POS-373 Advanced oxidation protein products activate oxidative stress in vitro via soluble (pro)renin receptor mediated intrarenal renin angiotensin system and Nox4/H2O2 pathway

Abstract

Advanced oxidation protein products (AOPPs) are newly identified as a novel oxidative stress biomarkers. Previous research has indicated potential associations between AOPPs and activation intrarenal local renin angiotensin system (RAS). However, no known empirical research has focused on exploring relationships between AOPPs and the new member of RAS, (pro) renin receptor (PRR). Aim of this study was to test the role of soluble PRR (sPRR) in AOPPs-induced oxidative stress responses in cultured human renal proximal tubular cell line HK-2 cells. The HK-2 cells were incubated for 24h in the presence of different concentrations of AOPPs, or incubated for different time lengths in the presence of 100 μg/ml of AOPPs. In HK-2 cells treated with PRO20, furin inhibitors, GM6001, PF-429242, sPRR-His, or transfected with PRR siRNA, site-1 protease (S1P) siRNA, Nox4 siRNA prior to exposure to AOPPs, the changes in the protein expressions of PRR/sPRR and Nox4 were examined with Western blotting, mRNA lelvels of PRR, angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), angiotensinogen (AGT), intercellular adhesion molecule (ICAM1), tumor necrosis factor-α (TNF-α) by using real-time quantitative PCR, the cell medium for renin activity, ACE activity, sPRR, Ang II, H2O2, thiobarbituric acid reactive substances (TBARS) concentrations by using ELISA Assay kits. AOPPs showed time-dependent and dose-independent effects on PRR cleavage produces sPRR. AOPP-albumin treatment induced a >4-times increase in medium sPRR due to enhanced cleavage of PRR but unmodified albumin has no effect. Inhibition of S1P with PF-429242 or siRNA remarkably suppressed AOPPs-induced sPRR production. Surprisingly, this cleavage event was unaffected by inhibition of furin or ADAM19. AOPPs (100 μg/ml) treatment for 24 h induced renin activity and oxidative stress, both of which were attenuated by a PRR decoy inhibitor PRO20 and S1P inhibitor PF429242. What’s more, PF-429242 significantly decreased AOPPs stimulated Nox4 and H2O2 expression and administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. Taken together, these findings suggest that AOPPs induce oxidative stress via S1P-derived soluble (pro)renin receptor activation intrarenal renin angiotensin system and Nox4/H2O2 pathway in vitro.