Exosomes are vesicles that are released from the kidney into urine. They contain 
protein and RNA from the glomerulus and all sections of the nephron and 
represent a reservoir for biomarker discovery. Current methods for the 
identification and quantification of urinary exosomes are time consuming and 
only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes 
particles by measuring their Brownian motion in solution. In this study, we 
applied NTA to human urine and identified particles with a range of sizes. Using 
antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated 
to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive 
particles of characteristic exosomal size. Extensive pre-NTA processing of urine 
was not necessary. However, the intra-assay variability in the measurement of 
exosome concentration was significantly reduced when an ultracentrifugation step 
preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 
upregulation induced by desmopressin stimulation of kidney collecting duct 
cells. Nanoparticle tracking analysis was also able to track changes in exosomal 
AQP2 concentration that followed desmopressin treatment of mice and a patient 
with central diabetes insipidus. When urine was stored at room temperature, 4°C 
or frozen, nanoparticle concentration was reduced; freezing at -80°C with the 
addition of protease inhibitors produced the least reduction. In conclusion, 
with appropriate sample storage, NTA has potential as a tool for the 
characterization and quantification of extracellular vesicles in human urine.