title: Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke.
abstract: OBJECTIVE
  WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage.
  
  
  METHODS
  Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method.
  
  
  RESULTS
  WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr(203)/Thr(207)/Thr(212), as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia.
  
  
  CONCLUSIONS
  These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Gulnaz
authors.initials: G
authors.lastname: Begum
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Kristopher T
authors.initials: KT
authors.lastname: Kahle
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Liaoliao
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Shaoxia
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Yejie
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Boris E
authors.initials: BE
authors.lastname: Shmukler
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Sung-Sen
authors.initials: SS
authors.lastname: Yang
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Shih-Hua
authors.initials: SH
authors.lastname: Lin
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Seth L
authors.initials: SL
authors.lastname: Alper
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Boston, MA (K.T.K.); Manton Center for Orphan Diseases, Harvard Medical School, MA (K.T.K.); Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (SS.Y., SH.L); Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA (D.S).
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
mesh_terms: D000818:Animals; D001930:Brain Injuries / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D002478:Cells, Cultured; D005260:Female; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009422:Nervous System Diseases / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000172:deficiency; D020127:Recovery of Function / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D020521:Stroke / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology
chemical_list: D017346:Protein Serine-Threonine Kinases; C507511:Wnk3 protein, mouse
keywords: KCC2 cotransporter; NKCC1 cotransporter; OSR1 kinase; SPAK Kinase; bumetanide