Circulating urinary RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations contain disease-specific miRNAs, which are currently explored as biomarkers in renal disease and bladder cancer. One of the major difficulties in the clinical application of uEV is the variety and technical complexity of the methods of uEV isolation. Previously we profiled the miRNA expression in the bladder biopsies of patients with bladder outlet obstruction (BOO)-induced LUTD and suggested a panel of miRNA biomarkers for different functional states of the bladder. Here we optimized different uEVs isolation methods to determine the miRNA profiles of total urine and urinary exosomes and correlated these data with urine composition and biopsy findings.
We used five different methods to isolate uEVs and compared the uEV morphology, presence of exosome protein markers, RNA content, uEV size distribution and yield. We present a combined ultracentrifugation and size exclusion chromatography for highly reproducible isolation of 50-150 nm uEVs, corresponding to the exosomes, from 50 ml of starting urine. We profiled miRNAs of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR.
Ultracentrifugation combined with size exclusion after protease inhibitor and DTT Treatment resulted in the best exosome yield and purity. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine, however, 15 miRNAs could only be detected in the total urine preparations, and might represent naked circulating miRNA species. Of the three-miRNA signature components, identified in the BOO patients’ biopsies, hsa-miR-103a-3p was detected in total urine and uEVs, hsa-10a-5p was present only in total urine and absent from exosomes, and the smooth muscle-specific hsa-miR-199a-3p was not detected. There was a significant correlation between particle count, exosome RNA content and protein quantification of exosome samples but no correlation between the particle parameters and the chemical composition of urine.
Ultracentrifugation combined with size exclusion following protease inhibitor and DTT treatment is an unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. The discrepancy between urinary and biopsy miRNAs might indicate selective packaging of miRNAs into uEVs.