Abstract 489: Hydrogen Peroxide Stimulates Renin Release From Mouse Juxtaglomerular Cells by Enhancing cAMP

Abstract

Renin plays an essential role in blood pressure control and the development of hypertension. Enhanced hydrogen peroxide (H 2 O 2 ) levels in the kidney are associated with hypertension enhancing tubular salt transport and vascular reactivity. However, it is not known whether H 2 O 2 can directly stimulate renin release from renal juxtaglomerular (JG) cells. Since cAMP is one of the most potent signaling messengers for renin release, we hypothesized that H 2 O 2 stimulates renin release by enhancing cAMP production in JG cells. Primary cultures of mouse JG cells were treated for 60 min with different agonists and inhibitors and renin released to the supernatant measured by radioimmunoassay. We first tested the effect of decreasing endogenous H 2 O 2 with catalase. Catalase (100U/ml) blunted baseline renin release by 45±9% ( n = 6; p < 0.05) indicating that endogenously produced H 2 O 2 tonically stimulates renin release. We then tested the effect of exogenous H 2 O 2 for 60 min on renin release. Physiological concentrations of H 2 O 2 (0.1 and 1μM) increased renin release by 101±43% ( p = 0.08) and 166±47% ( p < 0.04), respectively. To assure that exogenous H 2 O 2 crosses the plasma membrane to activate signaling, we monitored intracellular H 2 O 2 with a genetically encoded sensor (Hyper). We found that addition of exogenous H 2 O 2 enhanced intracellular H 2 O 2 within 5 minutes ( p < 0.02; n=5). cAMP is the most potent second messenger for stimulation of renin release. Thus, we tested whether H 2 O 2 stimulates intracellular cAMP levels. For this, JG cells were transduced with a cAMP-sensitive FRET (CFP/YFP) probe (Epac2camps) and cAMP measured by confocal microscopy in real-time. Treatment of JG cells with H 2 O 2 increased cAMP within 30 min as indicated by a 22±3% increase in FRET ( p < 0.03, n=3) whereas forskolin/IBMX (10μM/0.5mM) caused a maximal FRET stimulation of 44±13% ( p < 0.01; n=5). Inhibition of adenylyl cyclase with SQ22536 (100 μM) completely blocked H 2 O 2 -induced increase in cAMP (N.S from baseline; n=6). We concluded that H 2 O 2 is a potent stimuli for renin release from mouse JG cells and this effect is most likely mediated by enhanced production of cAMP. Our data suggest that enhances renal cortical reactive oxygen species may induce hypertension by enhancing renin release.