Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.

Loss-of-function mutations of the ClC-5 chloride channel lead to Dent's disease, a syndrome characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. We show that ClC-5 is expressed in renal proximal tubule cells, which normally endocytose proteins passing the glomerular filter. Expression is highest below the brush border in a region densely packed with endocytotic vesicles, where ClC-5 colocalizes with the H ؉ -ATPase and with internalized proteins early after uptake. In intercalated cells of the collecting duct it again localizes to apical intracellular vesicles and colocalizes with the proton pump in ␣-intercalated cells. In transfected cells, ClC-5 colocalizes with endocytosed ␣ 2 -macroglobulin. Cotransfection with a GTPasedeficient rab5 mutant leads to enlarged early endosomes that stain for ClC-5. We suggest that ClC-5 may be essential for proximal tubular endocytosis by providing an electrical shunt necessary for the efficient acidification of vesicles in the endocytotic pathway, explaining the proteinuria observed in Dent's disease.

The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane 2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their ion selectivity and conductance properties.

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.