Abstract P222: Oxidative Stress Causes Salt Sensitive Hypertension via Activation of Na + -Cl – Cotransporter (ncc)

Abstract

Background: We have reported that blockade of nitric oxide synthesis by L-NAME causes salt-sensitive hypertension via activating NCC. which was confirmed by the finding that systemic administration of L-NAME failed to cause salt-sensitive hypertension in NCC deficient mice. In vitro study showed when mDCT cells are treated with L-NAME, NCC phosphorylation is increased and NO donner reduced its phosphorylation. However, the precise direct effect of NO on NCC phosphorylation is not clear. In this study we clarified the role of oxidative stress but not angiotensin signaling in NCC phosphorylation Method: The passages 3 - 15 of mDCT cells were used in this study. The cells were starved 1 hour before the experiment. L-NAME (10-40 μM), SOD mimetic 4-Hydroxy-TEMPO (10 μM), NO donor sodium nitroprusside, (SNP 100 μM - 2 mM), pSPAK inhibitor STOCK2S-26016, and Losartan (10 μM) were used to treat the mDCT cells for 10 min and 30 min. Oxidative stress was measured by ROS production using the lucigenin method. To confirm the oxidative stress function, we performed vivo experiment with SOD mimetics--4-hydroxy-TEMPO. The C57BL/6J Kwl mice received implantation of subcutaneous osmotic mini-pump containing TEMPO (20 mg/kg/day) with 8 % high salt diet and L-NAME (3.7 μM/day in drinking water) for 4 weeks. Result: L-NA ME increased oxidative stress by ROS production (2.0 ± 0.15 times higher than control) and p-SPAK signaling, which was normalized by TEMPO, an SOD mimetic and p-SPAK inhibitor. In contrast, AT1R inhibitor Losartan did not affect L-NAME-induced activation of NCC in mDCT cells. Moreover, the function of TEMPO was also confirmed in vivo study, which attenuated the L-NAME- induced increases in superoxide, mean BP (HS 108.7 ± 3.7 mmHg; HSL 130.5 ± 2.9 mmHg; HSL + TEMPO 116.0 ± 1.7 mmHg) and p-NCC expression in the C57BL/6J mice. Conclusion: Oxidative stress - pSPAK pathway plays a pivotal role in the NO-induced inhibition of NCC.