New original research article from IVECAT members S. Inés Lozano, Anna Oliveira-Tercero and Dr Francesc Borràs. We have been working together with the Nephrology Department of the Germans Trias University Hospital (HUGTP), and also with Dr Armando Menezes and Dr Hernando A. del Portillo from the Barcelona Institute for Global Health (ISGlobal) to describe size-exclusion chromatography as a feasible isolation method for urinary Extracellular Vesicles. The study has been published in the Journal of Extracellular Vesicles on 28 May 2015.

Looking forward to hearing your comments, have a good and interesting read!

Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

Inés Lozano-Ramos, Ioana Bancu, Anna Oliveira-Tercero, María Pilar Armengol, Armando Menezes-Neto, Hernando A. Del Portillo, Ricardo Lauzurica-Valdemoros and Francesc E. Borràs

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.

Keywords: extracellular vesicles; size-exclusion chromatography; urine; biomarker

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