Effect Of Hypoxia Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat(FG-4592) On Acute Kidney Injury Induced By Ischemia Reperfusion

Objective:To investigate the protective effect and related mechanisms of hypoxia inducible factor（HIF）stabilizer FG-4592 pretreatment on acute renal injury induced by renal ischemia-reperfusion injury.Methods:Cell level:（1）To explore the effect of pretreatment with different concentrations of FG-4592 on the expression of HIF-1α.The HK-2 cells were divided into 5 groups,Control,IR,FG-4592（10u M）+IR,FG-4592（25u M）+IR and FG-4592（50u M）+IR groups.The three groups intervened in different concentrations of FG-4592were intervened 24 hours in advance according to the preset concentration,and then except the control group,the other four groups were all replaced with low-glucose medium,and then in three-gas incubator according to 5%CO₂,1%O₂,a hypoxia-reoxygenation model was established,and a total of 24 hours of hypoxia was established.After these four groups of cells were replaced with complete medium,they were reoxygenated for 6 hours under normal oxygen conditions of 5%CO2,21%O2.Subsequently,the expression of HIF-1αin each group was detected by Western blotting,and the 25u M group was selected for subsequent research according to the results.（2）The overall apoptosis rate of Control,IR and FG-4592（25u M）+IR groups was detected by flow cytometry.At the same time,the expression of related apoptotic proteins was detected by Western blotting.（3）The expression levels of PI3K/Akt signaling pathway-related proteins in Control,IR and FG-4592（25u M）+IR groups were detected by Western blotting.（4）The cells were divided into four groups:Control,IR,FG-4592（25u M）+IR and LY294002+FG-4592（25u M）+IR.The treatment methods of the first three groups were as described above.LY294002+FG-4592（25u M）+IR was pretreated with PI3K selective inhibitor LY294002（20u M）for 1 hour,and then FG-4592（25u M）was added to the original basis for 24 hours on the original basis,then replaced with low-glucose medium,and continued add LY294002（20u M）in a three-gas incubator for 24 hours of hypoxia intervention,and then replaced with normal complete medium for re-oxygenation for 6 hours.After the modeling was completed,the expression level of PI3K/Akt-related pathway proteins was detected by Western blotting to explore whether HIF-1αexerted the above-mentioned effects through the PI3K/Akt signaling pathway.Animal level:16 healthy male Wistar rats of SPF grade,weighing about 220 g,were randomly divided into 4 groups,4 rats in each group（n=4）.The Sham group and IR group were treated with 1ml/100g/day and intraperitoneal injection of 0.9%saline for 7 days,and the FG-4592 group and FG-4592+IR group were treated with 1mg/100g/day and intraperitoneal injection of FG-4592 for 7 days.Subsequently,the rat renal ischemia-reperfusion model was established in IR group and FG-4592+IR group.After ligation of the pedicle of the right kidney,the right kidney and the attached ureter were removed,and the left kidney was clipped for 45 minutes with a non-invasive vascular clip,and then removed.Blood perfusion was restored for24 hours.After the tissue samples were transected along the coronal plane,immunofluorescence samples and some wax block samples were collected respectively,and the remaining samples were stored in a-80°C refrigerator.Serum was extracted from blood samples after centrifugation,and serum creatinine（Scr）was detected to evaluate whether the model was successful and the kidney injury after modeling.HE staining and PAS staining were performed on the paraffin sections of kidney tissue to evaluate the degree of renal injury,and HIF-1αimmunofluorescence（IF）was used to detect the expression of HIF-1α,and TUNEL staining was used to detect the apoptosis of tissue cells.Results:Cell level:（1）The expression of HIF-1αpretreated with 25u M concentration of FG-4592 was relatively higher,and was significantly higher than that of HIF-1αin the IR group.（2）Flow cytometry showed that the overall apoptosis rate of IR group was much higher than that of Control group and FG-4592（25u M）+IR group,and the apoptosis-related proteins were detected by western blotting,which showed that the apoptosis of IR group was higher than that of FG-4592（25u M）+IR group.（3）The PI3K/Akt signaling pathway was activated in the FG-4592（25u M）+IR group,and the expression of p-Akt,the activated form of Akt,was significantly increased,and its expression level was higher than that in the IR group.（4）After LY294002 intervention,the expression level of p-Akt in LY294002+FG-4592（25u M）+IR group was significantly lower than that in FG-4592（25u M）+IR group,indicating that PI3K/Akt signaling pathway was successfully inhibited.The apoptosation-related proteins were detected by Western blotting,which showed that the apoptosis of the cells was higher than that of the FG-4592（25u M）+IR group.Animal level:The serum creatinine level of IR group was higher than that of the other three groups,and the observation under HE staining microscope after the wax section of the tissue showed that the cell disassembly tubule necrotic expansion,brush margin disappeared,tubular and calcium salt deposition occurred in IR group,while the damage of FG-4592+IR group was relatively light,and no obvious damage was found in the other two groups.The immunofluorescence of HIF-1αin renal tissue showed that pre-ischemic preconditioning with FG-4592 successfully inhibited the degradation of HIF-1α,and HIF-1αin the FG-4592+IR group was significantly higher than that in the other three groups.TUNEL staining showed that there was obvious apoptosis in the IR group,but no obvious apoptosis in the Sham group and the FG-4592 group,and the apoptosis in the FG-4592+IR group was significantly lower than that in the IR group.Conclusions:FG-4592 pretreatment can stabilize the expression of HIF-1α,and HIF-1αcan inhibit the apoptosis of HK-2 cells after renal ischemia-reperfusion injury through PI3K/Akt signaling pathway,thus alleviating the acute renal injury caused by renal ischemia-reperfusion injury.