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Lecithinized superoxide dismutase reduces cold ischemia-induced chronic allograft dysfunction
Kidney International, Vol. 61 (2002), pp. 1160–1169
Lecithinized superoxide dismutase reduces cold ischemia-induced chronic allograft dysfunction KEN NAKAGAWA, DICKEN D.H. KOO, DAVID R. DAVIES, DEREK W.R. GRAY, ANDREW J. MCLAREN, KENNETH I. WELSH, PETER J. MORRIS, and SUSAN V. FUGGLE Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; Department of Urology, Keio University, Japan; and Department of Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom
Lecithinized superoxide dismutase reduces cold ischemiainduced chronic allograft dysfunction. Background. Chronic renal allograft failure (CAF) is influenced by both allo-dependent and independent factors and is a major cause of graft loss in clinical renal transplantation. We evaluated a novel membrane-bound free radical scavenger, lecithinized superoxide dismutase (lec-SOD), to determine its potential in limiting the harmful effects of ischemia/reperfusion injury on CAF. Methods. Fisher rat kidneys were stored for either 1 hour or 18 hours in cold Marshall’s preservation solution either with or without lec-SOD and transplanted into Lewis recipients. Results. Within 3 days of transplantation, an early inflammatory response involving granulocytes and macrophages was detected in renal allografts exposed to 18 hours cold ischemia that was significantly reduced by preservation with lec-SOD. By 24 weeks post-transplantation, elevated proteinuria and detection of apoptotic cells was observed in kidneys exposed to 18 hours of cold ischemia, that was attenuated by preservation with lec-SOD (P ⬍ 0.05). However, up-regulated expression of intracellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) Class II together with a T lymphocyte infiltration were observed at 24 weeks that was not prevented by preservation with lec-SOD. Conclusions. These results demonstrate that ischemia/reperfusion injury, apoptotic cell death and allo-immune responses may be exacerbated by cold ischemia and accelerate the development of CAF. Preservation with lec-SOD may protect against the early damage induced by cold ischemia and reperfusion injury.
Despite progressive improvements in the short-term results of first cadaver renal allografts, the problems of long-term graft loss remain unresolved. It is becoming increasingly evident that chronic renal allograft dysfuncKey words: organ preservation, chronic rejection, superoxide dismutase, transplantation, kidney, apoptosis, antioxidant. Received for publication July 20, 2001 and in revised form October 19, 2001 Accepted for publication October 22, 2001
2002 by the International Society of Nephrology
tion is a major cause of late graft failure [1–3]. Chronic allograft failure has been defined as the progressive deterioration in graft function occurring at least 90 days after transplantation, and is associated with the histological features of interstitial fibrosis, arterial intimal thickening, vascular lesions and tubular atrophy [4–7]. A number of alloantigen-dependent and independent factors have been proposed as contributing to chronic allograft failure. Acute rejection episodes resulting from alloimmune responses are a major risk factor for chronic allograft failure, but damage to the organ as a result of alloindependent events also plays a key role in aggravating chronic renal allograft dysfunction and rejection [7–9]. In renal transplantation, prolonged cold ischemic storage of kidneys has been shown to be significantly associated with a higher incidence of delayed graft function (DGF) after transplantation [10–18]. DGF has been shown to have a significant impact on poor long-term graft survival, especially in association with acute rejection [17, 18]. We have previously demonstrated that in cadaver renal allografts with long cold ischemia times, a neutrophil and platelet-mediated inflammatory response and apoptotic endothelial and proximal tubular epithelial cells were observed after reperfusion [19, 20]. There is evidence to suggest that the initial inflammatory events arising from ischemia/reperfusion injury may up-regulate alloantigens and thus increase graft immunogenicity and exacerbate an alloimmune response [21–23]. In renal models of in situ cold ischemia and reperfusion, upregulated expression of MHC antigens, costimulatory molecules, cytokines, chemokines, adhesion molecules and leukocyte infiltration have been detected in association with progressive long-term deterioration in renal function [24, 25]. The mechanisms involved in the generation of an inflammatory response following ischemia/reperfusion are complex, but production of reactive oxygen species is thought to be a critical event [26]. Many studies have
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shown that ischemia/reperfusion injury is abolished by administration of an oxygen free radical scavenger, superoxide dismutase (SOD) [27–35]. Indeed, Land and colleagues demonstrated that administration of SOD at reperfusion significantly reduced the incidence of first acute rejection episodes and improved long-term graft survival in cadaver renal allografts [32]. These findings suggest that the production of reactive oxygen species during the initial period of transplantation may accelerate subsequent immune mediated events and affect longterm graft survival. To determine whether it was possible to limit the harmful effects of ischemia/reperfusion injury on chronic renal allograft failure, we have investigated the therapeutic potential of a novel form of lecithinized SOD (lecSOD). Lec-SOD was produced by the covalent linkage of recombinant human SOD with lecithin, enabling greater stability and high affinity binding to cell membranes [36]. We have previously demonstrated in vitro that in contrast to unmodified rhSOD, lec-SOD binds to microvascular and large vessel endothelial cells under cold hypoxic conditions in Marshall’s solution, thereby inhibiting hypoxia-induced cell death, endothelial adhesion molecule expression and neutrophil adhesion [37]. The aims of the current study were (1) to examine the effects of prolonged cold ischemia and reperfusion injury on chronic allograft failure in a rat renal allograft model, and (2) to determine whether preservation of kidneys with lec-SOD before transplantation exerted a beneficial effect on inflammatory and histological changes and on graft function.
the aorta and vena cava dissected and clamped. In the first instance only the left native kidney was removed and the donor kidney was transplanted by an end-toside anastomosis to the recipient aorta with a running 10-0 nylon suture. After release of the clamps an endto-end ureteric anastomosis was performed by an interrupted 10-0 suture. The remaining right native kidney was removed 10 days post-transplantation after providing initial support against the effects of delayed function of the allograft. Low-dose cyclosporine A (5 mg/kg/day) was administered by gavage during the initial 10-day post-operative period to prevent early rejection. In recipient animals whose renal allografts were harvested at either day 1 or day 3 post-transplantation, both native kidneys were removed at the time of transplantation so that the early effects of ischemia/reperfusion injury on histological changes could be determined. Between 4 and 7 animals were transplanted and harvested at each time point.
METHODS Experimental model Allogeneic renal transplants were performed from fasted, inbred male (250 to 300 g) donor Fisher (F344, RT1lv1) to recipient Lewis rats (LEW, RT1l; Harlan Olac Ltd, Bicester, UK) to recreate the model of chronic renal allograft failure described by Hancock and colleagues [38]. A midline abdominal incision was performed on anesthetized donor rats and the renal vessels and ureter mobilized after ligation of the adrenal and gonadal veins. The animals were heparinized and the main vessels clamped before both kidneys were flushed through the aorta with 5 mL of ice-cold Marshall’s hypertonic citrate solution (Baxter Healthcare Ltd, Berkshire, UK). Kidneys were removed and stored at 4⬚C. Four treatment groups were studied: (i) one hour cold ischemia in Marshall’s solution alone; (ii) one hour cold ischemia in Marshall’s solution ⫹ 50 g/mL lec-SOD (Seikagaku Corporation, Tokyo, Japan); (iii) 18 hours cold ischemia in Marshall’s solution alone; or (iv) 18 hours cold ischemia with Marshall’s ⫹ 50 g/mL lec-SOD. Recipient rats were fasted overnight, anesthetized and
Histopathology and Immunohistology Upon harvesting, kidneys were dissected longitudinally and half of the kidney fixed in formalin for paraffin embedding and the other half snap frozen and stored at –80⬚C for immunohistochemistry. Histopathological analysis for morphological changes to glomeruli, tubules, interstitium and vessels associated with chronic rejection was assessed by an experienced renal pathologist (DRD) under blinded conditions. Renal sections were graded on a semi-quantitative basis. Glomerular proliferation and sclerosis were scored as: Grade 0 ⫽ none; Grade 1 ⫽ occasional focal segmental proliferation and sclerosis; Grade 2 ⫽ areas of glomerular segmental proliferation and sclerosis. The degree of tubular atrophy was graded as: Grade 0 ⫽ none; Grade 1 ⫽ occasional tubular atrophy; Grade 2 ⫽ focal or multiple focal areas of atrophy. Tubular cast formation was assessed as: Grade 0 ⫽ none; Grade 1 ⫽ occasional hyaline cast; Grade 2 ⫽ areas of hyaline and/or granular casts. The level of interstitial infiltration was graded as: Grade 0 ⫽ none; Grade 1 ⫽ occasional focus; Grade 2 ⫽ multiple foci of infiltrating cells.
Measurement of renal allograft dysfunction Renal allograft dysfunction was determined by measurement of proteinuria in six animals in each of the four treatment groups. Urine samples were collected at four-week intervals from rats placed in metabolic cages for 24 hours, up to and including 24 weeks post-transplant. For this experiment there were six animals in each treatment group. Protein excretion was determined by measuring precipitation after interaction with 3% sulfosalicylic acid (Sigma, Poole, Dorset, UK). Turbidity was assessed by absorbance measurements at a wavelength of 595 nm.
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Frozen biopsy material was embedded in O.C.T. (Miles Inc., Shawnee Mission, KS, USA), and 7 m cryosections were stained using an indirect immunoperoxidase technique with the following monoclonal antibodies: IF4 (CD3 T lymphocytes), ED1 (macrophages), HIS48 (granulocytes), MRC OX1 (CD45 leukocyte common antigen), F16-4.4 [major histocompatibility complex (MHC) Class I] and TLD-4C9 [intracellular adhesion molecule-1 (ICAM-1)] were all obtained from Serotec (Oxford, UK) and MRC OX3 (MHC Class II) [39]. The signals for MHC Class I, ICAM-1, CD45 and ED1 were amplified with a further incubation step for 30 minutes using a peroxidase-conjugated swine anti-rabbit Ig (1:50 dilution; Serotec) pre-incubated with 20% rat serum. IgM isotype monoclonal antibodies against CD3 and HIS48 were developed with a peroxidase-conjugated goat anti-mouse IgM (1:50 dilution; Serotec) and enhanced with a rabbit anti-goat Ig (1:200 dilution; Sigma Ltd.), both pre-incubated with 20% rat serum. All sections were examined without knowledge of the treatment groups. Leukocyte infiltration was quantitated by determining the percentage area occupied by a particular cell type using an established morphometric point counting technique [40]. Semiquantitative grading for the induced expression of ICAM-1, MHC Class I and MHC Class II antigens on proximal tubular epithelial cells were assessed by two independent observers. The semiquantitative grades for ICAM-1 and MHC Class I were: Grade 1 ⫽ occasional weakly positive tubule; Grade 2 ⫽ isolated foci of positive tubules; Grade 3 ⫽ multiple foci of more extensive positive tubular staining. MHC Class II expression was graded as: Grade 1 ⫽ negative; Grade 2 ⫽ foci of weakly positive tubules. Measurement of apoptosis Identification of apoptotic cells was performed using the terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) technique as previously described [20]. Briefly, sections were obtained from paraffin embedded kidney specimens and deparaffinized, rehydrated and digested with 20g/mL Proteinase K (Sigma Ltd) at 37⬚C for 60 minutes. Following proteinase digestion, the specimens were stained with the Apoptag Plus peroxidase kit (Oncor, Cambridge, UK) according to the manufacturer’s protocol. TUNEL-positive apoptotic cells were identified as single positively stained cells, with associated chromatin condensation and positive chromatin fragments. The Apoptosis Index was calculated from the mean number of positive cells per ⫻250 field of view, following an analysis of 20 fields of view per section as previously described [20].
Statistical analyses Statistical analyses were performed using the Student t test to determine if there were significant differences between treatment groups when P ⬍ 0.05. RESULTS Renal allograft dysfunction Damage to renal allografts as determined by a rise in proteinuria level was detected in renal allografts exposed to 18 hours cold ischemia (Fig. 1). In contrast, kidneys preserved with lec-SOD for 18 hours remained relatively stable, with significantly lower proteinuria at 16, 20 and 24 weeks post-transplantation (P ⬍ 0.05). Furthermore, organ preservation for a minimal one-hour period in the presence or absence of lec-SOD had no detrimental effects on graft function, with stable proteinuria levels over the 24-week period (Fig. 1). Histopathology A histopathological analysis of rat renal allografts was performed on samples obtained at 24 weeks post-transplantation to determine the effects of long cold ischemia on chronic allograft failure. Glomerular mesangial deposition was equally prominent in all allografts and there was no evidence of interstitial fibrosis, glomerular basement membrane changes or arterial intimal proliferation and thickening. However, in renal allografts with 18 hours of cold ischemia, significantly higher mean grades were observed for focal segmental glomerular sclerosis and proliferation (0.75 ⫾ 0.62 vs. 0.25 ⫾ 0.45; P ⬍ 0.01), tubular atrophy (1.5 ⫾ 0.79 vs. 0.25 ⫾ 0.45; P ⬍ 0.001), tubular cast formation (1.58 ⫾ 0.67 vs. 0.33 ⫾ 0.65; P ⬍ 0.001) and interstitial infiltration (2.0 ⫾ 0.73 vs. 1.4 ⫾ 0.51; P ⬍ 0.05) compared with kidneys preserved for shorter periods of cold ischemia (Fig. 2). Preservation with lec-SOD had no effect on graft histology. Leukocyte infiltration Immunohistochemical analysis to detect infiltration by granulocytes was performed in the immediate post-transplant period. Significantly higher levels of HIS48⫹ granulocytes were present at day 1 in kidneys exposed to the 18-hour cold ischemia compared with renal allografts stored for only one hour (2.8 ⫾ 0.4% vs. 0.8 ⫾ 0.6%; P ⬍ 0.01), remaining at elevated levels three days after transplantation (2.7 ⫾ 0.9%; Fig. 3). The increase in granulocyte infiltration at day 1 in grafts exposed to 18 hours of cold ischemia was significantly attenuated by preservation with lec-SOD (2.8 ⫾ 0.4% vs. 1.9 ⫾ 0.7%; P ⫽ 0.017), but this protective effect was not evident at day 3 (2.2 ⫾ 0.6% vs. 2.8 ⫾ 0.8%; P ⬎ 0.05). At all time points following transplantation, the level of CD45⫹ leukocyte infiltration was increased by expo-
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Fig. 1. Preservation with lecithinized superoxide dismutase (lec-SOD) inhibits development of chronic renal allograft dysfunction induced by cold ischemia. Kidneys from Fisher donors were exposed to one hour or 18 hours of cold ischemic preservation in the presence or absence of lec-SOD. After transplantation, renal function was assessed by 24-hour proteinuria measurements taken at 4 weekly intervals up to 24 weeks post-transplantation. A significant increase in proteinuria was detected at 16, 20 and 24 weeks post-transplant in renal allografts stored for 18 hours compared with kidneys that were stored for only one hour (P ⬍ 0.05). This deterioration in longterm renal allograft function was delayed by preservation with lec-SOD (P ⬍ 0.05). Each time point represents the mean ⫾ SEM for 6 animals. Symbols are: (䊏) 1 hour no lec-SOD; (䊐) 1 hour ⫹lec-SOD; (䉱) 18 hours no lecSOD; (䉭) 18 hours ⫹lec-SOD; *P ⬍ 0.05, 18 hours cold ischemia time (CIT) vs. 1 hour CIT; §P ⬍ 0.05, 18 hours CIT ⫺ SOD vs. 18 hours CIT ⫹ lec-SOD.
3 was similar to that detected for total CD45⫹ leukocyte infiltration, demonstrating that macrophages and granulocytes (Fig. 3) are the major components of the infiltration at these time points (Fig. 4). Twenty-four weeks after transplantation, low levels of macrophages were detected in all allografts. In complete contrast, low levels of CD3⫹ T lymphocytes were detected on days 1 and 3 post-transplant, irrespective of the period of cold storage or preservation with lec-SOD. By 24 weeks post-transplant, increased levels of T lymphocytes were detected, an effect that was further exacerbated by 18 hours of cold storage. Lec-SOD did not influence the magnitude of this infiltration. Fig. 2. Long cold ischemia induces histological changes associated with chronic renal allograft failure. Paraffin sections from renal allografts at 24 weeks post-transplantation were analyzed for morphological changes characteristic of chronic rejection and assessed semiquantitatively to determine the severity of injury. In kidneys exposed to 18 hours of cold ischemia (䊏), there were significantly higher levels of focal segmental glomerular sclerosis and proliferation (FSGS), tubular atrophy, cast formation and interstitial infiltration at 24 weeks post-transplant than in kidneys stored for one hour ( ; P ⬍ 0.05). Preservation with lecSOD did not prevent the development of these chronic histological changes. *P ⬍ 0.01 vs. 1 hour CIT.
sing the kidney to 18 hours of cold ischemia. Preservation of kidneys with lec-SOD significantly reduced the level of CD45⫹ infiltration on day 1 (3.9 ⫾ 1.1% vs. 2.7 ⫾ 0.3%; P ⫽ 0.013), but not on day 3 or at 24 weeks post-transplantation (Fig. 4). To identify the leukocyte subpopulations that may be involved in contributing to the response, staining for CD3⫹ T lymphocytes and ED1⫹ macrophages was performed. The pattern of ED1⫹ macrophage infiltration observed at day 1 and day
Antigen induction In renal allografts stored for one hour, no increase in MHC Class I expression was detected over the 24 week period, irrespective of preservation with lec-SOD (Fig. 5). In contrast, kidneys stored for 18 hours had significantly up-regulated MHC Class I expression by day 1 (Grade 2.0 ⫾ 0.6), remaining elevated at day 3 (Grade 2.0 ⫾ 0.7) and declining by 24 weeks post-transplant (Grade 1.7 ⫾ 0.8). Preservation with lec-SOD significantly attenuated induction of MHC Class I antigens at day 1 post-transplant (Grade 1.3 ⫾ 0.5; P ⫽ 0.037; Fig. 5). MHC Class II antigens were not significantly upregulated by 18 hours of cold ischemia, nevertheless maximal expression was seen in all kidneys at 24 weeks, irrespective of cold storage time and preservation with lec-SOD (Fig. 5). Low levels of ICAM-1 expression were detected on day 1 following transplantation. Significantly increased levels of ICAM-1 were detected on day 3 in kidneys stored for 18 hours compared with kidneys stored for
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Fig. 3. Early infiltration of HIS48⫹ granulocytes following cold ischemia and reperfusion is attenuated by preservation with lec-SOD. Granulocytes were assessed by morphometric point counting following indirect immunoperoxidase staining of kidneys harvested one and three days post-transplant with a granulocytespecific monoclonal antibody, HIS48. A significant increase in granulocytes was detected on day 1 in kidneys stored for 18 hours, remaining at high levels on day 3 post-transplant (P ⬍ 0.01). Preservation with lec-SOD for 18 hours significantly inhibited the granulocyte infiltration 1 day after transplantation (P ⫽ 0.017). Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. preservation for one hour with no SOD at the same time point; §P ⫽ 0.017 vs. preservation for 18 hours with lec-SOD at the same time point.
one hour (2.6 ⫾ 0.6 vs. 1.5 ⫾ 0.6; P ⬍ 0.01; Fig. 5). At 24 weeks, elevated ICAM-1 expression was detected in all kidneys and this was significantly exacerbated by long cold ischemia. Preservation with lec-SOD did not influence expression of ICAM-1 at any time point (Fig. 5). Apoptosis At day 1 post-transplantation, low levels of tubular cell apoptosis were detected in kidneys stored for one hour and 18 hours (Fig. 6). However, by day 3 posttransplant, a significant increase in the level of tubular cell apoptosis was observed in kidneys that were stored for 18 hours compared with kidneys stored for one hour (1.61 ⫾ 0.39 vs. 0.44 ⫾ 0.08, respectively, P ⬍ 0.001). Preservation of kidneys with lec-SOD significantly reduced the tubular apoptosis observed at day 3 (0.81 ⫾ 0.37 vs. 1.61 ⫾ 0.39; P ⫽ 0.0032) and at 24 weeks (0.67 ⫾ 0.41 vs. 1.21 ⫾ 0.57; P ⬍ 0.01) compared with allografts stored with no SOD for 18 hours (Fig. 6). The results from this study suggest that following 18 hours of cold ischemia, an initial inflammatory response mediated by macrophages and granulocytes was evident within the first few days post-transplantation, but after 24 weeks, T lymphocytes were the predominant leukocyte population. Preservation with lec-SOD significantly inhibited the initial inflammatory responses generated by cold ischemia, but did not appear to be protective against subsequent alloimmune responses that were exacerbated by the initial ischemic injury. However, preservation with lec-SOD did appear to inhibit the induction of apoptotic cell death by cold ischemia in the later post-transplant period. DISCUSSION The results in this current study show that the additional effects of cold ischemia on allogeneic renal trans-
plantation elicit a non-specific inflammatory response in the immediate post-transplant period, accelerating the development of renal allograft failure and enhancing apoptotic cell death and alloimmune responses. Preservation with lec-SOD inhibited the development of postreperfusion inflammatory responses and delayed the onset of chronic renal allograft failure induced by prolonged cold ischemia, but had no apparent effect on later alloimmune events. This study provides evidence to show that production of reactive oxygen species at reperfusion plays an important role in the pathogenesis of chronic allograft failure and that preservation with lec-SOD may be a useful therapeutic agent for protecting against the initial post-transplant damage. In this rat model of chronic renal allograft failure, a progressive increase in proteinuria up to 24 weeks posttransplantation was detected in kidneys exposed to 18 hours of cold ischemia compared with those preserved for only one hour. The long-term damage to renal allografts exposed to prolonged cold ischemia was prevented by preservation of kidneys with lec-SOD. This progressive elevation in proteinuria arising from cold ischemic damage is similar to data reported from a rat kidney model of in situ cold ischemia, where proteinuria also developed 16 weeks after the initial ischemia/reperfusion injury [24, 25]. It may be surprising that deteriorating renal function was not observed in the renal allografts with only one hour cold ischemia (subjected to weak alloimmune responses) as has been previously described [38], but this may be due to optimal preservation of kidneys with Marshall’s solution in our study. In this Fisher to Lewis rat renal allograft model, it was possible to evaluate the effects of the weak alloimmune response in kidneys with a minimal one-hour ischemia time and assess the additional effects of prolonged cold ischemia. In renal allografts with minimal ischemia time,
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Fig. 4. Variations in leukocyte subpopulations in renal allografts exposed to long cold ischemia during the early and late post-transplantation period. Total CD45⫹ leukocytes, CD3⫹ lymphocytes and ED1⫹ macrophages were assessed by morphometric point counting following indirect immunoperoxidase staining of kidneys harvested 1 day, 3 days and 24 weeks post-transplant. At day 1 and day 3 post-transplantation, the increase in total CD45⫹ leukocyte infiltration correlated with a similar increase in macrophage and granulocytes induced by prolonged cold ischemia. By 24 weeks post-transplantation, the number of macrophages had declined, whereas the level of CD3⫹ T lymphocyte infiltration had increased in renal allografts and further exacerbated by 18 hours of cold ischemia. This was not prevented by preservation with lec-SOD. Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 versus preservation for 1 hour with no SOD at the same time point; §P ⫽ 0.013 versus 18 hours of preservation with lec-SOD at the same time point.
there was no evidence of an inflammatory response in the first three days following transplantation. In contrast, in renal allografts exposed to 18 hours of cold ischemia, an inflammatory response was detected in the early phase after transplantation (day 1 and day 3), mediated by granulocytes and macrophages, with up-regulated expression of MHC Class I antigens and ICAM-1. Furthermore, in kidneys exposed to long cold ischemia, an increase in apoptotic cell death was detected. Therefore,
these inflammatory events are likely to result from the effects of cold ischemia and reperfusion injury. Indeed, preservation with lec-SOD significantly inhibited the infiltration of granulocytes, induction of MHC Class I antigens at day 1 post-transplantation and the development of apoptotic cell death in the early post-transplantation period. The granulocyte infiltration detected at days 1 and 3 post-transplant is characteristic of an early inflammatory
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Fig. 5. Variation in expression of major histocompatible complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) in renal allografts exposed to long cold ischemia. MHC Class I, Class II and ICAM-1 expression was assessed semiquantitatively following indirect immunoperoxidase staining of kidneys harvested 1 day, 3 days and 24 weeks post-transplant. A significant increase in Class I expression was detected on day 1 in kidneys stored for 18 hours (P ⬍ 0.01) that was significantly attenuated by preservation with lecSOD (P ⫽ 0.037). Expression of MHC Class II antigens was not enhanced by cold ischemic injury, but maximal levels were detected in all renal allografts at 24 weeks post-transplant. ICAM-1 expression was minimal at day 1, but increased by day 3 in renal allografts exposed to 18 hours of cold ischemia. At 24 weeks post-transplant, ICAM-1 levels were elevated in all kidneys and this was exacerbated by cold ischemia. Preservation with lec-SOD did not prevent up-regulated ICAM-1 expression in kidneys exposed to the long cold ischemia. Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. preservation for 1 hour with no SOD at the same time point; §P ⫽ 0.037 vs. preservation for 18 hours with lec-SOD at the same time point; ¶P ⬍ 0.01 vs. preservation for 1 hour with no SOD at day 1.
response generated as a result of oxygen free radical damage following ischemia/reperfusion [41–47]. Reactive oxygen species are generated by endothelial cells following hypoxia/reoxygenation [48, 49], resulting in increased expression of adhesion molecules facilitating binding and activation of neutrophils [37, 50–56]. Experimental models of ischemia and reperfusion have demonstrated that organ function was markedly improved and neutrophil infiltration significantly inhibited following treatment with exogenously administered SOD or transfection with human SOD cDNA [27–35, 57, 58]. We have previously demonstrated that lec-SOD binds with high
affinity to endothelial cells under cold hypoxic conditions and remains on the cell surface up to three days following reperfusion [37]. It is likely that direct antioxidant activity by bound lec-SOD at the endothelial surface of vessels in the kidneys, prevented damage caused by an alloindependent ischemia/reperfusion injury. At 24 weeks following transplantation, histological changes associated with the diagnosis of chronic renal allograft failure were evident in renal allografts that were exposed to long cold ischemia, but were not observed in those allografts with short cold ischemia times. The inflammatory response involving granulocytes and mac-
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Fig. 6. Preservation with lec-SOD inhibited the late development of apoptosis in renal allografts exposed to long periods of cold ischemia. Apoptosis of tubular cells was assessed following TUNEL staining using the Apoptosis Index scoring system. Prolonged cold ischemia did not increase the level of apoptosis observed at day 1 post-transplant. However, the number of apoptotic cells increased significantly at 3 days and 24 weeks post-transplantation in kidneys stored for 18-hours cold ischemia. Preservation of kidneys with lecSOD significantly inhibited the development of apoptosis at 3 days and 24 weeks in kidneys exposed to long cold ischemia (P ⬍ 0.01). Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. 18 hours preservation with lecSOD at the same time point.
rophages was absent in all kidneys at 24 weeks posttransplantation. However, an increase in CD3⫹ T lymphocytes was detected in kidneys with minimal ischemia presumably as a result of alloimmune responses, and this was significantly enhanced in kidneys that were exposed to 18 hours of cold ischemia. Similarly, ICAM-1 expression was elevated by alloimmune responses at 24 weeks and was enhanced by cold ischemic injury. The harmful effects of cold ischemia also resulted in significantly higher levels of apoptotic cell death that was inhibited by preservation with lec-SOD. Although preservation with lec-SOD for 18 hours significantly improved longterm allograft function and reduced apoptotic cell death, no reduction in cellular infiltration, adhesion molecules or MHC antigen expression were observed at 24 weeks compared with untreated renal allografts. The induction of apoptosis as a result of ischemia/ reperfusion injury has been previously reported in a number of experimental models [45, 59–62]. In clinical renal allografts, correlations have been observed between delayed graft function and apoptosis following reperfusion [63]. Analysis of post-reperfusion biopsies from cadaver and living-related donor kidneys demonstrated a high number of apoptotic cells predominantly in cadaver renal allografts with long cold ischemia times [20]. The reduction in apoptotic cells following preservation with lec-SOD may reflect the reduction in the availability of reactive oxygen species known to cause direct DNA damage and activation of harmful intracellular endonucleases [64, 65]. These results suggest that preservation with lec-SOD may limit the initial inflammatory response generated by ischemia/reperfusion injury, and in so doing, prevent the accumulation of harmful events that lead to long-term apoptotic damage of the kidney. In summary, the results from this study strongly suggest that prolonged cold ischemia exacerbates the effects of alloimmune responses in the development of chronic
allograft failure. Preservation with lec-SOD for 18 hours significantly inhibited the early inflammatory response but not long-term alloimmune responses or histological changes associated with chronic allograft failure. However, chronic renal allograft dysfunction was prevented in allografts preserved with lec-SOD and was associated with a reduction in the level of apoptosis detected at 24 weeks. Taken together, these results suggest that the inflammatory response generated by ischemia/reperfusion injury is mediated by reactive oxygen species. If the harmful effects of reactive oxygen species and reperfusion injury can be attenuated, then this may prevent the onset of apoptosis within the graft and thus preserve renal function. Lec-SOD may be a suitable agent for protection of renal allografts against initial ischemia/reperfusion injury with potentially beneficial effects on chronic renal allograft dysfunction. ACKNOWLEDGMENTS This work was supported by the National Kidney Research Fund, UK and the Seikagaku Corporation, Japan. The results from this study were accepted for oral presentation and presented at the 2000 XVIII International Congress of the Transplantation Society, Rome, Italy. A.J.M. was in receipt of a Hanson Trust Research Fellowship. Reprint requests to Dr. Susan V. Fuggle, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom. E-mail: [email protected]
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5. Solez K, Axelsen RA, Benediktsson H, et al: International standardisation of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 44:411–422, 1993 6. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713–723, 1999 7. Vazquez MA: Chronic rejection of renal transplants: New clinical insights. Am J Med Sci 320:43–58, 2000 8. Massy Z, Guijarro C, Wiederkehr M, et al: Chronic renal allograft rejection: Immunologic and nonimmunologic risk factors. Kidney Int 49:518–524, 1996 9. McLaren A, Fuggle S, Welsh K, et al: Chronic allograft failure in human renal transplantation. A multivariate risk factor analysis. Ann Surg 232:98–103, 2000 10. Najarian JS, Gillingham KJ, Sutherland DE, et al: The impact of the quality of initial graft function on cadaver kidney transplants. Transplantation 57:812–816, 1994 11. Peters TG, Shaver TR, Ames JE, et al: Cold ischemia and outcome in 17,937 cadaveric kidney transplants. Transplantation 59:191–196, 1995 12. Troppmann C, Gillingham KJ, Benedetti E, et al: Delayed graft function, acute rejection, and outcome after cadaver renal transplantation. The multivariate analysis. Transplantation 59:962–968, 1995 13. Troppmann C, Gillingham KJ, Gruessner RWG, et al: Delayed graft function in the absence of rejection has no long-term impact. Transplantation 61:1331–1337, 1996 14. Shoskes DA, Halloran PF: Delayed graft function: Etiology, management and long-term significance. J Urol 155:1831–1840, 1996 15. Chertow GM, Milford EL, Mackenzie HS, et al: Antigen-independent determinants of cadaveric kidney transplant failure. JAMA 276:1732–1736, 1996 16. Nicholson ML, Wheatley TJ, Horsburgh T, et al: The relative influence of delayed graft function and acute rejection on renal transplant survival. Transpl Int 9:415–419, 1996 17. Ojo AO, Wolfe RA, Held PJ, et al: Delayed graft function: Risk factors and implications for renal allograft survival. Transplantation 63:968–974, 1997 18. Shoskes DA, Cecka JM: Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation 66:1697–1701, 1998 19. Koo DDH, Welsh KI, Roake JA, et al: Ischemia/reperfusion injury in human kidney transplantation: An immunohistochemical analysis of changes after reperfusion. Am J Pathol 153:557–566, 1998 20. Burns AT, Davies DR, McLaren AJ, et al: Apoptosis in ischemia/ reperfusion injury of human renal allografts. Transplantation 66:872–876, 1998 21. Land W, Messmer K: The impact of ischaemia/reperfusion injury on specific and non-specific, early and late chronic events after organ transplantation. Transplant Rev 10:108–127, 1996 22. Tilney NL, Guttman RD: Effects of inital ischemia/reperfusion injury on the transplanted kidney. Transplantation 64:945–947, 1997 23. Land W: Reactive oxygen species in chronic allograft dysfunction. Curr Opin Organ Transplant 14:16–22, 1999 24. Chandraker A, Takada M, Nadeau KC, et al: CD28–B7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury. Kidney Int 52:1678–1684, 1997 25. Takada M, Nadeau KC, Shaw GD, et al: Prevention of late renal changes after initial ischemia/reperfusion injury by blocking early selectin binding. Transplantation 64:1520–1525, 1997 26. Koo DDH, Fuggle SV: Impact of ischemia/reperfusion injury and early inflammatory responses in kidney transplantation. Transplant Rev 14:210–224, 2000 27. Koyama I, Bulkley GB, Williams GM, et al: The role of oxygen free radicals in mediating the reperfusion injury of cold-preserved ischaemic kidneys. Transplantation 40:590–595, 1985 28. Baker GL, Corry RJ, Autor AP: Oxygen free radical induced damage in kidneys subjected to warm ischaemia and reperfusion: Protective effect of superoxide dismutase. Ann Surg 202:628–641, 1985
29. Hoshino T, Maley WR, Bulkley GB, et al: Ablation of free radical mediated reperfusion injury for the salvage of kidneys taken from non-heart-beating donors. Transplantation 45:284–289, 1988 30. Marzi I, Knee J, Buhren V, et al: Reduction by superoxide dismutase of leucocyte-endothelial adherence after liver transplantation. Surgery 111:90–97, 1992 31. Lefer AM, Ma XL, Weyrich A, et al: Endothelial dysfunction and neutrophil adherence as critical events in the development of reperfusion injury. Inflamm Dis Ther 41:127–135, 1993 32. Land W, Schneeberger H, Schleibner S, et al: The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation 57:211–217, 1994 33. Gianello P, Saliez A, Bufkens X, et al: EUK-134, a synthetic superoxide dismutase and catalase mimetic, protects rat kidneys from ischemia-reperfusion-induced damage. Transplantation 62:1664– 1666, 1996 34. Caramelo C, Espinosa G, Manzarbetia F, et al: Role of endothelium-related mechanisms in the pathophysiology of renal ischemia/ reperfusion in normal rabbits. Circ Res 79:1031–1038, 1996 35. Kurose I, Argenbright LW, Wolf R, et al: Ischemia/reperfusioninduced microvascular dysfunction: Role of oxidants and lipid mediators. Am J Physiol 272:H2976–H2982, 1997 36. Igarashi R, Hoshino J, Ochiai A, et al: Lecithinized superoxide dismutase enhances its pharmacologic potency by increasing its cell membrane affinity. J Pharm Exp Ther 271:1672–1677, 1994 37. Koo DDH, Welsh KI, West NEJ, et al: Endothelial cell protection against ischemia/reperfusion injury by lecithinized superoxide dismutase. Kidney Int 60:786–796, 2001 38. Hancock WH, Whitley WD, Tullius SG, et al: Cytokines, adhesion molecules, and the pathogenesis of chronic rejection of rat renal allografts. Transplantation 56:643–650, 1993 39. McMaster WR, Williams AF: Identification of Ia glycoproteins in rat thymus and purification from rat spleen. Eur J Immunol 9:426–433, 1979 40. Fuggle SV, McWhinnie DL, Chapman JR, et al: Sequential analysis of HLA-class II antigen expression in human renal allografts. Induction of tubular class II antigens and correlation with clinical parameters. Transplantation 42:144–150, 1986 41. Granger DN, Kvietys PR, Perry MA: Leukocyte-endothelial cell adhesion induced by ischemia and reperfusion. Can J Physiol Pharmacol 71:67–75, 1993 42. Lefer AM, Ma XL, Weyrich AS, et al: Mechanism of the cardioprotective effect of transforming growth factor beta 1 in feline myocardial ischemia and reperfusion. Proc Natl Acad Sci USA 90:1018–1022, 1993 43. Seekamp A, Mulligan MS, Till GO, et al: Requirements for neutrophil products and L-arginine in ischemia-reperfusion injury. Am J Pathol 142:1217–1226, 1993 44. Lefer D, Flynn D, Phillips M, et al: A novel sialyl Lewis X analog attenuates neutrophil accumulation and myocardial necrosis after ischaemia and reperfusion. Circulation 90:2390–2401, 1994 45. Buerke M, Murohara T, Skurk C, et al: Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci USA 92:8031–8035, 1995 46. Raschke P, Becker B: Adenosine and PAF dependent mechanisms lead to myocardial reperfusion injury by neutrophils after brief ischaemia. Cardiovasc Res 29:569–576, 1995 47. Grisham MB, Granger DN, Lefer DJ: Modulation of leukocyteendothelial interactions by reactive metabolites of oxygen and nitrogen: Relevance to ischemic heart disease. Free Rad Biol Med 25:404–433, 1998 48. Zweier JL, Kuppusamy P, Thompson-Gorman S, et al: Measurement and characterization of free radical generation in reoxygenated human endothelial cells. Am J Physiol 266:C700–C708, 1994 49. Zweier JL, Broderick R, Kuppusamy P, et al: Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation. J Biol Chem 269:24156–24162, 1994 50. Arnould T, Michiels C, Remacle J: Increased PMN adherence on endothelial cells after hypoxia: Involvement of PAF, CD18/ CD11b and ICAM-1. Am J Physiol 264:C1102–C1110, 1993 51. Arnould T, Michiels C, Remacle J: Hypoxic human umbilical
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zinc-superoxide dismutase (SOD1) prevents post-ischemic injury. Proc Natl Acad Sci USA 95:4556–4560, 1998 Gottlieb RA, Burleson KO, Kloner RA, et al: Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621–1628, 1994 Fliss H, Gattinger D: Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79:949–956, 1996 Sasaki H, Matsuno T, Tanaka N, et al: Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transplant Proc 28:1908, 1996 Sasaki H, Matsuno T, Ishikawa T, et al: Activation of apoptosis during early phase of reperfusion after liver transplantation. Transplant Proc 29:406–407, 1997 Matsuno N, Sakurai E, Tamaki I, et al: The effect of machine perfusion preservation versus cold storage on the function of kidneys from non-heart beating donors. Transplantation 57:293–294, 1994 Hagar H, Ueda N, Shah SV: Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLCPK1 cells. Am J Physiol 271:F209–F215, 1996 Sato N, Iwata K, Nakamura TH, et al: Thiol mediated redox regulation of apoptosis: possible roles of cellular thiols other than glutathione in T cell apoptosis. J Immunol 154:3194–3203, 1995
Lecithinized superoxide dismutase reduces cold ischemia-induced chronic allograft dysfunction KEN NAKAGAWA, DICKEN D.H. KOO, DAVID R. DAVIES, DEREK W.R. GRAY, ANDREW J. MCLAREN, KENNETH I. WELSH, PETER J. MORRIS, and SUSAN V. FUGGLE Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; Department of Urology, Keio University, Japan; and Department of Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom
Lecithinized superoxide dismutase reduces cold ischemiainduced chronic allograft dysfunction. Background. Chronic renal allograft failure (CAF) is influenced by both allo-dependent and independent factors and is a major cause of graft loss in clinical renal transplantation. We evaluated a novel membrane-bound free radical scavenger, lecithinized superoxide dismutase (lec-SOD), to determine its potential in limiting the harmful effects of ischemia/reperfusion injury on CAF. Methods. Fisher rat kidneys were stored for either 1 hour or 18 hours in cold Marshall’s preservation solution either with or without lec-SOD and transplanted into Lewis recipients. Results. Within 3 days of transplantation, an early inflammatory response involving granulocytes and macrophages was detected in renal allografts exposed to 18 hours cold ischemia that was significantly reduced by preservation with lec-SOD. By 24 weeks post-transplantation, elevated proteinuria and detection of apoptotic cells was observed in kidneys exposed to 18 hours of cold ischemia, that was attenuated by preservation with lec-SOD (P ⬍ 0.05). However, up-regulated expression of intracellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) Class II together with a T lymphocyte infiltration were observed at 24 weeks that was not prevented by preservation with lec-SOD. Conclusions. These results demonstrate that ischemia/reperfusion injury, apoptotic cell death and allo-immune responses may be exacerbated by cold ischemia and accelerate the development of CAF. Preservation with lec-SOD may protect against the early damage induced by cold ischemia and reperfusion injury.
Despite progressive improvements in the short-term results of first cadaver renal allografts, the problems of long-term graft loss remain unresolved. It is becoming increasingly evident that chronic renal allograft dysfuncKey words: organ preservation, chronic rejection, superoxide dismutase, transplantation, kidney, apoptosis, antioxidant. Received for publication July 20, 2001 and in revised form October 19, 2001 Accepted for publication October 22, 2001
2002 by the International Society of Nephrology
tion is a major cause of late graft failure [1–3]. Chronic allograft failure has been defined as the progressive deterioration in graft function occurring at least 90 days after transplantation, and is associated with the histological features of interstitial fibrosis, arterial intimal thickening, vascular lesions and tubular atrophy [4–7]. A number of alloantigen-dependent and independent factors have been proposed as contributing to chronic allograft failure. Acute rejection episodes resulting from alloimmune responses are a major risk factor for chronic allograft failure, but damage to the organ as a result of alloindependent events also plays a key role in aggravating chronic renal allograft dysfunction and rejection [7–9]. In renal transplantation, prolonged cold ischemic storage of kidneys has been shown to be significantly associated with a higher incidence of delayed graft function (DGF) after transplantation [10–18]. DGF has been shown to have a significant impact on poor long-term graft survival, especially in association with acute rejection [17, 18]. We have previously demonstrated that in cadaver renal allografts with long cold ischemia times, a neutrophil and platelet-mediated inflammatory response and apoptotic endothelial and proximal tubular epithelial cells were observed after reperfusion [19, 20]. There is evidence to suggest that the initial inflammatory events arising from ischemia/reperfusion injury may up-regulate alloantigens and thus increase graft immunogenicity and exacerbate an alloimmune response [21–23]. In renal models of in situ cold ischemia and reperfusion, upregulated expression of MHC antigens, costimulatory molecules, cytokines, chemokines, adhesion molecules and leukocyte infiltration have been detected in association with progressive long-term deterioration in renal function [24, 25]. The mechanisms involved in the generation of an inflammatory response following ischemia/reperfusion are complex, but production of reactive oxygen species is thought to be a critical event [26]. Many studies have
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shown that ischemia/reperfusion injury is abolished by administration of an oxygen free radical scavenger, superoxide dismutase (SOD) [27–35]. Indeed, Land and colleagues demonstrated that administration of SOD at reperfusion significantly reduced the incidence of first acute rejection episodes and improved long-term graft survival in cadaver renal allografts [32]. These findings suggest that the production of reactive oxygen species during the initial period of transplantation may accelerate subsequent immune mediated events and affect longterm graft survival. To determine whether it was possible to limit the harmful effects of ischemia/reperfusion injury on chronic renal allograft failure, we have investigated the therapeutic potential of a novel form of lecithinized SOD (lecSOD). Lec-SOD was produced by the covalent linkage of recombinant human SOD with lecithin, enabling greater stability and high affinity binding to cell membranes [36]. We have previously demonstrated in vitro that in contrast to unmodified rhSOD, lec-SOD binds to microvascular and large vessel endothelial cells under cold hypoxic conditions in Marshall’s solution, thereby inhibiting hypoxia-induced cell death, endothelial adhesion molecule expression and neutrophil adhesion [37]. The aims of the current study were (1) to examine the effects of prolonged cold ischemia and reperfusion injury on chronic allograft failure in a rat renal allograft model, and (2) to determine whether preservation of kidneys with lec-SOD before transplantation exerted a beneficial effect on inflammatory and histological changes and on graft function.
the aorta and vena cava dissected and clamped. In the first instance only the left native kidney was removed and the donor kidney was transplanted by an end-toside anastomosis to the recipient aorta with a running 10-0 nylon suture. After release of the clamps an endto-end ureteric anastomosis was performed by an interrupted 10-0 suture. The remaining right native kidney was removed 10 days post-transplantation after providing initial support against the effects of delayed function of the allograft. Low-dose cyclosporine A (5 mg/kg/day) was administered by gavage during the initial 10-day post-operative period to prevent early rejection. In recipient animals whose renal allografts were harvested at either day 1 or day 3 post-transplantation, both native kidneys were removed at the time of transplantation so that the early effects of ischemia/reperfusion injury on histological changes could be determined. Between 4 and 7 animals were transplanted and harvested at each time point.
METHODS Experimental model Allogeneic renal transplants were performed from fasted, inbred male (250 to 300 g) donor Fisher (F344, RT1lv1) to recipient Lewis rats (LEW, RT1l; Harlan Olac Ltd, Bicester, UK) to recreate the model of chronic renal allograft failure described by Hancock and colleagues [38]. A midline abdominal incision was performed on anesthetized donor rats and the renal vessels and ureter mobilized after ligation of the adrenal and gonadal veins. The animals were heparinized and the main vessels clamped before both kidneys were flushed through the aorta with 5 mL of ice-cold Marshall’s hypertonic citrate solution (Baxter Healthcare Ltd, Berkshire, UK). Kidneys were removed and stored at 4⬚C. Four treatment groups were studied: (i) one hour cold ischemia in Marshall’s solution alone; (ii) one hour cold ischemia in Marshall’s solution ⫹ 50 g/mL lec-SOD (Seikagaku Corporation, Tokyo, Japan); (iii) 18 hours cold ischemia in Marshall’s solution alone; or (iv) 18 hours cold ischemia with Marshall’s ⫹ 50 g/mL lec-SOD. Recipient rats were fasted overnight, anesthetized and
Histopathology and Immunohistology Upon harvesting, kidneys were dissected longitudinally and half of the kidney fixed in formalin for paraffin embedding and the other half snap frozen and stored at –80⬚C for immunohistochemistry. Histopathological analysis for morphological changes to glomeruli, tubules, interstitium and vessels associated with chronic rejection was assessed by an experienced renal pathologist (DRD) under blinded conditions. Renal sections were graded on a semi-quantitative basis. Glomerular proliferation and sclerosis were scored as: Grade 0 ⫽ none; Grade 1 ⫽ occasional focal segmental proliferation and sclerosis; Grade 2 ⫽ areas of glomerular segmental proliferation and sclerosis. The degree of tubular atrophy was graded as: Grade 0 ⫽ none; Grade 1 ⫽ occasional tubular atrophy; Grade 2 ⫽ focal or multiple focal areas of atrophy. Tubular cast formation was assessed as: Grade 0 ⫽ none; Grade 1 ⫽ occasional hyaline cast; Grade 2 ⫽ areas of hyaline and/or granular casts. The level of interstitial infiltration was graded as: Grade 0 ⫽ none; Grade 1 ⫽ occasional focus; Grade 2 ⫽ multiple foci of infiltrating cells.
Measurement of renal allograft dysfunction Renal allograft dysfunction was determined by measurement of proteinuria in six animals in each of the four treatment groups. Urine samples were collected at four-week intervals from rats placed in metabolic cages for 24 hours, up to and including 24 weeks post-transplant. For this experiment there were six animals in each treatment group. Protein excretion was determined by measuring precipitation after interaction with 3% sulfosalicylic acid (Sigma, Poole, Dorset, UK). Turbidity was assessed by absorbance measurements at a wavelength of 595 nm.
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Frozen biopsy material was embedded in O.C.T. (Miles Inc., Shawnee Mission, KS, USA), and 7 m cryosections were stained using an indirect immunoperoxidase technique with the following monoclonal antibodies: IF4 (CD3 T lymphocytes), ED1 (macrophages), HIS48 (granulocytes), MRC OX1 (CD45 leukocyte common antigen), F16-4.4 [major histocompatibility complex (MHC) Class I] and TLD-4C9 [intracellular adhesion molecule-1 (ICAM-1)] were all obtained from Serotec (Oxford, UK) and MRC OX3 (MHC Class II) [39]. The signals for MHC Class I, ICAM-1, CD45 and ED1 were amplified with a further incubation step for 30 minutes using a peroxidase-conjugated swine anti-rabbit Ig (1:50 dilution; Serotec) pre-incubated with 20% rat serum. IgM isotype monoclonal antibodies against CD3 and HIS48 were developed with a peroxidase-conjugated goat anti-mouse IgM (1:50 dilution; Serotec) and enhanced with a rabbit anti-goat Ig (1:200 dilution; Sigma Ltd.), both pre-incubated with 20% rat serum. All sections were examined without knowledge of the treatment groups. Leukocyte infiltration was quantitated by determining the percentage area occupied by a particular cell type using an established morphometric point counting technique [40]. Semiquantitative grading for the induced expression of ICAM-1, MHC Class I and MHC Class II antigens on proximal tubular epithelial cells were assessed by two independent observers. The semiquantitative grades for ICAM-1 and MHC Class I were: Grade 1 ⫽ occasional weakly positive tubule; Grade 2 ⫽ isolated foci of positive tubules; Grade 3 ⫽ multiple foci of more extensive positive tubular staining. MHC Class II expression was graded as: Grade 1 ⫽ negative; Grade 2 ⫽ foci of weakly positive tubules. Measurement of apoptosis Identification of apoptotic cells was performed using the terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) technique as previously described [20]. Briefly, sections were obtained from paraffin embedded kidney specimens and deparaffinized, rehydrated and digested with 20g/mL Proteinase K (Sigma Ltd) at 37⬚C for 60 minutes. Following proteinase digestion, the specimens were stained with the Apoptag Plus peroxidase kit (Oncor, Cambridge, UK) according to the manufacturer’s protocol. TUNEL-positive apoptotic cells were identified as single positively stained cells, with associated chromatin condensation and positive chromatin fragments. The Apoptosis Index was calculated from the mean number of positive cells per ⫻250 field of view, following an analysis of 20 fields of view per section as previously described [20].
Statistical analyses Statistical analyses were performed using the Student t test to determine if there were significant differences between treatment groups when P ⬍ 0.05. RESULTS Renal allograft dysfunction Damage to renal allografts as determined by a rise in proteinuria level was detected in renal allografts exposed to 18 hours cold ischemia (Fig. 1). In contrast, kidneys preserved with lec-SOD for 18 hours remained relatively stable, with significantly lower proteinuria at 16, 20 and 24 weeks post-transplantation (P ⬍ 0.05). Furthermore, organ preservation for a minimal one-hour period in the presence or absence of lec-SOD had no detrimental effects on graft function, with stable proteinuria levels over the 24-week period (Fig. 1). Histopathology A histopathological analysis of rat renal allografts was performed on samples obtained at 24 weeks post-transplantation to determine the effects of long cold ischemia on chronic allograft failure. Glomerular mesangial deposition was equally prominent in all allografts and there was no evidence of interstitial fibrosis, glomerular basement membrane changes or arterial intimal proliferation and thickening. However, in renal allografts with 18 hours of cold ischemia, significantly higher mean grades were observed for focal segmental glomerular sclerosis and proliferation (0.75 ⫾ 0.62 vs. 0.25 ⫾ 0.45; P ⬍ 0.01), tubular atrophy (1.5 ⫾ 0.79 vs. 0.25 ⫾ 0.45; P ⬍ 0.001), tubular cast formation (1.58 ⫾ 0.67 vs. 0.33 ⫾ 0.65; P ⬍ 0.001) and interstitial infiltration (2.0 ⫾ 0.73 vs. 1.4 ⫾ 0.51; P ⬍ 0.05) compared with kidneys preserved for shorter periods of cold ischemia (Fig. 2). Preservation with lec-SOD had no effect on graft histology. Leukocyte infiltration Immunohistochemical analysis to detect infiltration by granulocytes was performed in the immediate post-transplant period. Significantly higher levels of HIS48⫹ granulocytes were present at day 1 in kidneys exposed to the 18-hour cold ischemia compared with renal allografts stored for only one hour (2.8 ⫾ 0.4% vs. 0.8 ⫾ 0.6%; P ⬍ 0.01), remaining at elevated levels three days after transplantation (2.7 ⫾ 0.9%; Fig. 3). The increase in granulocyte infiltration at day 1 in grafts exposed to 18 hours of cold ischemia was significantly attenuated by preservation with lec-SOD (2.8 ⫾ 0.4% vs. 1.9 ⫾ 0.7%; P ⫽ 0.017), but this protective effect was not evident at day 3 (2.2 ⫾ 0.6% vs. 2.8 ⫾ 0.8%; P ⬎ 0.05). At all time points following transplantation, the level of CD45⫹ leukocyte infiltration was increased by expo-
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Fig. 1. Preservation with lecithinized superoxide dismutase (lec-SOD) inhibits development of chronic renal allograft dysfunction induced by cold ischemia. Kidneys from Fisher donors were exposed to one hour or 18 hours of cold ischemic preservation in the presence or absence of lec-SOD. After transplantation, renal function was assessed by 24-hour proteinuria measurements taken at 4 weekly intervals up to 24 weeks post-transplantation. A significant increase in proteinuria was detected at 16, 20 and 24 weeks post-transplant in renal allografts stored for 18 hours compared with kidneys that were stored for only one hour (P ⬍ 0.05). This deterioration in longterm renal allograft function was delayed by preservation with lec-SOD (P ⬍ 0.05). Each time point represents the mean ⫾ SEM for 6 animals. Symbols are: (䊏) 1 hour no lec-SOD; (䊐) 1 hour ⫹lec-SOD; (䉱) 18 hours no lecSOD; (䉭) 18 hours ⫹lec-SOD; *P ⬍ 0.05, 18 hours cold ischemia time (CIT) vs. 1 hour CIT; §P ⬍ 0.05, 18 hours CIT ⫺ SOD vs. 18 hours CIT ⫹ lec-SOD.
3 was similar to that detected for total CD45⫹ leukocyte infiltration, demonstrating that macrophages and granulocytes (Fig. 3) are the major components of the infiltration at these time points (Fig. 4). Twenty-four weeks after transplantation, low levels of macrophages were detected in all allografts. In complete contrast, low levels of CD3⫹ T lymphocytes were detected on days 1 and 3 post-transplant, irrespective of the period of cold storage or preservation with lec-SOD. By 24 weeks post-transplant, increased levels of T lymphocytes were detected, an effect that was further exacerbated by 18 hours of cold storage. Lec-SOD did not influence the magnitude of this infiltration. Fig. 2. Long cold ischemia induces histological changes associated with chronic renal allograft failure. Paraffin sections from renal allografts at 24 weeks post-transplantation were analyzed for morphological changes characteristic of chronic rejection and assessed semiquantitatively to determine the severity of injury. In kidneys exposed to 18 hours of cold ischemia (䊏), there were significantly higher levels of focal segmental glomerular sclerosis and proliferation (FSGS), tubular atrophy, cast formation and interstitial infiltration at 24 weeks post-transplant than in kidneys stored for one hour ( ; P ⬍ 0.05). Preservation with lecSOD did not prevent the development of these chronic histological changes. *P ⬍ 0.01 vs. 1 hour CIT.
sing the kidney to 18 hours of cold ischemia. Preservation of kidneys with lec-SOD significantly reduced the level of CD45⫹ infiltration on day 1 (3.9 ⫾ 1.1% vs. 2.7 ⫾ 0.3%; P ⫽ 0.013), but not on day 3 or at 24 weeks post-transplantation (Fig. 4). To identify the leukocyte subpopulations that may be involved in contributing to the response, staining for CD3⫹ T lymphocytes and ED1⫹ macrophages was performed. The pattern of ED1⫹ macrophage infiltration observed at day 1 and day
Antigen induction In renal allografts stored for one hour, no increase in MHC Class I expression was detected over the 24 week period, irrespective of preservation with lec-SOD (Fig. 5). In contrast, kidneys stored for 18 hours had significantly up-regulated MHC Class I expression by day 1 (Grade 2.0 ⫾ 0.6), remaining elevated at day 3 (Grade 2.0 ⫾ 0.7) and declining by 24 weeks post-transplant (Grade 1.7 ⫾ 0.8). Preservation with lec-SOD significantly attenuated induction of MHC Class I antigens at day 1 post-transplant (Grade 1.3 ⫾ 0.5; P ⫽ 0.037; Fig. 5). MHC Class II antigens were not significantly upregulated by 18 hours of cold ischemia, nevertheless maximal expression was seen in all kidneys at 24 weeks, irrespective of cold storage time and preservation with lec-SOD (Fig. 5). Low levels of ICAM-1 expression were detected on day 1 following transplantation. Significantly increased levels of ICAM-1 were detected on day 3 in kidneys stored for 18 hours compared with kidneys stored for
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Fig. 3. Early infiltration of HIS48⫹ granulocytes following cold ischemia and reperfusion is attenuated by preservation with lec-SOD. Granulocytes were assessed by morphometric point counting following indirect immunoperoxidase staining of kidneys harvested one and three days post-transplant with a granulocytespecific monoclonal antibody, HIS48. A significant increase in granulocytes was detected on day 1 in kidneys stored for 18 hours, remaining at high levels on day 3 post-transplant (P ⬍ 0.01). Preservation with lec-SOD for 18 hours significantly inhibited the granulocyte infiltration 1 day after transplantation (P ⫽ 0.017). Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. preservation for one hour with no SOD at the same time point; §P ⫽ 0.017 vs. preservation for 18 hours with lec-SOD at the same time point.
one hour (2.6 ⫾ 0.6 vs. 1.5 ⫾ 0.6; P ⬍ 0.01; Fig. 5). At 24 weeks, elevated ICAM-1 expression was detected in all kidneys and this was significantly exacerbated by long cold ischemia. Preservation with lec-SOD did not influence expression of ICAM-1 at any time point (Fig. 5). Apoptosis At day 1 post-transplantation, low levels of tubular cell apoptosis were detected in kidneys stored for one hour and 18 hours (Fig. 6). However, by day 3 posttransplant, a significant increase in the level of tubular cell apoptosis was observed in kidneys that were stored for 18 hours compared with kidneys stored for one hour (1.61 ⫾ 0.39 vs. 0.44 ⫾ 0.08, respectively, P ⬍ 0.001). Preservation of kidneys with lec-SOD significantly reduced the tubular apoptosis observed at day 3 (0.81 ⫾ 0.37 vs. 1.61 ⫾ 0.39; P ⫽ 0.0032) and at 24 weeks (0.67 ⫾ 0.41 vs. 1.21 ⫾ 0.57; P ⬍ 0.01) compared with allografts stored with no SOD for 18 hours (Fig. 6). The results from this study suggest that following 18 hours of cold ischemia, an initial inflammatory response mediated by macrophages and granulocytes was evident within the first few days post-transplantation, but after 24 weeks, T lymphocytes were the predominant leukocyte population. Preservation with lec-SOD significantly inhibited the initial inflammatory responses generated by cold ischemia, but did not appear to be protective against subsequent alloimmune responses that were exacerbated by the initial ischemic injury. However, preservation with lec-SOD did appear to inhibit the induction of apoptotic cell death by cold ischemia in the later post-transplant period. DISCUSSION The results in this current study show that the additional effects of cold ischemia on allogeneic renal trans-
plantation elicit a non-specific inflammatory response in the immediate post-transplant period, accelerating the development of renal allograft failure and enhancing apoptotic cell death and alloimmune responses. Preservation with lec-SOD inhibited the development of postreperfusion inflammatory responses and delayed the onset of chronic renal allograft failure induced by prolonged cold ischemia, but had no apparent effect on later alloimmune events. This study provides evidence to show that production of reactive oxygen species at reperfusion plays an important role in the pathogenesis of chronic allograft failure and that preservation with lec-SOD may be a useful therapeutic agent for protecting against the initial post-transplant damage. In this rat model of chronic renal allograft failure, a progressive increase in proteinuria up to 24 weeks posttransplantation was detected in kidneys exposed to 18 hours of cold ischemia compared with those preserved for only one hour. The long-term damage to renal allografts exposed to prolonged cold ischemia was prevented by preservation of kidneys with lec-SOD. This progressive elevation in proteinuria arising from cold ischemic damage is similar to data reported from a rat kidney model of in situ cold ischemia, where proteinuria also developed 16 weeks after the initial ischemia/reperfusion injury [24, 25]. It may be surprising that deteriorating renal function was not observed in the renal allografts with only one hour cold ischemia (subjected to weak alloimmune responses) as has been previously described [38], but this may be due to optimal preservation of kidneys with Marshall’s solution in our study. In this Fisher to Lewis rat renal allograft model, it was possible to evaluate the effects of the weak alloimmune response in kidneys with a minimal one-hour ischemia time and assess the additional effects of prolonged cold ischemia. In renal allografts with minimal ischemia time,
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Fig. 4. Variations in leukocyte subpopulations in renal allografts exposed to long cold ischemia during the early and late post-transplantation period. Total CD45⫹ leukocytes, CD3⫹ lymphocytes and ED1⫹ macrophages were assessed by morphometric point counting following indirect immunoperoxidase staining of kidneys harvested 1 day, 3 days and 24 weeks post-transplant. At day 1 and day 3 post-transplantation, the increase in total CD45⫹ leukocyte infiltration correlated with a similar increase in macrophage and granulocytes induced by prolonged cold ischemia. By 24 weeks post-transplantation, the number of macrophages had declined, whereas the level of CD3⫹ T lymphocyte infiltration had increased in renal allografts and further exacerbated by 18 hours of cold ischemia. This was not prevented by preservation with lec-SOD. Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 versus preservation for 1 hour with no SOD at the same time point; §P ⫽ 0.013 versus 18 hours of preservation with lec-SOD at the same time point.
there was no evidence of an inflammatory response in the first three days following transplantation. In contrast, in renal allografts exposed to 18 hours of cold ischemia, an inflammatory response was detected in the early phase after transplantation (day 1 and day 3), mediated by granulocytes and macrophages, with up-regulated expression of MHC Class I antigens and ICAM-1. Furthermore, in kidneys exposed to long cold ischemia, an increase in apoptotic cell death was detected. Therefore,
these inflammatory events are likely to result from the effects of cold ischemia and reperfusion injury. Indeed, preservation with lec-SOD significantly inhibited the infiltration of granulocytes, induction of MHC Class I antigens at day 1 post-transplantation and the development of apoptotic cell death in the early post-transplantation period. The granulocyte infiltration detected at days 1 and 3 post-transplant is characteristic of an early inflammatory
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Fig. 5. Variation in expression of major histocompatible complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) in renal allografts exposed to long cold ischemia. MHC Class I, Class II and ICAM-1 expression was assessed semiquantitatively following indirect immunoperoxidase staining of kidneys harvested 1 day, 3 days and 24 weeks post-transplant. A significant increase in Class I expression was detected on day 1 in kidneys stored for 18 hours (P ⬍ 0.01) that was significantly attenuated by preservation with lecSOD (P ⫽ 0.037). Expression of MHC Class II antigens was not enhanced by cold ischemic injury, but maximal levels were detected in all renal allografts at 24 weeks post-transplant. ICAM-1 expression was minimal at day 1, but increased by day 3 in renal allografts exposed to 18 hours of cold ischemia. At 24 weeks post-transplant, ICAM-1 levels were elevated in all kidneys and this was exacerbated by cold ischemia. Preservation with lec-SOD did not prevent up-regulated ICAM-1 expression in kidneys exposed to the long cold ischemia. Each time point is representative of the mean ⫾ SD for 4 to 7 animals. Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. preservation for 1 hour with no SOD at the same time point; §P ⫽ 0.037 vs. preservation for 18 hours with lec-SOD at the same time point; ¶P ⬍ 0.01 vs. preservation for 1 hour with no SOD at day 1.
response generated as a result of oxygen free radical damage following ischemia/reperfusion [41–47]. Reactive oxygen species are generated by endothelial cells following hypoxia/reoxygenation [48, 49], resulting in increased expression of adhesion molecules facilitating binding and activation of neutrophils [37, 50–56]. Experimental models of ischemia and reperfusion have demonstrated that organ function was markedly improved and neutrophil infiltration significantly inhibited following treatment with exogenously administered SOD or transfection with human SOD cDNA [27–35, 57, 58]. We have previously demonstrated that lec-SOD binds with high
affinity to endothelial cells under cold hypoxic conditions and remains on the cell surface up to three days following reperfusion [37]. It is likely that direct antioxidant activity by bound lec-SOD at the endothelial surface of vessels in the kidneys, prevented damage caused by an alloindependent ischemia/reperfusion injury. At 24 weeks following transplantation, histological changes associated with the diagnosis of chronic renal allograft failure were evident in renal allografts that were exposed to long cold ischemia, but were not observed in those allografts with short cold ischemia times. The inflammatory response involving granulocytes and mac-
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Fig. 6. Preservation with lec-SOD inhibited the late development of apoptosis in renal allografts exposed to long periods of cold ischemia. Apoptosis of tubular cells was assessed following TUNEL staining using the Apoptosis Index scoring system. Prolonged cold ischemia did not increase the level of apoptosis observed at day 1 post-transplant. However, the number of apoptotic cells increased significantly at 3 days and 24 weeks post-transplantation in kidneys stored for 18-hours cold ischemia. Preservation of kidneys with lecSOD significantly inhibited the development of apoptosis at 3 days and 24 weeks in kidneys exposed to long cold ischemia (P ⬍ 0.01). Symbols are: ( ) no lec-SOD; (䊏) lec-SOD; *P ⬍ 0.01 vs. 18 hours preservation with lecSOD at the same time point.
rophages was absent in all kidneys at 24 weeks posttransplantation. However, an increase in CD3⫹ T lymphocytes was detected in kidneys with minimal ischemia presumably as a result of alloimmune responses, and this was significantly enhanced in kidneys that were exposed to 18 hours of cold ischemia. Similarly, ICAM-1 expression was elevated by alloimmune responses at 24 weeks and was enhanced by cold ischemic injury. The harmful effects of cold ischemia also resulted in significantly higher levels of apoptotic cell death that was inhibited by preservation with lec-SOD. Although preservation with lec-SOD for 18 hours significantly improved longterm allograft function and reduced apoptotic cell death, no reduction in cellular infiltration, adhesion molecules or MHC antigen expression were observed at 24 weeks compared with untreated renal allografts. The induction of apoptosis as a result of ischemia/ reperfusion injury has been previously reported in a number of experimental models [45, 59–62]. In clinical renal allografts, correlations have been observed between delayed graft function and apoptosis following reperfusion [63]. Analysis of post-reperfusion biopsies from cadaver and living-related donor kidneys demonstrated a high number of apoptotic cells predominantly in cadaver renal allografts with long cold ischemia times [20]. The reduction in apoptotic cells following preservation with lec-SOD may reflect the reduction in the availability of reactive oxygen species known to cause direct DNA damage and activation of harmful intracellular endonucleases [64, 65]. These results suggest that preservation with lec-SOD may limit the initial inflammatory response generated by ischemia/reperfusion injury, and in so doing, prevent the accumulation of harmful events that lead to long-term apoptotic damage of the kidney. In summary, the results from this study strongly suggest that prolonged cold ischemia exacerbates the effects of alloimmune responses in the development of chronic
allograft failure. Preservation with lec-SOD for 18 hours significantly inhibited the early inflammatory response but not long-term alloimmune responses or histological changes associated with chronic allograft failure. However, chronic renal allograft dysfunction was prevented in allografts preserved with lec-SOD and was associated with a reduction in the level of apoptosis detected at 24 weeks. Taken together, these results suggest that the inflammatory response generated by ischemia/reperfusion injury is mediated by reactive oxygen species. If the harmful effects of reactive oxygen species and reperfusion injury can be attenuated, then this may prevent the onset of apoptosis within the graft and thus preserve renal function. Lec-SOD may be a suitable agent for protection of renal allografts against initial ischemia/reperfusion injury with potentially beneficial effects on chronic renal allograft dysfunction. ACKNOWLEDGMENTS This work was supported by the National Kidney Research Fund, UK and the Seikagaku Corporation, Japan. The results from this study were accepted for oral presentation and presented at the 2000 XVIII International Congress of the Transplantation Society, Rome, Italy. A.J.M. was in receipt of a Hanson Trust Research Fellowship. Reprint requests to Dr. Susan V. Fuggle, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom. E-mail: [email protected]
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