[The role of calcium sensitive and vitamin D receptors in the pathogenesis of sporadic multiple parathyroid gland disease].

BACKGROUND: Sporadic multiple parathyroid gland disease is » cases of primary hyperparathyroidism (PHPT). However, a single tactic for diagnosing and operating volume in patients with this variant of PHPT has not yet been developed. One of the possible directions in the search for pathogenetically substantiated methods of diagnosis and treatment is the study of the molecular genetic features of the disease and associated clinical and laboratory factors. AIM: To study the features of the expression of calcium sensitive (CaSR) and vitamin D (VDR) receptors on the surface of parathyroid cells in primary hyperparathyroidism with solitary and multiple lesions of the parathyroid glands, as well as its changes under the influence of a decrease in the filtration function of the kidneys. MATERIALS AND METHODS: In a single center observational prospective study with retrospective data collection, there were patients who during 2019-2021. operated on for PHPT, secondary hyperparathyroidism (SHPT) and all cases of tertiary hyperparathyroidism (THPT) operated during 2014-2021. The expression of CaSR, VDR and its relationship with the main laboratory parameters, the clinical variant of hyperparathyroidism, and the morphological substrate were studied. RESULTS: The study included 69 patients: 19 with multiple and 25 with solitary PTG near PHPT, 15 with SHPT, 10 with THPT. A statistically significant decrease in the frequency of detection of normal expression of CaSR and VDR receptors occurs in any morphological variant of hyperparathyroidism and is observed in 93-60% of drugs. A decrease in the normal expression of CaSR in hyperplasia is detected statistically significantly less frequently than in adenoma (p≤0.01). The median expression intensity in adenoma was 2.5 (2:3), in hyperplasia 3.5 (3-4) (p≤0.01). The difference in the molecular mechanisms of the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma (PHPT with solitary adenoma) or hyperplasia (SHPT and PHPT with multiple PTG lesions) is realized in the frequency of maintaining normal CaSR expression in the PTG tissue. These mechanisms are implemented at the local level, their variability does not change under the influence of RRT. A common molecular genetic mechanism for the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma or hyperplasia has been found to reduce the frequency of maintaining normal VDR expression in PTG (up to 7-13%), p<0.01. This mechanism is implemented at the local level, its variability changes under the influence of RRT, reaching statistically significant differences in patients with THPT. CONCLUSION: The study demonstrates the features of changes in the expression of CaSR and VDR in PHPT with multiple lesions of the parathyroid glands. The relationship between the expression of these receptors and the clinical variant of hyperparathyroidism, the morphological substrate, the main laboratory parameters, and renal function was shown.

Analyzing Genetic Differences Between Sporadic Primary and Secondary/Tertiary Hyperparathyroidism by Targeted Next-Generation Panel Sequencing.

Secondary hyperparathyroidism (SHPT) is characterized by excessive serum parathyroid hormone levels in response to decreasing kidney function, and tertiary hyperparathyroidism (THPT) is often the result of a long-standing SHPT. To date, several genes have been associated with the pathogenesis of primary hyperparathyroidism (PHPT). However, the molecular genetic mechanisms of uremic hyperparathyroidism (HPT) remain uncharacterized. To elucidate the differences in genetic alterations between PHPT and SHPT/THPT, the targeted next-generation sequencing of genes associated with HPT was performed using DNA extracted from parathyroid tissues. As a result, 26 variants in 19 PHPT or SHPT/THPT appeared as candidate pathogenic mutations, which corresponded to 9 (35%) nonsense, 8 (31%) frameshift, 6 (23%) missense, and 3 (11%) splice site mutations. The MEN1 (23%, 6/26), ASXL3 (15%, 4/26), EZH2 (12%, 3/26), and MTOR (8%, 2/26) genes were frequently mutated. Sixteen of 25 patients with PHPT (64%) had one or more mutations, whereas 3 (21%) of 21 patients with SHPT/THPT had only 1 mutation (p = 0.001). Sixteen of 28 patients (57%) with parathyroid adenoma (PA) had one or more mutations, whereas 3 of 18 patients (17%) with parathyroid hyperplasia (PH) had just one mutation (p = 0.003). Known driver mutations associated with parathyroid tumorigenesis such as CCND1/PRAD1, CDC73/HRPT2, and MEN1 were identified only in PA (44%, 7/16 with mutations). Our results suggest that molecular genetic abnormalities in SHPT/THPT are distinct from those in PHPT. These findings may help in analyzing the molecular pathogenesis underlying uremic HPT development.

No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

Osteocytic osteolysis/perilacunar remodeling is thought to contribute to the maintenance of mineral homeostasis. Here, we utilized a reversible, adult-onset model of secondary hyperparathyroidism to study femoral bone mineralization density distribution (BMDD) and osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging. Male mice with a non-functioning vitamin D receptor (VDRΔ/Δ) or wild-type mice were exposed to a rescue diet (RD) (baseline) and subsequently to a low calcium challenge diet (CD). Thereafter, VDRΔ/Δ mice received either the CD, a normal diet (ND), or the RD. At baseline, BMDD and OLS characteristics were similar in VDRΔ/Δ and wild-type mice. The CD induced large cortical pores, osteomalacia, and a reduced epiphyseal average degree of mineralization in the VDRΔ/Δ mice relative to the baseline (-9.5%, p < 0.05 after two months and -10.3%, p < 0.01 after five months of the CD). Switching VDRΔ/Δ mice on the CD back to the RD fully restored BMDD to baseline values. However, OLS remained unchanged in all groups of mice, independent of diet. We conclude that adult VDRΔ/Δ animals on an RD lack any skeletal abnormalities, suggesting that VDR signaling is dispensable for normal bone mineralization as long as mineral homeostasis is normal. Our findings also indicate that VDRΔ/Δ mice attempt to correct a calcium challenge by enhanced osteoclastic resorption rather than by osteocytic osteolysis.

Post-transcriptional mechanisms regulating parathyroid hormone gene expression in secondary hyperparathyroidism.

Parathyroid hormone (PTH) regulates serum calcium levels and bone strength. Secondary hyperparathyroidism (SHP) is a common complication of chronic kidney disease (CKD) that correlates with morbidity and mortality. In experimental SHP, the increased PTH gene expression is due to increased PTH mRNA stability and is mediated by protein-PTH mRNA interactions. Adenosine-uridine-rich binding factor 1 (AUF1) stabilizes and K-homology splicing regulatory protein (KSRP) destabilizes PTH mRNA. The peptidyl-prolyl cis/trans isomerase Pin1 acts on target proteins, including mRNA-binding proteins. Pin1 leads to KSRP dephosphorylation, but in SHP, parathyroid Pin1 activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, leading to increased PTH mRNA stability and levels. A further level of post-transcriptional regulation occurs through microRNA (miRNA). Dicer mediates the final step of miRNA maturation. Parathyroid-specific Dicer knockout mice that lack miRNAs in the parathyroid develop normally. Surprisingly, these mice fail to increase serum PTH in response to both hypocalcemia and CKD, indicating that parathyroid Dicer and miRNAs are essential for stimulation of the parathyroid. Human and rodent parathyroids share similar miRNA profiles that are altered in hyperparathyroidism. The evolutionary conservation of abundant miRNAs and their regulation in hyperparathyroidism indicate their significance in parathyroid physiology and pathophysiology. let-7 and miR-148 antagonism modifies PTH secretion in vivo and in vitro, suggesting roles for specific miRNAs in parathyroid function. This review summarizes the current knowledge on the post-transcriptional mechanisms of PTH gene expression in SHP and the central contribution of miRNAs to the high serum PTH levels of both primary hyperparathyroidism and SHP.

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism.

Context: Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective: To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design: Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results: The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions: The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology.

ADAM17, a New Player in the Pathogenesis of Chronic Kidney Disease-Mineral and Bone Disorder.

The triad composed by α-Klotho, fibroblast growth factor-23, and its receptor are involved in the pathogenesis of chronic kidney disease-mineral and bone disorder. A disintegrin and metalloproteinase 17 (ADAM17) is a metalloproteinase causing the proteolytic shedding of α-Klotho from the cell membrane, and its role in chronic kidney disease-mineral and bone disorder is not yet known. We studied the circulating levels of the above-mentioned mediators in patients with secondary hyperparathyroidism due to uremia, compared to control subjects, as well as in patients with primary hyperparathyroidism. We also measured the immunofluorescence pattern of the relevant tissue proteins in specimens obtained from patients undergoing parathyroid surgery for secondary compared to primary hyperparathyroidism. Results showed that α-Klotho tissue levels are reduced, in the presence of increased ADAM17 tissue levels. In addition, we showed increased serum levels of the main product of ADAM17 proteolytic activity, tumor necrosis factor-α. Thus, we found a paradoxical situation, in secondary compared to primary hyperparathyroidism, that is, that in the face of increased tumor necrosis factor-α in circulation, both soluble and tissue α-Klotho are reduced significantly, despite increased tissue ADAM17. In conclusion, tissue and serum levels of α-Klotho seem to have become independent from the regulation induced by ADAM17, which constitutes therefore another tassel in the impaired α-Klotho-FGF23 receptor axis present in uremia.

Bsm1 vitamin D receptor polymorphism and calcium homeostasis following bariatric surgery.

PURPOSE/AIM: To evaluate the association between the Bsm1 vitamin D receptor polymorphism and the calcium-vitamin D-parathormone axis following bariatric surgery. MATERIALS AND METHODS: This cross-sectional study included 86 morbidly obese patients, who underwent either gastric bypass or sleeve gastrectomy, with a mean follow-up of four years. Calcium metabolism indices and bone turnover markers were assessed according to the presence of secondary hyperparathyroidism and the Bsm1 vitamin D receptor genotypes. RESULTS: Secondary hyperparathyroidism (42.2% of sample) was associated with lower levels of 25hydroxyvitamin D and elevated markers of bone turnover. In subjects without secondary hyperparathyroidism, presence of the unfavorable B allele resulted in higher levels of parathormone (Bb and BB vs. bb genotype: 50.3 ± 8.2 pg/dl vs. 44.4 ± 10.7 pg/dl, p = .011, adjusted for weight loss, baseline body mass index, 25hydroxyvitamin D, surgical procedure, and duration after surgery). In the whole sample, patients bearing the unfavorable B allele exhibited lower weight loss, a parameter that was negatively associated with markers of bone resorption. CONCLUSIONS: Secondary hyperparathyroidism is highly prevalent after bariatric surgery. Bsm1 vitamin D receptor polymorphism may have an effect in early stages of calcium metabolism imbalance, while no association is detected in patients who have already developed secondary hyperparathyroidism. Moreover, vitamin D receptor polymorphism is associated with post-surgery weight loss, a process related to bone turnover.

The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors.

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. The aim of this study was to investigate a potential role of the histone 3 lysine 27 methyltransferase EZH2 in parathyroid tumorigenesis. Parathyroid tumors from patients with pHPT included adenomas and carcinomas. Hyperplastic parathyroid glands from patients with HPT secondary to uremia and normal parathyroid tissue specimens were included in this study. Quantitative RT-PCR, western blotting, bisulfite pyrosequencing, colony formation assay, and RNA interference were used. EZH2 was overexpressed in a subset of the benign and in all malignant parathyroid tumors as determined by quantitative RT-PCR and western blotting analyses. Overexpression was explained by EZH2 gene amplification in a large fraction of tumors. EZH2 depletion by RNA interference inhibited sHPT-1 parathyroid cell line proliferation as determined by tritium-thymidine incorporation and colony formation assays. EZH2 depletion also interfered with the Wnt/ß-catenin signaling pathway by increased expression of growth-suppressive AXIN2, a negative regulator of ß-catenin stability. Indeed, EZH2 contributed to the total level of aberrantly accumulated transcriptionally active (nonphosphoylated) ß-catenin in the parathyroid tumor cells. To our knowledge EZH2 gene amplification presents the first genetic aberration common to parathyroid adenomas, secondary hyperplastic parathyroid glands, and parathyroid carcinomas. This supports the possibility of a common pathway in parathyroid tumor development.

Analysis of genotype-phenotype correlations and survival outcomes in patients with primary hyperparathyroidism caused by multiple endocrine neoplasia type 1: the experience at a single institution.

PURPOSE: To examine the clinical characteristics and survival outcomes of patients with primary hyperparathyroidism (PHPT) in multiple endocrine neoplasia type 1 (MEN1) in relation to the MEN1 gene mutation. METHODS: The study population included the patients, positive for the MEN1 gene mutation, who underwent parathyroidectomy between 1983 and 2009 at a single tertiary referral center. Manifestations of the syndrome, other tumors and causes of death were retrospectively correlated with the specific types and locations of MEN1 gene mutations. RESULTS: Thirty-two patients from 19 families were diagnosed as having MEN1 on genetic examinations. Mutations were most common in exons 2, 7 and 10. A phenotypic analysis of the main MEN1 tumor types among the 32 patients revealed that PHPT was the most common (100 %), followed in order by pancreatic neuroendocrine tumors (PNETs) (53 %) and pituitary tumors (38 %). Death due to MEN1-related disease occurred in five patients (16 %), including malignant PNET in three cases (exons 2, 3), pituitary crisis in one case (exon 2) and thymic cancer in one case (large deletion). CONCLUSIONS: Premature deaths related to MEN1 are due to the development of malignant PNET, pituitary crisis or thymic tumors associated with mutations in exons 2, 3 and a large deletion.

Risk factors for secondary hyperparathyroidism after bariatric surgery: a comparison of 4 different operations and of vitamin D-receptor-polymorphism.

OBJECTIVE: To determine the relative frequency of secondary hyperparathyroidism after 1 of 4 standard bariatric surgical procedures, with respect to vitamin D-receptor (VDR) Bsm1 genotype-polymorphism (VDRP). METHODS: Included were 141 obese men and women [aged 44.6±10.4 years, body mass index (BMI) 44.4±5.4 kg/m²], who had undergone either gastric banding (GB; n = 39), laparoscopic sleeve-gastrectomy (LSG; n = 31), Roux-en-Y-gastric-bypass (RYGB; n=43), or biliopancreatic-diversion with "duodenal switch" (BP-DS; n = 28)]. They were tested for VDR-genotype (Bsm1), vitamin D, and serum-PTH-levels postoperatively. RESULTS: Analysis of Covariance revealed a treatment effect, showing statistically significantly higher PTH-levels after BP-DS than after GB (mean difference = 32.14, p<0.001), after SG (mean difference = 25.18, p=0.001), or after RYGB (mean difference = 18.15, p=0.020). VDR-BSM1-genotype did not influence PTH-levels and vitamin-D postoperatively. Logistic regression indicated that the risk of developing SHPT after BP-DS was 12.5 times higher than after GB and 16.7 times higher than after SG. Beside other variables, VDR-genotype and the interaction between VDR-genotype and type of surgery did not attain statistical significance. CONCLUSIONS: In a comparison of the 4 most frequently performed bariatric operations vitamin-D-receptor polymorphism (VDRP) had no influence on the development of postoperative secondary hyperparathyroidism (SHPT) and is not useful as a predictor. SHPT occurs most often after BP-DS. Operation type, gender, VDRP, preoperative BMI, and relative postoperative BMI-loss, however, only explain 24% of the variance in postoperative PTH levels. Other gastral or intestinal factors physiologically promoting calcium-turnover and PTH regulation are postulated.

Characterization of patients with heterozygous cystinuria.

OBJECTIVE: To characterize a contemporary cohort of patients with heterozygous (TZ) cystinuria and compare them with a concurrent cohort of patients with homozygous (MZ) cystinuria. METHODS: A retrospective review of prospectively collected data was performed for 42 consecutive patients presenting with a positive cyanide-nitroprusside test from September 2009 to September 2011. Clinical data were collected, including the findings from a detailed metabolic stone workup that included two 24-hour urine collections with quantitative cystine. The patients were divided into 2 groups: those with TZ (30-400 mg/d) and those with MZ (>400 mg/d) cystinuria. RESULTS: One patient was excluded because the cystine excretion was within the normal range (<30 mg/d), 35 (83.3%) and 6 (14.3%) had TZ and MZ cystinuria, respectively. Compared with those with TZ cystinuria, those with MZ cystinuria were significantly younger at the first stone episode (median 48 years, range 14-67, vs 17, range 6-44, P = .002), more were female (20% vs 66.7%; P = .03), and more patients had bilateral stones (8.6% vs 50%; P = .03). Finally, the patients with MZ cystinuria had more stone episodes than those with TZ cystinuria (3 vs 1; P = .04). From the detailed metabolic stone evaluation, the incidence of hyperuricemia was significantly greater in the MZ patients (17.1% vs 66.7%; P = .02). Although all the MZ patients developed pure cystine stones, 18 (51.4%), 7 (20.0%), and 3 (8.6%) of the TZ patients developed calcium oxalate, uric acid, and cystine stones, respectively (P < .001). In the TZ group, 11 patients (31.4%) had false-negative results on subsequent cyanide-nitroprusside testing. CONCLUSION: Significant differences were found between the patients with TZ and MZ cystinuria in terms of age at the first stone episode, male/female ratio, incidence of hyperuricemia, and stone composition. The clinical significance remains to be elucidated.

Development and treatment of tertiary hyperparathyroidism in patients with pseudohypoparathyroidism type 1B.

CONTEXT: Pseudohypoparathyroidism type 1B (PHP1B) patients have PTH resistance at the renal proximal tubule and develop hypocalcemia and secondary hyperparathyroidism. Hyperparathyroid bone disease also develops in some patients. PHP1B patients are at theoretical risk of developing tertiary hyperparathyroidism. SETTING: Patients were studied in a clinical research center. PATIENTS: Five female PHP1B patients presented with hypercalcemia and elevated PTH. INTERVENTION: Patients either underwent parathyroidectomy (n = 4) or received cinacalcet (n = 1). MAIN OUTCOME MEASURES: Serum calcium and PTH were serially measured before and after intervention. RESULTS: Five PHP1B patients developed concomitantly elevated serum calcium and PTH levels (range, 235-864 ng/liter) requiring termination of calcium and vitamin D therapy (time after diagnosis, 21-42 yr; median, 34 yr), consistent with tertiary hyperparathyroidism. Four patients underwent parathyroidectomy with removal of one (n = 2) or two (n = 2) enlarged parathyroid glands. Calcium and vitamin D therapy was reinstituted postoperatively, and at 93-month median follow-up, PTH levels ranged between 56 and 182 (normal, <87) ng/liter. One patient was treated with cinacalcet, resulting in resolution of hypercalcemia. CONCLUSIONS: PHP1B patients are at risk of developing tertiary hyperparathyroidism and/or hyperparathyroid bone disease and should therefore be treated with sufficient doses of calcium and vitamin D to achieve serum calcium and PTH levels within or as close to the normal range as possible. Surgery is the treatment of choice in this setting. Cinacalcet may be a useful alternative in those who do not undergo surgery.

Control of bone resorption in mice by Schnurri-3.

Mice lacking the large zinc finger protein Schnurri-3 (Shn3) display increased bone mass, in part, attributable to augmented osteoblastic bone formation. Here, we show that in addition to regulating bone formation, Shn3 indirectly controls bone resorption by osteoclasts in vivo. Although Shn3 plays no cell-intrinsic role in osteoclasts, Shn3-deficient animals show decreased serum markers of bone turnover. Mesenchymal cells lacking Shn3 are defective in promoting osteoclastogenesis in response to selective stimuli, likely attributable to reduced expression of the key osteoclastogenic factor receptor activator of nuclear factor-κB ligand. The bone phenotype of Shn3-deficient mice becomes more pronounced with age, and mice lacking Shn3 are completely resistant to disuse osteopenia, a process that requires functional osteoclasts. Finally, selective deletion of Shn3 in the mesenchymal lineage recapitulates the high bone mass phenotype of global Shn3 KO mice, including reduced osteoclastic bone catabolism in vivo, indicating that Shn3 expression in mesenchymal cells directly controls osteoblastic bone formation and indirectly regulates osteoclastic bone resorption.

Increased parathyroid expression of klotho in uremic rats.

Klotho is a protein of significant importance for mineral homeostasis. It helps to increase parathyroid hormone (PTH) secretion and in the trafficking of Na+/K+-ATPase to the cell membrane; however, it is also a cofactor for fibroblast growth factor (FGF)-23 to interact with its receptor, FGFR1 IIIC, resulting in decreased PTH secretion. Studies on the regulation of parathyroid klotho expression in uremia have provided varying results. To help resolve this, we measured klotho expression in the parathyroid and its response to severe uremia, hyperphosphatemia, and calcitriol treatment in the 5/6 nephrectomy rat model of secondary hyperparathyroidism. Parathyroid klotho gene expression and protein were significantly increased in severely uremic hyperphosphatemic rats, but not affected by moderate uremia and normal serum phosphorus. Calcitriol suppressed klotho gene and protein expression in severe secondary hyperparathyroidism, despite a further increase in plasma phosphate. Both FGFR1 IIIC and Na+/K+-ATPase gene expression were significantly elevated in severe secondary hyperparathyroidism. Parathyroid gland klotho expression and the plasma calcium ion concentration were inversely correlated. Thus, our study suggests that klotho may act as a positive regulator of PTH expression and secretion in secondary hyperparathyroidism.

Development of a technique for introduction of an expressed complementary deoxyribonucleic acid into parathyroid cells by direct injection.

PTH is a major mediator of bone and mineral metabolism. However, physiological and pathological investigations of parathyroid cells (PTCs) have been limited because of the lack of available cell lines and because the organ is too small for detailed studies. Here, we describe a novel method for adenovirus-mediated cDNA transfer into PTCs, and we show the accuracy of the method in a rat model of uremia-induced secondary hyperparathyroidism. Rats underwent a 5/6-nephrectomy and were fed with a high-phosphate diet for 8 wk. The parathyroid glands were surgically exposed and adenoviruses containing LacZ or Ca-sensing receptor (CaSR) were directly injected into the glands under a zoom-stereo microscope. The parathyroid glands were analyzed for infection of adenovirus and immunohistochemically for expression of CaSR. The functional activity of exogenous CaSR in PTCs after this treatment was investigated based on changes of the calcium and PTH curve. A virus concentration of more than 10(9) plaque-forming units/ml was required for adequate infection of PTCs within 7 d after treatment. Marked increase of CaSR-positive PTCs by 2.39 +/- 0.72 times relative to control treatment, and significant colocalization of CaSR overexpression and virus labeling, were observed in glands after gene introduction. The calcium and PTH curve was shifted to the left from the basal position (set point, 1.10 +/- 0.09 to 0.76 +/- 0.12 mm; P < 0.0001), indicating successful introduction of a functionally active cDNA into the PTCs. This technique may facilitate an elucidation of biological effects through targeting and identification of specific features of PTCs, which may provide the basis for new clinical approaches.

Vitamin D receptor polymorphisms in secondary hyperparathyroidism after Scopinaro's biliopancreatic diversion.

BACKGROUND: Secondary hyperparathyroidism is a frequent metabolic complication of bariatric surgery. Individual differences in calcium absorption determine chronic secondary hyperparathyroidism after biliopancreatic diversion in half of the patients who have normal levels of 25-hydroxyvitamin D. We aimed to evaluate if certain vitamin D receptor polymorphisms may be responsible for the latter. Cases and controls study including 57 patients after biliopancreatic diversion with a mean serum 25-hydroxyvitamin D above 20 ng/mL, separated into those with secondary hyperparathyroidism (n = 26, cases) and those without it (n = 31, controls). METHODS: Genotyping for restriction-length-fragment polymorphisms of the vitamin D receptor gene was carried out for FOK1, BSM1, APA1, and TAQ1, and haplotype structure was also constructed. RESULTS: There were no differences in the allelic or genotypes distribution of the four studied polymorphisms between patients and controls (P = 0.352 and P = 0.301 for FOK1, P = 0.733 and P = 0.924 for BSM1, P = 0.974 and P = 0.992 for APA1, and P = 0.995 and P = 0.928 for TAQ1, respectively). Haplotype analysis showed no differences between patients and controls (P = 0.495 for BAT, P = 1.000 for BAt, P = 0.508 for Bat and P = 0.924 for bAT haplotypes, respectively). Furthermore, haplotypes were not associated with serum PTH levels or with the ratio between serum PTH and 25-hydroxyvitamin D levels. CONCLUSION: Chronic secondary hyperparathyroidism after biliopancreatic diversion in patients with normal levels of 25-hydroxyvitamin D is not dependent on vitamin D receptor gene polymorphisms.

Stimulating parathyroid cell proliferation and PTH release with phosphate in organ cultures obtained from patients with primary and secondary hyperparathyroidism for a prolonged period.

The pathogenesis of primary hyperparathyroidism (I degrees -HPT) and secondary hyperparathyroidism (II degrees -HPT) remains to be elucidated. To characterize their pathophysiology, we investigated the effects of calcium and phosphate on cell proliferation and PTH release in an organ culture of parathyroid tissues. Dissected parathyroid tissues obtained from patients with I degrees -HPT (adenoma) or II degrees -HPT (nodular hyperplasia) were precultured on a collagen-coated membrane for 1-4 week. After changing the medium for one containing various concentrations of phosphate, PTH release and [(3)H]thymidine incorporation were studied. In contrast to dispersed parathyroid cells cultured in a monolayer, calcium decreased PTH release in a concentration-dependent manner in parathyroid tissues. Furthermore, when parathyroid tissues obtained from II degrees -HPT were precultured for 1-4 weeks, PTH release and parathyroid cell proliferation were significantly increased in high-phosphate medium. These phosphate effects were also observed to a lesser extent in parathyroid tissues obtained from I degrees -HPT, but there was no significant difference between I degrees -HPT and II degrees -HPT. Microarray analyses revealed that mRNA levels of PTH, CaSR, and VDR were well preserved, and several growth factors (e.g. TGF-beta1-induced protein) were abundantly expressed in II degrees -HPT. Using organ cultures of hyperparathyroid tissues, in which PTH release and CaSR are well preserved for a prolonged period, we have demonstrated that phosphate stimulates parathyroid cell proliferation not only in II degrees -HPT but also in I degrees -HPT. Although the mechanism responsible for phosphate-induced cell proliferation remains to be elucidated, our in vitro findings suggest that both parathyroid tissues preserve to some extent a physiological response system to hyperphosphatemia as observed in normal parathyroid cells.

Regulation of PTH mRNA stability by the calcimimetic R568 and the phosphorus binder lanthanum carbonate in CKD.

Secondary hyperparathyroidism is characterized by increased parathyroid hormone (PTH) mRNA stability that leads to increased PTH mRNA and serum PTH levels. PTH gene expression is reduced by the calcimimetic R568 and the oral phosphorus binder lanthanum carbonate (La). Changes in PTH mRNA stability are regulated by the binding of trans-acting stabilizing and destabilizing factors to a defined cis element in the PTH mRNA 3'-untranslated region (UTR). Adenosine-uridine (AU)-binding factor 1 (AUF1) is a PTH mRNA-stabilizing protein, and K-homology splicing regulatory protein (KSRP) is a destabilizing protein that targets mRNAs, including PTH mRNA, to degradation by the ribonuclease complex exosome. We now show that KSRP-PTH mRNA binding is decreased in parathyroids from rats with adenine-induced chronic kidney disease (CKD) where PTH mRNA is more stable. KSRP-PTH mRNA binding is increased by treatment with both R568 and La, correlating with decreased PTH gene expression. In vitro degradation assays using transcripts for PTH mRNA and rat parathyroid extracts reproduce the differences in mRNA stability in vivo. Accordingly, PTH mRNA is destabilized in vitro by parathyroid extracts from CKD rats treated with R568 or La compared with parathyroid extracts from untreated CKD rats. This destabilizing effect of R568 and La is dependent on KSRP and the PTH mRNA 3'-UTR. Therefore, the calcimimetic R568 and correction of serum phosphorus by La determine PTH mRNA stability through KSRP-mediated recruitment of a degradation complex to the PTH mRNA, thereby decreasing PTH expression.

1α,24(S)(OH)2D2 normalizes bone morphology and serum parathyroid hormone without hypercalcemia in 25-hydroxyvitamin D-1-hydroxylase (CYP27B1)-deficient mice, an animal model of vitamin D deficiency with secondary hyperparathyroidism.

BACKGROUND: Vitamin D compounds are effective in managing elevated PTH levels in secondary hyperparathyroidism (SHPT) of renal failure. However, undesired increases in serum calcium and phosphorus associated with compounds such as calcitriol [1,25(OH)2D3] has prompted a search for compounds with improved safety profiles. 1alpha,24(S)(OH)2D2 (1,24(OH)2D2) is a vitamin D2 metabolite with low calcium-mo bilizing activity in vivo. We studied the efficacy of 1,24(OH)2D2 in mice lacking the CYP27B1 enzyme [25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase)], a novel vitamin D deficiency model with SHPT. MATERIALS AND METHODS: 1alpha-OHase-deficient (-/-) mice and normal (+/-) heterozygous littermates re ceived 1,24(OH)2D2 (100, 300, 1000, and 3000 pg/g/day) or 1,25(OH)2D3 (30, 300, and 500 pg/g/day) for 5 weeks via daily sc injection. Control groups received vehicle. RESULTS: Vehicle-treated 1alpha-OHase-deficient mice were hypocalcemic and had greatly elevated serum PTH. 1,24(OH)2D2 at doses above 300 pg/g/day normalized serum calcium, serum PTH, bone growth plate morphology, and other bone parameters. No hy percalcemia was observed at any dose of 1,24(OH)2D2 in normal or 1alpha-OHase-deficient animals. In contrast, 1,25(OH)2D3 at only 30 pg/g/day normalized calcemia, serum PTH, and bone parameters, but at higher doses completely suppressed PTH and caused hypercalcemia in both 1alpha-OHase-deficient and normal mice. Treatment with 500 pg/g/day of 1,25(OH)2D3 also induced osteomalacia in normal animals. CONCLUSION: 1,25(OH)2D3 was maximally active at 10-fold lower doses than 1,24(OH)2D2, but induced hypercalcemia and osteomalacia at high doses. 1,24(OH)2D2 normalized serum calcium, serum PTH, and bone histomorphometry without hypercalcemia in 1alpha-OHase-deficient mice with SHPT.

Mutational analysis of CDKN1B, a candidate tumor-suppressor gene, in refractory secondary/tertiary hyperparathyroidism.

Most patients with refractory secondary/tertiary hyperparathyroidism have monoclonal parathyroid tumors. Inactivating mutations of CDKN1B, encoding the p27 cyclin-dependent kinase inhibitor, were reported to cause hyperparathyroidism in a multiple endocrine neoplasia type 1-like syndrome. Further, there was decreased expression of CDKN1B in parathyroid tumors of patients with chronic kidney disease. We sequenced the entire coding region and splice sites of CDKN1B in 50 parathyroid tumors from 35 patients to see if inactivating mutations could cause monoclonal tumorigenesis in refractory secondary/tertiary hyperparathyroidism. No frameshift, nonsense, or other clearly inactivating mutations were found, nor was there evidence of homozygous deletion or loss of heterozygosity. The absence of clonal inactivating mutations suggests that CDKN1B is not a classical tumor-suppressor gene in secondary/tertiary parathyroid tumors.