Transcriptomic profiling during normothermic machine perfusion of human kidneys reveals a pro-inflammatory cellular landscape and gene expression signature associated with prolonged delayed graft function after transplantation

Assessment and treatment of severe ischaemia-reperfusion-injury (IRI) remains an unmet challenge in kidney transplantation. Normothermic machine perfusion (NMP) aims to resuscitate organs but also recapitulates IRI ex situ . Understanding transcriptional pathways, and associated cellular landscape, driving IRI during NMP could facilitate therapeutic targeting. Using tissue and urine from kidneys undergoing NMP pre-transplantation as part of a randomised controlled trial, we undertook in-depth transcriptomic analyses of kidneys developing prolonged delayed graft function (PDGF; clinical manifestation of severe IRI) versus immediate graft function (IGF). We validated upregulation of previously described pro-inflammatory and immune pathways and identified innate immune system driven processes at the core of the transcriptional signature in PDGF kidneys. Deconvolution using single-cell-RNAseq data showed PDGF kidneys were enriched for pro-inflammatory mononuclear phagocyte, myofibroblast and fibroblast, but depleted of tubuloepithelial, cell signatures. These findings were recapitulated in tissue biopsies from an external NMP kidney cohort with high versus low acute tubular injury, histologically similar to PDGF/IGF kidneys, respectively. Extracellular vesicles released in the urine of PDGF kidneys were enriched in miRNAs functionally annotated to transcriptional processes upregulated in the tissue compartment. Together, our study characterises the transcriptional signature of severe IRI during NMP, highlighting the role of pro-inflammatory innate and pro-fibrotic cells in this process.