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. 2022 Oct 9;12(16):7158–7179. doi: 10.7150/thno.78376

Figure 8.

Figure 8

PA-S14 induces autophagy activity and preserves mitochondrial homeostasis in ADR nephropathy through promoting LKB1/AMPK activation. (A-C) Representative western blot and quantitative data showing renal expression of p-LKB1, LKB1, p-AMPKα, and AMPKα in different groups. Numbers (1-3) indicate each individual animal in a given group. **P < 0.01, ***P < 0.001 versus control mice; ##P < 0.01, ###P < 0.001 versus ADR mice (n = 5). (D) Representative micrographs show the p-LKB1 and p-AMPKα expression in different groups as indicated. Kidney sections were stained with antibodies against p-LKB1 and p-AMPKα, respectively. Arrows indicate positive staining. Scale bar, 50 µm. (E) Representative micrographs show the expression of LC3B. The frozen kidney sections were stained with an antibody against LC3B. Arrows indicate positive staining. Scale bar, 50 µm. (F) Ultrathin kidney sections were studied using a transmission electron microscope (TEM). TEM analyses show that PA-S14 increased autophagic vacuoles (yellow arrowheads). Scale bar, 1 µm. (G-I) Representative western blot and quantitative data showing renal expression of the LC3BII/I ratio, SQSTM1, ATG5, p-mTOR, and mTOR in different groups. Numbers (1-3) indicate each individual animal in a given group. **P < 0.01, ***P < 0.001 versus control mice; ##P < 0.01, ###P < 0.001 versus ADR mice (n = 5). (J) Ultrathin kidney sections were studied using a transmission electron microscope. Arrow indicates the normal round or rod‐shaped mitochondria and regular arrangement of mitochondrial cristae in the group treated with PA-S14. Scale bar, 1 µm. (K-L) Representative western blot (K) and quantitative data (L) show an increased expression of PGC-1α, TFAM and TOMM20 proteins in ADR kidneys after PA-S14 treatment. Numbers (1-3) indicate each individual animal in given group. ***P < 0.001 versus control mice; #P < 0.05, ###P < 0.001 versus ADR mice (n = 5). (M) Representative micrographs show the expression of SQSTM1, p-mTOR, PGC-1α, TOMM20. Paraffin kidney sections were used for immunohistochemistry staining. The frozen kidney sections were stained with MitoTracker deep red (3 µm) probe. DAPI (4′,6‐diamidino‐2‐phenylindole) was used to stain the nuclei (blue). Arrows indicate positive staining. Scale bar, 50 µm. (N) Quantification of renal mitochondrial mass is shown. Mitochondrial mass was determined by the fluorescence intensity of MitoTracker deep red staining normalized to DAPI. ***P < 0.001 versus control mice; ###P < 0.001 versus ADR mice (n = 5).