Perfusate Liver Arginase 1 Levels After End-Ischemic Machine Perfusion Are Associated with Early Allograft Dysfunction

Abstract

Background/Objectives: The rising use of liver grafts from donation after circulatory death (DCD) has been enabled by advances in normothermic regional perfusion (NRP) and machine perfusion (MP) technologies. We aimed to identify predictive biomarkers in DCD grafts subjected to NRP, followed by randomization to either normothermic machine perfusion (NMP) or dual hypothermic oxygenated perfusion (D-HOPE). Methods: Among 57 DCD donors, 32 liver grafts were transplanted, and recipients were monitored for one week post-transplant. Biomarkers linked with oxidative stress, hepatic injury, mitochondrial dysfunction, inflammation, regeneration, and autophagy were measured during NRP, end-ischemic MP, and one week post-transplant. Results: Arginase-1 (ARG-1) levels were consistently higher in discarded grafts and in recipients who later developed early allograft dysfunction (EAD). Specifically, ARG-1 levels at the end of MP correlated with markers of hepatic injury. Receiver operating characteristic analysis indicated that ARG-1 at the end of MP had a good predictive accuracy for EAD (AUC = 0.713; p = 0.02). Lipid peroxidation (TBARS) elevated at the start of NRP, declined over time, with higher levels in D-HOPE than in NMP, suggesting a more oxidative environment in D-HOPE. Metabolites like flavin mononucleotide (FMN) and NADH exhibited significant disparities between perfusion types, due to differences in perfusate compositions. Inflammatory biomarkers rose during NRP and NMP but normalized post-transplantation. Regenerative markers, including osteopontin and hepatocyte growth factor, increased during NRP and NMP and normalized post-transplant. Conclusions: ARG-1 demonstrates strong potential as an early biomarker for assessing liver graft viability during perfusion, supporting timely and effective decision-making in transplantation.

Keywords: ARG-1; D-HOPE; DCD; EAD; FMN; NMP; arginase 1; donations after circulatory death; dual hypothermic oxygenated machine perfusion; early allograft dysfunction; flavin mononucleotide; normothermic regional perfusion.

Figure 1

(A) A schematic diagram of the donor and patient selection. (B) A timeline of blood sample collection: (a) at the initiation of NRP (T0), 2 h after initiation (T2h), and 4 h after initiation (T4h); (b) at the beginning, midpoint, and conclusion of the end-ischemic MP procedure; (c) in recipients: immediately post-reperfusion, one day after liver transplantation, and one week after liver transplantation.

Figure 2

Dynamic change of GST-α (A) and ARG-1 (B) during NRP, end-ischemic MPs and in recipients until a week post-transplant. Values are presented as mean ± SE. * Significant changes over time within each group (Friedman’s test with Bonferroni correction). Only values of p < 0.05, considered as significant, are reported. (C) Dynamic change of ARG-1 levels between the EAD group and non-EAD group during NRP, end-ischemic MPs of grafts and in recipients until a week post-transplant. (D) Differences in ARG-1 levels between the non-transplanted group and transplanted group during NRP and end-ischemic MPs of grafts.

Figure 3

(A) ROC curve and sensitivity/specificity graph for ARG-1 concentration differentiating groups at the end of ex situ MP. The threshold for differentiation between EAD group and non-EAD group was 77,471 pg/mL, with a sensitivity of 55.6% and a specificity of 99.9%. AUC, area under the ROC curve; ROC, receiver operator characteristic. (B) Correlation between Δ% ARG1 during end-ischemic MPs [(T_end − T_start)/T_start × 100] and L-GRAFT (r = 0.481, p < 0.01), and EASE (r = 0.448, p < 0.05). (C) Correlation between Δ% ATG5 tissue mRNA expression during end-ischemic MPs [(T_end − T_start)/T_start × 100] and L-GRAFT (r = 0.734, p < 0.01), and EASE (r = 0.830, p < 0.001) in D-HOPE group.

Figure 4

Dynamic change of: (A) TBARS, (B) succinate, (C) FMN, and (D) NADH during NRP, end-ischemic MPs and in recipients until a week post-transplant. Values are presented as mean ± SE. * Significant changes over time within each group (Friedman’s test with Bonferroni correction). Only values of p < 0.05, considered as significant, are reported.

Figure 5

Dynamic change of: (A) IL-6, (B) IL-8, and (C) TNF-α during NRP, end-ischemic MPs and in recipients until a week post-transplant. Values are presented as mean ± SE. * Significant changes over time within each group (Friedman’s test with Bonferroni correction). Only values of p < 0.05, considered as significant, are reported.

Figure 6

Dynamic change of: (A) OPN and (B) HGF during NRP, end-ischemic MPs and in recipients until a week post-transplant. * Significant changes over time within each group (Friedman’s test with Bonferroni correction). Only values of p <0.05, considered as significant, are reported.