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[K.sup.+] channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three [K.sup.+] channels with conductances of ~75, 40, and 20 pS were observed, but the [K.sup.+] channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an [Mg.sup.2+]-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal [Mg.sup.2+] had no influence on the inward conductance ([G.sub.in] = 35 pS) but reduced outward conductance ([G.sub.out]) to 13 pS, yielding a [G.sub.in]/[G.sub.out] of 3.2. The polycation spermine (6 x [10.sup.-7] M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH ([pH.sub.i]: a sigmoid relationship between [pH.sub.i] and channel normalized current ([NP.sub.o]) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying [K.sup.+] (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary [K.sup.+] had no influence on the properties of the intermediate-conductance channel, but a [Na.sup.+]-depleted diet increased its open probability by ~25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the [K.sup.+] conductance in the basolateral membrane of mouse CCD principal cells. kidney; patch clamp

To unravel the molecular regulation of renal transcellular [Ca.sup.2+] transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive [Na.sup.+]/[Cl.sup.-] cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the [Ca.sup.2+] channels TRPV5 and TRPV6 and other key players necessary for transcellular [Ca.sup.2+] transport, i.e., calbindin-[D.sub.9k], calbindin-[D.sub.28k], plasma membrane [Ca.sup.2+]-ATPase isoform lb, and [Na.sup.+]/[Ca.sup.2+] exchanger 1. Primary cultures of DCT cells exhibited net transcellular [Ca.sup.2+] transport of 0.4 [+ or -] 0.1 nmol x [h.sup.-1] x [cm.sup.-2], whereas net transeellular [Ca.sup.2+] transport across mpkDCT cells was significantly higher at 2.4 [+ or -] 0.4 nmol x [h.sup.-1] x [cm.sup.-2]. Transcellular [Ca.sup.2+] transport across mpkDCT cells was completely inhibited by mthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated [Ca.sup.2+] channel inhibitors felodipine and verapamil. With the use of patchclamp analysis, the I[C.sub.50] of ruthenium red on [Na.sup.+] currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin [D.sub.3], and 1-deamino-8-D-arginine vasopressin increased transcellular [Ca.sup.2+] transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular [Ca.sup.2+] transport in the kidney in vitro. TRPVS; TRPV6; vitamin D3; kidney; calcium reabsorption

The intrarenal expression and distribution of rat ClC-K1 and ClC-K2 chloride channel genes were analyzed via reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry using ClC-K1 and ClC-K2 antiserum. RT-PCR and immunocytochemical analysis indicated the expression of ClC-K1 and ClC-K2 chloride channel genes in the distal segments of rat renal tubules. Furthermore, the products of the genes were localized on the basolateral side of the cells from the medullary and cortical ascending limbs of the Henle's loop, distal tubules and collecting ducts.

The expression of the SV40 large T-antigen in the intestinal cells of transgenic mice establishes the m-IC(sub c12) intestinal cell line. These cells retain the main features of crypt cells involved in anti-microbial defence and form monolayers of cuboid cells separated by tight cells. The m-IC(sub c12) cells possess intracellular sucrase isomaltase, the expression of polymeric immunoglobulin receptor and the cystic fibrosis transmembrane conductance regulator gene. These cells help in the analysis of in vitro dimeric immunoglobulin A transport.

A study of two putative chloride channels of the ClC chloride channel family in both rat and human kidney reveals high similarity among all the channels. Recognition of glycosylation-occurring sites leads to a reconsideration of the transmembrane topology model for ClC channels. The high similarity within the ClC-K subfamily facilitates the generation of heterooligomers with enhanced functional features.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-v-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(-/-)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of [Na.sup.+] assessed by the short-circuit current ([I.sub.SC]) method in both CFTR(+/+) and CFTR(-/-) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in [I.sub.SC] induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter [I.sub.SC] in BAm-treated CFTR(-/-) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of [sup.36][Cl.sup.-] in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal [sup.36][Cl.sup.-] flux in CFTR(-/-) CCDs but not the basal-to-apical [sup.36][Cl.sup.-] flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted [Cl.sup.-] in mouse CCD. cyclic adenosine monophosphate; kidney; epithelial sodium channel; cystic fibrosis transmembrane conductance regulator; cystic fibrosis

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpk[CCD.sub.cl4]) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P [is less than] 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCC[D.sub.cl4] cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-[Alpha] (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Tissue distribution and subcellular localization of the ClC-5 chloride channel in rat intestinal cells. Am J Physiol Cell Physiol 280: C373-C381, 2001.--ClC-5 is the [Cl.sup.-] channel that is mutated in Dent's disease, an X-chromosome-linked disease characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this [Cl.sup.-] channel could also be expressed in intestinal tissues that have endocytotic machinery. ClC-5 mRNA was detected in the rat duodenum, jejunum, ileum, and colon. Western blot analyses revealed the presence of the 83-kDa ClC-5 protein in these tissues. Indirect immunofluorescence studies showed that ClC-5 was mainly concentrated in the cytoplasm above the nuclei of enterocytes and colon cells. ClC-5 partially colocalized with the transcytosed polymeric immunoglobulin receptor but was not detectable together with the brush-border-anchored sucrase isomaltase. A subfractionation of vesicles obtained by differential centrifugation showed that ClC-5 is associated with the vacuolar 70-kDa [H.sup.+] -ATPase and the small GTPases rab4 and rab5a, two markers of early endosomes. Thus these results indicate that ClC-5 is present in the small intestine and colon of rats and suggest that it plays a role in the endocytotic pathways of intestinal cells. intestine; endocytosis; [H.sup.+]-ATPase; tab