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Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease.

Author(s): Barreiro K, Dwivedi OP, Leparc G, Rolser M, Delic D, Forsblom C, Groop PH, Groop L, Huber TB, Puhka M, Holthofer H

Publication: J Extracell Vesicles, 2020, Vol. 10, Page e12038

PubMed ID: 33437407 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared results of size distribution, morphology, protein expression, and transcriptome analysis of uEVs that were isolated from urine by ultracentrifugation, hydrostatic filtration dialysis (HFD), and the Norgen Kit. Urine specimens were collected from patients diagnosed with type 1 diabetes and healthy volunteers. 24 h urine specimens were collected from five healthy volunteers and five patients with type 1 diabetes with macroalbuminuria. Calbiochem EDTA-free Protease Inhibitor Cocktail Set III was added to specimens before frozen storage at -80°C.  Specimens were stored at -80°C until isolation by ultracentrifugation, HFD, and the Urine Exosome Purification and RNA Isolation Midi Kit. For HFD isolation, urine was thawed at 37°C, centrifuged at 2000 g at 4°C, and the supernatant was poured into a cellulose ester membrane with a molecular weight cut-off of 1,000 kDa; the uEVs were then collected in LowBind tubes.  Isolation of uEVs by ultracentrifugation included thawing urine specimens at 37°C, centrifugation at 8000 g at 4°C for 15 min, filtering the supernatant through Whatman 1.2 μm cellulose acetate syringe filters, centrifugation at 100,000 g at 4°C for 90 min, washing the pellet in phosphate buffered saline, and centrifugation at 100,000 g at 4°C for 90 min; the uEVS were then resuspended in PBS and stored in LowBind tubes at -80°C. uEV protein levels were quantified by BCA and levels of Tamm-Horsfall protein, albumin,  podocalyxin, TSG-101, and CD9 were quantified by Western blot after concentration using Amicon Ultra-0.5 ml Centrifugal Filters 10k. uEVs were visualized by transmission electron microscopy and counted by nanoparticle tracking analysis. RNA was extracted from uEVS isolated by ultracentrifugation and HFD using TRIzol LS combined with the miRNeasy mini Kit and from uEVS isolated by Norgen Urine Exosome Purification with the Agilent RNA 6000 Pico Kit. RNA was profiled using a Bioanalyzer. mRNA was sequenced using the SMART-SeqR v4 Ultra Low Input RNA Kit for Sequencing and the Nextera XT Kit. miRNA was sequenced using the QIAseq miRNA Library Kit on an Illumina HiSeq 4000 instrument.

   Summary of Findings:

   All three isolation methods yielded uEVs of the expected morphology, which included filament structures compatible with Tamm-Horsfall protein (THP) complexes. uEV yields were significantly higher when isolation was by ultracentrifugation or HFD than with the Norgen Kit (P<0.05, both). Further, uEVs isolated using the Norgen Kit had a higher percentage of particles that were 0-100 nm in size than those isolated by HFD or by ultracentrifugation (4.0% versus 1.2% and 0.5%, respectively; P<0.05, both), and a lower percentage of particles that were ≥500 nm in size than those isolated by HFD (0.5% versus 4.3%, P<0.05). Protein content was greater when uEVs were isolated by HFD or with the Norgen Kit than by ultracentrifugation (P<0.05, both). Correspondingly, particle-to-protein ratio was significantly higher when uEV isolation was by ultracentrifugation than by HFD or with the Norgen Kit (P<0.01, both) and from uEVs isolated by HFD than with the Norgen Kit (P<0.01). uEVs isolated by HFD and ultracentrifugation expressed the uEV markers TSG101, CD9 and podocalyxin, but expression was lower or absent in uEVs isolated using the Norgen Kit.  The contaminating protein THP was found in uEVs isolated using each of the three methods but was lowest when uEVs were isolated using the Norgen Kit. In contrast, uEVs isolated with the Norgen Kit had the highest levels of albumin contamination, followed by those isolated by HFD. All three methods yielded RNA with a peak size of 25-200 nt, and the yield of RNA did not differ significantly among the isolation methods evaluated. The number of mRNA reads was lower from uEVs isolated with the Norgen Kit than those isolated by ultracentrifugation and HFD; in principal component analysis of the mRNA transcriptome, uEVs isolated with the Norgen Kit clustered separately than those isolated by HFD and ultracentrifugation, which clustered with each other. While only 16 mRNAs were differentially expressed between uEVs isolated by ultracentrifugation and those isolated by HFD, 3344 and 3414 mRNAs were differentially expressed between uEVs isolated with the Norgen Kit and those isolated by HFD and ultracentrifugation, respectively. mRNA expression was very strongly correlated between uEVs isolated by HFD and ultracentrifugation (r=0.97), but only weakly correlated between those isolated with the Norgen Kit and byHFD (r=0.45) and ultracentrifugation (r=0.44). The number of miRNA reads passing filters and raw miRNA counts were higher in uEVs isolated by ultracentrifugation compared to those isolated by HFD and with the Norgen Kit. Unlike the mRNA results, PCA of miRNA did not separate specimens based on uEV isolation method, and the correlations were strong between each pair of uEV isolation method evaluated (r=0.97-0.99). A similar number of miRNAs had >5 counts regardless of uEV isolation method, and 85-90% of miRNAs were shared between uEV isolation methods.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Diabetes Type 1

   Platform:

   Analyte	Technology Platform
   Morphology	Electron microscopy
   Cell count/volume	Light scattering
   Protein	Western blot
   Protein	Colorimetric assay
   RNA	Next generation sequencing
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Ultracentrifugation Hydrostatic filtration dialysis (HFD) Norgen Kit

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