Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1.

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate Cl(-) approximately Br(-) > I(-) > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK(a) of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.

Diet-dependent hypercalciuria in transgenic mice with reduced CLC5 chloride channel expression.

Dent's disease is an X-linked inherited disorder characterized by hypercalciuria, nephrocalcinosis, nephrolithiasis, low molecular weight proteinuria, Fanconi's syndrome, and renal failure. It is caused by inactivating mutations in CLC5, a member of the CLC voltage-gated chloride channel family. CLC5 is known to be expressed in the endosomal compartment of the renal proximal tubule, where it may be required for endosomal acidification and trafficking. Although the Fanconi's syndrome and low molecular weight proteinuria in Dent's disease can be explained by disruption of endosomal function in this nephron segment, the pathogenesis of the hypercalciuria in this disease is unknown. We have generated transgenic mice (RZ) with reduced CLC5 expression by introduction of an antisense ribozyme targeted against CLC5. RZ mice are markedly hypercalciuric compared with nontransgenic control mice, at a time when their serum electrolytes and renal function are otherwise normal. This suggests that hypercalciuria in Dent's disease is a direct consequence of CLC5 hypofunction and is not attributable to a gain of function by mutant CLC5, an effect of modifier genes, or a secondary result of nonspecific renal injury. Surprisingly, hypercalciuria in RZ mice is abolished by dietary calcium deprivation, suggesting that the hypercalciuria may be attributable to gastrointestinal hyperabsorption of calcium rather than a renal calcium leak.