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Shinichi Uchida - One of the best experts on this subject based on the ideXlab platform.
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WNK4 is indispensable for the pathogenesis of pseudohypoaldosteronism type ii caused by mutant klhl3
Biochemical and Biophysical Research Communications, 2017Co-Authors: Koichiro Susa, Daiei Takahashi, Eisei Sohara, Tomokazu Okado, Tatemitsu Rai, Shinichi UchidaAbstract:Abstract WNK-OSR1/SPAK-NCC signaling cascade is important for regulating salt balance and blood pressure. Activation of WNK-OSR1/SPAK-NaCl cotransporter (NCC) cascade increases sodium reabsorption in the kidney, leading to pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. It has been previously demonstrated that the amount of phosphorylated and total NCC markedly decreased in WNK4 −/− mice, indicating that WNK4 plays a major role for activation of OSR1/SPAK-NCC signaling. However, it is unclear whether absence of WNK4 can be compensated by other WNK kinases. We recently reported that KLHL3 R528H/+ knock-in mice, a PHAII model, exhibited augmented activation of OSR1/SPAK-NCC signaling by increased protein levels of both WNK1 and WNK4 due to impaired protein degradation by the mutant KLHL3. In this study, we sought to determine the contribution of WNK4 to OSR1/SPAK-NCC signaling using an in vivo model which shows extremely increased WNK1 with absence of WNK4. We generated WNK4 −/− KLHL3 R528H/+ mice and WNK4 −/− KLHL3 R528H/R528H mice by crossing WNK4 −/− mice with KLHL3 R528H/+ mice. Thereafter, WNK-OSR1/SPAK-NCC phosphorylation signal cascade was examined in kidneys from these mice. As expected, both WNK4 −/− KLHL3 R528H/+ mice and WNK4 −/− KLHL3 R528H/R528H mice demonstrated increased WNK1 in the kidney, due to the KLHL3 mutation, and WNK4 deficiency. However, phosphorylation of SPAK and NCC at distal convoluted tubules were almost completely absent even in WNK4 −/− KLHL3 R528H/R528H mice. In conclusion, increased WNK1 was unable to compensate for WNK4 deficiency and phosphorylate the NCC, indicating that WNK4 is indispensable for the onset of PHAII.
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a patient with pseudohypoaldosteronism type ii complicated by congenital hypopituitarism carrying a klhl3 mutation
Clinical Pediatric Endocrinology, 2016Co-Authors: Marie Mitani, Motoko Chiga, Shinichi Uchida, Munehiro Furuichi, Satoshi Narumi, Tomonobu Hasegawa, Seiji SatoAbstract:Pseudohypoaldosteronism (PHA) is a rare renal tubular disease that can be divided into two types based on their pathogenesis. PHA type I (PHA I) is caused by abnormalities in the aldosterone receptor or epithelial sodium channel and is inherited in an autosomal dominant or recessive manner, respectively. PHA I is clinically characterized by renal salt wasting and decreased response of the aldosterone receptor or epithelial sodium channel to aldosterone. PHA type II (PHA II, also referred to as Gordon syndrome(1)) is a heterogeneous syndrome inherited in an autosomal dominant or recessive manner. The best-characterized pathogenesis of PHA II is associated with increased membrane expression of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubules, resulting in excessive sodium resorption. Excretion of potassium and hydrogen is decreased because it is coupled with sodium resorption. As a result, patients with PHA II show early-onset hypertension, hyperkalemia, and metabolic acidosis. Plasma renin levels are suppressed and aldosterone levels are variable but are relatively low given the degree of hyperkalemia. Other clinical manifestations of PHA II include growth failure, muscle weakness, periodic paralysis, skeletal abnormalities, urinary calculus, and psychomotor retardation. Electrolyte abnormalities and hypertension can be treated with thiazide. Recently, the molecular pathogenesis of PHA II was partly elucidated. In 2001, Wilson et al. conducted positional cloning analyses of PHA II kindreds and identified mutations in the WNK family kinase genes (WNK1 and WNK4). The two kinases are involved in the plasma membrane expression of NCC (2). In 2012, Boyden et al. performed whole exome sequencing in patients with PHA II, identifying two additional causative genes (KLHL3 and CUL3) (3). The KLHL3-CUL3 complex acts as a negative regulator of WNK kinases through ubiquitination of the two kinases. Thus, mutations in KLHL3 or CUL3 are thought to increase the membrane expression of NCC via increased WNK kinases (4,5,6,7). Among the 52 PHA II kindreds, 24 showed KLHL3 mutations (eight were autosomal recessive inheritance, 16 were autosomal dominant inheritance), 17 showed CUL3 mutations, five showed WNK4 mutations, and two showed WNK1 mutations (3). Here, we report a patient with PHA II complicated by congenital hypopituitarism carrying a KLHL3 mutation. The patient exhibited growth failure. Two possible factors, PHA II and congenital hypopituitarism, were considered as the cause(s) of the growth failure.
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involvement of selective autophagy mediated by p62 sqstm1 in klhl3 dependent WNK4 degradation
Biochemical Journal, 2015Co-Authors: Yutaro Mori, Moko Zeniya, Mai Wakabayashi, Takayasu Mori, Eisei Sohara, Tatemitsu Rai, Yuki Yoshizaki, Shinichi UchidaAbstract:We reported that kelch-like protein 3 (KLHL3)–Cullin3 E3 ligase ubiquitinates with-no-lysine kinase 4 (WNK4) and that impaired WNK4 ubiquitination causes pseudohypoaldosteronism type II, a hereditary hypertensive disease. However, we also found that KLHL3-induced WNK4 degradation could not be inhibited completely by a proteasome inhibitor. Rather, on exposure, for 24 h, of HEK293T cells expressing WNK4 and KLHL3 to a proteasome inhibitor, epoxomicin, the WNK4 protein level was further decreased. As proteasome inhibition is known to activate p62-mediated selective autophagy, we investigated whether WNK4 degradation induced by KLHL3 is also mediated by such an autophagic mechanism. 3-Methyladenine, an autophagy inhibitor, blocked the epoxomicin-induced decrease in WNK4. Co-immunoprecipitation assays revealed that KLHL3 formed a complex not only with WNK4 but also with p62 via its kelch repeat domain. Under proteasome inhibition, p62 overexpression decreased KLHL3 and WNK4 protein levels, and p62 knockdown dramatically increased KLHL3 and WNK4 protein levels. Based on immunofluorescent staining, transiently overexpressed WNK4 showed punctate localization in the cytoplasm where it co-localized with KLHL3, p62 and light chain 3, a marker of autophagosomes. Thus, WNK4 was degraded not only by proteasomes but also by p62–KLHL3-mediated selective autophagy, which may be involved in WNK regulation under certain pathophysiological conditions.
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WNK4 is the major WNK positively regulating NCC in the mouse kidney
Portland Press Biochemical Society, 2014Co-Authors: Daiei Takahashi, Moko Zeniya, Yuya Araki, Naohiro Nomura, Takayasu Mori, Eisei Sohara, Sei Sasaki, Tatemitsu Rai, Muhammad zakir hossain Khan, Shinichi UchidaAbstract:By analysing the pathogenesis of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), we previously discovered that WNK (with-no-lysine kinase)–OSR1/SPAK (oxidative stress-responsive 1/Ste20-like proline/alanine-rich kinase) cascade regulates NCC (Na–Cl co-transporter) in the DCT (distal convoluted tubules) of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4−/− mice. Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4−/− mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4−/− mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys
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effect of heterozygous deletion of wnk1 on the wnk osr1 spak ncc nkcc1 nkcc2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Sung-sen Yang, Dario R Alessi, Akihito Ohta, Eisei Sohara, Sei Sasaki, Satomi Kita, Takahiro Iwamoto, Shinichi UchidaAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1+/− mice have not been performed.
David H. Ellison - One of the best experts on this subject based on the ideXlab platform.
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role of WNK4 and kidney specific wnk1 in mediating the effect of high dietary k intake on romk channel in the distal convoluted tubule
American Journal of Physiology-renal Physiology, 2018Co-Authors: Peng Wu, Juliette Hadchouel, Jacques Teulon, Xiaotong Su, David H. Ellison, Wenhui WangAbstract:With-no-lysine kinase 4 (WNK4) and kidney-specific (KS)-WNK1 regulate ROMK (Kir1.1) channels in a variety of cell models. We now explore the role of WNK4 and KS-WNK1 in regulating ROMK in the nativ...
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regulation of renal electrolyte transport by wnk and spak osr1 kinases
Annual Review of Physiology, 2016Co-Authors: Juliette Hadchouel, David H. Ellison, Gerardo GambaAbstract:The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na+:Cl− cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years.
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unique chloride sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis
Kidney International, 2016Co-Authors: Andrew S Terker, Chaoling Yang, Gerardo Gamba, Chong Zhang, Kayla J Erspamer, David H. EllisonAbstract:Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low-potassium diet. Recent data suggest that plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the with-no-lysine kinase (WNK)-Ste20p-related proline- and alanine-rich kinase (SPAK) pathway. As previous studies used extreme dietary manipulations, we sought to determine whether the relationship between potassium and NaCl cotransporter (NCC) is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect NCC in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride concentration. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable it to mediate effects of potassium on NCC in vivo.
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the effect of WNK4 on the na cl cotransporter is modulated by intracellular chloride
Journal of The American Society of Nephrology, 2015Co-Authors: Silvana Bazuavalenti, Maria Chavezcanales, Alejandro Rodriguezgama, Xochiquetzal Gonzalezrodriguez, Zesergio Melo, Norma Vazquez, Eduardo R. Argaiz, Lorena Rojasvega, Consuelo Plata, David H. EllisonAbstract:It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.
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the thiazide sensitive na cl cotransporter is regulated by a wnk kinase signaling complex
Journal of Clinical Investigation, 2007Co-Authors: Chaoling Yang, Xiaoman Zhu, David H. EllisonAbstract:The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.
Juliette Hadchouel - One of the best experts on this subject based on the ideXlab platform.
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kidney specific wnk1 isoform ks wnk1 is a potent activator of WNK4 and ncc
American Journal of Physiology-renal Physiology, 2018Co-Authors: Maria Chavezcanales, Mauricio Ostroskyfrid, Alejandro Rodriguezgama, Xochiquetzal Gonzalezrodriguez, Jesus Garciavaldes, Norma Vazquez, Eduardo R. Argaiz, Juliette HadchouelAbstract:Familial Hyperkalemic Hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the WNK1 and WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/SPAK/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylat...
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role of WNK4 and kidney specific wnk1 in mediating the effect of high dietary k intake on romk channel in the distal convoluted tubule
American Journal of Physiology-renal Physiology, 2018Co-Authors: Peng Wu, Juliette Hadchouel, Jacques Teulon, Xiaotong Su, David H. Ellison, Wenhui WangAbstract:With-no-lysine kinase 4 (WNK4) and kidney-specific (KS)-WNK1 regulate ROMK (Kir1.1) channels in a variety of cell models. We now explore the role of WNK4 and KS-WNK1 in regulating ROMK in the nativ...
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consequences of spak inactivation on hyperkalemic hypertension caused by wnk1 mutations evidence for differential roles of wnk1 and WNK4
Scientific Reports, 2018Co-Authors: Chloe Rafael, Perrine Frere, Christelle Soukaseum, Veronique Baudrie, Juliette HadchouelAbstract:Mutations of the gene encoding WNK1 [With No lysine (K) kinase 1] or WNK4 cause Familial Hyperkalemic Hypertension (FHHt). Previous studies have shown that the activation of SPAK (Ste20-related Proline/Alanine-rich Kinase) plays a dominant role in the development of FHHt caused by WNK4 mutations. The implication of SPAK in FHHt caused by WNK1 mutation has never been investigated. To clarify this issue, we crossed WNK1 +/FHHt mice with SPAK knock-in mice in which the T-loop Thr243 residue was mutated to alanine to prevent activation by WNK kinases. We show that WNK1 +/ FHHT :SPAK 243A/243A mice display an intermediate phenotype, between that of control and SPAK 243A/243A mice, with normal blood pressure but hypochloremic metabolic alkalosis. NCC abundance and phosphorylation levels also decrease below the wild-type level in the double-mutant mice but remain higher than in SPAK 243A/243A mice. This is different from what was observed in WNK4-FHHt mice in which SPAK inactivation completely restored the phenotype and NCC expression to wild-type levels. Although these results confirm that FHHt caused by WNK1 mutations is dependent on the activation of SPAK, they suggest that WNK1 and WNK4 play different roles in the distal nephron. Familial Hyperkaliaemic Hypertension (FHHt), also known as Gordon's syndrome or Pseudohypoaldosteronism type II (PHAII), is a rare genetic form of hypertension associated with hyperkalemia and metabolic hyperchlo-remic acidosis (OMIM #145260) 1. All these symptoms are corrected by thiazide diuretics, which inhibit the activity of the Na +-Cl − transporter NCC, encoded by the SLC12A3 gene 1. FHHt is caused by mutations in one of at least four genes: WNK1 [With No lysine (K) 1], WNK4, KLHL3 (Kelch-Like family member 3), and CUL3 (cullin-3) 2-4. The associated proteins belong to the same regulatory pathway, as WNK1 and WNK4 are recruited by KLHL3 for ubiquitination by the Cul3-E3 RING-type ubiquitin-ligase complex 5. The sensitivity of patients to thiazide diuretics strongly suggested that FHHt is mainly caused by a gain of activity of NCC in the distal nephron. The role of NCC in regulating blood pressure has been established by the discovery of inactivating mutations of the SLC12A3 gene, which cause Gitelman's syndrome, an inherited disorder that is the mirror image of FHHt, with arterial hypotension, renal salt wasting and hypokalemic metabolic alkalosis (OMIM #263800) 6,7. Therefore, several studies focused on the mechanisms underlying the regulation of NCC by WNK1 and WNK4. In vitro studies have demonstrated that the WNKs activate two kinases, SPAK (Ste20-related Proline/ Alanine-rich Kinase) and OSR1 (oxidative stress-responsive kinase 1) 8 , which can in turn phosphorylate and activate NCC 9. SPAK and OSR1 can also activate the Na +-K +-2Cl − cotransporters, NKCC1 and NKCC2, and inhibit the K +-Cl − cotransporters, KCC1-4 10. In vivo, the expression and phosphorylation of NCC is reduced
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reabsorption du sel et secretion du potassium par le nephron distal vision nouvelle du role regulateur des kinases de la famille wnk
M S-medecine Sciences, 2016Co-Authors: Chloe Rafael, Maria Chavezcanales, Juliette HadchouelAbstract:L’etude d’une forme mendelienne rare d’hypertension arterielle, l’hypertension hyperkaliemique familiale (FHHt), a permis des avancees remarquables dans la comprehension des mecanismes de regulation du transport renal du chlorure de sodium. Chez quelques patients, cette pathologie est due a des mutations touchant les genes codant WNK1 et WNK4, deux serine-threonine kinases de la famille WNK (with no lysine [K]). Les signes cliniques associes a la FHHt resultent, entre autres, de l’hyperactivite du co-transporteur Na+ -Cl- , NCC. De nombreuses equipes se sont interessees a la regulation de NCC par WNK1 et WNK4. Cependant, les donnees obtenues etaient tres souvent contradictoires. Recemment, deux de nos etudes ont permis d’expliquer en partie ces controverses et d’etablir un nouveau modele de regulation de NCC par les kinases de la famille WNK.
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regulation of renal electrolyte transport by wnk and spak osr1 kinases
Annual Review of Physiology, 2016Co-Authors: Juliette Hadchouel, David H. Ellison, Gerardo GambaAbstract:The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na+:Cl− cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years.
Gerardo Gamba - One of the best experts on this subject based on the ideXlab platform.
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wnk3 and WNK4 exhibit opposite sensitivity with respect to cell volume and intracellular chloride concentration
American Journal of Physiology-cell Physiology, 2020Co-Authors: Diana Pachecoalvarez, Maria Castanedabueno, Erika Moreno, Norma Vazquez, Adriana Mercado, Gerardo Gamba, Diego Luis Carrilloperez, Karla Leyvarios, Elisa HernandezmercadoAbstract:Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.
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regulation of renal electrolyte transport by wnk and spak osr1 kinases
Annual Review of Physiology, 2016Co-Authors: Juliette Hadchouel, David H. Ellison, Gerardo GambaAbstract:The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na+:Cl− cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years.
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Invited Review Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension
2016Co-Authors: Gerardo Gamba, Molecular Physiology UnitAbstract:Gamba, Gerardo. Role of WNK kinases in regulating tubular salt and potas-sium transport and in the development of hypertension. Am J Physiol Renal Physiol 288: F245–F252, 2005; doi:10.1152/ajprenal.00311.2004.—A recently discovered family of protein kinases is responsible for an autosomal-dominant disease known as Gordon’s syndrome or pseudohypoaldosteronism type II (PHA-II) that features hyperkalemia and hyperchloremic metabolic acidosis, accompanied by hyperten-sion and hypercalciuria. Four genes have been described in this kinase family, which has been named WNK, due to the absence of a key lysine in kinase subdomain II (with no K kinases). Two of these genes, WNK1 and WNK4 located in human chromosomes 12 and 17, respectively, are responsible for PHA-II. Immunohystochemical analysis revealed that WNK1 and WNK4 are predominantly expressed in the distal convoluted tubule and collecting duct. The physiological studies have shown that WNK4 downregulates the activity of ion transport pathways expressed in these nephron segments, such as the apical thiazide-sensitive Na-Cl cotransporter and apical secretory K channel ROMK, as well a
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unique chloride sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis
Kidney International, 2016Co-Authors: Andrew S Terker, Chaoling Yang, Gerardo Gamba, Chong Zhang, Kayla J Erspamer, David H. EllisonAbstract:Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low-potassium diet. Recent data suggest that plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the with-no-lysine kinase (WNK)-Ste20p-related proline- and alanine-rich kinase (SPAK) pathway. As previous studies used extreme dietary manipulations, we sought to determine whether the relationship between potassium and NaCl cotransporter (NCC) is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect NCC in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride concentration. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable it to mediate effects of potassium on NCC in vivo.
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the kinase wnk1 is a powerful inhibitor of the k cl cotransporters 1109 6
The FASEB Journal, 2014Co-Authors: Zesergio Melo, Maria Chavezcanales, Juliette Hadchouel, Adrian Murillo, Norma Vazquez, Gerardo Gamba, Adriana MercadoAbstract:The K+:Cl- cotransporters (KCC1-4) belong to the SLC12 family of electroneutral cation-chloride cotransporters that translocate ions outside the cells to regulate cell volume and intracellular chloride concentration. We previously shown that WNK3 and WNK4 inhibit the activity of KCCs and the elimination of kinase activity turned them into a powerful activators of all KCCs. WNK1 is a ubiquitously expressed kinase that produces two distinct hereditary diseases: pseudohypoaldosteronism type II and hereditary sensory neuropathy 2. Its effect on the regulation of KCCs activity is yet unknown. In the present study, we show using Xenopus laevis oocytes as expression system, that activation of all four KCCs by cell swelling was prevented by coinjection with the various WNK1 isoforms cRNA. The negative effect of WNK1 on the KCCs is kinase dependent, as it was not present when the kinase dead WNK1-D368A was used. Elimination of the SPAK binding site (WNK1-F316A) or the HQ motif of WNK1 (required for WNK to WNK inte...
Motoko Chiga - One of the best experts on this subject based on the ideXlab platform.
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WNK4 is an Adipogenic Factor and Its Deletion Reduces Diet-Induced Obesity in Mice
Elsevier, 2017Co-Authors: Daiei Takahashi, Moko Zeniya, Motoko Chiga, Naohiro Nomura, Takayasu Mori, Eisei Sohara, Miyako Tanaka, Yuichi Inoue, Hiroki Ochi, Shu TakedaAbstract:The with-no-lysine kinase (WNK) 4 gene is a causative gene in pseudohypoaldosteronism type II. Although WNKs are widely expressed in the body, neither their metabolic functions nor their extrarenal role is clear. In this study, we found that WNK4 was expressed in mouse adipose tissue and 3T3-L1 adipocytes. In mouse primary preadipocytes and in 3T3-L1 adipocytes, WNK4 was markedly induced in the early phase of adipocyte differentiation. WNK4 expression preceded the expression of key transcriptional factors PPARγ and C/EBPα. WNK4-siRNA-transfected 3T3-L1 cells and human mesenchymal stem cells showed reduced expression of PPARγ and C/EBPα and lipid accumulation. WNK4 protein affected the DNA-binding ability of C/EBPβ and thereby reduced PPARγ expression. In the WNK4−/− mice, PPARγ and C/EBPα expression were decreased in adipose tissues, and the mice exhibited partial resistance to high-fat diet-induced adiposity. These data suggest that WNK4 may be a proadipogenic factor, and offer insights into the relationship between WNKs and energy metabolism
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a patient with pseudohypoaldosteronism type ii complicated by congenital hypopituitarism carrying a klhl3 mutation
Clinical Pediatric Endocrinology, 2016Co-Authors: Marie Mitani, Motoko Chiga, Shinichi Uchida, Munehiro Furuichi, Satoshi Narumi, Tomonobu Hasegawa, Seiji SatoAbstract:Pseudohypoaldosteronism (PHA) is a rare renal tubular disease that can be divided into two types based on their pathogenesis. PHA type I (PHA I) is caused by abnormalities in the aldosterone receptor or epithelial sodium channel and is inherited in an autosomal dominant or recessive manner, respectively. PHA I is clinically characterized by renal salt wasting and decreased response of the aldosterone receptor or epithelial sodium channel to aldosterone. PHA type II (PHA II, also referred to as Gordon syndrome(1)) is a heterogeneous syndrome inherited in an autosomal dominant or recessive manner. The best-characterized pathogenesis of PHA II is associated with increased membrane expression of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubules, resulting in excessive sodium resorption. Excretion of potassium and hydrogen is decreased because it is coupled with sodium resorption. As a result, patients with PHA II show early-onset hypertension, hyperkalemia, and metabolic acidosis. Plasma renin levels are suppressed and aldosterone levels are variable but are relatively low given the degree of hyperkalemia. Other clinical manifestations of PHA II include growth failure, muscle weakness, periodic paralysis, skeletal abnormalities, urinary calculus, and psychomotor retardation. Electrolyte abnormalities and hypertension can be treated with thiazide. Recently, the molecular pathogenesis of PHA II was partly elucidated. In 2001, Wilson et al. conducted positional cloning analyses of PHA II kindreds and identified mutations in the WNK family kinase genes (WNK1 and WNK4). The two kinases are involved in the plasma membrane expression of NCC (2). In 2012, Boyden et al. performed whole exome sequencing in patients with PHA II, identifying two additional causative genes (KLHL3 and CUL3) (3). The KLHL3-CUL3 complex acts as a negative regulator of WNK kinases through ubiquitination of the two kinases. Thus, mutations in KLHL3 or CUL3 are thought to increase the membrane expression of NCC via increased WNK kinases (4,5,6,7). Among the 52 PHA II kindreds, 24 showed KLHL3 mutations (eight were autosomal recessive inheritance, 16 were autosomal dominant inheritance), 17 showed CUL3 mutations, five showed WNK4 mutations, and two showed WNK1 mutations (3). Here, we report a patient with PHA II complicated by congenital hypopituitarism carrying a KLHL3 mutation. The patient exhibited growth failure. Two possible factors, PHA II and congenital hypopituitarism, were considered as the cause(s) of the growth failure.
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impaired degradation of wnk1 and WNK4 kinases causes phaii in mutant klhl3 knock in mice
Human Molecular Genetics, 2014Co-Authors: Koichiro Susa, Moko Zeniya, Hidenori Nishida, Daiei Takahashi, Motoko Chiga, Naohiro Nomura, Yutaro Mori, Takayasu Mori, Eisei Sohara, Kiyoshi IsobeAbstract:Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na‐Cl cotransporter (NCC) were also reported tobeasubstrateofKLHL3-Cullin3E3ligasebyothergroups.Therefore,itremainsunclearwhichmoleculeisthe target(s)ofKLHL3.ToinvestigatethepathogenesisofPHAIIcausedbyKLHL3mutation,wegeneratedandanalyzedKLHL3 R528H/1 knock-inmice.KLHL3 R528H/1 knock-inmiceexhibitedsalt-sensitivehypertension,hyperkalemiaandmetabolicacidosis.Moreover,thephosphorylationofNCCwasincreasedintheKLHL3 R528H/1 mouse kidney,indicatingthattheKLHL3 R528H/1 knock-inmouseisanidealmousemodelofPHAII.Interestingly,theprotein expression of both WNK1 and WNK4 was significantly increased in the KLHL3 R528H/1 mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3 R528H/1 knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measuredthebindingofTAMRA-labeledWNK1andWNK4peptidestofull-lengthKLHL3usingfluorescencecor
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impaired degradation of wnk1 and WNK4 kinases causes phaii in mutant klhl3 knock in mice
Human Molecular Genetics, 2014Co-Authors: Koichiro Susa, Moko Zeniya, Hidenori Nishida, Motoko Chiga, Naohiro Nomura, Yutaro Mori, Takayasu Mori, Eisei Sohara, Tatemitsu Rai, Daiei TakahashiAbstract:Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.
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Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.
Cell Reports, 2013Co-Authors: Mai Wakabayashi, Eriko Kikuchi, Koichiro Susa, Yuya Araki, Motoko Chiga, Naohiro Nomura, Takayasu Mori, Kiyoshi Isobe, Eisei Sohara, Yutaro MoriAbstract:Summary Mutations in WNK kinases cause the human hypertensive disease pseudohypoaldosteronism type II (PHAII), but the regulatory mechanisms of the WNK kinases are not well understood. Mutations in kelch-like 3 ( KLHL3 ) and Cullin3 were also recently identified as causing PHAII. Therefore, new insights into the mechanisms of human hypertension can be gained by determining how these components interact and how they are involved in the pathogenesis of PHAII. Here, we found that KLHL3 interacted with Cullin3 and WNK4, induced WNK4 ubiquitination, and reduced the WNK4 protein level. The reduced interaction of KLHL3 and WNK4 by PHAII-causing mutations in either protein reduced the ubiquitination of WNK4, resulting in an increased level of WNK4 protein. Transgenic mice overexpressing WNK4 showed PHAII phenotypes, and WNK4 protein was indeed increased in WNK4 D561A/+ PHAII model mice. Thus, WNK4 is a target for KLHL3-mediated ubiquitination, and the impaired ubiquitination of WNK4 is a common mechanism of human hereditary hypertension.
