Abstract: SA-PO1037

Quantification of Urinary Extracellular Vesicles

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 702 Water/Urea/Vasopressin, Organic Solutes

Authors

  • Blijdorp, Charles J., Erasmus Medical Center, Rotterdam, Netherlands
  • Hartjes, Thomas, Erasmus Medical Center, Rotterdam, Netherlands
  • Van royen, Martin E., Erasmus Medical Center, Rotterdam, Netherlands
  • Zietse, Robert, Erasmus Medical Center, Rotterdam, Netherlands
  • Hoorn, Ewout J., Erasmus Medical Center, Rotterdam, Netherlands
Background

Urinary extracellular vesicles (uEVs) have emerged as a powerful non-invasive tool to study renal epithelial transport in humans. However, the optimal method to quantify and normalize uEVs remains unclear, especially for spot urines.

Methods

Four healthy subjects were subjected to overnight thirsting (10 pm-noon) followed by water loading (20 ml/kg in 30 min). Spot urines were collected during thirsting (T1-2) and after water loading (WL1-4, noon-7 pm). Subsequently, 4 uEV quantification techniques were compared: (1) nanoparticle tracking analysis (NTA), (2) uEV isolation by ultracentrifugation followed by immunoblotting of CD9, CD63, CD81, ALIX, and TSG101, (3) a time-resolved fluorescence immunoassay (TRFIA) that captures CD9+ uEVs, and (4) EVQuant, a novel technique which counts individual fluorescently labeled EVs after immobilization in a matrix. A Bland-Altman analysis was used to compare methods using NTA as reference.

Results

As expected, urine osmolality was near-maximal during thirsting, decreased after water loading and then increased again (Figure). The results of the 4 uEV quantification methods showed similar dynamics as urine osmolality suggesting that uEV number changes in proportion to urinary concentration (Figure). Of interest, EVQuant identified 2.4 ± 0.6 times more uEVs than NTA. Using NTA as reference, the Bland-Altman analysis showed that EVQuant had the lowest bias (% difference 6 ± 27) followed by TRFIA (10 ± 21). Of the uEV-markers, CD9 agreed best with NTA (-12 ± 34). uEV number correlated strongly with urine creatinine (Figure) and osmolality (r2 for both 0.9, P<0.0001).

Conclusion

uEV number is proportional to urinary concentration and both urine creatinine and osmolality can be used to normalize spot urines for uEV number. EVQuant is a promising alternative to NTA and appears more sensitive for uEV detection. These uEV quantification methods can be used to analyze if changes in a uEV protein of interest are the result of more protein per uEV or the excretion of more uEVs containing this protein.