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The ideal preservation temperature for donation after circulatory death kidney grafts is unknown. We investigated whether subnormothermic (22 °C) ex vivo kidney machine perfusion could improve kidney metabolism and reduce ischemia-reperfusion injury., Methods: To mimic donation after circulatory death procurement, kidneys from 45-kg pigs underwent 60 min of warm ischemia. Kidneys were then perfused ex vivo for 4 h with Belzer machine perfusion solution UW at 22 °C or at 4 °C before transplantation. Magnetic resonance spectroscopic imaging coupled with LCModel fitting was used to assess energy metabolites. Kidney perfusion was evaluated with dynamic-contrast enhanced MRI. Renal biopsies were collected at various time points for histopathologic analysis., Results: Total adenosine triphosphate content was 4 times higher during ex vivo perfusion at 22 °C than at 4 °C perfusion. At 22 °C, adenosine triphosphate levels increased during the first hours of perfusion but declined afterward. Similarly, phosphomonoesters, containing adenosine monophosphate, were increased at 22 °C and then slowly consumed over time. Compared with 4 °C, ex vivo perfusion at 22 °C improved cortical and medullary perfusion. Finally, kidney perfusion at 22 °C reduced histological lesions after transplantation (injury score: 22 °C: 10.5 ± 3.5; 4 °C: 18 ± 2.25 over 30)., Conclusions: Ex vivo kidney perfusion at 22°C improved graft metabolism and protected from ischemia-reperfusion injuries upon transplantation. Future clinical studies will need to define the benefits of subnormothermic perfusion in improving kidney graft function and patient's survival., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)