The Clinical Study Of Urinary Exosomal MiRNA As Novel Biomarkers In Idiopathic Pediatric Nephrotic Syndrome

BackgroundExosomes,extracellular vesicles secreted by cells,can regulate the biological activity of target cells by carrying miRNAs,proteins or lipids and play an important role in intercellular communication.It has been found that exosome-derived miRNAs can be used as a promising novel biomarker for a variety of diseases.However,the global miRNAs signature of urinary exosomes in children with idiopathic nephrotic syndrome(NS)has not been reported and its clinical value is unclear.ObjectiveTo investigate the expression profile of urinary exosomal miRNAs in children with idiopathic NS and to identify the significantly increased miRNAs in children with NS and to evaluate the clinical diagnostic value of miRNAs and the potential as biomarkers for the diagnosis and treatment of idiopathic NS.MethodsMorning urine samples were collected from 129 children with NS and 126 normal children matched in sex and age.We extracted exosomes from 5 pooled NS urine samples(n = 5/group)and 4 pooled healthy urine samples(n = 5/group)by ultrahigh-speed centrifugation.The miRNAs in these groups were detected by high-throughput Illumina SBS sequencing technique.After screening out the significantly elevated miRNAs in NS patients,the exosome extraction kit was used to extract the exosomes from all urine samples one by one.The quantitative reverse-transcription PCR(RT-qPCR)was used to validate the results in two independent sample groups(training set and validation set),and 65 paired urine samples from these patients were compared before and after treatment.Finally,ROC analysis,spearman correlation analysis and binary logistic regression analysis were used to evaluate the clinical value of the elevated miRNAs as biomarkers for NS.Results(1)Illumina SBS sequencing results showed that the expression levels of urinary exosomal miRNAs in NS patients were significantly different from that in normal controls.Of these,30 miRNAs were significantly up-regulated(?5-fold)and 15 miRNAs was significantly up-regulated by more than 9 times.(2)Five miRNAs(miR-194-5p,miR-146b-5p,miR-378a-3p,miR-23b-3p,miR-30a-5p)were identified to stably expressed and significantly increased in the urinary exosomes of NS patients in training set and validation set(3-13-fold,all P<0.01),and decreased significantly after remission(P<0.001).(3)ROC curve analysis indicated that the AUCs of five miRNAs for diagnosis of NS in training set and validation set were 0.770-0.890 and 0.658-0.803,respectively,miR-194-5p had the highest diagnostic value and the AUCs in training set and validation set were 0.890(95%CI = 0.749-0.986)and 0.803(95%CI = 0.744-0.862),followed by miR-23b-3p.Among all of the combinations of miRNAs,the combination of miR-194-5p and miR-146b-5p had a higher diagnostic accuracy,but lower than the independent diagnostic accuracy of miR-194-5p and miR-23b-3p.In addition,miR-23b-3p had higher diagnostic value for distinguishing post-treatment from pre-treatment and the AUC was 0.771(95%CI = 0.691-0.851)in the ROC analysis.(4)The expression levels of urinary exosomal miR-194-5p and miR-23b-3p in the high urine protein group were significantly higher than those in the low urine protein group(P<0.01).(5)Spearman correlation analysis suggested that the contents of 5 exosomal miRNAs in urine were positively correlated with serum cholesterol and triglyceride,but negatively correlated with serum albumin and total protein.However,only miR-194-5p and miR-23b-3p were positively correlated with 24-hour urinary protein.Furthermore,binary logistic regression analysis confirmed that 5 miRNAs were potential risk factors for NS(B>0,OR>1,P<0.05).ConclusionsWe identified 5 significantly up-regulated miRNAs in urinary exosomes of children with idiopathic NS,which are expected to be potential liquid biopsy indicators for NS.