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Keywords:

  • Brain death;
  • heme oxygenase;
  • kidney transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Brain death (BD) of the donor, a risk factor uniquely relevant for organs derived from cadaver donors, influences organ quality by induction of various inflammatory events. Consequently ischemia/reperfusion injury is deteriorated and acute and chronic rejections accelerated. Donor treatment might be an approach to improve the quality of the graft. The induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in living-donor transplantation models. Therefore, we examined the impact of donor treatment with the selective inducer of HO-1, cobalt protoporphyrin (CoPP), on organ quality and transplant outcome in a standardized BD model in a F344[RIGHTWARDS ARROW]LEW kidney transplant rat model. Immediately after BD induction, donor animals were administered a single dose of CoPP (5 mg/kg) and in control groups, HO-1 activity was blocked with zinc protoporphyrin (ZnPP, 20 mg/kg). Recipients of organs from brain-dead donors treated with CoPP survived significantly better than those from untreated brain-dead donors (p < 0.05) and intra-graft analysis showed improved histology (p < 0.05). Blockade of HO-1 with ZnPP decreased the survival rates (p < 0.05) comparable to untreated brain-dead donors. Our results demonstrate that HO-1 induction by one single treatment of CoPP in brain-dead donors leads to enhanced allograft survival.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Evolving from the precept that allograft rejection is an interaction between donor- and recipient-associated factors, the fields of transplantation biology and immunology have flourished, guided by the progressive understanding of functional dynamics and inter-relationships of immunologic factors. Among these, the systemic effects of donor brain death (BD) have received increasing attention (1). Experimentally, this catastrophic central injury has been shown to cause rapid and massive up-regulation of a variety of inflammatory mediators and other acute phase proteins in peripheral organs of the prospective organ donor (2). The tempo of acute rejection of heart, kidney and liver allografts from such donors after transplantation is accelerated because the inflamed organs increase host alloresponsiveness (3,4). Beside experimental findings, clinical studies showed that kidney grafts from brain-dead organ donors suffer more frequently acute rejection episodes and deteriorated ischemia/reperfusion injury after transplantation (2).

The lack of suitable organ donors and the increasing number of patients requiring an organ graft has resulted in the frequent acceptance of suboptimal donor organs for transplantation. In combination with the risk factor BD, these organs exhibit a greater risk of primary nonfunction compared to organs from optimal living donors (LD) where the phenomenon of primary nonfunction is almost completely unknown. There is consent about the fact that organ quality of the brain-dead organ donor should be improved before transplantation (5). Clinically, this concept has been emphasized by recent pooled United Network for Organ Sharing (UNOS) data which demonstrated clearly, that not only the survival rates of kidneys from living unrelated and one haplotype matched living related donors are similar despite genetic differences, but that organs from all LD demonstrate consistently superior results compared to those from deceased donors (6). Additionally, it was shown in clinical transplantation that inflammatory events are associated with BD and intensive care management (7). Elevated levels of ICAM-1, VCAM-1, E-Selectin and human leukocyte antigen (HLA) class II antigen were detected at an accelerated rate in cadavaric donor kidneys which experienced acute rejection and ischemia/reperfusion injury compared to organs from LD. These findings support the hypothesis that early nonspecific inflammatory responses contribute to the inferior graft outcome of kidneys derived from deceased donors (8).

We have previously shown that in allografts from brain-dead donors ischemia/reperfusion injury is associated with the incidence of delayed graft function (DGF) (4). These organs further underlie a higher risk to develop chronic graft dysfunction resulting in a significant risk of lower graft survival compared to organs from LD with an initial good function and shorter ischemia/reperfusion time (9). It is widely accepted that there is a necessity to improve the quality of brain-dead donor organs prior to transplantation in order to overcome the above limitations. Various attempts have been made including the substitution of hormones (10) or treatment with catecholamines (11,12) and recent experiments demonstrated the prevention of cellular infiltrates into the graft post-transplantation after the application of soluble P-selectin glycoprotein ligand to the brain-dead donor (13). This resulted in a decreased rate of acute rejections and an increased overall survival rate. Similar effects could be achieved with the application of a single dose of steroids to brain-dead organ donors (5). A further relevant mechanism to improve organ quality prior to transplantation could be the over-expression of cytoprotective and anti-oxidative stress proteins, like heme oxygenase 1 (HO-1). HO-1 induction exerts beneficial effects in a variety of living-donor transplantation models (14–17) and the up-regulation of HO-1 in vivo protects kidney allografts against chronic injury (18) resulting in significantly improved long-term function in transplants derived from marginal donors. Immunmodulatory effects associated with HO-1 over-expression, including reduced cytokine expression (TNFα, IFNγ), inhibition of cell-mediated cytotoxicity and apoptosis of endothelial cells have also been described (19–23). However, it was not clear whether induction of a stress protein like HO-1 is still protective in 'stressed' organs from brain-dead donors. Therefore, the objectives of the present study were to investigate the protective effects of HO-1 in brain-dead organ donors to improve transplant quality by reducing inflammatory processes in the graft. We demonstrate that the induction of the cytoprotective enzyme HO-1 following a single treatment with the potent HO-1 inducer cobalt protoporphyrin (CoPP) of the donor leads to the significant improvement of kidney graft survival in a standardized rat model of BD.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Operative techniques

Renal allografts from F344 donors were transplanted into unmodified LEW recipients (age 2–3 months, 200–250 g BW, Harlan Sprague-Dawley, IN, USA) using standard microsurgical techniques. The nonperfused kidneys were transplanted orthotopically to recipient renal vessels and ureter by end-to-end anastomoses using 10–0 prolene after the left kidney has been mobilized and removed. Right nephrectomy was performed 6 days later.

Brain death model

BD was produced in donor animals by gradually increasing intra-cranial pressure by slow inflation of a No. 3 Fogarty catheter balloon (Fogarty Arterial Embolectomy Catheter: 3F, Baxter Healthcare Co., Irvine, CA, USA) introduced into the intra-cranial cavity through an occipital burr hole. Herniation of the brain stem and BD was confirmed by electroencephalography, apnea, areflexia and maximally dilated and fixed pupils. All rats were intubated via a tracheostomy using a No. 13 blunt tipped cannula and mechanically respirated at a rate of 85/min and a tidal volume of 2.0 mL for 6 h (Rodent ventilator, model 683, Harvard Instruments, South Natick, MA, USA). Intra-arterial blood pressure was continuously monitored via a PE50 catheter placed in the left femoral artery and attached to a transducer and recorder. Only rats with stable mean arterial blood pressure (MAP > 80 mmHg) were accepted as donors in the study to preclude as much as possible the effects of peripheral ischemia secondary to hypotension. After 6 h, the left kidney was removed for transplantation. Brain-dead animals received no anesthesia as previous studies demonstrated that it did not influence physiologic parameters. Sham-operated rats served as LD controls. After ether anesthesia, a femoral artery catheter was placed and a tracheostomy performed for mechanical ventilation. A burr hole was drilled but no Fogarty catheter inserted. Maintenance anesthesia, pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL, USA, 40 mg/kg) was administered as needed for the 6 h period before kidney removal.

Experimental groups

In the experimental groups, kidneys were placed into unmodified hosts 6 h after induction of donor BD. In the first group, BD donors were treated immediately 1 h after BD induction with the administration of cobalt protoporphyrin (CoPP, 5 mg/kg intra-peritoneally, Porphyrin Products Inc., Logan, UT, USA) for 5 h (group 1) (Figure 1). In a second group, BD donors were treated concomitant with CoPP (5 mg/kg) and with zinc protoporphyrin (ZnPP, 20 mg/kg) (group 2), the latter for blocking HO-1 activity. CoPP (5 mg/kg) was applicated to anesthetized tracheotomized and ventilated (5 h) LD in a third group (group 3). BD donors (group 4) and anesthetized tracheotomized and ventilated LD rats (group 5) without treatment served as controls (Figure 1). The time of death of the graft recipients from renal failure secondary to complete and irreversible rejection of their allografts was compared between BD donors, LD/BD donors with CoPP treatment, and BD donors with CoPP/ZnPP treatment. Postoperative complications such as hydronephrosis secondary to ureteric obstruction were ruled out by autopsy. Engrafted organs (n = 5–10 animals/time interval/group) were analyzed immediately before transplantation (0 h), 6 and 24 h after placement and 3 and 10 days post-transplantation. All animal experiments were performed with the permission of the local authorities (Landesamt für Gesundheitsschutz, Arbeitsschutz und Technische Sicherheit, Berlin, Germany).

image

Figure 1. A standardized BD model in the rat was used. Brain-dead donors were treated immediately after BD induction with the administration of CoPP (5 mg/kg i.p., experimental group 1) for 5 h. In a second group, HO-1 induction (CoPP, 5 mg/kg, experimental group 2) was blocked by the application of ZnPP (20 mg/kg). In an additional group, CoPP (5 mg/kg, experimental group 3) was applicated to living donors (LD). Brain-dead donors (experimental group 4) and LD (experimental group 5) without treatment served as controls.

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Histology and immunohistology

Kidneys were fixed in 10% buffered formalin. Paraffin sections were stained with hematoxylin and eosin and assessed by light microscopy by 0 h, 6 h, 24 h, 3 and 10 days. For immunhistology cortical segments were immediately snap frozen in liquid nitrogen. Monoclonal antibodies (mAb) (Serotec, Wiesbaden, Germany), were directed against CD5+ T-cells (OX19), CD4+ helper cells (W3/25), CD8+ T-cytotoxic/suppressor cells (OX-8), monocytes/macrophages (ED1), and CD25 positive cells (activated T and B cells). Additional antibodies were directed against MHC class II antigens (OX-II). After specific mAb staining, the sections were then interacted with rabbit anti-mouse IgG, following mouse anti-alkaline phosphatase complex; sections were counterstained with hematoxylin. Numbers of labeled cells in 20 consecutive high power fields (×400 magnification) were determined in three kidneys/group.

Sample preparation and extraction of RNA

Kidney samples were removed and snap frozen in liquid nitrogen, then stored at −80°C until ready for use. Before analysis, thawing tissues were transferred in 700 μL guanidinium isothiocyanate solution plus 49 μL β-mercaptoethanol and homogenized with an Ultraturrax tissue homogenizer (Jahnke and Kunkel, Staufen im Breisgau, Germany). Total RNA was extracted by using the Absolutely RNA™ RT-PCR Microprep Kit (Stratagene, La Jolla, CA, USA) and its quality and quantity was controlled using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Palo Alto, CA, USA).

Quantification of gene expression

For cDNA synthesis a master solution was prepared by mixing 2 μL odT-Primer (0.1 mg/mL), 8 μL of 5× first strand buffer (GibcoBRL, Paisley, UK), 4 μL of dithiothreitol (0.1 M) (GibcoBRL), 4 μL of dNTP (Pharmacia Biotech, Uppsala, Sweden) (2.5 mM), 0.5 μL of RNasin ribonuclease inhibitor (Promega, Madison, WI, USA) (40 U/μL) and 2 μL of RQ1 RNase-free DNAse (Ambion, Austin, TX, USA) (2 U/μL). About 2 μg of total RNA dissolved in DEPC water were reverse transcribed. The mixture was incubated at 37°C for 30 min, thereafter for 5 min at 75°C to inactivate the DNAse. The reverse transcription was started by adding 1 μL of RNasin ribonuclease inhibitor (40 U/μL) and 1 μL of MMLV-reverse transcriptase (GibcoBRL) (200 U/μL). The mixture was incubated at 42°C for 1 h and the reaction was stopped by incubation at 95°C for 10 min.

To measure cytokine mRNA levels, the expression of each gene transcript was analyzed by real-time PCR using the ABI PRISM 7700 Sequence Detection System (TaqMan™, Perkin-Elmer Biosystems, Weiterstadt, Germany). Genes for the following products were investigated: CD25, INFγ, TNFα, TGFβ, IL-6 and HO-1. The cycle number at which the amplification plot crosses a fixed threshold above baseline is defined as the threshold cycle (Ct). Because preliminary experiments demonstrated amplification efficiencies in our system of nearly 1.0 for all panels, specific gene expression was normalized to the housekeeping gene β-actin for rat given by the formula 2−ΔCt. All primers and probes were designed and validated at the Institute of Medical Immunology, Universitätsmedizin Charité, Berlin and showed no cross reactivity with genomic DNA.

The PCR reaction was performed in a final volume of 25 μL containing 1 μL cDNA, 12.5 μL Master Mix (TaqMan™ Universal PCR Master Mix, Perkin Elmer, Applied Biosystems, Weiterstadt, Germany), 1 μL fluorogenic hybridization probe, 6 μL primer mix, and 5.5 μL distilled water. After an initial step of 2 min at 50°C involving activation of uracyl-n-glycosylase and degradation of any pre-existing contaminating RNA sequences, a denaturation and a hot start for AmpliTaq™ Gold DNA polymerase (Perkin Elmer Biosystems) was performed at 95°C for 10 min. The amplification took place in a two-step PCR (40 cycles; 15 s denaturation step at 95°C and 1 min annealing/extension step at 60°C). The mean Ct values for β-actin and the cytokines were calculated from double determinations. Samples were considered negative if the Ct values exceeded 40 cycles.

HO-1 activity test and HO-1 protein expression

Tissue samples of rat kidneys and livers were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% TritonX-100 and protease inhibitors. The assays for determination of HO-1 activity and HO-1 protein levels were carried out as described previously (18).

Alloantibody detection

For the detection of allo-specific IgG antibodies thymocytes of the donor cell type (LEW) were incubated with 1:20 diluted serum of the recipient (F344). After washing steps with PBS/2% FCS/0,1% NaN3 cells were further incubated with anti-rat-IgG-FITC labeled antibody (Serotec GmbH, Düsseldorf, Germany) and analyzed by flow cytometry using a FACS-Calibur (BD Biosciences, Heidelberg, Germany).

Alloreactive ELISPOT assay (alloreactive enzyme-linked immunospot assay)

Spleen cells were isolated by passing the spleen through a metal strainer. The spleen mononuclear cells were obtained after performing standard Ficoll density-gradient centrifugation (Ficoll 1083, Sigma, St. Louis, MO, USA). The ELISPOT was performed as previously described (24) using the Diaclone Research (Besançon, France) rat-IFNγ ELISPOT kit. LEW cells were cultivated with 3 × 105-irradiated F344 spleen cells for 24 h at 37°C. The plates were dried and measured using the Bioreader 3000 C/Biocounter system (Bioreader 3000 C; BIO-SYS GmbH, Karben, Germany). Results were calculated as IFNγ producing cells per 300 000 cells.

Statistical analysis

Statistical significance was calculated by applying the Student's t-test, log-rank-sum test for survival rates, and the Mann-Whitney test. Graft survival was expressed graphically using the Kaplan-Meier survival curve.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

HO-1 expression in rat kidneys and livers after HO-1 induction with CoPP

First we addressed the question whether endogenous HO-1 is already up-regulated in brain-dead donors compared to LD by the central injury itself. Therefore, we investigated HO-1 protein expression and HO-1 activity in whole tissues of kidneys and livers. The data revealed no significant differences in livers and kidneys between the two groups (Figure 2A,B). The next question to be answered was whether HO-1 is inducible in organs from brain-dead donors. For this purpose, brain-dead donors were treated with CoPP 1 h after BD induction and 5 h before engraftment (−5 h) (Figure 1). Induction of HO-1 protein and bioactivity levels were already observed in both kidneys and livers prior to transplantation (0 h), although the up-regulation was stronger in the latter (bioactivity: p < 0.05, Figure 2A, protein: p < 0.05, Figure 2B, respectively). The levels of HO-1 induction were comparable to those observed in kidneys and livers of LD. In contrast, HO-1 protein expression and bioactivity was reduced when brain-dead donors were treated simultaneously with CoPP and ZnPP (Figure 2A,B).

imageimageimage

Figure 2. Heme oxygenase 1 (HO-1) expression in rat kidneys and livers from cobalt protoporphyrin (CoPP-) treated and untreated brain-dead and living donors immediately before transplantation (0 h). (A) Kidney and liver tissue extracts were investigated for HO-1 activity by measurement of bilirubin formation (nmol bilirubin/mg protein × min) and (B) for HO-1 protein levels (ng HO-1/mg protein) measured by ELISA. Both HO-1 activity and protein expression were significantly increased following a single CoPP (5 mg/kg) administration in brain-dead and living-organ donors (*p < 0.05). (C) Renal allografts derived from treated brain-dead donors with CoPP (5 mg/kg) displayed significant higher levels of HO-1 activity (nmol bilirubin/mg protein × min) until day 10 post-transplantation compared to untreated donors (n = 10 animals/group/time point were analyzed and results are displayed as mean ± SD).

Kinetics of HO-1 activity following kidney transplantation

Because HO-1 protein expression and HO-1 bioactivity was already detectable in CoPP pre-treated BD donors, we analyzed HO-1 activity serially up to 10 days post-transplantation. The analysis demonstrated that renal allografts from brain-dead donors treated with CoPP displayed a significant higher activity of HO-1 (p < 0.05) not only before transplantation (0 h) but also during the whole observation period up to 10 days post-transplantation compared to kidneys derived from untreated brain-dead donors (Figure 2C).

Effect of HO-1 induction in brain-dead organ donors on graft survival

In agreement with our previous data, grafts from brain-dead donors showed a significantly reduced survival compared with living-donor grafts. Interestingly, the brain-dead donor graft survival was significantly (p < 0.05) improved after pre-transplant HO-1 induction by a single treatment of CoPP and reached the levels of living-donor transplants (>100 days). In contrast, organs derived from donors treated with CoPP and ZnPP showed a similar reduced survival comparable to untreated brain-dead donor grafts (Figure 3).

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Figure 3. Survival rates of renal allografts (n = 10 animals/group). Graft survival was significantly improved after HO-1 induction compared to untreated donor organs from brain-dead donors (p < 0.05) and showed a similar performance as untreated ideal living-donor organs (Kaplan-Meier survival curve).

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CoPP pre-treatment of the brain-dead donor leads to reduced graft infiltration

All renal allografts showed 6 h following engraftment and reperfusion infiltrates of CD4+ T cells and MHCII+ cells. Kidneys from CoPP-treated brain-dead donors showed no significant differences compared to CoPP/ZnPP and untreated brain-dead donors (Figure 4A) suggesting no influence of HO-1 and BD on early graft infiltration. By contrast, 3 days after transplantation kidneys derived from HO-1 activity blocked donors (CoPP/ZnPP-treated donors) displayed a significant higher number of ED-1+ monocytes/macrophages, CD4+ T-cells and MHCII+ cells in comparison to untreated brain-dead donors and CoPP-treated brain-dead donors (p < 0.05, Figure 4B). However, untreated and CoPP-treated HO-1-induced brain-dead grafts showed only differences in ED-1+ monocytes/macrophages infiltration (p < 0.05) (Figure 4B). At 10 days, the differences between the treated and untreated groups became more evident. CoPP-treated grafts showed significantly (p < 0.05) reduced infiltration for all investigated cell subsets, compared to untreated brain-dead donor grafts and CoPP/ZnPP pre-treated grafts (Figure 4C,D).

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Figure 4. Immunohistological examinations. Results are displayed as mean ± SD for n = 3 analyzed kidney grafts. (A) Immunohistology demonstrated no significant differences between untreated and treated brain-dead donors 6 h post-transplantation. (B) In contrast, after 3 days of engraftment organs derived from brain-dead donors treated with ZnPP/CoPP showed significantly higher infiltrates of ED1+, CD4+ and MHC II+ positive cells compared to organs after CoPP treatment only (p < 0.05). (C) After 10 days grafts with ZnPP/CoPP treatment displayed a higher infiltration rate of all investigated cell populations compared to kidneys with CoPP treatment, which demonstrated significant decreased cell infiltration (p < 0.05). (D) Particularly the expression of ED1+ (monocytes/macrophages), CD4+ (T cells) and CD8+ (T cells) (a, b, d, e, g and h) was decreased in CoPP-treated grafts from brain-dead donors. The number of CD25+ cells was comparable between CoPP-treated and untreated brain-dead donors (m, n). The inset in the upper right corner of the panel 'h' represents a magnified view of a single cell staining.

Gene expression in kidney allografts after HO-1 induction

The intra-graft mRNA expression of treated and untreated brain-dead donor grafts was analyzed by real-time RT-PCR before transplantation (0 h) and serially thereafter 24 h, 3 days and 10 days for the cytokines INFγ, TNFα, TGFβ, and IL-6, as well as for HO-1 and CD25. In addition to enhanced HO-1 levels, kidneys from CoPP-treated brain-dead donors showed slightly elevated mRNA levels of the anti-inflammatory cytokine TGFβ 5 hrs after drug administration (1.5-fold up-regulated compared to BD donor group). Furthermore, decreased TNFα could be observed in this group (5-fold down-regulated compared to untreated BD donor group). MRNA expression of TGFβ slightly increased over the observation period and reached its maximum at day 10. However, no statistical significance could be observed between the different groups at the transcriptional level at any time point (data not shown).

CoPP treatment leads to an impaired allo-immune response

To analyze the functional mechanisms of induction of HO-1 in brain-dead donors we performed ELISPOT analysis to measure the frequency of donor-specific T cells after 3 days of transplantation. The data demonstrate already lower frequencies of IFNγ secreting T cells in brain-dead donors after CoPP pre-treatment in comparison to the CoPP/ZnPP-treated group (Figure 5A), but this observation was not statistically significant. In contrast, measurement of donor-specific IgG alloantibodies after 10 days in recipient sera revealed a significant reduction in brain-dead organ donors compared to living- and brain-dead donors treated with CoPP and ZnPP, indicating a lower immune response toward the graft (Figure 5B).

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Figure 5. Studies on T- and B-cell responses in graft recipients. (A) ELISPOT assay for IFNγ-producing cells. Splenocytes of recipients (n = 5) were isolated. Control wells contained responder cells plus medium alone. Positive controls were stimulated with phorbol myristate acetate (2 ng/mL) and ionomycin (2 μg/mL; both from Sigma Aldrich GmbH, Munich, Germany) in RPMI medium. Cells were tested against donor antigen by adding 3 × 105 irradiated F344 spleen cells, and incubated for 24 h. Results are represented as IFNγ-producing cells/300 000 leukocytes ± SD. Rats receiving an allograft from brain-dead donors pre-treated with CoPP showed similar amounts of IFNγ producers in the spleen as recipients with grafts derived from living donors (Mann-Whitney, p = n.s.). (B) Results of IgG allo-antibody measurement are displayed as mean fluorescence intensity ± SD illustrating a significant decrease and IgG antibodies in the CoPP treatment group compared to recipients receiving either an allograft from living donors or donors pre-treated with CoPP and ZnPP.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

It is generally accepted that BD is an important donor associated risk factor which influences organ quality in the transplant process (3,25–27). Despite a sufficient systemic blood pressure, the central injury is regarded as an early onset of organ ischemia in the donor (28–30). It is proven in various experimental models and clinical investigations that the massive central damage leads to an increased expression of cytokines (31,32), adhesion molecules and a higher rate of leukocyte infiltration (7,33). These changes are consequences of a massive catecholamine release followed by subsequent hypotension and ischemia after donor BD (27,34). Consequently, organ ischemia as an integral part of BD, starts already before organ harvesting. These early nonspecific inflammatory events intensify subsequent immune responses after transplantation, leading to an accelerated rate of acute rejection episodes compared to grafts from more ideal living sources (8,35). A potential therapeutic approach to improve organ quality prior to transplantation is the treatment of the donor with various anti-inflammatory drugs before organ harvesting (5,36).

In this context, it is of valuable interest to use cytoprotective agents in order to diminish the detrimental effects of BD. In our study, we have examined the induction of the cytoprotective enzyme HO-1 in an established BD model in the rat and its putative role to improve organ quality. HO-1 up-regulation has already been shown to be protective in the treatment of marginal living-donor organs (18). However, in these models HO-1 up-regulation was induced before the stressful event (e.g. ischemia/reperfusion). Therefore, it was of outstanding interest whether the induction of a protective stress protein is still suitable in a BD donor model where the injury has taken place before onset of therapy. Although there was only a tendency of higher HO-1 expression in livers and kidneys of brain-dead donors compared to living-donor organs (Figure 2A,B), we could observe that the application of the selective HO-1 inducer CoPP leads to an up-regulated activity in brain-dead donors comparable to pre-treated LD (Figure 2A,B). After kidney transplantation, the differences in HO-1 activity were constantly higher up to 10 days in the CoPP group compared to untreated brain-dead donors (Figure 2C). Most importantly, this resulted in an improved 3-month graft survival and function in the brain-dead donor group similar to living-donor grafts (Figure 3).

While we could not observe clear differences in early graft infiltration (<day 3), CoPP-treated grafts showed significantly less infiltration of all investigated immune cell subsets compared to untreated brain-dead donor grafts 10 days after transplantation (Figure 4A–D). To investigate whether there is a difference in the alloresponse, we measured donor-specific T cells in the spleen at day 3 demonstrating a slight decrease of donor-specific T cells in the BD + CoPP group. In contrast, donor MHC-specific antibodies in the sera of recipient animals at day 10 revealed a significant decrease of alloantibodies in the BD + CoPP-treated group compared to the living-donor group supporting the histological data that after 10 days grafts with CoPP treatment displayed decreased infiltration of macrophages, CD4+ and CD8+ T cells. This effect was abolished by the simultaneous application of ZnPP, an inhibitor of HO-1 activity. Recently, it has been demonstrated that the induction of HO-1 expression with CoPP in human and rat dendritic cells inhibits lipopolysaccharide (LPS)-induced phenotypic maturation and secretion of pro-inflammatory cytokines, resulting in the inhibition of alloreactive T-cell proliferation (37). Furthermore, induction of HO-1 in an acute rejection model revealed reduced frequencies of donor-derived DCs in the graft and recipient compartments, which was associated with reduced frequencies of CD4+ T cells, CD8+ T cells and alloreactivity (38). Therefore, we hypothesize that the impaired immune response toward the graft in our model might be due to the targeting of donor-derived dendritic cells by the induction of HO-1 resulting in less alloreactive T cell priming and finally in less infiltration.

Another possibility of HO-1 action might be the prevention of inflammation by targeting chemokines and pro-inflammatory cytokines following very early ischemia/reperfusion. This is supported by observations demonstrating a HO-1-mediated inhibition of NFκB, which is necessary for the transcription of various pro-inflammatory cytokines (39). In contrast to reports demonstrating a strong suppression of cytokine transcription after HO-1 induction after ischemia/reperfusion injury, we could not observe significant differences in cytokine expression in our model (data not shown). As CoPP is administered 1 h after the induction of BD it is most likely that the up-regulation of HO-1 is too late in this context to prevent the expression of cytokines and the cascade is already started.

Our results are consistent with previous reports demonstrating prolonged kidney graft survival associated with up-regulation of HO-1 expression (18,40). In a chronic rejection model, HO-1 induction in graft recipients attenuated graft arteriosclerosis followed by induction of graft acceptance with monoclonal antibody therapy (17). Other authors demonstrated in a fatty liver isograft model that CoPP therapy or adenoviral HO-1 gene therapy prevented ischemia/reperfusion injury (41) and that in the brain of transgenic mice HO-1 over-expression attenuated cellular injury caused by ischemic stroke (42). Similarly, in a rodent model of renal failure, elevated HO-1 expression reduced tissue injury (43). Induction of HO-1 expression decreased in animal models of inflammation the inflammatory response, while inhibition of HO-1 exacerbated inflammation (44,45). In septic shock models, up-regulation of HO-1 decreased mortality. Consistent with these observations, HO-1/KO mice suffer from progressive chronic inflammatory disease and are extremely sensitive to stressful injury (46).

The potential role of HO-1 in protection from cellular injury and inhibition of inflammation in organs from brain-dead donors seems to be similar to ischemia/reperfusion injury as the mechanisms leading to organ injury after BD are early ischemic changes. In regard to BD the injury itself up-regulates HO-1 expression, which can be further amplified by timely application of HO-1 inducers. The application of CoPP leads within 6 h to a detectable up-regulation of HO-1 concentration and activity (Figure 2A,B). From the clinical point of view such a rapid induction of HO-1 would allow treatment of human organ donors after diagnosis of BD and prior to organ harvesting. This intention is in line with recent publications demonstrating that dopamine treatment is known to indirectly exert an anti-oxidative effect. Degradation of dopamine leads to the production of reactive oxygen species such as H2O2 and O2 resulting in the induction of HO-1. These studies illustrate that dopamine in clinically relevant dosages down-regulates inflammation and enhances the anti-oxidative capacity of kidneys in the experimental and clinical setting (47,48). In summary, we could clearly demonstrate that HO-1 induction in organs from brain-dead donors has beneficial effects on graft function after transplantation. These results stress the importance of donor treatment as a potential approach to improve cadaver donor organ quality particularly from marginal donors.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors thank Annelie Dernier, Sabine Brösel and Anke Jurisch for excellent technological assistance. The study was supported by DFG grant PR 578/2–2, PR 578/2–3. Katja Kotsch was supported by a grant from the German Kidney Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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