Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
Lozano-Ramos, Inés (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Bancu, Ioana (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Oliveira-Tercero, Anna (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Armengol Barnils, Maria del Pilar (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Menezes-Neto, Armando (Institut de Salut Global de Barcelona)
Del Portillo, Hernando A.. (Institució Catalana de Recerca i Estudis Avançats)
Lauzurica, Ricardo (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Borràs i Serres, Francesc Enric (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Universitat Autònoma de Barcelona

Data:	2015
Resum:	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.
Ajuts:	Ministerio de Economía y Competitividad PI13/00050 Agència de Gestió d'Ajuts Universitaris i de Recerca 2014SGR804 Ministerio de Economía y Competitividad CA12/00284
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Extracellular vesicles ; Size-exclusion chromatography ; Urine ; Biomarker
Publicat a:	Journal of extracellular vesicles, Vol. 4 (may 2015) , ISSN 2001-3078

DOI: 10.3402/jev.v4.27369
PMID: 26025625

11 p, 1.6 MB

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