Animal Models

Male C57BL/6 mice (2-, 18- and 24-month-old mice) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All experiments involving animals were approved by the Animal Ethics Committee at Southern Medical University. To testify the effects of O304 on the naturally aging mice, unilateral nephrectomy was firstly performed in the 18-month-old mice. One week after the surgery, some mice were sacrificed as control group, while other mice were injected intraperitoneally with saline solution or O304 (HY-112233; MedChemExpress) (10 mg/kg/d) every day for 12 weeks. The detailed experimental designs were shown in Figure 3A. Then all the mice were sacrificed and tissues were collected for various analyses.

O304 could activate AMPK, alleviate renal senescence and fibrosis, and inhibit β catenin in naturally aging mice. (A) Experimental design. White bar indicated the time after the surgery was 1 week. Green bar indicated mice were administered intraperitoneal injections of 0.9% sodium chloride solution for 12 weeks after surgery. Orange bar indicated mice were administered intraperitoneal injections of O304 at 10 mg/kg/day for 12 weeks after surgery. UNX: unilateral nephrectomy. (B) Representative micrographs showing renal expression of p-AMPK in different groups. Paraffin-embedded kidney sections were immunostained with antibody against p-AMPK. Arrows indicate positive staining. Scale bar, 50 μm. (C–E) Representative western blot and quantitative data showing renal expression of p-AMPK and AMPK in different groups. Numbers (1–3) indicate each individual animal in each given group. ***p < 0.001 versus 18-month-old mice group. ^## p < 0.01, ^### p < 0.001versus 21-month-old mice group alone (n = 5). (F–K) Representative western blot and quantitative data showing renal expression of active β-catenin, P16^INK4A, P19^ARF, α-SMA and fibronectin in different groups. Numbers (1–3) indicate each individual animal in each given group. **p < 0.01, ***p < 0.001 versus 18-month-old mice group. ^## p < 0.01, ^### p < 0.001versus 21-month-old mice group alone (n = 5). (L) Representative micrographs showing the expressions of active β-catenin (top), β-galactosidase activity (SA-β-gal) (middle) and fibronectin (bottom) in kidneys in different groups. Cryosections were subjected to fluorescence staining for active β-catenin or stained for SA-β-gal. Paraffin-embedded kidney sections were immunostained with antibody against fibronectin. Arrows indicate positive staining. Scale bar, 50 μm.