically, the advantage of PVP is that the length of stay in hospital is usually under 24 hours and it can be performed on anticoagulated patients. Outcomes have demonstrated a reduced frequency and severity of clinical complications, however it was limited to smaller prostate sizes (215).

In summary, several laser wavelengths (Potassium titanyl phosphate [KTP], Holmium:Yttrium aluminum garnet [Ho:YAG], Thulium), and delivery systems (end-firing; side-firing; interstitial) are available for PVP, and each has particular characteristics and potential advantages (171,219). In current practice, the use of 532nm 180W PVP (Greenlight) lasers is becoming increasingly more common due to significantly reduced operative times.

Holmium:YAG Laser for Enucleation (HoLEP) or Resection of the Prostate (HoLRP)

This laser may be used to enucleate the prostate and remove the tissue in pieces (HoLEP) or to vaporise the tissue (HoLRP). HoLRP is an operation involving laser resection of the prostate tissue via an endoscope, similar to a standard TURP using electrocautery as outlined above. The fragments of prostate tissue are made small enough to irrigate out prior to detachment from the prostate (220). HoLEP again uses a Holmium laser but the laser acts like a finger would at an open prostatectomy, shelling out tissue until it floats in the bladder. The tissue is then morcellated and extracted. This technique may be safely used in large prostate glands (those weighing >100g) as an alternative to open prostatectomy as discussed below (212). Initial studies have demonstrated that HoLEP improved flow rates by 56-119% and by TURP 96-127%, and symptom scores reduced in both groups by 60%. Further, these studies reported a reduced length of hospital stay, clot retention rates, the occurrence of hyponatremia, strictures but had a slightly higher risk of reoperation (221-223). Pooled data of recent randomized trials suggest HoLEP results in significantly improved maximal flow rate, IPSS, transfusion rate at a cost to operative time (224). Patients are usually kept in hospital a little longer with the Holmium:YAG compared to the PVP technique. However, the Holmium:YAG laser has a longer track record. The disadvantage is that treatment with the Holmium:YAG is quite a complex procedure to learn as it widely resects all the prostatic tissue. HoLRP with its inherent wavelength and laser properties is not photoselective for prostate tissue and as such causes more coagulation and necrosis and has not been popular as a therapeutic intervention.

Thulium Laser

A two-micron continuous-wave is produced with a wavelength of 2013nm. This wavelength is close to the water absorption peak in tissue. This provides several advantages including excellent hemostasis with minimal thermal injury to surrounding tissue. Tissue may be incised accurately or vaporized depending on the settings utilized. Initial reports in 2005 reported the use of a 50-Watt Thulium: YAG (Tm:YAG) laser (225). More recently an improved 120 W laser has been produced, allowing for up to 1.08g of vaporization per minute (226). With the high degree of accuracy of focal ablation, various resection techniques have been reported including: Tm laser resection of the prostate-tangerine technique (TmLRP-TT), Tm vaporization (ThuVaP), Tm vaporesection (ThuVaRP), Tm vapoenucleation (ThuVEP), Tm enucleation (ThuLEP). Combinations of the available techniques allow prostate removal rates to be increase to 2-3 grams per minute (227).

When compared to TURP, TmVaRP offered similar urinary symptom improvement however TURP was superior in improving max voiding velocity post operatively (228). Furthermore, no improvements in reduced blood loss or decreased length of hospital stay were observed (228). Similarly, a meta-analysis of four clinical trials comparing ThuVEP and HoLEP showed both lasers were effective in reducing BPH symptoms but found ThuVEP to have slightly reduced blood loss and shorter lasting urinary incontinence post procedure (229). This evidence largely suggests that the choice between TURP, HoLEP, and thulium laser is based on availability and surgeon experience.

Visual Laser Thermoablation of the Prostate (VLAP)

Alternate minimally invasive laser therapies such as VLAP rely on deep thermal coagulation of the prostate by Nd:YAG laser with later necrosis and sloughing of the prostate tissue (230). They are not photoselective for prostate tissue and do not vaporize the tissue as PVP lasers do. They require prolonged catheterization and have a failure rate of around 10% as reported by Chacko et al in a randomized trial in 2001 (231). Such therapies differ from debulking surgery and require a post-procedure period for resolution of symptoms with the advantages being lack of general or regional anesthesia. Durability and tolerability remain issues for such therapies with re-treatment rates between 10 and 49% (202). Certainly, further studies, using randomization, larger sample sizes, and comprehensive measures of outcomes and adverse events, are still needed to better define the role of laser techniques for treating benign prostatic obstruction (221).

OPEN SIMPLE PROSTATECTOMY

This is the oldest, most invasive therapy for BPH (232). This form of surgery was the standard for men with BPH for over a century however was often associated with complications and prolonged hospital stays. The number of simple prostatectomies being performed has declined since the introduction of TURP and laser energies.

It is commonly done through a transvesical approach, but may be done retropubically. Early complications of this operation include hemorrhage, blood transfusion, sepsis, and urinary retention with the most common late complication being bladder neck stricture (2-3%) (200). TURP has lower perioperative morbidity but open prostatectomy produces equivalent, if not superior improvement with a similar or lower re-operation rate (233). Sexual dysfunction is not likely to be altered by the surgery (74,234) however ED is still quoted at 3-5% risk (200). Retrograde ejaculation occurs in 90% patients. Other complications of surgery such as deep vein thrombosis, myocardial infarction, and stroke are less than 1% (200).

SUMMARY OF INVASIVE SURGICAL TECHNIQUES

Over the past decade, significant advances have been made regarding the invasive management for BPH. Traditionally, TURP has been reserved for refractory or complicated BPH. However, recent advances in laser technologies have resulted in a marked uptake in the use of laser prostatectomy. Novel approaches utilizing laser energy allows for the enucleation, resection, or ablation of prostatic tissue. Multiple meta-analyses demonstrate equivocal efficacy when comparing TURP and laser prostatectomy. In light of this information, in patients where prostatectomy is indicated it is reasonable to proceed with either of the energy sources discussed above based on surgeon and patient preferences.

Minimally Invasive Surgical Therapies (MIST)

Minimally invasive therapies for BPH have evolved in the past decade with the goal being to achieve symptomatic improvement that is durable, without the morbidity associated with surgery or the long-term side effects or compliance issues associated with medical therapies (202). The aim of such treatments is to achieve results similar to TURP but with minimal anesthesia, hospitalization, and morbidity. An overview of earlier randomized controlled trials in 2000 by Tubaro et al (235) comparing minimally invasive and invasive modalities of treatment found re-treatment rates to be higher in the minimally invasive group. They concluded that at the time, none of the minimally invasive treatments were superior to TURP from a cost and benefit standpoint and that TURP remains the gold standard of treatment.

More recently, an increasing number of therapeutic options have been developed to improve durability without limitation to the minimally-invasive approach. Multiple ablative (thermo or chemical) or mechanical options have been introduced with early data available. Accordingly, current practice suggests a markedly increasing use of MIST, particularly in the younger patients (236). While the precise role of MIST is not clear, some view such treatments as in-between medical and TURP and we await long term data on all proposed therapies.

TRANSURETHRAL INCISION OF THE PROSTATE (TUIP)

A similar approach to a TURP is used except that no surgical debulking is undertaken. Between one and three incisions are made into the prostate at the level of the bladder neck back almost to the insertion of the ejaculatory ducts. This releases the “ring” of BPH tissue at the bladder neck, creating a larger opening. There is a reduced risk of morbidity such as hemorrhage. In some instances, ejaculation may be preserved in younger men, especially if one incision is made. The procedure only works if the tissue in the periurethral area is not too bulky, otherwise a “ball-valve” mechanism of adenoma may develop. Therefore, TUIP should be recommended to men with smaller prostates (237). Laser may be used for incisions of the prostate, as well as standard electrocautery (212). Some studies have shown TUIP to have similar IPSS outcomes to TURP but lower urine peak flow rate. Understandably, TUIP has also been shown to give better outcomes in terms of ejaculatory function (238).

THERMO-ABLATIVE THERAPIES

Thermoablation is the principle underlying the several minimally invasive available treatments that have been introduced thus far (239) and these include transurethral microwave thermotherapy (TUMT), transurethral electrovaporization of the prostate (TUVP), and transurethral needle ablation (TUNA). Collectively, these therapies have been shown to have similar or decreased efficacy when compared to TURP but have a slightly better morbidity profile at this stage. Longer follow up data will determine the true efficacy and risk profiles for these thermo-ablative therapies.

Transurethral Microwave Therapy (TUMT)

An intraurethral antenna emits microwave radiation and delivers heat to a targeted region of the prostate. Histologically, this results in well-controlled coagulative necrosis. A number of series have been published reporting outcomes following TUMT. Multiple studies have compared TUMT versus TURP, which have demonstrated the sustained effect of mild symptom improvement when compared to TURP. A recent review reported a reduced efficacy when compared to TURP with regards to IPSS improvement at 12 months (65% decrease compared to 77% with TURP) and urinary flow rate (70% increase compared to 119% with TURP) (240,241). Retreatment rates are high, ranging between 10-22% compared to 4-8% following TURP. Despite this limitation to efficacy, TUMT provides significant benefits when compared to TURP including improved sexual function, hospitalization, hematuria, transfusions rates (242). Because of lower effectiveness compared to TURP, TUMT is considered a second line option at this stage (243).

Transurethral Electrovaporization of the Prostate (TUVP)

TUVP uses heat from a monopolar or bipolar high voltage electrical current to vaporize tissue (237). Theoretically this technique could have an ablative as well as coagulative effect. To date, a meta-analysis of randomized controlled trials comparing TUVP and TURP have shown no significant differences in IPSS, quality of life or post void residual volumes. Similar rates of complications have also been found however this is limited by short follow up durations (244,245). Furthermore, TUVP did not lead to a reduction in postoperative morbidity or shorter hospital stays (246).

Transurethral Needle Ablation (TUNA)

Radiofrequency ablation between two electrodes results in thermal ablation and resulting coagulative necrosis of tissue. Several randomized trials have been performed with only short-to-midterm follow up available. As with other forms of MIST, concerns regarding durability are present. A 5 year follow up demonstrated that 58% of patients had maintained symptom control, however 21% needed re-treatment (247). Meta-analytical data confirms an improved IPSS and urinary flow rate at one-year, however to a significantly lower magnitude when compared to TURP (248). Similar to TUMT and TUVP, TUNA has a favorable morbidity profile when compared to TURP.

MECHANICAL THERAPIES

Urolift

Prostatic urethral lift (PUL) is a novel procedure that is characterized by the placement of non-absorbable implants within the prostatic urethra. When placed correctly, these implants provide anterolateral traction to the lateral lobes of the prostate without necessitating tissue ablation. Advantages of PUL are that it is a short, simple procedure that can be done under local anesthesia and has low complication rates. However, the presence of an obstructing median lobe poses a hurdle for procedure due to the inability to place an implant to the median lobe safely. This exclusion criteria prevents a large portion of men with BPH from undergoing this procedure. The BPH6 was a randomized controlled trail that prospectively compared the PUL with TURP. This study reported that PUL improves IPSS to 52% compared to 72% following TURP. Maximal urinary flow rates improved to a modest degree (41% compared to 144% following TURP). Interestingly the preservation of native prostatic tissue results in preserved erectile and ejaculatory function (249). Pooled analysis of available studies confirm these modest improvements in urinary and sexual function (250,251). Further, this procedure is well-tolerated and is performed in the outpatient setting under local anesthetic in a vast majority of cases. Morbidity is representative of typical MIST procedures with small proportions of patients reporting dysuria, urinary tract infection, and hematuria. Durability is among the main concern surrounding this procedure. Only three-year data has been published at present, reporting a modest IPSS improvement (252). Further comparative robust studies are required to determine the role of the PUL in current practice.

Intra-Prostatic Stents

In keeping with the principles of minimal invasion, a stent or coil is placed into the urethra at the point of maximal obstruction under local anesthesia, endoscopic and radiographic guidance. Stents may be temporary/biodegradable or permanent. Although effective in the short term, they do have a significant complication rate raising concerns over safety and large randomized controlled trials are needed to establish their long-term efficacy and their true role in the management of BPH (204,253,254).

Transurethral Ethanol Ablation of the Prostate (TEAP)

Deep intra-prostatic injection of pure ethanol results in chemical ablation of the prostate. Of the limited studies available, 4 year follow up suggests sustained response in 73% of patient, with 23% requiring retreatment. More robust comparative data is required prior to more formal recommendations for the use of TEAP.

Fexapotide Triflutate – NX-1207

NX-1207 is injected into the transition zone of the prostate to ablate the tissue, but the precise mechanism by which NX-1207 acts has not been published to date. Trials of NX-1207 have shown a mean improvement of 5.7 points on IPSS score for men receiving one injection compared to placebo at mean 43 months follow up. When compared to men taking oral BPH medications, fewer in the NX-1207 group (8% vs 27%) required additional BPH intervention at 3 years (255). Long-term follow up of men receiving this chemical show durable reductions in symptom scores to 6.5-year follow-up (256). NX-1207 is well tolerated, with low rates of mild hematuria, dysuria and infection. No sexual dysfunction or incontinence has been reported for either agent.

Topsalysin - PRX-302

PRX-302 is a genetically modified bacterial pro-toxin that is activated by PSA within the prostatic tissue and forms transmembrane cellular pores that lead to apoptosis. Like TEAP and NX-1207, PRX-302 is injected into the transition zone of the prostate. PRX-302 results in a transient reduction in symptoms score that do not appear to be maintained at 12-month follow-up (257). Similar to NX-1207, it is well tolerated, with low rates of mild hematuria, dysuria and infection. No sexual dysfunction or incontinence has been reported.

Botulinum Toxin subtype A (botox)

Botox is a toxin produced by the bacterium Clostridium Botulinum. Its mechanism of action for intraprostatic injections is poorly understood however theories include glandular necrosis and the blockage of alpha-adrenergic receptors resulting in smooth muscle relaxation (258). Phase 2 single arm studies have shown that intraprostatic botox has minimal side effects but has a re-treatment rate as high as 29% (185).

OTHER THERAPIES

Aquablation

Aquablation involves a transrectal ultrasound guided, robot-assisted, high velocity saline stream. This results in the ability to ablate glandular tissue without the requirement of heat. Real-time monitoring is available and allows the surgeon to ensure sparing of the prostatic capsule. Early studies have demonstrated its safety and feasibility. The WATER trial showed that aquablation was not inferior to TURP for improving IPSS scores at 6 months follow up with slightly improved rates of anejaculation (259). Longer follow up data from this study is needed to prove long term efficacy and assess long term complication rates.

Prostatic Artery Embolization (PAE)

PAE is performed by a trained interventional radiologist. Unilateral or bilateral prostatic arteries are injected with an embolic agent - which is typically ethanol-based. With increasing experience, technical success has increased to greater than 90%. A metanalysis of 13 studies including 1,254 men found that PAE demonstrated a mean 16.2 increase in IPSS score and improved quality of life that remained statistically significant after 3 years follow up. Transient dysuria and urinary frequency were reported in 10% and 16% of men, respectively. Post embolization syndrome was reported in 3.6% of men and only three cases of major post-operative complications were recorded (260).

More recently, two-year follow up data from a randomized controlled trial of PAE vs TURP was published. Reduction in IPSS score was similar in both arms however TURP men showed better urinary flow, post void residual volume, reduced prostate volume but more erectile dysfunction. 21% of men who had PAE required subsequent TURP within the 2-year period. PAE adverse events were less frequent than TURP but distribution within the severity classes were similar (261).

Water Vapor Therapy - Rezum

Rezum uses radiofrequency to create thermal energy in the form of water vapor. This vapor is delivered transurethrally under cystoscopy to the prostate and causes instant cell necrosis through cell membrane disruption. It is frequently performed under local anesthetic in the outpatient setting. Two retrospective studies showed improvement in IPSS, urinary flow and post void residual volume at 6 and 12 months follow up (262,263). A randomized trial of Rezum vs sham procedure showed a mean improvement of 7 points on IPSS score and an improvement in quality of life in the Rezum group. Peak urinary flow rate was improved by 6.2ml/s in the rezum group and was sustained at 12 months (264). Morbidity is minimal and is in-line with those experienced following alternate MISTs (265).

Histotripsy

Histotripsy is the use of extracorporeal ultrasound energy that produces extreme pressure changes within the prostatic tissue. This pressure changes result in localized clusters of microbubbles which cause mechanical fractionation. Collapse of these microbubbles leads to cellular destruction and prostatic cavitation (266). The method of prostate injury allows the procedure to be monitored through ultrasound in a real-time setting. One safety and feasibility trial has been published to date reporting three cases of transient urinary retention, 1 case of minor anal abrasion, and one case of microscopic hematuria out of 25 men. No serious intraoperative complications occurred (267).

SUMMARY OF MINIMALLY INVASIVE THERAPIES

A myriad of minimally invasive therapies (MIST) has been developed to reduce the morbidity of surgical BPH management. Current evidence in MIST is characterized by improvements in symptom and urinary flow rates similar or slightly less than TURP with high rates of retreatment. Despite this, these procedures are very well tolerated and may be performed as an outpatient. Further, these therapies are highlighted by the significant reduced risk of sexual dysfunction. Some consider that MIST might be suitable in younger patients that are willing to accept less urinary improvement to preserve sexual function. Elderly or co-morbid men might also benefit given many of these procedures can be performed under local anesthetic or in the outpatient setting. However, the precise role for MIST has not become clear to date. It is clear that MISTs are emerging and will likely become a prevalent treatment option in the management of BPH.

MEASURING OUTCOMES AND EFFECTIVENESS OF TREATMENT

When considering the effectiveness of any treatment for BPH, one must consider the efficacy and tolerability of invasive or medical therapies (i.e., the effect on both subjective symptoms and urinary flow and incidence of adverse effects), the long-term effectiveness, the impact on daily life activities (quality of life) and the costs (268). Large scale randomized controlled trials provide information on the tolerability and efficacy of treatment options and evidence-based databases such as Cochrane reviews, may further analyze evidence-based data from multiple trials.

CONCLUSION

In conclusion, BPH is a common urological condition that is increasing in incidence in conjunction with the aging male population. If left untreated, BPH can lead to lower urinary tract obstructive symptoms that can significantly affect the quality of life of men.

As outlined in this chapter, the diagnosis of BPH begins with a detailed history of presenting complaint and interrogation of any lower urinary tract signs or symptoms. Questionnaires such as the IPSS score can help quantify the severity of these symptoms along with a uroflowmetry and PVR scan. A urinalysis, serum creatinine, and serum PSA should be ordered to investigate for prostate cancer, UTI, and renal failure. Imaging with USS or CT is not indicated unless there is concurrent hematuria, UTI, or urolithiasis.

Several options are now available for the treatment of BPH. Non-surgical management consists of medications such as alpha-blockers, 5-alpha reductase inhibitors, and PDE5 inhibitors which are available as monotherapy or in combination. BPH refractory to medical management is treated with surgical management which includes invasive and minimally invasive procedures. TURP remains the most widely used procedure for surgical BPH management and simple prostatectomies are reserved for larger prostates, complicated BPH or if TURP cannot be performed. These two procedures form the gold standard of treatment. Laser treatments when available offer good patient outcomes and have potential benefits when compared to TURP. The decision for either of these modalities of treatment is still largely dependent on availability and surgeon experience. The majority of minimally invasive treatment options are still experimental however may have a potential benefit for carefully selected men.

REFERENCES

Presti JC Jr. Neoplasms of the prostate gland. In: Tanagho EA, McAninch JW, eds. Smith's General Urology, 15th Edition. Lange Medical Books; 2000:399-421.
Hayward SW, Rosen MA, Cunha GR. Stromal-epithelial interactions in the normal and neoplastic prostate. Br J Urol. 1997;79(Suppl. 2):18-26.
Ball E, Risbridger G. New perspectives on growth factor-sex steroid interaction in the prostate. Cytokine Growth Factor Review. 2003;14:5-16.
Cuntha GR, Alarid ET, Turner T, et al. Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions and growth factors. J Androl. 1992;13:465-475.
Delmas V, Dauge MC. Embryology of the prostate- current state of knowledge. In: Khoury S CC, Murphy G, Denis L, ed. Prostate cancer in questions. ICI publications; 1991:16-17.
Moore KL. The Developing Human. 4th ed. W.B. Saunders Company; 1988.
McNeal JE. The Zonal Anatomy of the prostate. Prostate. 1981;2::35-49.
Berry SW, Coffey DS, Walsh PC, et al. The development of human benign prostate hyperplasia with age. J Urol. 1984;132:474-479.
McNeal JE. Normal and pathologic anatomy of the prostate. Urology. 1981;17 (suppl):11-16.
Algaba F. Lobar division of the prostate. In: Khoury S CC, Murphy G, Denis L, ed. Prostate cancer in questions. ICI publications; 1991:16-17.
Epstein JL. Non-neoplastic diseases of the prostate. In: Bostwick DG EJUSP, 1st Edition, ed. St Louis, USA. 307-340; 1997.
Warhol MJ, Longtine JA. The ultrastructural localization of prostatic specific antigen and prostatic acid phosphatase in hyperplastic and neoplastic human prostates. J Urol. 1985;134:607-613.
Di Sant'Agnese PA. Neuroendocrine differentiation in human prostatic carcinoma. Hum Pathol. 1992;23:287-296.
Donkervoort T, Sterling A, van Ness J, Donker PJ. A clinical and urodynamic study of tadenan in the treatment of benign prostatic hypertrophy. Eur Urol. 1977;3:218-25.
Brawer MK, Kirby R. Prostate Specific Antigen, 2nd edn. . Health Press Limited; 1999:7-14.
Partin AW, Rodriguez R. The molecular biology, endocrinology, and physiology of the prostate and seminal vesicles. In: Walsh PC, ed. Campbell's Urology. 8th ed. Saunders; 2002:1237-1296.
Ganong W. Review of Medical Physiology. 18th ed. Appleton and Lange; 1997:401.
Eaton CL. Aetiology and pathogenesis of benign prostatic hyperplasia. Curr Opin Urol. 2003;13:7-10.
Luo J, Dunn T, Ewing C, et al. Gene expression signature of Benign Prostatic Hyperplasia revealed by cDNA Microarray Analysis. Prostate. 2002;51:189-200.
Marengo SR, Chung LW. An orthotopic model for the study of growth factors in the ventral prostate of the rat: effects of epidermal growth factor and basic fibroblast growth factor. J Androl. Jul-Aug 1994;15(4):277-86.
Cooke PS, Young P, Cunha GR. Androgen receptor expression in developing male reproductive organs. Endocrinology. 1991;128:2867-2873.
Gao J, Arnold JT, Isaacs JT. Conversion from a paracrine to an autocrine mechanism of androgen-stimulated growth during malignant transformation of prostatic epithelial cells. Cancer Res. Jul 1 2001;61(13):5038-44.
Bartsch G, Rittmaster RS, Klocker H. Dihydrotestosterone and the concept of 5alpha-reductase inhibition in human benign prostatic hyperplasia. World J Urol. 2002;19:413-425.
Wright AS, Douglas RC, Thomas LN, et al. Androgen-induced regrowth in the castrated rat ventral prostate: role of 5alpha-reductase. Endocrinology. 1999;140:4509-4515.
Grino PB, Griffin JE, Wilson JD. Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology. 1990;126:1165-1172.
Wright AS, Thomas LN, Douglas RC, et al. Relative potency of testosterone and dihydrotestosterone in preventing atrophy and apoptosis in the prostate of the castrated rat. J Clin Invest. 1996;98:2558-2563.
Forti G, Salerno R, Moneti G, et al. Three-month treatment with a long-acting gonadotropin-releasing hormone agonist of patients with benign prostatic hyperplasia: effects on tissue androgen concentration, 5 alpha-reductase activity and androgen receptor content. J Clin Endocrinol Metab. Feb 1989;68(2):461-8.
Page ST, Lin DW, Mostaghel EA, et al. Persistent intraprostatic androgen concentrations after medical castration in healthy men. J Clin Endocrinol Metab. Oct 2006;91(10):3850-6. doi:jc.2006-0968 [pii] 10.1210/jc.2006-0968
Page ST, Lin DW, Mostaghel EA, et al. Dihydrotestosterone administration does not increase intraprostatic androgen concentrations or alter prostate androgen action in healthy men: a randomized-controlled trial. J Clin Endocrinol Metab. Feb 2011;96(2):430-7. doi:jc.2010-1865 [pii] 10.1210/jc.2010-1865
Barrack ER, Berry SJ. DNA synthesis in the canine prostate; effects of androgen induction and estrogen treatment. Prostate. 1987;10:45-56.
Gooren LJ, Toorians AW. Significance of oestrogens in male (patho)physiology. Ann Endocrinol (Paris). Apr 2003;64(2):126-35.
Risbridger GP, Bianco JJ, Ellem SJ, McPherson SJ. International Congress on Hormonal Steroids and Hormones and Cancer: Oestrogens and prostate cancer. Endocr Relat Cancer. Jun 2003;10(2):187-91.
Tsurusaki T, Aoki D, Kanetake H, et al. Zone-dependent expression of estrogen receptors alpha and beta in human benign prostatic hyperplasia. J Clin Endocrinol Metab. Mar 2003;88(3):1333-40.
Steiner MS, Raghow S. Antiestrogens and selective estrogen receptor modulators reduce prostate cancer risk. World J Urol. May 2003;21(1):31-6.
Jenkins DJ, Kendall CW, D'Costa MA, et al. Soy consumption and phytoestrogens: effect on serum prostate specific antigen when blood lipids and oxidized low-density lipoprotein are reduced in hyperlipidemic men. J Urol. Feb 2003;169(2):507-11.
Shibata K, Hirasawa A, Moriyma N, et al. alpha1a-Adrenoceptor polymorphism: pharmacological characterization and association with benign prostatic hypertrophy. Br J Pharm. 1996;118:1403-1408.
Ekman P. The prostate as an endocrine organ: androgens and estrogens. Prostate Suppl. 2000;10:14-8.
Verhamme KM, Dieleman JP, Bleumink GS, et al. Incidence and prevalence of lower urinary tract symptoms suggestive of benign prostatic hyperplasia in primary care--the Triumph project. Eur Urol. Oct 2002;42(4):323-8.
Lokeshwar SD, Harper BT, Webb E, et al. Epidemiology and treatment modalities for the management of benign prostatic hyperplasia. Translational andrology and urology. 2019;8(5):529.
Jacobsen SJ, Jacobson DJ, Girman CJ, et al. Treatment for benign prostatic hyperplasia among community dwelling men: the Olmsted County study of urinary symptoms and health status. J Urol. Oct 1999;162(4):1301-6.
Marberger MJ, Andersen JT, Nickel JC, et al. Prostate volume and serum prostate-specific antigen as predictors of acute urinary retention. Combined experience from three large multinational placebo-controlled trials. Eur Urol. Nov 2000;38(5):563-8.
Clifford GM, Logie J, Farmer RD. How do symptoms indicative of BPH progress in real life practice? The UK experience. Eur Urol. 2000;38 Suppl 1:48-53.
Lowe FC. Goals for benign prostatic hyperplasia therapy. Urology. Feb 2002;59(2 Suppl 1):1-2.
Moore RA. Benign prostatic hypertrophy and carcinoma of the prostate. Occurrence and experimental production in animals. Surgery. 1944;16:152-167.
Sanda MG, Beaty TH, Strutzman RE, et al. Genetic susceptibility of benign prostatic hyperplasia. J Urol. 1994;152:115-119.
Sanda MG, Beaty TH, Stutzman RE, Childs B, Walsh PC. Genetic susceptibility of benign prostatic hyperplasia. J Urol. Jul 1994;152(1):115-9.
Sidney S, Quesenberry CP, Jr., Sadler MC, Guess HA, Lydick EG, Cattolica EV. Incidence of surgically treated benign prostatic hypertrophy and of prostate cancer among blacks and whites in a prepaid health care plan. Am J Epidemiol. Oct 15 1991;134(8):825-9.
Choi J, Ikeguchi EF, Lee SW, Choi HY, Te AE, Kaplan SA. Is the higher prevalence of benign prostatic hyperplasia related to lower urinary tract symptoms in Korean men due to a high transition zone index? Eur Urol. Jul 2002;42(1):7-11.
Geller J, Sionit L, Partido C, et al. Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture. Prostate. Feb 1 1998;34(2):75-9.
Chyou PH, Nomura AM, Stemmermann GN, Hankin JH. A prospective study of alcohol, diet, and other lifestyle factors in relation to obstructive uropathy. Prostate. 1993;22(3):253-64.
Ryl A, Rotter I, Miazgowski T, et al. Metabolic syndrome and benign prostatic hyperplasia: association or coincidence? Diabetology & metabolic syndrome. 2015;7:94. doi:10.1186/s13098-015-0089-1
Xia BW, Zhao SC, Chen ZP, et al. The underlying mechanism of metabolic syndrome on benign prostatic hyperplasia and prostate volume. The Prostate. 2020;80(6):481-490.
De Nunzio C, Salonia A, Gacci M, Ficarra V. Inflammation is a target of medical treatment for lower urinary tract symptoms associated with benign prostatic hyperplasia. World journal of urology. 2020:1-9.
De Nunzio C, Kramer G, Marberger M, et al. The controversial relationship between benign prostatic hyperplasia and prostate cancer: the role of inflammation. European urology. 2011;60(1):106-117.
Nickel JC, Roehrborn CG, O'Leary MP, Bostwick DG, Somerville MC, Rittmaster RS. The relationship between prostate inflammation and lower urinary tract symptoms: examination of baseline data from the REDUCE trial. European urology. 2008;54(6):1379-1384.
Kahokehr A, Vather R, Nixon A, Hill AG. Non‐steroidal anti‐inflammatory drugs for lower urinary tract symptoms in benign prostatic hyperplasia: systematic review and meta‐analysis of randomized controlled trials. BJU international. 2013;111(2):304-311.
Schenk JM, Kristal AR, Arnold KB, et al. Association of symptomatic benign prostatic hyperplasia and prostate cancer: results from the prostate cancer prevention trial. Am J Epidemiol. Jun 15 2011;173(12):1419-28. doi:kwq493 [pii] 10.1093/aje/kwq493
Orsted DD, Bojesen SE, Nielsen SF, Nordestgaard BG. Association of clinical benign prostate hyperplasia with prostate cancer incidence and mortality revisited: a nationwide cohort study of 3,009,258 men. Eur Urol. Oct 2011;60(4):691-8. doi:S0302-2838(11)00640-3 [pii] 10.1016/j.eururo.2011.06.016
Isaacs JT, Coffey DS. Etiology and Disease Process of Benign Prostatic Hyperplasia. The Prostate Supplement. 1989;2:33-50.
McNeal JE. Origin and evaluation of benign prostatic enlargement. Invest Urol. 1978;15:340-345.
Rhodes T, Girman CJ, Jacobsen SJ, Roberts RO, Guess HA, Lieber MM. Longitudinal prostate growth rates during 5 years in randomly selected community men 40 to 79 years old. J Urol. Apr 1999;161(4):1174-9.
Caine M. The present role of alpha-adrenergic blockers in the treatment of benign prostatic hypertrophy. J Urol. Jul 1986;136(1):1-4.
Akduman B, Crawford ED. Terazosin, doxazosin, and prazosin: current clinical experience. Urology. Dec 2001;58(6 Suppl 1):49-54.
Peters TJ, Donovan JL, Kay HE, et al. The International Continence Society 'Benign Prostatic Hyperplasia Study': the bothersomeness of urinary symptoms. J Urol. 1997;157:885-889.
Scarpa RM. Lower urinary tract symptoms (LUTS): what are the implications for the patients? Eur Urol. 2001;40(Suppl. 4):12-20.
Garraway WM, Collins GN, Lee RJ. High prevalence of benign prostatic hypertrophy in the community. Lancet. 1991;338:469-471.
Lepor H. Alpha1-adrenoceptor selectivity: clinical or theoretical benefit? Br J Urol. 1995;76 (Suppl. 1):57-61.
Welch G, Kawachi I, Barry MJ, et al. Distinction between symptoms of voiding and filling in benign prostatic hyperplasia: findings from health professionals follow-up study. Urology. 1998;1998
Abrams P. New words for old: lower urinary tract symptoms for 'prostatism'. BMJ. 1994;308:929-930.
Puppo P. Do we know everything about alpha-blockade in the management of lower urinary tract symptoms? Eur Urol. Jan 2001;39 Suppl 2:38-41.
Price D. Potential mechanisms of action of superselective alpha1-adrenoceptor antagonists. Eur Urol. 2002;40(Suppl. 1):5-11.
Michel MC. Physiology and pathophysiology of lower urinary tract symptoms. Drugs of Today. 2001;37 (Suppl.D):7-11.
Van Moorselaar R, Emberton M, Harving N, et al. Alfuzosin 10mg once daily improves sexual function in men with luts and concomitant sexual dysfunction. J Urol. 2003;169 (Suppl 4):478.
Kassabian VS. Sexual function in patients treated for benign prostatic hyperplasia. Lancet. Jan 4 2003;361(9351):60-2.
Leliefeld HH, Stoevelaar HJ, McDonnell J. Sexual function before and after various treatments for symptomatic benign prostatic hyperplasia. BJU Int. Feb 2002;89(3):208-13.
Jacobsen SJ, Jacobsen DJ, Girman CJ, et al. Natural history of prostatism: risk factors for acute urinary retention. J Urol. 1997;158:481-487.
Douenias R, Rich M, Badlani M, et al. Predisposing factors in bladder calculi: a review of 100 cases. Urology. 1991;37:240-243.
McConnell JD. Benign prostatic hyperplasia. J Urol. 1994;152:459-460.
Westenberg A, Harper M, Zafirakis H, et al. Bladder and renal stones: management and treatment. Hospital Medicine. 2002;63:34-41.
Mebust WK, Holtgrewe HL, Cockett ATK, et al. Transurethral prostatectomy: immediate and postoperative complications. a cooperative study of 13 partcipating institutions evaluating 3885 patients. J Urol. 1989;141:243-247.
O'Connor RC, Laven BA, Bales GT, et al. Nonsurgical management of benign prostatic hyperplasia in men with bladder calculi. Urology. 2002;60:288-91.
Marshall S, Narayan P. Treatment of prostatic bleeding: suppression of angiogenesis by androgen deprivation. J Urol. 1993;149:1553.
Foley SJ, Bailey DM. Microvessel disease in prostatic hyperplasia. BJU Int. 2000;85:70-73.
Kashif KM, Foley SJ, Basketter V, Holmes SA. Haematuria associated with BPH-Natural history and a new treatment option. Prostate Cancer Prostatic Dis. Mar 1998;1(3):154-156.
Saez C, Cgonzalez-Baena AC, Japon MA, et al. Regressive changes in finasteride-treated human hyperplastic prostate correlate with an upregulation of TGF-beta receptor expression. Prostate. 1998;37:84-90.
Monti S, Sciarra F, Adaqmo EV, et al. Prevalent decrease in the EGF content in the periurethral zone of BPH. J Am. 1997;18:488-94.
Jepsen JV, Bruskewitz RC. Clinical manifestations and indications for treatment. In: Lepor H, ed. Prostatic Diseases. WB Saunders; 2000:127-142.
Andersson K-E. Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol Rev. 1993;45:253-308.
Barry MJ, Fowler FJ, Jr., O'Leary MP, et al. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol. Nov 1992;148(5):1549-57; discussion 1564.
Kakizaki H, Koyanagi T. Current view and status of lower urinary tract symptoms and neurogenic lower urinary tract dysfunction. BJU Int. 2000;85(Suppl. 2):25-30.
Lepor H, Nieder A, Feser J, et al. Effect of terazosin on prostatism in men with normal and abnormal peak urinary flow rates. Urology. 1997;49:476-80.
Serels S, Stein M. Prospective study comparing hyoscyamine, doxazosin and combination therapy for treatment of urgency and frequency in women. Neurourol Urodynam. 1998;17:31-36.
Hampel C, Dolber PC, Savic SL, et al. Changes in a1 adrenergic receptor (AR) subtype gene expression during bladder outlet obstruction of rats (abstract). J Urol. 2000;163:228.
Tubaro A, Trucchi A, Miano L. Investigation of benign prostatic hyperplasia. Current Opinion in Urology. 2003;13:17-22.
de la Rosette JJ, Alivizatos G, Madersbacher S, et al. EAU Guidelines on benign prostatic hyperplasia (BPH). Eur Urol. Sep 2001;40(3):256-63; discussion 264.
Roehrborn CG, Bartsch G, Kirby R, et al. Guidelines for the diagnosis and treatment of benign prostatic hyperplasia: a comparative, international overview. Urology. Nov 2001;58(5):642-50.
Recommendations of the International Science Committee. The evaluation and treatment of lower urinary tract symptoms suggestive of benign prostatic obstruction. Proceedings of the 4th International Consultation on BPH; 1997.
Chai TC, Belville WD, McGuire EJ, et al. Urological Association voiding symptom index: comparison of unselected and selected samples of both sexes. J Urol. 1993;150.
Lepor H, Machi G. Comparison of AUA symptom index in unselected males and females between fifty-five and seventy-nine years of age. Urology. 1993;42.:36-40.
Barry MJ. Evaluation of symptoms and quality of life in men with benign prostatic hyperplasia. Urology. 2001;58:25-32.
Yalla SV, Sullivan MP, Lecamwasam HS, DuBeau CE, Vickers MA, Cravalho EG. Correlation of American Urological Association symptom index with obstructive and nonobstructive prostatism. J Urol. Mar 1995;153(3 Pt 1):674-9; discussion 679-80.
Jensen KM, Jorgensen TB, Mogensen P. Long-term predictive role of urodynamics: an 8-year follow-up of prostatic surgery for lower urinary tract symptoms. Br J Urol. Aug 1996;78(2):213-8.
Roehrborn CG, Sech S, Montoya J, Rhodes T, Girman CJ. Interexaminer reliability and validity of a three-dimensional model to assess prostate volume by digital rectal examination: is there a place for combination medical therapy? Urology. Jun Jan 2001;57(6):1087-92.
Souverein PC, Erkens JA, de la Rosette JJ, Leufkens HG, Herings RM. Drug Treatment of Benign Prostatic Hyperplasia and Hospital Admission for BPH-Related Surgery. Eur Urol. May 2003;43(5):528-34.
Lobel B, Rodriguez A. Chronic prostatitis: what we know, what we do not know, and what we should do! World J Urol. Jun 2003;21(2):57-63.
Association for Genitourinary Medicine - Medical Specialty Society and Medical Society for the Study of Venereal Diseases - Disease Specific Society. 2002 national guideline for the management of prostatitis. 2002.
Krieger JN, Nyberg L, Jr., Nickel JC. NIH consensus definition and classification of prostatitis. Jama. Jul 21 1999;282(3):236-7.
Boyle P, Gould AL, Roehrborn CG. Prostate volume predicts outcome of treatment of benign prostatic hyperplasia with finasteride: meta-analysis of randomized clinical trials. Urology. Sep 1996;48(3):398-405.
McConnell JD, Roehrborn CG, Bautista OM, et al. The long-term effect of doxazosin, finasteride, and combination therapy on the clinical progression of benign prostatic hyperplasia. N Engl J Med. Dec 18 2003;349(25):2387-98.
Rovner ES, Wein AJ. Practical urodynamics, Part 1. AUA Update Series. 2002;21(19):146-151.
Feneley MR, Dunsmuir WD, Pearce J, Kirby RS. Reproducibility of uroflow measurement: experience during a double-blind, placebo-controlled study of doxazosin in benign prostatic hyperplasia. Urology. May 1996;47(5):658-63.
van Verooij GEPM, Eckhardt MD, Gisolf KWH, et al. Data from frequency-volume charts versus filling cystometric estimated capacities and prevalence of instability in men with lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Neurourol Urodyn. 2002;10:106-111.
Chen SS, Hong JG, Hsiao YJ, et al. The correlation between clinical outcome and residual prostatic weight ratio after transurethral resection of the prostate for benign prostatic hyperplasia. BJU Int. 2000;85:79-82.
MacDonald R, Ishani A, Rutks I, Wilt T J. A systematic review of Cernilton for the treatment of benign prostatic hyperplasia. BJU Int. May 2000;85(7):836-41.
Wilt TJ, Ishani A, MacDonald R, et al. Saw Palmetto extracts for treatment of benign prostatic hyperplasia: a systematic review. JAMA. 1998;280:1604-1609.
Lowe FC, et al. Phytotherapy in the treatment of benign prostatic hyperplasia: An update. Urology. 1999;53:671-678.
Foley CL, Kirby J. 5 Alpha-reductase inhibitors: what's new? Current Opinion in Urology. 2003;13:31-37.
Gerber GS. Phytotherapy for Benign Prostatic Hyperplasia. Current Urology Reports. 2002;3:285-291.
Fagelman E, Lowe FC. Herbal medications in the treatment of benign prostatic hyperplasia (BPH). Urol Clin North Am. Feb 2002;29(1):23-9.
Dreikorn K. The role of phytotherapy in treating lower urinary tract symptoms and benign prostatic hyperplasia. World J Urol. 2002;19:426-435.
Dreikorn K. Other medical therapies. Proceedings of the Fourth International Consultation on Benign Prostatic Hyperplasia (BPH). In: Paris, ed. July: 2-5; 1997.
Lowe FC, Dreikorn K, A B, et al. Review of recent placebo controlled trials utlizing phytotherapeutic agents for treatment of BPH. Prostate. 1998;37:187-193.
Yang Y, Ikezoe T, Zheng Z, Taguchi H, Koeffler HP, Zhu WG. Saw Palmetto induces growth arrest and apoptosis of androgen-dependent prostate cancer LNCaP cells via inactivation of STAT 3 and androgen receptor signaling. International journal of oncology. Sep 2007;31(3):593-600.
Delos S, Lehle C, Martin PM. Inhibition of the activity of basic 5-alpha reductase (type 1) detected in DU 145 cells and expressed in insect cells. J Steroid Biochem Mol Biol. 1994;48:347-352.
Bayne CW, Grant ES, Chapman K. Characterisation of a new coculture model for BPH which expresses 5-alpha reductase types 1 and 2: the effects of Permixon on DHT formation [abstract]. J Urol. 1997;157(Suppl 4):755.
Carilla E, Briley M, Fauran F. Binding of Permixon, a new treatment for prostatic benign hyperplasia, to the cytosolic androgen receptor in the rat prostate. J Steroid Biochem. 1984;20:521-523.
Sultan C, Terraza A, Devillier C. Inhibition of androgen metabolism and binding by liposterolic extract of 'Serenoa repens B' in human foreskin fibroblasts. J Steroid Biochem. 1984;20:515-519.
Minutoli L, Altavilla D, Marini H, et al. Inhibitors of apoptosis proteins in experimental benign prostatic hyperplasia: effects of serenoa repens, selenium and lycopene. Journal of biomedical science. 2014;21:19. doi:10.1186/1423-0127-21-19
Carraro JC, JP R, Koch G. Comparison of phytotherapy (Permixon) with finasteride in the treatment of benign prostate hyperplasia: a randomized international study of 1098 patients. Prostate. 1996;29:231-240.
Braeckman J. The extract of Serenoa repens in the treatment of benign prostatic hyperplasia: a multicenter open study. 1994;55:776-785.
Marks LS, Partin AP, Epstein JL. Effects of a saw palmetto herbal blend in men with symptomatic benign prostatic hyperplasia. J Urol. 2000;163:1451-1456.
Boyle P, C R, Lowe F, Roehrborn C. Meta-analysis of permixon in treatment of symptomatic benign prostatic hyperplasia: an update (abstract). J Urol. 2003;169 (Suppl 4):334-335.
Tacklind J, Macdonald R, Rutks I, Stanke JU, Wilt TJ. Serenoa repens for benign prostatic hyperplasia. The Cochrane database of systematic reviews. 2012;12:Cd001423. doi:10.1002/14651858.CD001423.pub3
Gerber GS, Kuznetsov D, Johnson BC, Burstein JD. Randomized, double-blind, placebo-controlled trial of saw palmetto in men with lower urinary tract symptoms. Urology. Dec 2001;58(6):960-4; discussion 964-5.
Carbin BE, Larsson B, Lindahl O. Treatment of benign prostatic hyperplasia with phytosterols. Br J Urol. 1990;66:639-641.
Zaida A, Rosenblum M, Crawford ED. Benign prostatic hyperplasia: an overview. Urology. 1999;53:1-6.
Cindolo L, Pirozzi L, Fanizza C, et al. Actual medical management of lower urinary tract symptoms related to benign prostatic hyperplasia: temporal trends of prescription and hospitalization rates over 5 years in a large population of Italian men. International urology and nephrology. Apr 2014;46(4):695-701. doi:10.1007/s11255-013-0587-8
Cornu JN, Cussenot O, Haab F, Lukacs B. A widespread population study of actual medical management of lower urinary tract symptoms related to benign prostatic hyperplasia across Europe and beyond official clinical guidelines. Eur Urol. Sep 2010;58(3):450-6. doi:10.1016/j.eururo.2010.05.045
Oesterling JE. Benign Prostatic hyperplasia: medical and minimally invasive treatment options. N Engl J Med. 1995;332:99-109.
Kobayashi S, Tang R, Shapiro E, et al. Characterisation of human alpha1 adrenoceptor using radioligand receptor binding on slide-mounted tissue section. J Urol. 1993;150:2002-2006.
Caine M, Perlberg S, Meretyk S. A placebo controlled double blind study of the effect of phenoxybenzamine in benign prostatic obstruction. Br J Urol. 1978; 50:551-554.
Foglar R, Shibata K, Horie K, et al. Use of recombinant alpha1-adrenoceptors to characterise subtype selectivity of drugs for the treatment of prostatic hypertrophy. Eur J Pharmacol. 1995;288:201-207.
Roehrborn CG. Efficacy and safety of once-daily alfuzosin in the treatment of lower urinary tract symptoms and clinical benign prostatic hyperplasia: a randomized, placebo-controlled trial. Urology. Dec 2001;58(6):953-9.
Michel MC, Flannery MT, Narayan P. Worldwide experience with alfuzosin and tamsulosin. Urology. Oct 2001;58(4):508-16.
Boyle P, Robertson C, Manski R, Padley RJ, Roehrborn CG. Meta-analysis of randomized trials of terazosin in the treatment of benign prostatic hyperplasia. Urology. Nov 2001;58(5):717-722.
Kirby RS, Roehrborn C, Boyle P, et al. Efficacy and tolerability of doxazosin and finasteride, alone or in combination, in treatment of symptomatic benign prostatic hyperplasia: the Prospective European Doxazosin and Combination Therapy (PREDICT) trial. Urology. Jan 2003;61(1):119-26.
Gillenwater JY, Conn RL, Chrysant SG, et al. Doxazosin for the treatment of benign prostatic hyperplasia in patients with mild to moderate essential hypertension: a double-blind, placebo-controlled, dose-response multicenter study. J Urol. Jul 1995;154(1):110-15.
Wilt TJ, MacDonald R, Nelson D. Tamsulosin for treating lower urinary tract symptoms compatible with benign prostatic obstruction: a systematic review of efficacy and adverse effects. J Urol. Jan 2002;167(1):177-83.
Kawabe K, Yoshida M, Homma Y. Silodosin, a new alpha1A-adrenoceptor-selective antagonist for treating benign prostatic hyperplasia: results of a phase III randomized, placebo-controlled, double-blind study in Japanese men. BJU Int. Nov 2006;98(5):1019-24. doi:10.1111/j.1464-410X.2006.06448.x
Chapple CR, Montorsi F, Tammela TL, Wirth M, Koldewijn E, Fernandez Fernandez E. Silodosin therapy for lower urinary tract symptoms in men with suspected benign prostatic hyperplasia: results of an international, randomized, double-blind, placebo- and active-controlled clinical trial performed in Europe. Eur Urol. Mar 2011;59(3):342-52. doi:10.1016/j.eururo.2010.10.046
Jung JH, Kim J, MacDonald R, Reddy B, Kim MH, Dahm P. Silodosin for the treatment of lower urinary tract symptoms in men with benign prostatic hyperplasia. The Cochrane database of systematic reviews. Nov 22 2017;11(11):Cd012615. doi:10.1002/14651858.CD012615.pub2
Ishizuka O, Nishizawa O, Hirao Y, Ohshima S. Evidence-based meta-analysis of pharmacotherapy for benign prostatic hypertrophy. Int J Urol. 2002;9:607-612.
Djavan B, Marberger M. A meta-analysis on the efficacy and tolerability of alpha1-adrenoceptor antagonists in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction. Eur Urol. 1999;36(1):1-13.
Wu YJ, Dong Q, Liu LR, Wei Q. A meta-analysis of efficacy and safety of the new alpha1A-adrenoceptor-selective antagonist silodosin for treating lower urinary tract symptoms associated with BPH. Prostate Cancer Prostatic Dis. Mar 2013;16(1):79-84. doi:10.1038/pcan.2012.36
Novara G, Tubaro A, Sanseverino R, et al. Systematic review and meta-analysis of randomized controlled trials evaluating silodosin in the treatment of non-neurogenic male lower urinary tract symptoms suggestive of benign prostatic enlargement. World J Urol. Aug 2013;31(4):997-1008. doi:10.1007/s00345-012-0944-8
Shim SR, Kim JH, Chang IH, et al. Is Tamsulosin 0.2 mg Effective and Safe as a First-Line Treatment Compared with Other Alpha Blockers?: A Meta-Analysis and a Moderator Focused Study. Yonsei medical journal. Mar 2016;57(2):407-18. doi:10.3349/ymj.2016.57.2.407
Norman RW, Coakes KE, Wright AS, et al. Androgen metabolism in men receiving finasteride before prostatectomy. J Urol. 1993;150:1736-1739.
Russell DW, Wilson JD. Steroid 5 alpha-reductase: two genes/two enzymes. Annu rev Biochem. 1994;63:25-61.
Thigpen AE, Silver RI, Guileyardo JM, et al. Tissue distribution and ontogeny of steroid 5 alpha-reductase isoenzyme expression. J Clin Invest. 1993;92:903-910.
Aumuller G, Eicheler W, Renneberg H, et al. Immunocytochemical evidence for differential subcellular localization of 5 alpha-reductase isoenzymes in human tissues. Acta Anat. 1996;156:241-252.
Coptcoat MJ. The Management of Advanced Prostate Cancer, 1st Edition. Blackwell Science Ltd; 1996.
Boyle P, Roehrborn C, Harkaway R, Logie J, De La Rosette J, Emberton M. 5-alpha reductase inhibitors provide superior benefits to alpha blockers by preventing aur and surgery (abstract). J Urol. 2003;169 (Suppl 4):479.
Zimmern P. Medical treatment modalities for lower urinary tract symptoms: what are the relevant differences in randomised controlled trials? Eur Urol. 2000;38 Suppl 1:18-24.
Roehrborn CG. Meta-analysis of randomized clinical trials of finasteride. Urology. Apr 1998;51(4A Suppl):46-9.
Stoner E, and the Finasteride Study Group. Three-year safety and efficacy data on the use of finasteride in the treatment of benign prostatic hyperplasia. Urology. 1994; 43:284-294.
Strauch G, Perles P, Vergult G. Comparison of finasteride (Proscar) and Serenoa repens (Permixon) in the inhibition of 5-alpha reductase in healthy male volunteers. Eur Urol. 1994;26:247-252.
Kaplan SA. 5alpha-reductase inhibitors: what role should they play? Urology. Dec 2001;58(6 Suppl 1):65-70; discussion 70.
Canby-Hagino E, Hernandez J, Brand TC, Thompson I. Looking back at PCPT: looking forward to new paradigms in prostate cancer screening and prevention. Eur Urol. Jan 2007;51(1):27-33. doi:S0302-2838(06)01022-0 [pii] 10.1016/j.eururo.2006.09.002
Thompson IM, Goodman PJ, Tangen CM, et al. The Influence of Finasteride on the Development of Prostate Cancer. N Engl J Med. Jun 24 2003;
Klotz L. Words of wisdom. Re: Effect of dutasteride on the risk of prostate cancer. Andriole G, Bostwick D, Brawley O, et al. N Engl J Med 2010;362:1192-202. Eur Urol. Aug 2010;58(2):313.
Nickel JC, Mendez-Probst CE, Whelan TF, Paterson RF, Razvi H. 2010 Update: Guidelines for the management of benign prostatic hyperplasia. Can Urol Assoc J. Oct 2010;4(5):310-6.
Gilling P, Jacobi G, Tammela T, van Erps P. Efficacy of dutasteride and finasteride for the treatment of benign prostate hyperplasia: results of the 1-year enlarged prostate international comparator study (EPICS) [abstract]. BJU Int. 2005;95:12.
Hagert J GP, Metro, MJ,et al. . A prospective, comparative study of the onset of symptomatic benefit of dutasteride versus finasteride in men with benign prostatic hyperplasia in everyday clinical practice [abstract no. 1353]. Journal of Urology. 2004;171(Suppl 4):356.
Naslund MJ, Miner M. A review of the clinical efficacy and safety of 5alpha-reductase inhibitors for the enlarged prostate. Clin Ther. Jan 2007;29(1):17-25. doi:S0149-2918(07)00032-X [pii] 10.1016/j.clinthera.2007.01.018
Roehrborn CG, Siami P, Barkin J, et al. The effects of dutasteride, tamsulosin and combination therapy on lower urinary tract symptoms in men with benign prostatic hyperplasia and prostatic enlargement: 2-year results from the CombAT study. J Urol. Feb 2008;179(2):616-21; discussion 621. doi:S0022-5347(07)02586-4 [pii] 10.1016/j.juro.2007.09.084
Crawford ED, Andriole GL, Marberger M, Rittmaster RS. Reduction in the Risk of Prostate Cancer: Future Directions After the Prostate Cancer Prevention Trial. Urology. Dec 24 2009;doi:S0090-4295(09)00931-5 [pii] 10.1016/j.urology.2009.05.099
Kavanagh LE, Jack GS, Lawrentschuk N. Prevention and management of TURP-related hemorrhage. Nat Rev Urol. Sep 2011;8(9):504-14. doi:10.1038/nrurol.2011.106 nrurol.2011.106 [pii]
Uckert S, Oelke M. Phosphodiesterase (PDE) inhibitors in the treatment of lower urinary tract dysfunction. Br J Clin Pharmacol. Aug 2011;72(2):197-204. doi:10.1111/j.1365-2125.2010.03828.x
Gonzalez RR, Kaplan SA. Tadalafil for the treatment of lower urinary tract symptoms in men with benign prostatic hyperplasia. Expert Opin Drug Metab Toxicol. Aug 2006;2(4):609-17. doi:10.1517/17425255.2.4.609
Donatucci CF, Brock GB, Goldfischer ER, et al. Tadalafil administered once daily for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a 1-year, open-label extension study. BJU Int. Apr 2011;107(7):1110-6. doi:10.1111/j.1464-410X.2010.09687.x
Gacci M, Andersson KE, Chapple C, et al. Latest Evidence on the Use of Phosphodiesterase Type 5 Inhibitors for the Treatment of Lower Urinary Tract Symptoms Secondary to Benign Prostatic Hyperplasia. Eur Urol. Jan 21 2016;doi:10.1016/j.eururo.2015.12.048
Warde N. Therapy: Two birds, one stone: tadalafil is an effective treatment for men with both BPH-LUTS and ED. Nat Rev Urol. Dec 2011;8(12):643. doi:10.1038/nrurol.2011.165 nrurol.2011.165 [pii]
Kaplan SA, Roehrborn CG, Rovner ES, Carlsson M, Bavendam T, Guan Z. Tolterodine and tamsulosin for treatment of men with lower urinary tract symptoms and overactive bladder: a randomized controlled trial. JAMA. Nov 15 2006;296(19):2319-28. oi:296/19/2319 [pii] 10.1001/jama.296.19.2319
Maria G, Brisinda G, Civello IM, Bentivoglio AR, Sganga G, Albanese A. Relief by botulinum toxin of voiding dysfunction due to benign prostatic hyperplasia: results of a randomized, placebo-controlled study. Urology. Aug 2003;62(2):259-64; discussion 264-5. doi:S0090429503004771 [pii]
Brisinda G, Cadeddu F, Vanella S, Mazzeo P, Marniga G, Maria G. Relief by botulinum toxin of lower urinary tract symptoms owing to benign prostatic hyperplasia: early and long-term results. Urology. Jan 2009;73(1):90-4. doi:S0090-4295(08)01505-7 [pii] 10.1016/j.urology.2008.08.475
Lepor H, Williford WO, Barry MJ, Haakenson C, Jones K. The impact of medical therapy on bother due to symptoms, quality of life and global outcome, and factors predicting response. Veterans Affairs Cooperative Studies Benign Prostatic Hyperplasia Study Group. J Urol. Oct 1998;160(4):1358-67.
Wessells H, Roy J, Bannow J, et al. Incidence and severity of sexual adverse experiences in finasteride and placebo-treated men with benign prostatic hyperplasia. Urology. Mar 2003;61(3):579-84.
Bruskewitz R, Girman CJ, Fowler J, et al. Effect of finasteride on bother and other health-related quality of life aspects associated with benign prostatic hyperplasia. PLESS Study Group. Proscar Long-term Efficacy and Safety Study. Urology. Oct 1999;54(4):670-8.
Albertsen PC, Pellissier JM, Lowe FC, Girman CJ, Roehrborn CG. Economic analysis of finasteride: a model-based approach using data from the Proscar Long-Term Efficacy and Safety Study. Clin Ther. Jun 1999;21(6):1006-24.
Kaplan SA, Roehrborn CG, McConnell JD, et al. Baseline symptoms, uroflow, and post-void residual urine as predictors of BPH clinical progression in the medically treated arms of the MTOPS trial (Abstract). J Urol. 2003;169 (Suppl 4):332h.
Roehrborn CG, Lukkarinen O, Mark S, Siami P, Ramsdell J, Zinner N. Long-term sustained improvement in symptoms of benign prostatic hyperplasia with the dual 5alpha-reductase inhibitor dutasteride: results of 4-year studies. BJU Int. Sep 2005;96(4):572-7. doi:BJU5686 [pii] 10.1111/j.1464-410X.2005.05686.x
McConnell JD, Roehrborn CG, Slawin KM, et al. Baseline Measures Predict the Risk of Benign Prostatic Hyperplasia Clinicalprogression In placebo-treated patients (abstract). J Urol. 2003;169 (Suppl 4):332.
Siami P, Roehrborn CG, Barkin J, et al. Combination therapy with dutasteride and tamsulosin in men with moderate-to-severe benign prostatic hyperplasia and prostate enlargement: the CombAT (Combination of Avodart and Tamsulosin) trial rationale and study design. Contemp Clin Trials. Nov 2007;28(6):770-9. doi:S1551-7144(07)00118-8 [pii] 10.1016/j.cct.2007.07.008
Roehrborn CG, Siami P, Barkin J, et al. The effects of combination therapy with dutasteride and tamsulosin on clinical outcomes in men with symptomatic benign prostatic hyperplasia: 4-year results from the CombAT study. Eur Urol. Jan 2010;57(1):123-31. doi:S0302-2838(09)00970-1 [pii] 10.1016/j.eururo.2009.09.035
Singh DV, Mete UK, Mandal AK, Singh SK. A comparative randomized prospective study to evaluate efficacy and safety of combination of tamsulosin and tadalafil vs. tamsulosin or tadalafil alone in patients with lower urinary tract symptoms due to benign prostatic hyperplasia. The journal of sexual medicine. Jan 2014;11(1):187-96. doi:10.1111/jsm.12357
Glina S, Roehrborn CG, Esen A, et al. Sexual function in men with lower urinary tract symptoms and prostatic enlargement secondary to benign prostatic hyperplasia: results of a 6-month, randomized, double-blind, placebo-controlled study of tadalafil coadministered with finasteride. The journal of sexual medicine. Jan 2015;12(1):129-38. doi:10.1111/jsm.12714
Zhang J, Li X, Yang B, Wu C, Fan Y, Li H. Alpha-blockers with or without phosphodiesterase type 5 inhibitor for treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia: a systematic review and meta-analysis. World journal of urology. 2019;37(1):143-153.
Jeong YB, Kwon KS, Kim SD, Kim HJ. Effect of discontinuation of 5alpha-reductase inhibitors on prostate volume and symptoms in men with BPH: a prospective study. Urology. Apr 2009;73(4):802-6. doi:S0090-4295(08)01821-9 [pii] 10.1016/j.urology.2008.10.046
Barkin J, Guimaraes M, Jacobi G, Pushkar D, Taylor S, van Vierssen Trip OB. Alpha-blocker therapy can be withdrawn in the majority of men following initial combination therapy with the dual 5alpha-reductase inhibitor dutasteride. Eur Urol. Oct 2003;44(4):461-6.
Han M, Alfert H, J.,, Partin AW. Retropubic and Suprapubic Open Prostatectomy. In: Walsh PC, ed. Campbell's Urology. 8th ed. Saunders; 2002:1423-1434.
Lu-Yao GL, Barry MJ, Chang CH, et al. Transurethral resection of the prostate among Medicare beneficiaries in the United States: time trends and outcomes. Prostate Patient Outcomes Research Team (PORT). Urology. 1994;44:692-696.
Blute M, Ackerman SJ, Rein AL, et al. Cost effectiveness of microwave thermotherapy in patients with benign prostatic hyperplasia: part II--results. Urology. Dec 20 2000;56(6):981-7.
Wasson JH, Reda DJ, Bruskewitz RC, Elinson J, Keller AM, Henderson WG. A comparison of transurethral surgery with watchful waiting for moderate symptoms of benign prostatic hyperplasia. The Veterans Affairs Cooperative Study Group on Transurethral Resection of the Prostate. N Engl J Med. Jan 12 1995;332(2):75-9.
Ogiste JS, Cooper K, Kaplan SA. Are stents still a useful therapy for benign prostatic hyperplasia? Curr Opin Urol. Jan 2003;13(1):51-7.
Fitzpatrick JM, Mebust WK. Minimally Invasive and Endoscopic Management of Benign Prostatic Hyperplasia. In: Walsh PC, ed. Campbell's Urology. 8th ed. Saunders; 2002:1379-1422.
Parsons CI. Impotence following transurethral resection of the prostate. In: Raifer J, ed. Common problems in infertility and impotence. Year Book Medical; 1990.
Cornu JN, Ahyai S, Bachmann A, et al. A Systematic Review and Meta-analysis of Functional Outcomes and Complications Following Transurethral Procedures for Lower Urinary Tract Symptoms Resulting from Benign Prostatic Obstruction: An Update. Eur Urol. Jun 2015;67(6):1066-96. doi:10.1016/j.eururo.2014.06.017
Chen Q, Zhang L, Fan QL, Zhou J, Peng YB, Wang Z. Bipolar transurethral resection in saline vs traditional monopolar resection of the prostate: results of a randomized trial with a 2-year follow-up. BJU Int. Nov 2010;106(9):1339-43. doi:10.1111/j.1464-410X.2010.09401.x BJU9401 [pii]
Naikai L, Jianjun Y. A Study Comparing Plasmakinetic Enucleation to Bipolar Plasmakinetic Resection of the Prostate for Benign Prostatic Hyperplasia. J Endourol. Nov 10 2011;26(7):884-8. doi:10.1089/end.2011.0358
Yip SK, Chan NH, Chiu P, Lee KW, Ng CF. A randomized controlled trial comparing the efficacy of hybrid bipolar transurethral vaporization and resection of the prostate with bipolar transurethral resection of the prostate. J Endourol. Dec 2011;25(12):1889-94. doi:10.1089/end.2011.0269
Wang K, Li Y, Teng JF, Zhou HY, Xu DF, Fan Y. Transurethral plasmakinetic resection of the prostate is a reliable minimal invasive technique for benign prostate hyperplasia: a meta-analysis of randomized controlled trials. Asian journal of andrology. Jan-Feb 2015;17(1):135-42. doi:10.4103/1008-682x.138191
Aho TF, Gilling PJ. Laser therapy for benign prostatic hyperplasia: a review of recent developments. Current Opinion in Urology. 2003;1:39-44.
Te AE, Malloy TR, Stein BS, et al. Photoselective vaporization of the prostate for the treatment of benign prostatic hyperplasia: 12-month results from the first United States multicenter prospective trial. J Urol. Oct 2004;172(4 Pt 1):1404-8. doi:00005392-200410000-00046 [pii]
Hai MA, Malek RS. Photoselective vaporization of the prostate: initial experience with a new 80 W KTP laser for the treatment of benign prostatic hyperplasia. J Endourol. Mar 2003;17(2):93-6. doi:10.1089/08927790360587414
Osterberg EC, Kauffman EC, Kang HW, Koullick E, Choi BB. Optimal laser fiber rotational movement during photoselective vaporization of the prostate in a bovine ex-vivo animal model. J Endourol. Jul 2011;25(7):1209-15. doi:10.1089/end.2010.0600
Campbell NA, Chung AS, Yoon PD, Thangasamy I, Woo HH. Early experience photoselective vaporisation of the prostate using the 180W lithium triborate and comparison with the 120W lithium triborate laser. Prostate international. 2013;1(1):42-5. doi:10.12954/pi.12006
Thomas JA, Tubaro A, Barber N, et al. A Multicenter Randomized Noninferiority Trial Comparing GreenLight-XPS Laser Vaporization of the Prostate and Transurethral Resection of the Prostate for the Treatment of Benign Prostatic Obstruction: Two-yr Outcomes of the GOLIATH Study. Eur Urol. Jan 2016;69(1):94-102. doi:10.1016/j.eururo.2015.07.054
Bachmann A, Ruszat R, Wyler S, et al. Photoselective vaporization of the prostate: the basel experience after 108 procedures. Eur Urol. Jun 2005;47(6):798-804. doi:S0302-2838(05)00076-X [pii] 10.1016/j.eururo.2005.02.003
Bouchier-Hayes DM, Anderson P, Van Appledorn S, Bugeja P, Costello AJ. KTP laser versus transurethral resection: early results of a randomized trial. J Endourol. Aug 2006;20(8):580-5.
Gilling P, Cass C, Cresswell M, et al. The use of the holmium laser in the treatment of BPH. J Endourol. 1996;10:459-461.
Hoffman RM, MacDonald R, Slaton JW, Wilt TJ. Laser prostatectomy versus transurethral resection for treating benign prostatic obstruction: a systematic review. J Urol. Jan 2003;169(1):210-215.
Gilling PJ, Mackey M, Cresswell M, Kennett K, Kabalin JN, Fraundorfer MR. Holmium laser versus transurethral resection of the prostate: a randomized prospective trial with 1-year followup. J Urol. Nov 1999;162(5):1640-4.
Hurle R, Vavassori I, Piccinelli A, Manzetti A, Valenti S, A. V. Holmium laser enucleation of the prostate combined with mechanical morcellation in 155 patients with benign prostatic hyperplasia. Urology. 2002;60(3):449-453.
Li S, Zeng XT, Ruan XL, et al. Holmium laser enucleation versus transurethral resection in patients with benign prostate hyperplasia: an updated systematic review with meta-analysis and trial sequential analysis. PloS one. 2014;9(7):e101615. doi:10.1371/journal.pone.0101615
Xia SJ, Zhang YN, Lu J, et al. [Thulium laser resection of prostate-tangerine technique in treatment of benign prostate hyperplasia]. Zhonghua yi xue za zhi. Nov 30 2005;85(45):3225-8.
Xia SJ. Two-micron (thulium) laser resection of the prostate-tangerine technique: a new method for BPH treatment. Asian journal of andrology. May 2009;11(3):277-81. doi:10.1038/aja.2009.17
Zhu Y, Zhuo J, Xu D, Xia S, Herrmann TR. Thulium laser versus standard transurethral resection of the prostate for benign prostatic obstruction: a systematic review and meta-analysis. World J Urol. Apr 2015;33(4):509-15. doi:10.1007/s00345-014-1410-6
Hashim H, Worthington J, Abrams P, et al. Thulium laser transurethral vaporesection of the prostate versus transurethral resection of the prostate for men with lower urinary tract symptoms or urinary retention (UNBLOCS): a randomised controlled trial. The Lancet. 2020;396(10243):50-61.
Hartung FO, Kowalewski KF, von Hardenberg J, et al. Holmium Versus Thulium Laser Enucleation of the Prostate: A Systematic Review and Meta-analysis of Randomized Controlled Trials. Eur Urol Focus. Apr 8 2021;doi:10.1016/j.euf.2021.03.024
Costello A, Bowsker W, Bolton G, et al. Laser ablation of prostate in patients with benign prostatic hyperplasia. Br J Urol. 1992;69:603-608.
Chacko K, Donovan J, Abrams P, et al. TURP or laser therapy for men with acute urinary retention: the ClasP randomised trial. J Urol. 2001;166:166-171.
Jepsen J, Bruskewitz R. Recent developments in the surgical management of benign prostatic hyperplasia. Urology. 1998;51(Suppl. 4A):23-31.
Serretta V, Morgia G, Fondacaro L, et al. Open prostatectomy for benign prostatic enlargement in southern Europe in the late 1990s: a contemporary series of 1800 interventions. Urology. Oct 2002;60(4):623-7.
Gacci M, Bartoletti R, Figlioli S, et al. Urinary symptoms, quality of life and sexual function in patients with benign prostatic hypertrophy before and after prostatectomy: a prospective study. BJU Int. Feb 2003;91(3):196-200.
Tubaro A, Vicentini C, Renzetti R, Miano L. Invasive and minimally invasive treatment modalities for lower urinary tract symptoms: what are the relevant differences in randomised controlled trials? Eur Urol. 2000;38 Suppl 1:7-17.
Yu X, Elliott SP, Wilt TJ, McBean AM. Practice patterns in benign prostatic hyperplasia surgical therapy: the dramatic increase in minimally invasive technologies. J Urol. Jul 2008;180(1):241-5; discussion 245. doi:10.1016/j.juro.2008.03.039
Christidis D, McGrath S, Perera M, Manning T, Bolton D, Lawrentschuk N. Minimally invasive surgical therapies for benign prostatic hypertrophy: the rise in minimally invasive surgical therapies. Prostate international. 2017;5(2):41-46.
Lourenco T, Shaw M, Fraser C, MacLennan G, N'Dow J, Pickard R. The clinical effectiveness of transurethral incision of the prostate: a systematic review of randomised controlled trials. World J Urol. Feb 2010;28(1):23-32. doi:10.1007/s00345-009-0496-8
Bostwick DG, Larson TR. Transurethral microwave thermal therapy: pathologic findings in the canine prostate. Prostate. 1995;26:116-122.
Thalmann G N, Mattei A, Treuthardt C, Burkhard FC, Studer UE. Transurethral microwave therapy in 200 patients with a minimum followup of 2 years: urodynamic and clinical results. J Urol. Jun 2002;167(6):2496-501.
Hoffman RM, Monga M, Elliott SP, et al. Microwave thermotherapy for benign prostatic hyperplasia. The Cochrane database of systematic reviews. 2012;9:Cd004135. doi:10.1002/14651858.CD004135.pub3
Gravas S, Laguna P, Ehrnebo M, Wagrell L, Mattiasson A, de la Rosette JJ. Seeking evidence that cell kill guided thermotherapy gives results not inferior to those of transurethral prostate resection: results of a pooled analysis of 3 studies of feedback transurethral microwave thermotherapy. J Urol. Sep 2005;174(3):1002-6; discussion 1006. doi:10.1097/01.ju.0000169266.20149.29
Klingler HC. New innovative therapies for benign prostatic hyperplasia: any advance? Curr Opin Urol. Jan 2003;13(1):11-5.
Schwartz RN, Couture F, Sadri I, et al. Reasons to believe in vaporization: a review of the benefits of photo-selective and transurethral vaporization. World Journal of Urology. 2020:1-6.
Ahyai SA, Gilling P, Kaplan SA, et al. Meta-analysis of functional outcomes and complications following transurethral procedures for lower urinary tract symptoms resulting from benign prostatic enlargement. European urology. 2010;58(3):384-397.
McAllister WJ, Karim O, Plail RO, et al. Transurethral electrovaporization of the prostate: is it any better than conventional transurethral resection of the prostate? BJU Int. Feb 2003;91(3):211-4.
Zlotta AR, Giannakopoulos X, Maehlum O, Ostrem T, Schulman CC. Long-term evaluation of transurethral needle ablation of the prostate (TUNA) for treatment of symptomatic benign prostatic hyperplasia: clinical outcome up to five years from three centers. Eur Urol. Jul 2003;44(1):89-93.
Bouza C, Lopez T, Magro A, Navalpotro L, Amate JM. Systematic review and meta-analysis of Transurethral Needle Ablation in symptomatic Benign Prostatic Hyperplasia. BMC urology. 2006;6:14. doi:10.1186/1471-2490-6-14
Sonksen J, Barber NJ, Speakman MJ, et al. Prospective, randomized, multinational study of prostatic urethral lift versus transurethral resection of the prostate: 12-month results from the BPH6 study. Eur Urol. Oct 2015;68(4):643-52. doi:10.1016/j.eururo.2015.04.024
Perera M, Roberts MJ, Doi SA, Bolton D. Prostatic urethral lift improves urinary symptoms and flow while preserving sexual function for men with benign prostatic hyperplasia: a systematic review and meta-analysis. Eur Urol. Apr 2015;67(4):704-13. doi:10.1016/j.eururo.2014.10.031
Roberts MJ, Perera M, Doi SA, Bolton D. Re: Marlon Perera, Matthew J. Roberts, Suhail A.R. Doi, Damien Bolton. Prostatic urethral lift improves urinary symptoms and flow while preserving sexual function for men with benign prostatic hyperplasia: a systematic review and meta-analysis. Eur Urol 2015;67:704-13. Eur Urol. Sep 2015;68(3):e53-4. doi:10.1016/j.eururo.2015.05.035
Roehrborn CG, Rukstalis DB, Barkin J, et al. Three year results of the prostatic urethral L.I.F.T. study. The Canadian journal of urology. Jun 2015;22(3):7772-82.
Traxer O, Anidjar M, Gaudez F, et al. A new prostatic stent for the treatment of benign prostatic hyperplasia in high-risk patients. Eur Urol. Sep 2000;38(3):272-8.
Gajewski JB, Chancellor MB, Ackman CF, et al. Removal of UroLume endoprosthesis: experience of the North American Study Group for detrusor-sphincter dyssynergia application. J Urol. Mar 2000;163(3):773-6.
Shore N, Tutrone R, Efros M, et al. Fexapotide triflutate: results of long-term safety and efficacy trials of a novel injectable therapy for symptomatic prostate enlargement. World journal of urology. 2018;36(5):801-809.
Shore N, Cowan B. The potential for NX-1207 in benign prostatic hyperplasia: an update for clinicians. Therapeutic advances in chronic disease. Nov 2011;2(6):377-83. doi:10.1177/2040622311423128
Nair SM, Pimentel MA, Gilling PJ. Evolving and investigational therapies for benign prostatic hyperplasia. The Canadian journal of urology. Oct 2015;22 Suppl 1:82-7.
Bailie J, McDonald S, Paez E. Novel intraprostatic injectable agents in the treatment of BPH. BJUI Knowledge. 2019;
Gilling P, Barber N, Bidair M, et al. WATER: a double-blind, randomized, controlled trial of Aquablation® vs transurethral resection of the prostate in benign prostatic hyperplasia. The Journal of urology. 2018;199(5):1252-1261.
Malling B, Røder M, Brasso K, Forman J, Taudorf M, Lönn L. Prostate artery embolisation for benign prostatic hyperplasia: a systematic review and meta-analysis. European radiology. 2019;29(1):287-298.
Abt D, Müllhaupt G, Hechelhammer L, et al. Prostatic Artery Embolisation Versus Transurethral Resection of the Prostate for Benign Prostatic Hyperplasia: 2-yr Outcomes of a Randomised, Open-label, Single-centre Trial. Eur Urol. Feb 18 2021;doi:10.1016/j.eururo.2021.02.008
Darson MF, Alexander EE, Schiffman ZJ, et al. Procedural techniques and multicenter postmarket experience using minimally invasive convective radiofrequency thermal therapy with Rezūm system for treatment of lower urinary tract symptoms due to benign prostatic hyperplasia. Research and reports in urology. 2017;9:159.
Mollengarden D, Goldberg K, Wong D, Roehrborn C. Convective radiofrequency water vapor thermal therapy for benign prostatic hyperplasia: a single office experience. Prostate cancer and prostatic diseases. 2018;21(3):379-385.
McVary KT, Gange SN, Gittelman MC, et al. Minimally invasive prostate convective water vapor energy ablation: a multicenter, randomized, controlled study for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia. The Journal of urology. 2016;195(5):1529-1538.
Mynderse LA, Hanson D, Robb RA, et al. Rezum System Water Vapor Treatment for Lower Urinary Tract Symptoms/Benign Prostatic Hyperplasia: Validation of Convective Thermal Energy Transfer and Characterization With Magnetic Resonance Imaging and 3-Dimensional Renderings. Multicenter Study

Research Support, Non-U.S. Gov't. Urology. Jul 2015;86(1):122-7. doi:10.1016/j.urology.2015.03.021

Lake AM, Hall TL, Kieran K, Fowlkes JB, Cain CA, Roberts WW. Histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model. Research Support, N.I.H., Extramural

Research Support, Non-U.S. Gov't. Urology. Sep 2008;72(3):682-6. doi:10.1016/j.urology.2008.01.037

Schuster TG, Wei JT, Hendlin K, Jahnke R, Roberts WW. Histotripsy treatment of benign prostatic enlargement using the Vortx Rx system: initial human safety and efficacy outcomes. Urology. 2018;114:184-187.
Speakman M. Lower Urinary Tract Symptoms Suggestive of Benign Prostatic Obstruction: What is the available evidence for rational management? Eur Urol. 2001;39 (Suppl 3):6-12.

Obesity In The Elderly

ABSTRACT

As the proportion of population above age 65 grows, so too increases the prevalence of those individuals who are obese. This phenomenon of an elderly population with obesity is the source of much research and debate with regards to treatment recommendations. It appears that older individuals on the extreme ends of the BMI spectrum, those who are underweight and those who are morbidly obese, have an increased risk of mortality. One major concern in the treatment of obese, elderly individuals is that many may have sarcopenic obesity which can be worsened with weight loss where some degree of lean body mass loss is inevitable. While various methods of weight loss may be recommended in some elderly who are obese, it is clear that any chosen method should be accompanied by a resistance training program in order to preserve muscle mass.

INTRODUCTION

The aging population in the U.S. is expected to more than double by 2050, increasing from 40.2 million to 88.5 million people (1). In tandem with this increase in elderly individuals is the high prevalence of those who are both elderly and obese. The significance of the increasing number of elderly individuals with obesity in terms of appropriate care and associated healthcare costs is the source of much debate.

PREVALENCE

Approximately 35% of adults in the U.S. aged 65 and over between 2007-2010 were obese as defined by body mass index (BMI, weight in kilograms over height in meters squared). In crude numbers this represents over 8 million adults aged 64-74 years and almost 5 million adults aged 75 and over (1). For individuals aged 75 and over there is a lower prevalence of obesity (27.8%) compared to those aged 65-74 years (40.8%) (1). A growing number of elderly are residing in nursing home (NH) facilities, and in line with this trend, researchers are examining the prevalence of obesity in NH facilities and its impact on healthcare utilization. Between 2000 and 2010, the prevalence of moderate to severe obesity in NHs increased from 14.7% to 23.9% (2). The rapid growth of the elderly population, which can largely be attributed to the aging baby boomers, will mark a change in the population’s composition in terms of sex ratios and ethnic diversity. Sex ratios of the population are projected to shift to include a larger share of elderly men (3). Moreover, the racial and ethnic make-up of this elderly cohort of patients is expected to develop to include more Hispanic individuals and a larger proportion of racial groups other than white. Between 2010 and 2050, the number of Hispanic people 65 years and older will increase from 2.9 to 17.5 million and the number of non-Hispanic individuals 65 years and older will increase from 37.4 to 71 million (3). These numbers of elderly individuals with obesity are also expected to increase as the population ages. Paradoxically, increased longevity does not necessarily translate to extra years spent in healthy living but may in fact result in more years spent in chronic poor health.

PATHOPHYSIOLOGY

Aging is accompanied by alterations in body composition. Fat free mass composed mostly of skeletal muscle declines by 40% between ages 20 and 70 years (4). Following age 70, both fat free mass and fat mass decrease together. With aging, there is also a redistribution of fat mass mainly in the visceral component but deposits are also observed in skeletal muscle and liver. The balance between energy intake and energy expenditure determines body fat mass. In the elderly, energy intake does not appear to increase significantly or may even decrease over time; therefore, decreased energy expenditure plays an important role in increasing fat mass with aging (4). After the age of 20, resting metabolic rate decreases by 2-3% per decade mainly due to a loss of fat free mass (4). In addition to a decrease in resting metabolic rate, physical activity declines and there is an increase in sedentary time, which accounts for approximately half the loss in total energy expenditure with aging (4).

The redistribution of body fat centrally leads to the production of pro-inflammatory cytokines (5). Pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) lead to muscle loss and sarcopenia due to their catabolic effects (6). This loss of muscle mass leads to adverse outcomes such as decreased mobility and increased frailty.

Endocrinologic changes that occur with aging also play a role in the pathophysiology of obesity including a decrease in growth hormone, testosterone, and DHEA in addition to resistance to leptin and insulin.

HEALTHCARE OUTCOMES: THE POSITIVE AND THE NEGATIVE

Limitations To BMI Measurements

The American College of Cardiology and the American Heart Association define adults as overweight if BMI ≥ 25 kg/m² and obese as BMI ≥ 30 kg/m² regardless of age range. Accurately assessing obesity outcomes in the elderly can be a challenge given the drawbacks of defining obesity by BMI. Other methods have been utilized including hydrostatic densitometry (underwater weighing), dual-energy x-ray absorptiometry (DXA), and waist circumference. Given that BMI can either underestimate or overestimate body fat mass in the elderly and the fact fat deposition in the elderly tends to be accumulated intraabdominally, measurement of waist circumstance may be a better way of assessment. Despite its drawbacks, most studies analyzing healthcare outcomes in the obese elderly have utilized BMI as an assessment tool.

The Obesity Paradox

According to existing studies and meta-analyses, a higher BMI can be protective in the elderly. In an analysis of 13 observational studies from 1966 to 1999 examining cardiovascular mortality in non-hospitalized subjects aged 65 and above, a U-shaped curve was observed with an increase in right curve only when BMI was above 31-32 kg/m² (7). A subsequent meta-analysis showed that BMI in overweight range did not confer an increased risk of mortality and a BMI in moderately obese range was only associated with a modest increase in mortality risk by 10% independent of gender, disease and smoking status (8). In a large, multi-ethnic study of community dwelling men and women aged 65 and above, the lowest hazard ratios (HRs) for mortality were seen in individuals with BMI 25 to less than 30 and BMI 30 to less than 35. HRs for mortality were increased when BMI was below 25 or higher than 35 (9). Similarly, in a large study of mortality in over 10,000 patients with type 2 diabetes mellitus and a median age of 63 years followed for a median of 10.6 years, a lower mortality risk was observed in overweight (BMI ≥ 25 kg/m²) and a higher mortality risk in those who were underweight (BMI ≤ 18.5 kg/m²) or obese (BMI ≥ 30 kg/m²) (10). A subsequent systemic review and meta-analysis evaluating the association of BMI with all-cause and cardiovascular mortality in subjects with type 2 diabetes mellitus, showed a strong non-linear relationship between BMI and all-cause mortality in both men and women. The lowest risk was seen in those with BMI 31-35 kg/m² and 28-31 kg/m². Lower BMI values were associated with higher mortality in both sexes (11). Combining available data suggests that BMI < 25 and > 35 kg/m² is associated with higher mortality (41) (Figure 1).

Figure 1. BMI and Mortality in Elderly

While there are positive effects of obesity including increased energy reserve and prevention of malnutrition, protection from bone mineral density loss and osteoporosis, and delay in cognitive decline, there are also potential biases which may account for the obesity paradox seen in the elderly. The survival effect is one such bias which postulates that the remaining living elderly with obesity are more resistant to the complications of obesity compared to those who were perhaps more susceptible and therefore died earlier. Many studies are epidemiologic in design with the limitation of reverse causation where an overestimation of mortality risk can occur if unintentional weight loss due to an underlying disease occurs prior to BMI measurements and are then compared to the BMI of healthy group. Finally, cohort effects can be seen as subjects in different environments practicing different lifestyles are compared to one another (12).

One of the most significant complications of obesity in the elderly is the metabolic syndrome. This clustering of risk factors including increased waist circumference, hypertension, dyslipidemia, and glucose intolerance increases the likelihood of diabetes and cardiovascular disease. Obesity can stress the joints leading to joint dysfunction and mobility impairment as well as lead to pulmonary dysfunction and obstructive sleep apnea. Certain cancers are associated with higher BMIs including breast, uterine, colon and leukemia.

Weight Loss

Numerous population-based studies have found that weight loss in older persons is associated with increased mortality (13, 42, 43, 44). This is also true in diabetes (14). Obviously, a part of this may be due to the disease itself causing weight loss, but a number of studies have used different approaches to control for this. The negative effects of weight loss are muscle loss (sarcopenia), the protective effect of fat (on hip fracture for example), lipolysis leading to accelerated atherosclerosis, and fat loss leading to release of fat-soluble toxins into circulation (15). Fat and protein loss can also lead to drug toxicity due to the alteration of the pharmacokinetics of medications that are either fat-soluble or protein-bound (15). The benefits of weight loss need to be weighed against the risks in older persons (Figure 2).

Figure 2. Risk and Benefits of Weight Loss in the Elderly

Sarcopenic Obesity

Diet-induced weight loss in both younger and elderly adults consist of 75% fat tissue loss and 25% is fat free mass loss (16, 17). Hypothetically, in the elderly with obesity the loss of lean body mass is buffered by the already increased muscle mass. This proved to be a falsely reassuring concept when sarcopenic obesity was first described in the early 2000s. Sarcopenia is defined as the loss of skeletal mass and function and leads to frailty, disability, and loss of independence in the elderly. Elderly individuals with obesity have the unique difficulty in that although weight gain causes increased lean body mass and fat mass, the increased muscle mass is of poor quality. In a study by Villareal and colleagues, 52 obese elderly adults, 52 nonobese frail adults and 52 nonobese, nonfrail subjects matched for age and sex were compared. Elderly adults with obesity showed lower muscle quality compared with the other two groups in addition to reduced functional performance, aerobic capacity, strength, balance, and walking speed (18). In essence, the elderly with obesity cohort were sarcopenic and their increased adiposity proved deleterious. Subsequent studies have continued to demonstrate that sarcopenic obesity is associated with and precedes the onset of instrumental activities of daily living (IADLs) disability in community dwelling elderly (19). However, elderly subjects who are obese with increased muscle mass have better outcomes compared to those with low muscle mass. Determining which individuals who are elderly and obese have sarcopenia is important clinically and can be accomplished inexpensively and easily by measuring muscle strength via handgrip dynamometry or gait speed. The brief SARC-F questionnaire (Table 1) can also be used to identify obese individuals with poor muscle function (20). Another method for measuring and monitoring skeletal muscle mass is the use of creatine (methyl-d₃) creatine dilution. In this noninvasive test, an oral tracer dose of D3-creatine is given and then subsequently measured in a fasting morning urine sample. Creatine dilution is a better measure of functional muscle mass than DXA (21).

Table 1. SARC-F Questionnaire
Component	Question	Scoring
Strength	How much difficulty do you have in lifting and carrying 10 pounds?	None = 0 Some = 1 A lot of unable = 2
Assistance in walking	How much difficulty do you have walking across a room?	None = 0 Some = 1 A lot, use of aids, or unable = 2
Rise from a chair	How much difficulty do you have transferring from a chair or bed?	None = 0 Some = 1 A lot or unable without help = 2
Climb stairs	How much difficult do you have climbing a flight of 10 stairs?	None = 0 Some = 1 A lot or unable = 2
Falls	How many times have you fallen in the past year?	None = 0 1-3 falls = 1 4 or more falls = 2

Score: ≥ 4 predictive of sarcopenia

TREATMENT

Who Should Be Recommended For Weight Loss?

Select elderly individuals with obesity and BMI ≥ 30 kg/m²who either have metabolic derangements or functional impairment may be recommended for weight loss provided that muscle and bone loss can be avoided (22).

Lifestyle Changes: Dietary Changes & Physical Exercise

Weight loss can be achieved alone by a moderate caloric deficit of 500-1000 kcal/day which leads to 1-2 pounds lost per week and 8-10% over 6 months (4). However, dietary changes should be prescribed in conjunction with an exercise program consisting of aerobic, resistance and balance training to promote functionality and improve frailty (23). In a study of 107 frail elderly subjects with obesity randomized to control, diet group with 500-750 kcal deficit with 1 gm protein/kg/day, and a multi-component exercise and diet group, the combined exercise and diet group was more effective. The combined group had better physical performance scores, functional status, and aerobic capacity. Subjects also lost less lean body mass and bone mineral density compared to the diet group (24). Additionally, lifestyle interventions can reduce disease burden. In the Diabetes Prevention Program, men and women ≥ 65 years with obesity were more likely to achieve 7% weight loss compared to their younger (age ≤ 45 years) counterparts with obesity, at 3 years, 63% and 27% respectively. For every kilogram lost through diet and physical activity, the incidence of T2DM was decreased by 16% over a 3-year period (25).

In order to prevent muscle catabolism, elderly individuals with obesity with or at risk for sarcopenic obesity should be counseled on a less restrictive caloric deficit of 200-500 kcal/day combined with a recommended protein intake of 1.0-1.5 gm/kg assuming normal renal function.

Pharmacotherapy

There is limited data on safety and efficacy of weight loss medications in the elderly as they have largely been excluded from clinical trials. The FDA has approved five medications for chronic weight management: Semaglutide, Liraglutide, Naltrexone/Bupropion, Phentermine/Topiramate, and Orlistat. Additionally, metformin has been studied as a weight loss medication in obese, non-diabetic subjects. There is also a study in progress of elderly Japanese patients with type 2 diabetes assessing the efficacy and safety of empagliflozin, a sodium-glucose cotransporter-2 inhibitor (SGLT2i), known to cause weight loss (EMPA-ELDERLY). In this population, the effects on skeletal muscle mass, muscle strength, and physical performance will be assessed in subjects age 65 and older with type 2 diabetes on Empagliflozin (26). Overall, drug interactions, affordability, efficacy, and safety are all potential drawbacks to pharmacotherapy for weight loss in the elderly. However, there are no studies of outcomes of anorectic drugs used with exercise to protect muscle and bone.

SEMAGLUTIDE

The weekly injectable glucagon-like peptide (GLP-1) receptor agonist was approved in 2021 for chronic weight management in adult patients with BMI of 30 kg/m2 or greater or 27 kg/m2 or greater plus a weight-related comorbid condition (hypertension, type 2 diabetes or dyslipidemia) as an adjunct to reduced calorie diet and increased physical activity. In the clinical trials, 233 (8.8%) of patients were between 65 and 75 years and 23 (0.9%) were 75 years or older and no differences in safety or efficacy were observed (27).

LIRAGLUTIDE

Liraglutide, a daily injectable GLP-1 receptor agonist was approved at doses of 3mg daily for weight loss by the FDA in 2014 for chronic weight management. This incretin-based therapy appears to have a short-term effect on decreasing gastric emptying but a long lasting central anorectic effect leading to a mean weight loss of 5.8kg in clinical studies (28, 29). The concern surrounding any weight loss in the elderly is the loss of skeletal muscle mass and sarcopenia. In a small study of elderly subjects who were either overweight or obese with type 2 diabetes mellitus treated with liraglutide 3mg daily in addition to metformin, reductions in fat mass and android fat were observed with the beneficial effect of preserved muscle tropism (30). A multicenter randomized, double-blind, parallel-group study of subjects with type 2 diabetes mellitus aged 18-80 years evaluated the effects of Liraglutide (as monotherapy or in combination with metformin) at various doses approved for treatment of diabetes mellitus (0.6mg, 1.2mg, 1.8mg daily) compared to individuals treated with Glimepiride or placebo. Mean body weight was reduced from baseline in all liraglutide treatment arms (up to 3.2 kg) and reduced fat tissue mass (1.0-2.4 kg) more than lean mass (1.5 kg) while glimepiride increased the mass of one or both tissue types (31). CT assessment also confirmed that reductions in fat tissue mass occurred in both abdominal subcutaneous and visceral fat compartments (31).

CONTRAVE

Contrave, the combination of naltrexone, an opioid antagonist, and bupropion, an aminoketone antidepressant, was FDA approved in 2014 for chronic weight management. Only 2% (62 of 3,239 subjects) in the Contrave clinical trials were over age 65 years and none older than 75 years (32). Data is lacking in terms of safety in older individuals, but given potential for neuropsychiatric disturbances, seizures, increased blood pressure and heart rate; extreme caution should be observed with this medication in the elderly.

QSYMIA

The combination of phentermine, a sympathomimetic amine anorectic, and topiramate extended release, an antiepileptic rug was FDA approved for chronic weight management in 2012. A small proportion of the subjects (254 total, 7%) studied in Qsymia clinical trials were aged 65 and older (33). While no differences in safety or effectiveness were observed, the adequate study numbers are also lacking. Given the side effect profile including risk of increased heart rate, acute myopia and secondary angle closure glaucoma, cognitive impairment and elevated creatinine, caution should be taken with starting this medication in elderly. Lower doses should be chosen and potential drug-drug interactions evaluated.

ORLISTAT

Orlistat acts as a pancreatic and gastric lipase inhibitor and leads to a 6.5-7.5 lb loss at one year. Its major side effects include steatorrhea, flatulence, fecal incontinence and malabsorption of fat-soluble vitamins. It appears to be equally efficacious with similar tolerance in a both the younger and elderly population (34).

METFORMIN

Metformin, a biguanide antidiabetic medication developed in the 1950s, may be a safe option to achieve modest weight loss even in nondiabetic individuals. In a small study of middle-aged nondiabetic subjects with obesity, metformin 2500mg daily without further caloric restriction or increased physical activity requirement resulted in a mean weight loss of 5.8 +/- 7kg (5.6+/-6.5%) compared to untreated controls (35). It may therefore be an efficacious and cost-effective strategy in elderly persons pending further studies.

Bariatric Surgery

According to the NIH, bariatric surgery procedures including sleeve gastrectomy, laparoscopic adjustable gastric banding (LAGB), Roux-en-Y gastric bypass (RYGB), and biliopancreatic diversion with or without duodenal switch are potential options for individuals with obesity between ages 18 and 64 with BMI ≥ 40 kg/m² or BMI ≥ 35 kg/m² with additional co-morbidities. The American Diabetes Association has recommended lower BMI cutoffs of ≥ 30 kg/m² for select individuals with uncontrolled hyperglycemia despite medical therapy (36). A retrospective review at a major surgical center in the U.S., found that of the 393 older patients (age > 65 years) who underwent bariatric surgery, older subjects had a higher comorbid burden compared to younger patients but exhibited comparable complication rates to patients under the age of 65 (37). In a systematic review of over 8,000 patients aged 60 years and older who underwent bariatric surgery, outcomes (resolution of hypertension, diabetes, lipid disorders) and complication rates were similar to a younger population, independent of type of procedure (38). While age should not necessarily be a barrier to recommending bariatric, this must be balanced against the limited existing data from pooled results of mostly small studies. Furthermore, bariatric surgery in the young and the elderly should always be coupled with resistance exercise.

Cryolipolysis

Cryolipolysis is FDA approved for treatment of focal fat deposits in the flanks, abdomen and thighs. In this procedure, fat cells are destroyed through a process of thermal reduction by which temperatures below normal but above freezing induce apoptosis-mediated cell death (39). Damaged adipocytes are then removed via an inflammatory response (39). This procedure has the advantage of being less invasive, does not require anesthesia with no downtime. In a retrospective review of a single surgery center with 528 subjects with age ranging from 18-79 years, the procedure was well tolerated with no adverse events and only 3 cases of mild or moderate pain reported to resolve in 4 or fewer days (40). However, there are limitations regarding the evaluation of the literature on this procedure thus far, including short follow-up time (typically 2-3 months), variability in cooling intensity factor (CIF) applied, differences in the evaluation of efficacy, and differences in the duration of procedure.

CONCLUSION

The landscape of the population is certainly changing and is marked by two significant trends: an increasingly elderly population and an ongoing obesity epidemic. This will undoubtedly impact families, social structures, and healthcare costs. How to appropriately care for these individuals will be the subject of much debate and further research. Physicians will need to balance the potential danger of weight loss in older persons against the complications of obesity to decide on the best patient centered approach. One clear recommendation is that all weight loss regimens in the elderly need to be coupled with a comprehensive resistance exercise program.

REFERENCES

Fakhouri THI, Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of Obesity Among Older Adults in the United States, 2007–2010. NCHS Data Brief 2012. No. 106.
Felix HC, Bradway C, Chisholm L, Pradhan R, Weech-Maldonado RW. Prevalence of Moderate to Severe Obesity Among U.S. Nursing Home Residents, 2000-2010. Res in Gerontol Nursing 2015;8(4):173-178.
Vincent GK, Velkoff VA. The next four decades, the older population in the United States: 2010 to 2050. Current Population Reports P25–1138. Washington, DC: U.S. Census Bureau. 2010.
Villareal DT, Apovian CM, Kushner RF, Klein S. Obesity in older adults: technical review and position statement of the American Society for Nutrition and NAASO, The Obesity Society. Obes Res 2005;82:923-934.
Schrager MA, Metter EJ, Simonsick E, Be A, Bandineli S. Lauretani F, et al. Sarcopenic obesity and inflammation in the InCHIANTI study. J Appl Physiol 2007;102:919-925.
Zamboni M, Mazzali G, Fantin F, Rossi A. Sarcopenic obesity: a new category of obesity in the elderly. Nutr Metab Cardiovasc Dis 2008;18:388-395.
Heiat A, Vaccarino V, Krumholz HM. An evidence-based assessment of federal guidelines for overweight and obesity as they apply to elderly persons. Arch Intern Med 2001;161:1194-1203.
Janssen I, Mark AE. Elevated body mass index and mortality risk in the elderly. Obes Rev 2007;8:41-59.
Al Snih S, Ottenbacher KJ, Markides KS, et al. The effect of obesity on disability vs mortality in older Americans. Arch Intern Med 2007;167:774-780.
Costanzo, P, Cleland JGF, Pellicori P et al. The obesity paradox in type 2 diabetes mellitus: relationship of body mass index to prognosis: a cohort study. Ann Intern Med 2015;162:610-618.
Zaccardi F, Dhalwani NN, Papamargaritis D, Webb DR, Murphy GJ, Davies MJ, Khunti K. Nonlinear association of BMI with all-cause and cardiovascular mortality in type 2 diabetes mellitus: a systematic review and meta-analysis of 414,587 participants in prospective studies. Diabetologia 2017;60:240-248.
Oreopoulos A, Kalantar-Zadeh K, Sharma AM, Fonarow GC. The obesity paradox in the elderly:potential mechanisms and clinical implications. Clin Geriatr Med 2009;25:643-659.
Yaari S, Goldbourt U. Voluntary and involuntary weight loss: associations with long term mortality in 9,228 middle-aged and elderly men. Am J Epidemiol 1998;148(6):546-555.
Wedick NM, Barrett-Connor E, Knoke JD, Wingard DL. The Relationship Between Weight Loss and All-Cause Mortality in Older Men and Women With and Without Diabetes Mellitus: The Rancho Bernardo Study. J Am Geriatr Soc 2002;50:1810-1815.
Rolland Y, Kim MJ, Gammack JK, Wilson MMG, Thomas DR, Morley JE. Office Management of Weight Loss in Older Persons. Amer Journal of Medicine 2006;119:1019-1026.
Dengel DR, Hagberg JM, Coon PJ, Drinkwater DT, Goldberg AP. Effects of weight loss by diet alone or combined with aerobic exercise on body composition in older obese men. Metabolism 1994;43:867-871.
Gallagher D, Kovera AJ, Clay-Williams G, Agin D, Leone P, Albu J, et al. Weight loss in postmenopausal obesity: no adverse alterations in body composition and protein metabolism. Am J Physiol Endocrinol Metab 2000;279:E124-131.
Villareal D, Banks M, Sienerc C, Sinacore D, Klein S. Physical Frailty and body composition in obese elderly men and women. Obes Res 2004;12:912-9.
Baumgartner RN, Wayne SJ, Waters DL, Janssen I, Gallagher D, Morley JE. Sarcopenia Obesity Predicts Instrumental Activities of Daily Living Disability in the Elderly 2004. Obes Res;12:1995-2004.
Malmstrom TK, Morley JE. SARC-F: A Simple Questionnaire to Rapidly Diagnose Sarcopenia. JAMDA 2014;14:531-532.
Clark RV, Walker AC, O’Connor-Semmes RL, Leonard MS, Miller RR, Stimpson SA, Turner SM, Ravussin E, Cefalu WT, Hellerstein MK, Evans WJ. Total body skeletal muscle mass: estimation of creatine (methyl-d3) dilution in humans. J Appl Physiol 2016;116:1605-1613.
Mathus-Vliegen EMH, Basdevant A, Finer N, Hainer V, Hauner H, Micic D, Maislos M, Roman G, Schutz Y, Tsigos C, Toplak H, Yumuk V, Zahorski-Markiewicz B. Prevalence, Pathophysiology, Health Consequences and Treatment Options of Obesity in the Elderly: A Guideline 2012. Obes Facts;5:460-483.
Villareal DT, Banks M, Sinacore DR, Siener C, Klein S. Effects of weight loss and exercise on frailty in obese older adults. Arch Intern Med 2006;166(8):860-6.
Villareal DT, Chode S, Parimi N, Sinacore DR, Hilton T, Armamento-Villareal R, et al. Weigth loss, exercise, or both and physical function in obese older adults. New Engl J Med 2011;364:1218-1229.
Crandall J, Schade D, Ma Y, Fujimoto WY, Barrett-Conner E, et al. The influence of age on the effects of lifestyle modification and metformin in prevention of diabetes. J Gerontol A Biol Sci Med Sci 2006;61:1075-1081.
Yabe D, Shiki K, Suzaki K, et al. Rationale and design of the EMPA-ELDERLY trial: a randomized, double-blind, placebo-controlled, 52-week clinical trial of the efficacy and safety of sodium-glucose cotransporter-2 inhibitor empagliflozin in elderly Japanese patients with type 2 diabetes. BMJ Open 2021;11:3045844.
Wegovy (semaglutide injection 2.4mg) [package insert]. Novo Nordisk Inc. New Jersey, U.S.
Jelsing J, Vrang N, Hansen G, Raun K, Tang-Christensen MT, Knudsen LB. Liraglutide: short-lived effect on gastric emptying-long lasting effects on body weight. Diabetes, Obesity and Metabolism 2012;14:531-538.
Apovian CM, Aronne LJ, Bessesen DH, McDonnell ME, Hassan Murad M, Pagotto U, Ryan DH, Still CD. Pharmacological Management of Obesity: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2015;100:342-362.
Perna S, Guido D, Bologna C, Solerte SB, Guerriero F, Isu A, Rondanelli M. Liraglutide and obesity in elderly: efficacy in fat loss and safety in order to prevent sarcopenia. A perspective case series study. Aging Clin Exp Res 2016;28(6):1251-1257.
Jendle J, Nauck MA, Matthews DR, Frid A, Hermansen K, During M, Zdravkovic M, Strauss BJ, Garber AJ. Weight loss with liraglutide, a once-daily human glucagon-like peptide-1 analogue for type 2 diabetes treatment as monotherapy or added to metformin, is primarily as a result of a reduction in fat tissue. Diabetes, Obesity and Metabolism 2009;11:1163-1172.
Contrave (naltrexone HCl and bupropion HCl extended release) [package insert]. Orexigen Therapeutics, Inc. California, U.S.
Qsymia (phentermine and topiramate extended-release) [package insert]. Vivus, Inc. California, U.S.
Segal KR, Lucas C, Boldrin M, Hauptman J. Weight loss efficacy of orlistat in obese elderly adults. Obes Res 1999;7(suppl):26A(abstract).
Seifarth C, Schehler B, Schneider HJ. Effectiveness of Metformin on Weight Loss in Non-Diabetic Individuals with Obesity. Exp Clin Endocrinol Diabetes 2013;121:27-31.
Obesity management for treatment of type 2 diabetes. Diabetes Care 2018;41(Supplement 1):S65-S72.
Quirante FP, Montorfano L, Rammohan R, Dhanabalsamy N, Lee A, Szomstein S, Lo Menzo E, Rosenthal RJ. Is bariatric surgery safe in the elderly population? Surg Endosc 2017;31:1538-1543.
Giordano S, Victorzon M. Bariatric surgery in elderly patients: a systematic review. Clin Interv Aging 2015;10:1627-1635.
Derrick CD, Shridharani SM, Broyles JM. The Safety and Efficacy of Cryolipolysis: A Systematic Review of Available Literature. Aesthetic Surg J 2015;35(7):830-836.
Stevens WG, Piertrzak LK, Spring MA. Broad overview of a clinical and commercial experience with CoolSculpting. Aesthetic Surg J 2013;33(6):835-46.
Winter JE, MacInnis RJ, Wattanapenpaiboon N, Nowson CA. BMI and all-cause mortality in older adults: a meta-analysis. Am J Clin Nutri 2014;99:875-90.
Reynolds MW, Fredman L, Langenberg P, Magaziner J. Weight, weight change, mortality in a random sample of older community-dwelling women. J Am Geriatr Soc 1999;47:1409-14.
Newman AB, Yanez D, Harris T, Duxbury A, Enright PL, Fried LP. Weight change in old age and its association with mortality. J Am Geriatr Soc 2001;49:1309-18.
Dey DK, Rothenberg E, Sundh V, Bosaeus I, Steen B. Body mass index, weight change and mortality in the elderly. A 15 y longitudinal population study of 70 y olds. Eur J Clin Nutr 2001;55:482-92.