Study Of Urinary Exosomal MiRNA In IgA Nephropathy And Evaluation Of Clinical Diagnostic Value

Objective:To analyze the difference of urinary exosomal miRNA expression profile between IgA nephropathy(IgAN)and healthy controls.Screening miRNA as biomarkers to evaluate patients with IgAN,The clinical diagnostic value of exosome miRNA in IgAN was evaluated.Methods:In this study,urinary exosomes were isolated from 29 IgAN and 29 healthy controls.The miRNA was analyzed by high-throughput sequencing.Urine of 9 IgAN and 9 healthy subjects was selected to screen miRNA with differential expression using high-throughput sequencing technology.The urine of 20 IgAN and 20 healthy controls was detected by real-time quantitative polymerase chain reaction(RT-PCR)to verify the candidate miRNA.We evaluated the diagnostic value of miRNAs by receiver operating characteristic(ROC)curves.We used Spearman correlation to analyze the correlation between candidate miRNA and clinical parameters.Results:We successfully extracted exosomes using exosome extraction kit and ultra-centrifuge process.The marker proteins CD9 and CD63 of exosomes were detected by Western Blot.The morphology and structure of exosomes were obviously observed under transmission electron microscope.Seven up-regulated miRNAs were identified in IgA nephropathy compared with the healthy subjects by high-throughput sequencing.RT-qPCR was used to verify the candidate miRNA,The expression of hsa-miR-451a and hsa-let-7d-3p in IgA nephropathy was significantly higher than the healthy control group.The results were consistent with high throughput sequencing.We evaluated the diagnostic value of hsa-miR-451a and hsa-let-7d-3p using ROC curves,hsa-miR-451a(Area under Curve AUC = 0.805,P = 0.001),hsa-let-7d-3p(AUC = 0.76,P = 0.0049),the combination of hsa-miR-451a and hsa-let-7d-3p(AUC = 0.8125,P = 0.0007).hsa-miR-451a has correlations with Lee’s grades(r = 0.511,P = 0.021),24-hour urinary protein quantification(UPQ;r = 0.557,P = 0.011);Hsa-let-7d-3p showed correlations with Lee’s grades(r = 0.6,P = 0.005),UPQ(r = 0.518,P = 0.019),Serum creatinine(Scr;r = 0.564,P = 0.01),Estimated glomerular filtration rate(e GFR;r =-0.532,P =0.016).According to the Oxford classification,for hsa-miR-451a,S0 had lower levels than S1(P = 0.016);For hsa-let-7d-3p,M0 had lower levels than M1(P = 0.05).Conclusion:The expression of urinary exosomal miRNA of IgA nephropathy and healthy controls was significantly different,hsa-miR-451a and hsa-let-7d-3p may serve as noninvasive biomarkers for the evaluation of IgAN.