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

Publisher: Wiley Country of Publication: United States NLM ID: 101610479 Publication Model: Print-Electronic Cited Medium: Print ISSN: 2001-3078 (Print) Linking ISSN: 20013078 NLM ISO Abbreviation: J Extracell Vesicles

Publication: 2020- : [Hoboken, NJ] : Wiley
Original Publication: Järfälla : Co-Action Pub.

Gene Expression Regulation* ; Transcriptome*; Biomarkers/*urine ; Diabetes Mellitus, Type 1/*complications ; Diabetic Nephropathies/*diagnosis ; Extracellular Vesicles/*genetics ; MicroRNAs/*genetics; Adult ; Aged ; Case-Control Studies ; Diabetic Nephropathies/etiology ; Diabetic Nephropathies/metabolism ; Diabetic Nephropathies/urine ; Extracellular Vesicles/metabolism ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Prognosis

Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit- based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA-rich uEV preparation components. Our data shows that HFD,UC and the kit-based method are suitable methods to isolate uEV for miRNA-seq. However, only HFD and UC were suitable for mRNA-seq in our settings.
Competing Interests: KB, ODP, GL, MR, DD, CF, PG, LG, MP and HH, have no conflict of interest to declare.
(© 2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Bioinformatics. 2014 Apr 1;30(7):923-30. (PMID: 24227677)
J Extracell Vesicles. 2019 Apr 01;8(1):1596016. (PMID: 30988894)
J Proteomics. 2016 Mar 16;136:89-98. (PMID: 26775013)
Nat Rev Mol Cell Biol. 2018 Apr;19(4):213-228. (PMID: 29339798)
J Am Soc Nephrol. 2017 Jan;28(1):56-63. (PMID: 27381844)
Kidney Int. 2014 May;85(5):1225-37. (PMID: 24196483)
J Diabetes Res. 2016;2016:5741518. (PMID: 28105442)
J Thromb Haemost. 2014 Jul;12(7):1182-92. (PMID: 24818656)
Nucleic Acids Res. 2019 Jan 8;47(D1):D516-D519. (PMID: 30395310)
Immun Ageing. 2020 Jan 04;17:1. (PMID: 31911808)
J Extracell Vesicles. 2019 Aug 27;8(1):1635420. (PMID: 31497264)
Theranostics. 2017 Aug 23;7(16):3824-3841. (PMID: 29109780)
Proteomics Clin Appl. 2019 Nov;13(6):e1900018. (PMID: 31424164)
Hum Pathol. 2010 Sep;41(9):1265-75. (PMID: 20447677)
Sci Rep. 2017 Oct 31;7(1):14444. (PMID: 29089562)
J Am Soc Nephrol. 2016 Jan;27(1):12-26. (PMID: 26251351)
Nat Rev Nephrol. 2017 Dec;13(12):731-749. (PMID: 29081510)
Kidney Int. 2010 Apr;77(8):736-42. (PMID: 20130532)
RNA Biol. 2015;12(8):810-23. (PMID: 26176991)
Sci Rep. 2020 Feb 28;10(1):3701. (PMID: 32111925)
Kidney Int. 2014 Aug;86(2):433-44. (PMID: 24352158)
Philos Trans R Soc Lond B Biol Sci. 2018 Jan 5;373(1737):. (PMID: 29158309)
Cell. 2019 Apr 4;177(2):446-462.e16. (PMID: 30951671)
Nat Methods. 2017 Feb 28;14(3):228-232. (PMID: 28245209)
Kidney Int Rep. 2017 Dec 01;3(3):555-572. (PMID: 29854963)
Cell Tissue Res. 2017 Jul;369(1):217-227. (PMID: 28429073)
Biomol Detect Quantif. 2019 Jun 04;17:100089. (PMID: 31194192)
J Circ Biomark. 2016 Mar 1;5:4. (PMID: 28936252)
Sci Rep. 2018 Jul 9;8(1):10340. (PMID: 29985427)
Mol Cell Proteomics. 2006 Oct;5(10):1760-71. (PMID: 16837576)
J Proteome Res. 2018 Jan 5;17(1):86-96. (PMID: 29090927)
Methods Mol Biol. 2014;1182:289-305. (PMID: 25055920)
Genome Biol. 2010;11(10):R106. (PMID: 20979621)
J Extracell Vesicles. 2013 Jan 10;2:. (PMID: 24009896)
Bioinformatics. 2016 Oct 1;32(19):3047-8. (PMID: 27312411)
Nat Rev Dis Primers. 2015 Jul 30;1:15018. (PMID: 27188921)
Diabetes Res Clin Pract. 2020 Feb;160:108010. (PMID: 31987752)
Methods Mol Biol. 2020;2067:175-188. (PMID: 31701453)
Can J Kidney Health Dis. 2017 May 22;4:2054358117705371. (PMID: 28616250)
Clin Med (Lond). 2009 Jun;9(3):284-7; quiz 288-9. (PMID: 19634399)
Sci Rep. 2014 Dec 23;4:7532. (PMID: 25532487)
Bioinformatics. 2013 Jan 1;29(1):15-21. (PMID: 23104886)
Kidney Int. 2010 Oct;78(8):810-6. (PMID: 20686450)
BMC Genomics. 2017 Jun 5;18(1):442. (PMID: 28583074)
J Extracell Vesicles. 2015 May 14;4:27066. (PMID: 25979354)
Nephrology (Carlton). 2017 Nov;22(11):854-863. (PMID: 27496221)
FASEB J. 2020 Mar;34(3):3616-3630. (PMID: 31957088)
J Extracell Vesicles. 2018 Nov 23;7(1):1535750. (PMID: 30637094)
World J Diabetes. 2013 Dec 15;4(6):245-55. (PMID: 24379914)
J Physiol. 2016 Oct 15;594(20):5735-5748. (PMID: 27104781)
J Extracell Vesicles. 2020 Dec;10(2):e12038. (PMID: 33437407)
Eur J Pharm Sci. 2017 Feb 15;98:30-39. (PMID: 27771514)
Acta Physiol (Oxf). 2020 Jan;228(1):e13346. (PMID: 31334916)
Blood Rev. 2013 Jan;27(1):31-9. (PMID: 23261067)
Nucleic Acids Res. 2006 May 31;34(10):2966-75. (PMID: 16738135)
Nat Methods. 2012 Jul;9(7):671-5. (PMID: 22930834)
Clin Kidney J. 2015 Feb;8(1):23-30. (PMID: 25713706)
Biomed Res Int. 2018 Jan 30;2018:8545347. (PMID: 29662902)
Brief Bioinform. 2019 Nov 27;20(6):2044-2054. (PMID: 30099484)

Keywords: diabetic kidney disease; exosomes; extracellular vesicles; isolation; mRNA sequencing; miRNA sequencing; urinary extracellular vesicles

0 (Biomarkers)
0 (MicroRNAs)

Date Created: 20210113 Date Completed: 20220110 Latest Revision: 20240330

20240330

PMC7789228

10.1002/jev2.12038

33437407