Fig 2: The HIF-1a-BNIP3 pathway was activated during hypoxia. (a) Analysis of mitophagy-related protein expression in Neuro-2a cells by immunoblotting. Neuro-2a cells were exposed to hypoxia and/or high glucose for 48 h. Total cellular extracts were analysed by immunoblotting with antibodies against HIF-1a, BNIP3, PINK1 and TOMM20. (b) The ImageJ densitometric analysis is shown. “C” represented control conditions (25 mM glucose), “G” represented high glucose conditions (75 mM glucose). The results are expressed as the mean ± SEM from three independent experiments. Two-way ANOVA, **p < 0.01, *p < 0.05, Hypoxia vs Normoxia. Full-length blots are presented in Supplementary Fig. S2.

Fig 3: BNIP3 deficiency exacerbated mitochondrial damage and apoptosis in the CI-AKI mice. (A) Survival of WT CI-AKI and bnip3-/- CI-AKI mice with iohexol treatment (10 mL/kg bodyweight) after 24 h (n = 10). (B) Diagrammatic representation of modified WT and bnip3-/- CI-AKI models. Briefly, the dose of iohexol reduced to 5 mL/kg bodyweight. (C) The renal function was evaluated by serum creatinine. (D and E) Representative histology and pathological tubular injury score in the renal cortex by H-E staining. The tubular injury was indicated by arrows. Scale bar: 50 µm. (F and G) Representative IHC images and quantification of BNIP3 in kidney cortex. Scale bar: 50 µm. (H and I) Immunoblotting analysis and quantification of BNIP3 and LC3B-II. (J-L) Representative images and quantification of immunofluorescence double-labeling LC3B and VDAC, VDAC and LAMP1. Scale bar: 20 µm. (M) Representative TEM images of mitochondrial morphology in renal tubular epithelial cells of WT and bnip3-/- mice. Scale bar: 500 nm. (N-Q) Apoptosis was measured by immunoblotting analysis of cleaved CASP3 and TUNEL staining of renal tubular epithelial cells of WT and bnip3-/- CI-AKI mice. Scale bar: 50 µm. n = 3–5. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Western blots of BNIP3, BNIP3 L, HIF-1a and LC3II. BNIP3, Bcl-2/adenovirus E1B 19-kDa interacting protein 3; BNIP3 L, BNIP3 like; HIF-1a, hypoxia-inducible factor 1a; LC3II, microtubule-associated protein 1A/1B light chain 3B; IR, ischemia-reperfusion; pre-LD, low-dose dexmedetomidine preconditioning; pre-HD, high-dose dexmedetomidine preconditioning; post-LD, low-dose dexmedetomidine postconditioning; post-HD, high-dose dexmedetomidine postconditioning.

Fig 5: Resveratrol treatment reversed the accumulation of P62 to enhance autophagy levels and attenuate fibrosis. (A) The mRNA level of LC3B was quantified. (B,C) The correlation between LC3B and FN1 and COL3A1 gene expression showed a stronger positive correlation. Spearman’s r and p values are indicated. (D) Levels of SOD2, HIF-1a, and LC3 in lung tissues were detected using Western blotting. (E) The immunoblotting data of LC3 II/I were quantified as described above. (F,G) The mRNA levels of P62 and BNIP3 were quantified. (H) The correlation between P62 and FN1 gene expression showed a stronger positive correlation. Spearman’s r and p values are indicated. (I) Levels of P62, p-P62(phospho-SQSTM1/p62(Ser349)), BNIP3, BECN1, and BCL2 in lung tissues were detected using Western blotting. (J) The relative intensity of P62 was quantified as described above using Western blotting. Values represent the mean ± SEM (n = 3–5). * p < 0.05 and ** p < 0.01 compared with the control group, and compared with the model group (CIA+BLM), # p < 0.05 and ## p < 0.01.