Extracellular Vesicles are Associated with Human Donor Kidney Characteristics 5 131 Concentrations of released EV subsets are correlated with donor demographics and NMP viability characteristics. To determine whether the identified EV subsets can be used as indicators of kidney quality prior to transplantation, we performed correlation analysis between the concentrations of EVs released by each individual kidney, and – in the absence of post-transplantation kidney function - donor kidney characteristics (specified in Table 1) and NMP viability characteristics (as a surrogate for kidney quality - specified in Table 2). Analysis of anti-CD9 single-positive EVs and CIT revealed a significant correlation after 360 minutes of NMP (R2 = 0.64, p = 0.017, Figure 4A), whilst no significant correlations with CIT were obtained for any of the other single-positive EV subsets. For CFSE and anti-CD9 double-positive EVs, significant correlations were observed for all time points analyzed (p < 0.05, Figure 4B). Additionally, CFSE and anti-CD63 double-positive EV were found to be significantly correlated after 60 minutes (R2 = 0.79, p = 0.003) – but not after 180 and 360 minutes – of NMP (Figure 4C). Analysis of CD81 and CD45 double-positive EVs revealed a positive correlation with donor age after the first 60 minutes of NMP only (R2 = 0.81 and p = 0.0023, Figure 4D). Anti-CD31 single-positive EVs were found to be the only EV subset that showed significant correlations with an NMP viability characteristic. For all time points analyzed, a positive correlation between concentrations of CD31+ EVs and renal blood flow was observed (Figure 4E), whilst negative correlations were found with intrarenal vascular resistance (Figure 4F). Although we did observe trends between some of the other donor kidney characteristics or NMP viability characteristics (e.g., kidney weight or impact of initial cold preservation – SCS vs HMP) and EV subset concentrations, none were found to be statistically significant. The differences between the individual ECD kidneys in terms of NMP viability characteristics (Table 2) did not correspond to e.g. matched kidney grafts retrieved from the same donor (K1 and K2, K7 and K8) or transplantability assessment post NMP (K3 and K5 were deemed of sufficient quality to be transplantable after 6 hours of NMP) when analyzing each kidney individually (as represented by the different shapes in Figure 5). Altogether, these findings indicate that the release of the identified EV subsets are differentially correlated to donor kidney characteristics and NMP viability characteristics.
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