OBJECTIVETo analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy adults.

METHODSThe 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking analysis (NTA).

RESULTSuEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed that the number of 24-hour uEVs amount ranged from 3.56 × 10¹² particles to 5.12 × 10¹² particles, with a CV of 14.23%. The proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49)× 10⁹ particles; the proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a number range of (1.00-1.77)× 10¹² particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86)× 10¹² particles.

CONCLUSIONThe established HFD method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies.