General Discussion and Future Perspectives 8 189 the perfusion dynamics and analysis of biomarkers (e.g., neutrophil gelatinaseassociated lipocalin, kidney injury molecule-1, and endothelin-1 33) in the perfusion fluids 29, 31, 34, 35. Probing Perfusion Fluids In the first-ever kidney NMP pilot trial in the Netherlands, we examined the release of nanoparticles by ECD kidneys during NMP with Nanoparticle Tracking Analysis (NTA) (chapter 2). Whilst 11 ECD kidney were included in this trial 36, we were able to demonstrate significant nanoparticle release (compared to baseline perfusion fluids) in 3 out of 11 kidneys. In the other 8 kidneys, we observed high concentrations of nanoparticles already present in the perfusion fluids before the perfusion procedure, which was attributed to the addition of Olimel (a mixture of glucose, amino acids, and lipids) as an energy source for cellular metabolism. Given that NTA is a technique which detects light scattering of individual particles in suspension, the addition of lipids to the perfusion fluids interfered with nanoparticle detection. In a second NMP study, Olimel was replaced with a non-lipid containing solution, and nanoparticle release during NMP was confirmed with NTA in 8 ECD kidneys (chapter 5). Characterization of these nanoparticles (using our single-EV IFCM protocol as developed in part I of this thesis) confirmed that these nanoparticles are representative of EVs, and showed that the detected EVs are representative of several subtypes based on their phenotypes. For example, after labelling the perfusion samples with a mixture of three common EV-markers (CD9, CD63, and CD81 – all belonging to the tetraspanin protein family), we found that the majority of detected EVs expressed CD81. This was a surprising find given that plateletpoor plasma or urine samples predominantly yielded CD9+ (chapter 3) or CD63+ (chapter 4) EVs, respectively. These findings suggest that the phenotypes and subsets of EVs differ dependent on the type of bio-fluid, representing a starting point for future researchers when examining EVs in different complex bio-fluids. To determine the cellular origin of the CD81+ EVs released during NMP, we labelled the perfusion fluids with anti-CD81 and anti-CD31 (endothelial marker). However, we found only marginal co-localization of these markers, suggesting that the majority of CD81+ EVs are either not of endothelial origin, or do not express CD31 on the vesicular surface.
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