High glucose concentration affects the oxidant-antioxidant balance in cultured mouse podocytes

Hyperglycemia is well‐recognized and has long‐term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (30 mM). We found long‐term (3 and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and SOD (Δ 39%) and decreased activities of GPx (Δ −40%) and CAT (Δ −35%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant–antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy. J. Cell. Biochem. 112: 1661–1672, 2011. © 2011 Wiley‐Liss, Inc.