CLDN16 genotype predicts renal decline in familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder caused by CLDN16 mutations. CLDN16 encodes the renal tight junction protein claudin-16, which is important for the paracellular reabsorption of calcium and magnesium in the thick ascending limb of Henle’s loop. That FHHNC is frequently associated with progressive renal failure suggests additional roles for claudin-16 in the maintenance of tight junction integrity. An investigation of 32 patients with FHHNC and 17 different mutations was previously reported; here, the analysis is expanded to 39 additional patients and 12 new mutations. Expression studies revealed that five of the 12 new mutations led to partial loss of claudin-16 function and the remaining seven led to complete loss of function. The 23 patients who had mutations resulting in complete loss of function of both alleles were significantly younger at the onset of symptoms than the 46 patients who had at least one mutant allele providing partial function (2.2 versus 5.6 years; P 0.01). In addition, those with complete loss of function had a more rapid decline in GFR (7.3 versus 2.9 ml/min per 1.72 m/y; P 0.01), leading to 54% requiring renal replacement therapy by age 15 compared with 20% of those with residual function (P 0.05). These data suggest that residual function of claudin-16 may delay the progression of renal failure in FHHNC. J Am Soc Nephrol 19: 171–181, 2008. doi: 10.1681/ASN.2007060709 Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM *248250) is a rare autosomal recessive tubular disorder. It is characterized by massive urinary losses of magnesium (Mg) and calcium (Ca) leading to hypomagnesemia and bilateral nephrocalcinosis. The disease usually presents with recurrent urinary tract infections and polyuria/polydipsia. Additional symptoms include rickets, nephrolithiasis, hematuria, muscular tetanies, seizures, failure to thrive, vomiting, and abdominal pain.1,2 Ocular abnormalities and hearing impairment have been described in a subset of patients with FHHNC.3,4 Additional biochemical abReceived June 25, 2007. Accepted July 24, 2007. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Martin Konrad, Department of Pediatrics, University of Münster, Waldeyerstrasse 22, 48149 Münster, Germany. Phone: 49-251-9813331; Fax: 49-251-9813336; Email: [email protected] Copyright © 2008 by the American Society of Nephrology C LI N IC A L R E SE A R C H CLINICAL RESEARCH www.jasn.org J Am Soc Nephrol 19: 171–181, 2008 ISSN : 1046-6673/1901-171 171 normalities include signs of incomplete distal renal tubular acidosis, hypocitraturia, increased parathyroid hormone (PTH) levels (independent of GFR), and hyperuricemia.3,5– 8 Unlike most other inherited tubular diseases affecting electrolyte homeostasis, FHHNC is generally complicated by progressive renal failure during childhood or adolescence, but the pathogenesis of chronic renal failure remains a matter of debate. Clearance studies in patients with FHHNC localized the site of disturbed handling of Mg and Ca to the thick ascending limb (TAL) of Henle’s loop.6 The TAL plays an important role in the tubular reabsorption of Mg and Ca, which occurs by paracellular flux, a process driven by the lumen-positive transepithelial potential in this nephron segment. In 1999, Simon et al.9 identified a new gene (CLDN16, formerly PCLN1) and characterized mutations in this gene as the underlying molecular defect in FHHNC. Since then, approximately 30 additional families with FHHNC as a result of CLDN16 defects have been described.2,10 –13 Hypercalciuria and nephrolithiasis have also been observed in heterozygous FHHNC mutation carriers.2,3 FHHNC is a genetically heterogeneous disease because recently CLDN19 mutations have been identified in a cohort of patients mainly originating from Spain.14 The renal phenotype of these patients is very similar to patients exhibiting CLDN16 mutations; however, patients with CLDN19 mutations also have severe ocular abnormalities in most cases. CLDN16 and CLDN19 encode the tight junction (TJ) proteins claudin-16 (paracellin-1) and claudin-19, both being members of the claudin multigene family. Claudins are important components of the TJ strands in various tissues.15 They are part of a complex protein network built up by a variety of different proteins, and there is clear evidence that claudins confer ion selectivity to the paracellular pathway.16 –18 In the kidney, claudin-16 expression is restricted to the TAL of Henle’s loop. It was speculated that claudin-16 constitutes the core of an intercellular pore, allowing paracellular reabsorption of Mg and Ca ions.9,19 Following the observation that a naturally occurring knockout model in Japanese black cattle shows early-onset renal failure with diffuse interstitial nephritis,20 it was recently speculated that claudin-16, like other claudins, may also be involved in the regulation of cell growth, proliferation, differentiation, and dedifferentiation.21 Hou et al.22 described the functional analysis of claudin-16 in polarized cell lines. They demonstrated that in LLC-PK1 cells, claudin-16 modulated the ion selectivity of the TJ by selectively increasing the permeability of Na with no effects on Cl , resulting in a high permeability ratio of Na to Cl . Instead, Mg flux across cell monolayers showed a far less pronounced change after claudin-16 expression. From these data, it might be concluded that claudin-16 defects lead to a loss of cation selectivity with a subsequent decrease in lumen-positive potential that is the driving force for the paracellular flux of cations. This hypothesis of a nonselective paracellular cation channel is supported by a mouse model using transgenic RNAi depletion of claudin-16. Loss of CLDN16 in this model caused TJ in TAL to lose the cation selectivity.23 Hou et al.22 also analyzed the consequences of most of the reported human CLDN16 mutations by heterologous expression in vitro. Whereas most mutations resulted in a complete loss of function, some mutations retained a substantial residual function. One of these mutations (L151F) is by far the most frequent FHHNC mutation, occurring in almost 50% of the patients described so far.2 Combining this information with additional functional analysis of new mutations using the same expression system, we present a genotype/phenotype correlation with a special focus on the progression of renal failure in a large cohort of patients with FHHNC. We provide clinical data indicating that homozygous or compound heterozygous patients who carry at least one CLDN16 mutation with residual function have a much more benign course of the disease than patients with a complete loss of function.