Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
Vall-Palomar, Mònica. (Hospital Universitari Vall d'Hebron)
Arévalo, Jazmine (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Ariceta Iraola, Gema (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Meseguer, A. (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Date:	2018
Abstract:	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.
Grants:	Instituto de Salud Carlos III PI14/01107
Rights:	Aquesta 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.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Urinary exosome-like vesicles ; Exosome isolation ; Exosomal miRNA ; FHHNC
Published in:	Journal of translational medicine, Vol. 16 (october 2018) , ISSN 1479-5876

DOI: 10.1186/s12967-018-1651-z
PMID: 30305086

9 p, 1.7 MB

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