Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.

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The iodide transporter of the thyroid (NIS) has been cloned by the group of Carrasco. The NIS-mediated transport was studied by electrophysiological methods in NIS-expressing Xenopus oocytes. Using this method, the anion selectivity of NIS was different from that previously reported for thyroid cells, whereas perchlorate and perrhenate were found not transported. In this study we compared the properties of human NIS, stably transfected in COS-7 cells to those of the transport in a thyroid cell line, the FRTL5 cells, by measuring the transport directly. We measured the uptake of (125)I(-), (186)ReO(4)(-), and (99m)TcO(4)(-) and studied the effect on it of known competing anions, i.e. ClO(4)(-), SCN(-), ClO(3)(-), ReO(4)(-), and Br(-). We conclude that the properties of the NIS transporter account by themselves for the properties of the thyroid iodide transporter as described previously in thyroid slices. The order of affinity was: ClO(4)(-) > ReO(4)(-) > I(-) >/= SCN(-) > ClO(3)(-) > Br(-). NIS is also inhibited by dysidenin (as in dog thyroid).

RT-PCR analysis of isolated colonic crypts revealed P2Y 2 , P2Y 4 and P2Y 6 specific transcripts. The luminal UTP-stimulated K + secretion was still present in P2Y 2 receptor knock-out mice, but significantly reduced (∆V te : 0.83 ± 0.26 mV) compared to wild-type littermates (∆V te : 2.08 ± 0.52 mV, n = 9). In P2Y 4 receptor knock-out mice the UTP-induced K + secretion was similarly reduced. Luminal UTP-stimulated K + secretion was completely absent in P2Y 2 /P2Y 4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y 2 and P2Y 4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K + secretion.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited nephropathy. The development and enlargement of cysts in ADPKD requires tubular cell proliferation, abnormalities in the extracellular matrix and transepithelial fluid secretion. Multiple studies have suggested that fluid secretion across ADPKD cyst-lining cells is driven by the transepithelial secretion of chloride, mediated by the apical CFTR channel and specific basolateral transporters. The whole secretory process is stimulated by increased levels of cAMP in the cells, probably reflecting modifications in the intracellular calcium homeostasis and abnormal stimulation of the vasopressin V2 receptor. This review will focus on the pathophysiology of fluid secretion in ADPKD cysts, starting with classic, morphological and physiological studies that were followed by investigations of the molecular mechanisms involved and therapeutic trials targeting these pathways in cellular and animal models and ADPKD patients. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

The P2Y 4 receptor is responsive to UTP in human and to ATP and UTP in rodents. With the aim of identifying its pharmacotherapeutic interest, we generated P2Y 4 -null mice by a classic gene targeting method. The proportion of genotypes was consistent with X-linked Mendelian transmission. Gene inactivation was checked by the complete disappearance of P2Y 4 receptor mRNA from liver, stomach, and intestine. The P2Y 4 -null mice had a grossly normal behavior, growth, and reproduction. Chloride secretion by the jejunal epithelium was assessed in Ussing chambers by the measurement of the short circuit current in the presence of phlorizin. We show here that the UTP-and ATPinduced chloride secretory responses observed in wild-type mice are abolished in P2Y 4 -null mice. This is the first clearcut demonstration of a biological role of the P2Y 4 receptor.

The distal convoluted tubule (DCT) plays an essential role in the reabsorption of NaCl by the kidney, a process that can be inhibited by thiazide diuretics. Parvalbumin (PV), a Ca 2؉ -binding protein that plays a role in muscle fibers and neurons, is selectively expressed in the DCT, where its role remains unknown. We therefore investigated the renal phenotype of PV knockout mice (Pvalb ؊/؊ ) vs. wild-type (Pvalb ؉/؉ ) littermates. PV colocalized with the thiazidesensitive Na ؉ -Cl ؊ cotransporter (NCC) in the early DCT. The Pvalb ؊/؊ mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Acute furosemide administration increased diuresis and natriuresis/kaliuresis, but, surprisingly, did not increase calciuria in Pvalb ؊/؊ mice. NaCl supplementation of Pvalb ؊/؊ mice increased calciuria at baseline and after furosemide. The Pvalb ؊/؊ mice showed no significant diuretic response to hydrochlorothiazide, but an accentuated hypocalciuria. A decreased expression of NCC was detected in the early DCT of Pvalb ؊/؊ kidneys in the absence of ultrastructural and apoptotic changes. The PV-deficient mice had a positive Ca 2؉ balance and increased bone mineral density. Studies in mouse DCT cells showed that endogenous NCC expression is Ca 2؉ -dependent and can be modulated by the levels of PV expression. These results suggest that PV regulates the expression of NCC by modulating intracellular Ca 2؉ signaling in response to ATP in DCT cells. They also provide insights into the Ca 2؉ -sparing action of thiazides and the pathophysiology of distal tubulopathies.distal convoluted tubule ͉ kidney ͉ salt-losing nephropathy ͉ sodium-chloride cotransport P arvalbumin (PV) belongs to the superfamily of EF-hand Ca 2ϩ -binding proteins that play a role in multiple cellular processes, including gene transcription, ion transport, protein phosphorylation, and enzymatic activities (1). These proteins possess well conserved helix-loop-helix motifs that bind Ca 2ϩ ions with high affinity, leading to conformational changes. The conformational plasticity and the cell-specific expression of these Ca 2ϩ sensor or buffer proteins contribute to the versatility of Ca 2ϩ signaling (2). PV is a 109-aa cytosolic protein that contains a pair of functional EF-hand motifs forming a stable unit that binds two Ca 2ϩ ions (3). This Ca 2ϩ buffer is expressed in a restricted number of vertebrate tissues, including fast-contracting/relaxing skeletal muscle fibers and GABA neurons in the brain (4). The generation of PV knockout (Pvalb Ϫ/Ϫ ) mice confirmed the important role played by PV in muscle and brain (5). The fast muscles of Pvalb Ϫ/Ϫ mice exhibit a decreased relaxation rate of the twitch (5), suggesting that PV facilitates Ca 2ϩ diffusion from myofibrils to the sarcoplasmic reticulum (6). The lack of PV in the brain induces changes in short-term synaptic plasticity and modified network properties, resulting in increased susceptibility to epileptic seizures (7). Although no human disease is ass...

The present data support the involvement of AA in the early PT dysfunction found in CHN; furthermore, they suggest a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptor-mediated endocytosis of LMWP.