Daiei Takahashi

WNK4 is the major WNK positively regulating NCC in the mouse kidney

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.

Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice

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.

Dietary Salt Intake Regulates WNK3–SPAK–NKCC1 Phosphorylation Cascade in Mouse Aorta Through Angiotensin II

Na–K–Cl cotransporter isoform 1 (NKCC1) is involved in the regulation of vascular smooth muscle cell contraction. Recently, the with-no-lysine kinase (WNK)–STE20/SPS1-related proline/alanine-rich kinase (SPAK)–NKCC1 phosphorylation cascade in vascular smooth muscle cells was found to be important in the regulation of vascular tone. In this study, we investigated whether the WNK–SPAK–NKCC1 cascade in mouse aortic tissue is regulated by dietary salt intake and the mechanisms responsible. Phosphorylation of SPAK and NKCC1 was significantly reduced in the aorta in high-salt–fed mice and was increased in the aorta in low-salt–fed mice, indicating that the WNK–SPAK–NKCC1 phosphorylation cascade in the aorta was indeed regulated by dietary salt intake. Acute and chronic angiotensin II infusion increased phosphorylation of SPAK and NKCC1 in the mouse aorta. In addition, valsartan, an antagonist of angiotensin II type 1 receptor, inhibited low-salt diet–induced phosphorylation of SPAK and NKCC1, demonstrating that angiotensin II activates the WNK–SPAK–NKCC1 phosphorylation cascade through the angiotensin II type 1 receptor. However, a low-salt diet and angiotensin II together did not increase phosphorylation of SPAK and NKCC1 in the aorta in WNK3 knockout mice, indicating that activation of the WNK–SPAK–NKCC1 phosphorylation cascade induced by a low-salt diet and angiotensin II is dependent on WNK3. Indeed, angiotensin II–induced increases in blood pressure were diminished in WNK3 knockout mice. In addition, decreased response to angiotensin II in the mesenteric arteries was observed in WNK3 knockout mice. Our data also clarified a novel mechanism for regulation of vascular tonus by angiotensin II. Inhibition of this cascade could, therefore, be a novel therapeutic target in hypertension.

KLHL2 interacts with and ubiquitinates WNK kinases.

Mutations in the WNK1 and WNK4 genes result in an inherited hypertensive disease, pseudohypoaldosteronism type II (PHAII). Recently, the KLHL3 and Cullin3 genes were also identified as responsible genes for PHAII. Although we have reported that WNK4 is a substrate for the KLHL3-Cullin3 E3 ligase complex, it is not clear whether all of the WNK isoforms are regulated only by KLHL3. To explore the interaction of WNKs and other Kelch-like proteins, we focused on KLHL2 (Mayven), a human homolog of Drosophila Kelch that shares the highest similarity with KLHL3. We found that KLHL2, as well as KLHL3, was co-immunoprecipitated with all four WNK isoforms. The direct interaction of KLHL2 with WNKs was confirmed on fluorescence correlation spectroscopy. Co-expression of KLHL2 and Cullin3 decreased the abundance of WNK1, WNK3 and WNK4 within HEK293T cells, and a significant increase of WNK4 ubiquitination by KLHL2 and Cullin3 was observed both in HEK293T cells and in an in vitro ubiquitination assay. These results suggest that KLHL2-Cullin3 also functions as an E3-ligase for WNK isoforms within the body.

Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target.

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3

ABSTRACT Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3−/− mice that expressed β-galactosidase (β-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of β-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3−/− mice but not in KLHL3+/− mice. KLHL3−/− mice also showed PHAII-like phenotypes, whereas KLHL3+/− mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.

WNK4 is an Adipogenic Factor and Its Deletion Reduces Diet-Induced Obesity in Mice

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.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 μM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Loop diuretics affect skeletal myoblast differentiation and exercise-induced muscle hypertrophy

Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl− cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks’ voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.

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.