Concentrating urine is mandatory for most mammals to prevent water loss from the body. Concentrated urine is produced in response to vasopressin by the transepithelial recovery of water from the lumen of the kidney collecting tubule through highly water-permeable membranes. In this nephron segment, vasopressin regulates water permeability by endo- and exocytosis of water channels from or to the apical membrane. CHIP28 is a water channel in red blood cells and the kidney proximal tubule, but it is not expressed in the collecting tubule. Here we report the cloning of the complementary DNA for WCH-CD, a water channel of the apical membrane of the kidney collecting tubule. WCH-CD is 42% identical in amino-acid sequence to CHIP28. WCH-CD transcripts are detected only in the collecting tubule of the kidney. Immunohistochemically, WCH-CD is localized to the apical region of the kidney collecting tubule cells. Expression of WCH-CD in Xenopus oocytes markedly increases osmotic water permeability. The functional expression and the limited localization of WCH-CD to the apical region of the kidney collecting tubule suggest that WCH-CD is the vasopressin-regulated water channel.

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance. WNK2 kinases (with no lysine (K)) comprise a family of novel serine/ threonine protein kinases conserved among multicellular organisms (1, 2). The kinase domain of this family is unique in that it lacks the conserved lysine residue previously known to be important for ATP binding in the catalytic site. A conserved lysine in subdomain I of the WNK kinases is thought to be essential for their catalytic activity (1, 3). There are four human WNK family members. WNK1 and WNK4 were identified as genes mutated in families of patients with pseudohypoaldosteronism type 2 (PHA II) human hypertension (4). The WNK1 gene mutation consists of a deletion within its first intron, leading to increased expression, whereas mutations in the WNK4 gene are found in the coding sequence near the coiled-coil domains.PHA II patients are treated by thiazide diuretics, which function as antagonists of the Na-Cl cotransporter (NCC, also known as thiazidesensitive cotransporter (TSC) or Na-Cl transporter (NCCT)), suggesting that the activity of NCC could be potentially involved in the development of PHA II. Previous studies using Xenopus oocytes have showed that wild-type WNK4 inhibits the surface expression and the activity of NCC, whereas one of the disease-causing mutants of WNK4 attenuated this inhibitory effect (5, 6). However, comparison of wild-type and mutant WNK4 revealed no differences in NCC surface expression in polarized epithelial cells (MDCK II cells), suggesting that the regulation of intracellular NCC localization by WNK4 might be unrelated to the pathogenesis of PHA II (7). WNK4 has ...

Water transport in highly water-permeable membranes is conducted by water-selective pores-namely, water channels. The recent cloning of water channels revealed the water-selective characteristics of these proteins when expressed in Xenopus oocytes or reconstituted in liposomes. Currently, It is as d that the function of water ch ls is to transport only water. We now report the cloning of a member of the water channel that also transports nonionic small molecules such as urea and glycerol. We named this channel aquaporin 3 (AQP3) for its predominant water permeabilit. AQP3 has amino acid sequence Identity with major intrinsic protein (MIP) family proteins including AQPchannel-forming integral membrane protein, AQP-colecting duct, MIP, AQP-ytonoplast intrinsic protein, nodulin 26, and glycerol facilitator (33-42%). Thus, AQP3 is an additional member of the MEP family. Osmotic water permeability of Xenopus oocytes measured by videomicroscopy was 10-fold higher in oocytes injected with AQP3 transcript than with water-injected oocytes. The increase in osmotic water permeability was inhibited by HgC12, and this effect was reversed by a reducing agent, 2-mercaptoethanol. Although to a smaller degree, AQP3 also facilitated the transport of nonionic small solutes such as urea and glycerol, while the previously cloned water channels are permeable only to water when expressed in Xenopus oocytes. AQP3 mRNA was expressed abundantly in kidney medulla and colon. In kidney, it was exclusively immunokoalzed at the baolateral membrane of collecting duct cells. AQP3 may functon as a water and urea exit hanism in antidlure in ollecting duct cells.Water channels have been postulated for the pathway of selective water permeation in highly water-permeable membranes. Recent

Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans. SPAK interacts with WNK kinases to regulate the Na ϩ -K ϩ -2Cl Ϫ and NaMutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans, but the physiologic role of SPAK in vivo is unknown. We generated and analyzed SPAK-null mice by targeting disruption of exons 9 and 10 of SPAK. Compared with SPAK ϩ/ϩ littermates, SPAK ϩ/Ϫ mice exhibited hypotension without significant electrolyte abnormalities, and SPAK Ϫ/Ϫ mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia, hypomagnesemia, and hypocalciuria. In the kidney tissues of SPAK Ϫ/Ϫ mice, the expression of total and phosphorylated (p-)NCC was markedly decreased, but that of p-OSR1, total NKCC2, and p-NKCC2 was significantly increased. We observed a blunted response to thiazide but normal response to furosemide in SPAK Ϫ/Ϫ mice. In aortic tissues, total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice. Both SPAK ϩ/Ϫ and SPAK Ϫ/Ϫ mice had impaired responses to the selective ␣ 1 -adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide, suggesting that impaired aortic contractility may contribute to the hypotension of SPAKnull mice. In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation. SPAK may be a promising target for antihypertensive therapy. 21: 186821: -187721: , 201021: . doi: 10.1681 Sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) 1,2 and oxidative stress-responsive kinase 1 (OSR1) 3 are serine/threonine kinases that share high homology in both their N-terminal catalytic and Cterminal regulatory domains and are widely distributed in the brain, pancreas, heart and kidney. Gene mutations of the NCC in the distal convoluted tubules (DCTs) and NKCC2 in the thick ascending limb of the loop of Henle (TAL) cause autosomal recessive Gitelman syndrome (GS) 12 and Bartter syndrome (BS), 13 respectively. These congenital renal tubular disorders are characterized by J Am Soc Nephrol

Stressful events during adulthood are potent adverse environmental factors that can predispose individuals to psychiatric disorders, including depression; however, many individuals exposed to stressful events can adapt and function normally. While stress vulnerability may influence depression, the molecular mechanisms underlying the susceptibility and adaptation to chronic stress within the brain are poorly understood. In this study, two genetically distinct mouse strains that exhibit different behavioral responses to chronic stress were used to demonstrate how the differential epigenetic status of the glial cell-derived neurotrophic factor (Gdnf) gene in the ventral striatum modulates susceptibility and adaptation to chronic stress. Our results suggest that the histone modifications and DNA methylation of the Gdnf promoter have crucial roles in the control of behavioral responses to chronic stress. Our data provide insights into these mechanisms, suggesting that epigenetic modifications of Gdnf, along with genetic and environmental factors, contribute to behavioral responses to stress.

WNK1 and WNK4 mutations have been reported to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. To elucidate the molecular pathophysiology of PHAII, we generated Wnk4(D561A/+) knockin mice presenting the phenotypes of PHAII. The knockin mice showed increased apical expression of phosphorylated Na-Cl cotransporter (NCC) in the distal convoluted tubules. Increased phosphorylation of the kinases OSR1 and SPAK was also observed in the knockin mice. Apical localization of the ROMK potassium channel and transepithelial Cl(-) permeability in the cortical collecting ducts were not affected in the knockin mice, whereas activity of epithelial Na(+) channels (ENaC) was increased. This increase, however, was not evident after hydrochlorothiazide treatment, suggesting that the regulation of ENaC was not a genetic but a secondary effect. Thus, the pathogenesis of PHAII caused by a missense mutation of WNK4 was identified to be increased function of NCC through activation of the OSR1/SPAK-NCC phosphorylation cascade.

Pseudohypoaldosteronism type II (PHAII) is caused by mutations in the WNK1 and WNK4 genes (WNK with-no-lysine kinase). In a mouse model of this disease where a mutant of Wnk4 D561A was knocked in, increased phosphorylation of the sodium chloride cotransporter (NCC) was found and the transporter was concentrated on the apical membrane of the distal tubules. In addition, we recently found that other kinases, such as the oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK), also showed increased phosphorylation in these mice. Here we determined whether this kinase cascade is regulated by dietary salt intake. We found that the phosphorylation states of NCC and OSR1/SPAK were increased by low-salt diets and decreased by high-salt diets; a regulation completely lost in the knock-in mice. Increased phosphorylation was reversed by spironolactone and this decreased phosphorylation was reversed by administration of exogenous aldosterone. These studies suggest that that the WNK-OSR1/SPAK-NCC cascade may be a novel effector system of aldosterone action in the kidney.

Vallon V, Schroth J, Lang F, Kuhl D, Uchida S. Expression and phosphorylation of the Na ϩ -Cl Ϫ cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1.