CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abstract

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p<0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p<0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p<0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.