Intermedin Protects Peritubular Capillary Endothelial Cells From Acute Kidney Injury To Chronic Kidney Disease By Reducing Oxidative Stress

Objective:To investigate whether intermedin(IMD)protects the endothelial cells of the peritubular capillary(PTC)by eliminating oxidative stress,preventing PTC destruction,and stabilizing the number of PTC,thereby inhibiting the transition of acute kidney injury(AKI)into chronic kidney disease(CKD).Methods:IMD knockout(IMD-KO)male mice and littermate wild-type(IMD-WT)male mice on the C57BL/6 background were divided into two groups: the mice in the control group were isolated the left renal pedicle and removed the right kidney 14 days later,while the left renal pedicle of the mice in model group were isolated and clipped for 21 minutes to create renal ischemia-reperfusion injury(IRI),and the right nephrectomy was also conducted 14 days later.The left nephrectomy of six mice were collected on 1 day and 42 days after right nephrectomy respectively.Masson staining was used to calculate the degree of renal fibrosis;immunocytochemistry was used to measure CD31 to determine the density of PTC endothelial cells;western blotting and q-PCR were used to quantify the expression of vascular endothelial cadherin(VE-cadherin),a marker of renal endothelial cells,so as to detect the number of endothelial cells;dihydroethidium(DHE)immunofluorescence assay was used to evaluate the expression of reactive oxygen species(ROS)in renal tissue;western blotting was also used to quantify the expression of caspase-3 active fragments in renal tissue to determine cell apoptosis; immunohistochemical staining was used to measure the expression of F4/80 and VCAM-1 to determine the inflammation of kidney tissue.Results:Masson staining showed that the area of renal fibrosis in the IMD-KO control group increased at 42 days after right nephrectomy,compared with the IMD-WT control group,but there was no statistical significance(P > 0.05).Compared with the corresponding control group,there were statistically significant increases in renal fibrosis outcome in the IMD-WT model group and the IMD-KO model group(P < 0.05),and the fibrosis results in the IMD-KO model group was more severe compared with the IMD-WT model group(P<0.05).Immunocytochemistry analysis showed that at 1 day after right nephrectomy,CD31 expression decreased in the kidney tissue of the IMD-KO control group,compared with the IMD-WT control group(P < 0.05).Compared with the corresponding control group,the expression of CD31 in the IMD-WT model group and the IMD-KO model group decreased significantly(P < 0.05),and CD31 expression in the IMD-KO model group decreased more obviously compared with IMD-WT model group(P<0.05).Data from western blotting and RT-PCR analysis showed that at 1 day after right nephrectomy,the expression of VE-cadherin in the IMD-KO control group was significantly down-regulated(P < 0.05),compared with IMD-WT control group.Compared with the corresponding control group,the IMD-WT model group and the IMD-KO model group had statistical decreases in the expression of VE-cadherin(P <0.05).Compared with the IMD-WT model group,the expression of VE-cadherin in the IMD-KO model group decreased more dramatically(P<0.05).The results of immunofluorescence showed that at 1 day after right nephrectomy,ROS expression in kidney tissue of the IMD-KO control group was higher than that of the IMD-WT control mice(P < 0.05).Compared with the corresponding control group,the ROS expression in the IMD-WT model group and the IMD-KO model group increased significantly(P <0.05),and the IMD-KO mice had more significant increases compared with the IMD-WT mice(P<0.05).Western blotting analysis showed that at 1 day after right nephrectomy,the expression of caspase-3 active fragments in the IMD-KO control mice had no significant difference(P>0.05)compared with the IMD-WT control mice.Compared with the corresponding control group,the expression of caspase-3 active fragments in the IMD-WT model group and the IMD-KO model group was significantly increased(P<0.05).Compared with the IMD-WT model group,the expression of caspase-3 active fragments in the IMD-KO model group increased more significantly(P<0.05).Immunohistochemistry analysis showed that at 1 day after right nephrectomy,the expression of F4/80 and VCAM-1 of kidney tissue in the IMD-KO control group was significantly higher than that in the IMD-WT control group(P<0.05).Compared with the corresponding control group,the expression of F4/80 and VCAM-1 in the IMD-WT model group and the IMD-KO model group increased significantly(P<0.05),of which in the IMD-KO model group increased more significantly(P<0.05).Conclusion:IMD protects PTC endothelial cells,prevents PTC destruction and AKI-CKD transition by inhibiting the renal ROS expression and its downstream inflammatory response and cell apoptosis.