Magnesium is an essential ion involved in many biochemical and physiological processes. Homeostasis of magnesium levels is tightly regulated and depends on the balance between intestinal absorption and renal excretion. However, little is known about specific proteins mediating transepithelial magnesium transport. Using a positional candidate gene approach, we identified mutations in TRPM6 (also known as CHAK2), encoding TRPM6, in autosomal-recessive hypomagnesemia with secondary hypocalcemia (HSH, OMIM 602014), previously mapped to chromosome 9q22 (ref. 3). The TRPM6 protein is a new member of the long transient receptor potential channel (TRPM) family and is highly similar to TRPM7 (also known as TRP-PLIK), a bifunctional protein that combines calcium- and magnesium-permeable cation channel properties with protein kinase activity. TRPM6 is expressed in intestinal epithelia and kidney tubules. These findings indicate that TRPM6 is crucial for magnesium homeostasis and implicate a TRPM family member in human disease.

The presence of CYP24A1 mutations explains the increased sensitivity to vitamin D in patients with idiopathic infantile hypercalcemia and is a genetic risk factor for the development of symptomatic hypercalcemia that may be triggered by vitamin D prophylaxis in otherwise apparently healthy infants.

Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg 2؉ -permeable cation channel TRPM7, resulting in the assembly of functional TRPM6͞TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6͞ TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.I nvestigations on Drosophila flies with impaired vision due to mutations in the transient receptor potential gene (trp) initiated a search for homologous proteins in mammals, leading to the discovery of three subfamilies of cation channels: TRPCs (canonical or classical TRPs), TRPVs (vanilloid receptor and related proteins), and TRPMs (melastatin and related proteins) (1, 2). TRPC channels mediate cation entry in response to phospholipase C activation, whereas TRPV proteins respond to physical and chemical stimuli, such as temperature, pH, and mechanical stress (3, 4). Within their respective subfamilies, TRPCs and TRPVs form homo-and heterotetramers displaying novel pore properties when compared to their homomultimeric counterparts (1, 5-9). The eight TRPM family members differ significantly from the aforementioned TRP channels in terms of domain structure, cation selectivity, and activation mechanisms (3, 10). Two TRPM proteins, TRPM6 and TRPM7, harbor serine͞threonine kinase domains in their C termini (11-16). Furthermore, TRPM7 displays unusual permeation properties by conducting a range of divalent metal ions including Mg 2ϩ and Mn 2ϩ (13,17,18).It was recently shown that autosomal recessive hypomagnesemia with secondary hypocalcemia (HSH) is caused by mutations in the TRPM6 gene (15,16). HSH is characterized by low serum Mg 2ϩ levels due to defective intestinal absorption or͞and renal wasting of Mg 2ϩ . Here we demonstrate that TRPM6 requires assembly with TRPM7 to form channel complexes in the cell membrane and that disruption of multimer formation by a mutated TRPM6 variant, TRPM6(S141L), results in human disease. MethodsMolecular Biology and Generation of TRPM6 Polyclonal Antisera. The cloning procedure of human TRPM6 isoforms (Table 1) as well as amplification of other TRPM cDNAs is described in detail in Supporting Methods, which is published as supporting information on the PNAS web site. For TRPM proteins C-terminally fused to cyan (CFP) or yellow (YFP) fluorescent proteins, STOP codons in TRPMs were replaced by XhoI restrictio...

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl − blocker, whereas CLDN16 functioned as a Na + channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl − permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. IntroductionThe human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM 248250) is characterized by progressive renal Mg 2+ and Ca 2+ wasting leading to impaired renal function and chronic renal failure. FHHNC has been genetically linked to mutations in the gene of claudin-16 (CLDN16, also known as paracellin-1) (1) and more recently to CLDN19 (2). The claudins comprise a 22-gene family that encodes essential structural proteins of the tight junction (TJ), which are the principal regulators of paracellular permeability. In vitro studies have shown that ion selectivity of the paracellular conductance (reviewed in ref.3) is a complex function of claudin subtype and cellular context (4, 5).In vitro analyses using cultured cell models show that CLDN16 plays a key role in maintaining the cation selectivity of the TJ and forms a nonselective paracellular cation channel (4). This hypothesis of a nonselective paracellular cation channel is supported by (a) a clinical study to correlate the cellular functions of CLDN16 mutations identified in FHHNC to the phenotypes of FHHNC patients, with a special focus on the progression of renal failure (6), and by (b) our mouse models using transgenic RNAi depletion of CLDN16 (7). Without CLDN16 expression in the kidney, TJs in the thick ascending limb (TAL) of the nephron lose cation selectivity, leading to the dissipation of the lumen-positive potential with a concomitant loss of the driving force for Mg 2+ reabsorption (7). While targeted deletion of CLDN19 in mice initially focused on its

Gitelman syndrome (GS) is a rare, salt-losing tubulopathy characterized by hypokalemic metabolic alkalosis with hypomagnesemia and hypocalciuria. The disease is recessively inherited, caused by inactivating mutations in the SLC12A3 gene that encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). GS is usually detected during adolescence or adulthood, either fortuitously or in association with mild or nonspecific symptoms or both. The disease is characterized by high phenotypic variability and a significant reduction in the quality of life, and it may be associated with severe manifestations. GS is usually managed by a liberal salt intake together with oral magnesium and potassium supplements. A general problem in rare diseases is the lack of high quality evidence to inform diagnosis, prognosis, and management. We report here on the current state of knowledge related to the diagnostic evaluation, follow-up, management, and treatment of GS; identify knowledge gaps; and propose a research agenda to substantiate a number of issues related to GS. This expert consensus statement aims to establish an initial framework to enable clinical auditing and thus improve quality control of care.

Antenatal Bartter syndrome (aBS) comprises a heterogeneous group of autosomal recessive salt-losing nephropathies. Identification of three genes that code for renal transporters and channels as responsible for aBS has resulted in new insights into renal salt handling, diuretic action and blood-pressure regulation. A gene locus of a fourth variant of aBS called BSND, which in contrast to the other forms is associated with sensorineural deafness (SND) and renal failure, has been mapped to chromosome 1p. We report here the identification by positional cloning, in a region not covered by the human genome sequencing projects, of a new gene, BSND, as the cause of BSND. We examined ten families with BSND and detected seven different mutations in BSND that probably result in loss of function. In accordance with the phenotype, BSND is expressed in the thin limb and the thick ascending limb of the loop of Henle in the kidney and in the dark cells of the inner ear. The gene encodes a hitherto unknown protein with two putative transmembrane alpha-helices and thus might function as a regulator for ion-transport proteins involved in aBS, or else as a new transporter or channel itself.

This is the first study that reports the long-term outcome of ARPKD patients with defined PKHD1 mutations. The 1- and 10-year survival rates were 85% and 82%, respectively. Chronic renal failure was first detected at a mean age of 4 years. Actuarial renal survival rates [end point defined as start of dialysis/renal transplantation (RTX) or by death due to end-stage renal disease (ESRD)] were 86% at 5 years, 71% at 10 years, and 42% at 20 years. All but six patients (92%) had a kidney length above or on the 97th centile for age. About 75% of the study population developed systemic hypertension. Sequelae of congenital hepatic fibrosis and portal hypertension developed in 44% of patients and were related with age. Positive correlations could further be demonstrated between renal and hepatobiliary-related morbidity suggesting uniform disease progression rather than organ-specific patterns. PKHD1 mutation analysis revealed 193 mutations (70 novel ones; 77% nonconservative missense mutations). No patient carried two truncating mutations corroborating that one missense mutation is indispensable for survival of newborns. We attempted to set up genotype-phenotype correlations and to categorize missense mutations. In 96% of families we identified at least one mutated PKHD1 allele (overall detection rate 76.6%) indicating that PKHD1 mutation screening is a powerful diagnostic tool in patients suspected with ARPKD.