Introduction: Kidney transplantation using donors after circulatory death can be complicated by warm ischaemic damage. The local production of carbon monoxide via the haemo-oxygenase (HMOX-1) pathway has been shown to improve outcomes. In addition, the sphingosine 1-phosphate receptor-1 (S1PR1) interacts with host immune cells and modulates immune reperfusion injury; both of these are controlled upstream by the microRNA( mir) 24-3-p. Ex situ normothermic perfusion (NMP) perfuses a kidney with oxygenated blood prior to implantation. We aimed to deliver beneficial microRNA blockers called anti-sense oligonucleotides (ASOs) during NMP and change the kidney micro-environment prior to impantation.
Methods: Human kidneys, deemed unsuitable for transplantation, were subjected to 6 hr of NMP using a red cell-based perfusate. Fluorescently labelled anti-miR-24-3p (n=5) or scrambled control ASO (n=5) were introduced in hypothermic perfusate and during NMP. Serial biopsies were taken at t=0, t=60 and t=360 minutes. Real time q-PCR was used to assess the expression of miR-24-3p, S1PR1 and HMOX-1. Other kidneys were perfused with ASO under hypothermic conditions.
Results: NMP facilitated uptake of ASOs faster than both previous in-vitro and in-vivo animal models (as early as 6 hrs, p= 0.0006), with fluorescent signal identified in both endothelium and proximal tubules. In kidneys perfused with anti-miR-24-3p (n=5) we observed a significant increase in HMOX-1 RNA(18 fold, p = 0.0033) and S1PR1 (4 fold, p =0.0217) at 6 hours. These effects were not seen when the ASO was introduced in the perfusate during hypothermic perfusion or in the scrambled control.
Conclusions: NMP is a highly effective platform for the pre-transplant delivery of ASO as a short acting genetic modification to human kidneys. NMP allows organ-specific targeting and reduces the risks of systemic side-effects in the transplant recipient.  This is a novel and attractive strategy to prevent renal transplant ischaemia-reperfusion injury.