Miguel Larraz

, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.

Background: , but there is limited understanding of the transcriptional pathways, and the associated cellular landscape, driving IRI during NMP and determining its severity. Such knowledge is essential for therapeutic targeting and organ resuscitation during machine perfusion. Methods: Using tissue obtained at the time of NMP from kidneys subsequently transplanted as part of a randomized controlled trial, we undertook in-depth transcriptomic analyses comparing kidneys suffering severe IRI, (manifesting clinically as the development of delayed graft function (DGF)), to kidneys with mild IRI (defined by immediate graft function, IGF) post-transplantation. Results: . Going further, we identified innate immune system driven processes at the core of the transcriptional signature in kidneys suffering severe IRI, such as recruitment and migration of myeloid leucocytes, macrophage activation, phagocytosis and inflammasome activation. Deconvolution using single-cell-RNAseq data showed kidneys with severe IRI and post-transplant DGF were enriched for pro-inflammatory mononuclear phagocytes, myofibroblasts and fibroblasts, but depleted of tubuloepithelial, cell signatures. These transcriptional findings were recapitulated in tissue biopsies obtained during NMP from an external cohort comparing kidneys with high acute tubular injury and severe IRI to kidneys with low acute tubular injury and mild IRI; these kidneys were histologically similar to the DGF/IGF kidneys, respectively. Discussion: Together, our study characterizes the transcriptional signature of severe IRI during NMP, suggesting the role of pro-inflammatory innate/pro-fibrotic cells in this process. We describe a transcriptomic signature that may support future prospective therapeutic trials as a potential efficacy endpoint, and highlight potential cellular targets for therapeutic intervention during NMP in an era of precision medicine.

Abstract 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.

Abstract Background Normothermic machine perfusion (NMP) preserves kidneys in a near-physiological state, allowing viability assessment and intervention. NMP typically uses red blood cell (RBC)-based perfusates, with associated challenges, including blood type compatibility, haemolysis, and free haem–driven ferroptotic tubular injury. Acellular perfusates avoid these risks. Subnormothermic machine perfusion (SMP, 32°C) may reduce metabolic demands while preserving organ viability, but the effect of perfusate type under these conditions remains unclear. Methods In a paired experimental design, kidneys from eight deceased donors were randomized 1:1 to 6 h of SMP with RBC-based (RBC-SMP) or acellular perfusate (SNAP), followed by 4 h of RBC-based NMP (37°C) to simulate reperfusion. Bulk RNA sequencing, transcriptomic pathway scoring, urinary biomarkers, and histology were used to compare perfusion methods. Publicly available RBC-NMP transcriptomic data were integrated for comparison. Results Transcriptional comparison showed no difference in pathways related to ischaemia-reperfusion injury (IRI; TNFα via NFκB, Allograft Rejection, Inflammatory Response) and Oxidative Phosphorylation, at 6 h SMP and 4 h reperfusion between RBC-SMP and SNAP kidneys. Similarly, metabolism-associated transcriptional pathways, urinary tubular injury biomarkers (NGAL, L-FABP, TIMP2, IGFBP7), and histological injury were similar between groups. Compared to 6 h of RBC-NMP at 37°C, both subnormothermic groups showed less depletion of oxidative phosphorylation with similar IRI levels, suggesting preserved mitochondrial function. Conclusions Acellular subnormothermic kidney perfusion is equivalent to RBC-based SMP at the transcriptomic, metabolic, and tubular injury level. Our data support the clinical feasibility of SNAP as a safe, logistically simple alternative for kidney preservation.

Abstract Normothermic machine perfusion (NMP) typically uses red blood cell (RBC)-based perfusates, with associated challenges, including free haem-driven ferroptotic tubular injury. Acellular perfusates avoid these risks. Subnormothermic machine perfusion (SMP, 32°C) may reduce metabolic demands while preserving organ viability, but the effect of perfusate type under these conditions remains unclear. In a paired experimental design, kidneys from eight deceased donors were randomized 1:1 to 6 hours of SMP with RBC-based (RBC-SMP) or acellular perfusate (SNAP), followed by 4 hours of RBC-based NMP (37°C) to simulate reperfusion. Upregulation of transcriptomic ischaemia-reperfusion injury (IRI) related pathways and downregulation of oxidative phosphorylation at 6 hours SMP and 4 hours reperfusion were similar between RBC-SMP and SNAP kidneys. No differences were observed between groups in transcriptional profiles, metabolic pathway scores, urinary tubular injury biomarker concentrations, or histology. We integrated publicly available NMP transcriptomic data for comparison, demonstrating that compared to 6 hours RBC-NMP at 37°C, both SMP groups showed equivalent pro-inflammatory pathway activation and oxidative phosphorylation depletion. Thus, we provide evidence that SNAP maintains kidneys with equivalent transcriptional and urinary tubular injury biomarker profiles to RBC-SMP. These findings support the feasibility of SNAP for clinical translation in future studies.