Chapter 5 136 EV subsets) are able to cross-decorate and activate alloreactive recipient B cells in a mouse skin-transplant model 29. In a human setting, this cross-decoration might be facilitated by CD81+ EVs. When determining the origin of detected CD81+ EVs, we found only marginal co-localization of CD81 with endothelial and hematopoietic markers CD31 and CD45. However, part of the CD81+CD31+ EV may represent CD45+ EVs derived from monocytic origin. Additionally, the low percentages of colocalization may be influenced by the usage of mAbs targeting extravesicular epitopes 20 (thus ignoring the presence of markers on the luminal side of EV). Moreover, the released particles were determined to be <400 nm (as shown by NTA, and selected in the IFCM analysis), and therefore were assumed to consist largely of exosomes. Exosomes form through inward budding of the membrane of early endosomes (forming multivesicular bodies in the process) which eventually fuse with the cell plasma membrane, releasing its content into the extracellular space 34. Consequentially, it is hypothesized that not all exosomes necessarily bear parental cell surface markers – which may explain why >90% of CD81+ EVs were found to not be colocalized with either anti-CD31 or anti-CD45. Correlation analysis of CFSE+ EVs and CIT revealed negative correlations for EVs bearing tetraspanins CD9 or CD63 (during the first 60 minutes of NMP) - but not CD81. Since CFDA-SE needs intravesicular esterases to acquire its fluorescent properties (CFSE+), these negative correlations may be explained by 1) the negative impact of CIT on cellular (and thus vesicular) enzyme (esterase) activity or 2) reduced release of EVs containing intravesicular esterases as a consequence of CIT. Diminished correlations of CFSE+ and CD9+/CD63+ double-positive EVs with CIT were found after 180 and 360 minutes of NMP – which might be explained by restoration of cellular metabolism during the course of NMP 5, 26. As the perfusion pressure during NMP is fixed, the perfused kidneys autoregulate their blood flow according to intrarenal vascular resistance. The inverse correlation between low renal blood flow and high intrarenal vascular resistance during NMP has been described in literature, and has been associated with increased vascular injury or interstitial oedema 35. We demonstrate that the release of CD31+ singlepositive EVs (likely to be of endothelial origin) is positively correlated with renal blood flow, and consequently inversely correlated with intrarenal vascular resistance. As it is well-known that the endothelial cell layer of microvessels is a key modulator
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