Simultaneous kidney-pancreas transplantation improves endothelial dysfunction induced by extracellular vesicles

Introduction: Extracellular vesicles (EVs) are membrane structures secreted by cells that have been described as potential mediators of pathogenic mechanisms in endothelial dysfunction (ED), causing stimulation or suppression of endothelial gene expression. The mechanisms of ED in patients with End stage kidney disease (ESKD) and type 1 diabetes mellitus (T1DM) are not well defined, although EVs may play an important role. In the present study, we report on the relevance of EVs on ED outcome in T1DM with ESKD and ESKD patients before and after one year after a simultaneous kidney-pancreas (SKPT), and compare with recipients of kidney transplantation alone (KdTx). Serum from 11patients/group were randomly pooled into 4 pools/group. EVs were isolated by differential centrifugation, and characterized by Nanoparticle Tracking Analysis (NTA), flow cytometry surface marker expression (MACSPlexã), and by transmission electron microscopy. HMEC-1 cells were exposed in vitro to patients’ pooled serum, with and without EVs, and results were compared to control serums. Expression of ICAM-1, VCAM-1 and von Willebrand Factor (vWF) by immunofluorescence (IF), and platelet adhesion by blood perfusion studies on extracellular matrices secreted by endothelial cells (ECMs) was used to evaluate ED in the in vitro cultures. Inflammatory cytokines (IL-6, TNF-α, CPR and IL-1β) and circulating endothelial markers (ICAM-1 and vWF) were analysed in total serum patients by immunoassay. ICAM-1 (p<0.0007), VCAM-1 (p<0.0001) and vWF (p<0.0038) IF expression on ECMs was significantly increased in HMEC-1 cells incubated with serum from T1DM+ESKD compared to controls. Depletion of EVs from T1DM+ESKD serum decreased the expression of these markers to levels comparable to controls. Serum from SPKT recipients was also associated with a significant inferior expression of ED markers compared to T1DM+ESKD pre-transplant. EVs depletion from SPKT only moderately improved expression of vWF alone (Figure 1A-C). The percentage of platelet-coated surface (%SC) over ECMs was increased in T1DM with ESKD (p<0.0016) compared to control serum. EVs depletion led to normalization of the %SC, which was similar to SPKT (Figure 1D). Serum levels of inflammatory cytokines IL-6, TNF-α, IL-1β, and CRP before and after SKPTx were similar levels to controls, as were ICAM1 and vWF levels. Conclusions: EVs from patients with ESRD are potent inducers of ED in vitro, particularly in patients with T1DM and ESRD. SPKT transplantation significantly improves EVs induced ED. Understanding the role of EVs and their cargo in inducing ED may be relevance to predict patient’s pre- and post-transplant cardiovascular risk. Study funded by a grant from the Portuguese Society of Nephrology.