Ischaemia–reperfusion injury (IRI) is an inevitable process in transplantation and results in inflammation and immune system activation. Alpha-1 antitrypsin (AAT) has anti-inflammatory properties. Normothermic machine perfusion (NMP) can be used to deliver therapies and may help in assessing the effects of IRI and immunity. This study investigated the effects of AAT on IRI and inflammation in pig kidneys when administered during preservation, followed by normothermic reperfusion (NR) with autologous whole blood, as a surrogate for transplant. Two different models were used to deliver AAT or placebo to paired slaughterhouse pig kidneys: Model 1: 7-h static cold storage (SCS) + 3-h NR (n = 5 pairs), where either AAT (10 mg/ml) or placebo was delivered in the flush following retrieval; Model 2: 4-h SCS + 3-h NMP + 3-h NR (n = 5 pairs), where either AAT or placebo was delivered during NMP. Injury markers and cytokines levels were analysed in the perfusate, and heat shock protein 70 KDa (HSP-70) was analysed in biopsies. AAT delivered to kidneys showed no adverse effects on perfusion parameters. HSP-70 fold changes were significantly lower in the AAT group during NMP (P < 0.01, paired t-test) but not during NR. Interleukin-1 receptor antagonist (IL-1ra) fold changes were significantly higher in the AAT group during NR model 1 (p < 0.05, two-way ANOVA). In contrast to the AAT group, significant upregulation of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) between t = 90 min and t = 180 min and interleukin-8 (IL-8) between baseline and t = 90 min was observed in the control group in NR model 2 (p < 0.05, Tukey’s multiple comparison test). However, overall inflammatory cytokines and injury markers showed similar levels between groups. Delivery of AAT to pig kidneys was safe without any detrimental effects. NMP and NR provided excellent methods for comparison of inflammation and immune activation in the delivery of a novel therapy.

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IntroductionCurrently there is an urgent need to translate interventions that may be beneficial to marginal donor kidneys prior to transplant, to improve their quality from bench to bedside. This project investigated the effects of CC-4066, a potent dual inhibitor of cyclophilin proteins A and D, treatment during static cold storage (SCS) in a porcine model of renal ischemia-reperfusion injury (IRI) using Normothermic Reperfusion (NR).Materials and methodsPorcine kidneys and autologous blood were retrieved in pairs from a local abattoir (n = 7). One kidney from each pair was randomly allocated to treatment and one allocated to control and flushed with preservation solution containing CC-4066 or vehicle. After 7 h of SCS kidneys underwent 3 h Normothermic Reperfusion (NR) with autologous whole blood while perfusion characteristics and samples were collected.ResultsPerfusion and metabolic parameters showed similar trends and no statistical differences were observed between the groups. IL-6 showed a significant increase over time but no significant difference between groups (p-value 0.009 and 0.14 respectively, two-way ANOVA). Oxygen consumption and lactate levels were similar between groups but there was increased vacuolation on histology in the control group.ConclusionsThe addition of CC-4066 during SCS of kidneys is safe and feasible and has no adverse or detrimental effects on perfusion during assessment on NR. There was no difference in cytokine levels although there was a trend towards less vacuolation on histology in the treatment group.