Regulation Of MicroRNA-21 On The Function Of Glomerular Endothelial Cells Under The Intervention Of High Insulin Or High Glucose

Objective:Diabetic kidney disease(DKD),as a common microvascular complication of type 2 diabetes mellitus,has become the primary cause of end-stage renal disease with the rapid increase of the prevalence of diabetes mellitus(DM).Therefore,early diagnosis and treatment are of great significance.Impaired glucose tolerance(IGT)is an important stage of pre-diabetic development and an independent risk factor for microvascular complications in DM.We observed the occurrence of kidney lesions in different stages of type 2 diabetes mellitus in the early stage of the study.We found that the rat model of IGT stage had different degrees of renal structure and function damage,mainly renal tubular microcirculation disturbance,and its specific mechanism needs further study.The early manifestation of microcirculation disorders around renal tubules is mainly endothelial cell dysfunction,which is an important part of renal microcirculation and the "first line of defense" against pathogenic factors.The main characteristics of abnormal glucose tolerance are insulin resistance and compensatory hyperinsulinemia.Compensatory elevated insulin may regulate the release of vasoconstrictive active substances in endothelial cells through PI3K/AKT and MAPK/ET-1 signaling pathways,resulting in reduced NO synthesis,over-secretion of ET-1 and renal tubular microcirculation disturbance.Therefore,vascular endothelial dysfunction caused by compensatory hyperinsulinemia in IGT stage may be an important pathogenic mechanism of renal injury in IGT stage.Various microRNAs are associated with the pathogenesis of DKD,in which the expression of microRNA-21 is significantly altered in pre-diabetic patients and insulin resistance animal models,and participates in the regulation of insulin signaling pathway.Therefore,microRNA-21 may mediate endothelial dysfunction caused by hyperinsulinemia by participating in insulin signaling pathways.Therefore,we propose an experimental hypothesis:high concentration of glucose and insulin interfere with the expression of microRNA-21 in peritubular capillary endothelial cells,regulate the downstream PTEN/PI3K/AKT/eNOS pathwayand the expression of MAPK/ET-1 pathway proteins,resulting in dysfunction of endothelial cell secretion and proliferation.In this study,the expression of microRNA-21 and downstream PTEN/AKT/eNOS and MAPK/ET-1 signaling pathway proteins in glomerular endothelial cells under the intervention of high concentration of glucose and insulin,and the expression of microRNA-21 and downstream pathway proteins in aorta of OLEFT rats during IGT phase were observed.At the same time,the expression of microRNA-21 and downstream pathway proteins in aorta tissues of OLEFT rats was detected under the intervention of high concentration of glucose and insulin.The secretion of NO and ET-1 in glomerular endothelial cells,the proliferation and migration of endothelial cells,and the effects of microRNA-21 on downstream pathway proteins and endothelial cell function were studied by transfection of microRNA-21 mimimics or inhibitor to explore the possible pathogenesis of renal injury in IGT stage.Method:1.The expression of microRNA-21 in glomerular endothelial cells was observed by qRT-PCR with different concentrations of glucose(5.5 mol/L,8.3 mmol/L,11.1 mmol/L,16.7 mmol/L,33.3 mmol/L)or insulin(0 ng/ml,5 ng/ml,10 ng/ml,25 ng/ml,50 ng/ml)intervening with time(0 h,12 h,24 h,36 h,48 h,72 h).2.Western blot was used to observe the expression of PTEN/AKT/eNOS pathway and MAPK/ET-1 pathway in the downstream of microRNA-21 48 hours after different concentrations of glucose(5.5mmol/L,16.7mmol/L,33.3mmol/L)or different concentrations of insulin(0ng/ml,5ng/ml,25ng/ml,50ng/ml).3.To investigate the role of microRNA-21 in the regulation of PTEN/AKT/eNOS and MAPK/ET-1 pathway,microRNA-21 was transfected into mimics or inhibitor for functional acquisition and functional deletion.4.At the same time,different concentrations of glucose and insulin were given:8.3 mmol/L of glucose or 33.3 mmol/L of glucose plus different concentrations of insulin(0ng/ml,5ng/ml,25ng/ml,50ng/ml)for 48h to observe the expression of microRNA-21 and downstream PTEN/AKT/eNOS pathway and MAPK/ET-1 pathway.5.The expression of microRNA-21 in the aorta of OLEFT rats at IGT stage was observed by qRT-PCR,and the expression of PTEN/AKT/eNOS and MAPK/ET-1 in the two groups was observed by Western blot.6.Nitrate reductase assay was used to observe the effects of different concentrations of glucose(5.5mmol/L,16.7mmol/L,33.3mmol/L)or different concentrations of insulin(0ng/ml,5ng/ml,25ng/ml,50ng/ml)on NO secretion in glomerular endothelial cells for 48 hours.Transfection of microRNA-21 MICs or inhibitor was used to investigate the effect of microRNA-21 on NO secretion.7.ELLSA was used to determine the effects of different concentrations of glucose(5.5mmol/L,16.7mmol/L,33.3mmol/L)or different concentrations of insulin(Ong/ml,5ng/ml,25ng/ml,50ng/ml)on the secretion of ET-1 in glomerular endothelial cells for 48 hours.The effects of microRNA-21 MICs or inhibitor on the secretion of ET-1 were investigated.8.Cell scratch assay was used to observe the effects of glucose 33.3 mmol/L or insulin 50 ng/ml for 48 hours on the proliferation and migration of glomerular endothelial cells.Transfection of microRNA-21 mimics or inhibitor to study the role of microRNA-21 in regulating the proliferation and migration of endothelial cells.Result:1.Effects of high concentration of glucose on the expression of microRNA-21,the activity of PTEN/AKT/eNOS and MAPK/ET-1 pathway in glomerular endothelial cells and the function of glomerular endothelial cells1.1.Compared with 5.5 mmmol/L normal glucose concentration group,the expression of microRNA-21 in glomerular endothelial cells was significantly increased in 16.7 mmol/L and 33.3 mmol/L glucose concentration groups,and the expression of microRNA-21 increased gradually with the increase of glucose concentration.The effect of high concentration of glucose on the expression of microRNA-21 was time-dependent,rising from 24 hours to 48 hours,reaching its peak.1.2.Compared with 5.5mmol/L glucose concentration group,16.7mmol/L and 33.3mmol/L glucose concentration group decreased PTEN expression,increased AKT phosphorylation,increased eNOS expression and phosphorylation,decreased P38-MAPK phosphorylation and decreased ET-1 expression in glomerular endothelial cells.1.3.Compared with 5.5mmol/L glucose concentration group,the NO secretion of glomerular endothelial cells in 16.7mmol/L and 33.3mmol/L glucose concentration group increased significantly,while the ET-1 secretion decreased significantly.With the increase of glucose concentration,the NO secretion increased gradually,and the ET-1 secretion decreased gradually.Compared with 33.3 mmol/L glucose concentration group,the NO secretion of glomerular endothelial cells decreased significantly and the ET-1 secretion increased significantly in 33.3 mmol/L+miR-21 inhibitor group.The migration rate of glomerular endothelial cells in 33.3 mmol/L glucose concentration group and 33.3 mmol/L glucose concentration+microRNA-21 inhibitor group was not significantly different from that in normal group.2.Effects of high concentration insulin intervention on the expression of microRNA-21,the activity of PTEN/AKT/eNOS and MAPK/ET-1 pathway and the function of glomerular endothelial cells2.1.Compared with the 0 ng/ml insulin concentration group,the expression of microRNA-21 in the 25 ng/ml and 50 ng/ml insulin concentration groups decreased significantly,and the expression of microRNA-21 decreased gradually with the increase of insulin concentration.The effect of high insulin concentration on the expression of microRNA-21 was time-dependent,which began to decrease from 24 hours and reached the lowest level at 48 hours.2.2.Compared with 0 ng/ml insulin concentration group,the expression of PTEN increased,the phosphorylation of AKT decreased,the expression of eNOS decreased,the phosphorylation of P38-MAPK increased and the expression of ET-1 increased in 25 ng/ml and 50 ng/ml insulin concentration groups.2.3.Compared with 0 ng/ml insulin concentration group,the NO secretion and ET-1 secretion of glomerular endothelial cells in 25 ng/ml and 50 ng/ml insulin concentration groups decreased significantly,and the ET-1 secretion increased gradually with the increase of insulin concentration.Compared with 50 ng/ml insulin concentration group,the NO secretion of glomerular endothelial cells increased significantly and the ET-1 secretion decreased significantly in 50 ng/ml insulin concentration+microRNA-21 mimics group.There was no significant difference in the migration rate of glomerular endothelial cells between 50 ng/ml insulin concentration group and 50 ng/ml insulin concentration+microRNA-21 mimimics group.3.The effect of microRNA-21 on PTEN/AKT/eNOS pathway and MAPK/ET-1 pathway3.1.Compared with 33.3 mmol/L glucose concentration group,the expression of PTEN,AKT,eNOS,P38-MAPK and ET-1 increased in 33.3 mmol/L glucose concentration+microRNA-21 inhibitor group.3.2.Compared with 50 ng/ml insulin concentration group,the expression of PTEN,AKT phosphorylation,eNOS expression and phosphorylation were increased,and the expression of P38-MAPK phosphorylation and ET-1 were decreased in 50 ng/ml insulin concentration+miR-21 mimimics group.4.The effects of glucose and insulin on the expression of microRNA-21 and PTEN/AKT/eNOS and MAPK/ET-1 pathways in glomerular endothelial cellsIn the group of 4.1.8.3 mmol/L glucose+different insulin concentrations(Ong/ml,5ng/ml,25ng/ml,50ng/ml),the expression of microRNA-21 in glomerular endothelial cells decreased with the increase of insulin concentration,the expression of PTEN increased,the phosphorylation of AKT decreased,the expression of eNOS decreased,the phosphorylation of P38-MAPK and ET-1 increased.The expression of microRNA-21 in glomerular endothelial cells in 4.2.33.3 mmol/L glucose+different insulin concentrations(Ong/ml,5ng/ml,25ng/ml,50ng/ml)group increased first and then decreased with the increase of insulin concentration.At 5ng/ml insulin concentration,the expression of microRNA-21 was the highest,the expression of PTEN was the lowest,the expression of AKT and eNOS reached the peak,and the phosphorylation of P38-MAPK was the highest.The level and ET-1 expression reached the lowest level.4.4.The expression of microRNA-21 in aorta of OLETF rats in IGT phase was significantly lower than that in normal group,the expression of PTEN increased,the phosphorylation of AKT decreased,the expression of eNOS and phosphorylation decreased,the phosphorylation of P38-MAPK increased,and the expression of ET-1 increased.Conclusion:1.High concentration of glucose can activate PTEN/AKT/eNOS pathway of glomerular endothelial cells by up-regulating microRNA-21,promote NO secretion,inhibit MAPK/ET-1 pathway and inhibit ET-1 secretion,but it has no significant effect on the proliferation and migration of glomerular endothelial cells.2.High concentration of insulin can inhibit the PTEN/AKT/eNOS pathway and NO secretion of glomerular endothelial cells by down-regulating microRNA-21,and activate the MAPK/ET-1 pathway to promote ET-1 secretion,but it has no significant effect on the proliferation and migration of glomerular endothelial cells.3.When high concentration of glucose and insulin acted together on glomerular endothelial cells,the expression of microRNA-21 and the activity of downstream PTEN/AKT/eNOS and MAPK/ET-1 pathway depended on the relative concentration of glucose and insulin.