Introduction: To figure out the role of metformin in acute renal ischemic reperfusion (IR) injury.
Methods: HK-2 cell was treated with, PBS, metformin, 3MA and rapamycin. Western-blot was used to observe cell autophagy and apoptosis level. 15 C57 mice were divided randomly into Control group, IR group, IR+ 3-MA group, IR+ rapamycin group, IR+ metformin group, with 3 mice in each group. In this research, the IR mouse model was established through clamping the bilateral renal artery and vein for 20min. The above mice were treated with PBS, metformin (200mg/kg), 3-MA (30mg/kg) and rapamycin (1mg/kg) by intraperitoneal injection respectively. All the mice were sacrificed after anesthesia 24h later, serum, the kidney was obtained for sequent experiments. The kidney was divided into two parts, one of which was fixed by 2.5% glutaraldehyde for autophagosome and apoptosis body observation under electron microscope, the other part was fixed by 10% formalin for the later histological experiments, including IHC, TUNEL staining and H&E.This study was approved by the Hospital Ethics Committee.
Results: Metformin inhibits autophagy and apoptosis level in HK-2 cell. In the IR model, metformin showed a more effective protective role compared to 3-MA and control group. After metformin treatment, IHC staining showed less LC3B and cleaved-caspase3 expression, and LAMP2 (lysosome) expression was inhibited. Metformin decreased serum creatinine and ß2-microglobulin. TUNEL staining showed apoptosis level decreased after metformin treatment. We also observed decreased autophagosome and apoptosis body in metformin group through electron microscope scan. In addition, H&E staining showed kidney necrosis was inhibited after metformin treatment.
Conclusions: Altogether, in this research, we established IR mouse model of kidney stone. We firstly found metformin inhibits autophagy and apoptosis in renal cell, which indicated metformin plays a protective role of metformin in acute renal ischemic reperfusion injury.
Source of Funding: National Natural Science Foundation of China (81860456, 81760462)