Enalaprilat inhibits hydrogen peroxide production by murine mesangial cells exposed to high glucose concentrations.

BACKGROUND Oxidative stress is considered to play a role in the pathogenesis of diabetic nephropathy. Angiotensin-converting enzyme (ACE) inhibitors are hypotensive drugs with a well-known effect in preventing the progression of chronic renal failure. Their mechanism of action is not clearly established. METHODS The effect of enalaprilat on hydrogen peroxide (H2O2) production by cultured murine mesangial cells exposed to 5.5 (basal condition), 30 and 50 mM glucose concentrations was examined over 8 h. A fluorimetric method quantifying, in arbitrary units, the intracellular dichlorofluorescein (DCFH) oxidation to the highly fluorescent compound 2'7'dichlorofluorescein (DCF) from the non-fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) was employed (a method not previously reported for cultured mesangial cells). Experiments were repeated three times in quadruplicate wells. RESULTS H2O2 production by mesangial cells exposed to 50 mM glucose was significantly increased after 1 h, compared to cells exposed to 5.5 and 30 mM glucose. This observation was not reproduced with 50 mM mannitol. Addition of 100 ng/ml enalaprilat to cells with 50 mM glucose significantly inhibited H2O2 production during the 8 h of the assay. This response was similar to that obtained with 100 ng/ml catalase. Increasing enalaprilat concentrations (10, 50 and 100 ng/ml) also significantly decreased the constitutive H2O2 generation in the presence of 5.5 mM glucose. Angiotensin II and saralasin, both at 1 microM, did not modify H2O2 production by cells exposed to 5.5 mM glucose. In contrast, 1 microM staurosporine, a protein kinase C (PKC) antagonist, significantly decreased H2O2 generation in the presence of 50 mM glucose. CONCLUSION Enalaprilat has an antioxidant effect in cultured mesangial cells. This action is not linked to ACE inhibition, but may be related to an inhibition of the PKC system.