Abstract 338: Kidney-specific SPAK Inhibits Activity of the Na-K-2Cl Cotransporter

Abstract

The WNK-SPAK/OSR1 pathway has been shown to play a critical role in the regulation of ion homeostasis, and the maintenance of extracellular fluid volume and blood pressure. SPAK and OSR1, members of the MAP kinase family, phosphorylate and activate the cation chloride cotransporters NKCC1, NKCC2 and NCC. SPAK and OSR1 are themselves phosphorylated and activated by members of the WNK kinase family. We recently identified a kidney-specific (KS-) SPAK isoform (KS-SPAK) which inhibits phosphorylation of the N-terminus of NKCC2 by SPAK or OSR1 in vitro . Since changes in NKCC2 phosphorylation status have been reported to incompletely correlate with changes in NKCC2 activity, we tested the ability of KS-SPAK to inhibit the activity of NKCC2 in Xenopus oocytes. First, 86 Rubidium ( 86 Rb, equivalent to potassium) uptake experiments were performed under hypertonic (300mOsM) conditions, to maximally activate endogenous SPAK and OSR1 in oocytes. KS-SPAK decreased 86 Rb uptake to 38% of that of oocytes injected with NKCC2 alone, and this inhibition required the conserved carboxy-terminus of SPAK, which interacts with cation cotransporters and WNK kinases. We next tested the ability of KS-SPAK to inhibit the activity of endogenous NKCC1, and observed a 67% reduction in 86 Rb uptake compared with water-injected oocytes, similar to the 73% reduction observed with the NKCC1/NKCC2 inhibitor bumetanide. This inhibition likely involves effects on endogenous SPAK or OSR1, since under hypotonic (160mOSM) conditions, when SPAK is relatively inactive, KS-SPAK did not inhibit endogenous NKCC1 activity, whereas bumetanide still inhibited 86 Rb uptake by water-injected oocytes by 50%. Co-injection of KS-SPAK with SPAK only reversed KS-SPAK inhibition of endogenous NKCC2 activity by 50%, despite a co-injection ratio of SPAK:KS-SPAK of 15:1, suggesting the mechanism of KS-SPAK inhibition may not be simple competition for NKCC2 binding. Another SPAK isoform, SPAK2, which has been proposed to be kinase-inactive, did not inhibit activity of co-injected NKCC2 or endogenous NKCC1 at any tonicity. Together, these data show that KS-SPAK is a potent inhibitor of both NKCC1 and NKCC2 activity, whereas the other putative inhibitory isoform SPAK2 had no effect on their activity.