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Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid hormone secretion depends on apical endocytosis of thyroglobulin (Tg), we investigated whether ClC-5 is expressed in the thyroid and affects its function, using Clcn5-deficient knockout (KO) mice. We found that ClC-5 is highly expressed in wild-type mouse thyroid ( approximately 40% of mRNA kidney level). The protein was immunolocalized at the apical pole of thyrocytes. In Percoll gradients, ClC-5 overlapped with plasma membrane and early endosome markers, but best codistributed with the late endosomal marker, Rab7. ClC-5 KO mice were euthyroid (normal T4 and TSH serum levels) but developed a goiter with parallel iodine and Tg accumulation (i.e. normal Tg iodination level). When comparing ClC-5 KO with wild-type mice, thyroid 125I uptake after 1 h was doubled, incorporation into Tg was decreased by approximately 2-fold, so that trichloroacetic acid-soluble 125I increased approximately 4-fold. Enhanced 125I- efflux upon perchlorate and presence of 125I-Tg as autoradiographic rings at follicle periphery demonstrated delayed iodide organification. Endocytic trafficking of 125I-Tg toward lysosomes was not inhibited. Expression of pendrin, an I-/Cl- exchanger involved in apical iodide efflux, was selectively decreased by 60% in KO mice at mRNA and protein levels. Thus, ClC-5 is well expressed in the thyroid but is not critical for apical endocytosis, contrary to the kidney. Instead, the goiter associated with ClC-5 KO results from impaired rate of apical iodide efflux by down-regulation of pendrin expression.

The vacuolar H(+)-ATPase (V-ATPase) is a ubiquitous multisubunit pump that is responsible for acidification of intracellular organelles. In the kidney, a particular form of V-ATPase, made of specific subunits isoforms, has been located at the plasma membrane of intercalated cells (IC). Mutations in genes encoding IC-specific subunits cause infant distal renal tubular acidosis (dRTA), suggesting that the segmental distribution of these subunits is acquired at birth or during early infancy. However, the comparative ontogeny of the IC-specific versus the ubiquitous subunits of V-ATPase and the mechanisms involved in their segmental expression remain unknown. Real-time reverse transcription-PCR, in situ hybridization, immunoblotting, immunostaining, and subcellular fractionation analyses characterized the expression and distribution of V-ATPase subunits, transcription factors, and differentiation markers during mouse nephrogenesis. Ubiquitous A, E1, B2, G1, and C1 subunits showed an early (embryonic day 13.5 [E13.5]) and stable expression throughout nephrogenesis, followed by a slight increase around birth. The developmental pattern of a1 was bimodal, with early induction, gradual decrease during organogenesis, and neonatal increase. These patterns contrasted with the later (from E15.5) and progressive expression of IC-specific a4, B1, G3, and C2 subunits, after the induction of the forkhead transcription factor Foxi1. From E15.5, Foxi1 mRNA was detected in IC, where it co-distributed with B1 in late nephrogenesis. Immunostaining showed that the distribution of ubiquitous E1 and B2 was acquired from E15.5, whereas a4 was located in IC during late nephrogenesis. Subcellular fractionation showed that in both fetal and mature (cortex and medulla) kidneys, E1 and a4 were located in endosomes. These data demonstrate a differential expression and a coordinate regulation of IC-specific versus ubiquitous V-ATPase subunits during nephrogenesis. They provide new insights into the complex regulation of V-ATPase subunits, the maturation of IC along the nephron, and the pathophysiology of hereditary dRTA.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Dent's disease is a hereditary renal tubular disorder characterized by low-molecular weight (LMW) proteinuria, hypercalciuria and nephrolithiasis. The disease is due to mutations of ClC-5, a member of the family of voltage-gated CLC chloride channels. ClC-5 is expressed in part in cells lining the proximal tubule (PT) of the kidney, where it colocalizes with albumin-containing endocytic vesicles belonging to the receptor-mediated endocytic pathway that ensures efficient reabsorption of ultrafiltrated LMW proteins. Since progression along the endocytic apparatus requires endosomal acidification, it has been suggested that dysfunction of ClC-5 in endosomes may lead to inefficient reabsorption of LMW proteins and dysfunction of PT cells. Analysis of a ClC-5 knockout (KO) mouse model, displaying all the characteristic renal tubular defects of Dent's disease, showed evidence of a severe LMW proteinuria. Cytochemical studies with the endocytic tracer, peroxidase, showed poor transfer into early endocytic vesicles, suggesting that impairment of receptor-mediated endocytosis in PT cells is the basis for the defective uptake of LMW proteins in patients with Dent's disease. Endocytosis and processing of LMW proteins involve the multiligand tandem receptors, megalin and cubilin, that are abundantly expressed at the brush border of PT cells. Characterization of the endocytic defect in ClC-5 KO mice revealed that ligands of both megalin and cubilin were affected. The total kidney content of megalin and especially cubilin at the protein level was decreased but, more importantly, using analytical subcellular fractionation and quantitative immunogold labelling we demonstrated a selective disappearance of megalin and cubilin at the brush border of PT cells. These observations allowed us to conclude that defective protein endocytosis linked to ClC-5 inactivation is due at least in part to a major and selective loss of megalin and cubilin at the brush border, reflecting a trafficking defect in renal PT cells. These results improve our understanding of Dent's disease, taken as a paradigm for renal Fanconi syndrome and nephrolithiasis, and demonstrate multiple roles for ClC-5 in the kidney. These studies also provided insights into important functions such as apical endocytosis, handling of proteins by renal tubular cells, calcium metabolism, and urinary acidification., (Copyright (c) 2005 S. Karger AG, Basel.)

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 alpha-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 alpha-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.