Mechanistic Studies of CLCN5 Point Mutations to Understand Genotype-Phenotype Correlations in Dent's Disease Pilot Project

This pilot project proposes three major aims to further the understanding of genotype-phenotype correlations and Dent's disease and gain insights into potential mechanisms of hypercalciuria, nephrolithiasis and nephrocalcinosis.

The specific aims of the project are:

  • To track CLC-5 and CLC-5 mutants in cellular expression
  • To determine Cl-/H+ coupling properties of CLC-5 mutants and transport stoichiometry
  • To determine functional interactions of CLC-5 with regulatory proteins

Dent's disease, an X-linked, recessive kidney disease affecting mostly males, is caused by mutations in the CLCN5 gene, and is clinically characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and progressive renal failure. CLCN5 encodes the CLC-5 protein, a voltage-gated H+/nCl- exchanger, found at the apical membrane in the kidney proximal tubule and endosome membranes of other tubule segments.

The Rare Kidney Stone Consortium (RKSC) has identified 20 patients with CLCN5 mutations in their cohort of patients with Dent's disease. Presently, the clinical data suggest that patients with the same genotypes can have markedly different clinical phenotypes.

In published series of patients with Dent's disease, S244L-CLCN5 is the most frequent mutation found. Q629X and R345W are two novel mutations found in newly diagnosed patients in the RKSC.

Two patients in the RKSC had the common S244L mutation. One had mild hypercalciuria, nephrocalcinosis and chronic kidney failure (eGFR 22.2 mL/min), while the other had preserved kidney function. Similarly, one brother with a Q629X mutation had a history of kidney stones and rickets with normal kidney function, while another was entirely asymptomatic. Another patient with a R345W mutation had impaired kidney function (eGFR 31 mL/min), but was without other symptoms.

T657S was previously reported as pathogenic in a published study of a patient with Dent's disease, but T657S is the only sequence variant we identified in the National Heart, Lung, and Blood Institute Exome Sequencing Project (ESP) database. Interestingly, the T657S variant had a high minor-allele frequency (MIF) of 0.3911 percent in the African-American population. The pathogenic significance of this T657S variation remains to be determined, although it is interesting to note that proteinuria and chronic kidney disease are common in African-Americans.

The laboratory of Minhwang Chang, Ph.D., has developed a xenopus oocyte overexpression system to recreate and characterize these coding mutations to better understand the clinical pathology of Dent's disease and the possible underpinnings of the associated nephrolithiasis. This system allows us to obtain mechanistic information to explain the divergence between genotype and phenotype.

Voltage-clamp experiments demonstrated that oocytes expressing the R345W mutant had a significant reduction (34.5 percent) in current amplitude compared with wild-type (WT) ClC-5. The Q629X and S244L mutants demonstrated even larger reductions of ClC-5 currents (IClC5): 95 percent and 90 percent, respectively. In contrast, IClC5 of oocytes expressing the T657S mutant (ESP variant) were not significantly different from WT ClC-5. IClC5 of the WT and ClC-5 mutants were decreased by reducing extracellular pH (pH 5.5) but were unaffected by elevating extracellular pH (pH 8.5).

The T657S and R345W mutants demonstrated the same anion selectivity as WT ClC-5 (SCN->NO3- ≈ Cl->Br->I-). However, the S244L and Q629X mutants had abnormal anion conductances.