Extracellular Vesicles are Associated with Human Donor Kidney Characteristics 5 123 INTRODUCTION The shortage of available grafts, the increasing number of patients on the waiting list and the general aging of the population has led to an increased use of expandedcriteria donor (ECD) grafts as well as grafts procured from donation after circulatory death (DCD) 1. Both ECD and DCD grafts are associated with poorer transplant outcomes when compared to organs from standard criteria donors 2, 3. This is in part because older grafts are more susceptible to ischemia reperfusion injury (IRI) as well as the inability to fully recover after transplantation as a consequence of natural loss of nephron mass 4. Moreover, an essential problem with the usage of these kidneys is the lack of quality measures needed to guide the clinician in deciding whether to accept or decline the organ. Combined, this has forced the transplant community to 1) investigate new methods of organ preservation aimed at reducing IRI and 2) to develop tools to evaluate transplant kidney quality. The most recent development in organ preservation is normothermic machine perfusion (NMP). In contrast to hypothermic machine perfusion (HMP), NMP aims to restore cellular metabolism and function to the organ, which is achieved through circulation of a warm, oxygenated red blood cell based solution through the organ prior to transplantation 5, 6. Because metabolism is activated, NMP offers the possibility to assess graft status prior to transplantation through monitoring of the perfusion dynamics and analysis of biomarkers in the perfusion fluids 2, 5, 7, 8. Potential candidates for the assessment of graft status are Extracellular vesicles (EVs). EVs are lipid bilayer membrane structures (30-8000 nm in diameter 9) involved in cellular communication 10. They express surface markers and carry a ‘cargo’ (e.g. DNA / RNA / Lipids / proteins 11), both of which are thought to be indicative for the status of its cell of origin. EVs are excreted by virtually all cell types and are considered an excellent, stable biomarker platform as their cargo is protected from fragmentation and degradation by the lipid bilayer 12. In transplantation, levels of (human) donor-specific EVs in animal models have been shown to be associated with acute rejection of the allograft 13, 14. Additionally, miRNA, RNA and proteomic profiling of EVs obtained from kidney preservation fluids 15 or the urine of kidney recipients 16, 17 suggest that EV analysis might enable kidney health assessment and prognostication in kidney transplantation. Despite the interest in EVs as biomarker, the analysis of EVs is hampered by their physical characteristics such as their small size, low epitope copy number 18, the
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