Synergistic effects of prolonged warm ischemia and donor age on the immune response following donation after cardiac death kidney transplantation

Synergistic effects of prolonged warm ischemia and donor age on the immune response following donation after cardiac death kidney transplantation Christian Denecke, MD,a,b Xiaodong Yuan, MD,a Xupeng Ge, MD,a Irene K. Kim, MD,a Daman Bedi, MD,a Olaf Boenisch, MD,c Anne Weiland,a Anke Jurisch,a Katja Kotsch, PhD,b Johann Pratschke, MD, PhD,b Anja Reutzel-Selke, PhD,d and Stefan G. Tullius, MD, PhD,a Boston, MA, Innsbruck, Austria, and Berlin, Germany

Background. Organs from DCD (donation after cardiac death) donors are increasingly used for transplantation. The impact of advanced donor age and warm ischemia on the immune response of the recipient has not been studied. We developed a novel and clinically relevant model of DCD kidney transplantation and investigated the effects of donor age and prolonged warm ischemia on the recipient immune response after following DCD kidney transplantation. Methods. DCD grafts from young and old F-344 donor rats were engrafted into LEW recipients who were nephrectomized bilaterally after a short (20 minutes) or prolonged (45 minutes) warm ischemia time. Results. Analysis of the recipient’s immune response early after transplantation showed an enhanced innate and adaptive immune response when old DCD kidneys were engrafted. Next, we studied DCD recipients with a supportive, contralateral native kidney in place, which allowed the recovery of the transplanted DCD kidney. Old DCD kidneys, demonstrated an impaired renal function associated with pronounced histomorphologic graft deterioration and an enhanced immune response by day 100 after transplantation. Interestingly, young DCD kidneys with a long warm ischemic time recovered from acute tubular necrosis and did not stimulate the long-term immune response. Conclusion. Our observations emphasize that prolonged warm ischemic time and advanced donor age augment the immune response after transplantation of DCD grafts. These results provide an experimental model and a mechanistic framework of clinically relevant aspects in DCD donation. (Surgery 2013;153:249-61.) From the Transplant Surgery Research Laboratory and Division of Transplant Surgery,a Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; Department of Visceral, Transplantation and Thoracic Surgery,b Medical University, Innsbruck, Austria; Transplantation Research Center,c Brigham and Women’s Hospital and Children’s Hospital of Boston, Boston, MA; and Department of General, Visceral and Transplantation Surgery,d Charite Virchow Clinic, Berlin, Germany

THE NUMBER OF DCD (DONATION AFTER CARDIAC DEATH) organs that are used for transplantation is constantly Supported by a grant from the Fundacio Carlos Slim (to SGT) and from the Deutsche Forschungsgemeinschaft (De 1452 /1-2) (to CD). Christian Denecke and Xiaodong Yuan contributed equally to this article. Current address of Xiaodong Yuan: Minimally Invasive Urology Center, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China. Current address of Xupeng Ge: Department of Anesthesia, Ronald Reagan Medical Center, UCLA, Los Angeles, CA. Accepted for publication July 30, 2012. Reprint requests: Stefan G. Tullius, MD, PhD, Division of Transplant Surgery, Brigham and Women’s Hospital, 15 Francis Street, Boston, MA 02115. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2012.07.035

increasing. Limitations and criteria in regard to donor age or duration of warm ischemia time are not yet clearly defined. It is well known that kidneys from DCD have greater rates of delayed and primary nonfunction as a result of extensive acute tubular necrosis (ATN).1 Provided a careful donor selection is made, long-term outcome of DCD kidneys has been equivalent to kidneys from brain-dead donors.1,2 Interestingly, DCD kidneys undergoing delayed graft function demonstrate a better long-term function than kidneys from brain-dead patients with delayed graft function, suggesting injury specific effects.3 Explanations for this phenomenon may include adverse consequences of brain death and an improved recovery of DCD organs also potentially linked to age-specific repair mechanisms. Nonetheless, the consequences of prolonged warm ischemia are closely associated with DCD donation. SURGERY 249

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Fig 1. (A) The DCD model (I). After asystole, 3- or 18-month-old donor F-344 kidneys were exposed to a either short (20 minutes) or prolonged (45 minutes) warm ischemia time and subsequently transplanted into 3-month-old native Lewis recipients. Recipients received a short course of clcyosporine A (CsA; 1.5 mg/kg for 10 days). Although the right native kidney was removed during transplantation, the contralateral kidney was left in situ for 1 week to allow for recovery from acute tubular injury. Recipients were sacrificed after 24 hours and 10 days; graft, spleen, and draining lymph nodes were collected for further analysis (n = 5 per group). In additional groups, the native contralateral kidney was kept in place for 4 weeks to allow the recovery of ATN after warm ischemia (n = 5/group). Living donors (3-monthold F-344) served as controls (n = 5). (B) The DCD model (II). Depicted is a characteristic blood pressure curve during the different stages of the DCD procedure. See text for methodology. After the intravenous administration of heparin, donor rats were paralyzed with rocuronium and the ventilator was withdrawn 3 minutes after the cessation of any respiratory activity. Ischemic asystole occurred after an average of 8 minutes and was defined as the start of warm ischemia time in this model. (Color version of figure is available online.)

Their effects on the recipient’s immune response remain to be elucidated. Brain death has been found to stimulate the early immune response in both, clinical and experimental studies.4-6 Likewise, advanced donor age is known as a major risk factor for primary nonfunction, delayed graft function, and long-term allograft dysfunction in organs originating from brain-dead donors.7-9 The correlation of these factors has not been investigated in DCD donors. Experimental studies on the impact of advanced donor age on the recipient’s immune response are inconsistent. Most but not all experimental studies suggested an increased immunogenicity of older donor kidneys, leading to an augmented adaptive

immune response and more frequent acute rejection rates.10-13 Prolonged warm ischemia is a unique feature of DCD organs linked to cellular damage---reflected by severe acute tubular necrosis---release of reactive oxygen species, up-regulation of cellular adhesion molecules, attraction of host leukocytes, and subsequent immunologic activation.14 On the basis of clinical experience, recommendations by the American Society of Transplant Surgeons suggest to limit warm ischemia times for liver grafts to 20 to 30 minutes and 45 to 60 minutes for kidney grafts.15 However, the longterm immunologic consequences of prolonged warm ischemia time in DCD kidneys have not

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Fig 2. Outcome of DCD grafts was influenced by ischemia and donor age. Although recipients of living donor grafts and DCD grafts with a short warm ischemia time (WIT) survived long term (>100 days), survival was decreased by 50% if the graft was exposed to prolonged warm ischemia time. Surprisingly, graft survival was donor age independent. Both, young and old DCD grafts recovered if a native kidney was kept in place for 4 weeks (n = 5 each group). Nx, Nephrectomy.

been studied. The correlation of increased donor age and warm ischemia in DCD organs is of great clinical importance because these factors may impact on the immunogenic potential and alter the immune response to DCD organs. In addition, recipients of DCD kidneys often receive the same immunosuppression as recipients of organs from brain-dead patients despite fundamental differences in organ quality, graft activation, and longterm graft outcome. Therefore, we thought that it was critical to examine the consequences of advanced donor age and prolonged ischemia time on the recipient’s immune activation. For this purpose, we developed an experimental DCD transplant model that we used to analyze the effects of advanced donor age and prolonged warm ischemia time on recipient immune response and long-term graft outcome. Specifically, we conducted a first set of experiments in which we investigated early immune responses after DCD transplantation and a second set of experiments that was designed to test recovery and long-term outcome of DCD grafts. To the best of our knowledge, this is the first experimental study to test the consequences of DCD donation and age on the adaptive immune response. MATERIALS AND METHODS Rats. Three-month-old F-344 and Lewis rats (150–200 g) were purchased from Charles River (Wilmington, MA), and 18-month-old F-344 rats were obtained from the National Institute of Aging (Bethesda, MD; 250–350 g). DCD donor procedure. To simulate a ‘‘controlled’’ DCD process (Maastricht Type III), young (3 months) and old (18 months) old F-344 donors were anesthetized, and the left femoral artery was canulated with a PE catheter (0.40 mm inner diameter) to monitor blood pressure (Fig 1). After a

tracheostomy, the donor was intubated using a 16G cannula and ventilated at a stroke rate of 47 per minute with a tidal volume of 2.0–2.5 mL. Once the animal showed stable ventilation and hemodynamics, heparin (100 IU/kg intravenously) and rocuronium (2 mg/kg intravenously) were administered. After paralysis was induced, the ventilator was withdrawn, and blood pressure was monitored until hypoxemic asystole occurred. Asystole was defined as the start of warm ischemic time. After a short (20 minutes) or long (45 minutes) warm ischemia time kidneys were then perfused in vivo with University of Wisconsin solution and transplanted into young Lewis recipients with a reproducible and short ischemic time of 20 compared with 45 minutes. Living donors (3-monthold F-344 into 3-month-old Lew recipients) served as controls. All kidney recipients were treated with low-dose cyclosporine A (1.5 mg/kg intraperitoneally for 10 days) to prevent early acute rejections. Orthotopic kidney transplantation. Orthotopic kidney transplants from 3- or 18-month-old F-344 donors into 3-month old Lewis recipients were performed by standard microvascular techniques (anastomosis time 25 ± 5 minutes). One week after transplantation, the contralateral native kidney was removed. Graft survival is shown as the median survival time in days. All kidney grafts were procured routinely on days 1 and 10, respectively. In additional groups, animals remained with a contralateral kidney in place until week 4 to allow graft recovery from ATN. Renal function. Serum and urine samples were collected 4, 8, and 12 weeks after transplantation. Creatinine clearance (CrCl) was calculated as: urine creatinine (mg/mL) 3 urine volume (mL)/serum creatinine (mg/mL) 3 time of urine collection (minutes). Proteinuria, the most

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* P<.05 Fig 3. Early innate immune responses following DCD donation. Grafts and draining lymph nodes (dLN) were collected 24 hours after transplantation, and frequencies of immune cells were assessed via FACS analysis (n = 5). Prolonged warm ischemia time (WIT; 45 minutes) in young DCD donors was associated with a significant expansion of MHC class II– expressing dendritic cells (OX62) in recipient dLN. In contrast, advanced donor age but not extended warm ischemia led to a marked increase of NK cells (CD161) in recipient dLN and spleens by 24 hours.

sensitive parameter for chronic renal allograft nephropathy in this model was tested at similar time intervals. Urinary excretion of protein (mg/ 24 h) was assessed by turbidimetry and benzethonium chloride (C27H42NO2Cl) precipitation. Flow cytometry. Cells were washed in phosphate-buffered saline containing 2% serum (Bio Whittaker, Walkersville, MD) to block nonspecific FcR-binding. Lymphocytes were stained with directly conjugated anti-CD8, anti-CD4, antiCD86, anti-CD45RA, anti-TCR, anti-CD28, anti-

CD45RC, anti-CD161, anti-OX62, anti-RT1B (MHC class II; All mAbs BD Biosciences (San Diego, CA), or anti-CD25 (Serotec, Raleigh, NC). For FoxP3-staining, a commercially available kit (eBioscience, San Diego CA) was used. ELISPOT assay. Splenocytes from naive or transplanted Lewis rats were procured and stimulated subsequently with na€ıve, wild-type F-344 splenocytes. The ELISPOT assay (using the Rat IFNg Elispot set, BD Biosciences) was used to measure the frequency of alloreactive T-cells producing

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* P<.05 Fig 4. T-cell stimulation after engraftment of old DCD kidneys. Although analysis of T-cell subsets in recipient dLN early after transplantation did not reveal differences between groups (n = 5), greater frequencies of effector/memory T cells (CD45RC ) and regulatory T-cells (CD25+FoxP3+) were observed in recipients spleens early after transplantation of old DCD grafts.

interferon-g (IFN-g) as previously described.16 The resulting spots were counted on a computer-assisted enzyme-linked immunospot image analyzer (Cellular Technology), and frequencies were expressed as the number of cytokine-producing spots per 0.5 3 106 splenocytes. Immunohistochemistry. Immunohistochemistry was performed using 4-mm-thick acetone-fixed, OCT-embedded rat tissue sections. All further steps were performed at room temperature in a

hydrated chamber. Slides were pretreated with Peroxidase Block (DAKO USA, Carpinteria, CA) for 5 minutes to quench endogenous peroxidase activity. Endogenous biotin was blocked using Biotin-Blocking System (DAKO) as per the manufacturer’s protocol. To minimize reactivity of antimouse secondary antibodies with endogenous immunoglobulin, all primary mouse anti-rat antibodies, including CD68 (clone: ED1, AbD Serotec, cat. no. MCA341GA), CD8alpha (clone:

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Fig 5. Alloreactive immune responses after DCD engraftment. To test alloreactive IFN-g secretion, recipient splenocytes were cocultured in ELISPOT plates with irradiated stimulator cells of donor origin, and the resulting IFN-g spots were counted. By 24 hours after transplantation, an early additive effect of prolonged warm ischemia time and advanced donor age on alloreactive IFN-g secretion was observed (P < .001). In contrast, by day 10 after transplantation, both recipients of young and old DCD grafts demonstrated an increased in vitro responsiveness compared with LD, suggesting an ongoing strong adaptive immune response after DCD transplantation at this time (n = 5 per group).

MRC-OX-8, AbD Serotec, cat. no. MCA48GA), CD4 (clone: W3/25, AbD Serotec, cat. no. MCA55GA), DC (OX62, clone: OX62, AbD Serotec, cat. no. MCA1029G), CD45RA (clone: OX33, AbD Serotec, cat. no. MCA340G) + MHC II (clone: OX3 AbD Serotec, cat. no. MCA45G) were preincubated with ARK (Animal Research Kit) peroxidase (DAKO) as per the manufacturer’s instructions; these were applied in DAKO diluent at 1:10 for CD4 and MHC IICD68 at 1:200, CD8alpha at 1:100, OX62 at 1:75, and CD45RA at 1:80 for 1 hour. Slides were washed in 50-mM Tris-Cl, pH 7.4, and detected with streptavidin-horseradish peroxidase (DAKO) for 15 minutes. After further washing, immunoperoxidase staining was developed with a DAB chromogen (DAKO) and counterstained with hematoxylin. Cells in 5 high-power fields were counted and stated as mean ± SD. Hematoxylin and eosin staining. Renal graft samples fixed in 10% formalin were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for the evaluation of acute graft lesions (glomerular infiltration, arteritis, acute tubular necrosis, and interstitial cellular infiltration). The severity of lesions was scored from 0 (no changes) to 4 (severe infiltration/destruction of parenchyma). The evaluation was conducted in a blinded fashion. Statistics. Comparisons between experimental groups were performed with the analysis

of variance one-way test. All results were generated with GraphPad Prism software (San Diego, CA). RESULTS The early immune response to DCD organs is augmented by prolonged warm ischemia time. To examine the effects of advanced donor age and prolonged warm ischemia time, we developed a novel DCD model (Fig 1, A and B). In a first set of experiments, recipients were bilaterally nephrectomized at the time of transplantation and the immune response was examined at 24 hours and 10 days after transplantation (Fig 1, A). Interestingly, graft survival was unaffected by donor age and prolonged warm ischemia time, leading to a graft failure rate of 50% (P < .05; Fig 2). Next, we tested the impact of prolonged warm ischemia and donor age as single and combined risk factors on the activation of the immune response 24 hours after transplantation. As shown in Figure 3, relative amounts of activated dendritic cells and natural killer cells were enhanced with prolonged warm ischemia time and advanced donor age, respectively. In parallel to an augmented innate immune response, prolongation of warm ischemia time and advanced donor age were also associated with an early activation of the CD4+ T-cell responses as demonstrated by greater percentages of

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* P<.05 Fig 6. Donor age and prolonged warm ischemia augment CD4+ T-cell responses after DCD donation. In line with the enhanced alloreactive IFN-g secretion, greater frequencies of effector/memory T cells (CD45RC ) were observed in dLNs and spleens after the transplantation of older DCD grafts with prolonged WIT. In parallel, greater frequencies of regulatory T cells (CD25+FoxP3+) were observed in recipient dLNs, suggesting that donation of old DCD grafts with prolonged warm ischemia time was associated with an overall enhanced T-cell response.

effector/memory CD4+ T cells (CD4+CD45RC T cells) and regulatory T cells (Fig 4). Moreover, Tcell alloreactivity as measured by IFN-g release in ELISPOT analysis was most pronounced in recipients of old DCD kidneys (Fig 5). Taken together, prolongation of warm ischemia time and advanced donor age caused early immune activation after DCD engraftment. Synergistic allostimulatory effects of donor age and warm ischemia can be observed 10 days after transplantation. Next, we analyzed the immune

response by 10 days. At this later time point, warm ischemia and advanced donor age were associated with increased frequencies of effector/memory T cells (CD4+CD45RC ) in draining lymph nodes and spleens, respectively (Fig 6). Similarly, in vitro alloreactivity as measured by IFN-g secretion upon allostimulation (ELISPOT) was more pronounced when the warm ischemic time was prolonged or when older DCD kidneys were transplanted (Fig 5). Moreover, augmented alloreactive T-cell responses toward older DCD grafts were paralleled by an

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* P<.05 Fig 7. Donor age and prolonged warm ischemia are associated with a stronger activation of dendritic cells and B cells after transplantation. In line with the more prominent T-cell response by day 10 after transplantation, greater frequencies of activated B cells were observed in recipients of old DCD grafts (n = 5). Likewise, activated DCs had an increased response, indicating an enhanced adaptive immune response in association with warm ischemia time and donor age.

expansion of CD4+CD25+FoxP3+ regulatory T cells in draining lymph nodes (Fig 6). Interestingly, old DCD grafts also provoked a significant expansion of B cells (CD45RA+MHCII+) and activated dendritic cells in recipient’s draining lymph nodes at this later time point (Fig 7). These data suggest that the early innate and CD4+T-cell response towards DCD grafts with prolonged warm ischemia time translated into a

strong adaptive immune response, causing graft rejection of 50% of transplanted DCD kidneys. To examine the recovery potential of DCD grafts undergoing delayed graft function and to further scrutinize the immune response long term, we set out to study a second set of experiments in which young and old DCD kidneys (45 minutes’ warm ischemia time) were engrafted under the protection of a native contralateral kidney.

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Fig 8. Recovery of DCD grafts. Long-term graft function of old DCD kidneys that had recovered in the presence of a native kidney was significantly impaired as measured by creatinine clearance (Crea Cl) and proteinuria by day 100 (A). In contrast, young DCD grafts with prolonged warm ischemia demonstrated an unimpaired creatinine clearance (P = n.s.) in the absence of a significant proteinuria, suggesting a potential for recovery from ATN. In line with the functional parameters, histologic deterioration was most advanced in old DCD organs whereas young DCD grafts showed only moderate cellular infiltrates and mild glomerular and tubular alterations (B). LD, Living donor. (Color version of figure is available online.)

DCD grafts recover from prolonged warm ischemia time. Next, we followed DCD recipients of either young or old kidneys in which a contralateral na€ıve kidney was kept in situ to allow the recovery from ATN. Interestingly, if the ‘‘protective’’ contralateral native kidney was removed by week 4 after transplantation, all recipients of DCD kidneys with prolonged warm ischemia time survived long-term (Fig 2). These results indicate that even very old DCD kidneys that were subject to a prolonged warm ischemia time harbor a potential for recovery after tubular injury. Older DCD grafts showed significant functional and structural deterioration and accelerated the recipient’s immune response. Although old DCD kidneys recovered, advanced donor age was associated with a worse renal function, as indicated by a decreased creatinine clearance and increased proteinuria (Fig 8, A). Next, long-term surviving recipients were sacrificed at day 100 after transplantation, and grafts were examined for structural changes. Indeed, impaired kidney function was associated with

advanced structural deterioration in old DCD organs (Fig 8, B). Young DCD organs recovered well after prolonged warm ischemia time with only mild structural changes. Old DCD kidneys, however, demonstrated advanced signs of chronic inflammatory changes including interstitial fibrosis, glomerular sclerosis, tubular atrophy and vasculopathy. In accordance with these findings, we observed intense T-cell, B-cell, and dendritic cell infiltration in grafts from older donors (CD4+T cells: LD vs DCD young vs DCD old: 5.25 ± 2.0 vs 6.21 ± 0.26 vs 39.55 ± 20.52; CD45RA+B cells: LD vs DCD young vs DCD old: 2.26 ± 1.22 vs 4.66 ± 0.85 vs 16.00 ± 1.79; CD8+T cells: LD vs DCD young vs DCD old: 14.74 ± 1.57 vs 18.90 ± 2.93 vs 31.46 ± 4.05; ED1+macrophages: LD vs DCD young vs DCD old: 14.65 ± 5.19 vs 26.68 ± 4.71 vs 68.32 ± 1.94; P < .05; Fig 9). Although cellular infiltrates were more prominent in older DCD kidneys, analysis via fluorescence-activated cell sorting (FACS) of recipient’s spleens did not reveal a different T-cell response to old DCD donor organs by day 100 (Fig 10, A). Although the frequencies of regulatory

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Fig 9. Increased cellular infiltrates in old DCD grafts. DCD grafts of long-term surviving recipients were procured by 100 days. Greater counts of cellular infiltrates were observed in old DCD grafts, suggesting that the systemic immune response translated into an accelerated graft rejection. (Color version of figure is available online.)

T cells had decreased after transplantation of old DCD kidneys, a trend towards greater numbers of effector/memory T cells was observed. The altered ratio of effector and regulatory T cells was further confirmed by a significantly stronger alloreactive IFN-g secretion (ELISPOT) in recipients of old DCD grafts (Fig 10, B). In contrast, young DCD organs subjected to a long warm ischemia time did not accelerate recipient systemic immune response. Taken together, we observed an age-dependent, more pronounced systemic immune response linked to a more rapid graft loss of old DCD kidneys and impaired renal function. Interestingly, young DCD organs recovered well after ATN, and the engraftment of old DCD kidneys was linked to a more intense immune response. These data suggest that donor age and prolonged warm ischemia time have synergistic adverse effects. DISCUSSION DCD donation has been established as a valuable source to increase the availability of organs.

Clinically, long-term outcome of DCD kidneys has been comparable with that seen in organs from brain-dead patients. Age limits for DCD donation, however, remain to be discussed controversially and little is known about the influence of prolonged ischemia on organ damage and the recipient’s immune response. We developed a novel DCD rat model that allowed us to study the combined influence of donor age and prolonged warm ischemia on the immune response to DCD organs in a clinically relevant fashion. Many experimental models of DCD use a vascular clamping approach to induce warm ischemia, which leads to a sympathetic storm and worsening of graft quality caused by cardiac hypoxia.17 In contrast, in our model, the technique of ventilator switch-off precedes a phase of hemodynamic instability followed by ischemic asystole and a period of warm ischemia, thus mirroring the clinical situation of DCD. Of note, warm ischemia was defined as the time from asystole to organ recovery. The period of relative hypoxia until asytole was quite consistent in our model and comparable between

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Fig 10. Systemic T-cell responses by day 100. Recipients of DCD kidneys who had a native kidney in place for 4 weeks were procured by day 100, and splenocytes were analyzed for T-cell subsets (FACS) (A). IFN-g secretion (ELISPOT) was measured after coculture of recipients splenocytes with irradiated donor cells and counting of IFN-g spots (n = 4 each) (B). Frequencies of activated CD4+ T cells expressing CD28 were not different between groups. In contrast, recipients of old DCD organs showed a trend towards increased effector/memory CD4+ T-cells with fewer numbers of regulatory T cells (CD4+CD25+FoxP3+). Transplantation of older DCD kidneys was associated with a more prominent IFN-g secretion, suggesting an important impact of donor age on the immune response to DCD organs long-term. In contrast, prolonged WIT in young DCD organs did not accelerate T-cell responses or IFN-g secretion. *P < .05; ***P < .001.

all experimental groups. Although this approach allowed us to focus on the variables of age and prolonged warm ischemia, a difference to the clinical situation is noted in which relative and varying degrees of hypoxia until asystole contribute to the observed changes in organs procured from DCD donors. The effects of donor age on delayed graft function and graft outcome have been evaluated in previous clinical studies.18-23 The association of age and prolonged warm ischemia and consequences on the recipient’s immune response after transplantation, however, have not been dissected so far. Donor age and prolonged warm ischemia time were associated in our experimental study with a reduced graft survival due to extensive ATN and an augmented early and late immune response. In contrast, young DCD kidneys recovered better

after prolonged WIT and demonstrated a wellpreserved graft function and structure long term, indicating an excellent potential for recovery after the initial ATN. Warm ischemia triggers a cascade of events that cumulate into innate immune activation on reperfusion of the ischemic graft. Reperfusion injury is associated with endothelial cell damage, which aggravates the inflammatory response by attracting host leukocytes. These cells release abundant inflammatory mediators, causing activation of Tolllike receptors on dendritic cells, which initiate the innate immune and subsequent T-cell stimulation.17,24 In young organs, energy restoration initiates cytoprotective mechanisms, leading to the repair of injured cells. In contrast, acute stress has a greater impact on older kidneys because of an augmented apoptosis, diminished proliferative reserve, and an altered growth factor profile. An

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impaired function of resident macrophages and diminished proliferation rates may further contribute to impaired healing.25-28 Injuries finally result into structural changes, such as tubular atrophy and fibrotic healing, which is accompanied by an increased influx of innate immune cells as demonstrated in our study. CD4+ T cells recently have been identified as mediating the consequences of warm ischemia; this observation suggests a well-orchestrated interplay of innate and adaptive immune cells.29 Likewise, our investigations showed an augmented innate and adaptive immune response early after transplantation. Interestingly, a greater B-cell and DC activation could be observed on day 10. B cells have humoral and nonhumoral regulatory functions such as regulation of DC function, T-cell priming, and antigen presentation.30-32 Although a donor origin of these cells cannot be excluded,12,33,34 an increased recipient B-cell stimulation in association with greater donor age recently has been reported. Reutzel-Selke et al have described a significantly enhanced systemic and intragraft B-cell response after transplantation of older grafts that was not associated with humoral effects (unpublished data). These results suggest a participation of B cells during acute rejection and early T-cell–mediated immune responses towards old grafts. Interestingly, young DCD grafts undergoing prolonged warm ischemic time showed a remarkable long-term outcome in our study. All grafts survived long-term with moderate structural damages, whereas in vitro data (FACS, ELISPOT) did not demonstrate an immunogenic effect on recipient immune response. This phenomenon also was observed in clinical studies. Gok et al1 found that in age-matched recipients of heart-beating and nonheart beating organs, graft function became equivalent at 3 months after transplantation, underlining the recovery potential of DCD grafts. This finding was further confirmed by Brook et al,3 who investigated the effects of delayed graft function on graft outcome after kidney transplantation from brain dead donors or DCD donors. Although graft outcome after DCD donation was superior to the outcome of organs from brain-dead patients, similar rates of acute rejection were observed in both groups. Interestingly, the overall severity of rejection episodes was milder in DCD kidneys. Our results showed an excellent long-term graft function of young DCD kidneys in the absence of an accelerated recipient immune response. In summary, young DCD grafts exposed to prolonged ischemia offer a greater potential for

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recovery from initial ATN, suggesting that it may be safe to accept young DCD organs with greater warm ischemia times. Yet, clinical studies are needed to confirm these findings. Of note, the correlation of initial graft damage and acute and chronic rejection as observed in organs from braindead patients may be less pronounced in young DCD grafts because early stimulation of the immune response did not affect graft immunogenicity and function long term. On the basis of these findings, one may argue that recipients of young DCD grafts may require a more tailored and potentially less potent induction treatment compared with recipients of organs from brain-dead patients. In contrast, recipients of old DCD organs may require a more potent induction compared to recipients of young DCD grafts as donor age was associated with an accelerated immune response. The proposed clinical applications require a more detailed analysis. REFERENCES 1. Gok MA, Buckley PE, Shenton BK, Balupuri S, El-Sheikh MA, Robertson H, et al. Long-term renal function in kidneys from non-heart-beating donors: a single-center experience. Transplantation 2002;74:664-9. 2. Saidi RF, Elias N, Kawai T, Hertl M, Farrell ML, Goes N, et al. Outcome of kidney transplantation using expanded criteria donors and donation after cardiac death kidneys: realities and costs. Am J Transplant 2007;7:2769-74. 3. Brook NR, White SA, Waller JR, Veitch PS, Nicholson ML. Non-heart beating donor kidneys with delayed graft function have superior graft survival compared with conventional heart-beating donor kidneys that develop delayed graft function. Am J Transplant 2003;3:614-8. 4. Francuski M, Reutzel-Selke A, Weiss S, et al. Donor brain death significantly interferes with tolerance induction protocols. Transpl Int 2009;22:482-93. 5. Weiss S, Kotsch K, Francuski M, Reutzel-Selke A, Mantouvalou L, Klemz R, et al. Brain death activates donor organs and is associated with a worse I/R injury after liver transplantation. Am J Transplant 2007;7:1584-93. 6. Pratschke J, Wilhelm MJ, Laskowski I, Kusaka M, Beato F, Tullius SG, et al. Influence of donor brain death on chronic rejection of renal transplants in rats. J Am Soc Nephrol 2001;12:2474-81. 7. Siddiqi N, McBride MA, Hariharan S. Similar risk profiles for posttransplant renal dysfunction and long-term graft failure: UNOS/OPTN database analysis. Kidney Int 2004; 65:1906-13. 8. Basar H, Soran A, Shapiro R, Vivas C, Scantlebury VP, Jordan ML, et al. Renal transplantation in recipients over the age of 60: the impact of donor age. Transplantation 1999; 67:1191-3. 9. Chavalitdhamrong D, Gill J, Takemoto S, Madhira BR, Cho YW, Shah T, et al. Patient and graft outcomes from deceased kidney donors age 70 years and older: an analysis of the Organ Procurement Transplant Network/United Network of Organ Sharing database. Transplantation 2008;85:1573-9.

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10. De Fijter JW, Mallat MJ, Doxiadis II, Ringers J, Rosendaal FR, Claas FH, et al. Increased immunogenicity and cause of graft loss of old donor kidneys. J Am Soc Nephrol 2001;12:1538-46. 11. Reutzel-Selke A, Jurisch A, Denecke C, Pascher A, Martins PNA, Kessler H, et al. Donor age intensifies the early immune response after transplantation. Kidney Int 2007;71:629-36. 12. Denecke C, Ge X, Jurisch A, Kleffel S, Kim IK, Padera RF, et al. Modified CD4(+) T-cell response in recipients of old cardiac allografts. Transpl Int 2012;25:328-36. 13. Melk A, Schmidt BM, Braun H, Vongwiwatana A, Urmson J, Zhu LF, et al. Effects of donor age and cell senescence on kidney allograft survival. Am J Transplant 2009;9:114-23. 14. Gok MA, Shenton BK, Pelsers M, Whitwood A, Mantle D, Cornell C, et al. Ischemia-reperfusion injury in cadaveric nonheart beating, cadaveric heart beating and live donor renal transplants. J Urol 2006;175:641-7. 15. Reich DJ, Mulligan DC, Abt PL, Pruett TL, Abecassis MM, D’Alessandro A, et al. ASTS Standards on Organ Transplantation Committee. ASTS recommended practice guidelines for controlled donation after cardiac death organ procurement and transplantation. Am J Transplant 2009;9:2004-11. 16. Pascher A, Reutzel-Selke A, Jurisch A, Bachmann U, Heidenhain C, Nickel P, et al. Alterations of the immune response with increasing recipient age are associated with reduced long-term organ graft function of rat kidney allografts. Transplantation 2003;76:1560-8. 17. Van de Wauwer C, Neyrinck AP, Geudens N, Rega FR, Verleden GM, Lerut TE, et al. The mode of death in the non-heart-beating donor has an impact on lung graft quality. Eur J Cardiothorac Surg 2009;36:919-26. 18. Hattori R, Ono Y, Yoshimura N, Hoshinaga K, Nishioka T, Ishibashi M, et al. Long-term outcome of kidney transplant using non-heart-beating donor: multicenter analysis of factors affecting graft survival. Clin Transplant 2003; 17:518-21. 19. Teraoka S, Nomoto K, Kikuchi K, Hirano T, Satomi S, Hasegawa A, et al. Outcomes of kidney transplants from nonheart-beating deceased donors as reported to the Japan Organ Transplant Network from April 1995-December 2003: a multi-center report. Clin Transpl 2004:91-102. 20. Matsuno N, Tashiro J, Uchiyama M, Iwamoto H, Hama K, Nakamura Y, et al. Early graft function in kidney transplantation from non-heart-beating donors. Ann Transplant 2004;9:21-2. 21. Asher J, Wilson C, Gok M, Balupuri S, Bhatti AA, Soomro N, et al. Factors predicting duration of delayed graft function in non-heart-beating donor kidney transplantation. Transplant Proc 2005;37:348-9. 22. Snoeijs MG, Schaefer S, Christiaans MH, van Hooff JP, van den Berg-Loonen PM, et al. Kidney transplantation using

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

elderly non-heart-beating donors: a single-center experience. Am J Transplant 2006;6(5 Pt 1):1066-71. Clavien PA, Selzner M, R€ udiger HA, Graf R, Kadry Z, Rousson V, et al. A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg 2003;238: 843-50; discussion 851-2. Boros P, Bromberg JS. New cellular and molecular immune pathways in ischemia/reperfusion injury. Am J Transplant 2006;6:652-8. Qiao X, Chen X, Wu D, Ding R, Wang J, Hong Q, et al. Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/ reperfusion injury. J Gerontol A Biol Sci Med Sci 2005;60: 830-9. Thomas SE, Anderson S, Gordon KL, Oyama TT, Shankland SJ, Johnson RJ. Tubulointerstitial disease in aging: evidence for underlying peritubular capillary damage, a potential role for renal ischemia. J Am Soc Nephrol 1998; 9:231-42. Danon D, Kowatch MA, Roth GS. Promotion of wound repair in old mice by local injection of macrophages. Proc Natl Acad Sci USA 1989;86:2018-20. Schmitt R, Zhu X, Cantley LG. Tubular epithelial cells contribute directly to the age-related increase in susceptibility to ischemic acute kidney injury (abstract). J Am Soc Nephrol 2006;17:530A. Zhai Y, Shen XD, Hancock WW, Gao F, Qiao B, Lassman C, et al. CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury. J Immunol 2006;176:6313-22. Zarkhin V, Li L, Kambham N, Sigdel T, Salvatierra O, Sarwal MM. Characterization of intragraft B cells during renal allograft rejection. Kidney Int 2008;74:664. Moulin V, Andris F, Thielemans K, Maliszewski C, Urbain J, Moser M. B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation. J Exp Med 2000;192:475. Stegall MD, Raghavaiah S, Gloor JM. The (re)emergence of B cells in organ transplantation. Curr Opin Organ Transplant 2010;15:451. Grau V, Fuchs-Moll G, Krasteva G, Hirschburger M, Steiniger B, Padberg W. Donor B cells in splenic follicles of experimental pulmonary allograft recipients. Langenbecks Arch Surg 2008;393:219. Okuda T, Ishikawa T, Azhipa O, Ichikawa N, Demeris AJ, Starzl TE, et al. Early passenger leukocyte migration and acute immune reactions in the rat recipient spleen during liver engraftment: with particular emphasis on donor major histocompatibility complex class II+ cells. Transplantation 2002;74:103.