ADAR1 plays a protective role in proximal tubular cells under high glucose conditions by attenuating the PI3K/AKT/mTOR signaling pathway

Figures

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  Figure 1. Detect the expression characteristics of ADAR1 in proximal renal tubular cells of db/db mice with spontaneous type 2 diabetes mellitus. (a) Detect FBG, (b) weight, (c) food intake, and (d) water intake of db/db mice with spontaneous type 2 diabetes mellitus and normal db/m mice. (e)–(i) IHC assays were presented for detecting the expression of (f) ADAR1, (g) p-PI3K, (h) p-AKT1, and (i) p-mTOR. (j)–(n) Western blot was presented for detecting the expression of (k) ADAR1, (l) p-PI3K, (m) p-AKT1, and (n) p-mTOR. Bar, 20 μm. *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 2. ADAR1 regulates SOD and MDA. (a) ADAR1-overexpression plasmids regulated ADAR1 mRNA expression level. The effects of different concentrations of (b) ADAR1 inhibitor 8-azaadenosine, (c) PI3K inhibitor LY294002, and (d) mTOR inhibitor rapamycin on the proliferation of HK-2 cells were screened by CCK8. (e) and (f) Detect SOD activity and MDA content under high-glucose conditions, ADAR1 inhibitor 8-azaadenosine, and ADAR1-overexpression. ***p < 0.001.

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  Figure 3. ADAR1 affects endoplasmic reticulum stress (a) and (b) ER-tracker dye detected effect of ADAR1 on the intensity of endoplasmic reticulum stress fluorescence probe under the high-glucose environment. (c) and (d) ER-tracker dye detected effect of ADAR1 on the intensity of endoplasmic reticulum stress fluorescence probe under high-glucose environment and rapamycin treatment. (e) and (f) ER-tracker dye detected effect of ADAR1 on the intensity of endoplasmic reticulum stress fluorescence probe under high-glucose environment and LY294002 treatment. Bar, 50 μm. *p < 0.05; ***p < 0.001.

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  Figure 4. ADAR1 affects cell apoptosis (a) and (b) TUNEL staining assessed the effect of ADAR1 on the ratio of red fluorescent-labeled cells under the high-glucose environment. (c) and (d) TUNEL staining assessed the effect of ADAR1 on the ratio of red fluorescent-labeled cells under high-glucose environment and rapamycin treatment. (e) and (f) TUNEL staining assessed the effect of ADAR1 on the ratio of red fluorescent-labeled cells under high-glucose environment and LY294002 treatment. Bar, 20 μm. (g) CCK8 assays detected cell proliferation ability of HK-2 cells with HG, ADAR1 inhibitor 8-azaadenosine, and ADAR1 overexpression. (h) CCK8 assays detected cell proliferation ability of HK-2 cells with HG, ADAR1 inhibitor 8-azaadenosine, ADAR1 overexpression, and rapamycin. (i) CCK8 assays detected cell proliferation ability of HK-2 cells with HG, ADAR1 inhibitor 8-azaadenosine, ADAR1 overexpression, and LY294002. *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 5. ADAR1 affects PI3K/AKT/mTOR signaling pathway under the high-glucose environment. (a)–(j) Expressions of (b) ADAR1, (c) PI3K, (d) p-PI3K, (e) AKT1, (f) p-AKT1, (g) mTOR, (h) p-mTOR, (i) cyclin D1, and (j) Ki67 were measured by western blot in HK-2 cells or ADAR1-overexpressed HK-2 cells under the high-glucose environment. ***p < 0.001.

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  Figure 6. ADAR1 affects PI3K/AKT/mTOR signaling pathway under high-glucose environment and LY294002 treatment. (a)–(h) Expressions of (b) ADAR1 (c) PI3K, (d) p-PI3K, (e) AKT1, (f) p-AKT1, (g) mTOR, and (h) p-mTOR were measured by western blot in HK-2 cells or ADAR1-overexpressed HK-2 cells under high-glucose environment and LY294002 treatment. **p < 0.01; ***p < 0.001.

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  Figure 7. ADAR1 affects PI3K/AKT/mTOR signaling pathway under high-glucose environment and rapamycin treatment. (a)–(h) Expressions of (a) ADAR1 (c) PI3K, (d) p-PI3K, (e) AKT1, (f) p-AKT1, (g) mTOR, and (h) p-mTOR were measured by western blot in HK-2 cells or ADAR1-overexpressed HK-2 cells under high-glucose environment and rapamycin treatment. ***p < 0.001.

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  Figure A1. Detect the expression characteristics of ADAR1 in proximal renal tubular cells of db/db mice with spontaneous type 2 diabetes mellitus.(a) Immunofluorescence assays were presented for detecting the expression of (b) ADAR1, (c) p-PI3K, (d) p-AKT1, and (e) p-mTOR. Bar, 20 μm. **p < 0.01.

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  Figure A2. Detect the characteristics of db/db mice with spontaneous type 2 diabetes mellitus. Representative images of tissue stained with (a) H&E, (b) and (c) Masson’s are listed. Bar, 20 μm. (d) Cr (e) BUN levels were determined in the control and db/db mice. The result of (f) insuin (INS), (g) OGTT. **p < 0.01; ***p < 0.001.