An association of VDR gene polymorphism in hypovitaminosis D mediated secondary hyperparathyroidism in adolescent girls; a tertiary hospital study in central India.

Vitamin D is required to maintain normal serum calcium and phosphate levels that help normal bone mineralization, nerve conduction, muscle contraction, immune function, cell proliferation, and differentiation. Interventions including vitamin D supplementation may not improve vitamin D deficiency, as various complex genomic actions could contribute to vitamin D deficiency in the Indian population. Thus, we assessed hypovitaminosis D's relationship with vitamin D receptor (VDR) gene polymorphism and evaluated parathyroid hormone (PTH) levels in seemingly healthy adolescent school-going girls. We included 100 school-going girls (aged 12-17 years) studying in four schools of different socio-economic strata of Bhopal, India. The selected participants were divided into four groups based on the school in which they were studying. Blood samples were tested for serum calcium, phosphorus, PTH, ALP, vitamin D 25(OH) D, and albumin levels.VDR polymorphism was detected through the PCR-RFLP. Data were analyzed using the chi-square test, ANOVA, and linear regression. The difference in the age, calcium, ALP, and vitamin D values between the four groups were significant (P < 0.05), whereas high PTH levels (80%) were found. A higher prevalence of homozygous polymorphic allele demonstrates a molecular signature for severe secondary hyperparathyroidism. Hypovitaminosis D ranged from 84.9% to 100%, and a high prevalence of VDR polymorphism was observed. Attention must be paid to the health of this age group of school-going girls as hypovitaminosis D and associated VDR gene polymorphism could be the reason for secondary hyperparathyroidism (SHPT), showing changes in bone mineral density in these adolescent girls to ensure their future health.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Discovery of a Long-Acting Parathyroid Hormone 1-34 Analogue to Treat Hypoparathyroidism.

Hypoparathyroidism (HP) is a rare disease with clinical manifestations of hypocalcemia and hyperphosphatemia, resulting from deficient or absent parathyroid hormone (PTH) secretion. Conventional treatment for patients with HP involves extensive calcium and vitamin D supplementation. In 2015, PTH1-84 was approved by the United States Food and Drug Administration as an adjunct for HP patients who cannot be well-controlled on conventional treatment. However, PTH1-84 therapy requires a daily injection, leading to poor patient compliance. The purpose of this study was to develop a long-acting PTH1-34 analogue by increasing its affinity to albumin. Three PTH1-34 variants were generated by substituting two of the three lysine (Lys) residues with arginine, reserving a single Lys as the modification site in each sequence. A series of side chains, containing fatty acid, deoxycholic acid, or biotin groups, were synthesized to modify these PTH1-34 variants by using a solid-liquid phase synthesis approach. In vitro bioactivity and albumin affinity tests were used to screen these new PTH1-34 analogues. Finally, Lys27-AAPC was selected from 69 synthesized analogues as a candidate therapeutic compound because it retained potency and exhibited a high albumin-binding capacity. In pharmacodynamic experiments, Lys27-AAPC demonstrated enhanced and prolonged efficacy in serum calcium elevating relative to PTH1-84. Moreover, a lyophilized powder for injection containing Lys27-AAPC was developed for further testing and represented a potential long-acting HP treatment.

Murine Fetal Serum Phosphorus is Set Independent of FGF23 and PTH, Except in the Presence of Maternal Phosphate Loading.

Fibroblast growth factor 23 (FGF23) appears to play no role until after birth, given unaltered phosphate and bone metabolism in Fgf23- and Klotho-null fetuses. However, in those studies maternal serum phosphorus was normal. We studied whether maternal phosphate loading alters fetal serum phosphorus and invokes a fetal FGF23 or parathyroid hormone (PTH) response. C57BL/6 wild-type (WT) female mice received low (0.3%), normal (0.7%), or high (1.65%) phosphate diets beginning 1 week prior to mating to WT males. Fgf23+/- female mice received the normal or high-phosphate diets 1 week before mating to Fgf23+/- males. One day before expected birth, we harvested maternal and fetal blood, intact fetuses, placentas, and fetal kidneys. Increasing phosphate intake in WT resulted in progressively higher maternal serum phosphorus and FGF23 during pregnancy, while PTH remained undetectable. Fetal serum phosphorus was independent of the maternal phosphorus and PTH remained low, but FGF23 showed a small nonsignificant increase with high maternal serum phosphorus. There were no differences in fetal ash weight and mineral content, or placental gene expression. High phosphate intake in Fgf23+/- mice also increased maternal serum phosphorus and FGF23, but there was no change in PTH. WT fetuses remained unaffected by maternal high-phosphate intake, while Fgf23-null fetuses became hyperphosphatemic but had no change in PTH, skeletal ash weight or mineral content. In conclusion, fetal phosphate metabolism is generally regulated independently of maternal serum phosphorus and fetal FGF23 or PTH. However, maternal phosphate loading reveals that fetal FGF23 can defend against the development of fetal hyperphosphatemia.

Physical Activity-Dependent Regulation of Parathyroid Hormone and Calcium-Phosphorous Metabolism.

Exercise perturbs homeostasis, alters the levels of circulating mediators and hormones, and increases the demand by skeletal muscles and other vital organs for energy substrates. Exercise also affects bone and mineral metabolism, particularly calcium and phosphate, both of which are essential for muscle contraction, neuromuscular signaling, biosynthesis of adenosine triphosphate (ATP), and other energy substrates. Parathyroid hormone (PTH) is involved in the regulation of calcium and phosphate homeostasis. Understanding the effects of exercise on PTH secretion is fundamental for appreciating how the body adapts to exercise. Altered PTH metabolism underlies hyperparathyroidism and hypoparathyroidism, the complications of which affect the organs involved in calcium and phosphorous metabolism (bone and kidney) and other body systems as well. Exercise affects PTH expression and secretion by altering the circulating levels of calcium and phosphate. In turn, PTH responds directly to exercise and exercise-induced myokines. Here, we review the main concepts of the regulation of PTH expression and secretion under physiological conditions, in acute and chronic exercise, and in relation to PTH-related disorders.

Calcium Ion Channels: Roles in Infection and Sepsis Mechanisms of Calcium Channel Blocker Benefits in Immunocompromised Patients at Risk for Infection.

Immunosuppression may occur for a number of reasons related to an individual's frailty, debility, disease or from therapeutic iatrogenic intervention or misadventure. A large percentage of morbidity and mortality in immunodeficient populations is related to an inadequate response to infectious agents with slow response to antibiotics, enhancements of antibiotic resistance in populations, and markedly increased prevalence of acute inflammatory response, septic and infection related death. Given known relationships between intracellular calcium ion concentrations and cytotoxicity and cellular death, we looked at currently available data linking blockade of calcium ion channels and potential decrease in expression of sepsis among immunosuppressed patients. Notable are relationships between calcium, calcium channel, vitamin D mechanisms associated with sepsis and demonstration of antibiotic-resistant pathogens that may utilize channels sensitive to calcium channel blocker. We note that sepsis shock syndrome represents loss of regulation of inflammatory response to infection and that vitamin D, parathyroid hormone, fibroblast growth factor, and klotho interact with sepsis defense mechanisms in which movement of calcium and phosphorus are part of the process. Given these observations we consider that further investigation of the effect of relatively inexpensive calcium channel blockade agents of infections in immunosuppressed populations might be worthwhile.

Effects of low phosphorus intake on serum calcium, phosphorus, alkaline phosphatase activity and parathyroid hormone in growing dogs.

A sufficient and balanced supply of calcium and phosphorus is important mainly for the skeletal health of dogs, especially during growth. Numerous reports and research results are quoted, which focus mainly on the effects of both deficient or excessive calcium supplies. The important role that phosphorus plays in the disruption to skeletal development is also explored. The aims of this study were to evaluate the effects of a low phosphorus supply on selected serum parameters of calcium and phosphorus metabolism in both medium and large breed growing dogs, alongside the effects on the apparent digestibility and retention of these minerals as well as the musculoskeletal development, which were published before. Beagle and Foxhound-crossbred puppies (n = 53) between the age of 6 and 24 weeks were either fed a balanced (control) or a low phosphorus diet, the latter one providing about one-third of P amount fed to the control group. In regular intervals, blood samples were obtained to measure serum levels of phosphorus, calcium, alkaline phosphatase activity (ALP) and parathyroid hormone (PTH). Functional Additive Mixed Models were used to analyse these serum concentrations per group and per diet. In the puppies being fed the low phosphorus diet, a distinct decline of serum phosphate concentration was detected with a more pronounced and prolonged effect in the smaller Beagles. At the same time, serum calcium concentration altered in the opposite direction, implying that the product of calcium multiplied by phosphorus was regulated more closely than the calcium to phosphorus ratio. The PTH concentrations were reduced and ALP activity increased at certain time points during the low phosphorus feeding compared to the puppies being fed the control diet. In the latter group, an effect of time or maturation could be demonstrated in relation to the serum phosphate concentration.

The intervention effect of zuogui pill on chronic kidney disease-mineral and bone disorder regulatory factor.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) play a critical role in the pathogenesis of cardiovascular complications in patients with chronic kidney disease (CKD). Zuogui pill as a traditional Chinese herbal drug has been used for nourish kidney essence improve bone malnutrition of renal bone disease by regulating the metabolism of calcium and phosphorus and participating in osteoblast metabolism. In the present study, 5/6 nephrectomy rat model was used to reveal the mechanism of zuogui pill in treatment of CKD-MBD. Compared with sham rats, the levels of serum phosphorus, PTH, iPTH and creatinine were significantly decreased, while the serum calcium level was significantly increased, and the Cbfa1 protein level was significantly decreased and FGF23 protein level was significantly increased by Zuogui pill treatment. Compared with model rats, the BMD of rat was significantly increased by Zuogui pill treatment. Histological analysis revealed that the kidney injury of rats with CKD was significantly reduced by zuogui pill treatment. Compared with model rats, the CYP27B1 mRNA level was significantly increased, and the PTH mRNA level and NaPiIIa protein level were significantly decreased in the kidney by zuogui pill treatment. We inferred that zuogui pill exhibited potential therapeutic effects on CKD-MBD in the rats by regulating bone metabolism and nourish kidney.

Fast and reliable dissection of porcine parathyroid glands - A protocol for molecular and histological analyses.

As calcium and phosphorus are of vital importance for life, physiological activity of the parathyroid glands (PTGs) is crucial to maintain mineral homeostasis and bone mineralization. However, PTG-specific molecular routes in response to environmental factors and intrinsic hormonal responses are not yet fully understood. Since nutrient requirements, pathophysiology and functional genomics of pigs are similar to those of humans, pigs might be a suitable model to study the holistic gene expression and physiological aspects of the parathyroid gland, which could be used in both animal sciences and biomedical research. However, due to their small size and hidden location, the dissection of the PTGs, particularly in pigs, is difficult. Therefore, a protocol for untrained dissectors has been established that allows a fast and reliable identification of the PTGs in domestic pigs. Based on their localization within the cranial thymus near the carotid bifurcation, sampling was verified by histological staining and mRNA expression pattern. Analyses revealed the prominence of parathyroid hormone (PTH)-producing chief cells. Moreover, the copy numbers of PTH differed substantially between the PTGs and their surrounding thymus tissue, as PTH was expressed virtually exclusively in the PTGs. The developed protocol will substantially facilitate a fast and reliable dissection of porcine PTGs which is essential for studies characterizing the molecular mechanisms of parathyroid glands, e.g. when applying new feeding strategies in pigs.

Transcription factor MafB may play an important role in secondary hyperparathyroidism.

The transcription factor MafB is essential for development of the parathyroid glands, the expression of which persists after morphogenesis and in adult parathyroid glands. However, the function of MafB in adult parathyroid tissue is unclear. To investigate this, we induced chronic kidney disease (CKD) in wild-type and MafB heterozygote (MafB+/-) mice by feeding them an adenine-supplemented diet, leading to secondary hyperparathyroidism. The elevated serum creatinine and blood urea nitrogen levels in heterozygous and wild-type mice fed the adenine-supplemented diet were similar. Interestingly, secondary hyperparathyroidism, characterized by serum parathyroid hormone elevation and enlargement of parathyroid glands, was suppressed in MafB+/- mice fed the adenine-supplemented diet compared to similarly fed wild-type littermates. Quantitative RT-PCR and immunohistochemical analyses showed that the increased expression of parathyroid hormone and cyclin D2 in mice with CKD was suppressed in the parathyroid glands of heterozygous CKD mice. A reporter assay indicated that MafB directly regulated parathyroid hormone and cyclin D2 expression. To exclude an effect of a developmental anomaly in MafB+/- mice, we analyzed MafB tamoxifen-induced global knockout mice. Hypocalcemia-stimulated parathyroid hormone secretion was significantly impaired in MafB knockout mice. RNA-sequencing analysis indicated PTH, Gata3 and Gcm2 depletion in the parathyroid glands of MafB knockout mice. Thus, MafB appears to play an important role in secondary hyperparathyroidism by regulation of parathyroid hormone and cyclin D2 expression. Hence, MafB may represent a new therapeutic target in secondary hyperparathyroidism.

Effect of Oral Nonionic Polymeric Micelles Delivered Parathyroid Hormone cDNA on Bone Density and Microarchitecture.

INTRODUCTION: Parathyroid Hormone (PTH) is an effective therapeutic agent for osteoporosis, but the treatment requires long-term daily injections. Oral gene delivery is a less invasive alternative to daily injections. OBJECTIVE: The aim of this study is to investigate the effect of orally administered nonionic polymeric micelles of plasmid cDNA containing human cytomegalovirus promoter (PCMV)-PTH (1-34) plus EDTA on body mineral density and bone microstructure in ovariectomized rats. MATERIAL AND METHODS: A total of 27 Spraque-Dawley female rats were subjected to a bilateral ovariectomy. One month following the ovariectomy, they were randomly assigned to three groups: (1) (PCMVPTH (1-34) cDNA in polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) polymeric micelle formations plus EDTA); (2) PCMV-PTH (1-34) cDNA and (3) drinking water. The treatment was administered by oral gavage on day 1, 2, 7, 14, and 21 at 8-hour intervals. Body mineral density, bone volume fraction, and trabecular thickness of the lumbar spine and femoral neck were examined with peripheral quantitative computed tomography at pre- and post-intervention (3 months after the start of the intervention) and analyzed using two-way repeated measures analysis of variance. RESULTS: Results showed that bone mineral density, bone volume fraction and trabecular thickness were significantly increased in Group 1 over time, compared with those in Group 2 and Group 3. CONCLUSION: In conclusion, significantly improved bone mineral density and bone microstructure were observed in ovariectomized rats treated with PTH (1-34) cDNA delivered by nonionic polymeric micelles.

[Algal oligosaccharides ameliorate osteoporosis via up-regulation of parathyroid hormone 1-84 and vascular endothelial growth factor].

OBJECTIVE: To determine whether algal oligosac- charide~ affects the levels of parathyroid hormone 1-84 (PTH1-84) and vascular endothelial growth fac- tor (VEGF). METHODS: An osteoporosis rat model was estab- lished via bilateral ovariectomy. The model rats were fed algal oligosaccharides (molecular weights: 600-1, 200 Da) for 4 months. Bone mineral density (BMD) was then measured. MG-63 human osteo- blastic cells were treated with algal oligosaccha- rides. The expression of PTH1-84 and VEGF was then examined. Oligosaccharide-treated cells were transfected with PTH1-84 short hairpin RNA (shR- NA), VEGF shRNA, and PTH1-84-VEGF small interfer- ing RNA (siRNA). The growth rates were then com- pared between transfected and non-transfected RESULTS: Algal oligosaccharides increased the BMD of the osteoporosis rat model compared with untreated controls (P < 0.05). When MG-63 cells were treated with algal oligosaccharides, the growth rate increased by 25% compared with the control group at day 3 (P < 0.05). In addition, the ex- pression of P.TH84 and VEGF was. enhanced. Con- versey w hen tecells were tranfected with PTH84 shRNA, VEGF shRNA, or PTH1-84-VEGF siR- NA, the growth rate was decreased by 17%, 35% and 70%, respectively, compared with controls at day 3 (P < 0.05). CONCLUSION: Algal oligosaccharides ameliorate osteoporosis via up-regulation of PTH1-84 and VEGF. Algal oligosaccharides should be developed as a potential drug for osteoporosis treatment.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Is calcium signaling relevant for long bone growth?

BACKGROUND: Neonatal severe hyperparathyroidism (NSHPT) is a rare autosomal recessive disorder of calcium homeostasis, more often induced by homozygous inactivating mutations of the calcium-sensing receptor gene. This rare syndrome can be lethal if total parathyroidectomy is not performed within the first weeks of life. CLINICAL REPORT: We report the clinical case of a male patient, son of consanguineous hypercalcemic parents, with clinical and biochemical features of NSHPT, followed until the age of 21 years. The patient underwent total parathyroidectomy, and then, due to the low compliance to calcium and calcitriol supplementation, an attempt was made with recombinant human parathyroid hormone [rhPTH (1-84)]. The patient did not reach the predicted height with an increased ratio of the upper and lower segments. CONCLUSIONS: While this case is unique for the length of follow-up, the continuous and detailed description of NSHPT after total parathyroidectomy in its adult phenotype, and the treatment of hypoparathyroidism with rhPTH (1-84). Following this first description of a statural defect due to shortening of long bones in NSHPT, future investigations will attempt to uncover the role of calcium signaling in growth plate cartilage in humans.

Inhibition of parathyroid hormone secretion by caffeine in human parathyroid cells.

CONTEXT AND OBJECTIVE: Caffeine is a highly consumed psychoactive substance present in our daily drinks. Independent studies have reported associations between caffeine consumption, low bone mineral density, and urinary calcium loss, as well as impaired bone development in vitro and in vivo. Calcium (Ca(2+)), vitamin D, and PTH are critical regulators of bone remodeling. A possible association between caffeine and parathyroid gland function has been suggested in the literature. DESIGN, SETTING, AND PATIENTS: Effects of caffeine on PTH secretion and Ca(2+) levels were determined by batch incubation and Fura-2, respectively, in pathological parathyroid cells. Protein expressions were studied by Western blot and immunohistochemistry in normal and parathyroid adenoma tissues. Alterations in gene expressions of adenosine receptor A1 (ADORA1) and A2 (ADORA2A) and PTH were quantified by PCR; intracellular cAMP levels and protein kinase A activity were analyzed by an antibody-based assay. RESULTS: We studied physiological concentrations of caffeine ranging from 1 to 50 µm and found that 50 µm caffeine caused a significant decrease of PTH secretion and PTH gene expression. This decrease occurred in parallel with a decrease of the intracellular cAMP level, protein kinase A activity, and ADORA1 gene expression, indicating a possible causal relationship. The intracellular level of Ca(2+) was unaffected even by high concentrations of caffeine. Protein expressions demonstrated two main targets for caffeine-ADORA1 and ADORA2A. CONCLUSION: A physiological high dose of caffeine inhibits PTH secretion in human parathyroid cells, possibly due to a decrease of the intracellular level of cAMP. The observation demonstrates a functional link between caffeine and parathyroid cell function.

Simultaneous expression analysis of vitamin D receptor, calcium-sensing receptor, cyclin D1, and PTH in symptomatic primary hyperparathyroidism in Asian Indians.

BACKGROUND: To explore underlying molecular mechanisms in the pathogenesis of symptomatic sporadic primary hyperparathyroidism (PHPT). MATERIALS AND METHODS: Forty-one parathyroid adenomas from patients with symptomatic PHPT and ten normal parathyroid glands either from patients with PHPT (n=3) or from euthyroid patients without PHPT during thyroid surgery (n=7) were analyzed for vitamin D receptor (VDR), calcium-sensing receptor (CASR), cyclin D1 (CD1), and parathyroid hormone (PTH) expressions. The protein expressions were assessed semiquantitatively by immunohistochemistry, based on percentage of positive cells and staining intensity, and confirmed by quantitative real-time PCR. RESULTS: Immunohistochemistry revealed significant reductions in VDR (both nuclear and cytoplasmic) and CASR expressions and significant increases in CD1 and PTH expressions in adenomatous compared with normal parathyroid tissue. Consistent with immunohistochemistry findings, both VDR and CASR mRNAs were reduced by 0.36- and 0.45-fold change (P<0.001) and CD1 and PTH mRNAs were increased by 9.4- and 17.4-fold change respectively (P<0.001) in adenomatous parathyroid tissue. PTH mRNA correlated with plasma PTH (r=0.864; P<0.001), but not with adenoma weight, while CD1 mRNA correlated with adenoma weight (r=0.715; P<0.001). There were no correlations between VDR and CASR mRNA levels and serum Ca, plasma intact PTH, or 25-hydroxyvitamin D levels. In addition, there was no relationship between the decreases in VDR and CASR mRNA expressions and the increases in PTH and CD1 mRNA expressions. CONCLUSIONS: The expression of both VDR and CASR are reduced in symptomatic PHPT in Asian Indians. In addition, CD1 expression was greatly increased and correlated with adenoma weight, implying a potential role for CD1 in adenoma growth and differential clinical expression of PHPT.

Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human 'knockout'.

OBJECTIVE: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'. CASE REPORT: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6  mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950  pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. DESIGN AND METHODS: The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells. RESULTS: CaR(R392X) expressed as a 54  kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. CONCLUSIONS: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

CaSR-mediated interactions between calcium and magnesium homeostasis in mice.

Calcium (Ca) and magnesium (Mg) homeostasis are interrelated and share common regulatory hormones, including parathyroid hormone (PTH) and vitamin D. However, the role of the calcium-sensing receptor (CaSR) in Mg homeostasis in vivo is not well understood. We sought to investigate the interactions between Mg and Ca homeostasis using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (double knockout, DKO). Serum Mg is lower in PTH KO and DKO mice than in WT mice on standard chow, whereas supplemental dietary Ca leads to equivalent Mg levels for all three genotypes. Mg loading increases serum Mg in all genotypes; however, the increase in serum Mg is most pronounced in the DKO mice. Serum Ca is increased with Mg loading in the PTH KO and DKO mice but not in the WT mice. Here, too, the hypercalcemia is much greater in the DKO mice. Serum and especially urinary phosphate are reduced during Mg loading, which is likely due to intestinal chelation of phosphate by Mg. Mg loading decreases serum PTH in WT mice and increases serum calcitonin in both WT and PTH KO mice but not DKO mice. Furthermore, Mg loading elevates serum 1,25-dihydroxyvitamin D in all genotypes, with greater effects in PTH KO and DKO mice, possibly due to reduced levels of serum phosphorus and FGF23. These hormonal responses to Mg loading and the CaSR's role in regulating renal function may help to explain changes in serum Mg and Ca found during Mg loading.

Skeletal effects of vitamin D supplementation in postmenopausal black women.

Black women have lower serum 25-hydroxyvitamin D (25[OH]D) levels and higher parathyroid hormone (PTH) levels than white peers but lower bone turnover, suggesting skeletal resistance to PTH. Our objective was to determine if vitamin D supplementation (1,000 IU/day) would prevent bone loss and whether vitamin D receptor (VDR) polymorphisms modify the response. We performed a 2-year randomized, controlled, double-blind study of 1,000 IU vitamin D(3) vs. placebo in postmenopausal black women with serum 25(OH)D levels <20 ng/mL (n = 103). Measurements of 25(OH)D, PTH, and bone turnover were evaluated at baseline and 3, 6, 12, 18, and 24 months. DNA was extracted from peripheral blood leukocytes, and genotyping was conducted using standard techniques. Spine and hip bone mineral density (BMD) was measured at baseline and every 6 months. Serum 25(OH)D increased 11 ng/mL with vitamin D supplementation (p < 0.001), with no change in the placebo group. Vitamin D supplementation produced a significant decline in PTH at 3 months only, with no differences in bone turnover between placebo and vitamin D at any time point. Two-year changes in BMD were not significantly different between placebo- and vitamin D-treated black women at any skeletal site. Despite similar elevations in 25(OH)D, femoral neck BMD was only responsive to vitamin D supplementation in FF subjects (n = 47), not Ff/ff subjects (n = 31). Vitamin D supplementation does not appear to influence bone loss in black women. However, in the FF polymorphism of the VDR gene group, vitamin D supplementation may retard the higher rate of bone loss.

Potential treatment of hypoparathyroidism with recombinant plasmids encoding preproparathyroid hormone.

Hypoparathyroidism cannot be cured by oral or intravenous calcium supplements. Using molecular biology techniques to induce the production of PTH is an ideal option to treat hypoparathyroidism. In this study, we established a recombinant plasmid encoding a mutant preproPTH with a skeletal muscle creatine kinase promoter (pCKM-mPTH). The sequence of the chimeric pCKM-mPTH gene was fully consistent with the DNA sequence reported previously and the site-directed mutagenesis was completed. Overlapping and nested PCR showed that PTH was highly expressed in and secreted from skeletal muscle cells transfected with the pCKM-mPTH plasmid: the PTH concentration in the culture medium 24 h after transfection was 26.37 pg/l. In the rat hypoparathyroidism model, serum PTH level significantly increased after injection with the pCKM-mPTH plasmid, compared with control groups (p<0.01). The effect lasted for about 30 days. Our results indicated that the recombinant mutant pCKM-mPTH plasmid was successfully constructed and was highly expressed in skeletal muscle cells. In vivo, the plasmid was introduced successfully into rat muscles and could express PTH for a decent period of time.