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Optimization of isolation protocol and characterization of urinary extracellular vesicles as biomarkers of kidney allograft injury.

Author(s): Sedej I, Tušek Žnidarič M, Dolžan V, Lenassi M, Arnol M

Publication: Clin Nephrol, 2021, Vol. 96, Page 107-113

PubMed ID: 34643501 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects associated with preserving urine supernatants with EDTA and/or performing a filtration step prior to the isolation of EVs by SEC on the yield and morphology of EV particles isolated from the urine of kidney transplant recipients. The size, protein and miRNA levels were further analyzed in EVs preserved with EDTA, and including a filtration step before SEC. Urine was collected by nine kidney transplant recipients and centrifuged at 2,000 g for 15 min at room temperature within 4 h of collection. Urine aliquots were stored at -80°C until EV isolation. Urine aliquots were thawed at room temperature and phosphate buffered saline with or without EDTA was added. Aliquots remained unfiltered or were filtered through a 0.22-μm filter before concentration using a 100 kDa membrane and EV isolation using qEV size exclusion chromatography columns. Fractions were eluted in PBS and analyzed for protein content by absorbance at 280 nm. Fractions were stored at -20°C before analysis of morphology by transmission electron microscopy, and EV concentration and size by nanoparticle tracking analysis. Fractions containing particles were combined and HSC70, CD63, tubulin, flotillin-1, CD9 and GAPDH levels were analyzed by Western blot analysis.  miRNA was isolated using the TRIzol reagent and the miRNeasy Mini Kit and levels of miR-126-3p, miR-451a, and let-7i were quantified by real-time PCR.

   Summary of Findings:

   While proteins were eluted in fractions 8-10, particles were released from the SEC columns in fractions 3-6. The three fractions containing particles were combined to produce the urine EV (uEV) sample.  Isolation of EVs by SEC without either EDTA preservation or urine filtration resulted in a higher particle count than when the urine was preserved with EDTA and filtered before EV isolation. However, there were many long and short filamentous structures in the absence of EDTA preservation and filtration, which the authors state were likely uromodulin polymers.  The authors conclude that including EDTA and filtration are necessary to obtain pure EVs. Characterization of the EVs isolated by SEC with EDTA and filtration revealed that the mean and mode size of the isolated EVs were 171.0 nm and 127.9 nm, respectively. The isolated particles were confirmed to express the EV markers, HSC70 and CD63 and the cytosolic proteins tubulin, flotiilin-1 and GAPDH. Let-7i was detected in the EVs, but miR-451a and -126-3p were not detected.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Morphology	Light scattering
   Morphology	Electron microscopy
   RNA	Real-time qRT-PCR
   Protein	Western blot
   Protein	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	EDTA Frozen
   Biospecimen Aliquots and Components	Filtration	Urine supernatant filtered through a 0.22-μm filter Urine supernatant not filtered

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