A variant in acis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones

Abstract

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria.

Claudin-14 Gene Polymorphisms and Urine Calcium Excretion

“…However, in vivo experiments are required to validate this in silico hypothesis. A previous in silico study hypothesized that rs78250838 might introduce a new transcription factor binding site within CLDN14, but this SNP was not associated with calcium excretion in our patients and was not in linkage with rs219780 and rs219755 (35).…”

Nephrolithiasis secondary to inherited defects in the thick ascending loop of henle and connecting tubules

“…In another study with 200 kidney SF and 200 healthy controls from the eastern part of India, the two variants rs219777 and rs219778 in the CLDN14 gene were found to be strongly associated with kidney stone disease, but no significant association was found with urinary or serum Ca [86]. A more recent study described the association of the CLDN14 gene variant rs78250838:C>T in an intronic cis-regulatory element with early-onset hypercalciuria and kidney stones in children [87]. In vitro studies revealed that this variant enhances claudin-14 expression, thereby increasing urinary Ca excretion and promoting calcium stone formation.…”

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability

“…Wild type littermate sections were taken as a negative control. Then, sections were rinsed with PBS twice for 5 min before mounting with DAKO (Carpinteria, CA, USA) or undergoing immunostaining, essentially as previously reported [19,32]. In brief, antigen retrieval was performed with sodium citrate, and then washed three times with TN buffer (0.1 M Tris/HCl and 0.15 M NaCl, pH 7.6).…”