The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Intracellular Ion Control of WNK Signaling.

The with no lysine (K) (WNK) kinases are an evolutionarily ancient group of kinases with atypical placement of the catalytic lysine and diverse physiological roles. Recent studies have shown that WNKs are directly regulated by chloride, potassium, and osmotic pressure. Here, we review the discovery of WNKs as chloride-sensitive kinases and discuss physiological contexts in which chloride regulation of WNKs has been demonstrated. These include the kidney, pancreatic duct, neurons, and inflammatory cells. We discuss the interdependent relationship of osmotic pressure and intracellular chloride in cell volume regulation. We review the recent demonstration of potassium regulation of WNKs and speculate on possible physiological roles. Finally, structural and mechanistic aspects of intracellular ion and osmotic pressure regulation of WNKs are discussed.

Circadian Rhythm Regulation by Pacemaker Neuron Chloride Oscillation in Flies.

Circadian rhythms in physiology and behavior sync organisms to external environmental cycles. Here, circadian oscillation in intracellular chloride in central pacemaker neurons of the fly, Drosophila melanogaster, is reviewed. Intracellular chloride links SLC12 cation-coupled chloride transporter function with kinase signaling and the regulation of inwardly rectifying potassium channels.

Chloride deregulation and GABA depolarization in MTOR related malformations of cortical development.

Focal Cortical Dysplasia, Hemimegalencephaly and Cortical Tuber are pediatric epileptogenic malformations of cortical development (MCDs) frequently pharmaco-resistant and mostly surgically treated by the resection of epileptic cortex. Availability of cortical resection samples allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes were identified. GABAa mediated paradoxical depolarization, related to altered chloride (Cl-) homeostasis, was shown to participate to ictogenesis in human pediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is still unclear. Here we studied the post translational interactions between the mTOR pathway and the regulation of cation-chloride cotransporters (CCC), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl- and ultimately GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo controls, diagnosed by histology plus genetic profiling) were operated for drug resistant epilepsy. Postoperative cortical tissues were recorded on multielectrode array (MEA) to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled Cl- conductance and equilibrium potential (EGABA) for GABA measurement. Of the 25 clinical cases, half harbored a somatic mutation in the mTOR pathway, while pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, that was reversed by WNK1 and SPAK antagonists (NEM and Staurosporine). mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR specific antagonist Rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in EGABA by rapamycin, measured after incorporation of MCD membranes to X. laevis oocytes, in line with a reestablishment of normal ECl-. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to chloride cotransporters deregulation, increased neuronal chloride levels and GABAa dysfunction in malformations of Cortical Development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.

Macrophage SPAK deletion limits a low potassium-induced kidney inflammatory program.

Inadequate dietary potassium (K+) consumption is a significant contributor to poor cardiovascular outcomes. A diet with reduced K+ content has been shown to cause salt-sensitive increases in blood pressure. More recently we have also shown that reductions in blood K+ can cause direct kidney injury, independent of dietary sodium (Na+) content. Here we investigated the role of the kinase, SPAK, in this kidney injury response. We observed that global SPAK deletion protected the kidney from damaging effects of a diet high in Na+ and low in K+. We hypothesized kidney macrophages were contributing to the injury response and that macrophage-expressed SPAK is essential in this process. We observed SPAK protein expression in isolated macrophages in vitro. Culture in K+-deficient medium increased SPAK phosphorylation and caused SPAK to localize to cytosolic puncta, reminiscent of WNK bodies identified along the distal nephron epithelium. WNK1 also adopted a punctate staining pattern under low K+ conditions and SPAK phosphorylation was prevented by treatment with the WNK inhibitor, WNK463. Macrophage-specific SPAK deletion in vivo protected against the low K+-mediated renal inflammatory and fibrotic responses. Our results highlight an important role for macrophages, and macrophage-expressed SPAK, in the propagation of kidney damage that occurs in response to reduced dietary K+ consumption.

Carotid artery vascular stenosis causes the blood-CSF barrier damage and neuroinflammation.

BACKGROUND: The choroid plexus (ChP) helps maintain the homeostasis of the brain by forming the blood-CSF barrier via tight junctions (TJ) at the choroid plexus epithelial cells, and subsequently preventing neuroinflammation by restricting immune cells infiltration into the central nervous system. However, whether chronic cerebral hypoperfusion causes ChP structural damage and blood-CSF barrier impairment remains understudied. METHODS: The bilateral carotid stenosis (BCAS) model in adult male C57BL/6 J mice was used to induce cerebral hypoperfusion, a model for vascular contributions to cognitive impairment and dementia (VCID). BCAS-mediated changes of the blood-CSF barrier TJ proteins, apical secretory Na+-K+-Cl- cotransporter isoform 1 (NKCC1) protein and regulatory serine-threonine kinases SPAK, and brain infiltration of myeloid-derived immune cells were assessed. RESULTS: BCAS triggered dynamic changes of TJ proteins (claudin 1, claudin 5) accompanied with stimulation of SPAK-NKCC1 complex and NF-κB in the ChP epithelial cells. These changes impacted the integrity of the blood-CSF barrier, as evidenced by ChP infiltration of macrophages/microglia, neutrophils and T cells. Importantly, pharmacological blockade of SPAK with its potent inhibitor ZT1a in BCAS mice attenuated brain immune cell infiltration and improved cognitive neurological function. CONCLUSIONS: BCAS causes chronic ChP blood-CSF damage and immune cell infiltration. Our study sheds light on the SPAK-NKCC1 complex as a therapeutic target in neuroinflammation.

The role of Cl- and K+ efflux in NLRP3 inflammasome and innate immune response activation.

Inflammation is part of innate immunity and is a natural response of the body to bacteria, virus, and any other pathogen infections or to damaged tissues. However, too much inflammation or chronic inflammation contributes to a wide variety of diseases such as inflammatory bowel disease, cancer, type 2 diabetes, heart disease, and autoimmune diseases such as rheumatoid arthritis. Recent studies underscored the critical role of K+ and Cl- efflux in the activation of the inflammasome. The NLRP3 inflammasome is a multiprotein complex that mediates the production of the proinflammatory cytokines IL-1ß and IL-18 and initiates the inflammatory cell death or pyroptosis. The NLRP3 inflammasome can be activated by multiple stimuli such as extracellular ATP, microbial toxins, ROS, mitochondrial DNA, or particulate matter. Although the precise mechanisms of NLRP3 activation and regulation by these diverse agonists remain unclear, multiple reports indicate that all NLRP3 agonists ultimately lead to a drop in intracellular concentration of potassium (K+ efflux) and chloride (Cl- efflux). The WNK-SPAK/OSR1-[N]KCC pathway plays a critical role in maintaining K+ and Cl- ion concentrations in the cell. Recent advances indicate that the WNK-SPAK-[N]KCC pathway plays a role in the activation of the innate immune response. This review highlights recent discoveries detailing how ion transport regulates innate immune cell response to inflammatory stimuli.

Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke.

BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. CONCLUSION: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains.

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinase are two serine/threonine protein kinases that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream 'With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. Interestingly, these crystal structures show a highly conserved primary pocket adjacent to a flexible secondary pocket. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to short peptides derived from WNK4 and NKCC1. Together, this work provides a platform that facilitates the design of CCT domain specific small molecule binders that inhibit SPAK- and OSR1-activation by WNK kinases, and these could be useful in treating hypertension and ischemic stroke.

DNMT1 Mediated CAHM Repression Promotes Glioma Invasion via SPAK/JNK Pathway.

Gliomas are the most common and fatal brain tumors worldwide. Abnormal DNA promoter methylation is an important mechanism for gene loss of tumor suppressors. A long non-coding RNA colorectal adenocarcinoma hypermethylated (CAHM) has been reported to be nearly deleted in glioblastomas (GBMs). Nevertheless, the roles of CAHM in gliomas remain unknown up to now. In the present study, 969 glioma samples downloaded from the CGGA and Gravendeel databases were included. We found that CAHM expression was correlated with glioma grades, molecular subtype, IDH mutation status, and 1q/19p codel status. In glioma cells, CAHM is hypermethylated by DNA methyltransferase1 (DNMT1) and the loss of CAHM expression could be reversed by 5-Aza-2'-deoxycytidine (5-Aza), a specific inhibitor of DNA methyltransferases. Besides, the expression of CAHM was negatively associated with overall survival in both primary and recurrent gliomas. Moreover, the result of Gene Ontology (GO) analysis suggested that CAHM participated in negatively regulating cell development, nervous system development, neurogenesis, and integrin-mediated signaling pathway. Overexpression of CAHM inhibited glioma cell proliferation, clone formation, and invasion. Further exploring results showed that CAHM overexpression suppressed glioma migration and invasion through SPAK/MAPK pathway. Collectively, this study disclosed that CAHM might be a suppressor in gliomas.

High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

Activation of the kidney sodium chloride cotransporter by the ß2-adrenergic receptor agonist salbutamol increases blood pressure.

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the ß2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to ß1-adrenergic receptors, the ß2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In ex vivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 µM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, ß2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

WNK-SPAK/OSR1-NCC kinase signaling pathway as a novel target for the treatment of salt-sensitive hypertension.

Hypertension is the most prevalent health condition worldwide, affecting ~1 billion people. Gordon's syndrome is a form of secondary hypertension that can arise due to a number of possible mutations in key genes that encode proteins in a pathway containing the With No Lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1). This pathway regulates the activity of the thiazide-sensitive sodium chloride cotransporter (NCC), which is responsible for NaCl reabsorption in the distal nephron. Therefore, mutations in genes encoding proteins that regulate the NCC proteins disrupt ion homeostasis and cause hypertension by increasing NaCl reabsorption. Thiazide diuretics are currently the main treatment option for Gordon's syndrome. However, they have a number of side effects, and chronic usage can lead to compensatory adaptations in the nephron that counteract their action. Therefore, recent research has focused on developing novel inhibitory molecules that inhibit components of the WNK-SPAK/OSR1-NCC pathway, thereby reducing NaCl reabsorption and restoring normal blood pressure. In this review we provide an overview of the currently reported molecular inhibitors of the WNK-SPAK/OSR1-NCC pathway and discuss their potential as treatment options for Gordon's syndrome.

OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant.

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K+ channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K+ channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.

Targeting the WNK-SPAK/OSR1 Pathway and Cation-Chloride Cotransporters for the Therapy of Stroke.

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl- and K+-Cl- cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.

MST3 Involvement in Na+ and K+ Homeostasis with Increasing Dietary Potassium Intake.

K+ loading inhibits NKCC2 (Na-K-Cl cotransporter) and NCC (Na-Cl cotransporter) in the early distal tubules, resulting in Na+ delivery to the late distal convoluted tubules (DCTs). In the DCTs, Na+ entry through ENaC (epithelial Na channel) drives K+ secretion through ROMK (renal outer medullary potassium channel). WNK4 (with-no-lysine 4) regulates the NCC/NKCC2 through SAPK (Ste20-related proline-alanine-rich kinase)/OSR1 (oxidative stress responsive). K+ loading increases intracellular Cl-, which binds to the WNK4, thereby inhibiting autophosphorylation and downstream signals. Acute K+ loading-deactivated NCC was not observed in Cl--insensitive WNK4 mice, indicating that WNK4 was involved in K+ loading-inhibited NCC activity. However, chronic K+ loading deactivated NCC in Cl--insensitive WNK4 mice, indicating that other mechanisms may be involved. We previously reported that mammalian Ste20-like protein kinase 3 (MST3/STK24) was expressed mainly in the medullary TAL (thick ascending tubule) and at lower levels in the DCTs. MST3 -/- mice exhibited higher ENaC activity, causing hypernatremia and hypertension. To investigate MST3 function in maintaining Na+/K+ homeostasis in kidneys, mice were fed diets containing various concentrations of Na+ and K+. The 2% KCl diets induced less MST3 expression in MST3 -/- mice than that in wild-type (WT) mice. The MST3 -/- mice had higher WNK4, NKCC2-S130 phosphorylation, and ENaC expression, resulting in lower urinary Na+ and K+ excretion than those of WT mice. Lower urinary Na+ excretion was associated with elevated plasma [Na+] and hypertension. These results suggest that MST3 maintains Na+/K+ homeostasis in response to K+ loading by regulation of WNK4 expression and NKCC2 and ENaC activity.

Low-dose L-NAME induces salt sensitivity associated with sustained increased blood volume and sodium-chloride cotransporter activity in rodents.

To investigate the cause of salt sensitivity in a normotensive animal model, we treated rats with a low-dose of the nitric oxide synthase inhibitor, L-NAME, that does not elevate blood pressure per se or induce kidney fibrosis. A high salt diet increased the circulating blood volume both in L-NAME-treated and nontreated animals for the first 24 hours. Thereafter, the blood volume increase persisted only in the L-NAME-treated rats. Blood pressure was higher in the L-NAME-treated group from the start of high salt diet exposure. Within the first 24 hours of salt loading, the L-NAME treated animals failed to show vasodilation and maintained high systemic vascular resistance in response to blood volume expansion. After four weeks on the high salt diet, the slope of the pressure-natriuresis curve was blunted in the L-NAME-treated group. An increase in natriuresis was observed after treatment with hydrochlorothiazide, but not amiloride, a change observed in parallel with increased phosphorylated sodium-chloride cotransporter (NCC). In contrast, a change in blood pressure was not observed in L-NAME-treated NCC-deficient mice fed a high salt diet. Moreover, direct L-NAME-induced NCC activation was demonstrated in cells of the mouse distal convoluted tubule. The vasodilatator, sodium nitroprusside, downregulated phosphorylated NCC expression. The effect of L-NAME on phosphorylated NCC was blocked by both the SPAK inhibitor STOCK2S-26016 and the superoxide dismutase mimetic TEMPO which also attenuated salt-induced hypertension. These results suggest that the initiation of salt sensitivity in normotensive rodents could be due to hyporeactivity of the vasculature and that maintaining blood pressure could result in a high circulating volume due to inappropriate NCC activity in the low-dose L-NAME model. Thus, even slightly impaired nitric oxide production may be important in salt sensitivity regulation in healthy rodents.

SPAK and OSR1 kinases bind and phosphorylate the ß2-Adrenergic receptor.

SPAK and OSR1 are two cytoplasmic serine/threonine protein kinases that regulate the function of a series of sodium, potassium and chloride co-transporters via phosphorylation. Over recent years, it has emerged that these two kinases may have diverse function beyond the regulation of ion co-transporters. Inspired by this, we explored whether SPAK and OSR1 kinases impact physically and phosphorylate the ß2-adrenergic receptor (ß2ADR). Herein, we report that the amino acid sequence of the human ß2ADR displays a SPAK/OSR1 consensus binding motif and using a series of pulldown and in vitro kinase assays we show that SPAK and OSR1 bind the ß2ADR and phosphorylate it in vitro. This work provides a notable example of SPAK and OSR1 kinases binding to a G-protein coupled receptor and taps into the potential of these protein kinases in regulating membrane receptors beyond ion co-transporters.

Structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK inhibitors blocking WNK kinase signaling.

We report here structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK (STE20/SPS1-related proline/alanine-rich kinase) inhibitors. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension, and therefore inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for antihypertensive drugs. Based on the structure of lead compound 2, we examined the SAR of N-(4-phenoxyphenyl)benzamide derivatives, and developed compound 20l as a potent SPAK inhibitor. Compounds 20l is a promising candidate for a new class of antihypertensive drugs.

Icariin alleviates hypoxia-induced damage in MC3T3-E1 cells by downregulating TALNEC2.

Osteonecrosis is a harmful musculoskeletal disease. We aim to detect the effects of icariin (ICA) in MC3T3-E1 cell. MC3T3-E1 cell was pretreated with ICA and was subjected to hypoxia stimuli. The tumor-associated long noncoding RNA expressed on chromosome 2 (TALNEC2) overexpression or silencing vectors (pTALNEC2 or si-TALNEC2) was utilized for MC3T3-E1 cell transfection. Viability and apoptosis rate were individually tested by cell counting kit-8 and Annexin V-fluorescein isothiocyanate/propidium iodide kit untied with flow cytometry. The alkaline phosphatase activity (ALP) activity was tested through ALP assay. The quantitative reverse transcription PCR or Western blot was performed for elements detection at the RNA or protein level. Hypoxia treatment induced viability inhibition and CyclinD1 reduction, but elevation of p53 and p16. It also promoted apoptosis by increasing apoptotic cells, Bax, and cleaved-poly ADP-ribose polymerase but decreasing Bcl-2. Also, hypoxia stimuli restrained ALP activity, and osteopontin, osteocalcin, and Runt-related transcription factor 2 expression. Those effects caused by hypoxia stimuli were all reversed by ICA. TALNEC2 was downregulated by ICA, whose impacts were subsequently abolished by pTALNEC2. Silencing TALNEC2 displayed similar effects with ICA. But the apoptosis was not affected by si-TALNEC2. ICA blocked ste20-related proline/alanine-rich kinase/c-Jun N-terminal kinase (SPAK/JNK) but triggered phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in MC3T3-E1 cell by suppressing TALNEC2. ICA relieved hypoxia-stimulated damage by restraining TALNEC2 through blocking SPAK/JNK and triggering PI3K/AKT/mTOR in the MC3T3-E1 cell.