Korean J Physiol Pharmacol.  2021 Mar;25(2):139-146. 10.4196/kjpp.2021.25.2.139.

IDH2 gene deficiency accelerates unilateral ureteral obstructioninduced kidney inflammation through oxidative stress and activation of macrophages

• Kim JI
• Noh MR
• Yoon GE
• Jang HS
• Kong MJ
• Park KM

• KMID: 2512957
• DOI: http://doi.org/10.4196/kjpp.2021.25.2.139

Abstract

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2^–/–). Eight- to 10-week-old female IDH2^–/– mice and wild type (IDH2^+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2^–/– mice, while UUO decreased IDH2 in IDH2^+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2^+/+ and IDH2^–/– mice, and these changes were greater in IDH2^–/– mice compared to IDH2^+/+ mice. Bone marrow-derived macrophages of IDH2^–/– mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2^+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2^–/– mice compared to IDH2^+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.

Keyword

IDH2; Inflammation; Macrophage; Oxidative stress; Ureteral obstruction

Figure

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  Fig. 1 Unilateral ureteral obstruction (UUO) decreased IDH2 expression. (A, B) IDH2+/+ and IDH2−/− female mice were subjected to either UUO or a sham operation. Kidneys were harvested 5 days after the operation. (A) Representative Western blots for IDH2 and GAPDH in the kidneys. (B) The graph summarizes IDH2 expression. Data are presented as mean ± SE (n = 4). **p < 0.01 vs. sham-operated IDH2+/+ group.

• 

  Fig. 2 IDH2 gene deficiency accelerated unilateral ureteral obstruction (UUO)-induced mitochondrial H2O2 production and lipid peroxidation. IDH2+/+ and IDH2−/− female mice were subjected to either UUO or a sham operation. Kidneys were harvested 5 days after the operation. (A) H2O2 level was measured in the kidney cell mitochondrial fraction. (B) Representative Western blots for HNE and COX IV in the mitochondrial fraction of kidneys. (C) The graph summarizes HNE expression. Data are presented as mean ± SE (n = 3). **p < 0.01 vs. its own sham-operated group; ##p < 0.01 vs. UUO-operated IDH2+/+ group.

• 

  Fig. 3 IDH2 gene deficiency accelerated unilateral ureteral obstruction (UUO)-induced leukocytes infiltration. IDH2+/+ and IDH2−/− female mice were subjected to either sham or UUO operation. Kidneys were harvested 5 days after the operation. (A) Representative Western blots for Ly6G and GAPDH in the kidneys. (B) The graph summarizes Ly6G expression. **p < 0.01 vs. sham-operated own control group. Data are presented as mean ± SE (n = 4). ##p < 0.01 vs. UUO-operated IDH2+/+ group. $$p < 0.01 vs. sham-operated IDH2+/+ group. (C) Representative pictures of immunohistochemically stained kidneys against F4/80 (brown). (D) The graph summarizes F4/80 expression. Data are presented as mean ± SE from 2 independent experiments. **p < 0.01 vs. sham-operated own control group. ##p < 0.01 vs. UUO-operated IDH2+/+ group.

• 

  Fig. 4 Migration and ruffle formation of bone marrow-derived macrophages were dependent on the IDH2 expression. (A) Representative pictures of bright field microscopy of cultured bone marrow-derived macrophages of IDH2+/+ and IDH2−/− mice. (B) Representative pictures of bright field microscopy of H&E-stained migrated bone marrow-derived macrophages of IDH2+/+ and IDH2−/− mice. Macrophages stained pink. Small circles are pores of the membrane filters. (C) The graph summarizes the number of migrated macrophages. Data are presented as mean ± SE (n = 3). **p < 0.01 vs. IDH2+/+. (D) Representative images of immunofluorescence staining for IDH2 (green), Rac1 (red), DAPI for nuclear staining (blue), and merge.

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