Retrospective cohort study of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis due to CLDN16 mutations.

BACKGROUND: Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder exhibiting a high risk for progressive chronic kidney disease (CKD). METHODS: This is a retrospective multicentre study of 25 paediatric cases with FHHNC in Poland. Median age at diagnosis was 4 years and median follow-up time was 4.8 years. RESULTS: All cases of FHHNC carried recessive mutations in CLDN16. The founder mutation in CLDN16, Leu151Phe, was the most frequent cause of FHHNC in Polish patients, with 13 (52%) cases being homozygous and 5 (20%) carrying Leu151Phe allele in compound heterozygosity. All cases showed nephrocalcinosis, increased urinary fractional excretion of magnesium and hypercalciuria. Other disease features included hypomagnesaemia (76%), hyperparathyroidism (76%), hyperuricaemia (56%) and hypocitraturia (60%). Treatment with thiazides effectively reduced hypercalciuria in most cases. During follow-up, renal function declined in 60% of patients; 12% of patients reached CKD stage 3 or 4 and one patient developed end-stage renal failure. CONCLUSIONS: We report one of the largest cohorts of FHHNC cases caused by CLDN16 mutations. A missense variant of CLDN16, Leu151Phe, is the most common mutation responsible for FHHNC in Poland. Additionally, we found that normomagnesaemia does not exclude FHHNC and the calculation of fractional excretion of Mg can be diagnostic in the setting of normomagnesaemia. We also demonstrate the efficacy of a treatment with thiazides in terms of hypercalciuria in the majority of patients.

Clinical and molecular characterization of Turkish patients with familial hypomagnesaemia: novel mutations in TRPM6 and CLDN16 genes.

BACKGROUND: Recent identification and characterization of novel renal Mg(2+) transporters and ion channels have greatly increased our understanding of the normal physiology of renal magnesium handling. METHODS: The present study deals with the clinical and molecular characterization of eight Turkish children (median age 10.6 years, range 3-16.2 years, five boys and three girls) with primary hypomagnesaemia from six families. RESULTS: All patients initially presented with tetany and convulsions. Laboratory evaluation yielded severely low serum magnesium levels and low serum calcium levels in all patients. While six patients exhibited inadequately low parathyroid hormone levels, the two remaining patients showed hyperparathyroidism, hypercalciuria and nephrocalcinosis. Genetic studies revealed familial hypomagnesaemia with secondary hypocalcaemia (HSH) due to a TRPM6 mutation in six patients and familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) due to a CLDN16 mutation in one patient. CONCLUSIONS: Among recently identified magnesium-wasting disorders, HSH and FHHNC represent two major entities also in the Turkish population. Besides clinical course and laboratory diagnosis of hypomagnesaemia, the detection of renal calcium wasting and parathyroid function are crucial to differentiate between these most prevalent forms of hereditary magnesium deficiency. While TRPM6 mutations underlying HSH almost uniformly lead to a complete loss of function of the TRPM6 protein, the severity of FHHNC phenotype depends on the residual function of the mutated claudin-16 protein.

The role of tight junctions in paracellular ion transport in the renal tubule: lessons learned from a rare inherited tubular disorder.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive renal tubular disorder that typically presents with disturbances in magnesium and calcium homeostasis, recurrent urinary tract infections, and polyuria and/or polydipsia. Patients with FHHNC have high risk of the development of chronic kidney disease and end-stage renal disease in early adolescence. Multiple distinct mutations in the CLDN16 gene, which encodes a tight junction protein, have been found responsible for this disorder. In addition, mutations in another member of the claudin family, CLDN19, were identified in a subset of patients with FHHNC with visual impairment. The claudins belong to the family of tight junction proteins that define the intercellular space between adjacent endo- and epithelial cells. Claudins are especially important for the regulation of paracellular ion permeability. We describe a Brazilian family with 2 affected siblings presenting with the typical FHHNC phenotype with ocular anomalies. The clinical diagnosis of FHHNC was confirmed using mutational analysis of the CLDN19 gene, which showed 2 compound heterozygous mutations. In the context of the case vignette, we summarize the clinical presentation, diagnostic criteria, and therapeutic options for patients with FHHNC. We also review recent advances in understanding the electrophysiologic function of claudin-16 and -19 in the thick ascending limb of the loop of Henle and implications for ion homeostasis in the human body.

Impaired paracellular ion transport in the loop of Henle causes familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare tubular disorder caused by mutations in genes coding for tight junction (TJ) proteins. TJs define the paracellular path between adjacent cells and thereby play a pivotal role for the regulation of the paracellular ion permeability of epithelia. The family of TJ proteins comprise a variety of transmembrane proteins, including the claudins. Multiple distinct mutations in the genes for claudin-16 and -19 have been described to be responsible for FHHNC. Both encoded proteins are especially important for the paracellular reabsorption of Mg(2+) and Ca(2+) in the thick ascending limb of Henle's loop. Interestingly, in addition to ion disturbances, FHHNC leads to chronic renal failure and may be associated with extrarenal symptoms.

Claudin function in the thick ascending limb of Henle's loop.

During the past decade, claudins have been established as major determinants of paracellular permeablilty in epithelia. In the kidney, each nephron segment expresses a distinct pattern of claudins. Cells of the thick ascending limb of Henle's loop (TAL), which is characterized by high paracellular cation permeability, co-express an unusually large number of different claudins: claudin-10, -16, and -19 and, depending on the species, also claudin-3, -4, -8, and/or -11. The function of most of these claudins has been investigated in vitro. We present a summary of their function with special emphasis on claudin-16 and -19. Mutations in the corresponding human genes lead to severely impaired renal Ca(2+) and Mg(2+) handling. To date, 42 different claudin-16 mutations and three claudin-19 mutations have been reported. These mutations prevent the claudins from reaching the surface membrane, decrease membrane residence time, or render them functionless. In spite of the clear clinical symptoms such as hypomagnesemia, hypercalciuria, nephrocalcinosis, and renal insufficiency, mechanisms that link claudin-16 and -19 to these symptoms are still unknown. Depending on the cell type used in overexpression studies, claudin-16 appears to cause a mild increase in paracellular Mg(2+)-permeability or a pronounced increase in Na(+) permeability. Claudin-19 selectively decreases Cl(-) permeability, thus synergistically increasing relative cation permeability, or indiscriminately decreases paracellular permeability. In the light of these results it is hypothesized that the renal Mg(2+)/Ca(2+) waste may not be solely due to reduced resorption in the TAL but at least in part to paracellular back-leak of Mg(2+)/Ca(2+) into the tubular lumen of the distal convoluted tubule.

Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function.

The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.