WNK3‐SPAK/OSR1‐NKCC1 Signaling Pathway in Ischemic Brain Injury

Abstract

Ischemia-mediated activation of Na-K-Cl cotransporter (NKCC1) leads to Na+ overload, cytotoxic edema, and cell death. However, the molecular mechanisms underlying these processes are not understood. Here we investigated the role of WNK (with no lysine = K)-SPAK/OSR1 kinase signaling pathway in regulating the function of NKCC1 in cerebral ischemia. We show that genetic deletion of WNK3, a WNK family member highly expressed in brain, or siRNA-mediated knockdown of SPAK or OSR1, protects against ischemic neuroglial death triggered by oxygen-glucose deprivation/reoxygenation. Using phospho specific antibodies, we detected robust up regulation of p-NKCC1 as well as p-SPAK/p-OSR1expression in peri-infarct cortex, striatum and corpus callosum in wild type mice subjected to transient middle cerebral artery occlusion. In WNK3 knockout mice, ischemia-induced hyper-phosphorylations of both the SPAK/OSR1 catalytic T-loop and NKCC1 are abolished, infarct volume and axonal demyelination are reduced, and neurobehavioral recovery is accelerated. These data provide fresh insight into the roles of ion transporters and their regulatory kinases in ischemic neuroglial injury, and identify the WNK3-SPAK/OSR1 kinase complex as a compelling target for novel neuroprotective strategies following stroke. Support: NIH R01NS38118