Effect Of Extracellular Vesicles From High Glucose-treated Podocytes On Proximal Tubular Epithelial Cells

BackgroundDiabetic nephropathy(DN)is one of the common and serious microvascular complications of diabetes mellitus(DM).DN is the leading cause of end stage renal disease and it predominantly accounts for the increased all-cause mortality and cardiovascular mortality in DM patients.DN is characterized by increasingproteinuria and declining renal function.Tubulointerstitial injury is closely correlated to renal function decline and disease progression in DN,but the mechanism of tubular injury in DN has not yet been fully elucidated.Studies have shown that the interplay between tubular epithelial cells and other resident cells is an important factor contributing to tubular injury.Extracellular vesicles(EVs),secreted into the extracellular space by cells,are membrane-bound vesicles that play a key role in mediating cell-to-cell communication.MicroRNA(miRNA)in EVs is emerging as crucial factor involved in the pathophysiological processes of many diseases.Recently,studies have revealed the important roles of EVs and EVs miRNA in the pathogenesis of DN.However,in DN,the effect and related mechanism of podocytes EVs on proximal tubular epithelial cells remain unclear.AimTo investigate the effect of EVs derived from high glucose-treated podocytes on proximal tubular epithelial cells and the related mechanism.MethodsEVs were isolated from the podocytes culture supernatants using ExoQuick-TC,and then were identified by transmission electron microscopy,nanoparticle tracking analysis(NTA)and western blot analysis.EVs were labeled with the green fluorescent dye PKH67 and then co-cultured with proximal tubular epithelial cells,and a confocal microscope was used to observe whether tubular epithelial cells could internalize podocytes EVs.Flow cytometry and TUNEL staining experiments were used to observe the effect of EVs from high glucose-treated podocytes on proximal tubular epithelial cells.High-throughput miRNA sequencing was performed on podocytes EVs to screen out the differentially expressed miRNA,and bioinformatics analysis was used to investigate their potential functions and signal pathways.EVs were isolated from high glucose-treated podocytes that transfected with let-7f-2-3p inhibitor or inhibitor NC,and then co-cultured with proximal tubular epithelial cells.Flow cytometry was used to observe the effect of EVs from high glucose-treated let-7f-2-3p inhibitor-transfected podocytes on proximal tubular epithelial cells.ResultsThe typical morphology of EVs was observed by transmission electron microscopy,a mean diameter of approximately 150 nm of EVs was detected by NTA,and EVs protein markers were detected by western blot analysis.Under a confocal microscope,green fluorescence was observed in the cytoplasm of proximal tubular epithelial cells.The results of flow cytometry and TUNEL staining showed that apoptotic tubular epithelial cells were increased after co-culture with EVs from high glucose-treated podocytes.From miRNA sequencing results,five miRNAs in podocytes EVs that differentially expressed in response to high glucose were screened out,including mmu-miR-1981-3p,mmu-miR-3474,mmu-miR-7224-3p,mmu-miR-6538 and mmu-let-7f-2-3p.Functional and pathway enrichment analysis revealed biological processes related to pro-apoptosis or pro-injury and pathways related to DN.As results from flow cytometry showed,apoptotic tubular epithelial cells were decreased after treatment with EVs from high glucose-treated podocytes that transfected with let-7f-2-3p inhibitor,compared to treatment with EVs from high glucose-treated podocytes that untransfected or transfected with inhibitor NC.ConclusionHigh glucose-treated podocytes release let-7f-2-3p-enriched EVs and induce apoptosis of proximal tubular epithelial cells.