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

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 bio...

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Autores: Lozano-Ramos, Inés, Bancu, Ioana|||0000-0002-6799-2169, Oliveira-Tercero, Anna, Armengol Barnils, Maria del Pilar|||0000-0002-9423-2966, Menezes-Neto, Armando, Del Portillo, Hernando A.|||0000-0002-5278-3452, Lauzurica, Ricardo|||0000-0002-8051-5199, Borràs i Serres, Francesc Enric|||0000-0003-4038-1912
Tipo de recurso: artículo
Fecha de publicación:2015
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:254375
Acceso en línea:https://ddd.uab.cat/record/254375
https://dx.doi.org/urn:doi:10.3402/jev.v4.27369
Access Level:acceso abierto
Palabra clave:Extracellular vesicles
Size-exclusion chromatography
Urine
Biomarker
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spelling Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samplesLozano-Ramos, InésBancu, Ioana|||0000-0002-6799-2169Oliveira-Tercero, AnnaArmengol Barnils, Maria del Pilar|||0000-0002-9423-2966Menezes-Neto, ArmandoDel Portillo, Hernando A.|||0000-0002-5278-3452Lauzurica, Ricardo|||0000-0002-8051-5199Borràs i Serres, Francesc Enric|||0000-0003-4038-1912Extracellular vesiclesSize-exclusion chromatographyUrineBiomarkerRenal 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.Universitat Autònoma de Barcelona 22015-01-0120152015-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/254375https://dx.doi.org/urn:doi:10.3402/jev.v4.27369reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengMinisterio de Economía y Competitividad https://doi.org/10.13039/501100003329 PI13/00050Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2014/SGR-804Ministerio de Economía y Competitividad https://doi.org/10.13039/501100003329 CA12/00284open accesshttp://purl.org/coar/access_right/c_abf2Aquest 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.https://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2543752026-06-06T12:50:31Z
dc.title.none.fl_str_mv Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
title Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
spellingShingle Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
Lozano-Ramos, Inés
Extracellular vesicles
Size-exclusion chromatography
Urine
Biomarker
title_short Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
title_full Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
title_fullStr Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
title_full_unstemmed Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
title_sort Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples
dc.creator.none.fl_str_mv Lozano-Ramos, Inés
Bancu, Ioana|||0000-0002-6799-2169
Oliveira-Tercero, Anna
Armengol Barnils, Maria del Pilar|||0000-0002-9423-2966
Menezes-Neto, Armando
Del Portillo, Hernando A.|||0000-0002-5278-3452
Lauzurica, Ricardo|||0000-0002-8051-5199
Borràs i Serres, Francesc Enric|||0000-0003-4038-1912
author Lozano-Ramos, Inés
author_facet Lozano-Ramos, Inés
Bancu, Ioana|||0000-0002-6799-2169
Oliveira-Tercero, Anna
Armengol Barnils, Maria del Pilar|||0000-0002-9423-2966
Menezes-Neto, Armando
Del Portillo, Hernando A.|||0000-0002-5278-3452
Lauzurica, Ricardo|||0000-0002-8051-5199
Borràs i Serres, Francesc Enric|||0000-0003-4038-1912
author_role author
author2 Bancu, Ioana|||0000-0002-6799-2169
Oliveira-Tercero, Anna
Armengol Barnils, Maria del Pilar|||0000-0002-9423-2966
Menezes-Neto, Armando
Del Portillo, Hernando A.|||0000-0002-5278-3452
Lauzurica, Ricardo|||0000-0002-8051-5199
Borràs i Serres, Francesc Enric|||0000-0003-4038-1912
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universitat Autònoma de Barcelona
dc.subject.none.fl_str_mv Extracellular vesicles
Size-exclusion chromatography
Urine
Biomarker
topic Extracellular vesicles
Size-exclusion chromatography
Urine
Biomarker
description 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.
publishDate 2015
dc.date.none.fl_str_mv 2
2015-01-01
2015
2015-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/254375
https://dx.doi.org/urn:doi:10.3402/jev.v4.27369
url https://ddd.uab.cat/record/254375
https://dx.doi.org/urn:doi:10.3402/jev.v4.27369
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Ministerio de Economía y Competitividad https://doi.org/10.13039/501100003329 PI13/00050
Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2014/SGR-804
Ministerio de Economía y Competitividad https://doi.org/10.13039/501100003329 CA12/00284
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by-nc/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by-nc/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
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repository.mail.fl_str_mv
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