Effect And Mechanism Of Peritoneal Dialysis Fluids Exosome MicroRNA With Peritoneal Membrane Function

Background:Peritoneal dialysis(PD)is an essential method of renal replacement therapy.Peritoneal mesothelial cells are affected by many factors,undergoing epithelial–mesenchymal transition(EMT)and resulting in fibrosis during PD.These changes deteriorate peritoneal membrane function and lead to treatment failure.Therefore,it is important to explore the influencing factors and pathogenic mechanisms of EMT in peritoneal mesothelial cells.Exosomes act as mediators in cell–cell communication and can affect disease occurrence and development by altering their contents,which include proteins and nucleic acids.Exosomes have been reported as biomarkers and potential therapeutic compounds in many diseases.We screened the micro RNAs(miRNAs)in PD fluid exosomes as diagnostic biomarkers for predicting peritoneal membrane function.Furthermore,we explored the effect of highly expressed miRNAs on EMT in peritoneal mesothelial cells and peritoneal membrane function in PD rats.Methods:Three separation methods were evaluated to isolate pure exosomes from PD fluid: an Exo Quick precipitation kit,high-speed centrifugation,and ultrafiltration-based centrifugation.The exosome samples were examined by transmission electron microscopy,particle size distribution analysis,and western blotting.In the discovery phase,exosome samples isolate from high-and low-transport status peritoneal membrane function patients(two groups,n = 4 per group)were tested by next-generation transcriptome sequencing assays.The gene expression of miRNAs in the PD fluid exosomes was established.Differentially expressed miRNAs were screened as candidate genes for further research.The downstream target genes were identified by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses.In the validation stage,high-and low-transport status peritoneal membrane function and high-and low-average transport status peritoneal membrane function patients(four groups,n = 10 patients per group)were enrolled.The PD fluid exosomes were separated by ultrafiltration-based centrifugation.The miRNA expression in the exosomes was determined by real-time PCR and analyzed by operating characteristic curve(ROC)to test the association between peritoneum membrane function and miRNA expression.To identify whether they could be taken up by cells,the exosomes purified from PD fluids were labeled with PKH26 and co-cultured with HMr SV5 cells.To explore the effect of miR-199a-3p on EMT,HMr SV5 cells were transfected with miR-199a-3p mimic,inhibitor,and negative control.The HMr SV5 cell proliferation ability and E-cadherin,cytokeratin,vimentin,?-SMA,and type I collagen expression levels were evaluated by Cell Counting Kit-8,western blotting,and q PCR.The dual-luciferase reporter assay was used to confirm that NEDD4 was the target gene of miR-199a-3p.NEDD4 m RNA and protein expression levels in the HMr SV5 cells were detected by western blotting and q PCR,respectively.The PD fluid exosomes with highly expressed miR-199a-3p were isolated and injected into the abdominal cavity of wistar rats.E-cadherin,vimentin,?-SMA,and NEDD4 expression in peritoneal tissue was detected by immunohistochemistry.The peritoneal membrane function was determined by the peritoneal equilibration test(PET).Results: Transmission electron microscopy;particle size distribution analysis;and the membrane proteins CD63,CD81,and TSG101 proved that exosomes existed in PD fluid.Ultrafiltration-based centrifugation was the preferred method for isolating exosomes from PD fluid.The miRNA sequencing determined that 123 exosomal miRNAs were differentially expressed between the high and low transporter status patients: 63 were upregulated and 60 were downregulated.The predicted target genes and signaling pathways of these miRNAs were involved in the biological process,cellular component,molecular function,c GMP–PKG,RAP1,and oxytocin signaling pathways.In the high transport status peritoneal membrane function PD patients,miR-199a-3p expression was significantly increased.The area under the ROC(AUC)was 0.788,(95%CI 0.629-0.947)when miR-199a-3p as a predictor used in high transport status peritoneal membrane function patients.The PKH26-labeled exosomes could be taken up and dispersed in the HMr SV5 cell cytoplasm.Transfection with miR-199a-3p mimic in vitro promoted HMr SV5 cell proliferation ability and EMT.It reduced E-cadherin and cytokeratin expression;and increased vimentin,?-SMA,and type I collagen expression compared with negative control.However,transfection with the miR-199a-3p inhibitor reversed these changes.The dual-luciferase reporter assay revealed that NEDD4 is a direct target of miR-199a-3p in HMr SV5 cells.Overexpressing miR-199a-3p decreased NEDD4 expression in the HMr SV5 cells.In vivo,rats injected with high-miR-199a-3p expression exosomes in vivo exhibited decreased E-cadherin and NEDD4 and increased vimentin and ?-SMA.These EMT changes in the peritoneum were the same as that in the HMr SV5 cells.The PET demonstrated that high-miR-199a-3p expression exosomes enhanced the dextrose-transporting ability in D2/D0 in the exosome injection group and the PD group(0.95±0.08,0.56±0.16,0.45±0.09,P = 0.004).Conclusion: PD fluids contain exosomes and ultrafiltration-based centrifugation is suitable for isolating the exosomes.In the PD exosomes,miR-199a-3p expression correlated with high peritoneal transport status in patients.Exosomes extracted from PD fluids were taken up and dispersed in the cytoplasm of HMr SV5 cells.miR-199a-3p overexpression inhibited NEDD4 expression and promoted EMT in HMr SV5 cells.miR-199a-3p-rich exosomes improved EMT changes in rat peritoneum and reduced peritoneal transport function.