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ABO blood group compatibility restrictions present the first barrier to donor-recipient matching in kidney transplantation. Here, we present the use of two enzymes, Fp GalNAc deacetylase and Fp Galactosaminidase, from the bacterium Flavonifractor plautii to enzymatically convert blood group A antigens from the renal vasculature of human kidneys to ‘universal’ O-type. Using normothermic machine perfusion (NMP) and hypothermic machine perfusion (HMP) strategies, we demonstrate blood group A antigen loss of approximately 80% in as little as 2 h NMP and HMP. Furthermore, we show that treated kidneys do not bind circulating anti-A antibodies in an ex vivo model of ABO-incompatible transplantation and do not activate the classical complement pathway. This strategy presents a solution to the donor organ shortage crisis with the potential for direct clinical translation to reduce waiting times for patients with end stage renal disease. ABO blood group compatibility restrictions limit the availability of organs for patients awaiting transplantation. Here, the authors show the rapid enzymatic removal of blood group A antigens from the vasculature of human kidneys using normothermic and hypothermic machine perfusion technologies to make universal blood group O organs for transplantation.

IntroductionThe introduction of perfusion technologies in kidney transplantation has the potential to improve graft function and survival and increase utilisation. Our previous work demonstrated that kidneys with an enhanced inflammatory and immune response during normothermic machine perfusion (NMP) had significant graft dysfunction after transplantation. The addition of a cytokine filter (CytoSorb) to the NMP circuit dramatically reduces both circulating inflammatory mediators and inflammatory gene expression, but this has not been trialled in clinical practice.Methods and analysisThis is a randomised phase 1 pilot study to evaluate the safety and feasibility of using a CytoSorb filter in clinical NMP to remove inflammatory and immune mediators. Eligible kidney transplant recipients on the waiting list in the East of England will be approached for consent. A total of 20 patients will be recruited and randomised in a 1:1 ratio for the donor kidney to receive either NMP or NMP with a CytoSorb filter pre-transplantation. The kidney will be transplanted according to standard practice after NMP. The primary endpoint is inflammatory and immune gene expression measured in a cortical biopsy from the kidney 60 min post-transplant. Secondary endpoints include rates and duration of delayed graft function and graft function as assessed by change in creatinine clearance and estimated glomerular filtration rate 2 days, 5 days, 1 month and 3 months post-transplant. Additionally, inflammatory mediators and injury markers will be measured in peripheral blood and urine samples taken pre-operatively and on days 2 and 5 after transplant.Ethics and disseminationThis study has been approved by the Health Research Authority Health and Care Research Wales Committee (REC 23/WM/0141) and by National Health Service (NHS) Blood and Transplant (Ref: Study 148). Findings will be published in a peer-reviewed journal and disseminated at scientific conferences. The dataset will be made available on request.Trial registrationThe study is prospectively registered on the ISCRTN registry (ID: 13698207).

Normothermic machine perfusion (NMP) is a technique of kidney preservation designed to restore cellular metabolism after cold ischemia. Kidneys are perfused with an oxygenated banked red blood cell (RBC) based solution for 1h at 36°C. During NMP, RBCs can become damaged, releasing free heme into the perfusate. This can act as a damage-associated molecular pattern (DAMP) activating inflammatory signalling pathways. The aim of this study was to measure the levels of free heme during NMP, assess the effect on kidney function and determine any association with inflammatory and stress related gene expression. Levels of free heme were measured in perfusate samples from a series of donation after circulatory death (DCD) kidneys undergoing NMP as part of a randomised controlled trial (RCT). The age of RBCs and levels of free heme were correlated with perfusion parameters. Changes in gene expression were analysed in a series of kidneys declined for transplantation using the NanoString nCounter Organ Transplant Panel and qRT-PCR. Older units of RBCs were associated with higher levels of free heme and levels increased significantly during NMP (Pre 8.56 ± 7.19µM vs 26.29 ± 15.18µM, P<0.0001). There was no association with levels of free heme and perfusion parameters during NMP (P > 0.05). Transcriptional and qPCR analysis demonstrated the upregulation of differentially expressed genes associated with apoptosis (FOS and JUN), inflammatory cytokines (IL-6, SOCS3, ATF3), chemokines (CXCL8, CXCL2, CC3/L1) and oxidative stress (KLF4) after NMP. However, these did not correlate with levels of free heme (P >0.05). A significant amount of free heme can be detected in the perfusate before and after NMP particularly when older units of red cells are used. Although transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways these were not associated with high levels of free heme.

Background Ex-vivo normothermic perfusion strategies are a promising new instrument in organ transplantation. The perfusion conditions are designed to be protective however the artificial environment can induce a local inflammatory response. The aim of this study was to determine the effect of incorporating a Cytosorb adsorber into an isolated kidney perfusion system. Methods Porcine kidneys were subjected to 22 h of cold ischaemia then reperfused for 6 h on an ex vivo reperfusion circuit. Pairs of kidneys were randomised to either control (n = 5) or reperfusion with a Cytosorb adsorber (n = 5) integrated into the circuit. Tissue, blood and urine samples were taken for the measurement of inflammation and renal function. Results Baseline levels of cytokines (IL-6, TNFα, IL-8, IL-10, IL-1β, IL-1α) were similar between groups. Levels of IL-6 and IL-8 in the perfusate significantly increased during reperfusion in the control group but not in the Cytosorb group (P = 0.023, 0.049). Levels of the other cytokines were numerically lower in the Cytosorb group; however, this did not reach statistical significance. The mean renal blood flow (RBF) was significantly higher in the Cytosorb group (162 ± 53 vs. 120 ± 35 mL/min/100 g; P = 0.022). Perfusate levels of prostaglandin E2 were significantly lower in the Cytosorb group (642 ± 762 vs. 3258 ± 980 pg/mL; P = 0.0001). Levels of prostacyclin were significantly lower in the Cytosorb group at 1, 3 and 6 h of reperfusion (P = 0.008, 0.003, 0.0002). Levels of thromboxane were also significantly lower in the Cytosorb group throughout reperfusion (P = 0.005). Haemoadsorption had no effect on creatinine clearance (P = 0.109). Conclusion Haemoadsorption can reduce the inflammatory response and improve renal blood flow during perfusion. Nonetheless, in this model haemoadsorption had no influence on renal function and this may relate to the broad-spectrum action of the Cytosorb adsorber that also removes potentially important anti-inflammatory mediators.

Assessment and treatment of severe ischemia-reperfusion-injury (IRI) remains an unmet challenge in kidney transplantation. Normothermic machine perfusion (NMP) recapitulates IRI , 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. 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. We validated upregulation of previously described pro-inflammatory and immune transcriptomic pathways, including and . 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. 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.

The survival of transplant kidneys using deceased donors (DD) is inferior to living donors (LD). In this study, we conducted a whole-transcriptome expression analysis of 24 human kidney biopsies paired at 30 minutes and 3 months post-transplantation using DD and LD. The transcriptome profile was found significantly different between two time points regardless of donor types. There were 446 differentially expressed genes (DEGs) between DD and LD at 30 minutes and 146 DEGs at 3 months, with 25 genes common to both time points. These DEGs reflected donor injury and acute immune responses associated with inflammation and cell death as early as at 30 minutes, which could be a precious window of potential intervention. DEGs at 3 months mainly represented the changes of adaptive immunity, immunosuppressive treatment, remodeling or fibrosis via different networks and signaling pathways. The expression levels of 20 highly DEGs involved in kidney diseases and 10 genes dysregulated at 30 minutes were found correlated with renal function and histology at 12 months, suggesting they could be potential biomarkers. These genes were further validated by quantitative polymerase chain reaction (qPCR) in 24 samples analysed by microarray, as well as in a validation cohort of 33 time point unpaired allograft biopsies. This analysis revealed that SERPINA3, SLPI and CBF were up-regulated at 30 minutes in DD compared to LD, while FTCD and TASPN7 were up-regulated at both time points. At 3 months, SERPINA3 was up-regulated in LD, but down-regulated in DD, with increased VCAN and TIMP1, and decreased FOS, in both donors. Taken together, divergent transcriptomic signatures between DD and LD, and changed by the time post-transplantation, might contribute to different allograft survival of two type kidney donors. Some DEGs including FTCD and TASPN7 could be novel biomarkers not only for timely diagnosis, but also for early precise genetic intervention at donor preservation, implantation and post-transplantation, in particular to effectively improve the quality and survival of DD.

Kidney transplantation is the optimal treatment for end-stage renal disease, but it is still severely limited by a lack of suitable organ donors. Kidneys from donation after circulatory death (DCD) donors have been used to increase transplant rates, but these organs are susceptible to cold ischemic injury in the storage period before transplantation, the clinical consequence of which is high rates of delayed graft function (DGF). Normothermic machine perfusion (NMP) is an emerging technique that circulates a warmed, oxygenated red-cell-based perfusate through the kidney to maintain near-physiological conditions. We conducted a randomized controlled trial to compare the outcome of DCD kidney transplants after conventional static cold storage (SCS) alone or SCS plus 1-h NMP. A total of 338 kidneys were randomly allocated to SCS ( n  = 168) or NMP ( n  = 170), and 277 kidneys were included in the final intention-to-treat analysis. The primary endpoint was DGF, defined as the requirement for dialysis in the first 7 d after transplant. The rate of DGF was 82 of 135 (60.7%) in NMP kidneys versus 83 of 142 (58.5%) in SCS kidneys (adjusted odds ratio (95% confidence interval) 1.13 (0.69–1.84); P  = 0.624). NMP was not associated with any increase in transplant thrombosis, infectious complications or any other adverse events. A 1-h period of NMP at the end of SCS did not reduce the rate of DGF in DCD kidneys. NMP was demonstrated to be feasible, safe and suitable for clinical application. Trial registration number: ISRCTN15821205 . In an open-label, randomized controlled trial, normothermic machine perfusion of kidneys from donation after circulatory death was found to be feasible and safe but did not reduce the rate of delayed graft function compared to static cold storage.

Renal ischemia–reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3β at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

Hypothermic and normothermic machine perfusion in kidney transplantation are purported to exert a beneficial effect on post-transplant outcomes compared to the traditionally used method of static cold storage. Kidney perfusion techniques provide a window for organ reconditioning and quality assessment. However, how best to deliver these preservation methods or improve organ quality has not yet been conclusively defined. This review summarises the promising advances in machine perfusion science in recent years, which have the potential to further improve early graft function and prolong graft survival.