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Apical bumetanide-sensitive [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), the kidney-specific 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.sup.+]-[Cl.sup.-] 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 [sup.86][Rb.sup.+] influx in Xenopus laevis oocytes was markedly reduced by methyl-[beta]-cyclodextrin (M[beta]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[beta]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. thick ascending limb; lipid raft; Xenopus oocyte; cholesterol depletion

Hypokalemia contributes to the progression of chronic kidney disease, although a definitive pathophysiological theory to explain this remains to be established. K+ deficiency results in profound alterations in renal epithelial transport. These include an increase in salt reabsorption via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes electroneutral K+ loss in downstream nephron segments. In experimental conditions of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases have been discovered in the murine DCT and termed "WNK bodies," referring to their component, with no K (lysine) kinases (WNKs). We hypothesized that in humans, WNK bodies occur in hypokalemia as well. Renal needle biopsies of patients with chronic hypokalemic nephropathy and appropriate controls were examined by histological stains and immunofluorescence. Segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely, WNKs and Ste20-related proline-alanine-rich kinase (SPAK). In both patients with hypokalemia, WNKs and SPAK concentrated in nonmembrane- bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region close to, but not within, the endoplasmic reticulum. They were closely adjacent to microtubules but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients. [ABSTRACT FROM AUTHOR]

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 μM 14,15- EET for 30 min reduced phosphorylation of NKCC2 at the AVPsensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis. [ABSTRACT FROM AUTHOR]

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gsmediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na+-K+-2Cl--cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissuespecific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications. [ABSTRACT FROM AUTHOR]

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport. [ABSTRACT FROM AUTHOR]

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization. [ABSTRACT FROM AUTHOR]

Apical bumetanide-sensitive Na+-K+-2C1- cotransporter (NKCC2), the kidney-specific 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+-Cl- 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 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-β-cyclodextrin (MβCD)-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/MβCD-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. [ABSTRACT FROM AUTHOR]

The Tamm-Horsfall protein (THP; uromodulin), the dominant protein in normal urine, is produced exclusively in the thick ascending limb of Henle's loop. THP mutations are associated with disease; however, the physiological role of THP remains obscure. We generated THP gene-deficient mice (THP -/-) and compared them with wild-type (WT) mice. THP -/- mice displayed anatomically normal kidneys. Steady-state electrolyte handling was not different between strains. Creatinine clearance was 63% lower in THP -/- than in WT mice (P < 0.05). Sucrose loading induced no changes between strains. However, water deprivation for 24 h decreased urine volume from 58 ± 9 to 28 ± 4 μ1· g body wt-1 ·24 h-1 in WT mice (P < 0.05), whereas in THP -/- mice this decrease was less pronounced (57 ± 4 to 41 ± 5 μl·g body wt-1 · 24 h-l; P < 0.05), revealing significant interstrain difference (P < 0.05). We further used RT-PCR, Northern and Western blotting, and histochemistry to study renal transporters, channels, and regulatory systems under steady-state conditions. We found that major distal transporters were upregulated in THP -/- mice, whereas juxtaglomerular immunoreactive cyclooxygenase-2 (COX-2) and renin mRNA expression were both decreased in THP -/- compared with WT mice. These observations suggest that THP influences transporters in Henle's loop. The decreased COX-2 and renin levels may be related to an altered tubular salt load at the macula densa, whereas the increased expression of distal transporters may reflect compensatory mechanisms. Our data raise the hypothesis that THP plays an important regulatory role in the kidney. [ABSTRACT FROM AUTHOR]

K + deficiency stimulates renal salt reuptake via the Na + -Cl - cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K + losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K + deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed "WNK bodies," the significance of which is undetermined. By feeding diets of varying salt and K + content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K + deprivation, and it was promptly suppressed by K + loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K + channel-forming protein Kir4.1, which is required for the DCT to sense plasma K + concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K + deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K + concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.