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AJP-Renal Physiol @BULLET
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ANG II, a mediator of renal injury in diabetic renal disease, promotes vascular endothelial growth factor (VEGF) mRNA translation in proximal tubular epithelial (MCT) cells (Feliers D, Duraisamy S, Barnes JL, Ghosh-Choudhury G, and Kasimath BS. Am J Physiol Renal Physiol 288: F521–F529, 2005). The mechanism by which ANG II elicits this effect is not known. ANG II is known to induce oxidative stress and the rapidity of the effect suggested a role for reactive oxygen species (ROS). 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 cells 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, and diphenyleneiodonium (DPI), an inhibitor of flavoproteins that include NAD(P)H oxidase, prevented ANG II-stimulated VEGF protein expression. NAC and DPI also inhibited phosphorylation of 4E-BP1 on Thr46 and association of eIF4E with eIF4G, steps that are important in the initiation phase of mRNA translation. NAC and DPI also blocked Akt activation which is required for 4E-BP1 phosphorylation. LY-294002, a selective phosphatidylinositol (PI 3-kinase) inhibitor, did not prevent ROS accumulation in response to ANG II, whereas DPI blocked ANG II activation of PI 3-kinase, demonstrating that ROS production is upstream of the PI 3-kinase signaling pathway. Preincubation with catalase abolished ANG II stimulation of VEGF expression and mRNA translation, suggesting involvement of hydrogen peroxide (H 2 O 2 ). H 2 O 2 reproduced the effects of ANG II on VEGF expression and aforementioned parameters of mRNA translation. Finally, neither preincubation of MCT cells with specific inhibitors of the mitochondrial respiratory chain nor inactivation of the mitochondrial respiratory chain in MCT cells prevented ANG II stimulation of VEGF expression. Inhibition of nitric oxide synthase by L -NAME had no effect on ANG II stimulation of VEGF expression. These data show that ROS, generated probably through activation of an NAD(P)H oxidase, mediate ANG II stimulation of VEGF mRNA translation. vascular endothelial growth factor; diabetic microvascular injury Address for reprint requests and other correspondence: D. Feliers, Dept. of Medicine, Mail Code 7882, Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (e-mail: feliers@uthscsa.edu )
AJP - Renal Physiology – The American Physiological Society
Published: Apr 1, 2006
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