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Synergistic interaction of angiotensin II and glucose in superoxide production by mesangial cells

Oxidative stress leads to glomerular mesangial changes, which are recognized in early stage of diabetic nephropathy. Previous studies showed either high concentration of D‐glucose (G) or angiotensin II (AII) stimulated superoxide (O2−) production in mesangial cells. However, direct interaction of AII and G on O2− production has not been clarified. We examined whether AII and G synergistically interact on O2− production in mesangial cells. Human or rat mesangial cells were cultured on cover slips and assessed under a real‐time fluorescent microscopy. Rate of O2− generation was determined by the slope of increases in the intensity of ethidium, that was converted from preloaded dihydroethidium. When the cells were preincubated with 10−7 M AII and 13.5mM G, O2− production was higher than vehicle (4.2±0.4 vs. 3.1±0.2, p<0.05). However, either AII or G alone had no effect on the production. Influence by osmolality was ruled out by L‐glucose. The O2− production was significantly inhibited by 1mM apocynin, an NADPH oxidase inhibitor, by 10μM RNH‐6270, an AII type 1 receptor antagonist, by 20μM thenoyltrifluoroacetone, a mitochondrial complex II inhibitor, or by 5μM carbonylcyanide m‐chlorophenylhydrazone, an uncoupler of oxidative phosphorylation that abolishes mitochondrial membrane proton gradient. These data suggests that a synergistic interaction of AII and G in O2− production via NADPH oxidase and mitochondrial pathway.