Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V 2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na ϩ -Cl Ϫ cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH 2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V 2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11␤-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V 2 receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT. antidiuretic hormone; distal convoluted tubule; phosphorylation; sodium-chloride cotransporter trafficking ANTIDIURETIC HORMONE [arginine vasopressin (AVP)] serves to control extracellular fluid homeostasis. The principal effect of AVP is found in the collecting duct where AVP increases water reabsorption. The prerequisite to ensure this function is the creation of a hypertonic interstitium via action of the thick ascending limb (TAL), which is also promoted by AVP. These epithelial effects of AVP are mediated by type 2 AVP receptors (V 2 R) (2, 29), whereas type 1a receptor signaling rather serves to limit the antidiuretic effects of AVP (31). Mapping receptorspecific probes to the tubular segments has revealed subtypeselective distribution along TAL, macula densa, distal convolutions, and collecting ducts (3, 28). In TAL, strong V 2 R expression has been observed, which agreed with increased abundance and phosphorylation of the furosemide-sensitive NaϪ cotransporter (NKCC2) and enhanced NaCl reabsorption in TAL in response to AVP (8, 13, 28). V 2 R signaling also activates epithelial Na ϩ channel (ENaC)-dependent Na ϩ reabsorption in collecting ducts (16); AVP-induced antinatriuresis in this segment has therefore been considered as a causal element in the context of salt-sensitive hype...

- ClϪ cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent. Am J Physiol Renal Physiol 295: F789 -F802, 2008. First published June 25, 2008 doi:10.1152/ajprenal.90227.2008.-Apical bumetanide-sensitive NaϪ cotransporter (NKCC2), the kidneyspecific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40 -70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na ϩ -Cl Ϫ cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86 Rb ϩ influx in Xenopus laevis oocytes was markedly reduced by methyl-␤-cyclodextrin (M␤CD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/M␤CD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

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