pH dependence of extracellular calcium sensing receptor activity determined by a novel technique

BACKGROUND: Increasing evidence points to the role of the extracellular Calcium Sensing Receptor (CaSR) as a multimodal receptor responding to diverse physiologic stimuli, such as extracellular divalent and polyvalent cations, amino acids, and ionic strength. Within the kidney, these stimuli converge on the CaSR to coordinate systemic calcium and water homeostasis. In this process, the impact of urinary pH changes on the activity of the CaSR has not yet been defined. We therefore performed the present study to analyze the pH sensitivity of the CaSR. METHODS: To assess the activation state of the CaSR, we developed a new method based on the functional coupling between CaSR activity and gating of calcium sensitive potassium currents mediated by SK4 potassium channels. Two-electrode voltage clamping was used to determine whole cell currents in Xenopus oocytes heterologously expressing rat CaSR and rat SK4 potassium channels. RESULTS: Coexpression of CaSR and SK4 gave rise to potassium currents that were dependent on CaSR-mediated intracellular calcium release, and thereby corresponded to the activation state of the CaSR. In presence of extracellular calcium, ambient alkalinization above pH 7.5 increased CaSR activity. Evaluation of the CaSR calcium sensitivity at various ambient proton concentrations revealed that this effect was due to a sensitization of the CaSR towards extracellular calcium. CONCLUSION: Coexpression with SK4 potassium channels provides a fast and sensitive approach to evaluate CaSR activity in Xenopus oocytes. As disclosed by this novel technique, CaSR activity is regulated by extracellular pH.     

Development and progression of secondary hyperparathyroidism in chronic kidney disease: lessons from molecular genetics

The identification of the calcium-sensing receptor (CaSR) and the clarification of its role as the major regulator of parathyroid gland function have important implications for understanding the pathogenesis and evolution of secondary hyperthyroidism in chronic kidney disease (CKD). Signaling through the CaSR has direct effects on three discrete components of parathyroid gland function, which include parathyroid hormone (PTH) secretion, PTH synthesis, and parathyroid gland hyperplasia. Disturbances in calcium and vitamin D metabolism that arise owing to CKD diminish the level of activation of the CaSR, leading to increases in PTH secretion, PTH synthesis, and parathyroid gland hyperplasia. Each represents a physiological adaptive response by the parathyroid glands to maintain plasma calcium homeostasis. Studies of genetically modified mice indicate that signal transduction via the CaSR is a key determinant of parathyroid cell proliferation and parathyroid gland hyperplasia. Because enlargement of the parathyroid glands has important implications for disease progression and disease severity, it is possible that clinical management strategies that maintain adequate calcium-dependent signaling through the CaSR will ultimately prove useful in diminishing parathyroid gland hyperplasia and in modifying disease progression.     

Aluminum is a weak agonist for the calcium-sensing receptor

BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+.     

Calcium-sensing receptors

It is now known that variations in extracellular calcium concentration exert diverse physiologic effects in a variety of tissues that are mediated by a calcium-sensing receptor (CaSRs). In parathyroid tissue, the CaSR represents the molecular mechanism by which parathyroid cells detect changes in blood ionized calcium concentration, modulate parathyroid hormone (PTH) secretion accordingly, and thus maintain serum calcium levels within a narrow physiologic range. In the kidney, the CaSR regulates renal calcium excretion and influences the transepithelial movement of water and other electrolytes. More generally, activation of the CaSR represents an important signal transduction pathway in intestine, placenta, brain, and perhaps bone. Some of these actions involve cell cycle regulation, changes that may be relevant to understanding the pathogenesis of parathyroid gland hyperplasia in secondary hyperparathyroidism caused by chronic kidney disease. The CaSR represents an appealing target for therapeutic agents designed to modify parathyroid gland function in vivo, offering the prospect of novel therapies for selected disorders of bone and mineral metabolism. Other receptors capable of responding to extracellular calcium ions also have been identified, but the functional importance of these interactions remains to be determined.     

Effect of Calcium and the Calcimimetic AMG 641 on Matrix-Gla Protein in Vascular Smooth Muscle Cells

Vascular calcification (VC) is frequently observed in patients with chronic renal failure and appears to be an active process involving transdifferentiation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Reports of VC prevention in uremic rodents by calcimimetics coupled with identification of the calcium-sensing receptor (CaSR) in VSMCs led us to hypothesize that CaSR activation in arterial cells and VSMCs may elicit expression of an endogenous inhibitor of VC. Toward this end, we determined the effects of calcium and the calcimimetic AMG 641 on arterial wall and isolated VSMC expression of matrix-Gla protein (MGP). Bovine VSMCs were incubated with increasing calcium chloride or AMG 641 concentrations, while in vivo experiments were carried out on healthy and uremic rats. Both AMG 641 and hypercalcemia induced MGP expression in the arterial wall in healthy and uremic rats. The results obtained in vitro supported those from in vivo experiments. In conclusion, selective CaSR activation, either by extracellular calcium or AMG 641, increased MGP expression in vivo in the arterial wall and in vitro in bovine VSMCs. This local upregulation of MGP expression provides one potential mechanism by which calcimimetics prevent VC.     

Association of decreased calcium-sensing receptor expression with proliferation of parathyroid cells in secondary hyperparathyroidism

BACKGROUND: The down-regulation of both calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in parathyroid (PT) glands of secondary hyperparathyroidism (HPT) caused by chronic renal failure has been associated with PT hormone hypersecretion as well as PT hypergrowth. To clarify the predominance of decreased expression of CaSR and VDR in the high proliferative activity of PT glands, we examined the relationship between the expression of both receptors and proliferative activity in human PT glands. METHODS: Serial sections of 56 PT glands, including 52 glands from secondary HPT and 4 normal PT glands resected together with thyroid carcinoma, were examined immunohistochemically with specific antibodies against CaSR, VDR, and Ki67. The Ki67-positive cell number was counted and expressed as the Ki67 score. The CaSR and VDR expressions were semiquantitatively analyzed. RESULTS: The expressions of both CaSR and VDR were markedly decreased in PT glands of secondary HPT, while the Ki67 score was significantly higher than it was in normal controls. When hyperplastic glands were classified into two subgroups, with [N(+)] or without [N(-)] nodular formation, CaSR expression was significantly decreased in N(+), while VDR expression was not different. Multiple regression analyses revealed that the decreased expression of CaSR could contribute significantly to the high proliferative activity, even if VDR expression was taken into account. CONCLUSION: The decrease in CaSR expression is associated with the high proliferative activity of PT glands in secondary HPT, independently of the decreased VDR expression. These findings provide a new insight into the pathogenesis of PT hyperplasia, which is refractory to vitamin D therapy in patients with severe secondary HPT.     

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium‐Sensing Receptor‐Mediated Signaling

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β‐catenin), the molecular basis of abnormal calcium sensing by calcium‐sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein‐coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3′,5′‐cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N‐terminally truncated human GPR64 is constitutively active and a 15–amino acid–long peptide C‐terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full‐length GPR64 physically interacts with CaSR and attenuates the CaSR‐mediated intracellular Ca²⁺ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.     

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation—an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca²⁺] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.     

Parathyroid growth and regression in experimental uremia

Early 1,25-dihydroxyvitamin D(3) (VD(3)) therapy during the course of renal failure prevents the downregulation of VD(3) receptor (VDR), calcium-sensing receptor (CaSR) or p21, and the parathyroid (PT) growth. We hypothesized that VD(3) could restore the decreased expressions of VDR and CaSR, and cause regression in enlarged PT glands. 5/6 nephrectomized rats fed high-phosphorus diet were killed at 1, 3, 5, or 7 days and at 2, 3, 4, 8, or 12 weeks. VD(3)-treated rats were given VD(3) intraperitoneally for 1, 2, 3, or 4 weeks, starting 8 weeks after 5/6 nephrectomy. PT glands were weighed and subjected to immunohistochemical analyses for VDR, CaSR, p21, Ki67, and Tdt-mediated dUTP nick end-labeling (TUNEL) assay. The area per cell was measured as the parameter of cell size. The expression of VDR and p21 began to decrease at day 1, and Ki67 increased at day 3, but decreased thereafter. There was a significant increase in PT gland weight to week 12 with the increase of cell size. VD(3) treatment significantly increased both VDR and CaSR expressions 2 weeks after the start of injection, and reduced the PT gland weight at week 3 with significant increase of TUNEL-positive cells and decrease of cell size. Our results suggest that PT growth in uremic rats involves both PT cell proliferation and hypertrophy, in association with the reduction of VDR, CaSR, and p21 expressions. In addition, VD(3) treatment could reverse PT hyperplasia and hypertrophy via restoration of these proteins.     

Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome

The extracellular Ca(2+)-sensing receptor (CaSR) plays an essential role in extracellular Ca(2+) homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca(2+) and ERK activity, showed a dramatic reduction in the EC(50) for extracellular Ca(2+) compared with the wild-type and a loss-of-function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.     

Calcium-sensing receptor attenuates AVP-induced aquaporin-2 expression via a calmodulin-dependent mechanism

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD(cl4)), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd(3+) attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V(2) receptor and adenylate cyclase via a calmodulin-dependent mechanism in cultured cortical collecting duct cells.     

Mapping of human autoantibody binding sites on the calcium‐sensing receptor

Previously, we have demonstrated the presence of anti‐calcium‐sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), a disease that is characterized in part by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum levels of parathyroid hormone. The aim of this study was to define the binding domains on the CaSR of anti‐CaSR antibodies found in APS1 patients and in one patient suspected of having autoimmune hypocalciuric hypercalcemia (AHH). A phage‐display library of CaSR peptides was constructed and used in biopanning experiments with patient sera. Selectively enriched IgG‐binding peptides were identified by DNA sequencing, and subsequently, immunoreactivity to these peptides was confirmed in ELISA. Anti‐CaSR antibody binding sites were mapped to amino acid residues 41–69, 114–126, and 171–195 at the N‐terminal of the extracellular domain of the receptor. The major autoepitope was localized in the 41–69 amino acid sequence of the CaSR with antibody reactivity demonstrated in 12 of 12 (100%) APS1 patients with anti‐CaSR antibodies and in 1 AHH patient with anti‐CaSR antibodies. Minor epitopes were located in the 114–126 and 171–195 amino acid domains, with antibody reactivity shown in 5 of 12 (42%) and 4 of 12 (33%) APS1 patients, respectively. The results indicate that epitopes for anti‐CaSR antibodies in the AHH patient and in the APS1 patients who were studied are localized in the N‐terminal of the extracellular domain of the receptor. The present work has demonstrated the successful use of phage‐display technology in the discovery of CaSR‐specific epitopes targeted by human anti‐CaSR antibodies. © 2010 American Society for Bone and Mineral Research     

雷尼酸锶治疗骨质疏松症的研究进展

目的 对雷尼酸锶在骨质疏松症中的临床应用、作用机制进行综述和评价.方法 从人群试验、动物试验、细胞培养和不良反应等方面进行综述.结果 雷尼酸锶具有抗骨吸收和增进骨形成双重作用,其作用可能通过CaSR和增加血清IGF-1、降低高半胱氨酸.结论 雷尼酸锶作为一种新型抗骨质疏松药物,对骨质疏松症具有良好的疗效,代表了骨质疏松症治疗的一个重要发展方向,但应注意其不良反应.     

Decreased Parathyroid Klotho Expression Is Associated With Persistent Hyperparathyroidism After Kidney Transplantation

Although successful kidney transplantation usually corrects hyperparathyroidism, the condition persists in some patients. The present study was designed to determine whether Klotho or fibroblast growth factor 23, the key regulator of parathyroid hormone, is involved in persistent hyperparathyroidism in kidney transplant recipients (KTRs). Nineteen hyperplastic parathyroid glands were obtained from end-stage renal disease (ESRD) patients and KTRs; 6 normal parathyroid glands were used as controls. We compared the expression of Klotho, fibroblast growth factor receptor 1 (FGFR1) and calcium-sensing receptor (CaSR) in the KTRs and ESRD patients. Expressions of Klotho, FGFR1, CaSR and vitamin D receptor, as evaluated by immunohistochemistry, were quantified as the number of positive cells per unit area. The Klotho, FGFR1 and CaSR expressions in parathyroid glands of the post–kidney transplantation (PSKT) and the ESRD groups were significantly decreased compared with normal controls. In the ESRD group, Klotho expression and number of proliferating cell nuclear antigen–positive cells in the parathyroid gland were significantly decreased in parathyroid adenomas as compared with parathyroid hyperplasia. The expression of FGFR1 and CaSR in the parathyroid glands was significantly increased in the PSKT compared with the ESRD group. There was no significant difference in Klotho expression between the PSKT and ESRD groups. Incomplete recovery of Klotho levels in the parathyroid gland may play a role in the pathogenesis of tertiary hyperparathyroidism after kidney transplantation.     

Lack of association between calcium-sensing receptor gene "A986S" polymorphism and bone mineral density in Hungarian postmenopausal women

Calcium-sensing receptor (CaSR) is an attractive candidate gene for osteoporosis susceptibility. The CaSR “A986S” genotype has been shown to have an effect on serum calcium. Recently, an association has been reported between the CaSR gene A986S polymorphism and bone mineral density in healthy white girls. In this study, we examined whether CaSR gene A986S polymorphism is associated with decreased bone mass in 230 Hungarian postmenopausal women. From this cohort, 108 osteoporotic patients were compared with 122 healthy control women. Bone mineral density (BMD) was measured at the lumbar spine (L2–4) and femoral neck using dual-energy X-ray absorptiometry. Allele-specific polymerase chain reaction was used to amplify A986S polymorphisms of the CaSR gene. We found no difference in the distribution of different alleles or genotypes between groups (p = 0.762). No significant effect of CaSR genotype on BMD was observed either in the whole population or in the subgroups. Our data do not support the idea that CaSR gene A986S polymorphism has an impact on bone mass.     

心肌钙敏感受体在高位脊髓损伤大鼠心肌损伤中的作用

目的 探讨心肌钙敏感受体(CasR)在高位脊髓损伤大鼠心肌损伤中的作用.方法 健康雄性SD大鼠18只,体重250 ～ 300 g,采用随机数字表法,将大鼠随机分为2组:假手术组(S组,n＝6)和高位脊髓损伤组(SCI组,n＝12).采用砝码(10 g)从5 cm高处沿中空玻璃管垂直自由落下撞击C7脊髓的方法制备高位脊髓损伤模型.SCI组于高位脊髓损伤后12和24h(T1,2)时,S组于T1时取血样,测定血清肌酸激酶(CK)和CK-MB活性,取心肌组织,在透射电镜下观察心肌超微结构,分别采用荧光定量PCR法和Western blot法检测心肌CaSR mRNA及其蛋白的表达水平.结果 与S组比较,SCI组血清CK和CK-MB活性升高,心肌CaSR mRNA及其蛋白表达上调(P<0.05);与T1时比较,SCI组T2时血清CK活性升高,CK-MB活性降低,心肌CaSR mRNA表达上调(P<0.05),CaSR蛋白表达差异则无统计学意义(P＞0.05).SCI组T1,2时心肌超微结构呈现不同程度的损伤改变.结论 大鼠高位脊髓损伤后心肌CaSR表达上调,该变化可能是高位脊髓损伤后心肌损伤的机制之一.     

The calcium-ion-sensing receptor

Conclusion The recent cloning, functional, morphologic, and genetic studies have established the CaSR as a vital component of the calcium homeostatic system. The CaSR provides both the sensing mechanism responsible for the regulation of parathyroid hormone secretion from parathyroid cells and the steep relationship between Ca²⁺ ₀ and urinary calcium excretion in the kidney. The renal CaSR appears to provide the crucial “sensing” mechanism in the thick ascending limb and papillary collecting duct for integrating and balancing salt, water, and divalent mineral loss. Direct interactions of extracellular Ca²⁺ with the renal CaSR could explain in large part the disordered water metabolism (ie, nephrogenic diabetes insipidus) observed under pathologic states of hypercalcemia (eg, with primary hyperparathyroidism or associated with certain malignancies). The promise of addition of new calcimimetic agents to our therapeutic arsenal is an exciting prospect. The CaSR story provides an nice example of going from bench to bedside. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology”, Tokyo, May 10, 1998.     

A case of gain-of-function mutation in calcium-sensing receptor: supplemental hydration is required for renal protection

AIMS: The calcium-sensing receptor (CaSR) regulates the extracellular calcium level, mainly by controlling parathyroid hormon secretion and renal calcium reabsorption. In gain-of-function CaSR mutations, the genetic abnormalities increase CaSR activity leading to the development of such clinical manifestations as hypercalciuric hypocalcemia and hypoparathyroidism. We report a Japanese case of CaSR gain-of-function mutation and represent a therapeutic intervention based on the functional characteristics of CaSR in renal tubule. METHODS AND RESULTS (CASE): DNA sequence analysis revealed a heterozygous G to T mutation identified in a 12-year-old Japanese girl presenting with sporadic onset of hypercalciuric hypocalcemia and hypoparathyroidism. The mutation is located in the N-terminal extracellular domain of the CaSR gene, one of the most important parts for the three-dimensional construction of the receptor, resulting in the substitution of phenylalanine for cysteine at amino acid 131 (C131F) in exon 3. Based on the diagnosis of the gain-of-function mutation in the CaSR, oral hydrochlorothiazide administration and supplemental hydration were started in addition to calcium supplementation. The combination therapy of thiazide and supplemental hydration markedly reduced both renal calcium excretion and urinary calcium concentration from 0.4-0.7 to less than 0.1 mg/mg (urinary calcium/creatinine ratio) and from 10-15 to 3-5 mg/dl (urinary calcium concentration), respectively. This therapy stopped the progression of renal calcification during the follow-up period. CONCLUSION: Supplemental hydration should be considered essential for the following reasons: (1) calcium supplementation activates the CaSR in the kidney and suppresses renal urinary concentrating ability, (2) the thiazide has a diuretic effect, (3) as calcium supplementation increases renal calcium excretion, the supplemental hydration decreases urinary calcium concentration by increasing urinary volume, thereby diminishing the risk of intratubular crystallization of calcium ion.     

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss‐of‐Function Gα11 Mutation

G‐protein subunit α‐11 (Gα₁₁) couples the calcium‐sensing receptor (CaSR) to phospholipase C (PLC)‐mediated intracellular calcium (Ca²⁺_i) and mitogen‐activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss‐of‐function Gα₁₁ mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα₁₁ germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild‐type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα₁₁, which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC‐mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα₁₁ proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα₁₁ protein to impair CaSR‐mediated Ca²⁺_i and extracellular signal‐regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα₁₁ cleft region also impaired signaling by PLC. The loss‐of‐function associated with the Ser220 Gα₁₁ mutant was rectified by treatment of cells with cinacalcet, which is a CaSR‐positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα₁₁ mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα₁₁ germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα₁₁ hydrophobic cleft region for CaSR‐mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss‐of‐function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.