Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.