Recurrent FXYD2 p.Gly41Arg mutation in patients with isolated dominant hypomagnesaemia

Background Magnesium (Mg2+) is an essential ion for cell growth, neuroplasticity and muscle contraction. Blood Mg2+ levels <0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps and epilepsy and disturbances in K+ and Ca2+ 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 letha

Functionomics of novel mutations in NCC and their relevance in development of Gitelman syndrome

<p>Gitelman syndrome is a genetic disease characterized by low blood pressure and salt wasting. In most cases, Gitelman syndrome results from loss-of-function mutations in the solute carrier family 12 (SLC12A3) gene, which encodes the thiazide-sensitive sodium chloride co-transporter (NCC). At present, more than 250 distinct loss-of-function mutations have been identified in patients with Gitelman syndrome. Functional analysis has been limited by the use of only Xenopus laevis oocytes as a model system. The aim of the present study is to understand the functional consequences of these NCC mutations in mammalian cell line systems. Recently, our group published a new technique to isolate primary cells using Complex Object Parametric Analyzer and Sorter (COPAS). We have pioneered with this COPAS sorting platform to isolate and culture distal convoluted tubules (DCT) based on parvalbumin-eGFP transgenic mice model. These primary cultures exhibit several characteristics of the original epithelium including thiazide-sensitive transepithelial NaCl transport. By generating crossbred of parvalbumin-eGFP mice with NCC knockout we aim to obtain DCT primary cell line without endogenous expression of wild type NCC, which in turn will be substituted by ectopic expression of mutated NCC. Generation of this model will create an excellent platform for further functional analysis of mutant NCC proteins. Currently, out of several patients with Gitelman syndrome symptoms eight new mutations were selected. The majority of these mutations exhibited a disturbed phosphorylation and glycosylation pattern as well as diminished expression of NCC. However, functional consequences of these mutations remain to be elucidated. Overall, functional screening of many NCC mutations in DCT cultures are now possible and will also allow to unravel a complex system of NCC regulatory mechanisms.</p

Enhanced passive Ca²⁺ reabsorption and reduced Mg²⁺ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia

Thiazide diuretics enhance renal Na⁺ excretion by blocking the Na⁺-Cl⁻ cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca²⁺ transport in the proximal tubule rather than active Ca²⁺ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na⁺ and Ca²⁺ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca²⁺ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5–knockout (Trpv5-knockout) mice, in which active distal Ca²⁺ reabsorption is abolished due to inactivation of the epithelial Ca²⁺ channel Trpv5. Third, HCTZ upregulated the Na⁺/H⁺ exchanger, responsible for the majority of Na⁺ and, consequently, Ca²⁺ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca²⁺ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na⁺ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg²⁺ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation

Supplementary figure 1-4.pdf

Supplementary figure 1 to 4 for manuscript:    Transcription factor HNF1β controls a transcriptional network regulating kidney cell structure and tight junction integrity </p

Supplementary Tables

Suppelemntary Table 1:   Differential expressed genes in RNA-seq data of mpkDCT transfected with siRNAs targeting Hnf1b or non-targeting siRNAs Supplementary Table 2:   HNF1β ChIP-sequencing peaks in mpkDCT cells annoted to closest gene. For manuscript:    Transcription factor HNF1β controls a transcriptional network regulating kidney cell structure and tight junction integrity </p

Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins.

Contains fulltext : 89044.pdf (publisher's version ) (Closed access)Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had higher urinary calcium excretion than female mice and their renal calcium transporters were expressed at a lower level. We also found that orchidectomized mice excreted less calcium in their urine than sham-operated control mice and that the hypocalciuria was normalized after testosterone replacement. Androgen deficiency increased the abundance of the renal mRNA and protein of both the luminal transient receptor potential vanilloid-subtype 5 (TRPV5) and intracellular calbindin-D(28K) transporters, which in turn were suppressed by testosterone treatment. There were no significant differences in serum estrogen, parathyroid hormone, or 1,25-dihydroxyvitamin D3 levels between control and orchidectomized mice with or without testosterone. Moreover, incubation of primary rabbit connecting tubule and cortical collecting duct cells with a nonaromatizable androgen, dihydrotestosterone, reduced transcellular calcium transport. Thus, our study shows that gender differences in renal calcium handling are, in part, mediated by the inhibitory actions of androgens on TRPV5-mediated active renal calcium transport.1 april 201

Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin.

Contains fulltext : 29907.pdf (publisher's version ) (Closed access