Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

Molecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disea...

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Autores: Vall-Palomar, Mònica|||0000-0002-4133-5814, Arévalo, Jazmine|||0000-0002-8824-424X, Ariceta Iraola, Gema|||0000-0003-1763-1098, Meseguer, A.
Tipo de recurso: artículo
Fecha de publicación:2018
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:235280
Acceso en línea:https://ddd.uab.cat/record/235280
https://dx.doi.org/urn:doi:10.1186/s12967-018-1651-z
Access Level:acceso abierto
Palabra clave:Urinary exosome-like vesicles
Exosome isolation
Exosomal miRNA
FHHNC
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spelling Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methodsVall-Palomar, Mònica|||0000-0002-4133-5814Arévalo, Jazmine|||0000-0002-8824-424XAriceta Iraola, Gema|||0000-0003-1763-1098Meseguer, A.Urinary exosome-like vesiclesExosome isolationExosomal miRNAFHHNCMolecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method. Characterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR. In summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria. 22018-01-0120182018-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/235280https://dx.doi.org/urn:doi:10.1186/s12967-018-1651-zreponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengMinisterio de Economía y Competitividad https://doi.org/10.13039/501100003329 PI14/01107open accesshttp://purl.org/coar/access_right/c_abf2Aquesta obra ha estat identificada com a lliure de restriccions d'ús conegudes segons la llei de propietat intel·lectual, incloent-hi els drets afins. Podeu copiar, modificar i distribuir l'obra, així com fer-ne comunicació pública, fins i tot amb finalitat comercial, sense necessitat de demanar permís.https://creativecommons.org/publicdomain/mark/1.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2352802026-06-06T12:50:31Z
dc.title.none.fl_str_mv Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
title Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
spellingShingle Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
Vall-Palomar, Mònica|||0000-0002-4133-5814
Urinary exosome-like vesicles
Exosome isolation
Exosomal miRNA
FHHNC
title_short Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
title_full Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
title_fullStr Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
title_full_unstemmed Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
title_sort Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
dc.creator.none.fl_str_mv Vall-Palomar, Mònica|||0000-0002-4133-5814
Arévalo, Jazmine|||0000-0002-8824-424X
Ariceta Iraola, Gema|||0000-0003-1763-1098
Meseguer, A.
author Vall-Palomar, Mònica|||0000-0002-4133-5814
author_facet Vall-Palomar, Mònica|||0000-0002-4133-5814
Arévalo, Jazmine|||0000-0002-8824-424X
Ariceta Iraola, Gema|||0000-0003-1763-1098
Meseguer, A.
author_role author
author2 Arévalo, Jazmine|||0000-0002-8824-424X
Ariceta Iraola, Gema|||0000-0003-1763-1098
Meseguer, A.
author2_role author
author
author
dc.subject.none.fl_str_mv Urinary exosome-like vesicles
Exosome isolation
Exosomal miRNA
FHHNC
topic Urinary exosome-like vesicles
Exosome isolation
Exosomal miRNA
FHHNC
description Molecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method. Characterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR. In summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria.
publishDate 2018
dc.date.none.fl_str_mv 2
2018-01-01
2018
2018-01-01
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/235280
https://dx.doi.org/urn:doi:10.1186/s12967-018-1651-z
url https://ddd.uab.cat/record/235280
https://dx.doi.org/urn:doi:10.1186/s12967-018-1651-z
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 PI14/01107
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/publicdomain/mark/1.0/
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/publicdomain/mark/1.0/
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
repository.name.fl_str_mv
repository.mail.fl_str_mv
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