Figure 7.

Apoptosis and its signal pathways in Idh2^+/+ and Idh2^−/− mice after I/R injury. Idh2^+/+ (Idh2 WT) and Idh2 ^−/− (Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. Twenty-four hours after ischemia, (A) Kidney tissue samples were analyzed by Western blotting using antibodies directed against Bax (6A7), Bax (5B7), Bcl-2, Bcl-xL, XIAP, and cleaved caspase-3. GAPDH was used as a loading control. (B–H) The densities of bands were measured using ImageJ software. (I) Mitochondrial (Mito.) and cytosolic (Cyto.) fractions were subjected to Western blot analysis using anti-cytochrome c (Cyto c) antibody. COX IV and GAPDH were used as a loading control of mitochondrial and cytosolic fraction, respectively. (J and K) The densities of bands were measured using ImageJ software. (L) Apoptotic cells in kidney tissue were evaluated by TUNEL assay 24 hours (upper panels) and 16 days (lower panels) after surgery. Arrows indicate TUNEL-positive cells. Images were obtained from the outer stripe of outer medulla. 4′,6-diamidino-2-phenylindole was used to stain the nuclei of cells. (M and N) TUNEL-positive cells were counted (ten fields per kidney). Results are expressed as mean±SEM (n=6). *P<0.05 versus sham-operated Idh2 WT mice; ^†P<0.05 versus ischemia-operated Idh2 WT mice; ^#P<0.05 versus sham-operated Idh2 WT. Isch, ischemia; KO, knockout; WT, wild type.