Ex Vivo Normothermic Machine Perfusion of Declined Kidneys: A Pilot Study of Perfusion Dynamics, Necroptosis Pathway Activation and Clinical Translation

BACKGROUND: Ex vivo normothermic machine perfusion (EV NMP) enables functional assessment and reconditioning of marginal donor kidneys. This pilot study aimed to establish an EV NMP platform, characterise perfusion parameters and cell death pathways and facilitate clinical translation. METHODS: Six kidneys declined for transplantation underwent 2 h of EV NMP with a red blood cell-based perfusate. Perfusion dynamics, biochemistry and urine output were monitored. Histological injury was graded using the Remuzzi scoring system. Immunohistochemistry evaluated necroptosis (RIPK1, RIPK3, MLKL, pMLKL) and apoptosis (cleaved caspase-3, caspase-8). RESULTS: All kidneys produced urine and achieved Remuzzi scores ≤ 3. Renal vascular resistance declined 78% over the perfusion period. Perfusate creatinine and potassium decreased significantly (reduction 69.7 μmol/L, 95% CI [22.4, 117.0], p = 0.006 and 2.4 mmol/L, 95% CI [0.4, 4.4], p = 0.021, respectively). Necroptosis markers were prominent within tubular epithelium; pMLKL positivity in 18.5% of proximal tubules at baseline indicated active necroptotic signalling prior to perfusion. Apoptotic markers were comparatively sparse. A marginal DCDD kidney declined by all allocated transplant units was assessed using EV NMP and accepted for transplantation. The recipient experienced delayed graft function resolving within 1 week and mixed rejection at 6 months that responded to treatment, with maintained graft function at 2-year follow-up (serum creatinine 159 μmol/L). CONCLUSION: EV NMP is feasible and clinically translatable within a kidney transplant programme. Prominent baseline necroptotic signalling, reflected by pMLKL burden at commencement of perfusion, identifies the RIPK3-MLKL axis as a rational therapeutic target during ex vivo organ reconditioning, warranting investigation in prospective trials.