Hypothermic Oxygenated Perfusion: a Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation

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Year: 2019

Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation

Kron, Philipp ; Schlegel, Andrea ; Muller, Xavier ; Gaspert, Ariana ; Clavien, Pierre-Alain ; Dutkowski, Philipp

Abstract: BACKGROUND Hypothermic oxygenated perfusion (HOPE) has been shown to protect liver recipients from acute rejection in an allogeneic model of liver transplantation in rats. Here we investigate the impact of HOPE on the T cell-mediated immune response following kidney transplantation. METH- ODS Kidneys from Lewis rats were transplanted into Brown Norway recipients to trigger acute rejection (allogeneic untreated group). Next, Brown Norway recipients were treated either with tacrolimus,= or donor kidneys underwent 1h-HOPE-treatment before implantation without additional immunosuppression in recipients. Syngeneic kidney transplants (Brown Norway to Brown Norway) served as controls. In a second set of experiments, the immune response was assessed in a donation after circulatory death model of kidney transplantation comparing standard cold storage with subsequent HOPE treatment and hypothermic nitrogenated perfusion, where oxygen was replaced during cold perfusion. RESULTS Allo- geneic kidney transplantation led to death in all untreated recipients within 10 days due to severe acute rejection. In contrast, immune activation was prevented by tacrolimus with significantly improved recipient survival. Similarly, HOPE treatment, without any immunosuppression, protected recipients from acute immune response, as measured by less cytokine release, T-cell, and macrophage activation. Addi- tionally, HOPE-treated kidneys showed better function and less early fibrosis leading to a significantly improved recipient survival, compared with untreated allogeneic controls. Similarly, HOPE treatment protected recipients of extended donation after circulatory death kidneys from immune activation. This effect was lost when deoxygenated perfusate was used. CONCLUSIONS In summary, this is thefirst study demonstrating the beneficial effects of HOPE on the immune response following kidney transplantation in an allogeneic rodent model.

DOI: https://doi.org/10.1097/TP.0000000000002634

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-171757 Journal Article Published Version

Originally published at: Kron, Philipp; Schlegel, Andrea; Muller, Xavier; Gaspert, Ariana; Clavien, Pierre-Alain; Dutkowski, Philipp (2019). Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation. Transplantation, 103(5):e128-e136. DOI: https://doi.org/10.1097/TP.0000000000002634 Original Basic Science—General

Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation Philipp Kron,1,2 Andrea Schlegel,1,3 Xavier Muller,1 Ariana Gaspert,4 Pierre-Alain Clavien,1 and Philipp Dutkowski, MD1

Background. Hypothermic oxygenated perfusion (HOPE) has been shown to protect liver recipients from acute rejection in an allogeneic model of liver transplantation in rats. Here we investigate the impact of HOPE on the T cell–mediated immune response following kidney transplantation. Methods. Kidneys from Lewis rats were transplanted into Brown Norway recipients to trigger acute rejection (allogeneic untreated group). Next, Brown Norway recipients were treated either with tacrolimus,= or donor kidneys underwent 1h-HOPE-treatment before implantation without additional immunosuppression in recipients. Syngeneic kidney transplants (Brown Norway to Brown Norway) served as controls. In a second set of experiments, the immune response was assessed in a donation after circulatory death model of kidney transplantation comparing standard cold storage with subsequent HOPE treatment and hypothermic nitrogenated perfusion, where oxygen was replaced during cold perfusion. Results. Allogeneic kidney transplantation led to death in all untreated recipients within 10 days due to severe acute rejection. In contrast, immune activation was prevented by tacrolimus with significantly improved recipient survival. Similarly, HOPE treatment, without any immunosuppression, protected recipients from acute immune response, as measured by less cytokine release, T-cell, and macrophage activation. Additionally, HOPE-treated kidneys showed better function and less early fibrosis leading to a significantly improved recipient survival, compared with untreated allogeneic controls. Similarly, HOPE treatment protected recipients of extended donation after circulatory death kidneys from immune activation. This effect was lost when deoxygenated perfusate was used. Conclusions. In summary, this is the first study demonstrating the beneficial effects of HOPE on the immune response following kidney transplantation in an allogeneic rodent model. (Transplantation 2019;103: e128–e136

INTRODUCTION T-cell–mediated rejections rates in KT are therefore cur- Kidney transplantation (KT) is the treatment of choice for rently low, for example, around 15%.3 However, due to end-stage renal disease with graft survival rates of more frequently used immunosuppressive drug combinations than 90% in the first year.1 Such excellent results have after KT, long-term side effects accumulate during the been achieved by several improvements during the past posttransplant course,5,6 such as infections, an increased 20 years, including advanced donor-recipient matching, number of cardiovascular events, the development of establishment of effective immunosuppressive regimens new cancers, and also chronic kidney dysfunction.5-7 by a combination of steroids, tacrolimus (TAC), mycophe- Accordingly, side effects of immunosuppressive treatment nolate mofetil, or mechanistic target of rapamycin inhibi- are nowadays a main reason for a higher death rate in KT tors, and induction therapy with Basiliximab.2-4 Acute and

Received 25 August 2018. Revision received 8 January 2019. P.K., A.S., and P.D. participated in research design. P.K., A.S., and X.M. participated in the performance of the research. P.K., A.S., A.G., P.D., and P.-A. Accepted 10 January 2019. wrote the article. No authors contributed new reagents or analytic tools. P.K, 1 Department of Surgery and Transplantation, Swiss HPB Centre, University A.S., X.M., and A.G. participated in data analysis. Hospital Zurich, Zürich, Switzerland. Supplemental digital content (SDC) is available for this article. Direct URL 2 HPB and Transplant Unit, St James’s University Hospital, Leeds, United citations appear in the printed text, and links to the digital files are provided in Kingdom. the HTML text of this article on the journal’s Web site (www.transplantjournal. 3 The Liver Unit, Queen Elizabeth Hospital Birmingham, Edgbaston, United Kingdom. com). 4 Department of Pathology and Molecular Pathology, University Hospital Zurich, Correspondence: Philipp Dutkowski, MD, Department of Surgery and Zürich, Switzerland. Transplantation, Swiss HPB Centre, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. ([email protected]). P.K. and A.S. contributed equally as first authors. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. The authors declare no funding or conflicts of interest. The interpretation and reporting of the here-included data are the responsibility ISSN: 0041-1337/19/10305-e128 of the authors. DOI: 10.1097/TP.0000000000002634

e128 Transplantation ■ May 2019 ■ Volume 103 ■ Number 5 www.transplantjournal.com

FIGURE 1. Kidney injury during ﬁrst 10 days after kidney transplantation. HOPE-treated allogeneic kidneys showed signiﬁcantly less release of several markers for oxidative injury and cytokine activation. 8-OHdG, 8-hydroxy-2-deoxy guanosine; ENO, enolase- phosphatase-1; HMGB, high-mobility group box protein-1; HOPE, hypothermic oxygenated perfusion; IL, interleukin; KT, kidney transplantation; PCR, polymerase chain reaction; TAC, tacrolimus; TLR-4, Toll-like receptor-4; TNF-α, tumor necrosis factor α. recipients, compared with the general, healthy population, therefore to test the effect of HOPE on the T-cell–mediated despite functioning graft.6,8-10 immune response in an allogeneic model of KT. Today, an increasing number of extended criteria grafts is used for transplantation, which has triggered an interest in new preservation techniques to improve organ MATERIALS AND METHODS quality and decrease severe complications.11 Various new concepts are currently explored including normothermic Animals regional perfusion in the donor, and ex-situ normother- Male Lewis and Brown Norway (BN) rats (250 to mic and hypothermic perfusion before implantation of 320 g) were used for all experiments. The rodents received 12-15 solid organs. While several groups have assessed the laboratory diet and water according to the Swiss Animal impact of machine perfusion upfront instead of cold stor- Health Care law and experiments were approved by the 16 age in liver and kidney transplantation, our group and animal’s ethics committee (009/2015). Anesthesia during others have introduced a short-term end-ischemic perfu- kidney procurement and transplantation was maintained sion approach, applied after cold storage, hypothermic with isoflurane. oxygenated perfusion (HOPE).17-22 HOPE can therefore be performed in the recipient centre, without transporting Study Design perfusion machines to donors, and is usually continued for 23 Transplantation of kidneys from Lewis to BN rats has only 1 to 2 hours at a temperature of 10°C. We used oxy- previously been shown to activate the immune system and genated Belzer machine perfusion solution (University of to induce acute rejection.27,28 Using this allogeneic renal Wisconsin [UW] gluconate) as perfusate and demonstrated transplant model, we assessed the impact of end-ischemic repeatedly a strong effect on mitochondrial metabolism 21,24 HOPE on immune response. Animals were randomly allo- and on upload of cellular nucleotides. Of note, how- cated to the following experimental groups (Figure S1, ever, while HOPE attenuated ischemia/reperfusion injury SDC, http://links.lww.com/TP/B686): in livers and kidneys,25 it was also effective against innate immune system activation in a recent rodent model of allo- 1. Syngeneic control: Kidneys from BN rats were procured geneic liver transplantation.26 The aim of this study was and transplanted into BN recipients (Syngeneic control).

FIGURE 2. Kidney macroscopy, animal survival, and kidney injury after transplantation. Tacrolimus and HOPE treatment protected kidneys from acute rejection, as demonstrated by a normal kidney size compared with the untreated allogeneic control (A). Consecutively, kidneys maintained their function in both groups throughout the observational period (B). HOPE treatment signiﬁcantly improved recipient survival to 70% (C). HOPE, hypothermic oxygenated perfusion; KT, kidney transplantation; TAC, tacrolimus.

2. Untreated group: Kidneys from Lewis rats were procured or end-ischemic HOPE treatment after cold storage (n = and transplanted into BN recipients without any additional 6). Importantly, we have added another group, in which treatment (allogeneic untreated). kidneys underwent hypothermic perfusion using a deox- 3. Immunosuppression: Kidneys from Lewis rats were pro- ygenated perfusate. This group was the hypothermic cured and transplanted into BN recipients, which received nitrogenated perfusion group (n = 6). Before perfusion, immunosuppressive treatment with TAC (0.1 mg/kg body- weight/day)29 (allogeneic TAC). the perfusate was actively deoxygenated by introducing nitrogen into the perfusate, resulting in a pO of ≤2 pKa. 4. HOPE group: Kidneys from Lewis rats were procured, 2 machine perfused (HOPE) for 1 hour, and transplanted Importantly, 6 animals were assigned to each group with without any additional recipient treatment, explicitly no the endpoint 10 days after transplantation (cold storage: additional immunosuppression (allogeneic HOPE). n = 6, HOPE group: n = 6, and hypothermic nitrogenated perfusion group: n = 6) (Figure S3, SDC, http://links.lww. On days of experiments, random decision about the com/TP/B686). allocation of a rat or respective kidney to experimental groups was performed before the preparation of the experimental settings. To avoid en-bloc experiments for Surgical Technique: Donor and Recipient each group, animals were assigned to syngeneic and allo- Donor kidneys were flushed in situ with 6 mL heparin- geneic groups of different treatments randomly. An overall ized (1 U/mL) saline at room temperature via aorta (renal number of 126 transplant experiments were done in this artery). Kidneys were then excised (weight 0.97 ± 0.053 g) set of experiments. Importantly, experiments were termi- and placed in precooled UW solution (4°C). In the per- nated at each time point (Figure S1, SDC, http://links.lww. fusion group, kidneys received stents for the aorta (renal com/TP/B686). artery). Ureters of all kidneys were stented (24G). To assess the impact of HOPE on reperfusion injury Before implantation, kidneys were flushed with 5 mL and subsequent immune system activation in a clinically cold UW solution and a cuff was inserted in the renal relevant scenario, we have added another set of experi- vein. Subsequently, KT was performed as previously ments, simulating donation after circulatory death (DCD) described.30,31 Before kidney implantation, all recipients KT. Rats were exposed to 30 minutes in situ asystolic underwent bilateral nephrectomy.32 Importantly, the cold warm ischemia time, followed by cold kidney flush and storage time was kept minimal in all groups (<60 min), by 18 hours of cold storage, performed in UW solution at means the time required to prepare the recipient and per- 4°C. No heparin was used in the donors. Rat kidneys form bilateral nephrectomy as mentioned above. HOPE- were randomly assigned to either cold storage (n = 6) treated kidneys experienced the longest out of body time

FIGURE 3. T-cell activation 10 days after kidney transplantation. In addition to treatment with tacrolimus, solitary graft treatment with HOPE achieved a protection from T-cell activation, circulation, and inﬁltration into transplanted allogeneic kidneys, as demonstrated by FACS analysis from blood samples (CD3, CD4, and CD8) and kidney histology (CD3 staining) (A–C). FACS, ﬂuorescence-activated cell sorting; HOPE, hypothermic oxygenated perfusion; KT, kidney transplantation; TAC, tacrolimus. by 1 hour cold perfusion. No animals died during the sur- α (TNF-α; R&D, RTA00), IL-1β (R&D, DLB 50) and eno- gical procedure of KT. lase-phosphatase-1 (ENO; Mybiosource, MBS9342292).35 Next, quantitative real-time polymerase chain reaction Hypothermic Oxygenated Perfusion (TaqMan gene expression assays) was done for TNF-α (Thermofisher, Rn00562055), Myd 88 (sinobiological, HOPE was performed for 1 hour through the renal RP 300783), IL-6 (Thermofisher, Rn01410330_m1), and artery (aortic patch) with a constant perfusion pressure of 33 IL-10 (Thermofisher, Rn01483987_m1). <18 mm Hg, and active oxygenation (pO2 60 to 80 kPa) 34 The following staining procedures were performed: as previously described. We used 50 mL recirculating Hematoxylin-Eosin (H&E) staining (tubular injury, necro- Kidney-Perfusion–Solution-1 as perfusate, which mainly sis), TLR-4-staining (macrophage and tubular activation; consists of hydroxyethyl starch (50 g/1000 mL), sodium LS-B2070), CD3 staining (T lymphocytes; Diagnostic gluconate (17.45 g/1000 mL), Mannitol (5.4 g/1000 mL), Biosystems, RMAB005), CD68 (macrophages and den- potassium phosphate (3.4 g/1000 mL), and HEPES dritic cells; macrosialin; MCA341R), C5 (activation of (2.38 g/1000 mL). Perfusion box and perfusate were pre- Complement 5 [C5]; Lifespan Bioscience, LS-C313394), cooled and maintained at 4°C by an open bath thermostat. and Sirius red staining (staining for fibrosis) to confirm tissue remodeling into fibrosis. Quantification of each Endpoints (Polymerase Chain Reaction, Assays, and marker was performed in 8 random fields of 2 slides per Fluorescence-activated Cell Sorting Analysis) animal. To further assess acute kidney rejection, the Banff Posttransplant kidney function and injury were analyzed classification was determined on the Periodic Acid Schiff by creatinine in recipient plasma samples (serum multi- staining. The histological evaluation and quantification ple biochemical analyzer DRI-CHEM4000i; FUJIFILM, was done in a blinded fashion by an expert pathologist Japan). Oxidative damage of DNA was detected in (A.G.), not involved in the experiments. plasma by 8-hydroxy-2-deoxy guanosine (8-OHdG) Additionally, the T-cell response was examined by ELISA (Abnova, KA0444) and nuclear injury was meas- fluorescence-activated cell sorting (FACS)-analysis in ured by release of high-mobility group box protein-1 (IBL recipient rat blood as described below: Following blood International GmbH, ST51011). In addition, plasma pro- sampling, 1.5 mL of heparinized full rat blood was used tein quantification by ELISA was performed for Toll-like for analysis and white blood cells were isolated using receptor-4 (TLR-4; R&D, 29260), Interleukin-6 (IL-6; Ficoll and DMEM + P/S buffer. Cells were then incu- R&D,R6000), IL-10 (R&D, R1000), tumor necrosis factor bated with various combinations of mAbs (anti-Rat CD3,

FIGURE 4. Tissue markers of rejection and ﬁbrosis 10 days after kidney transplantation. Allogeneic kidneys transplanted without any treatment showed severe activation of macrophages and dendritic cells (A). In this context, early signs of severe ﬁbrosis were found. In contrast, HOPE treatment or immunosuppression were equally protective (A and B). HOPE, hypothermic oxygenated perfusion; KT, kidney transplantation; TAC, tacrolimus.

PerCP-eFluor710 single staining, anti-rat CD4-PE com- and release of high-mobility group box protein-1, TLR-4, bined with anti-rat CD25-FITC, and anti-rat CD8-FITC MYD 88, TNF-α, IL-6, IL-10, and IL-1b (Figures 1 and combined with anti-rat CD28-PE) and afterward washed 6, Figure S2, SDC, http://links.lww.com/TP/B686).35,36 twice with FACS buffer and fixed with 1% paraformal- Consecutively, plasma creatinine levels demonstrated a dehyde. Three-color immunofluorescence staining was steady increase within the first 10 days after KT compared analyzed using a FACS Calibur instrument (FACS diva, with syngeneic controls (Figure 2B). In addition, untreated version 6.1.2). Lymphocytes were gated using forward and recipients of allogeneic kidneys developed a severe form side scatter to exclude debris and dead cells. Afterward, at of T-cell (CD3, CD4, and CD8)–mediated acute rejec- least 50 000 events were acquired in each assay for analy- tion underlined by significant increase in kidney size sis. Follow up for survival were 10 days after KT. (Figure 2A), macrophage activation, and T-cell infiltration (Figures 3A, B and 4A). Such histological findings were further underlined by quantification of high amounts of cir- Statistics culating and activated T cells in the recipient blood (FACS Data are presented as median and IQR or as frequency analysis, Figure 3C). Consistently, T-cell activation resulted in n and percent. Statistical analysis was performed using in an early graft fibrosis, expressed by an increased posi- the nonparametric Mann-Whitney-Wilcoxon U-test, the tivity in Sirius red staining (Figure 4B). Additionally, sig- 2-way Anova, and the log-rank test for animal survival nificant C5 expression was found in untreated allogeneic (GraphPad Prism, version 7.0, San Diego, CA). kidneys (Figure 5A). Histological grading for rejection, using the Banff classification, confirmed acute rejection in RESULTS untreated animals (Figure 5B and C), which was lethal to all recipients in the allogeneic untreated group (Figure 2C). Reperfusion Injury and Development of Acute To assess the effect of the regular immunosuppressive Rejection in the Allogeneic Model of KT With and treatment used in clinical practice, the next set of allo- Without Immunosuppression geneic kidney recipients received TAC (0.1mg/kg body- In the first step, we assessed reperfusion injury after weight/day; allogeneic TAC group), resulting in a trough KT in an allogeneic model. Untreated recipients of allo- level of 8 to 10 ng/L in accordance with earlier studies.37 geneic kidneys developed reperfusion injury within the Expectedly, TAC treatment decreased significantly the first day after transplantation as documented by 8-OHdG extent of immune system activation, such as the number

FIGURE 5. Complement 5 staining and histological scoring according to the Banff classiﬁcation 10 days after kidney transplantation. Expectedly, immunosuppressive treatment prevented development of acute rejection as shown by the Banff classiﬁcation and Complement 5 staining. Importantly, equal protection was achieved through 1-hour cold HOPE perfusion before kidney implantation (A-C). HOPE, hypothermic oxygenated perfusion; KT, kidney transplantation; PAS, Periodic Acid Schiff; TAC, tacrolimus. of activated macrophages, the number of T cells in blood fibrosis as demonstrated by less positivity for Sirius and transplanted kidneys, the Banff classification, and the red (Figures 4B, 5 and 6). Consistently, animal survival extent of graft fibrosis (Figures 2–5). TAC improved also significantly improved to 71% (5/7) in the allogeneic graft function and increased animal survival to 90% com- HOPE group (Figure 2C). Furthermore, and in contrast pared with untreated animals (Figure 2C). Of note, how- to the TAC group, HOPE treatment downregulated also ever, reactive oxygen species (ROS)-related nuclear injury the initial ROS release upstream to T-cell activation as quantified by 8-OHdG release was similar comparing (Figures 1A and 6). the allogeneic TAC group and the untreated allogeneic control group (Figure 1A). Protective Effect of HOPE in a DCD Model of KT To investigate the protective effect of HOPE treat- HOPE Treatment Protects From Acute Kidney ment in a transplant model of increased injury, we per- Rejection and Early Fibrosis formed another set of experiments where kidneys from In the third step, we investigated the effect of HOPE DCD donors were transplanted after 30 minutes of asys- treatment of kidney grafts on reperfusion injury and tolic warm ischemia time and 18 hours of cold storage consecutive immune response after allogeneic KT. One- (Figure S3, SDC, http://links.lww.com/TP/B686). In this hour HOPE treatment before implantation significantly group with maximal injury, we observed severe reperfu- decreased reperfusion injury, similarly as previously sion injury and immediate activation of the innate immune reported in a syngeneic model (Figure 1, Figure S2, SDC, system (Figure S4, SDC, http://links.lww.com/TP/B686). http://links.lww.com/TP/B686). More importantly for HOPE treatment before implantation conveyed protection this study, the number of activated macrophages and T of kidneys from the expected injury. Importantly, HOPE cells in recipient blood (CD3, CD4, and CD8) and kid- decreased also the activation of innate immune cells, as ney tissues were also significantly reduced after HOPE confirmed by less infiltrating T cells (CD3 positive) and treatment, despite the absence of immunosuppressive a reduced number of activated positive T cells in kidney treatment in recipients. This effect was almost compara- tissues as confirmed by CD4 and CD8 staining (Figure S4, ble to the allogeneic TAC group (Figures 3A–C and 4A). SDC, http://links.lww.com/TP/B686). In contrast, nonoxy- Such immune modulatory effects were further paralleled genated perfusion resulted in a similar reperfusion injury by a low Banff classification and less positive endothe- and T-cell activation when compared with the un-perfused, lial for C5 in kidney tissues with no signs of early graft cold storage group. These experiments confirmed the key

FIGURE 6. Underlying mechanism of reperfusion injury with the link to immune response. Mitochondrial ROS release results in nuclear DAMP release and activation of TLR4 receptors on macrophages and dendritic cells. TLR4 signaling activate proinflammatory cytokine production via MyD88 dependent pathways and triggers maturation of dendritic cells, which in turn present antigens to T cells. Subsequent activation and infiltration of T cells results in graft inflammation and further injury, including fibrosis. Hmgb1, high- mobility group box protein-1; HOPE, hypothermic oxygenated perfusion; IL, interleukin; IFN, interferon; KT, kidney transplantation; PCR, polymerase chain reaction; ROS, reactive oxygen species; TAC, tacrolimus; TLR-4, Toll-like receptor-4; TNF-α, tumor necrosis factor α. role of oxygen during hypothermic perfusion, similarly as high perfusion tradition in human kidneys, the optimal seen in livers, to achieve protection. method for improving best kidney quality and utilization rates remains under debate. Several competing and different strategies have been discussed. First, continuous hypo- DISCUSSION thermic perfusion instead of cold storage was shown to The use of machine perfusion approaches in organs decrease delayed graft function and increase graft survival before transplantation is currently a hot topic to opti- in a large randomized trial.41 Second, as a practical and mize organ function. Besides, however, direct effects on easy modification, hypothermic machine perfusion applied ischemia reperfusion injury, machine perfusion techniques after cold storage has recently been shown to be also supe- will likely also target immune pathways.38,39 Accordingly, rior, compared with cold storage alone.42 Third, normother- this study shows that a simple kidney graft treatment mic end-ischemic perfusion with a blood-based perfusate (HOPE) decreased the innate immune response in an was suggested to be even more protective and to offer the allogeneic model of KT. These effects were evident, first, advantage assessing kidney graft function before implan- by a decreased activation of macrophages and T cells in tation.43,44 The shortcoming, however, of this approach transplanted kidneys, and by less circulating CD3-, CD4-, is the potential increasing release of signaling cytokines and CD8-positive T cells in blood. Second, HOPE-treated throughout the entire normothermic kidney perfusion.45 allogeneic kidneys showed only minor complement acti- The finding of circulating mediators during normothermic vation and low Banff scores of rejections within the first kidney perfusion resulted therefore in attempts to include 10 days after transplantation, despite absence of any cytokine filters.45 Cytokine activation during end-ichemic immunosuppressive treatment. Finally, HOPE treatment normothermic perfusion after ischemia, however, indicates protected kidneys from fibrotic remodeling, achieved sig- reperfusion injury, and rather points to activation of dan- nificantly improved kidney function, and increased ani- ger signaling pathways, consistent to experiences in lung mal survival. and heart perfusion.45-48 Machine perfusion has a longstanding tradition in the In contrast, an end-ischemic cold and oxygenated perfu- field of KT, in contrast to other solid organs.40 Despite this sion strategy, for example, HOPE, appears to protect from

Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. © 2019 Wolters Kluwer Kron et al e135 initial release of ROS and DAMPs, as demonstrated in pre- gained through “traditional” immunosuppressive drugs. vious rodent transplant studies of kidney and liver.20,21,26,49 HOPE treatment alone prevented rejection despite high In addition to beneficial effects of HOPE treatment against immunogenic risk, evident by decreased T-cell and mac- oxidative stress, we show in the present study that HOPE rophage activation, and significantly improved survival. treatment protected rat kidneys from T-cell (CD3, CD4, Such cold perfusion approach, before transplantation, and CD8)–mediated rejection, without any additional appears therefore to be an easy but effective method to immunosuppression, throughout 10 days after transplan- modulate the immune response following allogeneic KT. tation. 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