Kidney Week

Abstract: FR-PO1017

Isolation of Extracellular Vesicles and Associated Biomarkers from Organ Preservation Fluid in Kidney Transplantation

Session Information

• Transplantation: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Transplantation

• 2101 Transplantation: Basic

Authors

• Kubitza, Leonie, Universitatsklinikum Essen Klinik fur Kinderheilkunde II, Essen, Nordrhein-Westfalen, Germany

Leonie Kubitza

• Tertel, Tobias, Universitatsklinikum Essen Institut fur Transfusionsmedizin, Essen, Nordrhein-Westfalen, Germany

Tobias Tertel, MSc

• Kaths, Moritz, Universitatsklinikum Essen, Essen, Nordrhein-Westfalen, Germany

Moritz Kaths, MD, MS

• Jaegers, Johannes, Universitatsklinikum Essen Klinik fur Kinderheilkunde II, Essen, Nordrhein-Westfalen, Germany

Johannes Jaegers, PhD

• Giebel, Bernd, Universitatsklinikum Essen Institut fur Transfusionsmedizin, Essen, Nordrhein-Westfalen, Germany

Bernd Giebel, MD, PhD

• Pape, Lars, Universitatsklinikum Essen Klinik fur Kinderheilkunde II, Essen, Nordrhein-Westfalen, Germany

Lars Pape, MD, PhD

• Droste, Marvin, Universitatsklinikum Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany

Marvin Droste, MD

• Büscher, Anja K., Universitatsklinikum Essen Klinik fur Kinderheilkunde II, Essen, Nordrhein-Westfalen, Germany

Anja K. Büscher, MD

Background

Donor kidneys are in short supply and many potentially preventable complications lead to loss of organ function. Biomarkers may help to detect these at an early stage. Analyzing extracellular vesicles (EVs) from organ perfusate could be a non-invasive way to obtain information about the donor organ prior to transplantation. Since there is no standardized method to isolate EVs from organ perfusate yet, we compared several common purification methods. Finally, to the best of our knowledge, we demonstrate the first in-depth proteomic analysis of perfusate-derived EVs.

Methods

Purification of EVs from kidney allograft perfusate was performed by ultrafiltration (UF), precipitation (PEG), size exclusion chromatography (SEC), and ultracentrifugation (UC), respectively, as well as a combination of these. Following purification, EVs were detected by electron microscopy, immunocapturing-based flow cytometry and nanoscale imaging flow cytometry. Particle-to-protein ratios were calculated to determine sample purity. EV protein composition was analyzed by mass spectrometry and bioinformatic analysis of the EV proteome.

Results

Electron micrographs demonstrated the successful purification of EVs. Microbead-based immunoprecipitation indicated the presence of the vesicular tetraspanins CD9, CD63 CD81 and of CD235a and semi-quantitative multiplex analysis the composition of the EV fraction of several subtypes. Imaging Flow Cytometry and nanoparticle tracking revealed that UC purified more EV marker-carrying nanoscale objects and bulk nanoparticles than the combinations UF-SEC and PEG-SEC, however, accepting a lower grade of purity. Impurity induced by hemolysis was associated with an increased ratio of CD63+/CD235a+-EVs. Proteomic analysis revealed that the preparations were highly enriched for vesicular proteins.

Conclusion

For the first time, we tested different EV purification methods for organ preservation fluid. Depending on the aim of research, the applied methods can be used according to their advantages to isolate EVs from organ perfusate. In addition, we have generated a proteome dataset from perfusate-derived EVs that can serve as a reference for further studies, for example in biomarker analyses.

Funding

• Private Foundation Support