EPC-MVs Carrying MiR-98-5p Are Involved In The Mechanism Of AngⅡ-Induced Renal Cell Damage Repair In Rats

Objective:To investigate the mechanism through which miR-98-5p in endothelial progenitor cell microvesicles(EPC-MVs)is involved in the repair of angiotensin II(Ang II)-induced injury of rat primary renal kidney cells(PRKs).Methods:Twelve 8-week-old Wistar-Kyoto male rats were selected and fed for 2 weeks.After isolation of rat renal cortical sections,PRKs were isolated by density gradient centrifugation and identified by immunofluorescence staining.The femur and tibia of the rats were treated,EPCs were separated,and identified by DIL-AC-LDL staining.PRKs were treated with AngⅡ to establish a PRK kidney injury model,used to simulate hypertensive nephropathy in vitro.The supernatants were separated and MVs were further extracted,EPC-MVs were labeled with PKH 26,and PRKs,AngⅡ,and EPCs,EPC-Medium,EPC-MVs constitute the co-culture system,after grouping,the expression levels of miR-98-5p and IGF1 R were determined by Rt-q PCR analysis.Then,miR-98-5p mimic,miR-98-5p inhibitor,miR-98-5p mimic NC,miR-98-5p inhibitor NC were transfected into EPCs.The IGF1 R plasmid and ov-NC plasmid was transfected into PRKs,and the supernatants were separated and MVs were further extracted.The transfected EPC-MVs and PRKs were added to the co-culture systemand and grouped for subsequent experiments.The Gene Expression Omnibus was used to analyze the differentially expressed genes between diabetic rats and normal rats,and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the signaling pathways involved in these differentially expressed genes.Dual luciferase assay was used to detect the binding of miR-98-5p to target genes.CCK-8,FCM,ELISA and Western blot were used to analyze the effects of by miR-98-5p / target gene which regulated PI3K/Akt/ endothelial nitric oxide synthase(e NOS)signaling on the cell viability,oxidative stress(ROS,MDA,GSH and SOD levels)and inflammation(IL-6,IL-1β and TNF-α levels)of PRKs induced by Ang II.All experiments were repeated three times.Results: The results revealed high expression of vimentin and α?SMA in PRKs(IF)under the fluorescence microscope,which suggested the successful isolation of PRKs.The superposition of highly positive red fluorescence(Dil?Ac?LDL staining)and blue fluorescence(nuclear staining with DAPI)under the fluorescence microscope,which suggested the successful isolation of EPCs.PKH26 fluores-cence was detected in the cytoplasm of PRKs,indicating that EPC-MVs had fused with PRKs.The results of Rt-q PCR analysis showed that compared with the control group,AngⅡ-induced down-regulation of miR-98-5p expression and up-regulation of IGF1 R expression were reversed in EPC-MVs group(P<0.05).Dual luciferase assay and Rt-q PCR analysis demonstrated that miR-98-5p targeted regulation of IGF1 R.The results of CCK-8,FCM and ELISA analysis revealed that treatment of PRKs with Ang II decreased cell viability,oxidative stress(ROS and MDA levels increased,GSH and SOD levels decreased)and inflammation(IL-1β,IL-6and TNF-α levels increased).However,EPC-MVs alleviated Ang II-induced damage of the PRKs.miR-98-5p mimic-MVs group protected PRKs from Ang II-induced inhibition of cell viability,oxidative stress and inflammation compared with the control group(P<0.05),and enhanced the effect of EPC-MVs.The results of miR-98-5p inhibitor-MVs group were opposite to those of miR-98-5p mimic-MVs group(P<0.05).Western blot,CCK-8,FCM and ELISA analysis results revealed that compared with AngⅡ group,miR-98-5p mimic-MVs group promoted the phosphorylation of PI3K/Akt/endothelial nitric oxide synthase(e NOS)(P<0.05).It also from Ang II-induced inhibition of cell viability,oxidative stress and inflammation compared with the control group(P<0.05).Compared with the control group,the effects of miR-98-5p mimic-MVs were reversed by miR-98-5p mimic-MVs +ov-IGF1 R group(P<0.05).Conclusion: EPC-MVs carrying high expression of miR-98-5p could promote phosphorylation of the signaling pathway PI3K/Akt/e NOS by downregulating IGF1 R,thereby protecting PRKs from Ang II-induced decrease in cell viability and oxidative stress and inflammation.To provide new targets for the treatment of hypertensive nephropathy.