Hyperglycemia‐induced Increases in Na Transporter Abundance and Activity in Blood‐Brain Barrier Endothelial Cells: Involvement of SGK1, PKCβII and SPAK/OSR1

Abstract

Diabetics have a higher risk of stroke and worse outcome following stroke. Studies have shown that cerebral edema is exacerbated in ischemic stroke of diabetics compared to non‐diabetics. However, the underlying mechanisms are not well understood. We have shown previously that during the early hours of ischemic stroke ischemic factors stimulate blood‐brain barrier (BBB) endothelial cell Na‐K‐2Cl cotransporter (NKCC) and Na‐H exchanger (NHE) activities, leading to “hypersecretion” Na, Cl and water from blood into brain and consequent edema formation.Recently our lab demonstrated that hyperglycemia (HG) exposures of 6 hr to 7 d significantly increase NKCC and NHE abundance and activity in cerebral microvascular endothelial cells (CMEC) and that subsequent exposure to ischemic factors results in more robust stimulation of NKCC and NHE activities compared to that of normoglycemic cells. Further, inhibiting BBB NKCC and NHE by IV administration of bumetanide and HOE‐642, respectively, attenuates the exacerbated edema for HG rats subjected to middle cerebral artery occlusion‐induced ischemia, suggesting that NKCC and NHE are good therapeutic targets for diabetic stroke.The present study was conducted to investigate signaling events involved in the HG‐induced increases in BBB Na transporter abundance and activity. Previous studies have provided evidence that PKCβII and SGK1 are involved in HG‐induced events in other cell types, suggesting the possibility that one or both of these kinases may also participate in HG‐induced effects on BBB NKCC and NHE. Here, we examined whether total and/or active/phosphorylated PKCβII and SGK1 are altered in CMEC subjected to HG exposures of min to hr. We also tested whether selective inhibitors of these kinases reduced HG effects on CMEC NKCC and NHE. Finally, we examined whether phosphorylation of SPAK/OSR1 and/or NKCC1 are altered in CMEC upon HG exposure.Bovine cerebral microvascular endothelial cells (CMEC) were exposed to normoglycemic, HG or osmotic control media for up to 48 hr. Protein abundance was determined using quantitate Western Blot analysis. NKCC activity was assessed as bumetanide‐sensitive K flux by radioisotopic assay and NHE activity was assessed as HOE‐642‐sensitive H flux by microspectrofluorometry assay using BCECF. Cells were also fixed and immunostained to determine protein localization following HG exposure.Application of HG medium (30 mM) to CMEC caused a transient increase in phosphorylation of PKCbetaII at 5 min and SGK1 at 5 and 30 min. CMEC showed increased abundance and activity of NKCC and NHE following 24 hr of HG exposure. SGK1 inhibition attenuated HG‐induced increases in NKCC and NHE abundance and activity. PKCβII inhibition attenuated the HG‐induced increases in NKCC and NHE activity with no effect on abundance. HG also caused a sustained increase in phosphorylation of SPAK/OSR1 through at least 6 hr with phosphorylation falling by 24 hr. Further, we found that phosphorylation of NKCC1 was increased at both 6 and 24 hr post HG exposure.Our findings indicate that HG causes rapid and transient activation of both SGK1 and PKCβII in CMEC. The HG‐induced increases in NKCC and NHE activity involves both SGK1 and PKCβII while HG‐induced increases in NKCC and NHE abundance require SGK1 activity but not PKCβII activity. They also suggest that HG exposure increases SPAK/OSR1 activity and phosphorylation of NKCC.Support or Funding InformationSupported by NINDS and American Heart Association