The main limitation to transplantation is an ever-increasing patient population requiring organ transplants, while the number of available organs has remained essentially stable. Donation after cardiac death (DCD) is possibly the main unexplored source of organs and may be the best solution to meet the demands of transplantation. In some programs, the number of DCD is higher than the number of cerebral death donors (1). When compared with organs recovered from donors with a beating heart, organs obtained after cardiac death have a higher degree of ischemia/reperfusion injury (IRI), and this may adversely affect both short-term and long-term allograft function (by causing primary nonfunction, delayed graft function, and/or chronic dysfunction).
Erythropoietin (EPO), in addition to its well-known erythropoietic effects, has been shown to exert important cytoprotective and antiapoptotic effects. The discovery by Westenfelder et al. (2) that the EPO receptor is expressed throughout the kidney-particularly in tubular epithelial cells, mesangial cells, and the glomerulus-led several groups to propose a role for EPO in protecting the kidney against IRI. Interestingly, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration, and promotes renal functional recovery, and-when used as a pretreatment-high doses of EPO preserve tubular function and reduce oxidative stress, in experimental models of renal ischemia and reperfusion (3, 4).
The extensive use of EPO in large animal models has been proven to be beneficial in several models of porcine IRI including aortic balloon occlusion (5), renal IRI (6), and in a controlled non-heart-beating donor kidney model (7). In each study, EPO was either administered before ischemia, at the onset of ischemia, or on reperfusion or infused throughout reperfusion, and in each case, EPO was found to significantly improve renal function. Thus, we have now taken those studies a step further and speculated that pretreatment of an organ donor before cardiac death with a single dose of EPO may improve both the quality of graft integrity and graft function. The aim of the present study was to determine the effects of EPO administration to the donor in DCD conditions on a porcine model of kidney transplantation.
MATERIALS AND METHODS
Unless otherwise stated, all compounds were purchased from Sigma-Aldrich Química S.A. (Sintra, Portugal). Pentobarbital sodium (Eutasil) was purchased from Sanofi Veterinária (Miraflores, Algés, Portugal). Celsior was purchased from Genzyme Corporation (Cambridge, Mass) and has a quantitative composition of mannitol (60 mM), lactobionic acid (80 mM), glutamic acid (20 mM), histidine (30 mM), calcium chloride (0.25 mM), potassium chloride (15 mM), sodium hydroxide (100 mM), reduced glutathione (3 mM), and water for injection (up to 1 L). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; B. Braun Medical Lda., Queluz, Portugal). Erythropoietin was obtained from Roche Farmacêutica Química (Amadora, Portugal). Celsior was obtained from Genzyme.
Animals and experimental protocol
Studies were carried out using 21 Landrace pigs weighing 40 to 50 kg. Pigs received a standard diet and water ad libitum and were cared for in accordance with the Institutional Animal Research Committee Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. Animals were randomly allocated into four groups as described: (i) reference group: data from unmanipulated animals-pigs that were not subjected to any surgical procedure; blood and urine samples were collected to obtain standard baseline values (n = 9); (ii) control group: pigs that were subjected to a conventional kidney transplant-grafts were preserved for 24 h in cold storage with Celsior (n = 6); (iii) DCD group: in this group, we mimicked the conditions of DCD donation and transplantation; donor pigs were subjected to cardiac arrest with a lethal injection of Eutasil (60 mg/kg i.v) 30 min before in situ cold preservation; pigs were subjected to kidney transplantation with grafts subjected to 30 min of warm ischemic time before explantation and then preserved in cold storage for 24 h with Celsior (n = 6); and (iv) EPO + DCD group: donor pigs were pretreated with EPO (1,000 IU/kg i.v.) 30 min before cardiac arrest. Pigs were subjected to kidney transplantation with grafts subjected to 30 min of warm ischemic time before explantation and then preserved in cold storage for 24 h with Celsior (n = 6).
Pigs were anesthetized with ketamine (100 mg/mL) and xylazine (20 mg/mL) mixture (2:1; 1.5 mL/kg). Anesthetized pigs were placed onto a surgical table, and body temperature maintained at 37 ± 0.5°C by means of a rectal probe. An endotracheal tube was introduced and was connected to a mechanical ventilator supplying isoflurane in a 2:1 mixture of air and oxygen. Appropriate artificial ventilation was verified by peripheral oximetry. MAP and heart rate (HR) were monitored for the duration of each experiment. For hemodynamic management, Ringer's lactate solution, saline, or dopamine (10 μg/kg per min) was administered. Two veins were cannulated for the administration of saline, drugs, or anesthesia as required. A midline laparotomy was performed to carefully expose the abdominal organs.
The aorta just above the diaphragm, terminal aorta, and vena cava were identified, isolated, and referenced. After systemic administration of 10,000 IU of heparin, a cannula was inserted through the terminal aorta. A vascular clamp was applied to the aorta near the diaphragm, and the abdominal organs were perfused with 4 L of Ringer's lactate solution at 4°C. Drainage was performed by an incision into the vena cava, and the peritoneal cavity was filled with ice. After perfusion in situ, the kidneys were carefully isolated, the hilus was exposed to find the renal artery and vein, and the kidneys were harvested with an arterial aorta patch. The harvested kidneys were then perfused with half a liter of Celsior and stored at 4°C for 24 h. In the DCD group, the perfusion of the organs began 30 min after cardiac arrest.
We used an experimental model of allograft kidney transplantation in the pig. Briefly, both kidneys were removed, and the terminal aorta and vena cava were identified, isolated, and referenced. A vascular clamp was applied to vena cava and aorta, and the heterotopic allotransplantation of the kidney using end-to-side aorta and vena cava anastomoses was performed with Prolene 5-0 suture. In all cases, the period of warm ischemia for completing anastomoses was 20 ± 4 min. The ureter was cannulated, and urine collected. After 4 h of reperfusion, the graft was removed and was cut in a sagittal section into two halves and fixed in formaldehyde and liquid nitrogen. A sample of blood and urine was collected, and the pig was killed with a lethal injection of Eutasil (60 mg/kg i.v.).
Measurement of biochemical parameters
At the end of the reperfusion period, 15 mL of blood was collected into serum SST gel and clot activator tubes (Becton Dickinson, Meylan, France) from the vena cava. The blood sample was centrifuged (3,000 revolutions/min for 10 min at room temperature) to separate serum. Urine samples were collected, and the volume of urine produced was recorded. Urine concentrations of Na+ were measured and were used in conjunction with serum Na+ concentrations to calculate fractional excretion of Na+ (FeNa) using standard formulas. Urine concentrations of creatinine were measured and were used in conjunction with serum creatinine concentrations to calculate creatinine clearance (CCr) using standard formulas. Serum and urine were analyzed by a certified laboratory for clinical chemistry (Laboratorio de Quimica Clinica, Hospital de Santa Maria, Lisboa, Portugal). Renal injury was assessed by measuring the levels of serum creatinine, CCr, and urinary flow (all markers of glomerular dysfunction); as well as FeNa, urinary N-acetyl-β-d-glucosaminidase (NAG), urinary total glutathione-S-transferase (GST), urinary aspartate aminotransferase (AST), and urinary urea (markers of tubular damage and dysfunction). Systemic injury was assessed by measuring the levels of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT). The inflammatory response was assessed by measuring the levels of IL-1 and IL-6.
Measurement of thiobarbituric acid-reactive substances
It has been well established that reactive oxygen species (ROS) play a key role in renal tissue damage caused by IRI, and these ROS attack polyunsaturated fatty acids within membrane lipids to produce malondialdehyde (MDA) and other thiobarbituric acid-reactive substances (TBARSs). Thiobarbituric acid-reactive substance levels in the kidneys were determined as a gross indicator of lipid peroxidation following a protocol described previously (8). In the thiobarbituric acid test reaction, MDA or MDA-like substances and thiobarbituric acid react with the production of a pink pigment. Briefly, a frozen renal tissue sample was weighed and homogenized in a 1.15% KCl solution. An aliquot (100 mL) of the homogenate was added to a reaction mixture containing 200 mL of 8.1% (wt/vol) lauryl sulfate, 1.5 mL 20% (vol/vol) acetic acid (pH 3.5), 1.5 mL of 0.8% (wt/vol) thiobarbituric acid, and 700 mL distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3,000g for 10 min. The absorbance of the supernatant was measured spectrophotometrically at 650 nm. Thiobarbituric acid-reactive substance levels were expressed as mmol/L of MDA per 100-mg wet tissue.
Determination of myeloperoxidase activity
Myeloperoxidase (MPO) activity in the kidneys was used as an indicator of polymorphonuclear (PMN) cell infiltration using a method previously described (9). Briefly, a frozen renal tissue sample was weighed and homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mmol/L potassium phosphate buffer (pH 7.4) and centrifuged for 30 min at 20,000g at 48°C. An aliquot of supernatant was then removed and added to a reaction mixture containing 1.6 mmol/L tetramethylbenzidine and 0.1 mmol/L hydrogen peroxide (H2O2). The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme required to degrade 1 mmol of H2O2 at 37°C and was expressed in milliunits per 100-mg wet tissue.
The histological and immunohistochemical evaluations were performed at Serviço de Anatomia Patologica, Hospital de Santa Maria, Lisboa, Portugal. At postmortem, a section of the kidney was removed and placed in formalin and processed through to wax. Four-micrometer sections were cut and stained with hematoxylin and eosin. Histological assessment of renal lesions was determined using a semiquantitative method. Using a magnification of 400×, 20 cortical fields were observed. Approximately 30 tubules per field were examined, and for each field, we used a classification from 0 to 3: 0 = normal histology, 1 = less than one third of the tubules with alterations, 2 = between one third and two thirds of the tubules with alterations, and 3 = more than two thirds of the tubules with alterations. The criteria used to classify tubular alterations were nuclear pyknosis, karyorrhexis, karyolysis, and cells inside the tubule. The presence of cells inside the tubules with nuclear alterations was given a classification of 2. The final classification for each kidney corresponded to the average of the classifications of the 20 fields. All sections were studied by using light microscopy (Dialux 22; Leitz, Frankfurt, Germany).
Immunohistochemical localization of poly(ADP-ribose) and iNOS
After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The 4-μm sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 25 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in phosphate-buffered saline for 25 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. Sections were incubated with (i) primary anti-poly(ADP-ribose) (PAR, P-248; Sigma), 1:1,500 for 30 min, or with (ii) anti-iNOS (MAB9502; R&D Systems, Minneapolis, Minn) 1:120 for 1 h. Specific labeling was detected with a biotin-conjugated goat anti-rabbit, donkey anti-goat, or goat anti-mouse IgG and avidin-biotin peroxidase complex (Milan, Italy). Kidney sections were then used for the immunohistochemical localization of poly (ADP-ribose) (PAR) (for Poly (ADP-ribose) polymerase [PARP] activation) and iNOS.
All data are presented as means ± SEM of n observations, where n represents the number of animals studied. For repeated measurements (hemodynamics), a two-factorial ANOVA was performed. Data without repeated measurements were analyzed by one-factorial ANOVA, followed by a Dunnett test for multiple comparisons using GraphPad Prism (version 5.03; GrapPad Software, Inc., La Jolla, Calif). P < 0.05 was considered to be statistically significant.
Effect of EPO on hemodynamic parameters after renal transplantation
There was no significant baseline difference in MAP or HR between any of the groups studied (Fig. 1). Ischemia resulted in a fall in MAP, but MAP recovered during reperfusion to mean values, which were not different from the ones obtained in the control group. No significant alterations in HR were observed in any of the groups studied (data not shown).
Effect of EPO on glomerular dysfunction after renal transplantation
When compared with reference animals, pigs that underwent renal transplantation only (control) demonstrated a significant increase in serum creatinine. Pigs that underwent renal transplantation with kidneys recovered under DCD conditions exhibited a significant increase in serum creatinine compared with control. This was reflected by a significant reduction in glomerular filtration rate, which was measured as CCr and urinary flow. The administration of EPO (1,000 IU/kg i.v.) to the donor 30 min before cardiac arrest significantly attenuated the renal/glomerular dysfunction associated with the subsequent transplantation of kidneys recovered after cardiac death (Fig. 2).
Effect of EPO on tubular dysfunction and injury after renal transplantation
When compared with reference animals, pigs that underwent renal transplantation only (control) demonstrated no change in the urine concentrations of NAG, AST, GST, and urea. Pigs that underwent renal transplantation with kidneys recovered under DCD conditions exhibited significant increases in urine concentrations of NAG, AST, GST, and urea, as well as fractional excretion of sodium (FENa) compared with control. The administration of EPO (1,000 IU/kg i.v.) to the donor 30 min before cardiac arrest significantly attenuated the tubular dysfunction and injury associated with the subsequent transplantation of kidneys recovered after cardiac death (Fig. 3).
Effect of EPO on systemic injury after renal transplantation
When compared with reference animals, pigs that underwent renal transplantation only (control) demonstrated no change in the serum concentrations of LDH and ALT. Pigs that underwent renal transplantation with kidneys recovered under DCD conditions exhibited significant increases in serum concentrations of LDH and ALT compared with control. The administration of EPO (1,000 IU/kg i.v.) to the donor 30 min before cardiac arrest abolished the systemic injury associated with the subsequent transplantation of kidneys recovered after cardiac death (Fig. 4).
Effect of EPO on the levels of inflammatory mediators after renal transplantation
When compared with reference animals, pigs that underwent renal transplantation only (control) demonstrated a significant increase in the serum concentration of IL-1 but not of IL-6. Pigs that underwent renal transplantation with kidneys recovered under DCD conditions exhibited significant increases in serum concentrations of IL-1 and IL-6 compared with control. The administration of EPO (1,000 IU/kg i.v.) to the donor 30 min before cardiac arrest abolished the increase of inflammatory mediators associated with the subsequent transplantation of kidneys recovered after cardiac death (Fig. 5).
Effect of EPO on the tissue levels of TBARS and MPO after renal transplantation
When compared with the control group, kidneys obtained from pigs subjected to renal transplantation with kidneys recovered after cardiac death demonstrated a significant increase in tissue TBARS levels, suggesting increased lipid peroxidation. When compared with the TBARS levels measured in the DCD group, treatment of the donor with EPO (1,000 UI/kg i.v.) resulted in a significant reduction in TBARS activity to levels that were not significantly different from the control group, suggesting marked reduction in lipid peroxidation by EPO treatment.
When compared with the control group, kidneys obtained from pigs subjected to renal transplantation with kidneys recovered after cardiac death demonstrated a significant increase in MPO activity, suggesting increased infiltration of the kidney with PMN cells. When compared with the MPO activity measured in the DCD group, treatment of the donor with EPO (1,000 UI/kg i.v.) resulted in a significant reduction in MPO activity to levels that were not significantly different from the control group, suggesting a marked reduction in PMN cell infiltration after EPO treatment (Fig. 6).
Effect of EPO on kidney histology after renal transplantation
Microscopic examination of the kidneys from the DCD group showed the recognized features of severe acute tubular damage. These features included nuclear condensation, loss of significant numbers of nuclei, cytoplasmic swelling, and cellular debris in the tubular lumen (Fig. 7). This was associated with infiltration of the interstitial space with neutrophils, while the glomerular structure was preserved. These findings are consistent with the biochemical alterations found in the DCD group. On comparison with the total severity score measured in kidneys obtained from the control group, renal transplantation with kidneys recovered after cardiac death produced a significant increase in total severity score. The treatment of the donor with EPO (1,000 IU/kg, i.v.) resulted in the preservation of renal tissue architecture and a significant reduction in the histological severity score (Fig. 8) associated with renal transplantation. These findings support the view that treatment of the donor with EPO reduces the tissue injury associated with IRI of the kidney.
Effect of EPO on the immunohistochemical localization of PAR and iNOS after renal transplantation
Kidneys obtained from the DCD group demonstrated marked staining for PAR and iNOS protein when compared with kidneys obtained from the control group, suggesting that renal transplantation after cardiac death resulted in PARP activation and iNOS protein expression. Treatment of the donor with EPO (1,000 IU/kg, i.v.) markedly reduced the staining for PAR and iNOS protein when compared with kidneys obtained from the DCD group, suggesting a reduction in the activation of PARP and expression of iNOS in response to renal injury (Figs. 9 and 10).
In our study, transplantation of kidneys recovered after cardiac death was associated with significant renal dysfunction and injury accompanied by a significant systemic inflammation (resulting in remote organ injury). We report here that pretreatment of the donor with EPO reduces the glomerular and tubular dysfunction as well as the renal injury (as indicated by histology) associated with renal transplantation. In addition, lipid peroxidation (assessed by tissue TBARS levels) within the kidneys collected from animals subjected to transplantation with organs recovered after cardiac death with EPO pretreatment was significantly reduced, suggesting that the beneficial effects of EPO may also be related to a decreased oxidative injury. In the kidney, PMN cells recruited during reperfusion are thought to be crucial mediators of early parenchymal injury (10), and indirect evidence suggests that PMN cell infiltration is related to the activity of MPO. Kidneys from donors pretreated with EPO before cardiac death and then transplanted to recipients demonstrated reduced tissue MPO levels, suggesting a reduction in PMN cell infiltration. Similar results were reported by Patel et al. (4), using the same dose of EPO (1,000 IU/kg) in a murine model of IRI. It was demonstrated that EPO, given either as a 3-day pretreatment or as a single bolus dose on reperfusion, significantly attenuated the increase in MPO levels. We have also demonstrated that EPO attenuates the histological changes associated with cell death. In all, these data suggest that the mechanisms underlying the renoprotective effects observed here may be the reduction in oxidative stress and cellular death.
The molecular signals by which EPO provides its beneficial actions in this model are currently unclear. Apoptosis and necrosis have been proposed as the mechanisms that produce cellular demise in ischemic kidneys (11), and either one may be the target of action for EPO. With respect to IRI of the kidney, previous evidence has also demonstrated that oxidative stress, among other mechanisms, is associated with the cell death in the rat kidney with IRI (12-14). It has been demonstrated that in cultured human proximal tubule cells, EPO dose-dependently reduced the cell death and increase in caspase-3 activity caused by either ATP depletion (simulated ischemia) or hydrogen peroxide (oxidative stress) (15). The same study also showed that, in the heart, the administration of EPO (300 IU/kg i.v.) before reperfusion also caused a significant reduction in infarct size after myocardial ischemia. In addition, cultured rat cardiac myoblasts (H9C2 cells) treated with EPO also reduced the increase in DNA fragmentation caused by either serum deprivation (simulated ischemia) or hydrogen peroxide (oxidative stress) (16). The fundamental concept that emerges from the above studies is that EPO induces tolerance to any subsequent insult involving IRI, and this induction may be the result of changes in the activation of various kinases (eg, MAPK, ERK1/2, or JNK1/2) that may promote cell survival.
In this study, transplantation of the kidney produced both biochemical and histological evidence of renal cell injury and PARP and iNOS activation in the kidney. The hypothesis that the generation of ROS contributes to renal IRI is supported by several studies that demonstrate the beneficial effects of various interventions, which either reduce the generation or the effects of ROS (12, 17, 18). NO, derived from NO synthase (NOS), plays an important role in renal function, both under normal and pathophysiological conditions (19). All three isoforms of NOS have been located in the kidney; the endothelial and neuronal (constitutive) isoforms have been identified in the renal vasculature and macula densa, respectively (19). iNOS can be induced in the kidney by cytokines or LPS and during IRI (20), leading to renal toxicity (21). Several in vivo and in vitro investigations have demonstrated that inhibition of iNOS activity, or absence of iNOS itself, can ameliorate or prevent renal IRI (20, 22-24), suggesting that NO, generated by iNOS, contributes to renal IRI. Furthermore, NO reacts with superoxide to form peroxynitrite (25), which causes injury via direct oxidant injury, protein tyrosine nitration, DNA damage, PARP activation, and, under certain conditions, formation of hydroxyl radicals (25, 26). It has been shown previously that reduction of the presence of peroxynitrite (eg, using the peroxynitrite scavenger ebselen) or the generation of peroxynitrite (eg, by reducing superoxide or NO levels) provides beneficial actions against renal IRI (26, 27).
Finally, a potential role for EPO on the regeneration of tubular epithelium after injury has been observed (28). There is some evidence that surviving cells retain the capacity to dedifferentiate, proliferate, and redifferentiate back to mature tubular epithelial cells and that non-organ-specific stem cells engraft in the kidney-particularly in the endothelium-under ischemic conditions (29), thus playing a role in regeneration, which is nevertheless an intriguing but interesting possibility.
Interestingly, pretreatment with EPO had no effect on the hematocrit level, suggesting that the renoprotective effect of this compound is independent of its growth-promoting effects. Although the effects of EPO on the hematocrit level seem to be of minor concern in these acute experiences, this would not be true if EPO would be used clinically in a chronic dosing regimen. Several large clinical trials of EPO carried out to assess potential utility of normalizing the marginally low hemoglobin levels of patients with breast or head and neck cancers unexpectedly found an increase in mortality within the EPO arm due to tumor progression or significant thrombosis (30-32). Additionally, a surgical trauma trial that showed increased survival in the EPO arm also showed that this was at the expense of a 40% increase in clinically significant thromboses (33). Thus, although administration of EPO has potentially valuable tissue-protective effects, clinical trials have shown EPO administration is accompanied by significant adverse complications. Notably, these complications seem to be more frequent with high doses of EPO, such as those used in the tissue protection proof-of-concept trials. There is, therefore, a need for the development of nonerythropoietic analogs of EPO that might allow for the avoidance of such undesirable effects in the clinical settings stated above. Although tissue-protective cytokine signaling needs to be further clarified, the availability of compounds such as carbamylated EPO (EPO where all the lysines were transformed to homocitrulline by carbamylation) that do not activate the homodimeric EPO receptor also opens up the possibility to distinguish experimentally between EPO's tissue-protective effects (eg, antiapoptotic) and its potentially detrimental effects (eg, procoagulant and prothrombotic effects within the microvasculature) and excessive erythropoiesis upon chronic dosing (34). This would enable triggering of the EPO-mediated tissue-protective pathways without cross-talk with the hematopoietic system.
The possibility of using EPO to induce ischemic tolerance suggests that there are advantages in its clinical application in organ transplantation. First, EPO is a safe drug in clinical practice. Second, the induction of ischemic tolerance seems to be relatively rapid after a single injection of EPO. Third, no additional or special equipment is required for the induction of tolerance in the patient.
Our results indicate for the first time that the administration of EPO to the donor causes a substantial reduction in renal dysfunction and injury (as determined by both biochemical markers and histology) as well as systemic inflammation associated with the transplantation of kidneys recovered after cardiac death. We speculate that the pretreatment of the donor with EPO may be useful in the prevention of graft injury and dysfunction (delayed graft function) in this 4-h reperfusion model associated with the transplantation of organs recovered after cardiac death. Further studies will be necessary to evaluate the therapeutic properties of EPO in preventing IRI not only in the kidney, but also when considering the transplantation of other solid organs.
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