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Comparative ontogeny, processing, and segmental distribution of the renal chloride channel, ClC-5. Background. The renal chloride channel ClC-5, which is responsible for Dent's disease, is coexpressed with the vacuolar H+-ATPase in proximal tubules (PT) and α-type intercalated cells (IC) of the mature kidney. Neonatal cases of Dent's disease suggest that ClC-5 distribution must be acquired before birth. However, the ontogeny of ClC-5, and its processing and segmental distribution with respect to related proteins during nephrogenesis remain unknown. Methods. Immunoblotting, real-time polymerase chain reaction (RT-PCR), immunostaining, and deglycosylation studies were used to investigate the expression, distribution, and maturation of ClC-5 during mouse and human nephrogenesis, in comparison with H+-ATPase, type II carbonic anhydrase (CAII), and aquaporin-1 (AQP1). Results. An early induction (E13.5-E14.5) of ClC-5 was observed in mouse kidney, with persistence at high levels through late nephrogenesis. This pattern contrasted with the progressive expression of H+-ATPase and AQP1, and the postnatal upregulation of CAII. Immunostaining showed expression of ClC-5 in ureteric buds and, from E14.5, its location in developing PT. From E15.5, ClC-5 codistributed with H+-ATPase in PT cells and α-type IC. In the human kidney, ClC-5 was detected from 12 gestation weeks; its distribution was similar to that observed in mouse, except for a later detection in IC. Although mouse and human ClC-5 proteins are glycosylated, biochemical differences between fetal and adult proteins were observed in both species. Conclusion. The segmental expression of ClC-5 and H+-ATPase is essentially achieved during early nephrogenesis, in parallel with the onset of glomerular filtration. These data give insight into PT and IC maturation, and explain early phenotypic variants of Dent's disease. [ABSTRACT FROM AUTHOR]

Altered polarity and expression of H+ -ATPase without ultrastructural changes in kidneys of Dent's disease patients. Background. Dent's disease is a proximal tubule (PT) disorder characterized by low-molecular-weight proteinuria (LWMP) that may be associated with hypercalciuria, nephrocalcinosis, and renal failure. It is caused by inactivating mutations of the renal chloride channel ClC-5, which colocalizes with the vacuolar H+ -ATPase in PT cells and α-type intercalated cells. Examinations of knockout mice have established the role of ClC-5 in PT endocytosis, but the consequences of ClC-5 mutations on the polarity of H+ -ATPase and other plasma membrane proteins remain unknown. Methods. We have studied renal biopsies from eight patients with Dent's disease, due to inactivating ClC-5 mutations, by light and electron microscopy, and by immunohistochemical staining. All patients exhibited LMWP, and renal function ranged from normal to end-stage renal failure. Results. Light microscopy revealed either normal renal architecture or glomerulosclerosis, tubular dedifferentiation and atrophy, and mild interstitial fibrosis. Focal, hyaline casts, sometimes calcified, were identified at all stages. Electron microscopy did not reveal any ultrastructural abnormalities in PT cells, and the endocytic apparatus was apparently normal. However, immunohistochemical studies demonstrated a consistent inversion of H+ -ATPase polarity in PT cells to a basolateral distribution contrasting with its apical location in the normal kidney. This inversion of polarity was specific for H+ -ATPase and did not affect distribution of aminopeptidase, megalin, and Na+ /K+ -ATPase. Furthermore, apical H+ -ATPase expression was absent in α-type intercalated cells. Conclusion. ClC-5 mutations are associated with modifications in the polarity and expression of H+ -ATPase, but not... [ABSTRACT FROM AUTHOR]

Functional characterization of renal chloride channel, CLCN5, mutations associated with Dent’s Japan disease. Background. The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis, and this represents a variant of Dent's disease. Hitherto, 12 mutations of the X-linked renal specific chloride channel, CLCN5, have been reported in the Dent’sJapan variant. To further identify such CLCN5 mutations and to define the structure-function relationships of this channel, we have investigated five unrelated, non-consanguinous Japanese families with this disorder. Methods. Leukocyte DNA from probands was used with CLCN5 primers for PCR amplification of the coding region, and the DNA sequences of the products determined. Functional studies were performed by expressing the mutants in Xenopus oocytes. Results. Five CLCN5 mutations consisting of two nonsense (R648X and R704X), two missense (S270R and L278F) and one acceptor splice site mutation (ag→cg) in intron 4 were identified. The missense and splice site mutations represent novel abnormalities. Heterologous expression in Xenopus oocytes of wild-type and the missense mutants demonstrated that the mutations, which were translated, either abolished or markedly reduced chloride conductance. Conclusions. These results expand the spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For example, both the missense mutations are located within a short putative loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation of channel activity. [ABSTRACT FROM AUTHOR]