Figure 2.

Expression and activity of IDH2 and IDH1 in Idh2^+/+ and Idh2^−/− mice after I/R. C57BL/6 mice were subjected to either renal ischemia or a sham operation. (A) Twenty-four hours after surgery, kidney sections were subjected to immunofluorescence staining using antibodies directed against IDH2, AQP1, and NCX1. 4′,6-diamidino-2-phenylindole (DAPI) staining was used to visualize the nuclei of cells. (B) Diagram showing IDH2 expression in normal kidney tubular segments. (C) Kidneys were harvested at the indicated times and analyzed by Western blotting using anti-IDH2 and anti-IDH1 antibodies. GAPDH was used as a loading control. (D) Idh2^+/+ and Idh2^−/− mice were subjected to 25 minutes of renal ischemia. Kidneys were harvested 24 hours after ischemia and then subjected analyzed by Western blotting using anti-IDH2 and anti-IDH1 antibodies. (E and F) The densities of bands were measured using ImageJ software. (G) The mitochondria and cytosol mitochondria were fractionated from kidney whole lysates, and the fractions were then confirmed by Western blot analysis using antibodies directed against IDH2, MnSOD, IDH1, CuZnSOD, and GAPDH. Ponceau S was used to determine equal loading. The activity of IDH2 (H), IDH1 (I), and IDH3 (J) was measured in the mitochondrial (H and J) and cytosolic fractions (I), respectively. Results are expressed as the mean±SEM (n=6). *P<0.05 versus respective sham-operated mice; ^†P<0.05 versus ischemia-operated Idh2 WT mice; ^#P<0.05 versus sham-operated Idh2 WT mice. CD, collecting duct; DT, distal tubule; G, glomerulus; Isch, ischemia; ISOM, inner stripe of outer medulla; KO, knockout; OSOM, outer stripe of outer medulla; S1–2 PT, segment 1–2 in the proximal tubule; S3 PT, segment 3 in proximal tubule; WT, wild type.