The role of claudin sequence variants in the formation of kidney stones

It is known that genetic risk factors contribute to the formation of calcium-based kidney stones. The majority of calcium is reabsorbed via paracellular transport through tight junctions along the epithelium of the nephron. Claudins are a family of integral membrane proteins that are expressed in tight junctions. Claudins determine the selectivity and permeability of different nephron segments. Variants of several CLDN genes have been found to be associated with kidney stones. Therefore, I hypothesize that sequence variants in claudin genes that regulate paracellular transport of calcium in the nephron will be associated with the formation of kidney stones.Ninety adult patients (45 male; 45 female) with recurrent calcium-based kidney stones were recruited from one urologist’s kidney stone clinic. The majority of the patients self-identified as Canadian-European for their ethnicity. Most of the patients presented with their first kidney stone at less than 40 years of age. Sixteen non-synonymous heterozygous variants were identified by next generation sequencing, and 13 were confirmed by Sanger sequencing. Of those variants, 4 are novel and 9 are rare (Minor Allele Frequency (MAF)<1%). In silico prediction software was used to predict the impact of the amino acid change on the protein structure and function. The novel variants were: CLDN11 S157F, CLDN16 K29E, CLDN17 A94V, and CLDN18 H212D. Based on the in silico results, CLDN11 S157F and CLDN17 A94V are predicted to be pathogenic. The rare variants are CLDN4 A82T, CLDN4 A113T, CLDN6 P211T, CLDN7 V55I, CLDN8 A94V, CLDN8 M97T, CLDN12 M98V, CLDN23 A90T, and CLDN24 V97I. Among these, CLDN4 A82T and CLDN8 A94V are predicted to be deleterious. The majority of the claudin variants are located in the second transmembrane domain of the claudin protein. The rest of the variants are located in different domains. CLDN7 V55I is located in the first extracellular loop, and CLDN11 S157F is located in the second extracellular loop. Finally, CLDN6 P211T and CLDN18 H212D are both located in the C-terminal tail.To evaluate the functional consequences of the claudin sequence variants, the claudin variants were generated by site-directed mutagenesis and cloned into a mammalian expression vector, pEGFP. Claudin variants and the corresponding wildtype (WT) sequences were transiently and/or stably transfected into HEK293 and MDCK II cells to determine if the protein encoded by the variant was able to co-localize to tight junctions. CLDN4 A82T, CLDN4 A113T, and CLDN8 A94V and their corresponding WT sequences were transiently transfected into HEK293 cells. Immunofluorescence (IF) showed that CLDN4 A82T and A113T proteins co-localize with ZO-1, a tight junction marker, like the CLDN4 WT protein. In contrast, the mutant protein CLDN8 A94V was unable to localize to tight junctions and exhibited submembranous expression in transiently transfected HEK293 cells. IF results of stably transfected MDCK cells that express CLDN4 WT and A82T showed that CLDN4 WT and A82T were able to localize to the tight junction. However, there was more cytoplasmic expression noted in MDCK II cells transfected with CLDN4 A82T. Measurement of transepithelial electrical resistance (TEER) of stably transfected MDCK II stable cells showed that CLDN4 A82T had no impact on the integrity and permeability of tight junctions. Similar future approaches including IF, TEER measurements, use of dextrans and assessment of relative paracellular calcium flux will be conducted to understand how claudin variants contribute to kidney stone formation

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