Extracellular Vesicles are Associated with Human Donor Kidney Characteristics 5 135 DISCUSSION A major benefit of machine perfusion is that it allows for the assessment of kidney quality prior to transplantation through analysis of biomarkers (such as EVs) in the perfusion fluid 2, 5, 7. Especially during NMP, where cellular metabolism becomes activated, the monitoring of EVs may be a promising tool to infer kidney quality prior to transplantation 26. Additionally, the characterization of EVs released during NMP may shed light on the origin and composition of EVs released into circulation of transplant recipients and increase our understanding of (distal) immune responses 27-29. However, although EVs are subject to intensive biomarker studies in various fields 13, 30, 31, little is currently known regarding EV release by kidney grafts during NMP and its association with kidney status. In this observational study we examined and characterized the release of EVs by discarded human ECD kidneys during NMP. In line with our previous findings 22, we found that the majority of released nanoparticles were <400 nm in size irrespective of the time of sampling and that total particle concentrations increased as NMP progressed. Using IFCM we found that EV concentrations significantly increased during the first 60 minutes of NMP and that concentrations remained relatively stable during the remainder of the NMP procedure (up to 6 hours). We reason that the observed stabilization of EV concentrations are due to the establishment of an equilibrium between EV biogenesis and breakdown during NMP and/or uptake by (endothelial) cells of the kidney. These findings may contribute to the debate regarding optimal NMP perfusion times: since EV release is considered an active process 32, release dynamics during NMP may be dependent on the metabolic status of the kidney graft. Examination of the tetraspanin profile on the released EVs revealed that the majority of detected EVs expressed tetraspanin CD81. Recently, CD81 has been shown to serve as a regulator of B cell signaling through complex formation with CD19 at the plasma membrane. Upon B cell activation CD19 dissociates from CD81 while in naïve B cells CD81 (epitope 5A6) is complexed by CD19 33. Given that the majority of detected EVs released during 6 hours of NMP expressed the CD81 epitope 5A6, the fusion of these CD81+ EVs with recipient B cells may have a dampening effect on B cell signaling as the addition of extra CD81 onto the B cell membrane may affect CD81-CD19 dissociation kinetics. Additionally, it has been shown that allograft-derived EVs bearing intact donor MHC molecules (CD63+ and CD9+CD81+
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