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.

The authors have declared no competing interest.

We acknowledge funding support from National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Organ Donation and Transplantation (NIHR203332), a partnership between NHS Blood and Transplant, University of Cambridge and Newcastle University. The views expressed are those of the authors and not necessarily those of the NIHR, NHS Blood and Transplant or the Department of Health and Social Care. HS acknowledges funding from a Sir Roy & Lady Calne Royal College of Surgeons Research Fellowship (G121988). IM acknowledges funding by the Wellcome Trust [203151/Z/16/Z] and the UKRI Medical Research Council [MC_PC_17230]. VK acknowledges funding from an NIHR Fellowship (PDF-2016-09-065) and as a Paul I. Terasaki Scholar (G106170).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethical approval for the previously completed trial and collection of biological samples was granted by the East of England Cambridge Central Research Ethics Committee (15/EE/0356). Ethical approval for the study (Research kidney NMP validation cohort) was granted by the national ethics committee in the UK REC (22/WA/0167).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All transcriptomic data produced within this study will be made available via GEO Accession Number upon publication of the manuscript.