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.Urinary extracellular vesicles (uEVs) are a medley of exosomes, exosome-like vesicles and microparticles/ microvesicles 1-4 . Confusing nomenclature aside 5,6 , all uEVs secreted in urine transport proteins, nucleic acid and small metabolites from all epithelial cells forming the nephron and lower urinary tract 7,8 . Thus, uEVs have become a valuable source of biomarkers for identifying any changes in the physio-pathological state of their parental cell. Moreover, uEVs are also bio-activators in renal diseases 9,10 . The most common method in use to enrich uEVs is a 2 or 3 step centrifugation protocol [11][12][13] . While it has been commonly discarded, the pellet obtained at relative low centrifugation force has proved to be an additional source of uEVs 14,15 . However this pellet has not been thoroughly characterized.In addition, the concomitant presence of multiple biomarker in uEVs offers the possibility to integrate multi-omic data analysis to better understand mechanism and possibly identify key role molecules implicated in the onset and progression of the disease 16 . However, no study has reported the amount of volume of urine that is necessary to collect to support multiple analyses. Hence, this study aims to: (1) estimate the minimum volume of urine necessary to yield uEVs for characterization according to both minimal information for studies of extracellular vesicles (MISEV) 17 and downstream analysis applying a very rigorous approach using several control sets for each analysis; (2) test the limit of detection of the techniques employed for downstream analysis and EV characterization before and after eliminati...

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