PROJECT ABSTRACT Stroke remains as the 5th leading cause of death and long-term adult disability in the USA because only ~15% patients can benefit from current standard therapies ( tissue plasminogen activator and endovascular recanalization) due to their short therapeutic windows. Therefore, identification of novel stroke therapeutic targets for neuroprotective drugs remains to be an unmet urgent need. Hypertension is the most significant risk factor for stroke epidemics and angiotensin II (Ang II)-induced neurogenic hypertension is associated with worsened ischemic brain damage. However, the clinical trial studies show no benefits of post-stroke blood pressure (BP)- lowering treatment in the acute stage of ischemic stroke on improvement of the risk of death or disability because it hinders cerebral perfusion. These findings challenge us to develop a novel strategy to block the AngII -mediated detrimental effects in the ischemic brains without lowering BP and cerebral perfusion. The serine-threonine WNK kinase family [with no lysine (K)], and its two downstream kinases SPAK (the STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) activate multiple ion transporters and channels via protein phosphorylation. Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is one of the major substrates of the WNK-SPAK/OSR1 kinases. Stimulation of the WNK-SPAK kinases increased brain NKCC1 activity via protein phosphorylation and led to ischemic cell damage through NKCC1- mediated Na+ and Cl- overload, cytotoxic edema and excitotoxicity. Our pilot study reveals that Ang II-induced hypertensive mice exhibited 2-5 fold increase in the WNK-SPAK-NKCC1 protein complex expression in ischemic brains, which was accompanied with worsened outcomes in infarct, edema, and neurological deficit in the permanent middle cerebral artery occlusion model (pdMCAO). Post-stroke administration of a novel, non-ATP competitive, selective SPAK inhibitor ZT-1a in these mice significantly reduced infarction and edema, and improved neurological function recovery. However, how Ang II signaling pathway regulates WNK-SPAK-NKCC1 protein complex expression and efficacy of ZT-1a in reducing ischemic brain injury in the Ang II-induced hypertensive mice remains unknown. We hypothesize that (1) Ang II stimulates Ang II receptor subtype 1 (AT1R)- NF-κB cascade which leads to upregulation of WNK-SPAK-NKCC1 signaling complex after stroke; (2) elevated WNK-SPAK-NKCC1 signaling directly contributes to the worsened ischemic neuronal damage and neurological deficits; (3) post-stroke administration of the novel SPAK kinase inhibitor ZT-1a reduces ischemic brain damage by preventing excessive activation of brain SPAK-NKCC1 signaling. These hypotheses will be tested in three specific aims. In summary, we investigate that Ang II-induced hypertension comorbidity causes worsened ischemic stroke outcome in part via stimulating the WNK-SPAK-NKCC1 signaling pathway in the CNS. C...