Ste20-related Proline-alanine-rich Kinase Research Articles

Consequences of SPAK inactivation on Hyperkalemic Hypertension caused by WNK1 mutations: evidence for differential roles of WNK1 and WNK4

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:SPAK243A/243A mice display an intermediate phenotype, between that of control and SPAK243A/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 SPAK243A/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.

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation

K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

Dietary K+ and Cl- independently regulate basolateral conductance in principal and intercalated cells of the collecting duct.

The renal collecting duct contains two distinct cell types, principal and intercalated cells, expressing potassium Kir4.1/5.1 (KCNJ10/16) and chloride ClC-K2 (ClC-Kb in humans) channels on their basolateral membrane, respectively. Both channels are thought to play important roles in controlling systemic water-electrolyte balance and blood pressure. However, little is known about mechanisms regulating activity of Kir4.1/5.1 and ClC-K2/b. Here, we employed patch clamp analysis at the single channel and whole cell levels in freshly isolated mouse collecting ducts to investigate regulation of Kir4.1/5.1 and ClC-K2/b by dietary K+ and Cl- intake. Treatment of mice with high K+ and high Cl- diet (6% K+, 5% Cl-) for 1week significantly increased basolateral K+-selective current, single channel Kir4.1/5.1 activity and induced hyperpolarization of basolateral membrane potential in principal cells when compared to values in mice on a regular diet (0.9% K+, 0.5% Cl-). In contrast, basolateral Cl--selective current and single channel ClC-K2/b activity was markedly decreased in intercalated cells under this condition. Substitution of dietary K+ to Na+ in the presence of high Cl- exerted a similar inhibiting action of ClC-K2/b suggesting that the channel is sensitive to variations in dietary Cl- per se. Cl--sensitive with-no-lysine kinase (WNK) cascade has been recently proposed to orchestrate electrolyte transport in the distal tubule during variations of dietary K+. However, co-expression of WNK1 or its major downstream effector Ste20-related proline-alanine-rich kinase (SPAK) had no effect on ClC-Kb over-expressed in Chinese hamster ovary (CHO) cells. Treatment of mice with high K+ diet without concomitant elevations in dietary Cl- (6% K+, 0.5% Cl-) elicited a comparable increase in basolateral K+-selective current, single channel Kir4.1/5.1 activity in principal cells, but had no significant effect on ClC-K2/b activity in intercalated cells. Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the stimulatory actions of high K+ intake on Kir4.1/5.1 channels in principal cells but was ineffective to alter ClC-K2/b activity and basolateral Cl- conductance in intercalated cells. In summary, we report that variations of dietary K+ and Cl- independently regulate basolateral potassium and chloride conductance in principal and intercalated cells. We propose that such discrete mechanism might contribute to fine-tuning of urinary excretion of electrolytes depending on dietary intake.

Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2 pan antibody but not by the N-terminal KCC2a antibody in KCC2b-deficient neurons. One possible cause for the epitope masking is the binding site of Ste20-related proline-alanine-rich kinase (SPAK) in the KCC2a N-terminus. SPAK, a known regulator of K-Cl cotransporters, was co-immunoprecipitated in a complex with KCC2a but not KCC2b isoform. Moreover, SPAK overexpression decreased the transport activity of KCC2a but not that of KCC2b, as revealed by rubidium flux assay in HEK293 cells. Thus, our data indicate that both KCC2 isoforms perform as chloride cotransporters in neuronal cells, while their N-terminal heterogeneity could play an important role in fine-tuning of the K-Cl transport activity.

Constitutively Active SPAK Causes Hyperkalemia by Activating NCC and Remodeling Distal Tubules.

Aberrant activation of with no lysine (WNK) kinases causes familial hyperkalemic hypertension (FHHt). Thiazide diuretics treat the disease, fostering the view that hyperactivation of the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is solely responsible. However, aberrant signaling in the aldosterone-sensitive distal nephron (ASDN) and inhibition of the potassium-excretory renal outer medullary potassium (ROMK) channel have also been implicated. To test these ideas, we introduced kinase-activating mutations after Lox-P sites in the mouse Stk39 gene, which encodes the terminal kinase in the WNK signaling pathway, Ste20-related proline-alanine-rich kinase (SPAK). Renal expression of the constitutively active (CA)-SPAK mutant was specifically targeted to the early DCT using a DCT-driven Cre recombinase. CA-SPAK mice displayed thiazide-treatable hypertension and hyperkalemia, concurrent with NCC hyperphosphorylation. However, thiazide-mediated inhibition of NCC and consequent restoration of sodium excretion did not immediately restore urinary potassium excretion in CA-SPAK mice. Notably, CA-SPAK mice exhibited ASDN remodeling, involving a reduction in connecting tubule mass and attenuation of epithelial sodium channel (ENaC) and ROMK expression and apical localization. Blocking hyperactive NCC in the DCT gradually restored ASDN structure and ENaC and ROMK expression, concurrent with the restoration of urinary potassium excretion. These findings verify that NCC hyperactivity underlies FHHt but also reveal that NCC-dependent changes in the driving force for potassium secretion are not sufficient to explain hyperkalemia. Instead, a DCT-ASDN coupling process controls potassium balance in health and becomes aberrantly activated in FHHt.

SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD.

Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

Future considerations based on the information from Barrter's and Gitelman's syndromes.

Bartter and Gitelman syndromes are typical normotensive salt losing hypokalaemic tubulopathies. Their pathogenesis was gradually deciphered in the past 5 decades, first by typical salt balance studies and histopathology, followed by genetic characterization and discovery of the affected different ion channels. Although the different genotypic subtypes were originally thought to show a similar phenotype, important clinical and biochemical differences can now be found. New findings on the regulation of these channels, as well as the recent discovery of newly affected genes, merit an update on this topic. Na-K-2CL cotransporter and NaCl cotransporter, the two main luminal channels in the thick ascending limb and distal convoluted tubule were found to be regulated by Ste 20-related proline alanine-rich kinase and oxidative stress response kinase. Knockout mice to these channels express a Bartter-like phenotype. MAGE-D2 is new gene found to cause severe polyhydramnios and transient postnatal Bartter-like syndrome. Variants in the different channels causing Bartter syndromes/Gitelman syndromes may also confer susceptibility for hypertension or protect against it. It remains to be determined if polymorphism or epigenetic changes in these genes and proteins may affect salt handling, explaining, apart from Bartter syndromes and Gitelman syndromes, also hypertension or stroke tendency, or both.

Sodium-chloride cotransporter activity regulated by extracellular potassium

The thiazide-sensitive sodium-chloride cotransporter (NCC) is exclusively expressed in the apical membrane of the renal distal convoluted tubule (DCT), and it is important for maintaining fluid and salt balance. NCC is responsible for electroneutral NaCl reabsorption, and its activity is determined by phosphorylation, which was reported to be regulated by WNK-stimulated Ste20-type kinases, Ste20-related proline alanine-rich kinase, and oxidative stress response 1 kinase. WNK kinases have chloride-binding sites, and WNK4 has the highest sensitivity to intracellular chloride concentration ([Cl−]i) compared with WNK1 and WNK3. NCC dysfunction often comes together with abnormal urinary potassium excretion, which is not only highlighted by Mendelian disorders such as familial hyperkalemic hypertension (FHHt) and Gitelman syndrome but also presented more frequently by long-term usage of thiazides, specific inhibitors of NCC. Recent studies have shown that extracellular potassium (K+) can modulate DCT cell membrane voltage and in turn intracellular Cl−, which regulates phosphorylation of WNK kinases. Additional Cl− independent mechanisms were also reported by several groups. This paper is a brief review of the recent discoveries on mechanisms of NCC regulation by extracellular potassium.

6-(3,4-Dihydro-1H-isoquinoline-2-yl)-N-(6-methoxypyridine-2-yl) nicotinamide-26 (DIMN-26) decreases cell proliferation by induction of apoptosis and downregulation of androgen receptor signaling in human prostate cancer cells

Previously, we reported that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl) nicotinamide (DIMN) analogues inhibited the growth of prostate cancer cells as an anti-androgenic compound. In the present study, we evaluated cytotoxic effects of these DIMN derivatives and found that DIMN-26 most potently inhibited the proliferation of the LNCap-LN3 androgen-dependent and DU145 androgen-independent prostate cancer cells through induction of G2/M phase cell cycle arrest and subsequent apoptosis. The G2/M phase arrest was found due to increases in the activation of cdc2 (also known as cyclin-dependent kinase 1, CDK1)/cyclin B1 complex. DIMN-26 also induced apoptosis in LNCap-LN3 and DU145 prostate cancer cells through activation of caspase-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). In addition, DIMN-26 caused the dephosphorylation and mitochondrial accumulation of Bad protein and induced the loss of mitochondria membrane potential, consequently releasing cytochrome c into the cytosol of the cell. Furthermore, overexpression of AKT protein significantly reduced DIMN-26-induced PARP-1 cleavage and p-Bad decrease and cdc2 activation. In addition, DIMN-26 inhibited the 5α-dihydrotestosterone (DHT)-induced cell growth and proliferation and nuclear translocation and transcriptional activities of androgen receptor (AR) in LNCap-LN3 prostate cancer cells. Consistent with these findings, DIMN-26 significantly inhibited the DHT-induced expression of AR-response genes (ARGs), such as prostate-specific antigen (PSA), AR, β2-microglobulin (B2M), selenoprotein P (SEPP1), and ste20-related proline-alanine-rich kinase (SPAK) in LNCap-LN3 prostate cancer cells. Taken together, these results suggest that DIMN-26 plays a therapeutic role not only in induction of G2/M arrest and apoptosis but also in suppression of androgen receptor signaling in androgen-dependent and androgen-independent prostate cancer cells.

The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

The regulation of Na+Cl- cotransporter by with-no-lysine kinase 4.

Abundant evidence supports that the NaCl cotransporter (NCC) activity is tightly regulated by the with-no-lysine (WNK) kinases. Here, we summarize the data regarding NCC regulation by WNKs, with a particular emphasis on WNK4. Several studies involving in-vivo and in-vitro models have provided paradoxical data regarding WNK4 regulation of the NCC. Although some studies show that WNK4 can activate the NCC, other equally compelling studies show that WNK4 inhibits the NCC. Recent studies have shown that WNK4 is regulated by the intracellular chloride concentration ([Cl]i), which could account for these paradoxical results. In conditions of high [Cl]i, WNK4 could act as an inhibitor via heterodimer formation with other WNKs. In contrast, when [Cl]i is low, WNK4 can activate Ste20-related, proline-alanine-rich kinase (SPAK)/oxidative stress responsive kinase 1 (OSR1) and thus the NCC. Modulation of WNK4 by [Cl]i has been shown to account for the potassium-sensing properties of the distal convoluted tubule. Other regulators of WNK4 include hormones and ubiquitination. Modulation of WNK4 activity by [Cl]i can account for its dual role on the NCC, and this has important physiological implications regarding the regulation of extracellular potassium concentration. Defective regulation of WNKs by ubiquitination explains most cases of familial hyperkalemic hypertension.

The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.

Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.

Abstract WP269: New Insights Into Worsened Cerebral Ischemia in Hypertensive Rats: Roles of WNK-SPAK/OSR1-NKCC1 Signaling

Background and Purpose: WNK kinases [with-no-lysine (K)], Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) are a family of evolutionarily conserved regulators of ion transporter proteins. The kinases alter the phosphorylation state of ion transporter proteins and so play an important role in renal salt handling and in the pathogenesis of hypertension. We previously reported that pharmacological inhibition or genetic knockout of Na+-K+-2Cl- cotransporter 1 (NKCC1) is neuroprotective against ischemic injury. Our current study investigated whether WNK-SPAK/OSR1-NKCC1 signaling is augmented in cerebral ischemia in hypertension. Methods: Cerebral ischemia was induced in adult male Wistar Kyoto (WKY) or spontaneously hypertensive rats (SHR) by 2-hour occlusion of the left middle cerebral artery (MCAO) followed by reperfusion. Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Another two cohorts of WKY and SHR were treated with saline or NKCC1 inhibitor bumetanide (BMT, 10 mg/kg/day, i.p.) after MCAO. In these animals, ischemic infarct volume was determined by TTC staining and behavioral deficits were analyzed by neurological scoring and turning-alley test. Results: Basal levels of total NKCC1 protein were significantly higher in the contralateral (CL) cortical tissues in SHR brains than WKY brains. Ischemia was associated with increased phosphorylation of NKCC1 in the ipsilateral (IL) cortex of SHR, but not WKY at 6 hours post-ischemia. WNK1 increased 6.1±1.5 fold and WNK4 increased 8.7±1.7 fold in IL cortex of SHR, but not in that of WKY, and phosphorylated SPAK/OSR1 was unchanged. Lastly, sensitivity to pharmacological NKCC1 inhibition was higher in SHR than WKY rats. BMT blunted the MCAO-induced infarction and improved neurobehavioral score and sensory motor function in SHR rats. Conclusion: These findings suggest that augmented WNK1/4-NKCC1 signaling is responsible, at least in part, for the increased susceptibility to ischemic brain damage associated with hypertension. They highlight the potential for inhibition of WNK1/4-NKCC1 signaling as part of anti-ischemic stroke therapy. Supported by NIH Grant R01 NS38118 (D Sun).

Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38MAPK-MK pathway.

The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway has been reported to be a crucial signaling pathway for triggering pseudohypoaldosteronism type II (PHAII), an autosomal dominant hereditary disease that is characterized by hypertension. However, the molecular mechanism(s) by which the WNK-SPAK/OSR1 pathway is regulated remain unclear. In this report, we identified WNK4 as an interacting partner of a recently identified MAP3K, apoptosis signal-regulating kinase 3 (ASK3). We found that WNK4 is phosphorylated in an ASK3 kinase activity-dependent manner. By exploring the ASK3-dependent phosphorylation sites, we identified Ser575 as a novel phosphorylation site in WNK4 by LC-MS/MS analysis. ASK3-dependent WNK4 Ser575 phosphorylation was mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway. Osmotic stress, as well as hypotonic low-chloride stimulation, increased WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway. ASK3 was required for the p38MAPK activation induced by hypotonic stimulation but was not required for that induced by hypertonic stimulation or hypotonic low-chloride stimulation. Our results suggest that the p38MAPK-MK pathway might regulate WNK4 in an osmotic stress-dependent manner but its upstream regulators might be divergent depending on the types of osmotic stimuli.

WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats

With-no-lysine kinase (WNK) and Na+-K+-2Cl− cotransporter 1 (NKCC1) are involved in the pathogenesis of hypertension. In this study, we investigated the WNK-NKCC1 signaling pathway in spontaneously hypertensive rats (SHR) and their associated susceptibility to stroke injury. Basal NKCC1 protein levels were higher in SHR than in normotensive Wistar Kyoto (WKY) rat brains. After inducing ischemic stroke, adult male WKY and SHR received either saline or NKCC1 inhibitor bumetanide (10 mg/kg/day, i.p.) starting at 3-h post-reperfusion. NKCC1 inhibition blunted the extent of ischemic infarction in SHR and improved their neurobehavioral functions. Interestingly, ischemia led to increased NKCC1 phosphorylation in SHR but not in WKY rats. Pronounced elevation of WNK1, WNK2 and WNK4 protein and downregulation of WNK3 were detected in ischemic SHR, but not in ischemic WKY rats. Upregulation of WNK-NKCC1 complex in ischemic SHR brain was associated with increased Ca2+-binding protein 39 (Cab39), without increases in Ste20-related proline alanine-rich kinase or oxidative stress-responsive kinase-1. Moreover, subacute middle cerebral artery stroke human brain autopsy exhibited increased expression of NKCC1 protein. We conclude that augmented WNK-Cab39-NKCC1 signaling in SHR is associated with an increased susceptibility to ischemic brain damage and may serve as a novel target for anti-hypertensive and anti-ischemic stroke therapy.

Kelch-like 3/Cullin 3 ubiquitin ligase complex and WNK signaling in salt-sensitive hypertension and electrolyte disorder.

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and thiazide sensitivity. Mutations in with-no-lysine kinase 1 (WNK1) and WNK4 genes are reported to cause PHAII. Rigorous studies have demonstrated that WNK kinases constitute a signaling cascade with oxidative stress-responsive gene 1 (OSR1), Ste20-related proline-alanine-rich kinase (SPAK) and the solute carrier family 12a (SLC12a) transporter, including thiazide-sensitive NaCl cotransporter. The WNK-OSR1/SPAK-SLC12a signaling cascade is present in the kidneys and vascular smooth muscle cells (VSMCs) and regulates salt sensitivity physiologically, i.e. urinary sodium excretion and arterial tone by various hormonal and dietary factors. However, although it was clear that the abnormal activation of this signaling cascade is the molecular basis of PHAII, the molecular mechanisms responsible for the physiological regulation of WNK signaling and the effect of WNK4 mutations on PHAII pathogenesis are poorly understood. Two additional genes responsible for PHAII, Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), were identified in 2012. WNK1 and WNK4 have been shown to be substrates of KLHL3-CUL3 E3 ubiquitin ligase both in vitro and in vivo In PHAII, the loss of interaction between KLHL3 and WNK4 induces increased levels of WNK kinases due to impaired ubiquitination. These results indicate that WNK signaling is physiologically regulated by KLHL3/CUL3-mediated ubiquitination. Here, we review recent studies investigating the pathophysiological roles of the WNK signaling cascade in the kidneys and VSMCs and recently discovered mechanisms underlying the regulation of WNK signaling by KLHL3 and CUL3.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells.

Angiotensin II (ANG II) increases thiazide-sensitive sodium-chloride cotransporter (NCC) activity both acutely and chronically. ANG II has been implicated as a switch that turns WNK4 from an inhibitor of NCC into an activator of NCC, and ANG II's effect on NCC appears to require WNK4. Chronically, ANG II stimulation of NCC results in an increase in total and phosphorylated NCC, but the role of NCC phosphorylation in acute ANG II actions is unclear. Here, using a mammalian cell model with robust native NCC activity, we corroborate the role that ANG II plays in WNK4 regulation and clarify the role of Ste20-related proline alanine-rich kinase (SPAK)-induced NCC phosphorylation in ANG II action. ANG II was noted to have a biphasic effect on NCC, with a peak increase in NCC activity in the physiologic range of 10(-11) M ANG II. This effect was apparent as early as 15 min and remained sustained through 120 min. These changes correlated with significant increases in NCC surface protein expression. Knockdown of WNK4 expression sharply attenuated the effect of ANG II. SPAK knockdown did not affect ANG II action at early time points (15 and 30 min), but it did attenuate the response at 60 min. Correspondingly, NCC phosphorylation did not increase at 15 or 30 min, but increased significantly at 60 min. We therefore conclude that within minutes of an increase in ANG II, NCC is rapidly trafficked to the cell surface in a phosphorylation-independent but WNK4-dependent manner. Then, after 60 min, ANG II induces SPAK-dependent phosphorylation of NCC.

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.