Urine beyond electrolytes: diagnosis through extracellular vesicles

Background and objective: Extracellular vesicles (EV) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EV present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EV in urine requires prior isolation, which slows down and hinders transition into clinical practice. The aim of this study is to show the applicability of the "single particle interferometric reflectance imaging sensor" (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EV and proteins involved in renal function.

Materials and methods: The ExoView® technology enables the quantification and phenotyping of EV present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EV and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5 μl of urine. Tubular expression was confirmed by immunohistochemistry.

Results: The mean size of the EV analysed was 59 ± 16 nm for those captured by tetraspanin CD63, 61 ± 16 nm for those captured by tetraspanin CD81, and 59 ± 10 for tetraspanin CD9, with CD63 being the majority EV subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1).

Conclusions: This work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EV and their protein content in relation to the renal tubule. The use of minimum volumes, 5 μl, and the total analysis time not exceeding three hours facilitate the transition of EV into daily clinical practice as sources of diagnostic information.