Abstract: SA-PO236
Characterization of Renal Tubule-Derived Extracellular Vesicles (EVs) and EVs Carrying Klotho
Session Information
- CKD-MBD: Basic and Translational
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bone and Mineral Metabolism
- 501 Bone and Mineral Metabolism: Basic
Authors
- Moe, Orson W., The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, Dallas, Texas, United States
- Salcedo Betancourt, Juan David, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
- Davidson, Taylor, The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, Dallas, Texas, United States
- Carroll, Isabelle, The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, Dallas, Texas, United States
- Pastor, Johanne Virginia, The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, Dallas, Texas, United States
Background
EVs are cell-derived vesicles that act as organ-to-organ messengers and are studied as potential biomarkers of physiology, disease diagnosis and therapy response. Previous studies found Klotho highly associated with membrane fragments in cell culture supernatants. We aimed to interrogate whether Klotho is carried in EVs in body fluids. Additionally, we try to quantify renal tubule-derived EVs in urine and serum.
Methods
Urine (60 ml) and serum (10 ml) from healthy subjects underwent ultracentrifugation for EV isolation. We compared the concentration of free soluble Klotho (supernatant) with EV-Klotho (EV-enriched pellet) in urine and serum. A mouse model with renal epithelium-derived EVs selectively labeled with GFP (KSP-Cad16-Cre/TIGER CD9) was developed to characterize renal tubule-originated EVs. Isolated urinary EVs underwent fluorescence-activated vesicle sorting based on GFP positivity.
Results
Three methods of EV isolation were compared- size exclusion chromatography, immunoprecipitation and ultracentrifugation- with the latter method being most efficient. EVs from human urine and serum were confirmed by EV-markers (ALIX, Flotillin-1) by immunoblot. A minimal amount of Klotho was found in human urinary EVs compared to free soluble urinary Klotho and no Klotho was detected in human serum EVs. Renal fluorescent histochemistry confirmed tubular apical membrane localization of the GFP-CD9 EV marker. Renal tubule-derived urinary EVs were identified by direct fluorescent microscopy and flow sorting. After flow sorting, only 23.1% of urinary EVs originated from the renal tubule (CD9+/GFP+ events = 2.8 million vs. GFP- events = 12.2 million). No renal tubule-derived EVs were observed in mice serum.
Conclusion
Only a minimal amount of Klotho is carried in urinary EVs, indicating that Klotho primarily exists in the soluble form in both human serum and urine. Only a subpopulation of urinary EVs in the mouse originated from the renal tubules. Tubular-derived EVs are present in the urine but not in serum, suggesting a selective destination for tubular EV secretion into the urine. One needs to take extreme caution to interpret data from urine EVs and draw conclusions about tubular function. Additional proteomic analysis is ongoing in our lab to assess the content of tubular vs non-tubular urinary-originated EVs.