| Objective: Peritoneal Dialysis(PD)is one of the alternative treatment modalities for patients with end-stage renal disease(ESDR),with more than 272,000 patients receiving PD worldwide,representing about 11% of the global dialysis population.The advantages of PD include affordability,simplicity,and protection of residual kidney function,but there are some problems,such as insufficient PD training and long-term dialysis leading to peritoneal damage,such as inflammation and fibrosis.Chronic stimulation by dialysis fluid leads to repeated damage and inflammatory responses in peritoneal tissue,with the involvement of oxidative stress and apoptosis of mesothelial cells,which eventually leads to epithelial-to-mesenchymal transition(EMT)trans-differentiation of mesothelial cells,causing fibrosis in peritoneal tissue and eventual withdrawal of the patient from PD.Therefore,the study of the mechanism of peritoneal EMT occurrence and its effective preventive interventions is of great theoretical and clinical significance to improve the survival quality of patients with ESRD.It has been proposed that mesenchymal stem cells(MSCs)can alleviate peritoneal fibrosis(PF),but the exact mechanism remains to be added.The aim of this study was to investigate whether exosomes(h UMSC-Exos)from human umbilical cord MSCs(h UMSCs)could alleviate peritoneal mesothelial cell EMT.Methods: Different concentrations(1.5%,2.5%,4.25%)of high glucose were used to stimulate peritoneal mesothelial cells(HMr SV5)to induce an in vitro peritoneal EMT model,and CCK-8 was applied to observe the optimal modeling conditions.WB and q RTPCR were taken to detect the changes in EMT occurring in HMr SV5 due to different concentrations of high glucose.According to three criteria for MSCs identification,the plastic adhesive ability of stem cells was observed under light microscopy;osteogenic differentiation,lipogenic differentiation,and chondrogenic differentiation were verified by alizarin red,oil red,and alizarin blue staining;and the surface-specific molecule expression of h UMSCs was detected by flow cytometry.The h UMSC-Exos concentration kit was applied to extract h UMSC-Exos,and then h UMSC-Exos was identified by electron microscopy,NTA,and WB.Different concentrations of h UMSC-Exos were applied to treat high glucose-induced HMr SV5,and the appropriate concentration of h UMSC-Exos exhibiting anti-EMT effects was detected by CCK-8.The effect of h UMSC-Exos on high glucose-induced HMr SV5 morphology was observed under light microscopy.PKH67-labeled h UMSC-Exos was applied to observe whether peritoneal mesothelial cells take up h UMSC-Exos under inverted fluorescence microscopy.Appropriate h UMSC-Exos was added to high glucose-treated peritoneal mesothelial cells to observe the expression of EMT markers.The expression of the Hippo signaling pathway-related gene YAP/TAZ was detected to investigate the anti-EMT mechanism of h UMSC-Exos in HMr SV5.Results: EMT expression of HMr SV5 was most evident after 2.5% high glucose stimulation for 48 hours.h UMSCs were successfully differentiated into bone,fat,and cartilage,while surface-specific molecules CD73(91.3%)and CD90(97.6%)were positive and CD45(0.3%)was negative,demonstrating that h UMSCs were successfully extracted.NTA analysis showed that the average particle size of h UMSC-Exos was 131.4nm,h UMSC-Exos showed a clear exosomal "teato-like" structure under electron microscopy and clearly expressed HSP70 and TSG101,demonstrating the successful isolation of h UMSC-Exos.CCK-8 results suggested that h UMSC-Exos at 100 μg/m L restored the most significant proliferative capacity of peritoneal cells treated with 2.5%high glucose.HMr SV5 was observed to uptake h UMSC-Exos under inverted fluorescence microscopy.Compared to the control group,the number of cells decreased and showed a shuttle shape after adding 2.5% high glucose for 48 hours;WB,q RT-PCR,and immunofluorescence suggested that E-Cadherin was significantly down-regulated and the expression of Vimentin and α-Sma was up-regulated in high glucose-induced peritoneal mesothelial cells with EMT changes;the protein expression of YAP and TAZ was significantly up-regulated,and YAP moving into the nucleus was increased.Conclusion: Experimental data suggest that 2.5% high glucose stimulation of HMr SV5 for 48 hours is optimal for EMT modeling.h UMSCs were successfully identified and h UMSC-Exos was successfully isolated and characterized.h UMSC-Exos can be taken up by HMr SV5 and can significantly alleviate high glucose-induced EMT in peritoneal mesothelial cells.Mechanistic studies suggest that h UMSC-Exos may alleviate EMT in peritoneal mesothelial cells via the YAP/ TAZ-mediated Hippo pathway. |
PDF Full Text Request