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

Abstract

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.

Urinary extracellular vesicles: Assessment of pre‐analytical variables and development of a quality control with focus on transcriptomic biomarker research

“…a. Barreiro et al, 2020. uEV were isolated from healthy controls and individuals with type 1 diabetes and macroalbuminuria. Raw counts were TMM normalized using edgeR.…”

“…However, most of the studies have focused on short storage times of up to 10 days at different temperatures (Armstrong et al., 2018 ; Oosthuyzen et al., 2013 ; Zhou et al., 2006 ) or up to 1 year at ‐80°C (Yuana et al., 2015 ; Zhou et al., 2006 ). Of note, while there is a handful of investigations comparing EV isolation methods using messenger RNA sequencing (mRNA‐seq) (Barreiro et al., 2020 ) and micro RNA sequencing (miRNA‐seq) (Barreiro et al., 2020 ; Cheng et al., 2014 ; Mussack et al., 2019 ; Park et al., 2020 ; Srinivasan et al., 2019 ), comparison of other pre‐analytical variables with sequencing as an end‐point are largely lacking (Miranda et al., 2014 ). With few exceptions (Armstrong et al., 2018 ; Barreiro et al., 2020 ; Park et al., 2020 ), studies have so far included only healthy control samples, which could bias results interpretation since diseases that influence urine composition could also alter composition and stability of uEV isolates.…”

Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy

“…To confirm these associations in FD, further studies are needed to investigate the molecular mechanisms associated with Fabry nephropathy. It should also be considered that the isolation protocol might influence the level of miRNAs [ 52 ].…”

Exosomes in Lung Cancer: Actors and Heralds of Tumor Development

“…Exosomes have been obtained from different biological fluids, such as serum, plasma, urine, cerebrospinal fluid or exudates [27][28][29][30]. Exosome isolation methods are mainly based on their physicochemical properties, such as size or density, or their biological characteristics and molecules expressed in their surface [31,32].…”