Hyperphosphorylation of Na-K-2Cl Cotransporter in Thick Ascending Limbs of Dahl Salt-Sensitive Rats

Abstract

Salt-sensitive hypertension involves a renal defect preventing the kidney from eliminating excess NaCl. The thick ascending limb of Henle loop reabsorbs ≈30% of filtered NaCl via the apical Na-K-2Cl cotransporter (NKCC2). Higher NKCC2 activity and Cl reabsorption have been reported in the thick ascending limbs from Dahl salt-sensitive rats (DSS) fed normal salt. NKCC2 activity is primarily regulated by protein trafficking and phosphorylation at Thr 96 /Thr 101 via STE20- and SPS1-related proline and alanine-rich kinases and oxidative stress-responsive kinase 1. However, the mechanism for enhanced NKCC2 activity in DSS is unclear. We hypothesized that DSS exhibit enhanced NKCC2 trafficking and higher NKCC2 phosphorylation compared with Dahl salt-resistant rats on normal salt diet. We measured steady state surface NKCC2 expression and phosphorylation at Thr 96 and Thr 101 by surface biotinylation and Western blot. In DSS, the surface:total NKCC2 ratio was enhanced by 25% compared with Dahl salt-resistant rats ( P <0.05) despite lower NKCC2 expression. Total NKCC2 phosphorylation at Thr 96 and Thr 101 was enhanced ≈5-fold in DSS thick ascending limbs. Moreover, total STE20- and SPS1-related proline and alanine-rich kinases expression, kidney-specific STE20- and SPS1-related proline and alanine-rich kinases, and oxidative stress-responsive kinase 1 were not different between strains, although STE20- and SPS1-related proline and alanine-rich kinases/oxidative stress-responsive kinase 1 phosphorylation was enhanced by 60% ( P <0.05) in DSS rats, suggesting increased activity. We concluded that phosphorylation of NKCC2 Thr 96 and Thr 101 and surface:total NKCC2 ratio are enhanced in DSS rats. These differences in NKCC2 may be, in part, responsible for higher NKCC2 activity and abnormally enhanced thick ascending limb NaCl reabsorption in DSS rats.