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.

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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

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

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow lightscattering method revealed that the osmotic water permeability (P f ) of LLC-PK1 cells transfected with WT was 69.6 ؎ 6.5 m/s (24.8 ؎ 2.2 m/s for mock-transfected), and stimulation by 500 M 8-(4-chlorophenylthio)-cAMP increased the P f by 85 ؎ 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the P f was only 8 ؎ 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 ؎ 10%, significantly less than that for WT (88 ؎ 5%). In addition, an in vivo [ 32 P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.The urine concentration system in the kidney is regulated mainly by vasopressin, an antidiuretic hormone that increases the osmotic water permeability of the collecting duct cells, resulting in bulk reabsorption of free water (1-3). The cellular actions of vasopressin in the collecting duct cell have been partially resolved since the identification of the vasopressin water channel aquaporin-2 (AQP2) 1 (4, 5). Vasopressin has been shown to induce regulatory redistribution of AQP2 from endosomal compartments to the apical membrane (6 -8). These observations have directly proved the shuttle hypothesis that exo-and endocytosis of water channel-containing vesicles account for the regulatory effects of vasopressin on water permeability of the collecting duct cells (9, 10).Despite elucidation of the regulatory translocation of AQP2, the potential interactions between vasopressin-induced intracellular cAMP accumulation and AQP2 trafficking remain unknown. It has been shown that cAMP-dependent phosphorylation of AQP2 expressed in the Xenopus oocyte slightly increased osmotic water permeability without changing AQP2 surface expression (11), but the increase was far too small to account for the dramatic increase in water permeability of collecting duct cells. Many channels have been shown to be functionally regulated by their phosphorylation (12), but the direct regulation of AQP2 channel functions through phosphorylation is minimal at best and is still controversial (13). On the other hand, the physiological significance of regulatory effects on AQP2 trafficking has been emphasized. Regulatory delivery and sorting of membrane proteins have been observed in many cell types (14, 15). Many vesicle-associated...

Asthma and atopy show epidemiological association and are biologically linked by T-helper type 2 (T(h)2) cytokine-driven inflammatory mechanisms. IL-4 operates through the IL-4 receptor (IL-4R, a heterodimer of IL-4Ralpha and either gammac or IL-13Ralpha1) and IL-13 operates through IL-13R (a heterodimer of IL-4Ralpha and IL-13Ralpha1) to promote IgE synthesis and IgE-based mucosal inflammation which typify atopy. Recent animal model data suggest that IL-13 is a central cytokine in promoting asthma, through the stimulation of bronchial epithelial mucus secretion and smooth muscle hyper-reactivity. We investigated the role of common genetic variants of IL-13 and IL-13Ralpha1 in human asthma, considering IgE levels. A novel variant of human IL-13, Gln110Arg, on chromosome 5q31, associated with asthma rather than IgE levels in case-control populations from Britain and Japan [peak odds ratio (OR) = 2.31, 95% CI 1.33-4.00]; the variant also predicted asthma and higher serum IL-13 levels in a general, Japanese paediatric population. Immunohistochemistry demonstrated that both subunits of IL-13R are prominently expressed in bronchial epithelium and smooth muscle from asthmatic subjects. Detailed molecular modelling analyses indicate that residue 110 of IL-13, the site of the charge-modifying variants Arg and Gln, is important in the internal constitution of the ligand and crucial in ligand-receptor interaction. A non-coding variant of IL-13Ralpha1, A1398G, on chromosome Xq13, associated primarily with high IgE levels (OR = 3. 38 in males, 1.10 in females) rather than asthma. Thus, certain variants of IL-13 signalling are likely to be important promoters of human asthma; detailed functional analysis of their actions is needed.

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.

Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.Aquaporins (AQPs) are a family of membrane proteins that facilitate the transport of water and small solutes (8,15,21). They are distributed widely in nature from bacteria to animals. Eleven aquaporins (AQP0 to AQP10) have been identified and functionally characterized in humans. We reported the most recent AQP, AQP10 (11, 13). Their physiological importance is documented by the targeted disruption in mice (knockout mice) and by the discovery of humans and mice with nonfunctioning mutations. Of nine AQPs disrupted in mice and humans (AQP0 to AQP7 and AQP10), only AQP2-null mice die due to massive polyuria from nephrogenic diabetes insipidus (23). The milder phenotypes in AQP disruptions in general are surprising, since water is vital for organisms. Therefore, AQPs seem to be not critically essential for the survival of mammals but seem to be involved in the quality of their lives.The completion of human genome projects has revealed two more aquaporin-like genes, which we have deposited in GenBank under the names of AQPX1 and AQPX2 (9). They are renamed AQP11 and AQP12 with the approval of the Human Gene Nomenclature Committee. Rat AQP11 (AQPX1) is highly expressed in the testis and moderately expressed in the kidney, liver, and brain. On the other hand, rat AQP12 (AQPX2) is selectively expressed in the pancreas. They share similar genome structures with three exons, which are distinct from other AQPs: AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6 have four exons; AQP3, AQP7, AQP8, AQP9, and AQP10 have six exons. In humans, AQP11 is mapped to chromosome 11q14 and AQP12 to chromosome 2q34-37, to which no diseases have been mapped. Moreover, we were not able to express them functionally in Xenopus oocytes. Therefore, their functions and physiological significance remain to be clarified.Previous AQPs have two highly conserved, short sequences named NPA (asparagine-proline-alanine) boxes. Each one of the NPA boxes has ...