Angiotensin II induces superoxide anion production by mesangial cells

Background. The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O₂^-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O₂^- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O₂^- production and (b) if increased O₂^- production elicits growth responses in MC. Methods. MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. Results. Ang II (10^-5 M to 10^-8 M) dose dependently increased MC O₂^- production up to 125% above control (ED 50 5 x 10^-7 M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O₂^- production. Ang II dose dependently increased MC ³H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as ³H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC ³H-leucine incorporation, thereby establishing the specificity of Ang II induced O₂^- in driving MC hypertrophy. Conclusions. The current studies demonstrate a previously unrecognized link between Ang II and MC O₂^- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.