Urinary extracellular vesicles (uEVs) are released by cells throughout the 
nephron and contain biomolecules from their cells of origin. Although 
uEV-associated proteins and RNA have been studied in detail, little information 
exists regarding uEV glycosylation characteristics. Surface glycosylation 
profiling by flow cytometry and lectin microarray was applied to uEVs enriched 
from urine of healthy adults by ultracentrifugation and centrifugal filtration. 
The carbohydrate specificity of lectin microarray profiles was confirmed by 
competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. 
Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. 
In both flow cytometry and lectin microarray assays, uEVs demonstrated surface 
binding, at low to moderate intensities, of a broad range of lectins whether 
prepared by ultracentrifugation or centrifugal filtration. In general, 
ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities 
than centrifugal filtration-prepared uEVs consistent with lesser amounts of 
co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs 
showed little inter-individual variation and were distinct from those of Tamm 
Horsfall protein, which bound a limited number of lectins. In a pilot study, 
lectin microarray was used to compare uEVs from individuals with autosomal 
dominant polycystic kidney disease to those of age-matched controls. The lectin 
microarray profiles of polycystic kidney disease and healthy uEVs showed 
differences in binding intensity of 6/43 lectins. Our results reveal a complex 
surface glycosylation profile of uEVs that is accessible to lectin-based 
analysis following multiple uEV enrichment techniques, is distinct from 
co-purified Tamm Horsfall protein and may demonstrate disease-specific 
modifications.