472-P: The Profiles of MicroRNAs from Urinary Extracellular Vesicles (EVS) Prepared by Various Isolation Methods and Their Correlation with Serum EV MicroRNAs in Patients with Type 2 Diabetes and Diabetic Nephropathy

Abstract

MicroRNAs that circulate in biological fluids are frequently enclosed in extracellular vesicles. However, urinary EVs and their cargo miRNAs have not been systematically studied according to their EV isolation methods. In type 2 diabetics with nephropathy (n=4), we compared miRNA species in urine EVs prepared by various isolation methods, including ultracentrifugation, qEV original size exclusion column, ExoQuick-TC Plus (ExoQuick), and simple ultrafiltration using Amicon Ultra centrifugal filter devices (Amicons) 10K and 100K. EV miRNAs were profiled by next-generation sequencing . We evaluated the correlations of EV miRNA expression between the urine and serum samples isolated by UC. From each of 100 ml of urine, the UC method yielded the highest number of EV miRNA species (233 ± 37.3), with the ExoQuick yielded the lowest (103 ± 17.4). Urine EV miRNA profiles were highly correlated between UC, qEV, ExoQuick and Amicon 10K methods. EV miRNA profiles between the urine and serum samples showed variable correlations between the patients (paired sample number=3, r =0.39-0.72). we examined EV miRNAs from urine isolated using two pore sizes with 10 kDa and 100 kDa filters. The expression level patterns of EV miRNAs isolated from urine were strongly correlated between Amicon 10K and the other three validated methods (UC, qEV, and ExoQuick). Pearson correlation coefficients between Amicon 100K and the three methods varied from 0.19 to 0.33. UC, qEV, ExoQuick, and Amicon 10K are suitable for urinary EV isolation to profile miRNAs. Urine- and serum-derived EV miRNA profiles have positive and variable correlations depending on specific patients. Further studies are required to identify a factor that influences these correlations and to determine the sample type that better describes the clinical and pathophysiological status of diabetic patients with diabetic kidney disease.