Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation

Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation Kazuhiko Yamada, Yu Hisadome, Daniel Eisenson, WeiLi Chen, Alex Schulick, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5220149/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Communications Medicine → Version 1 posted You are reading this latest preprint version Abstract Xenotransplantation (XTx) is an increasingly realistic solution to the organ shortage. Clinical XTx may require off-site procurement in a designated pathogen free (DPF) facility necessitating a period of cold ischemic time during transportation. This study evaluates the impact of different kidney preservation strategies on early graft function in pig-to-baboon XTx in a series of eight cases of pig-to-baboon xenotransplantation performed after five hours of cold ischemic time and compares these results to six cases of pig-to-baboon xenotransplantation performed with minimal ischemic time. Our data indicates that porcine kidneys appear to be particularly sensitive to IRI after cold preservation, especially across xenogeneic barriers, and routine static cold storage leads to hyperacute graft loss even in recipients with low levels of preformed antibodies. Hypothermic machine perfusion minimizes IRI and may prevent early xenograft loss. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Xenotransplantation (XTx) is an increasingly realistic solution to the organ shortage. Researchers have been making progress in XTx research in the pig-to-nonhuman primate (NHP) model over the past 20 years with recent reports showing long-term survival of life-supporting porcine kidney 1 – 3 and heart 4 , 5 xenografts using organs from genetically modified source pigs. These experiments paved the way for early studies in pig-to-human xenotransplantation. The Food and Drug Administration (FDA) granted case-by-case approval of xenografts for “compassionate use” in patients who were unlikely to receive conventional allografts, including two cases of pig-to-human heart transplantation 6 and two cases of pig-to-human kidney transplantation 7 . Porcine xenograft procurement occurred under variable conditions in each of these isolated clinical cases; however, xenograft procurement in future clinical trials in xenotransplantation will be more closely regulated, which will have important implications for procurement and xenograft ischemic time. Xenografts used in imminent clinical trials will be regulated as investigational new animal drugs (INADs). Source pigs must be maintained in “designated pathogen-free” (DPF) facilities from conception to procurement to avoid the potential transmission of swine-derived organisms to human patients 8 . This designation and the associated strict control of infectious pathogens will likely limit where procurements can occur. It is not feasible for each hospital offering solid organ transplants to have their own DPF pig facility for clinical XTx; therefore, clinical XTx will likely require off-site procurement in centralized DPF pig facilities followed by shipment of those porcine organs. While cold ischemic time (CIT) is well-tolerated in conventional allogeneic kidney transplantation, the impact of clinically relevant CIT on pig kidney grafts has not been investigated in xenogeneic kidney transplantation. Indeed, few studies have reported long-term xenograft survival with significant (> 3 hours) CIT, and none have reported > 1 year survival with > 3 hours CIT. Successful xenotransplantation requires overcoming both innate and adaptive immunological barriers 9 . Ischemia-reperfusion injury (IRI) is known to potentiate both innate and adaptive immunity 10 , and may thus raise the immunologic hurdles to effective xenotransplanation; while CIT is well-tolerated in conventional allogeneic kidney transplantation, IRI across xenogeneic barriers may lead to greater damage as species incompatibilities between porcine and primate inflammatory and coagulation systems may exacerbate underlying injury 11 . Moreover, while static cold storage (SCS) remains the gold standard for organ preservation in conventional allogeneic kidney transplantation, additional preservation modalities may be needed to minimize IRI across xenogeneic barriers. Hypothermic machine perfusion (HMP) is becoming widely used in clinical settings 12 with increasing evidence of improved graft outcomes with prolonged CIT as compared to SCS 13 ; while the benefit of HMP was first demonstrated with reduced incidence of delayed graft function (DGF) among higher risk kidneys 14 , subsequent studies have found reduced DGF all donor subgroups 15 , 16 . The mechanisms responsible for observed superior outcomes with HMP in conventional allogeneic transplantation are incompletely understood 17 , but mechanistic studies suggest that HMP may mitigate ischemia-reperfusion injury through reduction of hypoxia during preservation 18 , 19 and decreased inflammatory cell death 20 as well as improved endothelial cell function 21 after reperfusion. Existing data in transplantation across xenogeneic barriers indicate that mode of preservation may play a key role: Langin et al demonstrated that non-ischemic preservation with continuous perfusion was essential for successful orthotopic cardiac xenotransplantation 4 , 5 . Additionally, recent multi-omic data from two pig-to-human decedent cardiac xenotransplantation at New York University revealed evidence of ischemia reperfusion injury in one of two xenografts (Keating, TTS, 2024). However, no studies to date have investigated the impact of HMP on graft outcomes in kidney xenotransplantation 11 . We recently reported consistent survival of life-supporting kidney xenografts in pig-to-baboon model with clinically relevant ischemic time preserved with HMP as part of an IND-enabling study for the FDA 2 . Here we provide scientific justification for a nuanced preservation strategy in xenotransplantation, as used in that study. In this study, we investigated the impact of prolonged cold ischemic time on the outcomes of pig-to-baboon kidney transplantation, we compared the efficacy of different preservation strategies (SCS and HMP) on early graft function, and we explored the mechanistic underpinnings of the xenograft response to IRI. This study is the first to examine the effects of SCS versus HMP on xenografts under conditions of prolonged CIT. By addressing these objectives, we fill the existing knowledge gaps and contribute to the development of more effective organ preservation protocols for clinical xenotransplantation trials. Results Clinical impact of IRI across xenogeneic barriers The outcomes of the xenotransplantation cases with and without 5 hours of CIT are summarized in Table 1. We performed 8 cases with 5 hours of CIT: 4 cases with SCS and 4 cases with HMP. All grafts preserved using SCS lost function immediately after transplant. By visual inspection, these grafts looked healthy immediately following reperfusion but subsequently exhibited mottling that progressed to diffuse discoloration and anuria within 90 minutes post-reperfusion (Fig. 1 A). In contrast, all grafts preserved using HMP were reperfused without clinically apparent changes (Fig. 1 B) and survived for more than 14 days post-transplantation. All cases that did not undergo the 5-hour CIT accepted the kidney graft as expected, demonstrating similar outcomes to those preserved using HMP except for the slight difference in initial peak of serum creatinine levels (which were higher in the preservation arm) (Table 1). The outcomes of allotransplantation cases with 5 hours of CIT preserved using either HMP or SCS are detailed in Table 2. Unlike the xenotransplantation cases, no hyperacute graft loss was observed in allotransplantation cases preserved with SCS. These grafts did experience IRI with creatinine levels increasing up to 4.7 mg/dL after Tx, while HMP-preserved allografts maintained stable early graft function (Table 1, 2), but the differences were not statistically significant. Histopathological evaluation of the impact of IRI across xenogeneic barriers On histologic examination, SCS-preserved kidneys (hematoxylin and eosin (H&E) staining) showed extensive hemorrhage and hemostasis one hour after reperfusion (representative staining, Fig. 2 A), whereas HMP-preserved kidneys did not show significant pathological changes one hour after reperfusion (Fig. 2 B). There was a notable deposition of C3 in SCS-preserved kidneys, indicating complement activation, which was not observed in HMP-preserved kidneys (Fig. 3 ). SCS kidneys at two hours post-Tx had significantly higher infiltration of myeloperoxidase (MPO) positive cells compared with HMP-preserved kidneys (Fig. 4 ). There was no significant difference in CD56-positive cells (NK cells) and CD68-positive cells (macrophages) between SCS- and HMP-preserved kidneys in biopsies obtained one hour after transplantation. However, more CD68-positive cells and MPO-positive cells were seen at later time point in HMP-kidney on POD24 (Fig. 4 ). GeoMx Digital Spatial Profiling (DSP) Analysis GeoMx whole-transcriptome DSP analysis provided comparative insights between HMP and SCS preservation methods in the setting of xenotransplantation and standardized CIT in single-gene knockout (GalTKO) genetically modified pigs. A representative slide was prepared from surgical tissue biopsies obtained two hours after reperfusion in either preservation condition (n = 1 for HMP and SCS). Regions of interest (ROIs) were selected to stratify renal architecture into glomerular (n = 6 for SCS, n = 3 for HMP) and tubulointerstitial (n = 6 for SCS, and n = 3 for HMP) spatial compartments distinguished by immunofluorescent markers Syto 13 (nuclei) and pan-cytokeratin (epithelial cells). First, Unsupervised clustering of ROIs identified 2 distinct local communities that were primarily differentiated by renal compartment as opposed to preservation strategy. However, sub-clustering by preservation within each community is somewhat apparent, lending internal validity to the GeoMx assay and ROI selection process. (Supplementary Fig. 1). We then screened for differentially expressed genes across the HMP-SCS axis using cutoffs for fold change (abs(log 2 FC) > 2) and adjusted P value ( P adj < 0.01) (Fig. 5 ). Six genes were enriched in SCS ROIs, all of which were found to have relevance to ischemia and IRI. Specifically, FOS and JUNB are downstream effectors in non-canonical Wnt signaling pathways with crosstalk between the TGF-b and NF-Kb pathways leading to inflammation and apoptosis and have been implicated in renal IRI 22 . EGR1 is a transcription factor regulated by many external cell stress signals and is involved with inflammation and apoptosis signaling while NR4A1 and NR4A2 are nuclear receptors also involved in apoptotic pathways and implicated in IRI 23 ,24 . Only ATF3 has been shown to have a protective effect in renal IRI in an in-vitro model inducing oxidative stress 25 . On the other hand, HMP ROIs were highly enriched for genes protective against oxidative stress and hypoxia ( MDH1 , KCNJ1 ), wound healing ( B2M ), and mitochondrial as well as cellular metabolism and homeostasis ( UMOD , COX genes, ATP5 genes, CS ) 26–31 . Of these 17 genes, only one ( CD74 ) has been suggested to correlate with worse IRI-associated AKI 32 . Using gene sets defined by the Banff Human Organ Transplant (B-HOT) gene panel, we mapped DSP ROIs to phenotypic pathways specifically relevant to molecular studies in transplant immunology 33 . In general, IRI-associated pathways were enriched in SCS ROIs, including pro-inflammatory, cell death, and innate immune pathways such as cytokine signaling, inflammasomes, MAPK, Th17-mediated biology, TNF family signaling, and apoptosis & cell cycle regulation. On the other hand, HMP ROIs were enriched for pathways related to cellular metabolism and homeostasis, including cell-ECM interactions, Metabolism, and MHC Class I presentation (Fig. 6 and Supplementary Table 2). Those genes which were included in the panel were required to pass data quality control measures prior to inclusion in the analysis and are specifically detailed in Supplementary Table 3. Discussion This study is the first to demonstrate that clinically relevant CIT with routine SCS poses a significant risk for hyperacute graft loss in a pig-to-NHP kidney xenotransplantation model. Clinically relevant CIT (5 hours) with SCS uniformly led to hyperacute graft loss after transplantation across xenogeneic barriers; in contrast, kidney xenografts transplanted without CIT in immunologically matched donor-recipient pairs did not result in hyperacute graft loss, and porcine kidney allografts transplanted with clinically relevant CIT and preserved with SCS did not result in hyperacute graft loss. Importantly, we observed significant differences in the outcomes of kidney xenotransplantation based on the preservation method employed during CIT. All kidneys preserved using SCS were lost hyperacutely within 90 minutes, with clinically apparent vasoconstriction visible minutes after reperfusion and histologic evidence of severe endothelial injury on biopsies obtained one hour after reperfusion. Conversely, all kidneys preserved using HMP reperfused uneventfully with minimal histologic changes on one-hour biopsies and, after an initial elevation in creatinine corresponding to IRI associated with CIT, functioned for more than 14 days. While the underlying mechanisms for the observed differences between SCS and HMP remain unclear, spatial transcriptomic analyses suggest that HMP may confer benefit through modulation of initial ischemic injury as well as mitigation of reperfusion injury and subsequent inflammation. This study presents the first report of transcriptomic analyses with spatial resolution in an in vivo model of ischemia-reperfusion in kidney xenotransplantation. It is important to acknowledge some limitations of this analysis. First and foremost, there is no spatially resolved, species-specific transcriptomic technology widely available for porcine xenotransplant in an NHP background. However, GeoMx DSP probes were cross-referenced with the porcine genome and genetic similarity was found to be at least 85% across species. Furthermore, the GeoMx DSP analysis was limited by the statistical approach for hypothesis testing; Given the small sample size, it is difficult to account for the nested hierarchical nature of the experimental observations (the ROIs). Future, larger cohorts are required to confirm findings. Despite these limitations, transcriptomic analyses of two representative samples revealed that HMP-preserved kidney tissue was highly enriched for genes and pathways protective against oxidative stress and hypoxia, as well as genes/pathways promoting normal cellular and mitochondrial metabolism. The SCS tissue was enriched for genes and pathways associated with inflammation, cell death, and innate immunity. Altogether, molecular analysis substantiates clinical and histologic evidence in this study suggesting that HMP may minimize the initial hypoxic injury associated with CIT. A similar pattern has also been observed with HMP in conventional allogeneic transplantation, where studies have demonstrated reduced expression of hypoxia-related genes in HMP-stored donor organs 34 . Perhaps more importantly, the inflammatory response corresponding to this initial ischemic injury appears markedly different between SCS- and HMP-preserved kidney tissue. SCS-preserved kidneys were found to have significant increases in MPO-positive neutrophil infiltration on immunohistochemistry as-well-as enrichment of genes associated with innate immune activation and inflammation on transcriptomic analyses. While the trend is similar to preservation studies in conventional allogeneic transplantation 35 , differences between SCS and HMP preservation strategies appear more pronounced in xenotransplantation compared with conventional allogeneic transplantation. Indeed, innate immune activation may progress more rapidly across xenogeneic barriers, where species incompatibilities impair normal anti-inflammatory and anti-coagulant regulatory processes. For example, immunofluorescence of SCS-preserved kidneys demonstrated diffuse complement deposition which was not observed in the HMP-preserved kidneys despite identical levels of preformed antibodies (same recipient); once innate immunity is activated across xenogeneic barriers, it is likely more challenging to turn off without normal species-congruent inhibitory proteins. Targeted genetic modification of source pigs may minimize IRI-induced inflammation associated with species incompatibilities 11 . We recently demonstrated consistent and durable survival of life-supporting kidney xenografts in consecutive cases of pig-to-baboon transplantation using 10GE source pigs with transgenic expression of human anti-inflammatory (HO1), anti-coagulant (TBM, EPCR), and complement regulatory proteins (CD46, CD55) 2 . These xenografts incurred similar CIT to those in this study and were preserved with HMP, avoiding hyperacute graft loss. Still, IRI impacted each animal’s postoperative course and may have contributed to ultimate xenograft failure and preterminal euthanasia. Notably, porcine kidneys used in both decedent and clinical pig-to-human cases were preserved with SCS, incurred similar CIT, and did not undergo hyperacute graft loss. This may be a function of xenograft size – our experiments used small (< 80 grams) recipient size-matched porcine kidneys from juvenile pigs (< 20 kg), while the cases at NYU used larger kidneys from adult pigs. Juvenile pig kidneys may be more sensitive to acute injury 36 , which may be related to immature renal structures 37 . Similarly, kidneys from deceased pediatric donors less than five years old have a higher rate of vascular complications 38 in the early postoperative period. This may also be a function of the known immunologic 3 and physiologic differences in xenograft recipients (NHPs in our study versus humans). Although we were not able to salvage xenografts preserved with SCS in our transplantation studies, we did observe relative transient clinical improvement with increased blood pressure. While systolic blood pressures of > 130 mmHg are supraphysiologic in our pediatric NHP recipients, these pressures are normal in adult patients. Larger kidneys and larger recipients may result in better outcomes after CIT in xenotransplantation regardless of preservation modality. Still, available data indicates that xenograft function after SCS preservation in long-term pig-to-human decedent and clinical kidney xenotransplantation cases has not been normal. Early antibody mediated rejection was recently reported in the 61-day decedent (Keating, TTS, 2024). While there was no DGF reported in this case, IRI is known to amplify the humoral response to heterologous antigens 39 , and may have contributed to early and unexpected ABMR in this case. Our findings suggest that SCS preservation may increase the risk of IRI and acute graft loss after transplantation across xenogeneic barriers. Given these outcomes, HMP may be a safer preservation strategy for early clinical trials in xenotransplantation. Materials and methods We performed 23 cases of large animal kidney transplantation (19 cases of pig-to-baboon kidney xenotransplantation and 4 cases of pig-to-pig kidney allogeneic transplantation) at Columbia University (New York, NY, USA), Johns Hopkins University (Baltimore, MD, USA), and Kagoshima University (Kagoshima, Japan). All animal work was conducted in accordance with NIH and USDA guidelines and with approval from the Institutional Animal Care and Use Committee (IACUC). Animal husbandry Animal husbandry was conducted in accordance with each facilities’ SOPs. Swine were socially housed in individual pens on tenderfoot flooring in spaces with at least one and up to seven additional pigs. Swine were surveilled for pathogens of potential concern to NHP recipients. Baboons were housed both singly and in pairs in stainless-steel cages with access to an automatic watering valve. Temperature was maintained between 24 and 29°C, humidity maintained between 30–70%, and lighting was provided on a light/dark cycle for approximately 12 hours each day. Baboons were fed certified chow with LabDiet 5038—Monkey Diet, with enrichment in the form of fruits and vegetables provided once daily. Water was available ad libitum throughout the studies for both swine and baboons. MHC-matched pigs CLAWN-miniature swine, aged 9–12 months, were obtained from the Kagoshima Miniature Swine Research Center (Isa, Japan). CLAWN-miniature swine are genetically typed, MHC-inbred miniature swine. In this study, fully MHC-matched pairs of swine were used as donors and recipients. Swine were maintained, treated, and euthanized according to guidelines established by the Animal Welfare Act and in compliance with protocols approved by Kagoshima University Institutional Animal Care and Use Committees. Genetically modified source pigs Pig donors were provided by either Accuro Farms, Inc. (Southbridge, MA, USA) or National Swine Resource and Research Center, University of Missouri (Columbia, MO, USA). All donors were porcine cytomegalovirus (PCMV) negative. Genetic modification of donor pigs includes GalTKO or GalTKO with hCD55 Tg. Euthanasia was performed prior to organ procurement. Swine were maintained, treated, and euthanized according to guidelines established by the Animal Welfare Act and the NIH for the housing and care of laboratory animals and in compliance with protocols approved by Columbia University, Johns Hopkins University, and Kagoshima University Institutional Animal Care and Use Committees. Baboons Baboons (Papio spp.) were purchased as recipients of pig-to-baboon KXTx from the Mannheimer Foundation (Papio hamadryas, Homestead, FL, USA) or from the Michale E. Keeling Center for Comparative Medicine and Research, MD Anderson Cancer Center (Papio anubis, Bastrop, TX, USA). The baboons ranged from 2–4 years old and weighed between 8 to 12.5 kg. All baboons were maintained, treated, and euthanized at the study endpoint according to guidelines established by the Animal Welfare Act and the NIH for the housing and care of laboratory animals and in compliance with protocols approved by the Animal Care and Use Committee at Columbia University and at Johns Hopkins University. Pre-transplant screening All xenotransplant recipients were evaluated for anti-pig antibodies by complement-dependent cytotoxicity (CDC) using peripheral blood mononuclear cells (PBMCs) derived from GalTKO source pigs. The presence of donor-specific antibodies was also assessed by IgG and IgM antibody binding of baboon sera to GalTKO-derived PBMCs according to screening methodology developed by The Yamada Lab at Johns Hopkins University (Hisadome, Transplantation, 2024). Recipients are selected for complement-dependent cytotoxicity (CDC) < 20% at 1:4 dilution and serum IgG binding on antibody flow cytometry less than the pretransplant value of a known rejector recipient. Experimental groups • Pig-to-baboon xenogeneic kidney transplantation We performed 19 cases of pig-to-baboon XKTx. 8 cases were performed with 5-hours cold ischemic time (Xeno preservation group), which is consistent with likely duration of coastal transportation time in the United States. The kidney grafts were preserved for 5 hours with either (1) static cold storage (SCS) (n = 4) or (2) hypothermic machine perfusion (HMP) using the LifePort Kidney Transporter (Organ Recovery Systems; Itasca, IL, USA) (n = 4) (detail described in the section “Kidney graft preservation procedures”). The other cases (n = 11) were performed with minimal CIT less than 20 min (Xeno control group). Recipients’ pretransplant anti-pig natural antibody levels were evaluated using both complement-dependent cytotoxicity (CDC) and antibody binding assay using flow cytometry (AbFCM)(*Ref), and the risk for XKTx were stratified as previously reported (*Ref). We included either low Nab (low risk) or high Nab (exclusion) recipient into the study to assess effect of graft preservation on preformed Nab. • Pig-to-pig allogeneic kidney transplantation 4 cases of pig allo KTx were performed with 5-hours CIT (Allo preservation group). Pig donor and recipient were MHC-matched. 5-hours graft preservation was performed in the same manner as in Xeno preservation group: 2 cases with SCS and 2 cases with HMP. Surgical procedures A central venous catheter was placed in the recipient prior to Tx and used for drug administration and blood sampling throughout experiment. The kidney graft was transplanted as previously reported 40 with bilateral native nephrectomies, splenectomy (in the experimental group) and bilateral oophorectomy (for female baboons). Kidney graft preservation procedures Kidney grafts procured from the donor swine were immediately perfused with 50ml of the University of Wisconsin (UW) solution at 4C. For SCS arm, the grafts were immersed into 500ml of UW solution and stored at 4C for 5 hours. For the HMP arm, a disposable cannula was attached to renal artery and machine perfusion was initiated using the LifePort Kidney Transporter with an infusion pressure of 30mmHg. 1000ml of KPS-1 (Organ Recovery Systems, equivalent to UW solution) was used for the perfusion circuit. The grafts were continuously perfused at 2-8C for 5 hours prior to Tx. In the Xeno preservation group, one of the two kidneys from a donor pig was preserved with SCS and the other with HMP. Both grafts were transplanted into the same recipient baboon in order to compare differences between two preservation strategies while minimizing differences in other experimental conditions. After vascular anastomoses were completed, both kidneys were reperfused simultaneously. Immunosuppression All baboons received induction therapy with rabbit anti-thymocyte globulin (Thymoglobulin) and anti-CD20 monoclonal antibodies (Rituximab) prior to Tx. Maintenance therapy included anti-CD40 or anti-CD40L monoclonal antibodies, mycophenolate mofetil (MMF), and cytotoxic T lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig). Anti-C5 monoclonal antibody (Tesidolumab) was given concomitantly for the recipient who received GalTKO + hCD55 kidney. All recipients in the pig-to-pig allogeneic kidney transplantation group were treated with tacrolimus for 14 days, starting on the day of transplantation, with blood levels maintained at 15 to 25 ng/mL. Posttransplant outcomes Kidney graft function was assessed by urine volume and laboratory tests including serum creatinine (mg/dL) and blood urea nitrogen (BUN, mg/dL). We defined graft dysfunction based on macroscopic and/or microscopic/histologic findings of the kidney grafts, urine volume ( 8.0mg/dL). The primary endpoint was graft survival within 14 days post Tx. Histopathological analysis Graft kidney biopsies were obtained after procurement (after flushing with preservation solution, prior to transplant) and at 2 hours after reperfusion. Kidney biopsy specimens were either a) frozen or b) fixed in 10% formaldehyde and embedded in paraffin. Frozen samples were used for immunofluorescence (IF). Anti-human IgG, IgM, and C3 (DAKO, Carpentaria, CA) all conjugated to FITC; C5b (DAKO, Carpentaria, CA) and C4d (QUIDEL, San Diego, CA) were unconjugated ab detected by Alexa Fluor® 488 goat anti-mouse secondary antibody (Abcam, Cambridge, MA) to assess antibody binding and complement activation in the graft. Paraffin-embedded tissues were sectioned, stained using hematoxylin and eosin (H&E) and Periodic acid-Schiff, and examined by an experienced pathologist. Immunohistochemistry staining was performed to assess cell infiltration into grafts, including CD56, CD68 (sc20060 C1662, Santa Cruz Biotechnology, Dallas, TX) and myeloperoxidase (ab188211, Abcam, Cambridge, MA). Immunofluorescence staining was performed on frozen specimens for C3c (DAKO complement clone C3c FITC F0201, Carpentaria, CA) and C5b9 (DAKO complement clone C5b9 aE11 M0777, Carpentaria, CA) complement deposition using anti-mouse FITC secondary antibody (A2723, Invitrogen, Waltham, MA). GeoMx DSP data acquisition, processing and analysis Published experimental methods for DSP (Merritt et al., Nat Biotech. , 2020) were followed using standardized protocols on an in-house NanoString GeoMx instrument. 3 formalin-fixed, paraffin-embedded sections were processed for DSP with the human whole-transcriptome atlas (consisting of 18,676 UV-photocleavable barcode-conjugated RNA in situ hybridization probes and 139 negative control probes). 2 morphology markers were used to outline renal morphology prior to ROI selection: 400 nanomolar SYTO 13 (Invitrogen, Waltham, MA) and 1:100 anti-PanCK-Texas Red 594 (Clone: AE1/AE3; Novus Biologicals, Centennial, CO). Spatially indexed barcodes were collected from a total of 30 ROIs. Libraries were prepared according to manufacturer’s instructions and subsequently sequenced using a NovaSeq 5000 system. FASTQ files were converted to digital count conversion (DCC) files using NanoString software (GeoMx NGS Pipeline). DCC files were uploaded into R (version 4.4.0) and consolidated as a NanoStringGeoMxSet S4 object. Gene targets were removed from the object if they consistently fell below the limit of quantitation (LOQ; defined by the background signal for each ROI). The remaining dataset of 5,577 genes was quantile-normalized 41,42 . Differential expression analysis was performed using the DESeq2 R package 43 and pathway signatures were defined by the Banff Human Organ Transplant Gene Panel 33 . Pathway enrichment scores were obtained using single-sample gene set enrichment analysis (ssGSEA) 44 from the GSVA R package 45 , which were then scaled as z-scores. Reported P values were corrected for multiple testing using the Benjamini-Hochberg method. Statistical analysis Statistical analysis was performed using the Student’s t-test. All tests were 2-sided, and P < 0.05 was considered significant. All statistical analyses were performed using Prism 9 (GraphPad Software). Declarations Acknowledgements The authors would like to acknowledge David Sachs, Megan Sykes, Kristin Whitworth, Randall Prather, and Kevin Wells for providing genetically modified pigs used in this study. Disclosure This research was partially supported by NIH grant P01AI045897. K. Yamada reports institutional grant support from United Therapeutics Corporation outside the submitted work. E. Shenderov reports institutional grant support from AstraZeneca and institutional clinical trial support and non-financial reagent support from Macrogenics outside the submitted work. Data availability statement All data supporting this study are available within the article and its related supplementary information file. All raw data for graphs in this study and all microscopic images are available from the corresponding author on request. References Adams, A. B. et al. Anti-C5 Antibody Tesidolumab Reduces Early Antibody-mediated Rejection and Prolongs Survival in Renal Xenotransplantation. Ann Surg 274 , 473-480, doi:10.1097/SLA.0000000000004996 (2021). Eisenson, D. et al. Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10GE source pigs. Nat Commun 15 , 3361, doi:10.1038/s41467-024-47679-6 (2024). Anand, R. P. et al. Design and testing of a humanized porcine donor for xenotransplantation. Nature 622 , 393-401, doi:10.1038/s41586-023-06594-4 (2023). Langin, M. et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature 564 , 430-433, doi:10.1038/s41586-018-0765-z (2018). Mohiuddin, M. M. et al. Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months. Xenotransplantation 29 , e12744, doi:10.1111/xen.12744 (2022). Griffith, B. P. et al. Genetically Modified Porcine-to-Human Cardiac Xenotransplantation. N Engl J Med 387 , 35-44, doi:10.1056/NEJMoa2201422 (2022). Rabin, B. Noordergraaf, J. et al. Pathogen elimination and prevention within a regulated, Designated Pathogen Free, closed pig herd for long-term breeding and production of xenotransplantation materials. Xenotransplantation 25 , e12428, doi:10.1111/xen.12428 (2018). Eisenson, D. L., Hisadome, Y. & Yamada, K. Progress in Xenotransplantation: Immunologic Barriers, Advances in Gene Editing, and Successful Tolerance Induction Strategies in Pig-To-Primate Transplantation. Front Immunol 13 , 899657, doi:10.3389/fimmu.2022.899657 (2022). Tang, Q., Dong, C. & Sun, Q. Immune response associated with ischemia and reperfusion injury during organ transplantation. Inflamm Res 71 , 1463-1476, doi:10.1007/s00011-022-01651-6 (2022). Patel, P. M. et al. Minimizing Ischemia Reperfusion Injury in Xenotransplantation. Front Immunol 12 , 681504, doi:10.3389/fimmu.2021.681504 (2021). Malinoski, D. et al. Hypothermia or Machine Perfusion in Kidney Donors. N Engl J Med 388 , 418-426, doi:10.1056/NEJMoa2118265 (2023). Aubert, O. et al. Long term outcomes of transplantation using kidneys from expanded criteria donors: prospective, population based cohort study. BMJ 351 , h3557, doi:10.1136/bmj.h3557 (2015). Moers, C. et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 360 , 7-19, doi:10.1056/NEJMoa0802289 (2009). Callaghan, C. J. et al. Outcomes of transplantation of livers from donation after circulatory death donors in the UK: a cohort study. BMJ Open 3 , e003287, doi:10.1136/bmjopen-2013-003287 (2013). Tingle, S. J. et al. Hypothermic machine perfusion is superior to static cold storage in deceased donor kidney transplantation: A meta-analysis. Clin Transplant 34 , e13814, doi:10.1111/ctr.13814 (2020). Knijff, L. W. D., van Kooten, C. & Ploeg, R. J. The Effect of Hypothermic Machine Perfusion to Ameliorate Ischemia-Reperfusion Injury in Donor Organs. Front Immunol 13 , 848352, doi:10.3389/fimmu.2022.848352 (2022). Zhao, D. F., Dong, Q. & Zhang, T. Effects of Static Cold Storage and Hypothermic Machine Perfusion on Oxidative Stress Factors, Adhesion Molecules, and Zinc Finger Transcription Factor Proteins Before and After Liver Transplantation. Ann Transplant 22 , 96-100, doi:10.12659/aot.901897 (2017). Darius, T. et al. Influence of Different Partial Pressures of Oxygen During Continuous Hypothermic Machine Perfusion in a Pig Kidney Ischemia-reperfusion Autotransplant Model. Transplantation 104 , 731-743, doi:10.1097/TP.0000000000003051 (2020). Guarrera, J. V. et al. Hypothermic machine preservation facilitates successful transplantation of "orphan" extended criteria donor livers. Am J Transplant 15 , 161-169, doi:10.1111/ajt.12958 (2015). Burlage, L. C. et al. Oxygenated hypothermic machine perfusion after static cold storage improves endothelial function of extended criteria donor livers. HPB (Oxford) 19 , 538-546, doi:10.1016/j.hpb.2017.02.439 (2017). Zhang, M. et al. Ischemia-reperfusion injury: molecular mechanisms and therapeutic targets. Signal Transduction and Targeted Therapy 9 , doi:ARTN 12 10.1038/s41392-023-01688-x (2024). Jeong, K., Je, J., Dusabimana, T., Kim, H. & Park, S. W. Early Growth Response 1 Contributes to Renal IR Injury by Inducing Proximal Tubular Cell Apoptosis. Int J Mol Sci 24 , doi:10.3390/ijms241814295 (2023). Liu, J. et al. Molecular characterization of the transition from acute to chronic kidney injury following ischemia/reperfusion. JCI Insight 2 , doi:10.1172/jci.insight.94716 (2017). Yoshida, T. et al. ATF3 protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 19 , 217-224, doi:10.1681/ASN.2005111155 (2008). Burton, J. B. et al. Substantial downregulation of mitochondrial and peroxisomal proteins during acute kidney injury revealed by data-independent acquisition proteomics. Proteomics 24 , e2300162, doi:10.1002/pmic.202300162 (2024). Jang, H. S. et al. Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury. Kidney International 97 , 327-339, doi:10.1016/j.kint.2019.08.019 (2020). Gimelreich, D. et al. Regulation of ROMK and channel-inducing factor (CHIF) in acute renal failure due to ischemic reperfusion injury. Kidney Int 59 , 1812-1820, doi:10.1046/j.1523-1755.2001.0590051812.x (2001). Li, C. et al. Gp78 deficiency in hepatocytes alleviates hepatic ischemia-reperfusion injury via suppressing ACSL4-mediated ferroptosis. Cell Death Dis 14 , 810, doi:10.1038/s41419-023-06294-x (2023). El-Achkar, T. M. et al. Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression. Am J Physiol Renal Physiol 295 , F534-544, doi:10.1152/ajprenal.00083.2008 (2008). Karagiannidis, A. G., Theodorakopoulou, M. P., Pella, E., Sarafidis, P. A. & Ortiz, A. Uromodulin biology. Nephrol Dial Transplant 39 , 1073-1087, doi:10.1093/ndt/gfae008 (2024). Li, J. H. et al. Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion. Journal of Cellular and Molecular Medicine 23 , 3867-3877, doi:10.1111/jcmm.14234 (2019). Mengel, M. et al. Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation. Am J Transplant 20 , 2305-2317, doi:10.1111/ajt.16059 (2020). Krezdorn, N. et al. Reduced Hypoxia-Related Genes in Porcine Limbs in Ex Vivo Hypothermic Perfusion Versus Cold Storage. J Surg Res 232 , 137-145, doi:10.1016/j.jss.2018.05.067 (2018). Nieuwenhuijs-Moeke, G. J. et al. Ischemia and Reperfusion Injury in Kidney Transplantation: Relevant Mechanisms in Injury and Repair. J Clin Med 9 , doi:10.3390/jcm9010253 (2020). Robbins, M. E., Campling, D., Rezvani, M., Golding, S. J. & Hopewell, J. W. The effect of age and the proportion of renal tissue irradiated on the apparent radiosensitivity of the pig kidney. Int J Radiat Biol 56 , 99-106, doi:10.1080/09553008914551221 (1989). Iwase, H., Klein, E. C. & Cooper, D. K. Physiologic Aspects of Pig Kidney Transplantation in Nonhuman Primates. Comp Med 68 , 332-340, doi:10.30802/AALAS-CM-17-000117 (2018). Singh, A., Stablein, D. & Tejani, A. Risk factors for vascular thrombosis in pediatric renal transplantation: a special report of the North American Pediatric Renal Transplant Cooperative Study. Transplantation 63 , 1263-1267, doi:10.1097/00007890-199705150-00012 (1997). Fuquay, R. et al. Renal ischemia-reperfusion injury amplifies the humoral immune response. J Am Soc Nephrol 24 , 1063-1072, doi:10.1681/ASN.2012060560 (2013). Yamada, K. et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 11 , 32-34, doi:10.1038/nm1172 (2005). Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19 , 185-193, doi:10.1093/bioinformatics/19.2.185 (2003). van Hijfte, L. et al. Alternative normalization and analysis pipeline to address systematic bias in NanoString GeoMx Digital Spatial Profiling data. iScience 26 , 105760, doi:10.1016/j.isci.2022.105760 (2023). Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15 , 550, doi:10.1186/s13059-014-0550-8 (2014). Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462 , 108-112, doi:10.1038/nature08460 (2009). Hanzelmann, S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics 14 , 7, doi:10.1186/1471-2105-14-7 (2013). Tables Tables are available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.pdf Supplementary Figure 1. Unsupervised clustering of DSP Regions of Interest. (a) Scanned whole-slide image of stained HMP/SCS surgical biopsy sections from the GeoMx instrument. (b) Representative images of Glomerular and Tubulointerstitial regions of interest. (c) Uniform manifold approximation and projection (UMAP) embedding of HMP and SCS regions of interest, further stratified by renal compartment. Supplementary Table 1. Single Gene Analysis Summary of Differentially Enriched Genes Supplementary Table 2. Gene Set Enrichment Hypothesis Testing Summary Supplementary Table 3. B-HOT Panel Genes Used for Gene Set Enrichment Analysis Table1.pdf Table 1. Clinical outcomes of xenotransplantation cases with/without 5 hours of cold ischemic time (CIT) Table2.pdf Table 2. Clinical outcomes of allotransplantation cases with 5 hours of CIT Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Communications Medicine → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5220149","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":371307690,"identity":"686d7a73-32f7-49aa-9c5e-d7e11b4e91d5","order_by":0,"name":"Kazuhiko Yamada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACCTDJxiDHz3CGgSGBgYGxgVgtxpINpGpJNDjAA2YS1iI5I/nphh9ldgnGB88e3fCAwUZ2wwECWqQl0sxu9pxLzjM7cC7tRgJDmjFBLXISCWY3eNuYi80OnDEDajmcSISW9G83/7bVJ25uAGv5T1iLtESO2W3eNqDhDGAtBwhrkex5U3Zb5txxYwmwwwySjWcS0iJxPH3bzTdl1XL8M86Y3fxRYSfbR0gLg0ACTDNIqQEh5SDADzOUv4EY5aNgFIyCUTASAQCbk031HPOqIAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0510-5843","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kazuhiko","middleName":"","lastName":"Yamada","suffix":""},{"id":371307691,"identity":"7c5ed154-3798-4517-ace3-261cfa101f97","order_by":1,"name":"Yu Hisadome","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Hisadome","suffix":""},{"id":371307692,"identity":"6e09c3fa-2b9c-430a-9b7f-fd45aa41501d","order_by":2,"name":"Daniel Eisenson","email":"","orcid":"https://orcid.org/0000-0003-3868-0403","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Eisenson","suffix":""},{"id":371307693,"identity":"5afa9f4a-ce8e-4513-830c-165165ac2c08","order_by":3,"name":"WeiLi Chen","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"WeiLi","middleName":"","lastName":"Chen","suffix":""},{"id":371307694,"identity":"88e97fb9-3d4e-40e9-8a81-d2514c24ddff","order_by":4,"name":"Alex Schulick","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Schulick","suffix":""},{"id":371307695,"identity":"db389f4f-0625-405a-a205-2d74e4c1ca7d","order_by":5,"name":"Michelle Santillan","email":"","orcid":"https://orcid.org/0009-0005-9942-6240","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Santillan","suffix":""},{"id":371307696,"identity":"e7e79f17-c02e-46a9-a324-a34775648efd","order_by":6,"name":"Adam Luo","email":"","orcid":"https://orcid.org/0000-0002-3421-6913","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Luo","suffix":""},{"id":371307697,"identity":"d1c7faad-9a93-48eb-bef7-c2e4a0463d80","order_by":7,"name":"Kelly Casella","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Casella","suffix":""},{"id":371307698,"identity":"d109e4d4-571b-4967-89f6-e83b5d27b099","order_by":8,"name":"Du Gu","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Du","middleName":"","lastName":"Gu","suffix":""},{"id":371307699,"identity":"03458015-cff3-4978-9dce-291f8144ada6","order_by":9,"name":"Mitsuhiro Sekijima","email":"","orcid":"","institution":"Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Mitsuhiro","middleName":"","lastName":"Sekijima","suffix":""},{"id":371307700,"identity":"f12ab090-5c1b-4400-a282-6d3b23157f8d","order_by":10,"name":"Hisashi Sahara","email":"","orcid":"","institution":"Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Hisashi","middleName":"","lastName":"Sahara","suffix":""},{"id":371307701,"identity":"39d76d76-97ca-40ac-9869-cc01b317da92","order_by":11,"name":"Daniel Warren","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Warren","suffix":""},{"id":371307702,"identity":"92819c5d-f561-4817-ab38-9953101e6b7b","order_by":12,"name":"Andrew Cameron","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Cameron","suffix":""},{"id":371307703,"identity":"dfd4e3cb-3731-4846-a3fe-47e6d66ebaec","order_by":13,"name":"Hayato Iwase","email":"","orcid":"","institution":"The Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hayato","middleName":"","lastName":"Iwase","suffix":""},{"id":371307704,"identity":"27525386-48f9-4d49-806e-f31631fb8cc1","order_by":14,"name":"Eugene Shenderov","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Eugene","middleName":"","lastName":"Shenderov","suffix":""}],"badges":[],"createdAt":"2024-10-07 18:30:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5220149/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5220149/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s43856-025-00842-6","type":"published","date":"2025-04-15T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71743297,"identity":"659f99f1-9ff0-4000-85bf-d7e663d7e376","added_by":"auto","created_at":"2024-12-18 08:26:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5473465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of kidney xenografts with 5-hours CIT after reperfusion with low immunological risk (A and B) and high risk (C and D).\u003c/strong\u003e (A) SCS-preserved kidney with low risk looked healthy immediately following reperfusion but subsequently exhibited mottling that progressed to diffuse discoloration in 1 hour post-reperfusion (B) The graft preserved using HMP were reperfused without clinically apparent changes. (C) SCS-preserved graft with high risk showed severe discoloration in 1 hour post-reperfusion. (D) A graft preserved using HMP looked healthy immediately following reperfusion but lost function in 2 hours.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/c2a63b0bb849e3a193aaeb99.png"},{"id":71743290,"identity":"778f2a5b-f996-4234-83e3-d06c082585e8","added_by":"auto","created_at":"2024-12-18 08:26:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8234009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological findings of the kidney xenografts with 5-hours CIT. \u003c/strong\u003e(A) SCS-preserved kidney (low risk) showed extensive hemorrhage and hemostasis one hour after reperfusion (hematoxylin and eosin (H\u0026amp;E) staining). (B) HMP-preserved kidney (H\u0026amp;E) did not show significant pathological changes one hour after reperfusion. (C and D) In high-risk cases, severe interstitial hemorrhages were observed in both SCS- and HMP-preserved xenografts.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/b3b98c036db05e7b67d69c16.png"},{"id":71743599,"identity":"d943c575-ac77-4888-9393-875a5ca71da2","added_by":"auto","created_at":"2024-12-18 08:34:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3942787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComplement (C3) deposition on immunofluorescence staining. \u003c/strong\u003eNotable deposition of C3, indicating complement activation, was observed in SCS-preserved kidneys, but not in HMP-preserved kidneys.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/65c04895ad230fcc953cb715.png"},{"id":71743294,"identity":"33a8befb-f7ec-4253-af61-84dc89bd1674","added_by":"auto","created_at":"2024-12-18 08:26:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5300895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell infiltration immunohistochemistry in the kidney xenografts with 5-hours CIT.\u003c/strong\u003e SCS-preserved kidneys in 2 hours post-reperfusion had significantly higher infiltration of myeloperoxidase (MPO) positive neutrophils compared with HMP-preserved kidneys. There was no significant difference in CD56-positive cells (NK cells) and CD68-positive cells (macrophages) between SCS- and HMP-preserved kidneys in 1 hour after transplantation.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/13e913f00fda70934eb80fd0.png"},{"id":71743298,"identity":"0097fc33-8cc2-4969-adec-1523a7eeff7b","added_by":"auto","created_at":"2024-12-18 08:26:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":649268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional landscape of reperfused kidney xenografts by preservation strategy. \u003c/strong\u003eDifferential expression (x-axis) and significance (y-axis) of DSP genes between HMP (right) and SCS (left) regions of interest. P values were computed using the Wald test from the DESeq2 standard workflow and adjusted for multiple testing with the Benjamini-Hochberg approach.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/126c3e5274e50a7b3b1f123e.png"},{"id":71743601,"identity":"09593f76-eadc-4d92-bf7d-fbf4349405fd","added_by":"auto","created_at":"2024-12-18 08:34:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":332189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatially resolved B-HOT gene set enrichment of reperfused kidney xenografts by preservation strategy. \u003c/strong\u003eScaled single sample gene set enrichment scores for nine pathways defined by the Banff 2019 Meeting Report. Comparisons between HMP and SCS preservation are shown for combined (pooled), glomerular, and tubulointerstitial regions of interest for each pathway. P values were computed using the two-sided Wilcoxon rank-sum (Mann-Whitney U) test and adjusted for multiple testing with the Benjamini-Hochberg approach (***, P \u0026lt; 0.001; **, P \u0026lt; 0.01; *, P \u0026lt; 0.05; not significant, n.s.).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/091aa39c9f64b9efe8b85c22.png"},{"id":80697231,"identity":"5c2b8ddf-9a63-4be7-a20c-e3c18ff27ca5","added_by":"auto","created_at":"2025-04-16 07:06:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35020538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/2e15a5ad-dec4-4721-83c1-c1c7a43b3662.pdf"},{"id":71743292,"identity":"c6bb5ad3-1cf7-4c31-b8e0-1a763e89ff09","added_by":"auto","created_at":"2024-12-18 08:26:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":781290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Unsupervised clustering of DSP Regions of Interest.\u003c/strong\u003e (a) Scanned whole-slide image of stained HMP/SCS surgical biopsy sections from the GeoMx instrument. (b) Representative images of Glomerular and Tubulointerstitial regions of interest. (c) Uniform manifold approximation and projection (UMAP) embedding of HMP and SCS regions of interest, further stratified by renal compartment.\u003c/p\u003e\n\u003cp\u003eSupplementary Table 1. Single Gene Analysis Summary of Differentially Enriched Genes\u003c/p\u003e\n\u003cp\u003eSupplementary Table 2. Gene Set Enrichment Hypothesis Testing Summary\u003c/p\u003e\n\u003cp\u003eSupplementary Table 3. B-HOT Panel Genes Used for Gene Set Enrichment Analysis\u003c/p\u003e","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/9790b3ab432de956ed418ca6.pdf"},{"id":71743295,"identity":"b593e9d7-d218-48d5-a599-6a8d6d3ad346","added_by":"auto","created_at":"2024-12-18 08:26:49","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":152815,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1.\u003cstrong\u003e \u003c/strong\u003eClinical outcomes of xenotransplantation cases with/without 5 hours of cold ischemic time (CIT)\u003c/p\u003e","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/0e624f762e3ff54f24996399.pdf"},{"id":71743600,"identity":"58334ceb-1f42-4918-aae7-0f88c127e7ee","added_by":"auto","created_at":"2024-12-18 08:34:49","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":88750,"visible":true,"origin":"","legend":"\u003cp\u003eTable 2. Clinical outcomes of allotransplantation cases with 5 hours of CIT\u003c/p\u003e","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5220149/v1/86ace4b71547cc936cdb4f9e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eXenotransplantation (XTx) is an increasingly realistic solution to the organ shortage. Researchers have been making progress in XTx research in the pig-to-nonhuman primate (NHP) model over the past 20 years with recent reports showing long-term survival of life-supporting porcine kidney\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and heart\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e xenografts using organs from genetically modified source pigs. These experiments paved the way for early studies in pig-to-human xenotransplantation. The Food and Drug Administration (FDA) granted case-by-case approval of xenografts for \u0026ldquo;compassionate use\u0026rdquo; in patients who were unlikely to receive conventional allografts, including two cases of pig-to-human heart transplantation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and two cases of pig-to-human kidney transplantation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Porcine xenograft procurement occurred under variable conditions in each of these isolated clinical cases; however, xenograft procurement in future clinical trials in xenotransplantation will be more closely regulated, which will have important implications for procurement and xenograft ischemic time.\u003c/p\u003e \u003cp\u003eXenografts used in imminent clinical trials will be regulated as investigational new animal drugs (INADs). Source pigs must be maintained in \u0026ldquo;designated pathogen-free\u0026rdquo; (DPF) facilities from conception to procurement to avoid the potential transmission of swine-derived organisms to human patients\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This designation and the associated strict control of infectious pathogens will likely limit where procurements can occur. It is not feasible for each hospital offering solid organ transplants to have their own DPF pig facility for clinical XTx; therefore, clinical XTx will likely require off-site procurement in centralized DPF pig facilities followed by shipment of those porcine organs. While cold ischemic time (CIT) is well-tolerated in conventional allogeneic kidney transplantation, the impact of clinically relevant CIT on pig kidney grafts has not been investigated in xenogeneic kidney transplantation. Indeed, few studies have reported long-term xenograft survival with significant (\u0026gt;\u0026thinsp;3 hours) CIT, and none have reported\u0026thinsp;\u0026gt;\u0026thinsp;1 year survival with \u0026gt;\u0026thinsp;3 hours CIT.\u003c/p\u003e \u003cp\u003eSuccessful xenotransplantation requires overcoming both innate and adaptive immunological barriers\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Ischemia-reperfusion injury (IRI) is known to potentiate both innate and adaptive immunity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and may thus raise the immunologic hurdles to effective xenotransplanation; while CIT is well-tolerated in conventional allogeneic kidney transplantation, IRI across xenogeneic barriers may lead to greater damage as species incompatibilities between porcine and primate inflammatory and coagulation systems may exacerbate underlying injury\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, while static cold storage (SCS) remains the gold standard for organ preservation in conventional allogeneic kidney transplantation, additional preservation modalities may be needed to minimize IRI across xenogeneic barriers. Hypothermic machine perfusion (HMP) is becoming widely used in clinical settings\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e with increasing evidence of improved graft outcomes with prolonged CIT as compared to SCS\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; while the benefit of HMP was first demonstrated with reduced incidence of delayed graft function (DGF) among higher risk kidneys\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, subsequent studies have found reduced DGF all donor subgroups\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The mechanisms responsible for observed superior outcomes with HMP in conventional allogeneic transplantation are incompletely understood\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, but mechanistic studies suggest that HMP may mitigate ischemia-reperfusion injury through reduction of hypoxia during preservation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and decreased inflammatory cell death\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e as well as improved endothelial cell function\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e after reperfusion.\u003c/p\u003e \u003cp\u003eExisting data in transplantation across xenogeneic barriers indicate that mode of preservation may play a key role: Langin et al demonstrated that non-ischemic preservation with continuous perfusion was essential for successful orthotopic cardiac xenotransplantation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Additionally, recent multi-omic data from two pig-to-human decedent cardiac xenotransplantation at New York University revealed evidence of ischemia reperfusion injury in one of two xenografts (Keating, TTS, 2024). However, no studies to date have investigated the impact of HMP on graft outcomes in kidney xenotransplantation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe recently reported consistent survival of life-supporting kidney xenografts in pig-to-baboon model with clinically relevant ischemic time preserved with HMP as part of an IND-enabling study for the FDA\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Here we provide scientific justification for a nuanced preservation strategy in xenotransplantation, as used in that study. In this study, we investigated the impact of prolonged cold ischemic time on the outcomes of pig-to-baboon kidney transplantation, we compared the efficacy of different preservation strategies (SCS and HMP) on early graft function, and we explored the mechanistic underpinnings of the xenograft response to IRI. This study is the first to examine the effects of SCS versus HMP on xenografts under conditions of prolonged CIT. By addressing these objectives, we fill the existing knowledge gaps and contribute to the development of more effective organ preservation protocols for clinical xenotransplantation trials.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical impact of IRI across xenogeneic barriers\u003c/h2\u003e \u003cp\u003eThe outcomes of the xenotransplantation cases with and without 5 hours of CIT are summarized in Table\u0026nbsp;1. We performed 8 cases with 5 hours of CIT: 4 cases with SCS and 4 cases with HMP. All grafts preserved using SCS lost function immediately after transplant. By visual inspection, these grafts looked healthy immediately following reperfusion but subsequently exhibited mottling that progressed to diffuse discoloration and anuria within 90 minutes post-reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, all grafts preserved using HMP were reperfused without clinically apparent changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and survived for more than 14 days post-transplantation. All cases that did not undergo the 5-hour CIT accepted the kidney graft as expected, demonstrating similar outcomes to those preserved using HMP except for the slight difference in initial peak of serum creatinine levels (which were higher in the preservation arm) (Table\u0026nbsp;1). The outcomes of allotransplantation cases with 5 hours of CIT preserved using either HMP or SCS are detailed in Table\u0026nbsp;2. Unlike the xenotransplantation cases, no hyperacute graft loss was observed in allotransplantation cases preserved with SCS. These grafts did experience IRI with creatinine levels increasing up to 4.7 mg/dL after Tx, while HMP-preserved allografts maintained stable early graft function (Table\u0026nbsp;1, 2), but the differences were not statistically significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological evaluation of the impact of IRI across xenogeneic barriers\u003c/h3\u003e\n\u003cp\u003eOn histologic examination, SCS-preserved kidneys (hematoxylin and eosin (H\u0026amp;E) staining) showed extensive hemorrhage and hemostasis one hour after reperfusion (representative staining, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas HMP-preserved kidneys did not show significant pathological changes one hour after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). There was a notable deposition of C3 in SCS-preserved kidneys, indicating complement activation, which was not observed in HMP-preserved kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). SCS kidneys at two hours post-Tx had significantly higher infiltration of myeloperoxidase (MPO) positive cells compared with HMP-preserved kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There was no significant difference in CD56-positive cells (NK cells) and CD68-positive cells (macrophages) between SCS- and HMP-preserved kidneys in biopsies obtained one hour after transplantation. However, more CD68-positive cells and MPO-positive cells were seen at later time point in HMP-kidney on POD24 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGeoMx Digital Spatial Profiling (DSP) Analysis\u003c/h3\u003e\n\u003cp\u003eGeoMx whole-transcriptome DSP analysis provided comparative insights between HMP and SCS preservation methods in the setting of xenotransplantation and standardized CIT in single-gene knockout (GalTKO) genetically modified pigs. A representative slide was prepared from surgical tissue biopsies obtained two hours after reperfusion in either preservation condition (n\u0026thinsp;=\u0026thinsp;1 for HMP and SCS). Regions of interest (ROIs) were selected to stratify renal architecture into glomerular (n\u0026thinsp;=\u0026thinsp;6 for SCS, n\u0026thinsp;=\u0026thinsp;3 for HMP) and tubulointerstitial (n\u0026thinsp;=\u0026thinsp;6 for SCS, and n\u0026thinsp;=\u0026thinsp;3 for HMP) spatial compartments distinguished by immunofluorescent markers Syto 13 (nuclei) and pan-cytokeratin (epithelial cells).\u003c/p\u003e \u003cp\u003eFirst, Unsupervised clustering of ROIs identified 2 distinct local communities that were primarily differentiated by renal compartment as opposed to preservation strategy. However, sub-clustering by preservation within each community is somewhat apparent, lending internal validity to the GeoMx assay and ROI selection process. (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eWe then screened for differentially expressed genes across the HMP-SCS axis using cutoffs for fold change (abs(log\u003csub\u003e2\u003c/sub\u003eFC)\u0026thinsp;\u0026gt;\u0026thinsp;2) and adjusted \u003cem\u003eP\u003c/em\u003e value (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Six genes were enriched in SCS ROIs, all of which were found to have relevance to ischemia and IRI. Specifically, \u003cem\u003eFOS\u003c/em\u003e and \u003cem\u003eJUNB\u003c/em\u003e are downstream effectors in non-canonical Wnt signaling pathways with crosstalk between the TGF-b and NF-Kb pathways leading to inflammation and apoptosis and have been implicated in renal IRI\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eEGR1\u003c/em\u003e is a transcription factor regulated by many external cell stress signals and is involved with inflammation and apoptosis signaling while \u003cem\u003eNR4A1\u003c/em\u003e and \u003cem\u003eNR4A2\u003c/em\u003e are nuclear receptors also involved in apoptotic pathways and implicated in IRI\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,24\u003c/sup\u003e. Only \u003cem\u003eATF3\u003c/em\u003e has been shown to have a protective effect in renal IRI in an in-vitro model inducing oxidative stress\u003csup\u003e25\u003c/sup\u003e. On the other hand, HMP ROIs were highly enriched for genes protective against oxidative stress and hypoxia (\u003cem\u003eMDH1\u003c/em\u003e, \u003cem\u003eKCNJ1\u003c/em\u003e), wound healing (\u003cem\u003eB2M\u003c/em\u003e), and mitochondrial as well as cellular metabolism and homeostasis (\u003cem\u003eUMOD\u003c/em\u003e, \u003cem\u003eCOX\u003c/em\u003e genes, \u003cem\u003eATP5\u003c/em\u003e genes, \u003cem\u003eCS\u003c/em\u003e)\u003csup\u003e26\u0026ndash;31\u003c/sup\u003e. Of these 17 genes, only one (\u003cem\u003eCD74\u003c/em\u003e) has been suggested to correlate with worse IRI-associated AKI\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing gene sets defined by the Banff Human Organ Transplant (B-HOT) gene panel, we mapped DSP ROIs to phenotypic pathways specifically relevant to molecular studies in transplant immunology\u003csup\u003e33\u003c/sup\u003e. In general, IRI-associated pathways were enriched in SCS ROIs, including pro-inflammatory, cell death, and innate immune pathways such as cytokine signaling, inflammasomes, MAPK, Th17-mediated biology, TNF family signaling, and apoptosis \u0026amp; cell cycle regulation. On the other hand, HMP ROIs were enriched for pathways related to cellular metabolism and homeostasis, including cell-ECM interactions, Metabolism, and MHC Class I presentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Table\u0026nbsp;2). Those genes which were included in the panel were required to pass data quality control measures prior to inclusion in the analysis and are specifically detailed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to demonstrate that clinically relevant CIT with routine SCS poses a significant risk for hyperacute graft loss in a pig-to-NHP kidney xenotransplantation model. Clinically relevant CIT (5 hours) with SCS uniformly led to hyperacute graft loss after transplantation across xenogeneic barriers; in contrast, kidney xenografts transplanted without CIT in immunologically matched donor-recipient pairs did not result in hyperacute graft loss, and porcine kidney allografts transplanted with clinically relevant CIT and preserved with SCS did not result in hyperacute graft loss.\u003c/p\u003e \u003cp\u003eImportantly, we observed significant differences in the outcomes of kidney xenotransplantation based on the preservation method employed during CIT. All kidneys preserved using SCS were lost hyperacutely within 90 minutes, with clinically apparent vasoconstriction visible minutes after reperfusion and histologic evidence of severe endothelial injury on biopsies obtained one hour after reperfusion. Conversely, all kidneys preserved using HMP reperfused uneventfully with minimal histologic changes on one-hour biopsies and, after an initial elevation in creatinine corresponding to IRI associated with CIT, functioned for more than 14 days.\u003c/p\u003e \u003cp\u003eWhile the underlying mechanisms for the observed differences between SCS and HMP remain unclear, spatial transcriptomic analyses suggest that HMP may confer benefit through modulation of initial ischemic injury as well as mitigation of reperfusion injury and subsequent inflammation. This study presents the first report of transcriptomic analyses with spatial resolution in an \u003cem\u003ein vivo\u003c/em\u003e model of ischemia-reperfusion in kidney xenotransplantation.\u003c/p\u003e \u003cp\u003eIt is important to acknowledge some limitations of this analysis. First and foremost, there is no spatially resolved, species-specific transcriptomic technology widely available for porcine xenotransplant in an NHP background. However, GeoMx DSP probes were cross-referenced with the porcine genome and genetic similarity was found to be at least 85% across species. Furthermore, the GeoMx DSP analysis was limited by the statistical approach for hypothesis testing; Given the small sample size, it is difficult to account for the nested hierarchical nature of the experimental observations (the ROIs). Future, larger cohorts are required to confirm findings. Despite these limitations, transcriptomic analyses of two representative samples revealed that HMP-preserved kidney tissue was highly enriched for genes and pathways protective against oxidative stress and hypoxia, as well as genes/pathways promoting normal cellular and mitochondrial metabolism. The SCS tissue was enriched for genes and pathways associated with inflammation, cell death, and innate immunity. Altogether, molecular analysis substantiates clinical and histologic evidence in this study suggesting that HMP may minimize the initial hypoxic injury associated with CIT. A similar pattern has also been observed with HMP in conventional allogeneic transplantation, where studies have demonstrated reduced expression of hypoxia-related genes in HMP-stored donor organs\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePerhaps more importantly, the inflammatory response corresponding to this initial ischemic injury appears markedly different between SCS- and HMP-preserved kidney tissue. SCS-preserved kidneys were found to have significant increases in MPO-positive neutrophil infiltration on immunohistochemistry as-well-as enrichment of genes associated with innate immune activation and inflammation on transcriptomic analyses. While the trend is similar to preservation studies in conventional allogeneic transplantation\u003csup\u003e35\u003c/sup\u003e, differences between SCS and HMP preservation strategies appear more pronounced in xenotransplantation compared with conventional allogeneic transplantation. Indeed, innate immune activation may progress more rapidly across xenogeneic barriers, where species incompatibilities impair normal anti-inflammatory and anti-coagulant regulatory processes. For example, immunofluorescence of SCS-preserved kidneys demonstrated diffuse complement deposition which was not observed in the HMP-preserved kidneys despite identical levels of preformed antibodies (same recipient); once innate immunity is activated across xenogeneic barriers, it is likely more challenging to turn off without normal species-congruent inhibitory proteins.\u003c/p\u003e \u003cp\u003eTargeted genetic modification of source pigs may minimize IRI-induced inflammation associated with species incompatibilities\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We recently demonstrated consistent and durable survival of life-supporting kidney xenografts in consecutive cases of pig-to-baboon transplantation using 10GE source pigs with transgenic expression of human anti-inflammatory (HO1), anti-coagulant (TBM, EPCR), and complement regulatory proteins (CD46, CD55)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These xenografts incurred similar CIT to those in this study and were preserved with HMP, avoiding hyperacute graft loss. Still, IRI impacted each animal\u0026rsquo;s postoperative course and may have contributed to ultimate xenograft failure and preterminal euthanasia.\u003c/p\u003e \u003cp\u003eNotably, porcine kidneys used in both decedent and clinical pig-to-human cases were preserved with SCS, incurred similar CIT, and did not undergo hyperacute graft loss. This may be a function of xenograft size \u0026ndash; our experiments used small (\u0026lt;\u0026thinsp;80 grams) recipient size-matched porcine kidneys from juvenile pigs (\u0026lt;\u0026thinsp;20 kg), while the cases at NYU used larger kidneys from adult pigs. Juvenile pig kidneys may be more sensitive to acute injury\u003csup\u003e36\u003c/sup\u003e, which may be related to immature renal structures\u003csup\u003e37\u003c/sup\u003e. Similarly, kidneys from deceased pediatric donors less than five years old have a higher rate of vascular complications\u003csup\u003e38\u003c/sup\u003e in the early postoperative period. This may also be a function of the known immunologic\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and physiologic differences in xenograft recipients (NHPs in our study versus humans). Although we were not able to salvage xenografts preserved with SCS in our transplantation studies, we did observe relative transient clinical improvement with increased blood pressure. While systolic blood pressures of \u0026gt;\u0026thinsp;130 mmHg are supraphysiologic in our pediatric NHP recipients, these pressures are normal in adult patients. Larger kidneys and larger recipients may result in better outcomes after CIT in xenotransplantation regardless of preservation modality. Still, available data indicates that xenograft function after SCS preservation in long-term pig-to-human decedent and clinical kidney xenotransplantation cases has not been normal. Early antibody mediated rejection was recently reported in the 61-day decedent (Keating, TTS, 2024). While there was no DGF reported in this case, IRI is known to amplify the humoral response to heterologous antigens\u003csup\u003e39\u003c/sup\u003e, and may have contributed to early and unexpected ABMR in this case. Our findings suggest that SCS preservation may increase the risk of IRI and acute graft loss after transplantation across xenogeneic barriers. Given these outcomes, HMP may be a safer preservation strategy for early clinical trials in xenotransplantation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eWe performed 23 cases of large animal kidney transplantation (19 cases of pig-to-baboon kidney xenotransplantation and 4 cases of pig-to-pig kidney allogeneic transplantation) at Columbia University (New York, NY, USA), Johns Hopkins University (Baltimore, MD, USA), and Kagoshima University (Kagoshima, Japan). All animal work was conducted in accordance with NIH and USDA guidelines and with approval from the Institutional Animal Care and Use Committee (IACUC).\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eAnimal husbandry\u003c/h2\u003e\n \u003cp\u003eAnimal husbandry was conducted in accordance with each facilities\u0026rsquo; SOPs. Swine were socially housed in individual pens on tenderfoot flooring in spaces with at least one and up to seven additional pigs. Swine were surveilled for pathogens of potential concern to NHP recipients. Baboons were housed both singly and in pairs in stainless-steel cages with access to an automatic watering valve. Temperature was maintained between 24 and 29\u0026deg;C, humidity maintained between 30\u0026ndash;70%, and lighting was provided on a light/dark cycle for approximately 12 hours each day. Baboons were fed certified chow with LabDiet 5038\u0026mdash;Monkey Diet, with enrichment in the form of fruits and vegetables provided once daily. Water was available ad libitum throughout the studies for both swine and baboons.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMHC-matched pigs\u003c/h3\u003e\n\u003cp\u003eCLAWN-miniature swine, aged 9\u0026ndash;12 months, were obtained from the Kagoshima Miniature Swine Research Center (Isa, Japan). CLAWN-miniature swine are genetically typed, MHC-inbred miniature swine. In this study, fully MHC-matched pairs of swine were used as donors and recipients. Swine were maintained, treated, and euthanized according to guidelines established by the Animal Welfare Act and in compliance with protocols approved by Kagoshima University Institutional Animal Care and Use Committees.\u003c/p\u003e\n\u003ch3\u003eGenetically modified source pigs\u003c/h3\u003e\n\u003cp\u003ePig donors were provided by either Accuro Farms, Inc. (Southbridge, MA, USA) or National Swine Resource and Research Center, University of Missouri (Columbia, MO, USA). All donors were porcine cytomegalovirus (PCMV) negative. Genetic modification of donor pigs includes GalTKO or GalTKO with hCD55 Tg. Euthanasia was performed prior to organ procurement. Swine were maintained, treated, and euthanized according to guidelines established by the Animal Welfare Act and the NIH for the housing and care of laboratory animals and in compliance with protocols approved by Columbia University, Johns Hopkins University, and Kagoshima University Institutional Animal Care and Use Committees.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBaboons\u003c/h2\u003e\n \u003cp\u003eBaboons (Papio spp.) were purchased as recipients of pig-to-baboon KXTx from the Mannheimer Foundation (Papio hamadryas, Homestead, FL, USA) or from the Michale E. Keeling Center for Comparative Medicine and Research, MD Anderson Cancer Center (Papio anubis, Bastrop, TX, USA). The baboons ranged from 2\u0026ndash;4 years old and weighed between 8 to 12.5 kg. All baboons were maintained, treated, and euthanized at the study endpoint according to guidelines established by the Animal Welfare Act and the NIH for the housing and care of laboratory animals and in compliance with protocols approved by the Animal Care and Use Committee at Columbia University and at Johns Hopkins University.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003ePre-transplant screening\u003c/h2\u003e\n \u003cp\u003eAll xenotransplant recipients were evaluated for anti-pig antibodies by complement-dependent cytotoxicity (CDC) using peripheral blood mononuclear cells (PBMCs) derived from GalTKO source pigs. The presence of donor-specific antibodies was also assessed by IgG and IgM antibody binding of baboon sera to GalTKO-derived PBMCs according to screening methodology developed by The Yamada Lab at Johns Hopkins University (Hisadome, Transplantation, 2024). Recipients are selected for complement-dependent cytotoxicity (CDC)\u0026thinsp;\u0026lt;\u0026thinsp;20% at 1:4 dilution and serum IgG binding on antibody flow cytometry less than the pretransplant value of a known rejector recipient.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eExperimental groups\u003c/h2\u003e\n \u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e\u0026bull; Pig-to-baboon xenogeneic kidney transplantation\u003c/h2\u003e\n \u003cp\u003eWe performed 19 cases of pig-to-baboon XKTx. 8 cases were performed with 5-hours cold ischemic time (Xeno preservation group), which is consistent with likely duration of coastal transportation time in the United States. The kidney grafts were preserved for 5 hours with either (1) static cold storage (SCS) (n\u0026thinsp;=\u0026thinsp;4) or (2) hypothermic machine perfusion (HMP) using the LifePort Kidney Transporter (Organ Recovery Systems; Itasca, IL, USA) (n\u0026thinsp;=\u0026thinsp;4) (detail described in the section \u0026ldquo;Kidney graft preservation procedures\u0026rdquo;). The other cases (n\u0026thinsp;=\u0026thinsp;11) were performed with minimal CIT less than 20 min (Xeno control group). Recipients\u0026rsquo; pretransplant anti-pig natural antibody levels were evaluated using both complement-dependent cytotoxicity (CDC) and antibody binding assay using flow cytometry (AbFCM)(*Ref), and the risk for XKTx were stratified as previously reported (*Ref). We included either low Nab (low risk) or high Nab (exclusion) recipient into the study to assess effect of graft preservation on preformed Nab.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e\u0026bull; Pig-to-pig allogeneic kidney transplantation\u003c/h2\u003e4 cases of pig allo KTx were performed with 5-hours CIT (Allo preservation group). Pig donor and recipient were MHC-matched. 5-hours graft preservation was performed in the same manner as in Xeno preservation group: 2 cases with SCS and 2 cases with HMP.\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eSurgical procedures\u003c/h2\u003e\n \u003cp\u003eA central venous catheter was placed in the recipient prior to Tx and used for drug administration and blood sampling throughout experiment. The kidney graft was transplanted as previously reported\u003csup\u003e40\u003c/sup\u003e with bilateral native nephrectomies, splenectomy (in the experimental group) and bilateral oophorectomy (for female baboons).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eKidney graft preservation procedures\u003c/h2\u003e\n \u003cp\u003eKidney grafts procured from the donor swine were immediately perfused with 50ml of the University of Wisconsin (UW) solution at 4C. For SCS arm, the grafts were immersed into 500ml of UW solution and stored at 4C for 5 hours. For the HMP arm, a disposable cannula was attached to renal artery and machine perfusion was initiated using the LifePort Kidney Transporter with an infusion pressure of 30mmHg. 1000ml of KPS-1 (Organ Recovery Systems, equivalent to UW solution) was used for the perfusion circuit. The grafts were continuously perfused at 2-8C for 5 hours prior to Tx. In the Xeno preservation group, one of the two kidneys from a donor pig was preserved with SCS and the other with HMP. Both grafts were transplanted into the same recipient baboon in order to compare differences between two preservation strategies while minimizing differences in other experimental conditions. After vascular anastomoses were completed, both kidneys were reperfused simultaneously.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eImmunosuppression\u003c/h2\u003e\n \u003cp\u003eAll baboons received induction therapy with rabbit anti-thymocyte globulin (Thymoglobulin) and anti-CD20 monoclonal antibodies (Rituximab) prior to Tx. Maintenance therapy included anti-CD40 or anti-CD40L monoclonal antibodies, mycophenolate mofetil (MMF), and cytotoxic T lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig). Anti-C5 monoclonal antibody (Tesidolumab) was given concomitantly for the recipient who received GalTKO\u0026thinsp;+\u0026thinsp;hCD55 kidney.\u003c/p\u003e\n \u003cp\u003eAll recipients in the pig-to-pig allogeneic kidney transplantation group were treated with tacrolimus for 14 days, starting on the day of transplantation, with blood levels maintained at 15 to 25 ng/mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003ePosttransplant outcomes\u003c/h2\u003e\n \u003cp\u003eKidney graft function was assessed by urine volume and laboratory tests including serum creatinine (mg/dL) and blood urea nitrogen (BUN, mg/dL). We defined graft dysfunction based on macroscopic and/or microscopic/histologic findings of the kidney grafts, urine volume (\u0026lt;\u0026thinsp;0.5ml/kg/hr) or serum creatinine level (\u0026gt;\u0026thinsp;8.0mg/dL). The primary endpoint was graft survival within 14 days post Tx.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eHistopathological analysis\u003c/h2\u003e\n \u003cp\u003eGraft kidney biopsies were obtained after procurement (after flushing with preservation solution, prior to transplant) and at 2 hours after reperfusion. Kidney biopsy specimens were either a) frozen or b) fixed in 10% formaldehyde and embedded in paraffin. Frozen samples were used for immunofluorescence (IF). Anti-human IgG, IgM, and C3 (DAKO, Carpentaria, CA) all conjugated to FITC; C5b (DAKO, Carpentaria, CA) and C4d (QUIDEL, San Diego, CA) were unconjugated ab detected by Alexa Fluor\u0026reg; 488 goat anti-mouse secondary antibody (Abcam, Cambridge, MA) to assess antibody binding and complement activation in the graft. Paraffin-embedded tissues were sectioned, stained using hematoxylin and eosin (H\u0026amp;E) and Periodic acid-Schiff, and examined by an experienced pathologist. Immunohistochemistry staining was performed to assess cell infiltration into grafts, including CD56, CD68 (sc20060 C1662, Santa Cruz Biotechnology, Dallas, TX) and myeloperoxidase (ab188211, Abcam, Cambridge, MA). Immunofluorescence staining was performed on frozen specimens for C3c (DAKO complement clone C3c FITC F0201, Carpentaria, CA) and C5b9 (DAKO complement clone C5b9 aE11 M0777, Carpentaria, CA) complement deposition using anti-mouse FITC secondary antibody (A2723, Invitrogen, Waltham, MA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eGeoMx DSP data acquisition, processing and analysis\u003c/h2\u003e\n \u003cp\u003ePublished experimental methods for DSP (Merritt et al., \u003cem\u003eNat Biotech.\u003c/em\u003e, 2020) were followed using standardized protocols on an in-house NanoString GeoMx instrument. 3 formalin-fixed, paraffin-embedded sections were processed for DSP with the human whole-transcriptome atlas (consisting of 18,676 UV-photocleavable barcode-conjugated RNA \u003cem\u003ein situ\u003c/em\u003e hybridization probes and 139 negative control probes). 2 morphology markers were used to outline renal morphology prior to ROI selection: 400 nanomolar SYTO 13 (Invitrogen, Waltham, MA) and 1:100 anti-PanCK-Texas Red 594 (Clone: AE1/AE3; Novus Biologicals, Centennial, CO). Spatially indexed barcodes were collected from a total of 30 ROIs. Libraries were prepared according to manufacturer\u0026rsquo;s instructions and subsequently sequenced using a NovaSeq 5000 system.\u003c/p\u003e\n \u003cp\u003eFASTQ files were converted to digital count conversion (DCC) files using NanoString software (GeoMx NGS Pipeline). DCC files were uploaded into R (version 4.4.0) and consolidated as a NanoStringGeoMxSet S4 object. Gene targets were removed from the object if they consistently fell below the limit of quantitation (LOQ; defined by the background signal for each ROI). The remaining dataset of 5,577 genes was quantile-normalized\u003csup\u003e41,42\u003c/sup\u003e. Differential expression analysis was performed using the \u003cem\u003eDESeq2\u003c/em\u003e R package\u003csup\u003e43\u003c/sup\u003e and pathway signatures were defined by the Banff Human Organ Transplant Gene Panel\u003csup\u003e33\u003c/sup\u003e. Pathway enrichment scores were obtained using single-sample gene set enrichment analysis (ssGSEA)\u003csup\u003e44\u003c/sup\u003e from the \u003cem\u003eGSVA\u003c/em\u003e R package\u003csup\u003e45\u003c/sup\u003e, which were then scaled as z-scores. Reported \u003cem\u003eP\u003c/em\u003e values were corrected for multiple testing using the Benjamini-Hochberg method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using the Student\u0026rsquo;s t-test. All tests were 2-sided, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All statistical analyses were performed using Prism 9 (GraphPad Software).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge David Sachs, Megan Sykes, Kristin Whitworth, Randall Prather, and Kevin Wells for providing genetically modified pigs used in this study.\u003c/p\u003e\n\n\u003cp\u003eDisclosure\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by NIH grant P01AI045897. K. Yamada reports institutional grant support from United Therapeutics Corporation outside the submitted work. E. Shenderov reports institutional grant support from AstraZeneca and institutional clinical trial support and non-financial reagent support from Macrogenics outside the submitted work.\u003c/p\u003e\n\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll data supporting this study are available within the article and its related supplementary information file. All raw data for graphs in this study and all microscopic images are available from the corresponding author on request.\u003c/p\u003e\n\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams, A. B.\u003cem\u003e et al.\u003c/em\u003e Anti-C5 Antibody Tesidolumab Reduces Early Antibody-mediated Rejection and Prolongs Survival in Renal Xenotransplantation. \u003cem\u003eAnn Surg\u003c/em\u003e \u003cstrong\u003e274\u003c/strong\u003e, 473-480, doi:10.1097/SLA.0000000000004996 (2021).\u003c/li\u003e\n\u003cli\u003eEisenson, D.\u003cem\u003e et al.\u003c/em\u003e Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10GE source pigs. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 3361, doi:10.1038/s41467-024-47679-6 (2024).\u003c/li\u003e\n\u003cli\u003eAnand, R. P.\u003cem\u003e et al.\u003c/em\u003e Design and testing of a humanized porcine donor for xenotransplantation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e622\u003c/strong\u003e, 393-401, doi:10.1038/s41586-023-06594-4 (2023).\u003c/li\u003e\n\u003cli\u003eLangin, M.\u003cem\u003e et al.\u003c/em\u003e Consistent success in life-supporting porcine cardiac xenotransplantation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e564\u003c/strong\u003e, 430-433, doi:10.1038/s41586-018-0765-z (2018).\u003c/li\u003e\n\u003cli\u003eMohiuddin, M. M.\u003cem\u003e et al.\u003c/em\u003e Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, e12744, doi:10.1111/xen.12744 (2022).\u003c/li\u003e\n\u003cli\u003eGriffith, B. P.\u003cem\u003e et al.\u003c/em\u003e Genetically Modified Porcine-to-Human Cardiac Xenotransplantation. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e387\u003c/strong\u003e, 35-44, doi:10.1056/NEJMoa2201422 (2022).\u003c/li\u003e\n\u003cli\u003eRabin, B.\u003c/li\u003e\n\u003cli\u003eNoordergraaf, J.\u003cem\u003e et al.\u003c/em\u003e Pathogen elimination and prevention within a regulated, Designated Pathogen Free, closed pig herd for long-term breeding and production of xenotransplantation materials. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, e12428, doi:10.1111/xen.12428 (2018).\u003c/li\u003e\n\u003cli\u003eEisenson, D. L., Hisadome, Y. \u0026amp; Yamada, K. Progress in Xenotransplantation: Immunologic Barriers, Advances in Gene Editing, and Successful Tolerance Induction Strategies in Pig-To-Primate Transplantation. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 899657, doi:10.3389/fimmu.2022.899657 (2022).\u003c/li\u003e\n\u003cli\u003eTang, Q., Dong, C. \u0026amp; Sun, Q. Immune response associated with ischemia and reperfusion injury during organ transplantation. \u003cem\u003eInflamm Res\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 1463-1476, doi:10.1007/s00011-022-01651-6 (2022).\u003c/li\u003e\n\u003cli\u003ePatel, P. M.\u003cem\u003e et al.\u003c/em\u003e Minimizing Ischemia Reperfusion Injury in Xenotransplantation. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 681504, doi:10.3389/fimmu.2021.681504 (2021).\u003c/li\u003e\n\u003cli\u003eMalinoski, D.\u003cem\u003e et al.\u003c/em\u003e Hypothermia or Machine Perfusion in Kidney Donors. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e388\u003c/strong\u003e, 418-426, doi:10.1056/NEJMoa2118265 (2023).\u003c/li\u003e\n\u003cli\u003eAubert, O.\u003cem\u003e et al.\u003c/em\u003e Long term outcomes of transplantation using kidneys from expanded criteria donors: prospective, population based cohort study. \u003cem\u003eBMJ\u003c/em\u003e \u003cstrong\u003e351\u003c/strong\u003e, h3557, doi:10.1136/bmj.h3557 (2015).\u003c/li\u003e\n\u003cli\u003eMoers, C.\u003cem\u003e et al.\u003c/em\u003e Machine perfusion or cold storage in deceased-donor kidney transplantation. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e360\u003c/strong\u003e, 7-19, doi:10.1056/NEJMoa0802289 (2009).\u003c/li\u003e\n\u003cli\u003eCallaghan, C. J.\u003cem\u003e et al.\u003c/em\u003e Outcomes of transplantation of livers from donation after circulatory death donors in the UK: a cohort study. \u003cem\u003eBMJ Open\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, e003287, doi:10.1136/bmjopen-2013-003287 (2013).\u003c/li\u003e\n\u003cli\u003eTingle, S. J.\u003cem\u003e et al.\u003c/em\u003e Hypothermic machine perfusion is superior to static cold storage in deceased donor kidney transplantation: A meta-analysis. \u003cem\u003eClin Transplant\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e13814, doi:10.1111/ctr.13814 (2020).\u003c/li\u003e\n\u003cli\u003eKnijff, L. W. D., van Kooten, C. \u0026amp; Ploeg, R. J. The Effect of Hypothermic Machine Perfusion to Ameliorate Ischemia-Reperfusion Injury in Donor Organs. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 848352, doi:10.3389/fimmu.2022.848352 (2022).\u003c/li\u003e\n\u003cli\u003eZhao, D. F., Dong, Q. \u0026amp; Zhang, T. Effects of Static Cold Storage and Hypothermic Machine Perfusion on Oxidative Stress Factors, Adhesion Molecules, and Zinc Finger Transcription Factor Proteins Before and After Liver Transplantation. \u003cem\u003eAnn Transplant\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 96-100, doi:10.12659/aot.901897 (2017).\u003c/li\u003e\n\u003cli\u003eDarius, T.\u003cem\u003e et al.\u003c/em\u003e Influence of Different Partial Pressures of Oxygen During Continuous Hypothermic Machine Perfusion in a Pig Kidney Ischemia-reperfusion Autotransplant Model. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 731-743, doi:10.1097/TP.0000000000003051 (2020).\u003c/li\u003e\n\u003cli\u003eGuarrera, J. V.\u003cem\u003e et al.\u003c/em\u003e Hypothermic machine preservation facilitates successful transplantation of \u0026quot;orphan\u0026quot; extended criteria donor livers. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 161-169, doi:10.1111/ajt.12958 (2015).\u003c/li\u003e\n\u003cli\u003eBurlage, L. C.\u003cem\u003e et al.\u003c/em\u003e Oxygenated hypothermic machine perfusion after static cold storage improves endothelial function of extended criteria donor livers. \u003cem\u003eHPB (Oxford)\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 538-546, doi:10.1016/j.hpb.2017.02.439 (2017).\u003c/li\u003e\n\u003cli\u003eZhang, M.\u003cem\u003e et al.\u003c/em\u003e Ischemia-reperfusion injury: molecular mechanisms and therapeutic targets. \u003cem\u003eSignal Transduction and Targeted Therapy\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, doi:ARTN 12 10.1038/s41392-023-01688-x (2024).\u003c/li\u003e\n\u003cli\u003eJeong, K., Je, J., Dusabimana, T., Kim, H. \u0026amp; Park, S. W. Early Growth Response 1 Contributes to Renal IR Injury by Inducing Proximal Tubular Cell Apoptosis. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, doi:10.3390/ijms241814295 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, J.\u003cem\u003e et al.\u003c/em\u003e Molecular characterization of the transition from acute to chronic kidney injury following ischemia/reperfusion. \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, doi:10.1172/jci.insight.94716 (2017).\u003c/li\u003e\n\u003cli\u003eYoshida, T.\u003cem\u003e et al.\u003c/em\u003e ATF3 protects against renal ischemia-reperfusion injury. \u003cem\u003eJ Am Soc Nephrol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 217-224, doi:10.1681/ASN.2005111155 (2008).\u003c/li\u003e\n\u003cli\u003eBurton, J. B.\u003cem\u003e et al.\u003c/em\u003e Substantial downregulation of mitochondrial and peroxisomal proteins during acute kidney injury revealed by data-independent acquisition proteomics. \u003cem\u003eProteomics\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, e2300162, doi:10.1002/pmic.202300162 (2024).\u003c/li\u003e\n\u003cli\u003eJang, H. S.\u003cem\u003e et al.\u003c/em\u003e Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury. \u003cem\u003eKidney International\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 327-339, doi:10.1016/j.kint.2019.08.019 (2020).\u003c/li\u003e\n\u003cli\u003eGimelreich, D.\u003cem\u003e et al.\u003c/em\u003e Regulation of ROMK and channel-inducing factor (CHIF) in acute renal failure due to ischemic reperfusion injury. \u003cem\u003eKidney Int\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 1812-1820, doi:10.1046/j.1523-1755.2001.0590051812.x (2001).\u003c/li\u003e\n\u003cli\u003eLi, C.\u003cem\u003e et al.\u003c/em\u003e Gp78 deficiency in hepatocytes alleviates hepatic ischemia-reperfusion injury via suppressing ACSL4-mediated ferroptosis. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 810, doi:10.1038/s41419-023-06294-x (2023).\u003c/li\u003e\n\u003cli\u003eEl-Achkar, T. M.\u003cem\u003e et al.\u003c/em\u003e Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression. \u003cem\u003eAm J Physiol Renal Physiol\u003c/em\u003e \u003cstrong\u003e295\u003c/strong\u003e, F534-544, doi:10.1152/ajprenal.00083.2008 (2008).\u003c/li\u003e\n\u003cli\u003eKaragiannidis, A. G., Theodorakopoulou, M. P., Pella, E., Sarafidis, P. A. \u0026amp; Ortiz, A. Uromodulin biology. \u003cem\u003eNephrol Dial Transplant\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 1073-1087, doi:10.1093/ndt/gfae008 (2024).\u003c/li\u003e\n\u003cli\u003eLi, J. H.\u003cem\u003e et al.\u003c/em\u003e Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion. \u003cem\u003eJournal of Cellular and Molecular Medicine\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 3867-3877, doi:10.1111/jcmm.14234 (2019).\u003c/li\u003e\n\u003cli\u003eMengel, M.\u003cem\u003e et al.\u003c/em\u003e Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 2305-2317, doi:10.1111/ajt.16059 (2020).\u003c/li\u003e\n\u003cli\u003eKrezdorn, N.\u003cem\u003e et al.\u003c/em\u003e Reduced Hypoxia-Related Genes in Porcine Limbs in Ex Vivo Hypothermic Perfusion Versus Cold Storage. \u003cem\u003eJ Surg Res\u003c/em\u003e \u003cstrong\u003e232\u003c/strong\u003e, 137-145, doi:10.1016/j.jss.2018.05.067 (2018).\u003c/li\u003e\n\u003cli\u003eNieuwenhuijs-Moeke, G. J.\u003cem\u003e et al.\u003c/em\u003e Ischemia and Reperfusion Injury in Kidney Transplantation: Relevant Mechanisms in Injury and Repair. \u003cem\u003eJ Clin Med\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, doi:10.3390/jcm9010253 (2020).\u003c/li\u003e\n\u003cli\u003eRobbins, M. E., Campling, D., Rezvani, M., Golding, S. J. \u0026amp; Hopewell, J. W. The effect of age and the proportion of renal tissue irradiated on the apparent radiosensitivity of the pig kidney. \u003cem\u003eInt J Radiat Biol\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 99-106, doi:10.1080/09553008914551221 (1989).\u003c/li\u003e\n\u003cli\u003eIwase, H., Klein, E. C. \u0026amp; Cooper, D. K. Physiologic Aspects of Pig Kidney Transplantation in Nonhuman Primates. \u003cem\u003eComp Med\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 332-340, doi:10.30802/AALAS-CM-17-000117 (2018).\u003c/li\u003e\n\u003cli\u003eSingh, A., Stablein, D. \u0026amp; Tejani, A. Risk factors for vascular thrombosis in pediatric renal transplantation: a special report of the North American Pediatric Renal Transplant Cooperative Study. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 1263-1267, doi:10.1097/00007890-199705150-00012 (1997).\u003c/li\u003e\n\u003cli\u003eFuquay, R.\u003cem\u003e et al.\u003c/em\u003e Renal ischemia-reperfusion injury amplifies the humoral immune response. \u003cem\u003eJ Am Soc Nephrol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1063-1072, doi:10.1681/ASN.2012060560 (2013).\u003c/li\u003e\n\u003cli\u003eYamada, K.\u003cem\u003e et al.\u003c/em\u003e Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 32-34, doi:10.1038/nm1172 (2005).\u003c/li\u003e\n\u003cli\u003eBolstad, B. M., Irizarry, R. A., Astrand, M. \u0026amp; Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 185-193, doi:10.1093/bioinformatics/19.2.185 (2003).\u003c/li\u003e\n\u003cli\u003evan Hijfte, L.\u003cem\u003e et al.\u003c/em\u003e Alternative normalization and analysis pipeline to address systematic bias in NanoString GeoMx Digital Spatial Profiling data. \u003cem\u003eiScience\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 105760, doi:10.1016/j.isci.2022.105760 (2023).\u003c/li\u003e\n\u003cli\u003eLove, M. I., Huber, W. \u0026amp; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 550, doi:10.1186/s13059-014-0550-8 (2014).\u003c/li\u003e\n\u003cli\u003eBarbie, D. A.\u003cem\u003e et al.\u003c/em\u003e Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e462\u003c/strong\u003e, 108-112, doi:10.1038/nature08460 (2009).\u003c/li\u003e\n\u003cli\u003eHanzelmann, S., Castelo, R. \u0026amp; Guinney, J. GSVA: gene set variation analysis for microarray and RNA-seq data. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 7, doi:10.1186/1471-2105-14-7 (2013).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5220149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5220149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eXenotransplantation (XTx) is an increasingly realistic solution to the organ shortage. Clinical XTx may require off-site procurement in a designated pathogen free (DPF) facility necessitating a period of cold ischemic time during transportation. This study evaluates the impact of different kidney preservation strategies on early graft function in pig-to-baboon XTx in a series of eight cases of pig-to-baboon xenotransplantation performed after five hours of cold ischemic time and compares these results to six cases of pig-to-baboon xenotransplantation performed with minimal ischemic time. Our data indicates that porcine kidneys appear to be particularly sensitive to IRI after cold preservation, especially across xenogeneic barriers, and routine static cold storage leads to hyperacute graft loss even in recipients with low levels of preformed antibodies. Hypothermic machine perfusion minimizes IRI and may prevent early xenograft loss.\u003c/p\u003e","manuscriptTitle":"Routine cold storage leads to hyperacute graft loss in pig-to-primate kidney xenotransplantation; hypothermic machine perfusion may be preferred preservation modality in xenotransplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-18 08:26:43","doi":"10.21203/rs.3.rs-5220149/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7cfdc62-e3f0-4ba2-b7ea-60dbeb9325f4","owner":[],"postedDate":"December 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-16T07:06:11+00:00","versionOfRecord":{"articleIdentity":"rs-5220149","link":"https://doi.org/10.1038/s43856-025-00842-6","journal":{"identity":"communications-medicine","isVorOnly":false,"title":"Communications Medicine"},"publishedOn":"2025-04-15 04:00:00","publishedOnDateReadable":"April 15th, 2025"},"versionCreatedAt":"2024-12-18 08:26:43","video":"","vorDoi":"10.1038/s43856-025-00842-6","vorDoiUrl":"https://doi.org/10.1038/s43856-025-00842-6","workflowStages":[]},"version":"v1","identity":"rs-5220149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5220149","identity":"rs-5220149","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}