Over-stimulation of Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) activity contributes to cerebral ischemic damage. NKCC1 transports 1Na+, 1K+, and 2Cl- ions into cells and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system under physiological conditions. Under ischemic conditions, NKCC1 activation causes intracellular Na+ and Cl- overload in astrocytes and neurons. The intracellular Na+ overload subsequently stimulates the reverse mode operation of Na+/Ca2+ exchange and leads to a delayed, secondary cytosolic Ca2+ rise and Ca2+ dysregulation in ER and mitochondria. Most importantly, either pharmacological inhibition or genetic ablation of NKCC1 shows significant neuroprotective effects in in vivo focal ischemia model and in vitro ischemia model. Despite of the neuroprotective effects in ischemic brain damage by blocking NKCC1 activity, it remains unknown how NKCC1 protein is stimulated in ischemic brains and what are the up- stream regulatory mechanisms. The recent research reveals that a novel WNK kinase family (with no lysine = K) and its two key down-stream substrates SPAK (Ste20/SPS1-related proline/alanine-rich kinase) and its homolog OSR1 (oxidative stress-responsive kinase 1) are evolutionarily conserved regulators of ion transporters by altering their net phosphorylation state. Our preliminary study shows that triansient focal ischemia triggered a significant stimulation of the key proteins (p-SPAK, p-OSR1 and p-NKCC1) in neurons and in white matter oligodendrocytes of peri-infarct regions during 6- 72 h reperfusion. Most importantly, inhibition of the WNK-SPAK/OSR1 signaling pathway with siRNA or transgenic knockout approaches is protective against ischemic cell death. In addition, spontaneously hypertensive rats (SHRs) exhibited higher sensitivity to NKCC1 inhibition. These new findings led us to hypothesize that: 1) the WNK-SPAK/OSR1 signaling pathway is activated following cerebral ischemia and functions as up-stream regulators of NKCC1 through protein phosphorylation;2) the activation of the WNK-SAPK/OSR1-NKCC1 signaling cascade contributes to both grey and white matter damage after ischemia;3) augmentation of the WNK- SPAK/OSR1-NKCC1 signaling pathway in hypertensive brains is in part responsible for the worsened ischemic brain damage in hypertension. These hypotheses will be tested in four Specific Aims. A positive outcome of this project will generate new knowledge on whether the WNK-SPAK/OSR1-NKCC1 signaling pathway is a novel target for developing more effective stroke therapy. This will pave a foundation for testing future novel inhibitors of WNK- SPAK/OSR1 in stroke therapy.

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This proposal is to study roles of Na-K-Cl cotransporter and its kinases in cerebral ischemic damage. The long-term goal of the research is to understand how ion transport proteins and their kinases contribute to disruption of ionic homeostasis and brain damage following ischemia and to determine whether these proteins are potential targets for developing more effective stroke treatments.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Brain Injury and Neurovascular Pathologies Study Section (BINP)
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Bosetti, Francesca
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University of Pittsburgh
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Wu, Connie; Sun, Dandan (2015) GABA receptors in brain development, function, and injury. Metab Brain Dis 30:367-79
Leng, Tiandong; Shi, Yejie; Xiong, Zhi-Gang et al. (2014) Proton-sensitive cation channels and ion exchangers in ischemic brain injury: new therapeutic targets for stroke? Prog Neurobiol 115:189-209
Zhu, Wen; Begum, Gulnaz; Pointer, Kelli et al. (2014) WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration. Mol Cancer 13:31
Cengiz, Pelin; Kintner, Douglas B; Chanana, Vishal et al. (2014) Sustained Na+/H+ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation. PLoS One 9:e84294
Shi, Yejie; Kim, Dong; Caldwell, Marie et al. (2013) The role of Na(+)/h (+) exchanger isoform 1 in inflammatory responses: maintaining H(+) homeostasis of immune cells. Adv Exp Med Biol 961:411-8
Ferrazzano, Peter; Chanana, Vishal; Uluc, Kutluay et al. (2013) Age-dependent microglial activation in immature brains after hypoxia- ischemia. CNS Neurol Disord Drug Targets 12:338-49
Lam, Tina I; Brennan-Minnella, Angela M; Won, Seok Joon et al. (2013) Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase. Proc Natl Acad Sci U S A 110:E4362-8
Shi, Yejie; Yuan, Hui; Kim, Dong et al. (2013) Stimulation of Na(+)/H(+) exchanger isoform 1 promotes microglial migration. PLoS One 8:e74201
Begum, Gulnaz; Harvey, Lloyd; Dixon, C Edward et al. (2013) ER stress and effects of DHA as an ER stress inhibitor. Transl Stroke Res 4:635-42
Uluc, Kutluay; Kendigelen, Pýýnar; Fidan, Emin et al. (2013) TrkB receptor agonist 7, 8 dihydroxyflavone triggers profound gender- dependent neuroprotection in mice after perinatal hypoxia and ischemia. CNS Neurol Disord Drug Targets 12:360-70

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