Figure 2. EPR spectra and ROS release rate in control and diabetic kidney mitochondria.

Dissociated kidney cell suspensions from control (A) or diabetic (B) animals were prepared as described and analyzed by EPR using DPMPO as the spin probe 10 minutes after addition of the mitochondrial substrates malate and pyruvate. The characteristic spectrum for the superoxide-DPMPO was observed in both control and diabetic samples at baseline. However, addition of high glucose (bottom traces) did not increase mitochondrial superoxide production and, in the diabetic kidney samples, actually reduced superoxide production. (C) Analysis of the EPR signal intensity using the upfield peak at 334 mT, normalized to mg protein. Values are mean ± SEM, n = 6 per condition, *P < 0.05 vs control. The second set of bars to the right show data derived from Supplemental Figure 3, which shows additional spectra from samples after addition of the mitochondrial complex III inhibitor, antimycin A. Antimycin A produced the expected increase in superoxide, confirming the ability of this technique to detect increased mitochondrial superoxide production if present. (D) Decreased ROS release from kidney mitochondria isolated from diabetic animals Measurements were made in the presence of succinate (Succ), succinate and FCCP (Succ + FCCP), glutamate plus malate (G/M) or glutamate plus malate with rotenone (G/M + Rot) by Amplex Red assay. Rates were calculated using calibration curve as described in Methods. Values are mean ± SEM, n = 5–8. *P < 0.001 vs. corresponding control; ^#P < 0.05 vs. G/M.