Angiotensin II (Ang II) is a mediator of renal hypertrophy in diabetic renal disease, and vascular endothelial growth factor (VEGF) could mediate some of its effects in diabetic nephropathy. We have shown that Ang II causes a rapid increase in VEGF protein expression, due to increased mRNA translation. Ang II has been shown to increase reactive oxygen species (ROS) production in renal cells. The aim of this study is to test the hypothesis that Ang II regulates VEGF mRNA translation in MCT cells through ROS production. In MCT exposed to 1 nM Ang II, ROS production was increased in a time-dependent manner. Inhibition of ROS production by N-acetylcysteine (NAC), a precursor of glutathione, prevented Ang II stimulation of VEGF protein expression. Pre-incubation of MCT cells with NAC also inhibited phosphorylation of 4E-BP1 on Thr 46 and association of eIF4E with eIF4G, steps that we have previously shown to be necessary for increased VEGF mRNA translation. Phosphorylation of 4E-BP1 on this residue is under the control of a PI-3K-AKT-mTOR pathway, so we measured the effect of NAC on AKT activation, and found that Ang II dependent AKT activation was totally blocked by NAC. Pre-incubation of MCT cells with LY294002, a specific PI-3K inhibitor, did not prevent ROS accumulation in response to Ang II, which shows that ROS production is upstream of the PI-3K/AKT signaling pathway. Ang II is known to stimulate production of ROS in kidney cells through NADPH-oxidases, so we used diphenyleneidonium (DPI), an inhibitor of flavoproteins to which the NADPH-oxidase family belongs. Pre-incubation of MCT cells with DPI prevented Ang II-induced accumulation of ROS, VEGF protein expression and VEGF mRNA translation, as well as 4E-BP1 phosphorylation and eIF4E/ eIF4G association. Finally, incubation of MCT cells with 200 μM H2O2 reproduced the effects of Ang II on VEGF expression and mRNA translation, and pre-incubation with catalase (that catalyzes the degradation of H2O2 in water) totally prevented Ang II stimulation of VEGF expression and mRNA translation. These data show that Ang II rapidly stimulates production of reactive oxygen species in MCT cells, and that ROS production is upstream of the PI-3K/AKT pathway that leads to increased VEGF protein expression.
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