Abstract Body: Up to 50% of hypertensive patients are salt-sensitive. Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) in the kidney contributes to the development of salt-sensitivity. NaCl reabsorption in the TAL is entirely mediated by the apical Na/K/2Cl cotransporter NKCC2. We showed that NKCC2 phosphorylation at Thr-96,101 is increased in TALs from Dahl salt-sensitive (DSS) rats. We discovered a new kinase, TNIK (Traf2-and-NCK-interacting kinase), that binds the amino-terminus of NKCC2 and phosphorylates NKCC2 at Thr-96,101. TNIK is abundantly expressed in TALs and other nephron segments. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 phosphorylation in TALs from DSS rats. First, we measured TNIK expression in isolated TALs. TNIK expression was 8±4-fold higher in TALs from DSS compared to SD rats (n=5, p<0.05). Then, we used a pharmacological specific inhibitor of TNIK, NCB-0846. We implanted renal medullary catheters connected to minipumps to directly infuse NCB-0846 or vehicle into the left kidney of uninephrectomized DSS rats. After recovery rats were fed 4% NaCl diet and measured systolic blood pressure (SBP) via tail-cuff. NCB-0846 decreased SBP by 21±6 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs vehicle). To specifically decrease TNIK gene expression in medullary TALs, we used Cas9-mediated gene editing. We injected the renal medulla of the left kidney with AAV-gRNA-TNIK simultaneously with AV-NKCC2promoter-Cas9 (drives Cas9 expression in TALs) or AV-NKCC2promoter-Cas9 alone (AV-control) in DSS rats. After 10 days, rats were fed a 4% NaCl diet and SBP was measured. Rats transduced with AAV-gRNA-TNIK had a 18±5 mmHg lower BP compared to AV-control group (AV-control:147 ± 4.7 vs AAV-gRNA-TNIK:129 ± 5.6 mmHg n=5, p<0.03 vs control) which remained lower after 14 days (AV-control:163 ± 4.8 vs AAV-gRNA-TNIK:140 ± 4.9 mmHg, n=5, p<0.01). After 14 days on high salt, TNIK expression was decreased by 30 ± 4% (n=4, p<0.05), and pThr-96,101-NKCC2 decreased by 43 ± 11% in AAV-gRNA-TNIK transduced TALs compared to AV-control transduced TALs (n=4, p<0.02). We conclude that hypertension and enhanced NKCC2 phosphorylation in the DSS rat is partly due to higher TNIK expression in TALs and that inhibition of this novel kinase could be a target for salt-sensitive hypertension.