Analysis Of Urinary Exosomal MiRNA Expression Profile And Diagnostic Value In Patients With Type 2 Diabetic Kidney Disease

Diabetic kidney disease?DKD?has become the primary cause of chronic kidney disease in China.At present,the diagnosis of DKD based on the glomerular filtration rate?eGFR?<60ml·min-1·1.73m-2 or UACR?>30mg/g?has certain limitations,and it is susceptible to the influence of strenuous exercise,high-protein diet,infection,fever and other factors and false positive results.Therefore,a non-invasive biomarker is needed to more accurately reflect renal function in diabetic patients.Exosomes are small bilayer microvesicles formed by the Exosomes in cells and the Exosomes fall off after the fusion with the cell membrane.They contain a large number of RNA,miRNA,specific proteins and other substances with biological functions.They play an important role in various pathophysiological processes by participating in inter-cell information transmission.Recent studies have shown that the urinary exosome miRNA is a potential biomarker and carrier for the diagnosis and treatment of DKD,which is helpful for us to further study the pathogenesis of DKD.ObjectiveTo study the difference between the expression profiles of urinary exosomal miRNA in patients with DM and DKD,observe the correlation between miRNA and renal function indexes,and find novel biomarkers for diagnosis of DKD.MrthodsUrine samples of 17 DM patients and 17 DKD patients from The First Affiliated Hospital of Zhengzhou University were collected and divided into screening sequence?DM-A group:n=3,DKD-A group:n=3?and confirmation sequence?DM-B group:n=14,DKD-B group:n=14?.MiRNA microarray detection technology was used to analyze the miRNA microarray expression profile of urinary exosomes of patients with screened sequences.MiRNA with large expression differences were screened according to Fold change>2 and P value?<0.05?,and RT-PCR was used for further quantitative analysis in the confirmed sequences.Statistically difference of micrornas with renal function indexes such as glomerular filtration rate?glomerular filtration rate,eGFR?,24-hour urine trace albumin?microalbuminuria,UALB?correlation analysis and assessment of diagnostic value,clear the biological markers in the diagnosis of DKD.Finally,bioinformatics was used to obtain the target genes and enrichment pathways of differentially expressed mirnas and to investigate their possible involvement in the pathogenesis of DKD.SPSS 20.0 was used for statistical processing and analysis,and GraphPad Prism 8.0 was used to generate graphs.All clinical data were expressed as mean±standard deviation.The mann-whitney U test was used to compare the miRNA expression levels between the groups,expressed as median?25%-75%?.Spearman order correlation was used to analyze the correlation between differential miRNA and eGFR and 24hUALB respectively.The sensitivity and specificity of biomarkers were calculated by ROC curve to evaluate their diagnostic efficacy.Using miRWalk?http://mirwalk.umm.uni-heidelberg.de/?,TargetScan?http://www.targetscan.org/vert₇2/?and miRDB?http://www.mirdb.org/?to predict the miRNA target genes,DAVID?https://david.ncifcrf.gov/?to predict concentration pathways.Results1.There was no significant difference in age,course of disease and BMI between the DM group and the DKD group in the screening sequence or the confirmation sequence?P>0.05?.DKD group had worse renal function with higher micro-albuminuric?UALB?and glomerular filtration rate?eGFR?than controls and DM group?P<0.01?.Furthermore,DKD patients were associated with diabetic retinopathy;2.According to the results of urinary exosomal microarray,a total of 2085 miRNA were detectable from DKD patients?Fold change>5,P<0.05?.326 miRNAs showed significant upregulation in the exosomes of DKD group compared to DM group;3.Among these miRNAs,212 are up-regulated and 14 are down-regulated;14 up-regulated miRNAs?miR-4491,miR-2117,miR-4507,miR-5088-5P,miR-1587,miR-219a-3p,miR-5091,miR-498,miR-4687-3p,miR-516b-5p,miR-4534,miR-1275,miR-5007-3p,miR-4516?were screened according to Fold change>2 and P values<0.05;4.The relative expression level of 7 miRNA species miR-2117,miR-5091,miR-4687-3p,miR-516b-5p,miR-4534,miR-1275,miR-5007-3p exhibited increased in the DKD group compared with DM group?P<0.05?in confirmation cohort;5.Up-regulated urinary exosomal miR-516b-5p?r=0.4037,P=0.0331?and miR-4534?r=0.5604,P=0.006?expression positively correlated with microalbuminuria?UALB?;6.ROC analysis revealed that miR-4534 had an area under the curve?AUC?of 0.786?95%confidence interval,0.607-0.965?,which was better than miR-516b-5p with an AUC of 0.750?95%confidence interval,0.561-0.939?and the combination of miR-4534 and miR-516b-5p with an AUC of 0.781?95%confidence interval,0.602-0.959?in the confirmation cohort;7.As predicted by the database,the results showed that there were 733 mRNAs involved in the intersection and the enrichment pathway contained 10 pathways including glutamatergic synapses,axon guidance,neurotrophic signal pathways,transcriptional misregulation in cancer,and FOXO signal pathways etc.Conclusions1.The expression of miRNA in urinary exosomes in the DKD group was different from that in the DM group;2.Urinary exosomes miR-516b-5p and miR-4534 were positively correlated with 24hUALB in patients with DKD;3.The urinary exosome miR-4534 maybe be a biomarker for the diagnosis of DKD.