Comparison of Hydrostatic Filtration Dialysis with Ultracentrifugation Methods for the Identification and Proteomic Profiling of Urinary Extracellular Vesicles

Background: Urinary extracellular vesicles (UEVs) carry rich markers of their parent cells, so they can serve as possible biomarkers of kidney diseases. Methods: In this study, we isolated urinary extracellular vesicles from five individuals using a simple, clinically applicable method called hydrostatic filtration dialysis (HFD) and compared it to the gold-standard ultracentrifugation (UC) with transmission electron microscopy (TEM). We also employed a proteomic approach using pooled human urine samples from the same five individuals to profile the protein composition of UEVs to evaluate the effectiveness of these two methods. Results: Notably, using TEM, we found that all isolations contained 0 - 400 nm vesicles with the traditionally reported morphology, although the TEM results showed that the UEVs isolated from HFD compared to those from UC are larger and more extensive. We obtained a total of 2,564 UEV proteins in the two methods. We showed a large overlap (2,185 > 85%) between the proteins identified by both isolation methods. The result also showed that the obtained proteins in extracellular vesicles, which are isolated with these methods, are consistent with the results in currently available databases. However, in the associated gene ontologies, the enriched proteins found by the two methods showed some differences. Conclusions: The HFD method is clinically feasible and allows large-scale protein profiling of UEV biomarkers. The results of this study also provide valuable UEV protein data from the methodological comparison, which might be valuable to other researchers.