Tumor necrosis factor alpha activates RhoA and decreases NO synthase type 3 expression in rat thick ascending limbs

Abstract

NO produced by NO synthase type 3 (NOS3) decreases thick ascending limb (THAL) NaCl reabsorption and induces natriuresis. Tumor necrosis factor alpha (TNF) stimulates RhoA GTPase and Rho kinase (ROCK) and decreases NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF decreases NOS3 expression via Rho/ROCK in rat THALs. We measured: 1) NOS3 expression by Western blot and 2) RhoA activity by ELISA. Treatment of THAL primary cultures with 1 nM TNF for 24 hrs decreased NOS3 expression by 54 ± 4% (n= 6, p<0.001). Treatment with TNF for 10 min increased RhoA activity from 0.520 ± 0.038 OD to 0.816 ± 0.090 (Δ= 60±23%, p<0.03). Blockade of RhoA with 0.05 ug/mL C3 exoenzyme inhibited TNF‐induced reductions in NOS3 expression by 30 ± 9% (n=7, p<0.02) while C3 exoenzyme alone did not change NOS3 expression. Blockade of ROCK with 10 μM H‐1152 blunted TNF‐mediated reductions in NOS3 expression by 66 ± 15 % (n=6 p<0.001) while H‐1152 alone did not affect NOS3 expression. Simultaneous blockade of RhoA and ROCK were not additive (inhibition of TNF's effect = 56 ± 19 % n=5, p<0.05). We conclude that: 1) TNF decreases NOS3 expression in THALs; and 2) this effect is partially mediated by Rho/ROCK. Blockade of Rho/ROCK may improve renal function in hypertension by preventing TNF‐induced decreases in NOS3 expression.Sources of funding: NIH HL 028982 to J.L.G. and AHA 11PRE7510005 to V.D.R