Lorsung, Rebecca M.

Dent disease I is a rare X‐linked recessive chronic kidney disorder caused by mutations of the CLCN5 gene, which encodes the electrogenic Cl−/H+ exchanger (transporter) ClC‐5. CLC‐5 protein has been localized to the apical membrane and endosomal vesicles of the kidney in the proximal tubule as well as other tubule segments. Clinical manifestations of patients with Dent disease include low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, bone disease (rickets), and often progressive renal failure. While it is known that the CLC‐5 protein functions in the formation and maturation of endosomes, the etiology of the nephrocalcinosis and kidney stones often observed in Dent disease patients is not known. Therefore, in this project we investigated the effects of several CLC‐5 mutations identified in Dent disease patients, including S244L, Q629X, and R345W, on cellular processing of calcium oxalate (CaOx) crystals. Monolayers of renal proximal tubular cells (LLC‐PK1) and renal cortical tubular epithelial cells (RCTE) transfected with GFP tagged WT and mutated CLC‐5 were subjected to a CaOx crystal adhesion/agglomeration assay for thirty minutes then imaged by DIC and epifluorescence microscopy. The total area of CaOx crystal formation around each transfected cell within a 35, 55, and 75 μm diameter concentric circle was analyzed using ImageJ, and compared to adjacent un‐transfected cells which served as an internal control. A significant increase in CaOx aggregation was observed around cells expressing CLC‐5 mutants compared to wild type cells, especially for S244L. In LLC‐PK1 cells crystal adhesion/agglomeration decreased in this order: S224L > R345W > Q629X = WT; while in RCTE it decreased in this order: S224L = Q629X > R345W = WT. These findings support the hypothesis that mutated CLC‐5 proteins alter the apical surface of epithelial nephron cells and encourage CaOx aggregation/adhesion. It is also possible that decreased internalization and dissolution of CaOx crystals which form on the cell surface plays a role, as the greatest crystal adhesion/agglomeration was found within the 35 μm diameter circle around cells transfected with mutated CLC‐5 (35 μm > 55 μm > 75 μm). Further investigation into the impact of specific CLC‐5 mutation of protein structure and function, including protein‐protein interactions, is needed. Support or Funding Information Supported by O'Brien Urology Research Center U54‐DK100227 summer student supplement and the Rare Kidney Stone Consortium (U54‐DK083908)