NMP may serve while a bridge across this valley by giving a system for direct, nonsystemic drug treatment from the kidney whilst it really is undergoing normal metabolic procedures15,16. Between the multiple anti-IRI real estate agents tested in pre-clinical versions, Compact disc47-blocking antibody (CD47Ab), recombinant thrombomodulin (rTM), and soluble complement receptor 1 (sCR1) are especially translatable as they have been safely employed for other clinical applications1725. although further efficacy needs to be proven in the transplantation setting. Subject terms:Physiology, Translational research, Renal replacement therapy, Urology == Introduction == End-stage renal disease (ESRD) has a sizeable global burden of disease, causing at least 1.2 million global annual deaths1. Kidney transplantation is the best available treatment for ESRD, conferring a significant survival benefit over dialysis24. However, there is a perpetual supply-demand gap between patients awaiting transplantation and the availability of deceased donor kidneys. This has necessitated expansion of the donor pool to include more marginal organs, including donation after circulatory death (DCD) kidneys, which are subjected to greater warm ischemia57. Short-term transplantation outcomes, including delayed graft function (DGF), are inferior in DCD kidneys in comparison to kidneys from brain-dead (DBD) donors with no significant comorbidities6. This increased susceptibility to ischemia-reperfusion injury (IRI) and DGF can translate into poorer long-term graft survival8. As such, an improved method of kidney assessment, repair and preservation is required above and beyond the currently accepted gold standard of cold static storage (CS), particularly in this donor kidney subset. Machine perfusion (MP) preservation is an important alternative that has regained prominence9. Normothermic MP (NMP) is especially promising, and is now the subject of a multi-center randomized control trial (RCT) comparing it to CS alone in DCD kidneys1013. Most pharmacotherapeutics shown to ameliorate renal IRI have been unable to bridge the valley of death (translational gap) to the clinic. This is at least partly attributable to the inherent difficulties and ethical considerations associated with the systemic use of such therapies in donors or recipients14,15. NMP can serve as a bridge across this valley by providing a platform for direct, non-systemic drug treatment of the kidney whilst it is undergoing normal metabolic processes15,16. Amongst the multiple anti-IRI agents tested in pre-clinical models, CD47-blocking antibody (CD47Ab), recombinant thrombomodulin (rTM), and soluble complement receptor 1 (sCR1) are especially translatable as they have been safely employed Epristeride for other clinical applications1725. However, the comparative efficacy of these agents has not been established. CD47Ab ameliorates thrombospondin (TSP)-1 mediated IRI signaling, including inhibition of nitric oxide and promotion of oxidative stress21. sCR1 is an inhibitor of the Epristeride classical and alternative complement pathways, activation of which is important in IRI26. rTM is an anti-coagulant molecule involved in the generation of activated protein C, although its efficacy in IRI may be more attributable to anti-inflammatory effects17,27. Because IRI is characterized by the activation of multiple intersecting pathways28,29, it is also plausible that synergistic anti-IRI effects may be derived by delivering two or more of these agents together. The aims of this study were to directly compare the acute effects of CD47Ab, sCR1, and rTM in a murine model Epristeride of renal IRI and to establish the combined efficacy of two of the best agents. We then show that the chosen drug could be directly delivered to porcine DCD kidneys using NMP to enhance renal perfusion parameters and ameliorate IRI. The primary focus of this study is the immediate phase of IRI, which correlates to the immediate post-transplant setting and the risk of delayed graft function in Epristeride higher risk renal allografts. == Methods == == Animal work ethics == All protocols were approved by the Western Sydney Local Health District Animal Ethics Committee, in accordance with the Australian code for the care and use of animals for scientific purposes (8thEd., 2013), developed by the National Health and Medical Research Council. Animal experiments adhere to the ARRIVE guidelines. == PART 1. Comparison of IRI targets murine model == == Animals == Male C57BL/6 mice (1012 weeks age) were obtained from the Animal Resources Centre (Canning Vale, Australia), acclimatized and allowed free access to food and water until surgery. The use male C57BL/6 mice is commonly accepted and performed in kidney IRI experiments30,31. == IRI model == General anesthesia (GA) was induced using intra-peritoneal ketamine (100 mg/kg) and xylazine (8 mg/kg). The abdomen was shaved, prepared with povidone-iodine, and a midline laparotomy performed. The small intestine was wrapped in gauze moistened with 3 ml of 0.9% sodium chloride (NaCl) (3637 C), and placed Fzd10 outside the operating field. The right renal pedicle was ligated (60 silk tie) prior to a right nephrectomy. Each drug/combination was diluted to.