LRBA signalosomes activate vasopressin-induced AQP2 trafficking at recycling endosomes.

Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba-/- mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.

LRBA is essential for urinary concentration and body water homeostasis.

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells.

We previously showed that in polarized Madin-Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.

The proteasome inhibitor bortezomib attenuates renal fibrosis in mice via the suppression of TGF-ß1.

Kidney fibrosis and fibrogenesis significantly exacerbate chronic kidney disease (CKD) progression and are essential therapeutic targets. Bortezomib (BZM) is a proteasome inhibitor used for the treatment of multiple myeloma (MM). Several studies have demonstrated that BZM attenuates renal impairment in patients with MM, although this effect is generally considered to be the result of MM remission. Recently, several studies on BZM reported anti-fibrotic effects on liver and skin in experimental animal models. However, its effect on renal fibrosis has yet to be examined. Here, we investigated the anti-fibrotic effects of BZM in an experimental mouse model of fibrosis that uses aristolochic acid I (AA). Ten weeks of AA administration with BZM treatment twice a week significantly attenuated AA-induced renal dysfunction and albuminuria, reduced the expression of renal fibrosis-related proteins and kidney injury markers, such as αSMA, Kim1, and Ngal, and prevented renal fibrosis at the level of histopathology. Furthermore, pathological activation of TGFß1-Smad3 signaling and apoptosis, essential pathophysiological causes of AA-induced nephropathy (AAN), were ameliorated by BZM, suggesting this mechanism may be involved in improving fibrosis in AAN. In conclusion, BZM directly inhibits renal fibrosis in CKD via suppression of TGFß1-Smad3 signaling and is promising in terms of drug repositioning.

Ezrin directly interacts with AQP2 and promotes its endocytosis.

The water channel aquaporin-2 (AQP2) is a major regulator of water homeostasis in response to vasopressin (VP). Dynamic trafficking of AQP2 relies on its close interaction with trafficking machinery proteins and the actin cytoskeleton. Here, we report the identification of ezrin, an actin-binding protein from the ezrin/radixin/moesin (ERM) family as an AQP2-interacting protein. Ezrin was first detected in a co-immunoprecipitation (co-IP) complex using an anti-AQP2 antibody in a proteomic analysis. Immunofluorescence staining revealed the co-expression of ezrin and AQP2 in collecting duct principal cells, and VP treatment caused redistribution of both proteins to the apical membrane. The ezrin-AQP2 interaction was confirmed by co-IP experiments with an anti-ezrin antibody, and by pulldown assays using purified full-length and FERM domain-containing recombinant ezrin. By using purified recombinant proteins, we showed that ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain. Knocking down ezrin expression with shRNA resulted in increased membrane accumulation of AQP2 and reduced AQP2 endocytosis. Therefore, through direct interaction with AQP2, ezrin facilitates AQP2 endocytosis, thus linking the dynamic actin cytoskeleton network with AQP2 trafficking.

Ser-261 phospho-regulation is involved in pS256 and pS269-mediated aquaporin-2 apical translocation.

Vasopressin catalyzes aquaporin-2 phosphorylation at several serine sites in the C-terminal region. Compared with Ser-256 and Ser-269 phosphorylation, the role of Ser-261 phospho-regulation on vasopressin-regulated AQP2 apical translocation is largely unknown. In addition, recent discovery of transcytotic apical delivery of AQP2 made the concept of its intracellular trafficking even more complicated. In this study, we evaluated how intact phospho-AQP2 signals fit with the transcytosis trafficking model in Madin-Darby canine kidney cells. PS256 and pS269 signals were intracellularly detectable in wild-type AQP2 at the beginning of forskolin stimulation (1 min). These phospho-signals were detectable in basolateral membranes even after 10 min of stimulation. AQP2 stably inserted in the apical membrane increased pS269 and decreased pS261 signals. In an NDI-causing mutant P262L-AQP2, in which Ser-261 phospho-regulation is impaired, the pS256 and pS269 signals were detectable in the basolateral membranes with increased pS261 signals after forskolin stimulation. These results suggest that Ser-261 phospho-regulation is involved in pS256- and pS269-mediated AQP2 apical translocation.

Aquaporin-2 Ser-261 phosphorylation is regulated in combination with Ser-256 and Ser-269 phosphorylation.

Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phospho-regulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256- and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation.

Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway.

Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI.

Bence-Jones Protein λ-type Multiple Myeloma Patient Withdrawn from Maintenance Hemodialysis after Long-term Bortezomib and Dexamethasone Therapy.

The effectiveness of bortezomib treatment for multiple myeloma (MM) is well established. However, the protocol by which maintenance therapy using bortezomib should be continued for myeloma patients requiring regular hemodialysis remains to be established. We herein report a case of MM with severe renal insufficiency requiring hemodialysis for nearly 30 months which was finally withdrawn from renal replacement therapy during monthly maintenance treatment with bortezomib and dexamethasone for two years. The details of this case are essential for establishing clinical guidelines for applying intermittent low-frequency bortezomib therapy in dialysis-dependent myeloma patients.

Two Cases of Hemodialysis-associated Chronic Portal-systemic Shunt Encephalopathy (CPSE) with Opposite Changes in the Blood Ammonia Concentrations during Hemodialysis: A Case Report and Literature Review.

The onset of hyperammonemia due to the flow of ammonia-rich portal vein blood through a portal-systemic shunt causes a type of encephalopathy known as chronic portal-systemic shunt encephalopathy (CPSE). We herein report two cases of CPSE that presented with opposite changes in the blood ammonia concentrations during hemodialysis. It is curious that the encephalopathy was ameliorated by hemodialysis in case 1, but not case 2. Therefore, it is necessary to recognize CPSE and assess the blood ammonia concentrations in dialysis patients who develop a disturbance of consciousness, even if the serum transaminase level is normal.

Anaemia management and mortality risk in newly visiting patients with chronic kidney disease in Japan: The CKD-ROUTE study.

AIM: To investigate the association between iron deficiency anaemia and mortality risk and assess the changes in anaemia and iron status after primary management by a nephrologist. METHODS: In this prospective cohort study, we stratified 951 non-dialysis chronic kidney disease (CKD) G2-G5 patients newly visiting 16 nephrology centres into four groups according to the presence of anaemia with or without iron deficiency. All-cause mortality, cardiovascular (CV)-related mortality, and a change in anaemia and iron status after specialized primary care were the endpoints evaluated. RESULTS: During a median follow-up time of 19 months, the number of all-cause deaths and CV-related deaths were 56 and 26, respectively. Compared with the control group, the groups with isolated anaemia and iron deficiency anaemia had significantly higher all-cause mortalities (isolated anaemia: hazard ratio (HR), 3.37; 95% confidence intervals (CI), 1.76-6.44; iron deficiency anaemia: HR, 3.11; 95% CI, 1.21-8.01) and CV-related mortalities (isolated anaemia: HR, 3.64; 95% CI, 1.36-9.73; iron deficiency anaemia: HR, 3.86; 95% CI, 1.11-13.41). In the isolated anaemia group, erythropoietin-stimulating agent (ESA) prescriptions significantly increased to approximately 70%. However, in patients with both anaemia and iron deficiency, iron prescriptions only increased to 48.1%. CONCLUSIONS: Iron deficiency anaemia and isolated anaemia were associated with all-cause and CV-related mortality. The absence of relative increase in iron prescriptions suggests that iron deficiency should be accurately assessed and iron supplementation should be appropriately used to manage anaemia in non-dialysis patients with CKD.

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model.

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Treatment with 17-allylamino-17-demethoxygeldanamycin ameliorated symptoms of Bartter syndrome type IV caused by mutated Bsnd in mice.

Mutations of BSND, which encodes barttin, cause Bartter syndrome type IV. This disease is characterized by salt and fluid loss, hypokalemia, metabolic alkalosis, and sensorineural hearing impairment. Barttin is the ß-subunit of the ClC-K chloride channel, which recruits it to the plasma membranes, and the ClC-K/barttin complex contributes to transepithelial chloride transport in the kidney and inner ear. The retention of mutant forms of barttin in the endoplasmic reticulum (ER) is etiologically linked to Bartter syndrome type IV. Here, we report that treatment with 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, enhanced the plasma membrane expression of mutant barttins (R8L and G47R) in Madin-Darby canine kidney cells. Administration of 17-AAG to Bsnd(R8L/R8L) knock-in mice elevated the plasma membrane expression of R8L in the kidney and inner ear, thereby mitigating hypokalemia, metabolic alkalosis, and hearing loss. These results suggest that drugs that rescue ER-retained mutant barttin may be useful for treating patients with Bartter syndrome type IV.

Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na(+) transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel "noncanonical" regulatory pathway for GPCR activation and response termination, via the sequential action of ß-arrestin and the retromer complex.

Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

AQP2 is necessary for vasopressin- and forskolin-mediated filamentous actin depolymerization in renal epithelial cells.

Remodeling of the actin cytoskeleton is required for vasopressin (VP)-induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)-mediated F-actin depolymerization depends on AQP2 expression. Using various MDCK and LLC-PK1 cell lines with different AQP2 expression levels, we performed F-actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F-actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F-actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC-PK(1) cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F-actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane-associated F-actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F-actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion.

FAPP2 is required for aquaporin-2 apical sorting at trans-Golgi network in polarized MDCK cells.

FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.

Targeted disruption of the Wnk4 gene decreases phosphorylation of Na-Cl cotransporter, increases Na excretion and lowers blood pressure.

We recently generated Wnk4(D561A/+) knockin mice and found that a major pathogenesis of pseudohypoaldosteronism type II was the activation of the OSR1/SPAK kinase-NaCl cotransporter (NCC) phosphorylation cascade by the mutant WNK4. However, the physiological roles of wild-type WNK4 on the regulation of Na excretion and blood pressure, and whether wild-type WNK4 functions positively or negatively in this cascade, remained to be determined. In the present study, we generated WNK4 hypomorphic mice by deleting exon 7 of the Wnk4 gene. These mice did not show hypokalemia and metabolic alkalosis, but they did exhibit low blood pressure and increased Na and K excretion under low-salt diet. Phosphorylation of OSR1/SPAK and NCC was significantly reduced in the mutant mice as compared with their wild-type littermates. Protein levels of ROMK and Maxi K were not changed, but epithelial Na channel appeared to be activated as a compensatory mechanism for the reduced NCC function. Thus, wild-type WNK4 is a positive regulator for the WNK-OSR1/SPAK-NCC cascade, and WNK4 is a potential target of anti-hypertensive drugs.