Role of the Novel Kinase TNIK on NKCC2 Surface Expression, Phosphorylation and Na Reabsorption in the Thick Ascending Limb

Abstract

NaCl reabsorption by the thick ascending limb (TAL) of the loop of Henle maintains NaCl balance and is important for blood pressure regulation. The apical cotransporter NKCC2 mediates NaCl reabsorption in the TAL. In animal models of hypertension, NKCC2 phosphorylation at amino‐terminal Thr‐96,101 and its trafficking to the plasma membrane are enhanced. The kinases SPAK and OSR1 bind NKCC2 and phosphorylate Thr‐96,101 and are important for blood pressure control. In addition to these, we recently showed that a novel kinase, Traf2 and NCK‐interacting kinase (TNIK), also binds and phosphorylates Thr‐96,101 in NKCC2. However, the role of TNIK on NKCC2 trafficking, activity and blood pressure regulation is unknown. We hypothesized that TNIK enhances NKCC2 phosphorylation, surface NKCC2 expression and NKCC2‐dependent water and Na transport. To test this, we generated TNIK knockout (TNIK−/−) mice and measured phosphorylation of NKCC2 and surface expression in isolated TALs. We observed that NKCC2 phosphorylation at Thr‐96,101 was decreased by 23 ± 6% (p<0.05) in TALs from TNIK−/− mice on a normal Na diet. Feeding mice a low‐Na diet increased NKCC2 phosphorylation by 179 ± 38% in WT mice, but only by 56 ± 5% in TNIK−/− mice (p<0.05). The NKCC2 surface‐to‐intracellular ratio was decreased by 58 ± 4% in TALs from TNIK−/− on a normal diet and by 55 ± 16% on a low‐Na diet. When placed in metabolic cages TNIK−/− mice excreted 99% more urine (WT = 1.03 ± 0.01 ml vs. TNIK−/− = 2.05 ± 0.13 ml; p<0.05). Baseline Na excretion was similar between strains on normal Na diet. However, after 48 hours of switching to low‐Na diet, TNIK−/− mice excreted 75% more Na compared to WT (WT = 15 ± 0.7 μmol/day vs. TNIK−/− = 26 ± 5 μmol/day; p<0.05). To test whether TNIK mediates NKCC2‐dependent Na excretion, we measured bumetanide‐induced diuresis and natriuresis in TNIK−/− mice over a six‐hour period following oral administration of bumetanide (1.66 mg/g food). On a normal diet, we did not observe any difference between strains. However, when placed on a low‐Na diet, TNIK−/− mice showed decreased bumetanide‐induced diuresis (WT = 2.66 ± 0.12 ml vs. TNIK−/− = 2.14 ± 0.11 ml; p<0.05) and natriuresis (WT = 239 ± 9 μmol vs. TNIK−/−= 201 ± 7 μmol; p<0.05). Finally, TNIK−/− mice had lower systolic blood pressure than WT mice when fed a low‐Na diet (WT = 123 ± 3.9 mmHg vs. TNIK−/−= 105 ± 3 mmHg; p<0.05). We conclude that TNIK mediates NKCC2 phosphorylation, surface expression and is involved in NKCC2‐mediated Na reabsorption during adaptation to low salt diet. TNIK‐mediated activation of NKCC2 may also be involved in blood pressure control.Support or Funding InformationAmerican Heart Association