SPAK‐Independent NKCC2 Phosphorylation in Dahl SS Rats: A Role for TNIK in NKCC2 Hyperphosphorylation

Abstract

Increased NaCl reabsorption in the Thick Ascending Limb (TAL) is implicated in salt sensitive hypertension. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. We reported that NKCC2 phosphorylation at Tre 96,101 and surface expression are enhanced in Dahl Salt sensitive rats (Dahl SS) on normal or high salt diets. The phosphorylation of SPS1‐related proline/alanine‐rich kinase (SPAK), an upstream kinase for Thre 96,101 in NKCC2, was found to be higher in Dahl SS TALs. Deletion of SPAK in Dahl SS, lowers NKCC2 phosphorylation and expression and blunts salt‐sensitive hypertension, but does not completely prevent this. 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 Thre 96,101 in NKCC2. We hypothesize that TNIK is in part responsible for enhanced NKCC2 phosphorylation in Dahl SS rats. We first measured total expression of SPAK, OSR1 and TNIK by Western blot in TALs isolated form Dahl SS and control rats. SPAK and OSR1 expression (normalized to GAPDH) was similar between strains whereas TNIK expression was 3‐fold higher in Dahl SS TALs (control: 100, Dahl SS: 396±80%, n =5, p<.025). We then tested whether a novel TNIK inhibitor (KY‐05009) decreased NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with KY‐05009 (1 µM) for 25 min decreased NKCC2 Thre 96,101 phosphorylation by 40±4% (p<0.05, n=5). To study whether NKCC2 phosphorylation can be stimulated in the absence of SPAK, we used SPAK knockout Dahl SS rats. Baseline NKCC2 expression and phosphorylation in TALs was 50% lower in Dahl SS‐SPAK KO TALs, however, stimulation of Beta‐adrenergic receptors with Isoproterenol increased NKCC2 phosphorylation by 192±49% (p<0.01, n =4) in SPAK‐KO. Similarly, the cAMP analogue db‐cAMP (500 µM) increased NKCC2 phosphorylation by 939±183% in SPAK KO (p<0.01 n=4) and by 497±105% (p<0.01 n=4) in control rat TALs, indicating that a kinase other than SPAK is essential for baseline and cAMP‐stimulated NKCC2 phosphorylation in Dahl SS rats. We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK, which show 3‐fold higher expression in this strain. SPAK also contributes to higher baseline NKCC2 phosphorylation, whereas a kinase other than SPAK (either TNIK or OSR1) is involved in cAMP‐stimulated NKCC2 Thre 96,101 phosphorylation in Dahl SS TALs.