Relevance of leukaemia inhibitory factor to anti-melanoma differentiation-associated gene 5 antibody-positive interstitial lung disease.

OBJECTIVES: Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive rapidly progressive interstitial lung disease (RP-ILD) is a life-threatening disease, the aetiology of which remains unclear. To detect potential diagnostic markers, a transcriptome analysis of the lung sample from a patient with anti-MDA5 antibody-positive RP-ILD was performed. METHODS: RNA sequencing analyses of an autopsy lung sample from a 74-year-old woman with anti-MDA5 antibody-positive RP-ILD was performed and compared with an age- and sex-matched normal lung sample. Genes with changes of gene expression ≥5-fold were considered differentially expressed genes and analysed by Metascape. The levels of leukaemia inhibitory factor (LIF) were measured in the serum samples from 12 cases of anti-MDA5 antibody-positive ILD, 12 cases of anti-aminoacyl tRNA synthetase (ARS) antibody-positive ILD, 10 cases of anti-transcription intermediary factor 1γ/anti-Mi-2 antibody DM and 12 healthy volunteers. RESULTS: Gene ontology enrichment analysis on the RNA sequencing data showed a strong association with antigen binding. Upregulated expressions of IL-1ß, IL-6 and LIF were also detected. Serum LIF levels were significantly elevated in anti-MDA5 antibody-positive ILD patients {median 32.4 pg/ml [interquartile range (IQR) 13.2-125.7]} when compared with anti-ARS antibody-positive ILD patients [4.9 pg/ml (IQR 3.1-19.7), P < 0.05] and DM patients [5.3 pg/ml (IQR 3.9-9.7), P < 0.05]. CONCLUSION: Our present study suggested that upregulation of LIF might be a new potential disease marker specific for anti-MDA5 antibody-positive ILD.

Renal TNFα activates the WNK phosphorylation cascade and contributes to salt-sensitive hypertension in chronic kidney disease.

The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

Encephalopathy Induced by High Plasma and Cerebrospinal Fluid Ceftriaxone Concentrations in a Hemodialysis Patient.

Encephalopathy is a rare side effect of cephalosporin treatment. We herein present a case of encephalopathy induced by ceftriaxone, a third-generation cephalosporin, in a patient with renal failure. An 86-year-old woman on maintenance hemodialysis received ceftriaxone for Helicobacter cinaedi bacteremia. Her mental status deteriorated during antibiotic treatment, and an electroencephalogram revealed triphasic waves predominantly in the frontal area. Her consciousness improved after the discontinuation of the antibiotic due to the suspicion of ceftriaxone-induced encephalopathy. This is the first reported case of encephalopathy associated with high plasma and cerebrospinal fluid ceftriaxone concentrations, and provides significant evidence for a causal relationship between the administration of ceftriaxone and the onset of encephalopathy.

Dialysis Case Volume Associated With In-Hospital Mortality in Maintenance Dialysis Patients.

INTRODUCTION: Accumulating evidence suggests that a large hospital volume (HV) is associated with favorable outcomes in various diseases or surgical procedures. The aim of this study is to clarify the correlation of HV and dialysis case volume (DCV) with in-hospital death in patients on maintenance dialysis. METHODS: The study cohort was derived from the Diagnosis Procedure Combination database, a national inpatient database in Japan, from 2012 to 2014. We included 382,689 admissions of maintenance dialysis patients over the age of 20 years in the analysis. HV was defined as the mean number of daily hospitalized patients, and DCV was defined as the mean number of annually hospitalized patients on maintenance dialysis. The primary outcome was in-hospital all-cause mortality, evaluated using multivariable logistic regression models across the respective quartiles of HV and DCV. RESULTS: The mean age of participants was 69 ± 12 years; 94% were receiving hemodialysis, and 21,182 patients (5.5%) died after hospitalization. In unadjusted models, larger HV and DCV were both associated with lower in-hospital mortality. However, this association remained significant only for DCV after adjustment for potential confounders, with multivariable-adjusted odds ratios of 0.82 (95% confidence interval [CI], 0.79-0.85), 0.76 (95% CI, 0.73-0.80), and 0.68 (95% CI, 0.65-0.72) for DCV 249 to 432, 433 to 713, and ≥714 (vs. ≤ 248) admissions per year, respectively. Multivariable subgroup analyses determined that this association was independent of age, sex, dialysis modality, Charlson Comorbidity Index, and emergency admission. CONCLUSION: Selective admission to hospitals with a large DCV may improve outcomes of dialysis patients.

Loop diuretics are associated with greater risk of sarcopenia in patients with non-dialysis-dependent chronic kidney disease.

INTRODUCTION: Sarcopenia, the age-related loss of muscle mass and function, frequently accompanies chronic kidney disease. The aim of this study was to clarify the prevalence and the risk factors for sarcopenia among patients with non-dialysis-dependent chronic kidney disease (NDD-CKD), focusing on the use of drugs. METHODS: We conducted a cross-sectional analysis on a cohort of 260 patients with NDD-CKD in a university hospital, recruited between June 2016 and March 2017. We extracted data on patient gender, age, cause of chronic kidney disease, use of drugs, and comorbidities that could potentially affect the prevalence of sarcopenia. Sarcopenia was diagnosed using the criteria of the Asian Working Group for Sarcopenia. Logistic regression analysis was performed to analyze the association of each factor on the prevalence of sarcopenia. RESULTS: 25.0% of our study subjects had sarcopenia. Multivariable analysis revealed that an increased risk of sarcopenia was significantly associated with age, male gender, body mass index, diabetes mellitus, and loop diuretic use (odds ratio, 4.59: 95% confidence interval, 1.81-11.61: P-value 0.001). CONCLUSIONS: In our cohort, the prevalence of sarcopenia in patients with NDD-CKD was high, and diuretics use, particularly loop diuretic use, was suggested to be a risk factor of sarcopenia. Although loop diuretics are commonly used in patients with CKD, careful consideration of the risk of sarcopenia may be necessary.

Role of ClC-K and barttin in low potassium-induced sodium chloride cotransporter activation and hypertension in mouse kidney.

The sodium chloride cotransporter (NCC) has been identified as a key molecule regulating potassium balance. The mechanisms of NCC regulation during low extracellular potassium concentrations have been studied in vitro. These studies have shown that hyperpolarization increased chloride efflux, leading to the activation of chloride-sensitive with-no-lysine kinase (WNK) kinases and their downstream molecules, including STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NCC. However, this mechanism was not studied in vivo Previously, we developed the barttin hypomorphic mouse (Bsndneo/neo mice), expressing very low levels of barttin and ClC-K channels, because barttin is an essential ß-subunit of ClC-K. In contrast with Bsnd-/- mice, Bsndneo/neo mice survived to adulthood. In Bsndneo/neo mice, SPAK and NCC activation after consuming a low-potassium diet was clearly impaired compared with that in wild-type (WT) mice. In ex vivo kidney slice experiment, the increase in pNCC and SPAK in low-potassium medium was also impaired in Bsndneo/neo mice. Furthermore, increased blood pressure was observed in WT mice fed a high-salt and low-potassium diet, which was not evident in Bsndneo/neo mice. Thus, our study provides in vivo evidence that, in response to a low-potassium diet, ClC-K and barttin play important roles in the activation of the WNK4-SPAK-NCC cascade and blood pressure regulation.

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.

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.

Impaired degradation of medullary WNK4 in the kidneys of KLHL2 knockout mice.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), and Cullin3 (CUL3) genes were identified as being responsible for hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). Normally, the KLHL3/CUL3 ubiquitin ligase complex degrades WNKs. In PHAII, the loss of interaction between KLHL3 and WNK4 increases levels of WNKs because of impaired ubiquitination, leading to abnormal over-activation of the WNK-OSR1/SPAK-NCC cascade in the kidney's distal convoluted tubules (DCT). KLHL2, which is highly homologous to KLHL3, was reported to ubiquitinate and degrade WNKs in vitro. Mutations in KLHL2 have not been reported in patients with PHAII, suggesting that KLHL2 plays a different physiological role than that played by KLHL3 in the kidney. To investigate the physiological roles of KLHL2 in the kidney, we generated KLHL2-/- mice. KLHL2-/- mice did not exhibit increased phosphorylation of the OSR1/SPAK-NCC cascade and PHAII-like phenotype. KLHL2 was predominantly expressed in the medulla compared with the cortex. Accordingly, medullary WNK4 protein levels were significantly increased in the kidneys of KLHL2-/- mice. KLHL2 is indeed a physiological regulator of WNK4 in vivo; however, its function might be different from that of KLHL3 because KLHL2 mainly localized in medulla.

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/EBPß 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.

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

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 ß-galactosidase (ß-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ß-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.

Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.

Involvement of selective autophagy mediated by p62/SQSTM1 in KLHL3-dependent WNK4 degradation.

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.

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.

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters.

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.

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.

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.

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.