ADK maintains tissue homeostasis by preventing RIPK1 kinase activation–driven liver injury in physiological conditions and IRI. (A–D)Adkf/f, Adkf/f;Alb-Cre, or Adkf/f;Alb-Cre;Ripk1D138N/D138N mice underwent 1-h ischemia/6-h reperfusion operation at the age of 6 wk. Liver TUNEL staining (A), CC3 immunostaining (B), and MPO immunostaining (C) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (D). Scale bar: 100 μm (A–C). (E–H) Vehicle or 5-ITu (20 mg/kg) was injected intraperitoneally to WT mice at the age of 8 wk. 1 h after the injection, the mice underwent 1-h ischemia/6-h reperfusion operation. Liver TUNEL staining (E), CC3 immunostaining (F), and MPO immunostaining (G) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (H). Scale bar: 100 μm (E–G). (I–L) AAV8 that was expressing Adk-L or control was injected to WT mice through a tail vein. 1 mo after the injection, the mice underwent 1-h ischemia/6-h reperfusion operation. Liver TUNEL staining (I), CC3 immunostaining (J), and MPO immunostaining (K) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (L). Scale bar: 100 μm (I–K). (M) Postreperfusion clinical donor liver injury and inflammation was analyzed with H&E staining, TUNEL staining, CC3 staining, MPO staining, and p-RIPK1 (S166) staining. The donor livers were divided into postreperfusion ADK high- or low-expression groups according to immunoblotting data in Fig. 8 E. The representative images of the two groups are shown. Scale bar: 100 μm. (N) Levels of TUNEL+ cells, CC3+ cells, neutrophils, and p-RIPK1 (S166)–positive cells in postreperfusion clinical donor livers were quantified from staining images in Fig. S4 M. Serum levels of ALT and AST of donor liver recipients on the first day after liver transplantation were collected from their medical records. Postreperfusion ADK expression level was analyzed according to immunoblotting data in Fig. 8 E. The correlations between levels of postreperfusion TUNEL+ cells, CC3+ cells, neutrophils, ALT, AST, p-RIPK1 (S166)–positive cells, and ADK expression level were analyzed. Data are represented as the mean ± SD (A–N). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A–L) or Pearson’s correlation test (N). ***P < 0.001.
Figure S4.

ADK maintains tissue homeostasis by preventing RIPK1 kinase activation–driven liver injury in physiological conditions and IRI. (A–D) Adk f/f, Adkf/f;Alb-Cre, or Adkf/f;Alb-Cre;Ripk1D138N/D138N mice underwent 1-h ischemia/6-h reperfusion operation at the age of 6 wk. Liver TUNEL staining (A), CC3 immunostaining (B), and MPO immunostaining (C) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (D). Scale bar: 100 μm (A–C). (E–H) Vehicle or 5-ITu (20 mg/kg) was injected intraperitoneally to WT mice at the age of 8 wk. 1 h after the injection, the mice underwent 1-h ischemia/6-h reperfusion operation. Liver TUNEL staining (E), CC3 immunostaining (F), and MPO immunostaining (G) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (H). Scale bar: 100 μm (E–G). (I–L) AAV8 that was expressing Adk-L or control was injected to WT mice through a tail vein. 1 mo after the injection, the mice underwent 1-h ischemia/6-h reperfusion operation. Liver TUNEL staining (I), CC3 immunostaining (J), and MPO immunostaining (K) were performed. Serum levels of proinflammatory cytokines TNFα and IL-6 were detected with ELISA (L). Scale bar: 100 μm (I–K). (M) Postreperfusion clinical donor liver injury and inflammation was analyzed with H&E staining, TUNEL staining, CC3 staining, MPO staining, and p-RIPK1 (S166) staining. The donor livers were divided into postreperfusion ADK high- or low-expression groups according to immunoblotting data in Fig. 8 E. The representative images of the two groups are shown. Scale bar: 100 μm. (N) Levels of TUNEL+ cells, CC3+ cells, neutrophils, and p-RIPK1 (S166)–positive cells in postreperfusion clinical donor livers were quantified from staining images in Fig. S4 M. Serum levels of ALT and AST of donor liver recipients on the first day after liver transplantation were collected from their medical records. Postreperfusion ADK expression level was analyzed according to immunoblotting data in Fig. 8 E. The correlations between levels of postreperfusion TUNEL+ cells, CC3+ cells, neutrophils, ALT, AST, p-RIPK1 (S166)–positive cells, and ADK expression level were analyzed. Data are represented as the mean ± SD (A–N). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A–L) or Pearson’s correlation test (N). ***P < 0.001.

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