A comprehensive proteomic and transcriptomic mapping study found porcine and human kidneys exhibit a profoundly altered biological profile during warm ex vivo perfusion.

INTRODUCTION

Organ shortages remain a major limitation to kidney transplantation. Normothermic machine perfusion (NMP) is a technique to address this challenge by improving quality assessment and ex vivo organ preservation. However, the molecular state of the organ during NMP, and how it differs from in vivo physiology, remains poorly defined.

METHODS

To address this, we performed RNA sequencing and LC-MS/MS analyses of porcine and human kidneys during NMP. Cortical kidney tissue was collected from 60 porcine and 24 human donor kidneys at multiple time-points, including an in vivo baseline (porcine only), cold preservation, and four time points during NMP yielding 480 paired transcriptomic and proteomic profiles of donor kidneys.

RESULTS

NMP triggered extensive, time-dependent transcriptional reprogramming (86% of genes differentially expressed) and more gradual proteomic alterations. NMP consistently activated inflammatory TNF-α/NF-κB signaling and suppressed oxidative phosphorylation, reflecting ischemia-reperfusion-like injury and impaired mitochondrial metabolism. Transcriptomic responses were highly similar between porcine and human kidneys, while proteomic responses showed greater inter-individual and interspecies variability. Warm ischemia induced persistent proteotoxic stress, characterized by activation of protein folding and chaperone pathways and sustained metabolic suppression. This suggests that the delayed and variable proteomic responses could result from stress-induced dysregulation of protein homeostasis. Additionally, conventional donor characteristics explained only a minor fraction of the change in biological behavior during NMP.

CONCLUSION

The molecular profile of donor kidneys is extensively remodeled during NMP, creating an ex vivo state that diverges considerably from in vivo physiology. These molecular changes cannot be explained by differences in donor characteristics, indicating that other NMP-specific or injury-related factors dominate these deviating responses. Our findings deliver a high-resolution molecular map of kidneys during NMP and challenge the use of in vivo biomarkers for ex vivo assessment. Ex vivo specific responses should be considered when evaluating or resuscitating kidneys during perfusion.