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Abstract: SA-PO1042

Investigation of Temporal Patterns of Biomarker Expression from Different Segments of the Kidneys in Healthy Subjects

Session Information

Category: Pathology and Lab Medicine

  • 1800 Pathology and Lab Medicine


  • Upson, Samantha G., University of Virginia, Charlottesville, Virginia, United States
  • Greig, Morgan, University of Virginia, Charlottesville, Virginia, United States
  • Selesky, Margaret, University of Virginia, Charlottesville, Virginia, United States
  • Yavuz, Hayrettin, University of Virginia, Charlottesville, Virginia, United States
  • Alwagdani, Abdulrahman A., University of Virginia, Charlottesville, Virginia, United States
  • Harding, Michael A., University of Virginia, Charlottesville, Virginia, United States
  • Musante, Luca, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Erdbruegger, Uta, University of Virginia, Charlottesville, Virginia, United States

Urinary extracellular vesicles (uEVs) may parallel physiologic and pathologic processes taking place in their kidney cell of origin. Combined with their ease of access, this makes uEVs excellent candidate biomarkers. It is known that kidney physiology and expression of some proteins follow a circadian pattern. Therefore, the normal daily variation in uEV excretion and the content of specific protein cargoes must be understood. The goal of this study is to examine changes over a 24 hour period of uEV markers of glomerula and tubular origin. In addition, we examined TSG101 (Tumor susceptibility gene protein 101) as an EV marker.


Each void during a 24-hour period for 13 healthy individuals (108 samples total) was collected. uEVs from each void was isolated by differential centrifugation at 20,000g, and washed with a low ionic strength buffer to remove uromodulin. uEV count and sizing was performed on each final pellet using Nanoparticle Tracking Analysis (ZetaView; Particle Metrix). Flow Cytometry using SpectralFlow was performed on uEVs stained with an antibody panel including CD35 (CR1), CD26 (DPP4), CD9 (Tetraspanin) and CD10 (Neprilysin). uEV protein lysates were immunoblotted for SLC12A3 and TSG101.


TSG101 signal normalized to total protein has a positive linear correlation with creatinine (r=0.62, p<0.001); indicating it may be expressed steadily. Normalizing to creatinine or TSG101 expression of SLC12A3 vary over a day in a temporal pattern. The antibody panel of glomerular and tubular markers detected with single flow cytometry did not express a temporal pattern when normalized to TSG101.


uEV biomarker expression may be normalized to TSG101 signal as it correlates with creatinine. SLC12A3 has a critical role in kidney function with sodium reabsorption and has shown circadian pattern of expression previously in rodent models. Our results demonstrate a similar temporal pattern of a large range of uEVs carrying SLC12A3 in humans. This temporal variation of uEV markers needs to be further analyzed on other kidney markers in healthy individuals and disease. However, characterizing temporal protein expression patterns of uEVs has potential to accelerate uEV biomarker discovery for kidney diseases.