IDH2 deficiency increases the liver susceptibility to ischemia-reperfusion injury via increased mitochondrial oxidative injury

Abstract

Mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2 (IDH2) is a major producer of mitochondrial NADPH, required for glutathione (GSH)-associated mitochondrial antioxidant systems including glutathione peroxidase (GPx) and glutathione reductase (GR). Here, we investigated the role of IDH2 in hepatic ischemia-reperfusion (HIR)-associated mitochondrial injury using Idh2-knockout (Idh2^-/-) mice and wild-type (Idh2^+/+) littermates. Mice were subjected to either 60min of partial liver ischemia or sham-operation. Some mice were administered with 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (mito-TEMPO, a mitochondria-targeting antioxidant). HIR induced severe histological and functional damages of liver in both Idh2^+/+ mice and Idh2^-/- mice and those damages were more severe in Idh2^-/- mice than in wild-type littermates. HIR induces dysfunction of IDH2, leading to the decreases of NADPH level and mitochondrial GR and GPx functions, consequently resulting in mitochondrial and cellular oxidative injury as reflected by mitochondrial cristae loss, mitochondrial fragmentation, shift in mitochondrial fission, cytochrome c release, and cell death. These HIR-induced changes were greater in Idh2^-/- mice than wild-type mice. The mito-TEMPO supplement significantly attenuated the aforementioned changes, and these attenuations were much greater in Idh2^-/- mice when compared with wild-type littermates. Taken together, results have demonstrated that HIR impairs in the IDH2-NADPH-GSH mitochondrial antioxidant system, resulting in increased mitochondrial oxidative damage and dysfunction, suggesting that IDH2 plays a critical role in mitochondrial redox balance and HIR-induced impairment of IDH2 function is associated with the pathogenesis of ischemia-reperfusion-induced liver failure.

Keywords: Apoptosis; IDH2; Liver ischemia; Mitochondria; Oxidative stress.

Graphical abstract

Fig. 1

Histological and functional damages to the liver ofIdh2^+/+andIdh2^-/-mice after HIR. Livers were harvested 5 h after surgery. Liver sections were stained with PAS reagent (A) and the damaged area (B) were determined. Plasma concentrations of AST (C) and ALT (D) were measured. (E) Liver sections were subjected to immunohistochemical staining using an anti-F4/80 antibody. Hematoxylin was used to stain the nuclei. (F) F4/80-positive cells were counted. (G) Whole liver lysates were subjected to western blot analysis using an anti-Ly6G antibody. GAPDH was used as a loading control. (H) The densities of the Ly6G bands on the blot were determined using ImageJ software. The results are expressed as the means ± SEM (n = 6). *, p < 0.05 vs. sham-operated Idh2^+/+. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, NADP⁺-dependent isocitrate dehydrogenase 2; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAS, Periodic acid–Schiff.

Fig. 2

Changes in IDH2 expression and activity, and NADPH levels in theIdh2^+/+andIdh2^-/-mice livers after HIR. Livers were harvested 5 h after surgery. (A, B) Whole liver lysates were subjected to western blot analysis using anti-IDH2 antibodies. GAPDH was used as a loading control. (B) The densities of the IDH2 bands on the blot were determined using ImageJ software. (C) The enzyme activity of IDH2 was measured in the liver mitochondrial fractions. (D) The mitochondrial (Mito) and cytosolic (Cyto) fractions were isolated and the efficient separation of the fractions was confirmed by western blot analysis using anti-COX IV and GAPDH antibodies, respectively. β-actin was used as the loading control. (E) Liver sections were subjected to immunohistochemical staining using an anti-NADPH antibody. (F) The intensity of NADPH staining was measured using i-solution software. The results are expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. respective sham-operated mice. #, p < 0.05 vs. sham-operated Idh2^+/+. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2.

Fig. 3

The ratio of GSSG to total glutathione and the activities of glutathione reductase (GR), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), and catalase in the Idh2^+/+ and Idh2^-/- mouse livers after HIR. Livers were harvested 5 h after surgery. In the liver mitochondrial (Mito) fractions, the ratio of GSSG to total glutathione (GSSG/tGSH) (A), and the activities of GR (B), and GPx (C) were determined. In the liver cytosolic (Cyto) fractions, the ratio of GSSG to total glutathione (GSSG/tGSH) (D), and the activities of GR (E), and GPx (F), G6PD (G), and catalase (H) were determined. The results are expressed as the mean ± SEM (n = 6). *, P < 0.05 vs. respective sham-operated mice. #, p < 0.05 vs. sham-operated Idh2^+/+. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2; GSSG, oxidized glutathione; tGSH, total glutathione.

Fig. 4

Levels of reactive oxidative species (ROS) and oxidative stress inIdh2^+/+andIdh2^-/-mouse livers after HIR. Livers were harvested 5 h after surgery. (A) Hydrogen peroxide formation was determined in the whole liver tissue. (B) Liver sections were subjected to immunohistochemical staining using an anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) antibody. (C, D) The number of 8-OHdG positive cells and the intensity of 8-OHdG staining were measured. (E) Liver mitochondrial fractions were subjected to western blot analysis using an anti-oxidized peroxiredoxin (Prx-SO₃) antibody. Cox IV was used as the mitochondrial loading control. (F) The density of the Prx-SO₃ band on the blot was determined using ImageJ software. (G) Lipid peroxidation was determined in the whole liver tissue. The results are expressed as the means ± SEM (n = 6). *, p < 0.05 vs. respective sham-operated mice. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2.

Fig. 5

Mitochondrial fragmentation and damage in theIdh2^+/+andIdh2^-/-mouse livers after HIR. Livers were harvested 1 h or 5 h after surgery. (A) Mitochondrial structures were evaluated under a transmission electron microscope (TEM). Upper panels are at low magnification. Lower panels are at high magnification and are the regions that are within the rectangular box in the upper panels. The extent of mitochondrial abnormal enlargement (B) and loss of cristae (C) were measured. (D) The mitochondrial aspect ratio [(major axis) to (minor axis)] was measured. (E) Livers samples 5 h after surgery were subjected to western blot analysis using anti-Optic atrophy 1 (Opa1) and Fission 1 (Fis1) antibodies. GAPDH was used as a loading control. (F, G) Densities of the bands on the blots were measured using ImageJ software. (H) Five hours after surgery, ATP levels were measured. The results are expressed as the mean ± SEM (n = 6). *, p < 0.05 vs. respective sham-operated mice. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2.

Fig. 6

Apoptosis in the livers ofIdh2^+/+andIdh2^-/-mice after HIR. Livers were harvested 5 h after surgery. (A) Liver mitochondrial (Mito) and cytosolic (Cyto) fractions were subjected to western blot analysis using an anti-cytochrome c (cyto-c) antibody. COX IV and GAPDH were used as loading controls for mitochondrial and cytosolic fractions, respectively. (B, C) The densities of the bands were measured using ImageJ software. (D) Whole liver tissue samples were analyzed by western blot using anti-Bax, -Bcl2, -Bcl-xL, and -cleaved caspase-3 (c-casp. 3) antibodies. GAPDH was used as a loading control. (E to I) The densities of the bands were measured using ImageJ software. (J) Apoptotic cell death was evaluated using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Arrows (green) in box indicate TUNEL-positive cells. DAPI (blue) was used to stain the nuclei. (K) TUNEL-positive cells were counted. The results are expressed as the mean ± SEM (n = 6). *, p < 0.05 vs. respective sham-operated mice. †, p < 0.05 vs. ischemia-operated Idh2^+/+. IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase 2; DAPI, 4′,6-diamidino-2-phenylindole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 7

Effect of Mito-TEMPO treatment on the survival rate, and functional and morphological damage to the livers of Idh2^+/+ and Idh2^-/- mice after HIR. Mice were treated with mito-TEMPO (mito-T), twice, 17 h and 1 h before either hepatic ischemia or sham-operation and livers were harvested 5 h after surgery. (A) Cumulative survival rates were determined until 5 h after HIR. Data are expressed as the cumulated survival rates (B, C) Five hours after surgery, plasma concentrations of AST (B) and ALT (C) were determined. (D, E) Liver sections were stained with PAS reagent and the damaged areas were evaluated. The results are expressed as the mean ± SEM. *, p < 0.05 vs. respective sham-operated mice. †, p < 0.05 vs. respective vehicle-I/R. #, p < 0.05. NS, no significance.

Fig. 8

Effect of Mito-TEMPO treatment on H₂O₂ formation, morphological oxidative damage, and expression of Opa1 and Fis1 in the livers of Idh2^+/+ and Idh2^-/- mice after HIR. Mice were treated with mito-TEMPO (mito-T), twice, 17 h and 1 h before either hepatic ischemia or sham-operation and livers were harvested 5 h after surgery. (A) Levels of H₂O₂ (I) was measured in mitochondrial fraction. (B, C) Liver mitochondrial fractions were subjected to western blot analysis using an anti-Prx-SO₃ antibody. (C) The density of the Prx-SO₃ band was determined using ImageJ software. (D) Liver sections were subjected to immunohistochemical staining using an anti-8-OHdG antibody. (F) Liver whole lysates were subjected to western blot analysis using an anti-optic atrophy 1 (Opa1) antibody. GAPDH was used as a loading control. (G) Densities of the bands on the blots were measured using ImageJ software. The results are expressed as the mean ± SEM. *, p < 0.05 vs. respective sham-operated mice. †, p < 0.05 vs. respective ischemia-operated Idh2^+/+.