The ClC-5 chloride channel knock-out mouse – an animal model for Dent's disease

Abstract

Mutations in the gene CLCN5 encoding the vesicular chloride channel ClC-5 lead to Dent's disease, an X-linked renal disorder. Dent's disease is characterised by proteinuria, hyperphosphaturia and hypercalciuria, which eventually lead to kidney stones and nephrocalcinosis. As it was unclear how mutations in a chloride channel might cause these symptoms, we and others have generated genetic mouse models to elucidate the underlying pathophysiological mechanisms. We review results obtained from these three mouse models and present new data on endosomal acidification and vitamin D metabolism in ClC-5 knock-out (KO) mice. ClC-5 is expressed in apical endosomes of proximal tubular cells where it co-localizes with endocytosed proteins and the proton ATPase. ClC-5 may provide an electric shunt for the efficient operation of the electrogenic H(+)-ATPase. We confirmed this hypothesis by showing that endosomes from CLCN5 KO mice are acidified at a significantly lower rate than wild-type endosomes. This probably results in the drastic impairment of endocytosis observed in ClC-5 KO mice. Parathyroid hormone (PTH) is filtered into the lumen of the nephron, where it is endocytosed and degraded by proximal tubular cells. The defective endocytosis in ClC-5 KO mice entails an increased luminal concentration of PTH, subsequent stimulation of apical PTH receptors which causes an increased endocytosis of the phosphate transporter NaPi and phosphaturia. We now show that it also results in up-regulation of proximal tubular alpha-hydroxylase that generates the active form of vitamin D from its precursor. We discuss how the primary defect in endocytosis leads via secondary changes in calciotropic hormones to the tertiary symptoms hyperphosphaturia, hypercalciuria and kidney stones.

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

“…A very similar phenotype is observed with the loss of Ostm1, an essential ␤-subunit of ClC-7 (21). Despite the belief that vesicular CLCs are generally involved in the acidification of intracellular organelles (22)(23)(24), lysosomal pH was normal in these KO mouse models (16,20,21). This observation led to the hypothesis (5) that these phenotypes are rather a consequence of impaired proton gradient-driven Cl Ϫ accumulation in those vesicles, similar to impaired nitrate accumulation in plant vacuoles that lack AtClC-a (7).…”

“…These vesicular CLCs may provide counterion conductances for electrogenic proton pumping, thereby facilitating acidification of the respective compartment. Indeed, ClC-3, -4, and -5 have been shown to be required for proper acidification of endosomes (6,(22)(23)(24)41). The severe reduction in renal endocytosis observed with a loss of ClC-5 function may be attributed to reduced endosomal acidification (33) or Cl Ϫ accumulation (6).…”

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

“…4B). Because other vesicular CLC proteins facilitate vesicular acidification (5,6,8), we determined the lysosomal pH of cultured hippocampal neurons by ratiometric fluorescence imaging (see Fig. 10, which is published as supporting information on the PNAS web site).…”

“…In fact, ClC-4 and -5 function not as Cl Ϫ channels but as electrogenic Cl Ϫ ͞H ϩ exchangers (3,4). An impaired acidification of endosomes or synaptic vesicles was found in mice lacking ClC-3 (5, 6) or -5 (7,8). Probably as a consequence of defective endosomal acidification, the loss of ClC-5 leads to a stark reduction in proximal tubular endocytosis (7,9) and to human Dent's disease (10).…”

Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5’ UTR end of ClC-5 mRNA in human renal tissue and leukocytes

“…ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established (Gunther et al 1998;Devuyst et al 1999;Sakamoto et al 1999;Sayer and Simmons 2002). Animal models of knockout ClC-5 mice demonstrated that impairment of the endocytotic traffic is the major cause not only of tubular proteinuria but also of hypercalciuria and consequently kidney stones (Piwon et al 2000;Wang et al 2000;Gunther et al 2003). However, given the high interfamilial and intrafamilial phenotypic variability of DentÕs disease and its variants, it is conceivable that modifying genes could determine the phenotypic manifestation of the disease.…”