Search

Sirtuin1 (Sirt1) activation significantly attenuated calcium oxalate (CaOx) crystal deposition and renal inflammatory injury by regulating renal immune microenvironment. Here, to elucidate the molecular mechanism underlying the therapeutic effects of Sirt1 on macrophage related inflammation and tubular epithelial cells (TECs) necrosis, we constructed a macrophage and CaOx monohydrate (COM)-stimulated tubular cell co-culture system to mimic immune microenvironment in kidney and established a mouse model of CaOx nephrocalcinosis in wild-type and myeloid-specific Sirt1 knockout mice. Target prediction analyses of Gene Expression Omnibus Datasets showed that only miR-34b-5p is regulated by lipopolysaccharides and upregulated by SRT1720 and targets the TLR4 3'-untranslated region. In vitro, SRT1720 suppressed TLR4 expression and M1 macrophage polarization and decreased reactive oxygen species (ROS) production and mitochondrial damage in COM-stimulated TECs by targeting miR-34b-5p. Mechanically, Sirt1 promoted miR-34b-5p expression by suppressing the tri-methylation of H3K27, which directly bound to the miR-34b-5p promoter and abolished the miR-34b-5p transcription. Furthermore, loss of Sirt1 aggravated CaOx nephrocalcinosis-induced inflammatory and oxidative kidney injury, while AgomiR-34b reversed these effects. Therefore, our data suggested that Sirt1 inhibited TLR4 signaling and M1 macrophage polarization and decreased inflammatory and oxidative injury of TECs in vitro and in vivo., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interest exists., (Copyright © 2023. Published by Elsevier Inc.)

Aims: Calcium oxalate (CaOx) crystal deposition induces damage to the renal tubular epithelium, increases epithelial adhesion, and contributes to CaOx nephrocalcinosis. The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 ( NEAT1 ) is thought to be involved in this process. In this study, we aimed to investigate the mechanism by which NEAT1 regulates renal tubular epithelium in response to inflammatory and oxidative injury triggered by CaOx crystals. Results: As CaOx crystals were deposited in mouse kidney tissue, the expression of NEAT1 was significantly elevated and positively correlated with interferon regulatory factor 1 ( IRF1 ), Toll-like receptor 4 ( TLR4 ), and NF-κB . NEAT1 targets and inhibits miR-130a-3p as a competitor to endogenous RNA. miR-130 binds to and exerts inhibitory effects on the 3'-untranslated region of IRF1 . After transfected with silence-NEAT1, IRF1 , TLR4 , and NF-κB were also variously inhibited, and oxidative damage in renal calcinosis was subsequently attenuated. When we simultaneously inhibited NEAT1 and miR-130 , renal tubular injury was exacerbated. Innovation and Conclusion: We found that the lncRNA NEAT1 can enhance IRF1 signaling through targeted repression of miR-130a-3p and activate TLR4/NF-κB pathways to promote oxidative damage during CaOx crystal deposition. This provides an explanation for the tubular epithelial damage caused by CaOx crystals and offers new ideas and drug targets for the prevention and treatment of CaOx nephrocalcinosis. Antioxid. Redox Signal. 38, 731-746.

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.)

Intrarenal calcium oxalate (CaOx) crystals induce renal tubular epithelial cell (TEC) inflammatory and oxidative injury. This study is aimed at exploring potential therapeutic lipid components in kidney stones because lipids are involved in the development of several diseases and indicate the risk of kidney stones. Serum specimens were collected from 35 kidney stone patients and 35 normal controls. The lipid components in serum were measured, and differences were analyzed. The documented biological importance was comprehensively reviewed to identify lipids that differed significantly between the two groups to find potential agents associated with kidney stones. CaOx nephrocalcinosis mouse model was established to examine the therapeutic effects of specific lipids on CaOx deposition and CaOx-induced oxidative renal injury. Several lipids with significantly different levels were present in the serum of patients with stones and normal controls. Resolvin D1 (RvD1) (4.93-fold change, P < 0.001) and protectin D1 (PD1) (5.06-fold change, P < 0.001) were significantly decreased in the serum of patients with kidney stones, and an integrative review suggested that these factors might be associated with inflammatory responses, which is a crucial mechanism associated with stone damage. The administration of RvD1 and PD1 significantly inhibited kidney CaOx deposition and suppressed CaOx-induced renal tubular cell inflammatory injury and necrosis in a CaOx nephrocalcinosis mouse model. Furthermore, RvD1 and PD1 facilitated the expression of the oxidative indicator superoxide dismutase 2 (SOD2), inhibited NADPH oxidase 2 (NOX2) expression, and diminished intracellular reactive oxygen species (ROS) levels. This study preliminarily elucidated the role of lipids in kidney stones. The inhibitory effects of RvD1 and PD1 on oxidative damage induced by CaOx deposition provide a promising perspective for kidney stone treatment strategies., Competing Interests: The authors have declared that no conflicts of interest exist., (Copyright © 2022 Bohan Wang et al.)

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.)

The roles of the lncRNA X inactive specific transcript (XIST) in many diseases, including cancers and inflammatory sickness, have been previously elucidated. However, renal calculus remained poorly understood. In this study, we revealed the potential effects of XIST on kidney stones that were exerted via inflammatory response and oxidative stress mechanisms. We established a glyoxylate-induced calcium oxalate (CaOx) stone mouse model and exposed HK-2 cells to calcium oxalate monohydrate (COM). The interactions among XIST, miR-223-3p, and NOD-like receptor protein 3 (NLRP3) and their respective effects were determined by RNAs and protein expression, luciferase activity, and immunohistochemistry (IHC) assays. Cell necrosis, reactive oxygen species (ROS) generation, and inflammatory responses were detected after silencing XIST, activating and inhibiting miR-223-3p, and both knocking down XIST and activating miR-223-3p in vitro and in vivo. The XIST, NLRP3, caspase-1, and IL-1 β levels were notably increased in kidney samples from glyoxylate-induced CaOx stone model mice. XIST knockdown significantly suppressed the inflammatory damage and ROS production and further attenuated oxalate crystal deposition. miRNA-223-3p mimics also exerted the same effects. Moreover, we verified the interactions among XIST, miRNA-223-3p and NLRP3, and the subsequent effects. Our results suggest that the lncRNA XIST participates in the formation and progression of renal calculus by interacting with miR-223-3p and the NLRP3/Caspase-1/IL-1 β pathway to mediate the inflammatory response and ROS production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Peng Lv et al.)

Primary hyperoxaluria type 1 (PH1) is a severe inherited disorder caused by a genetic defect in alanine-glyoxylate aminotransferase ( AGXT ), which results in recurrent urolithiasis and renal failure. Animal models that precisely reflect human PH1 phenotypes are lacking. We aimed to develop a novel PH1 rat model and study the mechanisms involved in PH1 deterioration. One cell stage Sprague-Dawley embryos were injected with the CRISPR/Cas9 system to introduce a Q84X mutation in Agxt . Liver tissues were harvested to determine Agxt expression. Urine oxalate, crystals, and electrolyte levels in Agxt Q84X and wild-type (WT) littermates were evaluated. Kidney tissues were used for Pizzolato staining and kidney injury evaluation. Data showed that Agxt mRNA and protein were absent in Agxt Q84X rats. At 4 and 24 wk, Agxt Q84X rats displayed 2.1- and 2.9-fold higher urinary oxalate levels, respectively, compared with WT littermates. As a result, calcium oxalate (CaOx) crystals in urine were revealed in all Agxt Q84X rats but in none of the WT rats. We also observed bladder stones in 36.4% of Agxt Q84X rats, of which 44.4% had renal CaOx deposition. Moreover, the elevated serum urea and creatinine levels indicated the impaired renal function in Agxt Q84X rats. Further investigation revealed significantly increased expression of inflammation-, necroptosis-, and fibrosis-related genes in the kidneys of Agxt Q84X rats with spontaneous renal CaOx deposition, indicating that these pathways are involved in PH1 deterioration. Collectively, these results suggest that this rat model has broad applicability in mechanistic studies and innovative therapeutics development for PH1 and other kidney stone diseases. NEW & NOTEWORTHY Primary hyperoxaluria type 1 is a severe inherited disorder that results in recurrent urolithiasis and renal failure. We generated an alanine-glyoxylate aminotransferase ( Agxt ) Q84X nonsense mutant rat model that displayed an early onset of hyperoxaluria, spontaneous renal CaOx precipitation, bladder stone, and kidney injuries. Our results suggest an interaction of renal CaOx crystals with the activation of inflammation-, fibrosis-, and necroptosis-related pathways. In all, the Agxt Q84X rat strain has broad applicability in mechanistic studies and the development of innovative therapeutics.

SHORT syndrome is a rare developmental disorder frequently associated with growth failure and insulin resistant diabetes mellitus (IRDM). Since GH has a diabetogenic effect, GH therapy has been regarded as a contraindication. We observed a Brazilian girl with SHORT syndrome who received GH therapy from 4 6/12 years of age for SGA short stature. GH dosage was increased from 0.23 to 0.36 mg/kg/week, but statural response to GH therapy remained poor. Her blood HbA1c level, though it remained 5.5-6.0% in childhood, began to elevate with puberty and increased to 9.2% at 10 6/12 years of age, despite the discontinuation of GH therapy at 9 11/12 years of age. Laboratory studies indicated antibody-negative IRDM. She was treated with metformin and canagliflozin (a sodium glucose co-transporter 2 (SGLT2) inhibitor), which ameliorated overt diurnal hyperglycemia and mild nocturnal hypoglycemia and reduced her blood HbA1c around 7%. Whole exome sequencing revealed a de novo heterozygous pathogenic variant (c.1945C>T:p.(Arg649Trp)) in PIK3R1 known as the sole causative gene for SHORT syndrome. Subsequent literature review for patients with molecularly confirmed SHORT syndrome revealed the development of IRDM in 10 of 15 GH-untreated patients aged ≥12 years but in none of three GH-treated and six GH-untreated patients aged ≤10 years. These findings imply a critical role of pubertal development and/or advanced age rather than GH therapy in the development of IRDM, and a usefulness of SGLT2 inhibitor in the treatment of IRDM.

The short telomere syndromes encompass a spectrum of clinical manifestations that present from infancy to late adulthood. They are caused by mutations in telomerase and other telomere maintenance genes and have a predominantly degenerative phenotype characterized by organ failure across multiple systems. They are collectively one of the most common inherited bone marrow failure syndromes; however, their most prevalent presentations are extrahematopoietic. This review focuses on these common nonhematologic complications, including pulmonary fibrosis, liver pathology, and immunodeficiency. The short telomere syndrome diagnosis informs clinical care, especially in guiding diagnostic evaluations as well as in the solid organ transplant setting. Early recognition allows an individualized approach to screening and management. This review illustrates a myriad of extrahematopoietic presentations of short telomere syndromes and how they impact clinical decisions., Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests., (© 2020 by The American Society of Hematology.)

When extrapolating data from animal toxicological studies a default factor (dUF) of 100 is applied to derive a heath based guidance value. The UF takes into account the interspecies differences (ID) and the intraspecies variability (IV). When re-evaluating the safety of phosphates used as food additives nephrocalcinosis was identified as the critical endpoint. The underlying mechanism for nephrocalcinosis was attributed to the precipitation of calcium phosphate in the kidney, depending on its solubility, irrespective of the species and the population. Based on the mechanism, the volume of primary urine, for which the glomerular filtration rate (GFR) was used as a proxy, was considered to be the only parameter relevant for ID and IV. Median value of GFR in rats was 4.0 ml/min/kg bw. In humans it was 1.6 ml/min/kg bw in healthy adults and 0.9 in elderly. These values were calculated from the distribution of the GFR data from 8 studies in rats (n = 191), 16 studies in adults (n = 1540) and 5 studies in elderly (n = 2608). Multiplying the distribution of the ratio rat/healthy humans (ID) with the distribution of the ratio healthy humans/elderly human (IV) resulted in a phosphate specific factor of 4.5 (3.3-6.7) (median; 25th - 75th percentile)., (Copyright © 2020. Published by Elsevier Inc.)

Objective: Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance., Methods: The human pathogenic Pik3r1 Y657 ∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically., Results: Like humans with SHORT syndrome, Pik3r1 WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia., Conclusions: Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Kidney stone disease is a crystal concretion formed in the kidneys that has been associated with an increased risk of chronic kidney disease. MicroRNAs are functionally involved in kidney injury. Data mining using a microRNA array database suggested that miR-21 may be associated with calcium oxalate monohydrate (COM)-induced renal tubular cell injury. Here, we confirmed that COM exposure significantly upregulated miR-21 expression, inhibited proliferation, promoted apoptosis, and caused lipid accumulation in an immortalized renal tubular cell line (HK-2). Moreover, inhibition of miR-21 enhanced proliferation and decreased apoptosis and lipid accumulation in HK-2 cells upon COM exposure. In a glyoxylate-induced mouse model of renal calcium oxalate deposition, increased miR-21 expression, lipid accumulation, and kidney injury were also observed. In silico analysis and subsequent experimental validation confirmed the peroxisome proliferator-activated receptor (PPAR)-α gene (PPARA) a key gene in fatty acid oxidation, as a direct miR-21 target. Suppression of miR-21 by miRNA antagomiR or activation of PPAR-α by its selective agonist fenofibrate significantly reduced renal lipid accumulation and protected against renal injury in vivo. In addition, miR-21 was significantly increased in urine samples from patients with calcium oxalate renal stones compared with healthy volunteers. In situ hybridization of biopsy samples from patients with nephrocalcinosis revealed that miR-21 was also significantly upregulated compared with normal kidney tissues from patients with renal cell carcinoma who underwent radical nephrectomy. These results suggested that miR-21 promoted calcium oxalate-induced renal tubular cell injury by targeting PPARA, indicating that miR-21 could be a potential therapeutic target and biomarker for nephrolithiasis.

Excessive phosphorus intake adversely affects bone and mineral metabolism. Estrogen is one of the factors affecting fibroblast growth factor 23 (FGF23), a phosphorus-regulating hormone. However, the interaction between excess phosphorus and estrogen status has not been fully elucidated. This study investigated the involvement of estrogen in the effects of high phosphorus intake on bone metabolism and ectopic calcification in ovariectomized (OVX) rats. The interaction between high phosphorus diet and OVX was not observed in bone mineral density and aortic calcium. In contrast, high phosphorus intake markedly increased renal calcium concentration in sham rats, whereas the effect was attenuated in OVX rats, which was reversed by a selective estrogen-receptor modulator treatment. A strong positive correlation between renal calcium and serum FGF23 was observed. In addition, fibroblast growth factor receptor 1 (FGFR1: a predominant receptor of FGF23) inhibitor treatment partially decreased renal calcium concentrations in rats with high phosphorus intake. In conclusion, the effect of high phosphorus intake on bone metabolism and aortic calcification did not depend on the estrogen status; in contrast, high phosphorus intake synergistically induced nephrocalcinosis in the presence of estrogenic action on the bone. Furthermore, FGF23 was involved in the nephrocalcinosis induced by high phosphorus intake partially through FGFR1 signaling.

Claudins are integral proteins expressed at the tight junctions of epithelial and endothelial cells. In the mammalian kidney, every tubular segment express a specific set of claudins that give to that segment unique properties regarding permeability and selectivity of the paracellular pathway. So far, 3 claudins (10b, 16 and 19) have been causally traced to rare human syndromes: variants of CLDN10b cause HELIX syndrome and variants of CLDN16 or CLDN19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The review summarizes our current knowledge on the physiology of mammalian tight junctions and paracellular ion transport, as well as on the role of the 3 above-mentioned claudins in health and disease. Claudin 14, although not having been causally linked to any rare renal disease, is also considered, because available evidence suggests that it may interact with claudin 16. Some single-nucleotide polymorphisms of CLDN14 are associated with urinary calcium excretion and/or kidney stones. For each claudin considered, the pattern of expression, the function and the human syndrome caused by pathogenic variants are described.

Background: Intrarenal calcium oxalate (CaOx) crystals induce inflammation and kidney tubular cell injury, which are processes that involve TLR4/NF-κB signalling. A recent genome-wide gene expression profile analysis of Randall's plaques in CaOx stone patients revealed that the expression of the long noncoding RNA H19 was significantly upregulated. However, to date, its role in kidney CaOx stones has not been reported., Method: A Gene Expression Omnibus (GEO) dataset was utilized to analyse gene expression profiles. Luciferase reporter, Western blotting, qRT-PCR, immunofluorescence staining and reactive oxygen species (ROS) assays were employed to study the molecular mechanism of HMGB1/TLR4/NF-κB regulation by H19 and miR-216b. In vitro and in vivo assays were performed to further confirm the proinflammatory and prooxidative stress effects., Finding: H19 expression was significantly increased and positively correlated with the expression levels of HMGB1, TLR4 and NF-κB in Randall's plaques and glyoxylate-induced CaOx nephrocalcinosis mouse models. H19 interacted with miR-216b and suppressed its expression. Additionally, miR-216b inhibited HMGB1 expression by directly binding its 3'-untranslated region. Moreover, H19 downregulation inhibited HMGB1, TLR4 and NF-κB expression and suppressed CaOx nephrocalcinosis-induced renal tubular epithelial cell injury, NADPH oxidase, and oxidative stress in vivo and in vitro. Interestingly, miR-216b inhibition partially reversed the inhibitory effect of H19 knockdown on HMGB1 expression., Interpretation: We determined that H19 might serve as a facilitator in the process of CaOx nephrocalcinosis-induced oxidative stress and renal tubular epithelial cell injury, and we revealed that the interaction between H19 and miR-216b could exert its effect via the HMGB1/TLR4/NF-κB pathway., Funding: This work was supported by the National Nature Science Foundation of China (Nos. 8196030190, 8190033175, 81370805, 81470935, 81900645, 81500534, and 81602236)., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Objective: To determine whether multiple daily injections of parathyroid hormone (PTH) 1-34 are safe and effective as long-term therapy for children with hypoparathyroidism., Study Design: Linear growth, bone accrual, renal function, and mineral homeostasis were studied in a long-term observational study of PTH 1-34 injection therapy in 14 children., Methods: Subjects were 14 children with hypoparathyroidism attributable to autoimmune polyglandular syndrome type 1 (N = 5, ages 7-12 years) or calcium receptor mutation (N = 9, ages 7-16 years). Mean daily PTH 1-34 dose was 0.75 ± 0.15 µg/kg/day. Treatment duration was 6.9 ± 3.1 years (range 1.5-10 years). Patients were evaluated semiannually at the National Institutes of Health Clinical Center., Results: Mean height velocity and lumbar spine, whole body, and femoral neck bone accretion velocities were normal throughout the study. In the first 2 years, distal one-third radius bone accrual velocity was reduced compared with normal children (P < .003). Serum alkaline phosphatase correlated with PTH 1-34 dose (P < .006) and remained normal (235.3 ± 104.8 [SD] U/L, N: 51-332 U/L). Mean serum and 24-hour urine calcium levels were 2.05 ± 0.11 mmol/L (N: 2.05-2.5 mmol/L) and 6.93 ± 1.3 mmol/24 hour (N: 1.25-7.5 mmol/24 hour), respectively-with fewer high urine calcium levels vs baseline during calcitriol and calcium treatment (P < .001). Nephrocalcinosis progressed in 5 of 12 subjects who had repeated renal imaging although renal function remained normal., Conclusions: Twice-daily or thrice-daily subcutaneous PTH 1-34 injections provided safe and effective replacement therapy for up to 10 years in children with hypoparathyroidism because of autoimmune polyglandular syndrome type 1 or calcium receptor mutation., (Published by Elsevier Inc.)

Ectopic calcification occurs during development of chronic kidney disease and has a negative impact on long-term prognosis. The precise molecular mechanism and prevention strategies, however, are not established. Fibulin-7 (Fbln7) is a matricellular protein structurally similar to elastogenic short fibulins, shown to bind dental mesenchymal cells and heparin. Here, we report that Fbln7 is highly expressed in renal tubular epithelium in the adult kidney and mediates renal calcification in mice. In vitro analysis revealed that Fbln7 bound heparin at the N-terminal coiled-coil domain. In Fbln7-expressing CHO-K1 cells, exogenous heparin increased the release of Fbln7 into conditioned media in a dose-dependent manner. This heparin-induced Fbln7 release was abrogated in CHO-745 cells lacking heparan sulfate proteoglycan or in CHO-K1 cells expressing the Fbln7 mutant lacking the N-terminal coiled-coil domain, suggesting that Fbln7 was tethered to pericellular matrix via this domain. Interestingly, Fbln7 knockout (Fbln7 -/- ) mice were protected from renal tubular calcification induced by high phosphate diet. Mechanistically, Fbln7 bound artificial calcium phosphate particles (aCPP) implicated in calcification and renal inflammation. Binding was decreased significantly in Fbln7 -/- primary kidney cells relative to wild-type cells. Further, overexpression of Fbln7 increased binding to aCPP. Addition of heparin reduced binding between aCPP and wild-type cells to levels of Fbln7 -/- cells. Taken together, our study suggests that Fbln7 is a local mediator of calcium deposition and that releasing Fbln7 from the cell surface by heparin/heparin derivatives or Fbln7 inhibitory antibodies may provide a novel strategy to prevent ectopic calcification in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nephrocalcinosis often begins on a calcium phosphate deposit, at the tip of the medullo-papillary complex (MPC) known as Randall's plaque (RP). Contextualizing proximally observed biominerals within the MPC has led us to postulate a mechanobiological switch that can trigger interstitial biomineralization at the MPC tip, remote from the intratubular biominerals. Micro X-ray computed tomography scans of human MPCs correlated with transmission and scanning electron micrographs, and X-ray energy dispersive spectrometry demonstrated novel findings about anatomically-specific biominerals. An abundance of proximal intratubular biominerals were associated with emergence of distal interstitial RP. The fundamental architecture of the MPC and mineral densities at the proximal and distal locations of the MPC differed markedly. A predominance of plate-like minerals or radially oriented plate-like crystallites within spheroidal minerals in the proximal intratubular locations, and core-shell type crystallites within spheroidal minerals in distal interstitial locations were observed. Based on the MPC anatomic location of structure-specific biominerals, a biological switch within the mineral-free zone occurring between the proximal and distal locations is postulated. The "on" and "off" switch is dependent on changes in the pressure differential resulting from changes in tubule diameters; the "Venturi effect" changes the "circumferential strain" and culminates in interstitial crystal deposits in the distal tubule wall in response to proximal tubular obstruction. These distal interstitial mineralizations can emerge into the collecting system of the kidney linking nephrocalcinosis with nephrolithiasis.

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 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.)

Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the β-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45 + F4/80 + CD11b + CX3CR1 + CD206 - ) and pro-fibrotic (CD45 + F4/80 + CD11b + CX3CR1 + CD206 + TGFβ + ) to an anti-inflammatory (CD45 + F4/80 + CD11b + CD206 + TGFβ - ) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-β signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Primary hyperoxaluria is a rare genetic disorder characterized by oxalate overproduction, leading to kidney failure due to nephrocalcinosis, and is eventually responsible for systemic oxalosis. Bone impairment, secondary to oxalate deposits, is one of the many complications that may occur. Skeletal involvement can be difficult to diagnose because of lack of clinical symptoms and therefore needs to be confirmed by invasive testing, such as transiliac bone biopsy. If confirmed, bone oxalosis is the proof of disease severity and that combined liver-kidney transplantation should be performed., (Copyright © 2017. Published by Elsevier Masson SAS.)

Experimental induction of hyperoxaluria by ethylene glycol (EG) administration is disapproved as it causes metabolic acidosis while the oral administration of chemically synthesized potassium oxalate (KOx) diet does not mimic our natural system. Since existing models comprise limitations, this study is aimed to develop an improved model for the induction of dietary hyperoxaluria, and nephrocalcinosis in experimental rats by administration of naturally available oxalate rich diet. Male albino Wistar rats were divided into five groups. Group I, control; group II rats received 0.75% EG, group III rats fed with 5% KOx diet and group IV and V rats were administered with spinach extract of 250 and 500 mg soluble oxalate/day respectively, for 28 d. Urine and serum biochemistry were analyzed. After the experimental period, rats were sacrificed, liver and kidney tissue homogenates were used for antioxidant and lipid peroxidation assay. Relative change in expression of kidney injury molecule-1 (KIM-1) and crystal modulators genes in kidney tissues were evaluated. Tissue damage was assessed by histology studies of liver and kidney. Experimental group rats developed hyperoxaluria and crystalluria. Urine parameters, serum biochemistry, antioxidant profile, lipid peroxidation levels and gene expression analysis of experimental group II and III rats reflected acute kidney damage compared to group V rats. Histopathology results showed moderate hyperplasia in liver and severe interstitial inflammation in kidneys of group II and III than group V rats. Ingestion of naturally available oxalate enriched spinach extract successfully induced dietary hyperoxaluria and nephrocalcinosis in rats with minimal kidney damage.

Nephrocalcinosis involves the deposition of microscopic crystals in the tubular lumen or interstitium. While the clinical, biochemical, and genetic aspects of the diseases causing nephrocalcinosis have been elucidated, little is known about the cellular events in this calcification process. We previously reported a phenomenon involving the spontaneous formation of Ca 2 PO 4 nodules in primary papillary renal cells from a patient with medullary nephrocalcinosis harboring a rare glial cell-derived neurotrophic factor ( GDNF ) gene variant. We also demonstrated that cultivating GDNF -silenced human kidney-2 (HK-2) cells in osteogenic conditions for 15 days triggered Ca 2 PO 4 deposits. Given the reportedly close relationship between cell death and pathological calcification, aim of the present study was to investigate whether apoptosis is involved in the calcification of GDNF -silenced HK-2 cells under osteogenic conditions. Silenced and control cells were cultured in standard and osteogenic medium for 1, 5, and 15 days, and any Ca 2 PO 4 deposition was identified by means of von Kossa staining and environmental SEM (ESEM) analyses. Based on the results of annexin V and propidium iodide (PI) analysis, and terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay, the silenced cells in the osteogenic medium showed a significant increase in the percentage of cells in the late phase of apoptosis and an increased Ca 2 PO 4 deposition at 15 days. The results of quantitative real-time PCR (qRT-PCR) of BAX and BCL2 , and in-cell Western analysis of caspases indicated that the cell death process was independent of caspase-3, -6, -7, and -9 activation, however. Using this model, we provide evidence of caspase-independent cell death triggering the calcification process in GDNF -silenced HK-2 cells., (© 2018 The Author(s).)

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.

SHORT syndrome is a rare, recognizable syndrome resulting from heterozygous mutations in PIK3R1 encoding a regulatory subunit of phosphoinositide-3-kinase (PI3K). The condition is characterized by short stature, intrauterine growth restriction, lipoatrophy and a facial gestalt involving a triangular face, deep set eyes, low hanging columella and small chin. PIK3R1 mutations in SHORT syndrome result in reduced signaling through the PI3K-AKT-mTOR pathway. We performed whole exome sequencing for an individual with clinical features of SHORT syndrome but negative for PIK3R1 mutation and her parents. A rare de novo variant in PRKCE was identified. The gene encodes PKCε and, as such, the AKT-mTOR pathway function was assessed using phospho-specific antibodies with patient lymphoblasts and following ectopic expression of the mutant in HEK293 cells. Kinase analysis showed that the variant resulted in a partial loss-of-function. Whilst interaction with PDK1 and the mTORC2 complex component SIN1 was preserved in the mutant PKCε, it bound to SIN1 with a higher affinity than wild-type PKCε and the dynamics of mTORC2-dependent priming of mutant PKCε was altered. Further, mutant PKCε caused impaired mTORC2-dependent pAKT-S473 following rapamycin treatment. Reduced pFOXO1-S256 and pS6-S240/244 levels were also observed in the patient LCLs. To date, mutations in PIK3R1 causing impaired PI3K-dependent AKT activation are the only known cause of SHORT syndrome. We identify a SHORT syndrome child with a novel partial loss-of-function defect in PKCε. This variant causes impaired AKT activation via compromised mTORC2 complex function., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.)

Nephrocalcinosis describes the ectopic deposition of calcium salts in the kidney parenchyma. Nephrocalcinosis can result from a number of acquired causes but also an even greater number of genetic diseases, predominantly renal but also extrarenal. Here we provide a review of the genetic causes of nephrocalcinosis, along with putative mechanisms, illustrated by human and animal data., (Copyright © 2016 the American Physiological Society.)

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.

Pathologic calcification is a significant cause of increased morbidity and mortality in patients with chronic kidney disease. The precise mechanisms of ectopic calcification are not fully elucidated, but it is known to be caused by an imbalance of procalcific and anticalcific factors. In the chronic kidney disease population, an elevated phosphate burden is both highly prevalent and a known risk factor for ectopic calcification. Here we tested whether osteopontin, an inhibitor of calcification, protects against high phosphate load-induced nephrocalcinosis and vascular calcification. Osteopontin knockout mice were placed on a high phosphate diet for 11 weeks. Osteopontin deficiency together with phosphate overload caused uremia, nephrocalcinosis characterized by substantial renal tubular and interstitial calcium deposition, and marked vascular calcification when compared with control mice. Although the osteopontin-deficient mice did not exhibit hypercalcemia or hyperphosphatemia, they did show abnormalities in the mineral metabolism hormone fibroblast growth factor-23. Thus, endogenous osteopontin plays a critical role in the prevention of phosphate-induced nephrocalcinosis and vascular calcification in response to high phosphate load. A better understanding of osteopontin's role in phosphate-induced calcification will hopefully lead to better biomarkers and therapies for this disease, especially in patients with chronic kidney disease and other at-risk populations., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Purpose: Nephrocalcinosis is commonly present in primary hyperparathyroidism, distal renal tubular acidosis and medullary sponge kidney disease. To our knowledge it has not been studied in patients with calcium phosphate stones who do not have systemic disease., Materials and Methods: We studied patients undergoing percutaneous nephrolithotomy who had calcium phosphate or calcium oxalate stones and did not have hyperparathyroidism, distal renal tubular acidosis or medullary sponge kidney disease. On postoperative day 1 all patients underwent noncontrast computerized tomography. If there were no residual calcifications, the patient was categorized as not having nephrocalcinosis. If there were residual calcifications, the patient underwent secondary percutaneous nephrolithotomy. If the calcifications were found to be stones, the patient was categorized as not having nephrocalcinosis. If the calcifications were not stones, the patient was categorized as having nephrocalcinosis. Patients were grouped based on the type of stones that formed, including hydroxyapatite, brushite and idiopathic calcium oxalate. The extent of nephrocalcinosis was quantified as 0--absent nephrocalcinosis to 3--extensive nephrocalcinosis. Patients with residual calcifications on postoperative day 1 noncontrast computerized tomography who did not undergo secondary percutaneous nephrolithotomy were excluded from analysis. The presence or absence of nephrocalcinosis was correlated with metabolic studies., Results: A total of 67 patients were studied, including 14 with hydroxyapatite, 19 with brushite and 34 with idiopathic calcium oxalate calculi. Nephrocalcinosis was present in 10 of 14 (71.4%), 11 of 19 (57.9%) and 6 of 34 patients (17.6%) in the hydroxyapatite, brushite and idiopathic calcium oxalate groups, respectively (chi-square p = 0.01). The mean extent of nephrocalcinosis per group was 1.98, 1.32 and 0.18 for hydroxyapatite, brushite and idiopathic calcium oxalate, respectively (p ≤0.001). The presence of nephrocalcinosis positively correlated with urine calcium excretion (mean ± SD 287.39 ± 112.49 vs 223.68 ± 100.67 mg per day, p = 0.03)., Conclusions: Patients without systemic disease who form hydroxyapatite and brushite stones commonly have coexistent nephrocalcinosis. Nephrocalcinosis can occur in calcium oxalate stone formers but the quantity and frequency of nephrocalcinosis in this group are dramatically less., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Diagnosis and management of pediatric nephrolithiasis/nephrocalcinosis is a very complex and challenging task for every pediatrician. It is based on correct. disease history taking, which may guide to the mode of inheritance (dominant, recessive, x-linked). Ethnicity and consanguinity should also be investigated since they predispose to high prevalence of certain disorders. One should always begin with cheap and available screening tests. Herein we will review clinical, biochemical, metabolic and genetic characteristics of the inherited diseases which lead to nephrolithiasis/nephrocalcinosis, such as: idiopathic hypercalciuria, renal hypophosphatemia, renal tubular acidosis, idiopathic infantile hypercalcemia, Dent disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis, hypocitraturia, cystinuria, primary hyperoxaluria and renal hypouricemia. Modern genetic techniques such as next generation sequencing enable nowadays diagnosis of rare disease using only a blood sample, trough massive parallel resequencing of many genes. This is very helpful for anuric patients or on dialysis where blood and urine biochemistry are not informative. Genetic testing also replaces invasive liver biopsy or unpleasant acidification tests and enables prenatal or early postnatal diagnosis.

The available publications on nephrocalcinosis are wide-ranging and have documented multiple causes and associations of macroscopic or radiological nephrocalcinosis, most often located in the renal medulla, with various metabolic and genetic disorders; in fact, so many and various are these that it is difficult to define a common underlying mechanism. We have reviewed nephrocalcinosis in relation to its definition, genetic associations, animal models, and putative mechanisms. We have concluded, and hypothesized, that nephrocalcinosis is primarily a renal interstitial process, resembling metastatic calcification, and that it may have some features in common with, and pathogenic links to, vascular calcification.

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.)

Loss-of-function mutations of CYP24A1, the enzyme that converts the major circulating and active forms of vitamin D to inactive metabolites, recently have been implicated in idiopathic infantile hypercalcemia. Patients with biallelic mutations in CYP24A1 present with severe hypercalcemia and nephrocalcinosis in infancy or hypercalciuria, kidney stones, and nephrocalcinosis in adulthood. We describe a cohort of 7 patients (2 adults, 5 children) presenting with severe hypercalcemia who had homozygous or compound heterozygous mutations in CYP24A1. Acute episodes of hypercalcemia in infancy were the first symptom in 6 of 7 patients; in all patients, symptoms included nephrocalcinosis, hypercalciuria, low parathyroid hormone (PTH) levels, and higher than expected 1,25-dihydroxyvitamin D levels. Longitudinal data suggested that in most patients, periods of increased sunlight exposure tended to correlate with decreases in PTH levels and increases in calcemia and calciuria. Follow-up of the 2 adult patients showed reduced glomerular filtration rate and extrarenal manifestations, including calcic corneal deposits and osteoporosis. Cases of severe PTH-independent hypercalcemia associated with hypercalciuria in infants should prompt genetic analysis of CYP24A1. These patients should be monitored carefully throughout life because they may be at increased risk for developing chronic kidney disease., (Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: The tight control of blood magnesium (Mg) levels is of central importance for numerous physiological processes. A persistent low Mg status (hypomagnesemia) is associated with severe health risks and is involved in the pathogenesis of type 2 diabetes mellitus, osteoporosis, asthma, and heart and vascular diseases. The current view has expanded significantly as a result of the identification of novel genes and regulatory pathways involved in hypomagnesemic disorders. This review aims to give an up-to-date overview of transient receptor potential melastatin 6 (TRPM6) regulation and its role in the maintenance of Mg homeostasis., Recent Findings: The epithelial Mg channel TRPM6 is considered to be the Mg entry pathway in the distal convoluted tubule of the kidney, where it functions as gatekeeper for controlling the body's Mg balance. Various factors and hormones contribute not only to the function, but also to the dysregulation of TRPM6, which has a substantial impact on renal Mg handling. Recent genetic and molecular studies have further elucidated the signaling processes of epithelial Mg transport, including their effect on the expression and function of TRPM6., Summary: Knowledge of TRPM6 functioning is of vital importance to decipher its role in Mg handling and will, in particular, provide a molecular basis for achieving a better understanding of Mg mal(re)absorption and hence systemic Mg balance.

Background and Aims: It has been suggested that magnesium deficiency is associated with the triggering of acute phase response, which may contribute to type 2 diabetes and cardiovascular disease risk. We undertook this study to determine whether oral magnesium supplementation modifies serum levels of high-sensitivity C-reactive protein (hsCRP) in apparently healthy subjects with prediabetes and hypomagnesemia., Methods: A total of 62 men and non-pregnant women aged 18-65 year, with new diagnosis of prediabetes (glucose 5.6 <7.0 mmol/L and/or post-load glucose ≥7.7 <11.1 mmol/L) and hypomagnesemia (serum magnesium levels <0.74 mmol/L) were enrolled in a clinical double-blind placebo-controlled trial and randomly allocated to receive either magnesium chloride (30 mL of MgCl2 5% solution) or NaHCO3 0.1% solution, once daily for 3 months., Results: At basal conditions, anthropometric and biochemical variables were similarly distributed in both groups. At the end of follow-up, participants who received magnesium chloride showed higher serum magnesium levels (0.86 ± 0.08 vs. 0.69 ± 0.16 mmol/L, p = 0.002) and lower hsCRP levels (4.8 ± 15.2 vs. 17.1 ± 21.0 nmol/L, p = 0.01) compared with participants in the control group., Conclusions: Oral magnesium supplementation decreases hsCRP levels in apparently healthy subjects with prediabetes and hypomagnesemia., (Copyright © 2014 IMSS. Published by Elsevier Inc. All rights reserved.)

Purpose: The objective was to identify the risk factors associated with nephrocalcinosis (NC) in preterm infants., Methods: NC was diagnosed by renal sonography at 4 or 8 weeks of life, and 10 infants who had findings of type 3 or 4 NC were classified as the NC group. Various clinical and laboratory factors were compared between NC and control groups., Results: Serum sodium (Na) on day 1, serum creatinine and fractional excretion of calcium (FeCa) at 1 and 2 weeks, and serum calcium (Ca), fractional excretion of sodium (FeNa), and urine Na on 2 weeks of life were significantly different between the two groups: the NC group showed significantly higher serum creatinine, FeNa, and FeCa than the control group, suggesting a greater decrease in renal function in the NC group. Differences of the laboratory findings disappeared after 4 weeks of life. The strongest risk factor was birth weight., Conclusion: A transient decrease in renal function during the first 2 weeks of life was associated with development of NC in preterm very low-birth-weight infants, and the risk of NC increased as birth weight decreased., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Mutations in PCBD1 are causative for transient neonatal hyperphenylalaninemia and primapterinuria (HPABH4D). Until now, HPABH4D has been regarded as a transient and benign neonatal syndrome without complications in adulthood. In our study of three adult patients with homozygous mutations in the PCBD1 gene, two patients were diagnosed with hypomagnesemia and renal Mg(2+) loss, and two patients developed diabetes with characteristics of maturity onset diabetes of the young (MODY), regardless of serum Mg(2+) levels. Our results suggest that these clinical findings are related to the function of PCBD1 as a dimerization cofactor for the transcription factor HNF1B. Mutations in the HNF1B gene have been shown to cause renal malformations, hypomagnesemia, and MODY. Gene expression studies combined with immunohistochemical analysis in the kidney showed that Pcbd1 is expressed in the distal convoluted tubule (DCT), where Pcbd1 transcript levels are upregulated by a low Mg(2+)-containing diet. Overexpression in a human kidney cell line showed that wild-type PCBD1 binds HNF1B to costimulate the FXYD2 promoter, the activity of which is instrumental in Mg(2+) reabsorption in the DCT. Of seven PCBD1 mutations previously reported in HPABH4D patients, five mutations caused proteolytic instability, leading to reduced FXYD2 promoter activity. Furthermore, cytosolic localization of PCBD1 increased when coexpressed with HNF1B mutants. Overall, our findings establish PCBD1 as a coactivator of the HNF1B-mediated transcription necessary for fine tuning FXYD2 transcription in the DCT and suggest that patients with HPABH4D should be monitored for previously unrecognized late complications, such as hypomagnesemia and MODY diabetes.

Rationale: Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences., Methods: In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin., Results: By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis., Conclusions: Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis is a rare tubulopathy leading to renal calcification and progressive renal failure., Case Presentation: We report a consanguineous Arab family (of Qatari origin) with 7 affected siblings with variable phenotypes including hypomagnesaemia, hypercalciuria, nephrocalcinosis and renal stones. Presenting features included haematuria and recurrent urinary tract infections. As the biochemical and clinical phenotypes of this family resembled familial hypomagnesaemia with hypercalciuria and nephrocalcinosis, we performed genetic investigation in order to provide a precise molecular diagnosis. We screened all coding regions of the CLDN16 gene and identified a novel mutation (c.G647A, p.R216H) which was found homozygously in the six severely affected cases, who manifested significant nephrocalcinosis, often nephrolithiasis and sometimes reduced GFR. Parents were both heterozygous for the mutation and, together with children carrying the mutation in its heterozygous state, exhibited mild or no biochemical phenotypes., Conclusion: Mutations in CLDN16 underlie familial hypomagnesaemia with hypercalciuria and nephrocalcinosis but remain a rare cause of nephrocalcinosis and nephrolithiasis. Management includes reduction of hypercalciuria with thiazide diuretics, correction of serum magnesium and close monitoring of renal function given the significant risk of end stage renal failure with this inherited form of nephrocalcinosis.

Hypomagnesemia is known to occur for a variety of renal, gastrointestinal and other causes, and is often associated with other electrolyte and metabolic disturbances. We present a case of isolated hypomagnesemia in a patient who had been treated with the chemotherapy agent capecitabine. The approach to diagnosis and treatment is discussed. We postulate that capecitabine may cause isolated hypomagnesemia, possibly due to renal magnesium loss.

Background and Objectives: Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals., Design, Setting, Participants, & Measurements: Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis., Results: Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%-20%., Conclusions: The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

The serum glycoprotein fetuin-A is an important inhibitor of extraosseous calcification. The importance of fetuin-A has been confirmed in fetuin-A null mice, which develop widespread extraosseous calcification including the kidney. However, the mechanism how fetuin-A protects kidneys from nephrocalcinosis remains uncertain. Here, we demonstrate that intratubular fetuin-A plays a role in the prevention of nephrocalcinosis in the proximal tubules. Although normal rat kidney did not express mRNA for fetuin-A, we found punctate immunohistochemical staining of fetuin-A mainly in the S1 segment of the proximal tubules. The staining pattern suggested that fetuin-A passed through the slit diaphragm, traveled in the proximal tubular lumen, and was introduced into proximal tubular cells by megalin-mediated endocytosis. To test this hypothesis, we inhibited the function of megalin by intravenous injection of histidine-tagged soluble receptor-associated protein (His-sRAP), a megalin inhibitor. His-sRAP injection diminished fetuin-A staining in the proximal tubules and led to urinary excretion of fetuin-A. We further analyzed the role of fetuin-A in nephrocalcinosis. Continuous injection of parathyroid hormone (PTH) 1-34 induced nephrocalcinosis mainly in the proximal tubules in rats. His-sRAP retained fetuin-A in renal tubular lumen and thereby protected the kidneys of PTH-treated rats from calcification. Our findings suggest that tubular luminal fetuin-A works as a natural inhibitor against calcification in the proximal tubules under PTH-loaded condition.