title: Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples.
abstract: Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.
authors.affiliation: IVECAT-Group, Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona, Spain.|Universitat Autònoma de Barcelona - Campus Can Ruti, Badalona, Spain.
authors.forename: Inés
authors.initials: I
authors.lastname: Lozano-Ramos
authors.affiliation: Universitat Autònoma de Barcelona - Campus Can Ruti, Badalona, Spain.|Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.
authors.forename: Ioana
authors.initials: I
authors.lastname: Bancu
authors.affiliation: IVECAT-Group, Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona, Spain.|Genomic Platform, Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Oliveira-Tercero
authors.affiliation: Genomic Platform, Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona, Spain.
authors.forename: María Pilar
authors.initials: MP
authors.lastname: Armengol
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.
authors.forename: Armando
authors.initials: A
authors.lastname: Menezes-Neto
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
authors.forename: Hernando A
authors.initials: HA
authors.lastname: Del Portillo
authors.affiliation: Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Lauzurica-Valdemoros
authors.affiliation: IVECAT-Group, Institut d'Investigació Germans Trias i Pujol (IGTP), Badalona, Spain.|Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain; feborras@igtp.cat.
authors.forename: Francesc E
authors.initials: FE
authors.lastname: Borràs
keywords: biomarker; extracellular vesicles; size-exclusion chromatography; urine