Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease

Dent's disease, which is a renal tubular disorder characterized by low mole cular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5, Howev er, the manner in which a functional loss of CLC-5 leads to such diverse re nal abnormalities remains to be defined. In order to elucidate this, we per formed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characte rized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla, Immunoh istochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in inte rcalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with tha t of Rab4, which is a marker of recycling early endosomes, In addition, con focal microscopy studies using the proximal tubular cell model of opossum k idney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-loc alized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-med iated endocytic pathway. Furthermore, the functional loss of CLC-5 in the p roximal tubules and the thick ascending limbs provides an explanation for t he occurrences of low molecular weight proteinuria and hypercalciuria, resp ectively. These results help to elucidate further the pathophysiological ba sis of the renal tubular defects of Dent's disease.