Materials and Methods: Landrace pigs underwent either preharvest immunosuppression plus left kidney ischemic training (group 1, n = 6) or ischemic training alone (group 2, n = 6). Immunosuppression was composed of mycophenolate mofetil (20 mg/kg) and tacrolimus (0.1 mg/kg) administered intravenously 30 minutes before training. Training comprised 2 cycles of left renal pedicle occlusion for 5 minutes followed by release (reperfusion) for 10 minutes. Warm renal ischemia was then induced by clamping the left renal pedicle for 30 minutes, followed by heterotopic left kidney transplantation. Blood from the transplanted kidney renal vein was sampled directly at 0, 10, 20, 40, and 60 minutes posttransplantation for malondialdehyde (a reactive oxygen species marker), tumor necrosis factor-alpha (TNFα), interleukins 6 and 8 (inflammatory cytokines), and erythrocyte-reduced glutathione (an antioxidant). Renal histology was graded on a 3-point scale.

Results: Reperfusion levels of malondialdehyde, TNFα, and interleukin 6 were significantly lower in group 1 at both 40 and 60 minutes. None of the animals in group 1 (0/6) that received preharvest immunosuppression showed severe interstitial inflammation, compared with 4 of 6 animals in group 2 that did (P < .03).

Conclusions: Preharvest immunosuppression with mycophenolate mofetil and tacrolimus significantly decreases immediate posttransplant reactive oxygen species and inflammatory cytokine production, enhances the protective effect of ischemic training, and should not only reduce ischemia-reperfusion injury in transplanted kidneys but also should enhance immediate and long-term graft function while preventing acute rejection.

Key words : Graft function, Immunosuppression, Ischemia-reperfusion injury, Ischemic training, Kidney transplantation

Ischemia-reperfusion injury (IRI) is a major determinant of immediate posttransplant renal function. The extent of IRI is also directly proportional to the probability of acute rejection, and thus, is associated with chronic rejection and impaired long-term function. Our previous studies in an animal model show a close relationship between IRI development and the production of reactive oxygen species and inflammatory cytokines in the immediate posttransplant kidney. Additionally, in a non–heart-beating donor model, we have shown that IRI can be moderated by administering immunosuppressants to the donor immediately before kidney procurement [1, 2].

Mycophenolate mofetil (MMF) inhibits inosine-5-monophosphate dehydrogenase, an enzyme controlling guanosine nucleotide biosynthesis, to inhibit de novo T-lymphocyte and B-lymphocyte proliferation. MMF also blocks lymphocyte and monocyte penetration through the endothelium by inhibiting endothelial cell adhesion molecule expression. MMF also may affect inflammatory cytokine production [3].

Tacrolimus binds to the cytoplasmic 12 kDa cis-trans rotamase, FK506 binding protein (FKBP12). This tacrolimus-FKBP12 complex binds to the phosphatase, calcineurin, and inhibits dephosphorylation of the nuclear factor of activated T cells (N-FAT), preventing translocation of N-FAT to the nucleus. Tacrolimus also inhibits the production of interleukin (IL)-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, granulocyte macrophage colony stimulating factor, tumor necrosis factor-α, interferon-γ, and the IL-2 receptor [4]. Thus, both of these immunosuppressants, which are in routine use in transplantation medicine, may influence the development of IRI.

Pretransplant induction of brief ischemia followed by reperfusion heightens graft resistance to warm or cold ischemia and enhances immediate posttransplant graft function [5-7]. Most studies in ischemic training or preconditioning have been in cardiovascular surgery or transplantation of the heart, brain, liver, intestine, and recently, the kidney [8,9]. We based the present study on the hypothesis that a combination of ischemic training and immunosuppression prior to renal harvest would maximize the transplanted kidney’s resistance to IRI.

Materials and Methods

All procedures complied with the Guidelines of the European Community Committee on Care and Use of Laboratory Animals and Good Laboratory Practice. Twelve male Landrace pigs weighing 20-25 kg were divided into 2 groups to undergo either ischemic training preceded by 30 minutes of immunosuppression with mycophenolate mofetil (MMF; 20 mg/kg IV) and tacrolimus (0.1 mg/kg IV; group 1; n = 6), or ischemic training alone (group 2; n = 6).

After premedication with azaperone (5-6 mg/kg) and atropine (0.075 mg) intramuscularly, the entire procedure was performed under anesthesia with regulated ventilation. A peripheral venous cannula was inserted into the auricular vein, and a central venous catheter was placed into the internal jugular vein. Before tracheal intubation, thiopental (7.5-10 mg/kg) was administered IV. Conventional pressure-controlled ventilation then was maintained at the following average settings: T, 0.8 s; inspiratory P, 16 cm H₂O; respiratory rate, 28/min; positive end expiratory pressure, 5 cm H₂O; and FiO₂, 0.3%. Anesthesia was maintained with IV ketamine (≈0.2 mg/kg) and pancuronium bromide (0.1 mg/kg). Volume expansion was performed with 10% hydroxyethyl starch IV and a 1:1 ratio of Ringer’s lactate (50-80 mL/hour). Dopamine (2-3 µg/kg/min IV) was infused for inotropic support.

After a single dose of sodium heparin (100 IU/kg IV), ischemic preconditioning was performed in 2 cycles comprising occlusion of the left renal pedicle for 5 minutes followed by release (reperfusion) for 10 minutes. Warm renal ischemia was then induced by clamping the left renal pedicle for 30 minutes, after which the left kidney was harvested, washed immediately in 200 mL of histidine-tryptophan-ketoglutarate solution at 4°C, and immediately transplanted heterotopically. The donor artery and vein were anastomosed in an end-to-side manner to the recipient aorta and vena cava. Surgery did not exceed 30 minutes. The right kidney was left intact because small animals are very sensitive and do not survive removal of both kidneys.

Posttransplantation venous blood samples were taken directly from the renal vein of the transplanted kidney at 0, 10, 20, 40, and 60 minutes. Sodium heparin was used as an anticoagulant. Malondialdehyde, tumor necrosis factor-alpha (TNFα), and interleukins (IL) 6 and 8 were measured in plasma, and reduced glutathione was measured in erythrocytes. Plasma samples and erythrocytes were stored at -80°C for no more than 2 weeks until they were assayed.

Malondialdehyde was determined indirectly and photometrically in heparinized plasma as thiobarbituric acid reactive substances after reacting with thiobarbituric acid [10]. Although nonspecific for malondialdehyde, most thiobarbituric acid-reactive substances are produced by lipid peroxidation only. The expected plasma value was 2.34 ± 0.59 µmol/L.

Erythrocyte glutathione was determined by spectrophotometry. Thiols react with 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulphate to form thioethers; under strong alkaline pH (pH > 7.5), the glutathione thioether is converted to a color thione. Measurements were performed on an AU 400 Analyzer (Olympus, Ishikawa, Japan) using a glutathione 400 reagent kit (OXIS International, Inc; Foster City, Calif, USA). The expected value was 1.91 ± 0.34 mmol/L of erythrocytes.

Serum TNFα was determined using a swine TNFα enzyme linked immunosorbent assay (ELISA) kit (Biosource International, Camarillo, Calif, USA) on an Auto-EIA II microtiter plate reader (Labsystems Oy, Espoo, Finland). The swine TNFα kit was a solid-phase sandwich ELISA, using a monoclonal antibody specific for swine TNFα precoated onto strip-well plates. After binding TNFα, a second biotinylated monoclonal anti-swine TNFα was added. The detection system used streptavidin-peroxidase and hydrogen peroxide, with tetramethylbenzidine as the substrate. The plasma value was 67.1 ng/L (expected values, 0-168 ng/L).

Serum IL6 was determined using the Quantikine porcine IL6 ELISA kit (R&D Systems, Minneapolis, Minn, USA). In this case, the second antibody was not biotinylated but was directly labeled with peroxidase. Enzyme activity was detected using the same substrates. Expected plasma values were below 39.1 ng/L.

Serum IL8 was determined using the swine IL8 ELISA kit (Biosource International, Camarillo, Calif, USA). A specific anti-IL8 antibody was bound to the wells of the microtiter plate. After binding IL8, a secondary swine biotinylated anti-IL8 was added. The detection system used streptavidin-peroxidase and hydrogen peroxide, with tetramethylbenzidine as the substrate. The manufacturer did not provide expected values.

After 60 minutes of reperfusion, kidney upper poles were wedge-biopsied for histologic examination, and the animals were killed using 40 mL of potassium chloride (7.5% IV). Tissue specimens were fixed in 4% formaldehyde, routinely processed, and stained with hematoxylin and eosin, blue trichrome, Jones silver stain, and periodic acid Schiff. Tissue for ultrastructural examination was fixed in 4% paraformaldehyde, contrasted in 1% osmium tetroxide, and embedded in epoxy resin (Durcupan-Epon; Fluka, Steinheim, Germany); 1-µm sections were stained with uranyl acetate and lead citrate and viewed under a Philips EM 208S electron microscope (FEI Company, Brno, Czech Rep). Glomerular damage, tubular epithelial damage, and interstitial inflammation were graded on a 3-point scale by the same blinded histopathologist (OH) - (Table 1). The method of evaluation has been validated in our previous studies [11,12].

The STATISTICA ’98 edition (StatSoft, Tulsa, Okla, USA) was used for the statistical analyses. Descriptive statistics were computed for the groups and subgroups. Trends and interactions were evaluated using repeated measures univariate analyses of variance. Graded histologic damage was evaluated by testing the difference between 2 proportions. All evaluations were performed by the same histopathologist. Values for P less than .05 were considered significant.

Results

Malondialdehyde levels (Figure 1) changed in parallel for the first 20 minutes after reperfusion. Malondialdehyde levels significantly decreased by 60 minutes in group 1, but increased in group 2 (P < .004). TNFα levels (Figure 2) followed an identical pattern (P < .00139). IL6 levels (Figure 3) increased in both groups throughout reperfusion; however, this increase was significantly lower in group 1 between 40 and 60 minutes (P < .00025). IL8 levels decreased in both groups throughout reperfusion but were significantly higher at baseline in group 2 (P < .00292). Glutathione levels did not differ significantly between the 2 groups.

Group-2 kidneys showed significantly greater severe interstitial inflammation, with 4/6 animals affected at 60 minutes compared with 0/6 animals in group 1 (P < .03; Table 2, Figure 4).

Discussion

IRI leads to delayed graft function or primary nonfunction of the transplanted kidney, the extent of IRI is directly proportional to the probability of acute rejection, and IRI also plays an important role in the development of chronic graft failure and graft loss [13, 14]. By drawing lymphocytes into the graft [15], reperfusion generates reactive oxygen species that cause lipid peroxidation, damage to the cell membrane, and production of the marker malondialdehyde [16, 17]. During reperfusion, macrophages, monocytes, T cells, natural killer cells, and neutrophils express genes that encode inflammatory cytokines such as IL-2, 4, 6, and 8, as well as TNFα, which in turn activate leukocytes and increase release of reactive oxygen species. TNFα in particular also has a direct cytostatic effect on renal cells [18, 19]. Our previous animal study documented significant reactive oxygen species and inflammatory cytokine production in kidneys transplanted from non–heart-beating donors [20].

Several clinical and experimental studies [21-25] have demonstrated that development of IRI can be effectively reduced either through inhibition of lymphocyte activation via monoclonal antibodies or down-regulation of inflammation through administration of other immunosuppressants [19–23]. Preconditioning ischemic training has a similar effect, although the mechanism of this enhancement of graft resistance has yet to be elucidated [26]; putative mediators include heat shock proteins, endothelin, nitric oxide, and adenosine [27]. The timing of ischemic training and ischemic “window” lengths differ between individual organs [28, 29]. For example, in a transplanted kidney, the benefit of ischemic training disappears if the ischemic window exceeds 15 minutes, regardless of the number of preconditioning cycles [30, 31].

Based on our experimental evidence for the potential effect of IRI in transplanted kidneys from marginal donors [30], we sought to enhance the effect of ischemic training by pretreating the donor with MMF and tacrolimus.

MMF inhibits inosine-5-monophosphate dehydrogenase, an enzyme controlling guanosine nucleotide biosynthesis, to inhibit de novo T-lymphocyte and B-lymphocyte proliferation. MMF also blocks lymphocyte and monocyte penetration through the endothelium by inhibiting endothelial cell adhesion molecule expression. MMF may also affect inflammatory cytokine production.

Tacrolimus binds to the cytoplasmic 12 kDa cis-trans rotamase, FK506 binding protein (FKBP12). This tacrolimus-FKBP12 complex binds to the phosphatase, calcineurin, and inhibits dephosphorylation of N-FAT, preventing translocation of N-FAT to the nucleus. Tacrolimus also inhibits the production of interleukin (IL)-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, granulocyte macrophage colony stimulating factor, tumor necrosis factor-α, interferon-γ, and the IL-2 receptor. Thus, both of these immunosuppressants, which are in routine use in transplantation medicine, may influence development of IRI.

This study extends our previous work showing the benefit of ischemic training to reduce IRI in transplanted kidneys. Immunosuppression with co-administered MMF and tacrolimus potentiates the effect of ischemic training on IRI during renal transplant reperfusion. Our results suggest that a significant reduction in the production of reactive oxygen species and inflammatory cytokines in the early reperfusion phase will lead to improved immediate and long-term graft function. Further studies are needed to determine whether down-regulation of inflammation in the early posttransplant period also prolongs graft survival.

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