Fig. 4.

Increased oxidative stress in old diabetic kidneys and mesangial cells. A: levels of protein oxidation in the kidneys of Y (9 mo old, n = 5), YD (n = 7), O (22 mo old, n = 5), and OD (22 mo old, n = 8) mice were determined by Western blot using a specific anti-oxidized protein antibody. Representative gels of 2 animals from each group showed that, while the bands of oxidized protein are visible in samples of the kidneys from young mice (top gel, Y, lane 1, 2), stronger bands with a similar pattern of proteins were seen in kidney samples from YD and O mice (top gel, YD, lane 3, 4; O, lane 5, 6). Protein oxidation was markedly increased in kidneys from OD mice (top gel, OD, lane 7, 8). A, bottom: same gel stained with Ponceau red before Western blotting. Note that the regular protein markers shown on the bottom gel were not detected by the anti-oxidized protein antibody and therefore cannot be seen in top gel. B: oxidized protein levels were measured in 2 isolates of mesangial cells from O (lane 1, 2) and OD (lane 3, 4). Bottom gel was the staining of Ponceau red before Western blotting with the anti-oxidized protein antibody. C: H₂O₂ levels in O/MC and OD/MC were determined by DCF fluorescence intensity. *P < 0.05 vs. O/MC. Data were obtained from the measurements of 2 cell lines each of OD/MC and O/MC (n = 2). The experiment was repeated 3 times. D: DCF intensity was directly observed under a fluorescence microscopy. Left: image of old control and diabetic mesangial cells under light microscopy (×40). Right: same field of mesangial cells under fluorescence microscopy. An obvious increase in DCF fluorescence was shown in mesangial cells of old diabetic mice.