RNA/Exosome
Veronica Dimuccio, Andrea Ranghino, Giovanni Camussi, and Benedetta Bussolati (2012) Exosome isolation, count and characterization from normal urine, Nephrol. Dial. Transplant. (2012) 27(suppl 2): ii3ii4 doi:10.1093/ndt/gfs196, FO010
ntroduction and Aims: Exosomes are microvescicles secreted from various types of activated cells. In the kidney, exosomes are mainly released into urine by cells of the nephron. Recent studies demonstrated that exosomes express surface receptors, as well as selected patterns of mRNA and microRNA of the cell of origin. Since it is a readily available fluid, urine may be regarded as the ideal source of information on renal conditions. In particular, exosomes appear to bet the perfect mirror of the pathological conditions of the different segments of the nephron. In this perspective, membrane proteins and RNA content of exosomes could be useful markers of renal pathology. The purpose of the present study is the set up of a protocol for exosomes isolation, and the quantification and analysis of surface markers and microRNA (miRNA) content.
Methods: Exosomes were isolated from a pool of urine from healthy subjects. A total of four protocols of exosome isolation were tested in this study, based on 1) ultracentrifugation (100.000g at 4°C for 1h); 2) nanomembrane concentrator Amicon (100k); 3) nanomembrane concentrator Vivaspin 500 (Sartorius); 4) or denaturation of TammHorsfall Protein (THP) with DTT (200mg/ml) followed by ultracentrifugation. Exosome quantification was performed with Bradford for protein content, or with Nanosigh count. mRNA was extracted using mirVana kit (Ambion) and miRNA analysis was performed using quantitative RTPCR. As exosomes are smaller than the lower limit of sensitivity of the cytofluorimetric, cytofluorimetric analysis was performed after adsorption of isolated vesicles on 4 Ym aldehyde–sulphate latex beads.
Results: The protein concentration tested with a Bradford assay only showed a very low exosomes concentration for protocol n.2. Nanosight analysis showed high concentration of exosomes in samples obtained using protocols n.12 (4.7 × 108 and 3,5 × 108 exosomes/ml) while other two were very poor. RNA extracted from exosomes from protocol n.12 showed the presence of a panel of 20 miRNAs. A citofluorimetric assay, set up with latexbeads, was able to identify the presence of the classical exosome markers (CD63, CD24, CD9) as well as of markers of the renal cell of origin (Aquaporins, integrins, membrane transporters).
Conclusions: In the present study we identified a protocol based on ultracentrifugation as the most suitable to obtain exosomes from urine. Exosome count using the Nanosight analysis was more reliable than protein quantification possibly due to a contamination by urinary proteins. In addition, we identified in exosomes from normal urine surface markers involved in the process of exocytosis and in renal cell physiology using a latexbeads based FACS analysis and a panel of miRNAs possibly relevant for renal cell functions. These findings can be a valid starting point for the further development of studies in a wide variety of renal pathologies.