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Abstract: PO2229

Urinary Excretion of Extracellular Vesicles in 24 Hours: Time Point Collection and Normalization Strategy

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Musante, Luca, University of Virginia Health System, Charlottesville, Virginia, United States
  • La salvia, Sabrina, University of Virginia Health System, Charlottesville, Virginia, United States
  • Erdbruegger, Uta, University of Virginia Health System, Charlottesville, Virginia, United States
Background

Urinary extracellular vesicles (uEVs) are an ideal source of biomarkers for kidney diseases. Despite the interest generated, little is known about collection time and normalization approach. The majority of the studies on uEVs focus on spot urine collection based on the assumption that it accurately reflects the renal function. However, the practice to collect spot urine does not allow for calculating and standardizing the uEV excretion rate and then assessing the renal function. Moreover, no research has been carried out yet to show the difference between spot urine and 24h collections. The aim of this study is to compare uEVs excreted in all 24 hour urine void as single spot and compare it with 24 hour collection.

Methods

Each single spot void urine was collected and 20% of the volume was used to to create the 24 hours collection. uEVs were enriched by differential centrifugation. Transmission electron microscopy (TEM), western blot (WB), nanoparticle tracking analysis (NTA), tuneable resistive pulse sensing (TRPS), imaging flow cytometry (iFC) and miRNA (miR16 and miR200b) quantitation by qPCR were used to quantify uEVs markers variation during the 24 hour. Creatinine, urine osmolality and particle concentration (NTA, TRPS ) were used to normalize the analytes.

Results

TEM showed a heterogeneous population of EVs and WB confirmed the presence of EVs marker (TSG101, ALIX and CD9). RNA was extracted by a column-based method (miRNA extraction kit Qiagen) and cel-39 miRNA was spiked in each sample. A multiparametric detection of nephron markers podocalyxin (PODXL), aquaporin-2 (AQP2) and uEVs pan tetraspanins (CD9 + DC63 + CD81) was performed in imaging flow cytometry. Whereas the uEV composition of did not change across the 24 hours analysis, the quantity of uEVs and related markers (miRNA and protein) fluctuated during the day depending on the hydration and excretion rate. Creatinine, urinary osmolarity and particle count normalization failed to normalize “outliers”

Conclusion

In conclusion, this study represents the very first report which compares single void urine versus 24 hour uEV analysis. We concluded that the 24 hour collection is the preferred choice for a robust and rigorous assessment of uEVs and its associated markers.