INTRARENAL AND SUBCELLULAR-LOCALIZATION OF RAT CLC5

Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight prot einuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unkno wn. We have investigated the intrarenal and subcellular localization o f CLC5 in rat kidney by in situ hybridization and immunohistochemistry . By in situ hybridization, CLC5 mRNA was detected predominantly in co rtical medullary ray and outer medullary tubule epithelial cells. Poly clonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 is oform-specific antiserum. By immunohistochemistry, CLC5 protein was lo calized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending L imb. By subcellular membrane fractionation and flow cytometry, CLC5 ex pression was found in outer medullary endosomes. These findings are co nsistent with a model in which CLC5 encodes an endosomal chloride chan nel that facilitates acidification and trafficking of renal epithelial endosomes.