The recent breakthrough in hypertension 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 and play an important role in renal salt handling and in the pathogenesis of hypertension. Na+-K+-2Cl- cotransporter isoform 1 (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 (CNS) under physiological conditions. Previous studies from our lab and others' clearly indicate that over-stimulation of NKCC1 activity contributes to cerebral ischemic damage. 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 (middle cerebral artery occlusion, MCAO) 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. Our pilot study revealed robust stimulation of the WNK-SPAK/OSR1 signaling pathway in ischemic brains. The goal of this project is to investigate whether the cerebral WNK3-SPAK-NKCC1 signaling pathway contributes to ischemic brain damage and whether blocking the WNK3/SPAK kinases with a newly discovered WNK-SPAK pathway inhibitor STOCK1S-50699 or transgenic knockout of WNK3 (WNK3 KO), or SPAK (SPAK KO) is neuroprotective. Our preliminary study shows that WNK3 KO mice exhibited significantly reduced infarct volume, less axonal demyelination, and accelerated neurobehavioral recovery. New data in this resubmission illustrates a ~70% reduction in infarct volume in SPAK KO mice. These data provide fresh insight into the role of ion transporters and their regulatory kinases in ischemic neuroglial injury. Completion of this project will help us to determine whether the WNK3-SPAK kinase complex presents a compelling target for novel neuroprotective strategies for ischemic brain injury. Our study will pave a foundation for developing new WNK-SPAK inhibitors for ischemic brain damage therapy.

Public Health Relevance

Ischemic brain injury is a major problem in the veteran population and there are no effective treatments proven to improve long-term recovery. 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 ischemic brain injury treatments. Therefore, a positive outcome from this study could be beneficial for development of novel therapies for ischemic brain injury treatment.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX002891-02
Application #
9206093
Study Section
Neurobiology C (NURC)
Project Start
2015-10-01
Project End
2019-09-30
Budget Start
2016-10-01
Budget End
2017-09-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Veterans Health Administration
Department
Type
Independent Hospitals
DUNS #
033127569
City
Pittsburgh
State
PA
Country
United States
Zip Code
15240