Effect of High Glucose on Superoxide in Human Mesangial Cells: Role of Angiotensin II

Abstract

Background/Aims: Reactive oxygen species, and especially superoxide (O₂·–),have been implicated in diabetic nephropathy. O₂·– accumulation in cells is dependent on O₂·– production (by NADH/NADPH oxidase) as well as scavenging by superoxide dismutase (SOD) activity. This study was designed to investigate the effects of high glucose (HG) on O₂·– accumulation and SOD activity in human mesangial cells (HMC) and to determine if these effects are mediated by angiotensin II (Ang II). Methods: HMC were incubated in media containing 10 mMglucose (control, C), 30 mM glucose (HG), 10 mM glucose + either 20 mM 2-deoxy-D-glucose (2-DG) or 20 mM mannitol (high mannitol, HM) (osmotic controls), or Ang II (10^–5M). Ang II action was antagonized by employing 10^–4M of Ang II receptor antagonists (losartan or irbesartan) or 10^–4M of NADH/NADPH oxidase inhibitors [diphenyleneiodonium chloride (DPI) or apocynin]. Superoxide and total SOD activity were assayed using chemiluminescence of lucigenin. Results: Incubation of HMC in HG resulted in a 1.6-fold increase in Ang I (p < 0.05) and a 1.4-fold increase in Ang II levels (p < 0.05) in cell lysates. These changes were accompanied by a >2-fold increase in O₂·– accumulation (p < 0.01), which was inhibited by losartan and irbesartan. Exogenous Ang II increased net O₂·– accumulation by 2.7-fold (p < 0.01), which was normalized by losartan and irbesartan. DPI and apocynin blocked the HG and Ang II-induced increases in O₂·– (p < 0.01). HG but not exogenous Ang II inhibited total SOD activity by 30%, which was not affected by losartan. Conclusion: High glucose increases O₂^–· accumulation in HMC primarily by increasing its production via the Ang II-NADH/NADPH oxidase system.