WNK-SPAK-NKCC1 Cascade Activation Contributes to Worsened Brain Damage in Mice With Hypertension Co-Morbidity after Ischemic Stroke

Objectives: The WNK-SPAK/OSR1 kinase complex plays an important role in renal salt handling and pathogenesis of hypertension by regulating ion transporters and channels. Hypertension is the most common risk factor for stroke and stroke patients with hypertension comorbidity have worsened outcome with an increased risk of dependency or death. However, the mechanisms underlying the worsened ischemic stroke pathophysiology with hypertension comorbidity remain poorly defined. In this study, we investigated roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain damage in mice with hypertension comorbidity. Methods: Hypertension was induced in C57BL/6j male mouse (12-15 weeks) by subcutaneous infusion of 1000 ng/kg/min angiotensin II (AngII, mini-osmotic pump) for two weeks. Permanent ischemic stroke was induced by permanent occlusion of the distal branches of the left middle cerebral artery (pd-MCAO). Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Infarct volume and hemisphere swelling were determined by TTC staining, and behavioral deficits were analyzed by foot fault test, cylinder test and adhesive tape removal test. Results: pd-MCAO stimulated expression of WNK proteins (isoforms 1, 2, 4), total and phosphorylated SPAK/OSR1 and NKCC1 proteins in ischemic brains of the AngII-infused hypertensive mice compared to normotensive saline controls. In parallel with the increased activation of WNK-SPAK-NKCC1 signaling, hypertensive mice displayed significantly larger infarct volume and hemispheric swelling at 24 h after pd-MCAO compared to normotensive controls. Moreover, hypertensive mice exhibited a slow recovery of neurological function after ischemic stroke compared to normotensive counterparts as assessed by sensory-motor sensitive tests. Conclusions: These results suggest that activation of the WNK-SPAK-NKCC1 complex in hypertensive ischemic brains associates, at least in part, with the worsened brain damage and neurological deficits. Pharmacological inhibition of WNK-SPAK complex has therapeutic potentials for stroke therapy with hypertension comorbidity.