High Glucose Induces HK-2 Cell-derived Small Extracellular Vesicles To Promote Renal Tubular Phenotypic Alterations Via MiR-21-5p

Objective:Diabetic nephropathy(DN)is one of the most common and serious microvascular complications of diabetes mellitus,and 20-30% of patients with diabetes mellitus will develop DN.The number one cause of most end-stage renal disease(ESRD)is DN,which is also the leading cause of death in end-stage renal disease patients.The clinical manifestations of DN are persistent proteinuria and decreased glomerular filtration rate,followed by irreversible pathological changes of renal fibrosis in advanced stages,eventually leading to ESRD.Studies have shown that renal fibrosis in DN is associated with extracellular matrix(ECM)deposition in the renal tubular interstitium,which in turn is associated with epithelial mesenchymal transition(EMT).The mechanisms of EMT in DN are not fully understood.Small extracellular vesicles(s EVs)are bilayer membrane structured vesicles secreted by cells that carry various genetic materials to the recipient cells and can affect intercellular communication.Micro RNAs(mi RNAs)are important loaders of s EVs and have been shown to be involved in the progression of various diseases.In recent years,various studies have shown that s EVs play an important role in the diagnosis and disease progression of DN.The role of s EVs-loaded mi RNAs in DN has also been heavily investigated,however,the pathophysiological role of s EVs crosstalk within proximal renal tubular epithelial cells in the development and progression of DN is unclear.In this study,we investigated the mechanism of action of s EVs secreted by HK-2 cells under high glucose stimulation on renal tubular phenotypic transformation via mi R-21-5p.Methods:1.Plasma and cellular s EVs were separated using ultracentrifugation,and the extracted s EVs were identified by nano-flowmetry,transmission electron microscopy and Western Blot(WB).2.The expression levels of mi R-21-5p in plasma s EVs from DN group and healthy physical examination group were detected by RT-q PCR,and the difference in expression levels was calculated.3.DN models were constructed using glucose-stimulated HK-2 cells,and the expression levels of EMT marker proteins in each group were detected using WB,and the expression levels of mi R-21-5p in cells and cellular s EVs were detected by RT-q PCR.4.HK-2 cell-derived s EVs were induced using high glucose and co-incubated with recipient HK-2 cells,and s EVs uptake was observed by confocal microscopy.5.Lentiviral infection of HK-2 cells was used to construct stable overexpression and knockdown of mi R-21-5p in a stably transduced HK-2 cell line.6.Normal glucose(NG)group and high glucose(HG)group cell-derived s EVs were co-cultured with HK-2 cells and knockdown mi R-21-5p cells,respectively,and the changes of proliferation,migration and EMT were detected by CCK8,scratch experiment and WB experiment.7.The functional changes of the cells in NG group,HG group,overexpression group and knockdown group were detected by CCK8,scratch assay and WB.Results:1.s EVs were distributed between 45-200 nm by nano-flowmetry;s EVs were detected by transmission electron microscopy as biconcave discs with intact envelope;three positive marker proteins ALIX,TSG101 and CD63 bands were detected by WB for s EVs,and GM130 band was not detected.2.The expression of mi R-21-5p in plasma s EVs was significantly higher in the diabetic nephropathy group compared with the healthy physical examination group,and the difference was statistically significant(P<0.0001).3.Glucose-stimulated HK-2 cells were used to construct a diabetic nephropathy model,and the expression of fibronectin was significantly higher and E-cadherin was significantly lower in the HG group compared with the NG group,and the difference was statistically significant(P<0.0001).4.mi R-21-5p expression was significantly higher in cells of HG group compared with NG group,and the difference was statistically significant(P<0.05).mi R-21-5p expression was significantly higher in cell-derived s EVs of HG group,and the difference was statistically significant(P<0.001).5.CCK8,scratch and WB experiments showed that HK-2 cells co-cultured with HG-s EVs had strong proliferative,migration and EMT capabilities(P<0.05).HK-2 cells knocked down mi R-21-5p were co-cultured with HG-s EVs,which reversed the ability of HG-s EVs to proliferate,migrate,and EMT of HK-2 cells(P<0.05).6.CCK8,scratch and WB experiments showed that HK-2 cells overexpressing mi R-21-5p had stronger proliferation,migration and epithelial mesenchymal transition ability(P<0.05);HK-2 cells knocking down mi R-21-5p in high glucose culture could reverse the proliferation,migration and epithelial mesenchymal transition ability of HK-2 cells induced by high glucose(P<0.05).Conclusion:1.Ultracentrifugation method can successfully extract morphologically intact s EVs from peripheral blood plasma and cell culture supernatants.2.mi R-21-5p was consistent with plasma expression in cell-derived s EVs,both higher than those in the control group.3.High glucose-induced HK-2 cell-derived s EVs can promote the proliferation,migration and epithelial-mesenchymal transformation of HK-2 cells.4.The ability of HK-2 cells overexpressing mi R-21-5p to proliferate,migrate and metathelial-mesenchymal transformation is enhanced.5.The effects of s EVs and mi R-21-5p on HK-2 cells in high-glycemic groups were similar,indicating that high-glucose-induced HK-2 cell-derived s EVs can promote HK-2 cells proliferation,migration and epithelial-mesenchymal transformation through mi R-21-5p.