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Context: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown., Objective: This work aimed to investigate the role of CaSR in human spermatozoa., Methods: We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors., Results: CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology., Conclusion: CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Hypercalciuria is a frequent characteristic of hypertension. In this report we extend our earlier studies investigating the role of renal interstitial fluid calcium (ISF(Ca))(2+) as a link between urinary calcium excretion and blood pressure in the Dahl salt-sensitive (DS) hypertensive model., Methods: Dahl salt-sensitive and salt-resistant (DR) rats were placed on control (0.45%) and high (8%) salt diets to determine if changes in renal cortical and medullary ISF(Ca)(2+)correlated with changes in urinary calcium excretion and blood pressure., Results: We observed that renal ISFCa(2+) was predicted by urinary calcium excretion (P < 0.05) in DS rats but not DR rats. Renal cortical ISF(Ca)(2+) was negatively associated with blood pressure (P < 0.03) while renal medullary ISF(Ca)(2+) was positively associated with blood pressure in DS rats (P < 0.04). In contrast, neither urinary calcium excretion nor renal ISF(Ca)(2+) was associated with blood pressure in the DR rats under the conditions of this study., Conclusion: We interpret these findings to suggest that decreased renal cortical ISF(Ca)(2+) plays a role in the increase in blood pressure following a high salt diet in salt hypertension perhaps by mediating renal vasoconstriction; the role of medullary calcium remains to be fully understood. Further studies are needed to determine the mechanism of the altered renal ISF(Ca)(2+) and its role in blood pressure regulation., (© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Magnesium (Mg(2+)) is an essential ion for cell growth, neuroplasticity and muscle contraction. Blood Mg(2+) levels <0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps and epilepsy and disturbances in K(+) and Ca(2+) homeostasis. Over the last decade, the genetic origin of several familial forms of hypomagnesaemia has been found. In 2000, mutations in FXYD2, encoding the γ-subunit of the Na(+)-K(+)-ATPase, were identified to cause isolated dominant hypomagnesaemia (IDH) in a large Dutch family suffering from hypomagnesaemia, hypocalciuria and chondrocalcinosis. However, no additional patients have been identified since then., Methods: Here, two families with hypomagnesaemia and hypocalciuria were screened for mutations in the FXYD2 gene. Moreover, the patients were clinically and genetically characterized., Results: We report a p.Gly41Arg FXYD2 mutation in two families with hypomagnesaemia and hypocalciuria. Interestingly, this is the same mutation as was described in the original study. As in the initial family, several patients suffered from muscle cramps, chondrocalcinosis and epilepsy. Haplotype analysis revealed an overlapping haplotype in all families, suggesting a founder effect., Conclusions: The recurrent p.Gly41Arg FXYD2 mutation in two new families with IDH confirms that FXYD2 mutation causes hypomagnesaemia. Until now, no other FXYD2 mutations have been reported which could indicate that other FXYD2 mutations will not cause hypomagnesaemia or are embryonically lethal., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective: Autosomal dominant hypocalcemia (ADH) is a congenital isolated hypoparathyroidism caused by activating mutations in the calcium-sensing receptor (CASR) gene. The clinical features of ADH are heterogeneous; some patients are asymptomatic, and others show severe hypocalcemia with Bartter's syndrome. We therefore recruited 12 patients with ADH to clarify the determinants of their clinical presentation., Design and Methods: We studied two sporadic and 10 familial cases of ADH. Serum concentrations of calcium, intact PTH, and magnesium (Mg(2+)) were measured in each patient. Fractional excretion of Mg (FE(Mg)) was calculated in spot urine samples. A nuclear factor of activated T cells luciferase assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca(2+)., Results: Genomic analysis revealed five known activating mutations and a novel mutation, E481K, in the CASR. Patients with the A843E, C131W, or F788C mutation showed hypomagnesemia with elevated FE(Mg). Intact PTH in these patients was consistently near the detection limit. In contrast, patients with the P221L, K47N, or E481K mutation exhibited normal Mg(2+) levels. In these patients, intact PTH increased in response to low calcium, and their maximum intact PTH exceeded the lower limit of the reference range. Functional analysis showed an association between the disease severity and the in vitro activity of the mutant CaSR., Conclusions: The functional activity of mutant CaSR determines the serum Mg(2+) level, renal Mg(2+) handling, and intact PTH in patients with ADH. The presence of hypomagnesemia with elevated FE(Mg) may indicate the diagnosis of ADH among patients with PTH-deficient hypoparathyroidism.

Background: CaSR gene is a candidate for calcium nephrolithiasis. Single-nucleotide polymorphisms (SNPs) encompassing its regulatory region were associated with calcium nephrolithiasis., Aims: We tested SNPs in the CaSR gene regulatory region associated with calcium nephrolithiasis and their effects in kidney., Subjects and Methods: One hundred sixty-seven idiopathic calcium stone formers and 214 healthy controls were genotyped for four CaSR gene SNPs identified by bioinformatics analysis as modifying transcription factor binding sites. Strontium excretion after an oral load was tested in 55 stone formers. Transcriptional activity induced by variant alleles at CaSR gene promoters was compared by luciferase reporter gene assay in HEK-293 and HKC-8 cells. CaSR and claudin-14 mRNA levels were measured by real-time PCR in 107 normal kidney medulla samples and compared in patients with different CaSR genotype., Results: Only rs6776158 (A>G), located in the promoter 1, was associated with nephrolithiasis. Its minor G allele was more frequent in stone formers than controls (37.8% vs 26.4%, P = .001). A reduced strontium excretion was observed in GG homozygous stone formers. Luciferase fluorescent activity was lower in cells transfected with the promoter 1 including G allele at rs6776158 than cells transfected with the A allele. CaSR mRNA levels were lower in kidney medulla samples from homozygous carriers for the G allele at rs6776158 than carriers for the A allele. Claudin-14 mRNA levels were also lower in GG homozygous subjects., Conclusions: Minor allele at rs6776158 may predispose to calcium stones by decreasing transcriptional activity of the CaSR gene promoter 1 and CaSR expression in kidney tubules.

Context: Many inherited disorders of calcium and phosphate homeostasis are unexplained at the molecular level., Objective: The objective of the study was to identify the molecular basis of phosphate and calcium abnormalities in two unrelated, consanguineous families., Patients: The affected members in family 1 presented with rickets due to profound urinary phosphate-wasting and hypophosphatemic rickets. In the previously reported family 2, patients presented with proximal renal tubulopathy and hypercalciuria yet normal or only mildly increased urinary phosphate excretion., Methods: Genome-wide linkage scans and direct nucleotide sequence analyses of candidate genes were performed. Transport of glucose and phosphate by glucose transporter 2 (GLUT2) was assessed using Xenopus oocytes. Renal sodium-phosphate cotransporter 2a and 2c (Npt2a and Npt2c) expressions were evaluated in transgenically rescued Glut2-null mice (tgGlut2-/-)., Results: In both families, genetic mapping and sequence analysis of candidate genes led to the identification of two novel homozygous mutations (IVS4-2A>G and R124S, respectively) in GLUT2, the gene mutated in Fanconi-Bickel syndrome, a rare disease usually characterized by renal tubulopathy, impaired glucose homeostasis, and hepatomegaly. Xenopus oocytes expressing the [R124S]GLUT2 mutant showed a significant reduction in glucose transport, but neither wild-type nor mutant GLUT2 facilitated phosphate import or export; tgGlut2-/- mice demonstrated a profound reduction of Npt2c expression in the proximal renal tubules., Conclusions: Homozygous mutations in the facilitative glucose transporter GLUT2, which cause Fanconi-Bickel syndrome, can lead to very different clinical and biochemical findings that are not limited to mild proximal renal tubulopathy but can include significant hypercalciuria and highly variable degrees of urinary phosphate-wasting and hypophosphatemia, possibly because of the impaired proximal tubular expression of Npt2c.

Background: Hypercalciuria is frequent in patients with acromegaly, but it is unclear how GH/IGF-I regulate renal calcium handling. Elevated fasting plasma calcium levels despite increased glomerular filtration suggest enhanced renal calcium reabsorption., Objective: The aim of this study was to investigate the impact of acromegaly on phosphocalcium metabolism., Design and Setting: We conducted a prospective sequential study at a tertiary referral medical center and clinical investigation center (www.ClinicalTrials.gov Identifier: NCT00531908)., Intervention: Sixteen consecutive patients (five females/11 males) with acromegaly received a single iv infusion of 25 mg of furosemide to induce an acute increase in calcium and magnesium delivery to distal tubular segments during a high-sodium diet with stable dietary calcium, magnesium, and phosphate intake., Measurements: Baseline plasma and urine electrolytes, plasma calciotropic hormones, and furosemide-induced changes in the fractional excretion and tubular reabsorption of Na, Ca, and Mg were measured before and 6 months (range, 1-12) after effective treatment of acromegaly., Results: Serum IGF-I concentrations normalized in all the patients after acromegaly treatment. Compared with controlled acromegaly, active acromegaly was associated with higher fasting plasma (P = 0.0002) and urinary calcium (P = 0.0003) levels, lower PTH levels (P = 0.0075), higher calcitriol levels (P = 0.0137), higher phosphatemia (P<0.0001) and tubular phosphate reabsorption (P = 0.0002), and a lower calciuric (P = 0.0327) but not magnesiuric response to furosemide related to higher baseline and postfurosemide tubular calcium (P = 0.0034 and P = 0.0081, respectively), but not magnesium reabsorption., Conclusion: The IGF-I-mediated and PTH-independent increase in calcitriol synthesis in acromegaly is responsible for both absorptive hypercalciuria and increased fasting plasma calcium linked to enhanced distal tubular calcium reabsorption, as shown by the selectively diminished calciuric response to furosemide.

Context and Objective: Dietary intake of animal proteins is associated with an increase in urinary calcium and nephrolithiasis risk. We tested the hypothesis that the acid load imposed by dietary proteins causes this hypercalciuria., Design and Setting: In a short-term crossover metabolic study, an alkali salt was provided with a high-protein diet (HPD) to neutralize the acid load imparted by dietary proteins., Participants and Interventions: Eleven healthy volunteers were evaluated at the end of each of four phases while consuming metabolic diets with fixed calcium and sodium content. Phases 1 and 3 consisted of a control diet (CD). Phases 2 and 4 consisted of a eucaloric HPD (60 g/d animal proteins added to CD). Along with HPD in phases 2 and 4, subjects ingested 30 mEq twice daily of either potassium citrate (KCitrate, alkaline salt) or potassium chloride (KCl, control neutral salt)., Results: KCitrate completely neutralized the acid load imparted by HPD (based on changes in urine pH and net acid excretion) and increased urinary citrate. Urinary calcium increased during both HPD phases compared with CD but was not significantly different between the HPD + KCl and HPD + KCitrate phases (182 ± 85 vs. 170 ± 85 mg/d; P = 0.28). Increased urinary saturation with respect to calcium oxalate and uric acid with HPD was abrogated by KCitrate., Conclusions: This study suggests that, at least in the short-term, mechanism(s) other than acid load account for hypercalciuria induced by HPD. The beneficial effect of KCitrate on nephrolithiasis risk with HPD is through correction of declines in urine pH and citrate.

The mechanisms underlying hypercalciuria in pseudohypoaldosteronism type II (PHAII) caused by WNK4 mutations remain unclear. In this study, we used Wnk4(D561A/+) knock-in mice as a model of human PHAII for investigating the pathogenesis of hypercalciuria in PHAII. Serum and urine biochemistries were obtained from Wnk4(+/+) and Wnk4(D561A/+) littermates. Expression of the epithelial Ca(2+) channels [transient receptor potential channel vanilloid subtype 5 (TRPV5) and TRPV6] and calbindin-D28k (CBP-D28k) in the distal nephron and two upstream Na(+) transporters, Na(+)/H(+) exchanger 3 and Na(+)-K(+)-2Cl(-) cotransporter 2 involved in paracellular Ca(2+) reabsorption, were examined by real-time PCR, immunofluorescent staining, and immunoblotting. Compared with Wnk4(+/+) littermate controls, Wnk4(D561A/+) mice manifested hypercalciuria despite no significant differences in serum creatinine, ionized Ca(2+), PTH, and 1,25 hydroxylvitamin D(3) levels. There was no significant difference in TRPV5 expression, but a significant increase in TRPV6 and CBP-D28k was observed in Wnk4(D561A/+) mice. Despite no significant change in Na(+)/H(+) exchanger 3 expression, Na(+)-K(+)-2Cl(-) cotransporter 2 expression was significantly attenuated and urine Ca(2+) excretion rate in response to furosemide was blunted in Wnk4(D561A/+) mice. Decreased Ca(2+) reabsorption in the upstream nephron, especially in the thick ascending loops of Henle, with a secondary adaptive increase in TRPV6 and CBP-D28k expression in the distal tubules might be involved in the hypercalciuria of PHAII.

Background: Kidney stone formation is a major socioeconomic problem in humans, involving pain, recurrent treatment and renal insufficiency. As most renal precipitates contain calcium as a major component, hypercalciuria is the main risk factor for renal stone formation. Different forms of hypercalciuria can be classified, which primarily arise from defects in the main organs involved in calcium homeostasis. A distinction can be made between renal, absorptive and resorptive hypercalciuria, originating from disturbed calcium handling in kidney, intestine and bone, respectively. A positive family history predisposes individuals to an increased risk of stone formation, which strongly indicates the involvement of genetic susceptibility factors. TRPV5 is the renal epithelial calcium channel that is the gatekeeper protein in active calcium reabsorption in the kidney. TRPV5 gene ablation in mice leads to severe hypercalciuria, implying that TRPV5 is an interesting candidate gene for renal hypercalciuria in humans. This study aims to identify and functionally characterize TRPV5 gene aberrations in patients with renal hypercalciuria., Methods: The TRPV5 coding region and intron-exon boundaries were screened for gene mutations in 20 subjects displaying renal hypercalciuria after which identified non-synonymous polymorphisms were functionally characterized by patch-clamp analysis. Wild-type and TRPV5 channels including polymorphisms were transiently expressed in human embryonic kidney (HEK) 293 cells and functionally characterized by path-clamp analysis., Results: Genotyping TRPV5 in renal hypercalciuria patients revealed three non-synonymous and five synonymous polymorphisms. Electrophysiological characterization of the TRPV5 mutants did not reveal significant functional changes compared to wild-type TRPV5 channel recordings., Conclusions: In this specific patient cohort, our data do not support a primary role for TRPV5 in the pathogenesis of renal hypercalciuria. However, TRPV5 cannot be excluded as a candidate gene in hypercalciuria.

Background: Renal calcium stones and hypercalciuria are associated with a reduced bone mineral density (BMD). Therefore, the effect of changes in calcium homeostasis is of interest for both stones and bones. We hypothesized that the response of calciuria, parathyroid hormone (PTH) and 1.25 vitamin D to changes in dietary calcium might be related to BMD., Methods: A single-centre prospective interventional study of 94 hyper- and non-hypercalciuric calcium stone formers consecutively retrieved from our stone clinic. The patients were investigated on a free-choice diet, a low-calcium diet, while fasting and after an oral calcium load. Patient groups were defined according to lumbar BMD (z-score) obtained by dual X-ray absorptiometry (group 1: z-score <-0.5, n = 30; group 2: z-score -0.5-0.5, n = 36; group 3: z-score >0.5, n = 28). The effect of the dietary interventions on calciuria, 1.25 vitamin D and PTH in relation to BMD was measured., Results: An inverse relationship between BMD and calciuria was observed on all four calcium intakes (P = 0.009). On a free-choice diet, 1.25 vitamin D and PTH levels were identical in the three patient groups. However, the relative responses of 1.25 vitamin D and PTH to the low-calcium diet were opposite in the three groups with the highest increase of 1.25 vitamin D in group 1 and the lowest in group 3, whereas PTH increase was most pronounced in group 3 and least in group 1., Conclusion: Calcium stone formers with a low lumbar BMD exhibit a blunted response of PTH release and an apparently overshooting production of 1.25 vitamin D following a low-calcium diet.