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Calcium-containing stones represent the most common form of kidney calculi, frequently linked to idiopathic hypercalciuria, though their precise pathogenesis remains elusive. This research aimed to elucidate the molecular mechanisms involved by employing urinary exosomal microRNAs as proxies for renal tissue analysis. Elevated miR-148b-5p levels were observed in exosomes derived from patients with kidney stones. Systemic administration of miR-148b-5p in rat models resulted in heightened urinary calcium excretion, whereas its inhibition reduced stone formation. RNA immunoprecipitation combined with deep sequencing identified miR-148b-5p as a suppressor of calcitonin receptor (Calcr) expression, thereby promoting urinary calcium excretion and stone formation. Mice deficient in Calcr in distal epithelial cells demonstrated elevated urinary calcium excretion and renal calcification. Mechanistically, miR-148b-5p regulated Calcr through the circRNA-83536/miR-24-3p signaling pathway. Human kidney tissue samples corroborated these results. In summary, miR-148b-5p regulates the formation of calcium-containing kidney stones via the circRNA-83536/miR-24-3p/Calcr axis, presenting a potential target for novel therapeutic interventions to prevent calcium nephrolithiasis., (© 2024. The Author(s).)

Purpose of Review: Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood., Recent Findings: Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation., Summary: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca 2+ ], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies., (Published by Elsevier Inc.)

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca 2+ entry triggers a transepithelial Ca 2+ flux to regulate proximal tubular (PT) luminal [Ca 2+ ], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca 2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca 2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca 2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca 2+ entry) inhibitors (Pyr10), Ca 2+ entry by WTT cells was moderately inhibited, suggesting that the Ca 2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Idiopathic hypercalciuria is defined as excessive urine calcium excretion in the absence of an identifiable cause. It has been strongly associated with the risk of calcium kidney stone formation. Animal and human studies have suggested excessive bone mineral loss or increased gastrointestinal calcium absorption with abnormal renal calcium excretion may contribute to this process. In this article we will review the complex pathophysiology of idiopathic hypercalciuria and discuss clinical management and challenges.

Magnesium is the fourth most abundant cation in the body. It plays a critical role in many biological processes, including the process of energy release. Paracellular transport of magnesium is mandatory for magnesium homeostasis. In addition to intestinal absorption that occurs in part across the paracellular pathway, magnesium is reabsorbed by the kidney tubule. The bulk of magnesium is reabsorbed through the paracellular pathway in the proximal tubule and the thick ascending limb of the loop of Henle. The finding that rare genetic diseases due to pathogenic variants in genes encoding specific claudins (CLDNs), proteins located at the tight junction that determine the selectivity and the permeability of the paracellular pathway, led to an awareness of their importance in magnesium homeostasis. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is caused by a loss of function of CLDN16 or CLDN19. Pathogenic CLDN10 variants cause HELIX syndrome, which is associated with a severe renal loss of sodium chloride and hypermagnesemia. The present review summarizes the current knowledge of the mechanisms and factors involved in paracellular magnesium permeability. The review also highlights some of the unresolved questions that need to be addressed., (© 2023 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

Aim: Perturbed calcium homeostasis limits life expectancy in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). This rare disease occurs by loss-of-function mutations in CLDN16 or CLDN19 genes, causing impaired paracellular reabsorption of divalent cations along the cortical thick ascending limb (cTAL). Only partial compensation takes place in the ensuing late distal convoluted tubule, connecting tubule, and collecting duct, where the luminal transient receptor potential channel V5 (TRPV5), as well as basolateral plasma membrane calcium ATPase (PMCA) and sodium-potassium exchanger (NCX1) mediate transcellular Ca 2+ reabsorption. The loop diuretic furosemide induces compensatory activation in these distal segments. Normally, furosemide enhances urinary calcium excretion via inhibition of the aforementioned cTAL. As Ca 2+ reabsorption in the cTAL is already severely impaired in FHHNC patients, furosemide may alleviate hypercalciuria in this disease by activation of the distal transcellular Ca 2+ transport proteins., Methods: Cldn16-deficient mice (Cldn16 -/- ) served as a FHHNC model. Wild-type (WT) and Cldn16 -/- mice were treated with furosemide (7 days of 40 mg/kg bw) or vehicle. We assessed renal electrolyte handling (metabolic cages) and key divalent transport proteins., Results: Cldn16 -/- mice show higher Ca 2+ excretion than WT and compensatory stimulation of Cldn2, TRPV5, and NCX1 at baseline. Furosemide reduced hypercalciuria in Cldn16 -/- mice and enhanced TRPV5 and PMCA levels in Cldn16 -/- but not in WT mice., Conclusions: Furosemide significantly reduces hypercalciuria, likely via upregulation of luminal and basolateral Ca 2+ transport systems in the distal nephron and collecting duct in this model for FHHNC., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia and inappropriately low PTH concentrations. ADH type 2 (ADH2) is caused by a heterozygous gain-of-function mutation in GNA11 that encodes the subunit of G11, the principal G protein that transduces calcium-sensing receptor signaling in the parathyroid. Clinical features related to hypocalcemia in ADH2 range from asymptomatic to tetany and seizures. We report the clinical and molecular analysis of an infant with ADH2. Exome sequencing identified a de novo heterozygous missense variant, c. G548C (p. Arg183Pro) in GNA11. This is the youngest Korean case to be diagnosed with ADH 2. In addition, we summarized the literature related to eight mutations in GNA11 from 10 families., (© 2022 by the Association of Clinical Scientists, Inc.)

The majority of calcium filtered by the glomerulus is reabsorbed along the nephron. Most is reabsorbed from the proximal tubule (> 60%) via a paracellular pathway composed of the tight junction proteins claudins-2 and -12, a process driven by sodium and consequently water reabsorption. The thick ascending limb reabsorbs the next greatest amount of calcium (20-25%), also by a paracellular pathway composed of claudins-16 and -19. This pathway is regulated by the CaSR, whose activity increases the expression of claudin-14, a protein that blocks paracellular calcium reabsorption. The fine tuning of urinary calcium excretion occurs in the distal convoluted and connecting tubule by a transcellular pathway composed of the apical calcium channel TRPV5, the calcium shuttling protein calbindin-D 28K and the basolateral proteins PMCA1b and the sodium calcium exchanger, NCX. Not surprisingly, mutations in a subset of these genes cause monogenic disorders with hypercalciuria as a part of the phenotype. More commonly, "idiopathic" hypercalciuria is encountered clinically with genetic variations in CLDN14, the CASR and TRPV5 associating with kidney stones and increased urinary calcium excretion. An understanding of the molecular pathways conferring kidney tubular calcium reabsorption is employed in this review to help explain how dietary and medical interventions for this disorder lower urinary calcium excretion., (© 2021. IPNA.)

Background: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis., Methods: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD , a gene that encodes a small Rag guanosine triphosphatase (GTPase)., Results: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo . Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD , plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro ., Conclusions: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function., (Copyright © 2021 by the American Society of Nephrology.)

Calcium (Ca 2+ ) is an important mediator of multicellular homeostasis and is involved in several diseases. The interplay among the kidney, bone, intestine, and parathyroid gland in Ca 2+ homeostasis is strictly modulated by numerous hormones and signaling pathways. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, that is expressed in calcitropic tissues such as the parathyroid gland and the kidney, plays a pivotal role in Ca 2+ regulation. CaSR is important for renal Ca 2+ , as a mutation in this receptor leads to hypercalciuria and calcium nephrolithiasis. In addition, CaSR is also widely expressed in the vascular system, including vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) and participates in the process of vascular calcification. Aberrant Ca 2+ sensing by the kidney and VSMCs, owing to altered CaSR expression or function, is associated with the formation of nephrolithiasis and vascular calcification. Based on emerging epidemiological evidence, patients with nephrolithiasis have a higher risk of vascular calcification, but the exact mechanism linking the two conditions is unclear. However, a dysregulation in Ca 2+ homeostasis and dysfunction in CaSR might be the connection between the two. This review summarizes renal calcium handling and calcium signaling in the vascular system, with a special focus on the link between nephrolithiasis and vascular calcification.

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.)

Urinary calcium and magnesium wasting is a characteristic feature of metabolic acidosis, and this study focused on the role of the thick ascending limb of Henle's loop in metabolic acidosis-induced hypercalciuria and hypermagnesiuria because thick ascending limb is an important site of paracellular calcium and magnesium reabsorption. Male Sprague-Dawley rats were used to determine the effects of acid loading (by adding NH 4 Cl, 7.2 mmol/220 g body wt/day to food slurry for 7 days) on renal expression of claudins and then to evaluate whether the results were reversed by antagonizing calcium-sensing receptor (using NPS-2143). At the end of each animal experiment, the kidneys were harvested for immunoblotting, immunofluorescence microscopy, and quantitative PCR (qPCR) analysis of claudins and the calcium-sensing receptor. As expected, NH 4 Cl loading lowered urinary pH and increased excretion of urinary calcium and magnesium. In NH 4 Cl-loaded rats, renal protein and mRNA expression of claudin-16, and claudin-19, were decreased compared with controls. However, claudin-14 protein and mRNA increased in NH 4 Cl-loaded rats. Consistently, the calcium-sensing receptor protein and mRNA were up-regulated in NH 4 Cl-loaded rats. All these changes were reversed by NPS-2143 coadministration and were confirmed using immunofluorescence microscopy. Hypercalciuria and hypermagnesiuria in NH 4 Cl-loaded rats were significantly ameliorated by NPS-2143 coadministration as well. We conclude that in metabolic acidosis, claudin-16 and claudin-19 in the thick ascending limb are down-regulated to produce hypercalciuria and hypermagnesiuria via the calcium-sensing receptor. NEW & NOTEWORTHY This study found that the thick ascending limb of Henle's loop is involved in the mechanisms of hypercalciuria and hypermagnesiuria in metabolic acidosis. Specifically, expression of claudin-16/19 and claudin-14 was altered via up-regulation of calcium-sensing receptor in NH 4 Cl-induced metabolic acidosis. Our novel findings contribute to understanding the regulatory role of paracellular tight junction proteins in the thick ascending limb.

Vitamin D supplementation in patients with urolithiasis and hypercalciuria is considered to be unsafe. We analyzed the impact of vitamin D supplementation on selected health status parameters in children with idiopathic hypercalciuria. The study included 36 children with urolithiasis resulting from excessive calcium excretion. The level of calcium and 25(OH)D (hydroxylated vitamin D - calcidiol) in serum, urinary calcium excretion and the presence of stones in urinary tract were assessed prospectively. Blood and urine samples were collected at the time when the patient was qualified for the study and every three months up to 24 month of vitamin D intake at a dose of 400 or 800 IU/day. At time zero and at 12, and 24 months of vitamin D supplementation, densitometry was performed. Supplementation with vitamin D caused a statistically significant increase in the concentration of 25(OH)D in serum. There were no significant changes in calcium concentration in serum, excretion of calcium in urine but also in bone density. There was no significant increase in the risk of formation or development of stones in the urinary tract. Supplementation with vitamin D (400-800 IU/day) in children with idiopathic hypercalciuria significantly increases 25(OH)D concentration, does not affect calciuria, but also does not improve bone density.

Background: Gitelman syndrome is a rare salt-losing renal tubular disorder associated with mutation of SLC12A3 gene, which encodes the Na-Cl co-transporter (NCCT). Gitelman syndrome is characterized by hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria, and renin-angiotensin-aldosterone system (RAAS) activation. Different SLC12A3 variants may lead to phenotypic variability and severity., Methods: In this study, we reported the clinical features and genetic analysis of a Chinese pedigree diagnosed with Gitelman syndrome., Results: The proband exhibited hypokalaemia, hypomagnesemia, metabolic alkalosis, but hypercalciuria and kidney stone formation. The increased urinary calcium excretion made it confused to Bartter syndrome. The persistent renal potassium wasting resulted in renal tubular lesions, and might affect urinary calcium reabsorption and excretion. Genetic analysis revealed mutations of SLC12A3 gene with c.433C > T (p.Arg145Cys), c.1077C > G (p.Asn359Lys), and c.1666C > T (p.Pro556Ser). Potential alterations of structure and function of NCCT protein due to those genetic variations of SLC12A3 are predicted. Interestingly, one sibling of the proband carried the same mutant sites and exhibited similar clinical features with milder phenotypes of hypokalemia and hypomagnesemia, but hypocalciuria rather than hypercalciuria. Family members with at least one wild type copy of SLC12A3 had normal biochemistry. With administration of spironolactone, potassium chloride and magnesium supplement, the serum potassium and magnesium were maintained within normal ranges., Conclusions: In this study, we identified compound mutations of SLC12A3 associated with varieties of clinical features. Further efforts are needed to investigate the diversity in clinical manifestations of Gitelman syndrome and its correlation with specific SLC12A3 mutations.

Introduction: There is much debate about whether idiopathic hypercalciuria (IH) affects kidney water management. For the first time in the literature, we carried out a longitudinal study of kidney water management (KWM) in patients diagnosed with IH in childhood and followed-up until adulthood (mean follow-up 17.7±1.4 years)., Methods: Twenty-nine patients (7 M, 22 F) over the age of 24 years (mean 28.2±2.9 years, range: 24.1-35.9) who were diagnosed with IH in childhood (mean 7.6±3.2 years, range: 1-14) were included. Maximum urine osmolality (UO) and/or urine volume adjusted for 100ml of glomerular filtration rate (V/GFR) in both age groups (paediatric and adult) were determined. Moreover, whenever possible, in both age groups plasma creatinine levels, plasma sodium levels, uric acid levels, the citrate/creatinine ratio and the calcium/citrate ratio were recorded and a renal and bladder ultrasound was performed., Results: In the paediatric age group, KWM was altered in 9/29 cases (31%) (4 with reduced maximum UO and 5 with elevated V/GFR). In adulthood, KWM was found to be affected in 7/29 cases (24.1%) (6 with reduced UO and one with elevated V/GFR). Compared to the paediatric age group, adult patients had lower V/GFR, calcium/creatinine and citrate/creatinine values, as well as higher plasma creatinine, uric acid and calcium/citrate. There were no differences in the maximum UO in both age groups. However, UO in adulthood was significantly lower in subjects who had renal colic compared to those who did not (P=.04)., Conclusions: KWM was affected in approximately one third of patients with IH, which persisted 20 years after diagnosis. We think that these results may be due to adherence to the recommended protective diet and to the pharmacological treatment administered at the diagnosis of IH during childhood., (Copyright © 2019. Published by Elsevier España, S.L.U.)

Background: The incidence of nephrolithiasis in children and adolescents is increasing and appears to double every 10 years. The most important role of the pediatric nephrologist is to diagnose and modify various metabolic and non-metabolic risk factors, as well as prevent long-term complications especially in the case of recurrent nephrolithiasis., Objective: The purpose of this review is to summarize the existing literature on the etiology and management of pediatric nephrolithiasis., Results: The incidence of kidney stones is increasing; dietary and environmental factors are probably the main causes for this increased incidence. In most pediatric patients, the etiology for the kidney stones can be identified. Metabolic factors, such as hypercalciuria and hypocitraturia, urinary tract infection, and urinary stasis, constitute leading causes. Herein, we review the etiologies, diagnostic work-up, and treatment options for the most prevalent causes of kidney stones. The detrimental effects of excessive dietary sodium, reduced fluid intake, and the benefits of plant-based over animal-based protein consumption on urinary crystal formation are discussed. We also review the long-term complications., Conclusions: Pediatric nephrologists have an important role in the diagnostic work-up and prevention of recurring nephrolithiasis.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new-onset diabetes mellitus, and hypercalciuria, which may contribute to post-transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off-target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus-treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX-1 and Calbindin-D 28k ). However, qPCR also showed decreased mRNA expression of NCX-1 and Calbindin-D 28k , and TRPM6. In contrast, KS-FKBP12 -/- mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Calcium metabolism alterations are quite common in sarcoidosis and have been correlated with disease activity., Objectives: The aim of the study was to investigate the clinical significance of calcium metabolism alterations in patients with chronic sarcoidosis. We paid particular attention to associations with specific disease phenotypes and chitotriosidase (CTO) expression., Methods: 212 chronic sarcoidosis patients (mean age 56.07 ± 12 years; 97 males) were retrospectively recruited. Demographic, clinical, functional, and radiological data, and serum-urinary calcium metabolism were entered into an electronical database for analysis. Levels of CTO and angiotensin-converting enzyme (ACE) were measured and bone mineral density and lung function tests were conducted., Results: Hypercalciuria and hypercalcemia were observed in 18.8 and 1.8% of patients, respectively. Urinary calcium levels correlated with CTO activity (r = 0.33, p = 0.0042). Patients with worsening persistent disease showed the highest levels of urinary calcium. Diffusing capacity of the lung for carbon monoxide (DLCO) percentage correlated inversely with urinary calcium (r = 0.1482; p = 0.0397)., Conclusions: Calcium metabolism alteration, particularly hypercalciuria, was observed in a significant percentage of patients of sarcoidosis. Urinary calcium was correlated with clinical status, DLCO, and serum CTO activity, suggesting its potential role as a biomarker of the activity and severity of sarcoidosis., (© 2020 S. Karger AG, Basel.)

The proximal tubule (PT) is responsible for the majority of calcium reabsorption by the kidney. Most PT calcium transport appears to be passive, although the molecular facilitators have not been well established. Emerging evidence supports a major role for PT calcium transport in idiopathic hypercalciuria and the development of kidney stones. This review will cover recent developments in our understanding of PT calcium transport and the role of the PT in kidney stone formation.

The role of the calcium-sensing receptor (CaSR) as a regulator of parathyroid hormone secretion is well established, but its function in bone is less well defined. In an effort to elucidate the CaSR's skeletal role, bone tissue and material characteristics from patients with autosomal dominant hypocalcemia (ADH), a genetic form of primary hypoparathyroidism caused by CASR gain-of-function mutations, were compared to patients with postsurgical hypoparathyroidism (PSH). Bone structure and formation/resorption indices and mineralization density distribution (BMDD), were examined in transiliac biopsy samples from PSH (n = 13) and ADH (n = 6) patients by histomorphometry and quantitative backscatter electron imaging, respectively. Bone mineral density (BMD by DXA) and biochemical characteristics were measured at the time of the biopsy. Because both study groups comprised children and adults, all measured biopsy parameters and BMD outcomes were converted to Z-scores for comparison. Histomorphometric indices were normal and not different between ADH and PSH, with the exception of mineral apposition rate Z-score, which was higher in the ADH group. Similarly, average BMD Z-scores were normal and not different between ADH and PSH. Significant differences were observed for the BMDD: average Z-scores of mean and typical degree of mineralization (CaMean, CaPeak, respectively) were lower (p = 0.02 and p = 0.03, respectively), whereas the heterogeneity of mineralization (CaWidth) and percentage of lower mineralized areas (CaLow) were increased in ADH versus PSH (p = 0.01 and p = 0.002, respectively). The BMDD outcomes point toward a direct, PTH-independent role of the CaSR in the regulation of bone mineralization. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Background/aims: Tributyltin (TBT) is an organotin (OTs) and biohazard organometallic pollutant. Recently our group has shown that TBT, even in very low doses, has deleterious effects on several tissues most likely due to its role as an endocrine-disrupting molecule. Other studies have confirmed that OT exposure could be responsible for neural, endocrine, and reproductive dysfunctions via in vitro and in vivo models. However, TBT effects on bone lack concise data despite the fact that bone turnover is regulated by endocrine molecules, such as parathormone (PTH), estrogen (E2), etc. Our group has already shown that TBT disrupts adrenal and female gonadal functions., Methods: We studied the effects of TBT on bone metabolism and structure using DXA, microCT scan, and SEM. We also determined the calcium (Ca²⁺) and phosphate (Pi) metabolism in TBT-treated rats as well as some biomarkers for bone formation and resorption., Results: Surprisingly, we found that TBT leads to higher bone mineral density (BMD) although lesions in spinal bone were observed by either microCT scan or SEM. Biomarkers for bone resorption, such as the urinary deoxipyridinolines (DPD) excretion ratio was increased in TBT-treated animals versus mock-treated controls. Osteocalcin (OC) and alkaline phosphatase (AP) are markers of bone formation and are also elevated suggesting that the bone matrix suffers from a higher turnover. Serum Ca²⁺ (total and ionized) do not changed by TBT treatment although hypercalciuria is observed., Conclusion: It is known that Sn atoms have three valence states (Sn²⁺, Sn³⁺, and Sn⁴⁺); hence, we hypothesized that Sn (more likely Sn²⁺) could be competing with Ca²⁺ and/or Mg²⁺ in hydroxyapatite mineral matrix to disturb bone turnover. Further work is needed to confirm this hypothesis., Competing Interests: The authors declare that they have no conflicts of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Background: A limited number of studies have evaluated biochemical bone metabolism markers in children with idiopathic hypercalciuria, which in adults has been linked with osteopenia. Our aim was to investigate in children with idiopathic hypercalciuria biochemical markers of bone formation and resorption and the osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kB ligand (sRANKL) system which is involved in the osteoclastogenesis process., Methods: A prospective study was conducted on 50 children with idiopathic hypercalciuria and 50 healthy age-, sex-, and Tanner stage-matched control subjects. Following the diagnosis, patients were requested to follow a 3-month dietary recommendation for idiopathic hypercalciuria. In patients, at diagnosis and at 3 months of follow-up, and in controls, bone-related hormones and serum/urine biochemical parameters were studied. The bone formation markers (total ALP and osteocalcin) and the bone resorption markers (β-Crosslaps) and the OPG and sRANKL levels were determined., Results: No differences were found in the bone formation markers or OPG and sRANKL between the children with idiopathic hypercalciuria and controls. The β-Crosslaps and the β-Crosslaps/osteocalcin ratio were higher in the patients at diagnosis than in controls (p = 0.019 and p = 0.029, respectively), with a trend to decrease after the 3-month dietary intervention. The initially increased 24-h urinary Ca in the patients decreased after the 3-month dietary intervention (p = 0.002)., Conclusions: Children with idiopathic hypercalciuria had biochemical markers compatible with normal bone formation but increased bone resorption. After a 3-month dietary intervention, the trend observed towards decrease in the serum β-Crosslaps may reflect a beneficial response.

The calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five 'disease-switch' residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias.

Purpose of Review: The greatest risk factor for kidney stone formation is increased urinary calcium excretion. Most filtered calcium is reabsorbed from the proximal tubule and the thick ascending limb (TAL) of Henle's loop via a paracellular pathway. Claudins are tight junction proteins that confer the permeability properties of an epithelium. We review the contribution of renal claudins to nephron calcium permeability and how perturbations in these pathways cause alterations in tubular calcium transport, hypercalciuria, nephrocalcinosis, or nephrolithiasis., Recent Findings: Claudin-16 and Claudin-19 form a complex with claudin-3 enabling divalent cation permeability in the TAL. Claudin-14 interacts with claudin-16 to attenuate calcium permeability through this pore. Intronic mutations in claudin-14 increase expression causing hypercalciuria and kidney stones. A different type of TAL tight junction pore is composed of claudin-10b, which does not preferentially permeate calcium. Deletion of claudin-10b results in increased expression of the claudin-16/claudin-19 complex expressed in the medullary TAL and nephrocalcinosis., Summary: Alterations to claudins expressed in the TAL tight junction greatly affects calcium homeostasis as highlighted by point mutations in claudin-16 or claudin-19 causing FHHNC or gain of function mutations in claudin-14 causing kidney stones.

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the G q/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders., (© 2018 Society for Endocrinology.)

Hypercalciuria is a main risk factor for kidney stone  formation. TRPV5 is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28 days after injecting the negative control saRNA, 1% EG for 28 days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28 days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Most patients with idiopathic hypercalciuria and calcium nephrolithiasis have a family history of the disease. Idiopathic hypercalciuria is a metabolic abnormality with various causes and developmental pathways. The systematic review describes specific mutations associated with idiopathic hypercalciuria and nephrolithiasis. Detection of these mutations may provide a better understanding of the pathogenesis of this heterogeneous disease and personalize patient management depending on the detected polymorphisms. A promising treatment option for a mutation in the vitamin D receptor gene is thiazide diuretics in combination with bisphosphonates. Among bisphosphonates, the drug of choice which has been most strongly supported by research evidence is alendronate.

In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6's close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.

Hyponatremia is associated with bone demineralization. We hypothesized that, during hyponatremia, calciuria and calcium balance depend on volemic status. We evaluated calciuria in patients with hyponatremia, secondary to SIAD or hypovolemia. Patients with SIAD exhibited a volemic expansion that was associated with hypercalciuria. Calciuria was proportional to markers of volemia., Introduction: Chronic mild hyponatremia has been associated with bone demineralization of unknown mechanisms. During chronic hyponatremia, arginine-vasopressin secretion can result from hypovolemia or from syndrome of inappropriate anti-diuresis (SIAD) that leads to a slightly volemic expansion. Since volemia determines renal calcium excretion and balance, we evaluated calcium homeostasis in patients with chronic hyponatremia, related to SIAD or to hypovolemia., Methods: We retrospectively included all patients referred to our Department between May 2006 and May 2014 for hyponatremia, resulting from SIAD or chronic hypovolemia. None had edema, cirrhosis, cardiac, or renal insufficiency. Exploration included estimation of volemia, extracellular fluid volume (ECFV) measurement with inulin, and calcium homeostasis., Results: In total, the SIAD and hypovolemic groups included 22 and 7 patients, respectively. The SIAD group exhibited signs of increased volemia: higher glomerular filtration rate, higher fractional excretion of uric acid, and lower plasma renin. ECFV exceeded that of the hypovolemic group and was above usual values. There was no difference between the two groups regarding plasma calcium, PTH, and vitamin D. However, in the SIAD group, calciuria was higher than in the hypovolemic group, reaching levels of hypercalciuria. Furthermore, there was a positive correlation between calciuria and markers of volemia., Conclusions: Our results show that SIAD results in a volemic expansion tendency that is associated with a decrease in renal calcium reabsorption and thus hypercalciuria, whereas in the hypovolemic group, calciuria was not increased. Therefore, renal loss of calcium and bone demineralization in SIAD patients could be partly induced by volemic expansion.

CYP24A1, encoding the vitamin D-24-hydroxylase, is of major clinical and physiologic importance, serving to regulate the catabolism of 1,25-(OH) 2 D, the physiologically active vitamin D metabolite. In addition to facilitating catabolism of 1,25-(OH) 2 D, CYP24A1 also enhances the turnover and elimination of 25-OHD, the abundant precursor metabolite and storage form of the vitamin. CYP24A1 can be stimulated hormonally by 1,25-(OH) 2 D and by FGF23, whereas CYP27B1, encoding the vitamin D-1α-hydroxylase, is stimulated hormonally by parathyroid hormone (PTH) and downregulated by FGF23. Thus CYP24A1 and CYP27B1, together, provide for alternate and regulated fates of 25-OHD, and control the availability of the active metabolite, 1,25-(OH) 2 D, depending upon physiologic needs. These two enzymes, are therefore central to the homeostatic control of vitamin D metabolism, and as a result affect calcium metabolism in critical ways. Disruption of CYP24A1 in mice results in elevated circulating 1,25-(OH) 2 D, substantiating the importance of the enzyme in the maintenance of vitamin D metabolism. The consequential skeletal phenotype in these mice further demonstrates the biologic sequelae of the disruption of the vitamin D pathway, and illustrates a specific developmental pathology mediated largely by oversupply of 1,25-(OH) 2 D. More recent evidence has identified loss of function mutations in CYP24A1 in association with hypercalcemia, hypercalciuria and nephrolithiasis in humans. Initial reports described certain variant mutations in CYP24A1 as an unrecognized cause of "Idiopathic Infantile Hypercalcemia," and more recently older children and adults have been identified with a similar phenotype. Over 25 likely disease-causing variants are described. Homozygous and compound heterozygote mutations account for the overwhelming majority of cases, however the heterozygous loss-of-function mutations of CYP24A1 do not appear to consistently result in symptomatic hypercalcemia. Considerations ripe for exploration include the potential role for such mutations in the tolerance to challenges to the calcium homeostatic system, such as changes in dietary calcium intake, vitamin D supplementation, sunlight exposure or pregnancy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Using a proteomic approach, we aimed to identify and compare the urinary excretion of proteins involved in lipid transport and metabolism in children with kidney stones and hypercalciuria (CAL), hypocitraturia (CIT), and normal metabolic work-up (NM), and in healthy controls (HCs). Additionally, we aimed to confirm these results using ELISA, and to examine the relationship between the urinary excretion of selected proteins with demographic, dietary, blood, and urinary parameters., Methods: Prospective, controlled, pilot study of pooled urine from CAL, CIT, and NM versus age- and gender-matched HCs, using liquid chromatography-mass spectrometry. Relative protein abundance was estimated using spectral counting. Results were confirmed by ELISA performed on individual samples., Results: Of the 1,813 proteins identified, 230 met the above criteria. Of those, 5 proteins (apolipoprotein A-II [APOA2]; apolipoprotein A-IV [APOA4]; apolipoprotein C-III [APOA3]; fatty acid-binding protein, liver [FABPL]; fatty acid-binding protein, adipocyte [FABP4]) involved in lipid metabolism and transport were found in the CAL group, with significant differences compared with HCs. ELISA analysis indicated statistically significant differences in the urinary excretion of APOC3, APOA4, and FABPL in the CAL group compared with HCs. Twenty-four-hour urinary calcium excretion correlated significantly with concentrations of ApoC3 (r = 0.77, p < 0.001), and FABPL (r = 0.80, p = 0.005)., Conclusions: We provide proteomic data showing increased urinary excretion of lipid metabolism/transport-related proteins in children with kidney stones and hypercalciuria. These findings suggest that abnormalities in lipid metabolism might play a role in kidney stone formation.

Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5 Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5 Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5 Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5 Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The study aims to observe the urinary excretion of monocyte chemoattractant-1 (MCP-1) and high-mobility group box 1 (HMGB1) in patients with calcium nephrolithiasis and to determine the influence of hypercalciuria on the production of the two cytokines. 81 cases of patients with calcium nephrolithiasis (group CN) and 30 healthy controls (group C) were involved in this study. To observe the influence of urinary calcium on the excretion of those cytokines, the patients were subdivided according to their 24-h urinary calcium level: ≥4 mg/kg/day (group H) and <4 mg/kg/day (group N). MCP-1 and HMGB1 in urina sanguinis were determined for all subjects. In addition, in vitro study was done to determine the production of the two cytokines and index of apoptosis and oxidative injuries in human kidney epithelial cells (HK-2) exposed to three high levels of calcium. Data showed that both urinary MCP-1 and HMGB1 in group CN were higher than that of group C. When the patients were subdivided, comparisons among the three groups showed that both MCP-1 and HMGB1 in group H and group N were higher than group C, but there was no significant statistical difference between the two stone groups. In vitro study, the apoptosis rate of cells, the lactate dehydrogenase activities, the hydrogen peroxide, and 8-isoprostane concentrations in the medium all increased in accordance with the increased concentration of calcium supplemented. Compared with the control, mRNA expressions of MCP-1 and HMGB1 in cells and the protein concentrations of the two cytokines in the medium of calcium-supplemented groups increased significantly. Results showed that urinary MCP-1 and HMGB1 increased in calcium nephrolithiasis patients and hypercalciuria might affect the identical pathways (through the reactive oxygen species) with other factors in stimulating the production of MCP-1 and HMGB1 in vivo.

Heterozygous germline gain-of-function mutations of G-protein subunit α 11 (Gα 11 ), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant ( Dsk7 ) - which has a germline hypermorphic Gα 11 mutation, Ile62Val - may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα 11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα 11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy., Competing Interests: The authors have declared that no conflict of interest exists.

Heterotrimeric G proteins play critical roles in transducing extracellular signals generated by 7-transmembrane domain receptors. Somatic gain-of-function mutations in G protein α subunits are associated with a variety of diseases. Recently, we identified gain-of-function mutations in Gα 11 in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11 R6OC is partly dependent on coupling to the CASR. Treatment with the Gα 11/q -specific inhibitor YM-254890 increased blood calcium in heterozygous but not in homozygous GNA11 R60C mice, consistent with published crystal structure data showing that Arg60 forms a critical contact with YM-254890. This animal model of ADH2 provides insights into molecular mechanism of this G protein-related disease and potential paths toward new lines of therapy., Competing Interests: MM is associated with Shire Pharmaceuticals (unrelated to this work). RT is associated with Shire/NPS Pharmaceuticals Inc. (USA), GlaxoSmithKline, Novartis Pharma AG, and Marshall-Smith Syndrome Foundation. KS is associated with Avilex Pharma (unrelated to this work).

The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The diseases caused by an abnormality of the CaSR are genetically determined or are more rarely acquired. The genetic diseases consist of hyper- or hypocalcemia disorders. Hypercalcaemia disorders are related to inactivating mutations of the CASR gene either heterozygous (autosomal dominant familial benign hypercalcaemia, still named hypocalciuric hypercalcaemia syndrome type 1) or homozygous (severe neonatal hyperparathyroidism). The A986S, R990G and Q1011E variants of the CASR gene are associated with higher serum calcium levels than in the general population, hypercalciuria being also associated with the R990G variant. The differential diagnosis consists in the hypocalciuric hypercalcaemia syndrome, types 2 (involving GNA11 gene) and 3 (involving AP2S1 gene); hyperparathyroidism; abnormalities of vitamin D metabolism, involving CYP24A1 and SLC34A1 genes; and reduced GFR. Hypocalcemia disorders, which are more rare, are related to heterozygous activating mutations of the CASR gene (type 1), consisting of autosomal dominant hypocalcemia disorders, sometimes with a presentation of pseudo-Bartter's syndrome. The differential diagnosis consists of the hypercalciuric hypocalcaemia syndrome type 2, involving GNA11 gene and other hypoparathyroidism aetiologies. The acquired diseases are related to the presence of anti-CaSR antibodies, which can cause hyper- or especially hypocalcemia disorders (for instance in APECED syndromes), determined by their functionality. Finally, the role of CaSR in digestive, respiratory, cardiovascular and neoplastic diseases is gradually coming to light, providing new therapeutic possibilities. Two types of CaSR modulators are known: CaSR agonists (or activators, still named calcimimetics) and calcilytic antagonists (or inhibitors of the CasR). CaSR agonists, such as cinacalcet, are indicated in secondary and primary hyperparathyroidism. Calcilytics have no efficacy in osteoporosis, but could be useful in the treatment of hypercalciuric hypocalcaemia syndromes.

Mutations in the renal sodium-dependent phosphate cotransporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis. Oral phosphate supplementation is currently thought to reduce risk by reversing the hypercalciuria, but the exact mechanism remains unclear and the relative contribution of modifiers of mineralization such as osteopontin (Opn) to the formation of renal mineral deposits in renal phosphate wasting disorders has not been studied. We observed a marked decrease of renal gene expression and urinary excretion of Opn in Npt2a -/- mice, a mouse model of these disorders, at baseline. Following supplementation with phosphate Opn gene expression was restored to wild-type levels in Npt2a -/- mice; however, urine excretion of the protein remained low. To further investigate the role of Opn, we used a double-knockout strategy, which provides evidence that loss of Opn worsens the nephrocalcinosis and nephrolithiasis observed in these mice on a high-phosphate diet. These studies suggest that impaired Opn gene expression and urinary excretion in Npt2a -/- mice may be an additional risk factor for nephrolithiasis, and normalizing urine Opn levels may improve the therapy of phosphaturic disorders., (Copyright © 2017 the American Physiological Society.)

Activating mutations of the G protein-coupled receptor, calcium-sensing receptor (CaSR), cause autosomal dominant hypocalcemia and Bartter syndrome type 5. These mutations lower the set-point for extracellular calcium sensing, thereby causing decreased parathyroid hormone secretion and disturbed renal calcium handling with hypercalciuria. Available therapies increase serum calcium levels but raise the risk of complications in affected patients. Symptom relief and the prevention of adverse outcome is currently very difficult to achieve. Calcilytics act as CaSR antagonists that attenuate its activity, thereby correcting the molecular defect of activating CaSR proteins in vitro and elevating serum calcium in mice and humans in vivo, and have emerged as the most promising therapeutics for the treatment of these rare and difficult to treat diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background and Objectives: Idiopathic hypercalciuria is a frequent defect in calcium kidney stone formers that is associated with high intestinal calcium absorption and osteopenia. Characteristics distinguishing hypercalciuric stone formers from hypercalciuric patients without kidney stone history (HNSFs) are unknown and were explored in our study., Design, Setting, Participants, & Measurements: We compared 172 hypercalciuric stone formers with 36 HNSFs retrospectively selected from patients referred to outpatient clinics of the San Raffaele Hospital in Milan from 1998 to 2003. Calcium metabolism and lumbar bone mineral density were analyzed in these patients. A strontium oral load test was performed: strontium was measured in 240-minute urine and serum 30, 60, and 240 minutes after strontium ingestion; serum strontium concentration-time curve and renal strontium clearance were evaluated to estimate absorption and excretion of divalent cations., Results: Serum strontium concentration-time curve (P<0.001) and strontium clearance (4.9±1.3 versus 3.5±2.7 ml/min; P<0.001) were higher in hypercalciuric stone formers than HNSFs, respectively. The serum strontium-time curve was also higher in hypercalciuric stone formers with low bone mineral density (n=42) than in hypercalciuric stone formers with normal bone mineral density (n=130; P=0.03) and HNSFs with low (n=22; P=0.01) or normal bone mineral density (n=14; P=0.02). Strontium clearance was greater in hypercalciuric stone formers with normal bone mineral density (5.3±3.4 ml/min) than in hypercalciuric stone formers and HNSFs with low bone mineral density (3.6±2.5 and 3.1±2.5 ml/min, respectively; P=0.03). Multivariate regression analyses displayed that strontium absorption at 30 minutes was positively associated calcium excretion (P=0.03) and negatively associated with lumbar bone mineral density z score (P=0.001) in hypercalciuric stone formers; furthermore, hypercalciuric patients in the highest quartile of strontium absorption had increased stone production risk (odds ratio, 5.06; 95% confidence interval, 1.2 to 20.9; P=0.03)., Conclusions: High calcium absorption in duodenum and jejunum may expose hypercalciuric patients to the risk of stones because of increased postprandial calcium concentrations in urine and tubular fluid. High calcium absorption may identify patients at risk of bone loss among stone formers., (Copyright © 2016 by the American Society of Nephrology.)

Contribution of salt wasting and volume depletion to the pathogenesis of hypercalciuria and hyperphosphaturia is poorly understood. Pendrin/NCC double KO (pendrin/NCC-dKO) mice display severe salt wasting under basal conditions and develop profound volume depletion, prerenal renal failure, and metabolic alkalosis and are growth retarded. Microscopic examination of the kidneys of pendrin/NCC-dKO mice revealed the presence of calcium phosphate deposits in the medullary collecting ducts, along with increased urinary calcium and phosphate excretion. Confirmatory studies revealed decreases in the expression levels of sodium phosphate transporter-2 isoforms a and c, increases in the expression of cytochrome p450 family 4a isotypes 12 a and b, as well as prostaglandin E synthase 1, and cyclooxygenases 1 and 2. Pendrin/NCC-dKO animals also had a significant increase in urinary prostaglandin E2 (PGE-2) and renal content of 20-hydroxyeicosatetraenoic acid (20-HETE) levels. Pendrin/NCC-dKO animals exhibit reduced expression levels of the sodium/potassium/2chloride co-transporter 2 (NKCC2) in their medullary thick ascending limb. Further assessment of the renal expression of NKCC2 isoforms by quantitative real time PCR (qRT-PCR) reveled that compared to WT mice, the expression of NKCC2 isotype F was significantly reduced in pendrin/NCC-dKO mice. Provision of a high salt diet to rectify volume depletion or inhibition of PGE-2 synthesis by indomethacin, but not inhibition of 20-HETE generation by HET0016, significantly improved hypercalciuria and salt wasting in pendrin/NCC dKO mice. Both high salt diet and indomethacin treatment also corrected the alterations in NKCC2 isotype expression in pendrin/NCC-dKO mice. We propose that severe salt wasting and volume depletion, irrespective of the primary originating nephron segment, can secondarily impair the reabsorption of salt and calcium in the thick ascending limb of Henle and/or proximal tubule, and reabsorption of sodium and phosphate in the proximal tubule via processes that are mediated by PGE-2.

Purpose of Review: Identification of the mechanisms of magnesium absorption and reabsorption has markedly enhanced our understanding of the causes of hypomagnesemia., Recent Findings: New gastrointestinal and renal causes of hypomagnesemia have been recently documented., Summary: The recognition of new mechanisms and causes of magnesium absorption and reabsorption should enhance the ability to monitor patients at risk for hypomagnesemia and improve our ability to mitigate the serious symptoms associated with this disorder.

Purpose of Review: The tight junction conductance made of the claudin-based paracellular channel is important in the regulation of calcium and magnesium reabsorption in the kidney. This review describes recent findings of the structure, the function, and the physiologic regulation of claudin-14, claudin-16, and claudin-19 channels that through protein interactions confer calcium and magnesium permeability to the tight junction., Recent Findings: Mutations in two tight junction genes - claudin-16 and claudin-19 - cause the inherited renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. A recent genome-wide association study has identified claudin-14 as a major risk gene of hypercalciuric nephrolithiasis. The crystal structure of claudin-19 has recently been resolved allowing the reconstruction of a claudin assembly model from cis-dimers made of claudin-16 and claudin-19 interaction. MicroRNAs have been identified as novel regulators of the claudin-14 gene. The microRNA-claudin-14 operon is directly regulated by the Ca sensing receptor gene in response to hypercalcemia., Summary: The paracellular pathway in the kidney is particularly important for mineral metabolism. Three claudin proteins - claudin-14, claudin-16, and claudin-19 - contribute to the structure and function of this paracellular pathway. Genetic mutations and gene expression changes in these claudins may lead to alteration of the paracellular permeability to calcium and magnesium, ultimately affecting renal mineral metabolism.

The role of the kidney in calcium homeostasis has been reshaped from a classic view in which the kidney was regulated by systemic calcitropic hormones such as vitamin D3 or parathyroid hormone to an organ actively taking part in the regulation of calcium handling. With the identification of the intrinsic renal calcium-sensing receptor feedback system, the regulation of paracellular calcium transport involving claudins, and new paracrine regulators such as klotho, the kidney has emerged as a crucial modulator not only of calciuria but also of calcium homeostasis. This review summarizes recent molecular and endocrine contributors to renal calcium handling and highlights the tight link between calcium and sodium reabsorption in the kidney., (Copyright © 2016 the American Physiological Society.)

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain-of-function mutations of the G-protein coupled calcium-sensing receptor (CaSR), and ADH2 caused by germline gain-of-function mutations of G-protein subunit α-11 (Gα11 ). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole-exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three-dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C-terminal α5 helix, which may affect G-protein coupled receptor binding and G-protein activation. In vitro functional expression of wild-type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration-response curve with a significantly (p < 0.0001) reduced mean half-maximal concentration (EC50 ) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild-type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain-of-function mutation. A novel His403Gln variant in transforming growth factor, beta-induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain-of-function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C-terminal region for G-protein function and CaSR signal transduction. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

Objective: To address whether hypercalciuria can stimulate calcium oxalate (CaOx) crystal deposition in kidney through osteopontin (OPN)., Materials and Methods: Rat tubular epithelial NRK cells were exposed to calcium. The cell viability, the cellular malondialdehyde content as a marker of reactive oxygen species (ROS), and the release of lactate dehydrogenase as markers of injury were detected. The production and gene expression of OPN were also examined. CaOx crystal attachment to cells was accomplished by measuring the calcium concentration of the cell lysates with atomic absorption analysis., Results: Exposure to calcium produced signs of cell injury and ROS-induced lipid peroxidation. The messenger ribonucleic acid expression and production of OPN increased significantly. OPN knockdown can significantly decrease the amount of CaOx crystal attachment to NRK cells., Conclusion: In response to exposure to high concentration of calcium, renal tubular epithelial NRK cells increase the production of OPN, which may have a promoting role in CaOx crystal adhesion to NRK cells by calcium stimulation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na(+)-HCO3 (-) cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H(+)-ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K(+) and Ca(2+) wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H(+)-ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K(+) and Ca(2+) in the distal nephron., (Copyright © 2015 the American Physiological Society.)