153 research outputs found
Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport.
No full textThis is the author accepted manuscript. The final version is available from Taylor & Francis via the DOI in this record.Introduction: The mammalian SPS1-related proline/alanine-rich serine-threonine kinase SPAK (STK39) modulates ion transport across and between epithelial cells in response to environmental stimuli such osmotic stress and inflammation. Research over the last decade has established a central role for SPAK in the regulation of ion and water transport in the distal nephron, colonic crypts, and pancreatic ducts, and has implicated deregulated SPAK signaling in NaCl-sensitive hypertension, ulcerative colitis and Crohn's disease, and cystic fibrosis. Areas covered: We review recent advances in our understanding of the role of SPAK kinase in the regulation of epithelial transport. We highlight how SPAK signaling - including its upstream Cl- sensitive activators, the WNK kinases, and its downstream ion transport targets, the cation- Cl- cotransporters contribute to human disease. We discuss prospects for the pharmacotherapeutic targeting of SPAK kinase in specific human disorders that feature impaired epithelial homeostasis. Expert opinion: The development of novel drugs that antagonize the SPAK-WNK interaction, inhibit SPAK kinase activity, or disrupt SPAK kinase activation by interfering with its binding to MO25α/β could be useful adjuncts in essential hypertension, inflammatory colitis, and cystic fibrosis.This paper was supported in part by NIH grant R21GM118944 to E. Delpire; and the March of Dimes Basil O’Connor Award, Simons Foundation SFARI Awardand NIH award (NRCDP K12) to K. T. Kahle
Expression of SPAK in mouse hippocampus at various stages of PISE.
No full text<p>(a) (b), (c) Evaluation of SPAK expression in the CA1 and CA3 regions of mice hippocampi after PISE by immunohistochemistry. A: CA1 or CA3 region of hippocampus indicated with a box. B-F: The region selected in “A” from the blank group (B), sham group (C), day after PISE (D), day 14 after PISE (E), and day 45 after PISE (F). (d) (e) Evaluation of SPAK protein expression in mice hippocampi after PISE by Western blotting analysis. (f) Evaluation of SPAK mRNA expression in mice hippocampi after PISE by RT-PCR. Values are mean ± SD, <sup># </sup><i>P</i>< 0.05 versus the blank and sham groups, <sup>## </sup><i>P</i><0.05 versus the blank sham groups, * <i>P</i><0.05 versus day 1 and day 45 in the PISE-affected group. Scale bars in “F” apply to B-F.</p
Immunofluorescence images of Ncc and Nkcc2 in kidneys. (Spak experiment series )
No full text<p>Representative immunofluorescence image of (A) total Ncc, (B) p-Ncc (T53), (C) p-Ncc (T58), (D) p-Ncc (S71), (E) total Nkcc2, and (F) p-Nkcc2 (T96) in kidneys of WT, <i>Wnk4</i><sup>D561A/+</sup>, <i>Spak</i><sup>−/−</sup>, and <i>Wnk4</i><sup>D561A/+</sup>.<i>Spak</i><sup>−/−</sup> mice. The scale bars indicate 20 µm.</p
Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke.
No full textThis is the final version. Available from BMC via the DOI in this record. Availability of data and materials:
Supporting data and information about used material are available from the
corresponding author on reasonable request.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.National Institutes of HealthVeterans AdministrationVA Research Career Scientist awardUPMC Endowed Chair professorship for Brain Disorders Researc
Enhanced FGF23 Serum Concentrations and Phosphaturia in Gene Targeted Mice Expressing WNK-Resistant Spak
No full textContains fulltext :
107752.pdf (Publisher’s version ) (Open Access)Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na(+),K(+),2Cl(-) cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak(tg/tg)) and in mice expressing wild type SPAK (spak(wt/wt)). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak(tg/tg) mice than in spak(wt/wt) mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak(tg/tg) mice compared to spak(wt/wt) mice. Treatment of spak(wt/wt) mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca(2+) and phosphate excretion
<i>Spak</i> gene deletion normalizes Ncc phosphorylation but not Nkcc2 phosphorylation in PHA II.
No full text<p>Shown is the representative immunoblot (top, n = 3/group) and densitometry (bottom, n = 6/group) of (A) total Ncc, (B) p-Ncc (T53), (C) p-Ncc (T58), (D) p-Ncc (S71), (E) total Nkcc2, and (F) p-Nkcc2 (T96) in the whole kidneys of WT, <i>Wnk4</i><sup>D561A/+</sup>, <i>Spak</i><sup>−/−</sup>, and <i>Wnk4</i><sup>D561A/+</sup>.<i>Spak</i><sup>−/−</sup> mice, representively. The results are shown as mean ± SD. <b><sup>a</sup></b><i>p</i><0.05 vs. WT. <b><sup>b</sup></b><i>p</i><0.05 vs. <i>Wnk4</i><sup>D561A/+</sup>. <b><sup>c</sup></b><i>p</i><0.05 vs. <i>Spak</i><sup>−/−</sup>.</p
Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways
No full textIon cotransporters, such as the Na+/Cl-cotransporter (NCC), control renal salt re-absorption and are regulated by the WNK-signalling pathway, which is over-stimulated in patients suffering from Gordon's hypertension syndrome. Here, we study the regulation of the NKCC2 (SLC12A1) ion cotransporter that contributes towards similar to 25% of renal salt re-absorption and is inhibited by loop-diuretic hypertensive drugs. We demonstrate that hypotonic low-chloride conditions that activate the WNK1-SPAK and OSR1 pathway promote phosphorylation of NKCC2 isoforms (A, B and F) at five residues (Ser91, Thr95, Thr100, Thr105 and Ser130). We establish that the SPAK and OSR1 kinases activated by WNK interact with an RFQV motif on NKCC2 and directly phosphorylate Thr95, Thr100, Thr105 and, possibly, Ser91. Our data indicate that a SPAK-OSR1-independent kinase, perhaps AMP-activated protein kinase (AMPK), phosphorylates Ser130 and that phosphorylation of Thr105 and Ser130 plays the most important roles in stimulating NKCC2 activity. In contrast with NCC, whose membrane translocation is triggered by SPAK-OSR1 phosphorylation, NKCC2 appears to be constitutively at the membrane. Our findings provide new insights into how NKCC2 is regulated and suggest that inhibitors of SPAK and/or OSR1 for the treatment of hypertension would be therapeutically distinct from thiazide or loop diuretics, as they would suppress the activity of both NCC and NKCC2.</p
NF-κB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice
No full textThis is the author accepted manuscript. The final version is available from Lippincott, Williams & Wilkins via the DOI in this recordData availability: The data that support the findings of this study are available within the article and its Data Supplement.BACKGROUND: Worsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes. METHODS: Saline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined. RESULTS: Stroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow. CONCLUSIONS: The ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.Veteran AffairsNational Institutes of Health (NIH)AH
Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1
No full textMutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl- cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.</p
Effects of cadmium on the cuttlefish Sepia pharaonis' arginine kinase: unfolding kinetics integrated with computational simulations
No full textArginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd2+ on the cuttlefish Sepia pharaonis’ arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band？on？SDS-PAGE. Cd2+ effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd2+ induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd2+ induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd2+ showed that Cd2+ partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd2+-induced inactivation of SPAK. These results demonstrate the effect of Cd2+ on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd2+ in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefishopen
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