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Kidney International, Vol. 61 (2002), pp. 1160–1169

Lecithinized superoxide dismutase reduces coldischemia-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 ischemia- tion is a major cause of late graft failure [1–3]. Chronicinduced chronic allograft dysfunction. allograft failure has been defined as the progressive dete-

Background. Chronic renal allograft failure (CAF) is influ- rioration in graft function occurring at least 90 days afterenced by both allo-dependent and independent factors and istransplantation, and is associated with the histologicala major cause of graft loss in clinical renal transplantation. Wefeatures of interstitial fibrosis, arterial intimal thickening,evaluated a novel membrane-bound free radical scavenger,

lecithinized superoxide dismutase (lec-SOD), to determine its vascular lesions and tubular atrophy [4–7]. A numberpotential in limiting the harmful effects of ischemia/reperfusion of alloantigen-dependent and independent factors haveinjury on CAF.

been proposed as contributing to chronic allograft fail-Methods. Fisher rat kidneys were stored for either 1 hourure. Acute rejection episodes resulting from alloimmuneor 18 hours in cold Marshall’s preservation solution either with

or without lec-SOD and transplanted into Lewis recipients. responses are a major risk factor for chronic allograftResults. Within 3 days of transplantation, an early inflam- failure, but damage to the organ as a result of allo-

matory response involving granulocytes and macrophages was independent events also plays a key role in aggravatingdetected in renal allografts exposed to 18 hours cold ischemiachronic renal allograft dysfunction and rejection [7–9].that was significantly reduced by preservation with lec-SOD.

In renal transplantation, prolonged cold ischemic stor-By 24 weeks post-transplantation, elevated proteinuria anddetection of apoptotic cells was observed in kidneys exposed age of kidneys has been shown to be significantly associ-to 18 hours of cold ischemia, that was attenuated by preserva- ated with a higher incidence of delayed graft functiontion with lec-SOD (P � 0.05). However, up-regulated expres- (DGF) after transplantation [10–18]. DGF has beension of intracellular adhesion molecule-1 (ICAM-1) and major

shown to have a significant impact on poor long-termhistocompatibility complex (MHC) Class II together with a Tlymphocyte infiltration were observed at 24 weeks that was graft survival, especially in association with acute rejec-not prevented by preservation with lec-SOD. tion [17, 18]. We have previously demonstrated that in

Conclusions. These results demonstrate that ischemia/reper- cadaver renal allografts with long cold ischemia times, afusion injury, apoptotic cell death and allo-immune responses

neutrophil and platelet-mediated inflammatory responsemay be exacerbated by cold ischemia and accelerate the devel-and apoptotic endothelial and proximal tubular epithe-opment of CAF. Preservation with lec-SOD may protect

against the early damage induced by cold ischemia and reperfu- lial cells were observed after reperfusion [19, 20]. Theresion injury. is evidence to suggest that the initial inflammatory events

arising from ischemia/reperfusion injury may up-regulatealloantigens and thus increase graft immunogenicity and

Despite progressive improvements in the short-term exacerbate an alloimmune response [21–23]. In renalresults of first cadaver renal allografts, the problems of models of in situ cold ischemia and reperfusion, up-long-term graft loss remain unresolved. It is becoming regulated expression of MHC antigens, costimulatoryincreasingly evident that chronic renal allograft dysfunc- molecules, cytokines, chemokines, adhesion molecules

and leukocyte infiltration have been detected in associa-tion with progressive long-term deterioration in renalKey words: organ preservation, chronic rejection, superoxide dismu-

tase, transplantation, kidney, apoptosis, antioxidant. function [24, 25].The mechanisms involved in the generation of an in-Received for publication July 20, 2001

flammatory response following ischemia/reperfusion areand in revised form October 19, 2001Accepted for publication October 22, 2001 complex, but production of reactive oxygen species is

thought to be a critical event [26]. Many studies have 2002 by the International Society of Nephrology

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Nakagawa et al: Cold ischemia and allograft dysfunction 1161

shown that ischemia/reperfusion injury is abolished by the aorta and vena cava dissected and clamped. In thefirst instance only the left native kidney was removedadministration of an oxygen free radical scavenger, su-

peroxide dismutase (SOD) [27–35]. Indeed, Land and and the donor kidney was transplanted by an end-to-side anastomosis to the recipient aorta with a runningcolleagues demonstrated that administration of SOD at

reperfusion significantly reduced the incidence of first 10-0 nylon suture. After release of the clamps an end-to-end ureteric anastomosis was performed by an inter-acute rejection episodes and improved long-term graft

survival in cadaver renal allografts [32]. These findings rupted 10-0 suture. The remaining right native kidneywas removed 10 days post-transplantation after provid-suggest that the production of reactive oxygen species

during the initial period of transplantation may acceler- ing initial support against the effects of delayed functionof the allograft. Low-dose cyclosporine A (5 mg/kg/day)ate subsequent immune mediated events and affect long-

term graft survival. was administered by gavage during the initial 10-daypost-operative period to prevent early rejection. In recip-To determine whether it was possible to limit the

harmful effects of ischemia/reperfusion injury on chronic ient animals whose renal allografts were harvested ateither day 1 or day 3 post-transplantation, both nativerenal allograft failure, we have investigated the therapeu-

tic potential of a novel form of lecithinized SOD (lec- kidneys were removed at the time of transplantation sothat the early effects of ischemia/reperfusion injury onSOD). Lec-SOD was produced by the covalent linkage of

recombinant human SOD with lecithin, enabling greater histological changes could be determined. Between 4and 7 animals were transplanted and harvested at eachstability and high affinity binding to cell membranes [36].

We have previously demonstrated in vitro that in con- time point.trast to unmodified rhSOD, lec-SOD binds to microvas-

Measurement of renal allograft dysfunctioncular and large vessel endothelial cells under cold hyp-oxic conditions in Marshall’s solution, thereby inhibiting Renal allograft dysfunction was determined by mea-

surement of proteinuria in six animals in each of thehypoxia-induced cell death, endothelial adhesion mole-cule expression and neutrophil adhesion [37]. The aims four treatment groups. Urine samples were collected at

four-week intervals from rats placed in metabolic cagesof the current study were (1) to examine the effectsof prolonged cold ischemia and reperfusion injury on for 24 hours, up to and including 24 weeks post-trans-

plant. For this experiment there were six animals in eachchronic allograft failure in a rat renal allograft model,and (2) to determine whether preservation of kidneys treatment group. Protein excretion was determined by

measuring precipitation after interaction with 3% sulfo-with lec-SOD before transplantation exerted a beneficialeffect on inflammatory and histological changes and on salicylic acid (Sigma, Poole, Dorset, UK). Turbidity was

assessed by absorbance measurements at a wavelengthgraft function.of 595 nm.

METHODS Histopathology and ImmunohistologyExperimental model Upon harvesting, kidneys were dissected longitudi-

nally and half of the kidney fixed in formalin for paraffinAllogeneic renal transplants were performed fromfasted, inbred male (250 to 300 g) donor Fisher (F344, embedding and the other half snap frozen and stored

at –80�C for immunohistochemistry. HistopathologicalRT1lv1) to recipient Lewis rats (LEW, RT1l; Harlan OlacLtd, Bicester, UK) to recreate the model of chronic renal analysis for morphological changes to glomeruli, tubules,

interstitium and vessels associated with chronic rejectionallograft failure described by Hancock and colleagues[38]. A midline abdominal incision was performed on was assessed by an experienced renal pathologist (DRD)

under blinded conditions. Renal sections were gradedanesthetized donor rats and the renal vessels and uretermobilized after ligation of the adrenal and gonadal veins. on a semi-quantitative basis. Glomerular proliferation

and sclerosis were scored as: Grade 0 � none; Grade 1 �The animals were heparinized and the main vesselsclamped before both kidneys were flushed through the occasional focal segmental proliferation and sclerosis;

Grade 2 � areas of glomerular segmental proliferationaorta with 5 mL of ice-cold Marshall’s hypertonic citratesolution (Baxter Healthcare Ltd, Berkshire, UK). Kid- and sclerosis. The degree of tubular atrophy was graded

as: Grade 0 � none; Grade 1 � occasional tubular atro-neys were removed and stored at 4�C. Four treatmentgroups were studied: (i) one hour cold ischemia in Mar- phy; Grade 2 � focal or multiple focal areas of atrophy.

Tubular cast formation was assessed as: Grade 0 � none;shall’s solution alone; (ii) one hour cold ischemia in Mar-shall’s solution � 50 �g/mL lec-SOD (Seikagaku Corpo- Grade 1 � occasional hyaline cast; Grade 2 � areas of

hyaline and/or granular casts. The level of interstitialration, Tokyo, Japan); (iii) 18 hours cold ischemia inMarshall’s solution alone; or (iv) 18 hours cold ischemia infiltration was graded as: Grade 0 � none; Grade 1 �

occasional focus; Grade 2 � multiple foci of infiltratingwith Marshall’s � 50 �g/mL lec-SOD.Recipient rats were fasted overnight, anesthetized and cells.

Nakagawa et al: Cold ischemia and allograft dysfunction1162

Frozen biopsy material was embedded in O.C.T. Statistical analyses(Miles Inc., Shawnee Mission, KS, USA), and 7 �m cryo- Statistical analyses were performed using the Studentsections were stained using an indirect immunoperoxi- t test to determine if there were significant differencesdase technique with the following monoclonal antibod- between treatment groups when P � 0.05.ies: IF4 (CD3 T lymphocytes), ED1 (macrophages),HIS48 (granulocytes), MRC OX1 (CD45 leukocyte com-

RESULTSmon antigen), F16-4.4 [major histocompatibility complexRenal allograft dysfunction(MHC) Class I] and TLD-4C9 [intracellular adhesion

Damage to renal allografts as determined by a rise inmolecule-1 (ICAM-1)] were all obtained from Serotecproteinuria level was detected in renal allografts exposed(Oxford, UK) and MRC OX3 (MHC Class II) [39]. Theto 18 hours cold ischemia (Fig. 1). In contrast, kidneyssignals for MHC Class I, ICAM-1, CD45 and ED1 werepreserved with lec-SOD for 18 hours remained relativelyamplified with a further incubation step for 30 minutesstable, with significantly lower proteinuria at 16, 20 andusing a peroxidase-conjugated swine anti-rabbit Ig (1:5024 weeks post-transplantation (P � 0.05). Furthermore,dilution; Serotec) pre-incubated with 20% rat serum.organ preservation for a minimal one-hour period inIgM isotype monoclonal antibodies against CD3 andthe presence or absence of lec-SOD had no detrimentalHIS48 were developed with a peroxidase-conjugatedeffects on graft function, with stable proteinuria levelsgoat anti-mouse IgM (1:50 dilution; Serotec) and en-over the 24-week period (Fig. 1).hanced with a rabbit anti-goat Ig (1:200 dilution; Sigma

Ltd.), both pre-incubated with 20% rat serum. HistopathologyAll sections were examined without knowledge of the

A histopathological analysis of rat renal allografts wastreatment groups. Leukocyte infiltration was quantitated performed on samples obtained at 24 weeks post-trans-by determining the percentage area occupied by a partic- plantation to determine the effects of long cold ischemiaular cell type using an established morphometric point on chronic allograft failure. Glomerular mesangial depo-counting technique [40]. Semiquantitative grading for sition was equally prominent in all allografts and therethe induced expression of ICAM-1, MHC Class I and was no evidence of interstitial fibrosis, glomerular base-MHC Class II antigens on proximal tubular epithelial ment membrane changes or arterial intimal proliferationcells were assessed by two independent observers. The and thickening. However, in renal allografts with 18semiquantitative grades for ICAM-1 and MHC Class I hours of cold ischemia, significantly higher mean gradeswere: Grade 1 � occasional weakly positive tubule; were observed for focal segmental glomerular sclerosis

and proliferation (0.75 � 0.62 vs. 0.25 � 0.45; P � 0.01),Grade 2 � isolated foci of positive tubules; Grade 3 �tubular atrophy (1.5 � 0.79 vs. 0.25 � 0.45; P � 0.001),multiple foci of more extensive positive tubular staining.tubular cast formation (1.58 � 0.67 vs. 0.33 � 0.65; P �MHC Class II expression was graded as: Grade 1 �0.001) and interstitial infiltration (2.0 � 0.73 vs. 1.4 �negative; Grade 2 � foci of weakly positive tubules.0.51; P � 0.05) compared with kidneys preserved forshorter periods of cold ischemia (Fig. 2). PreservationMeasurement of apoptosiswith lec-SOD had no effect on graft histology.Identification of apoptotic cells was performed using

the terminal deoxytransferase-mediated dUTP nick-end Leukocyte infiltrationlabeling (TUNEL) technique as previously described

Immunohistochemical analysis to detect infiltration by[20]. Briefly, sections were obtained from paraffin em-granulocytes was performed in the immediate post-trans-

bedded kidney specimens and deparaffinized, rehydra-plant period. Significantly higher levels of HIS48� gran-

ted and digested with 20�g/mL Proteinase K (Sigma Ltd) ulocytes were present at day 1 in kidneys exposed to theat 37�C for 60 minutes. Following proteinase digestion, 18-hour cold ischemia compared with renal allograftsthe specimens were stained with the Apoptag Plus perox- stored for only one hour (2.8 � 0.4% vs. 0.8 � 0.6%;idase kit (Oncor, Cambridge, UK) according to the man- P � 0.01), remaining at elevated levels three days afterufacturer’s protocol. transplantation (2.7 � 0.9%; Fig. 3). The increase in

TUNEL-positive apoptotic cells were identified as sin- granulocyte infiltration at day 1 in grafts exposed to 18gle positively stained cells, with associated chromatin hours of cold ischemia was significantly attenuated bycondensation and positive chromatin fragments. The preservation with lec-SOD (2.8 � 0.4% vs. 1.9 � 0.7%;Apoptosis Index was calculated from the mean number P � 0.017), but this protective effect was not evident atof positive cells per �250 field of view, following an day 3 (2.2 � 0.6% vs. 2.8 � 0.8%; P � 0.05).analysis of 20 fields of view per section as previously At all time points following transplantation, the level

of CD45� leukocyte infiltration was increased by expo-described [20].


Fig. 1. Preservation with lecithinized super-oxide dismutase (lec-SOD) inhibits develop-ment of chronic renal allograft dysfunctioninduced by cold ischemia. Kidneys from Fisherdonors were exposed to one hour or 18 hoursof cold ischemic preservation in the presenceor absence of lec-SOD. After transplantation,renal function was assessed by 24-hour pro-teinuria measurements taken at 4 weekly in-tervals up to 24 weeks post-transplantation.A significant increase in proteinuria was de-tected at 16, 20 and 24 weeks post-transplant inrenal allografts stored for 18 hours comparedwith kidneys that were stored for only onehour (P � 0.05). This deterioration in long-term renal allograft function was delayed bypreservation with lec-SOD (P � 0.05). Eachtime point represents the mean � SEM for 6animals. Symbols are: (�) 1 hour no lec-SOD;(�) 1 hour �lec-SOD; (�) 18 hours no lec-SOD; (�) 18 hours �lec-SOD; *P � 0.05, 18hours cold ischemia time (CIT) vs. 1 hour CIT;§P � 0.05, 18 hours CIT SOD vs. 18 hoursCIT � lec-SOD.

3 was similar to that detected for total CD45� leukocyteinfiltration, demonstrating that macrophages and granu-locytes (Fig. 3) are the major components of the infiltra-tion at these time points (Fig. 4). Twenty-four weeksafter transplantation, low levels of macrophages weredetected in all allografts. In complete contrast, low levelsof CD3� T lymphocytes were detected on days 1 and 3post-transplant, irrespective of the period of cold storageor preservation with lec-SOD. By 24 weeks post-trans-plant, increased levels of T lymphocytes were detected,an effect that was further exacerbated by 18 hours ofcold storage. Lec-SOD did not influence the magnitudeof this infiltration.

Antigen inductionFig. 2. Long cold ischemia induces histological changes associated withchronic renal allograft failure. Paraffin sections from renal allografts at In renal allografts stored for one hour, no increase in24 weeks post-transplantation were analyzed for morphological changes

MHC Class I expression was detected over the 24 weekcharacteristic of chronic rejection and assessed semiquantitatively todetermine the severity of injury. In kidneys exposed to 18 hours of cold period, irrespective of preservation with lec-SOD (Fig.ischemia (�), there were significantly higher levels of focal segmental 5). In contrast, kidneys stored for 18 hours had signifi-glomerular sclerosis and proliferation (FSGS), tubular atrophy, cast

cantly up-regulated MHC Class I expression by day 1formation and interstitial infiltration at 24 weeks post-transplant thanin kidneys stored for one hour ( ; P � 0.05). Preservation with lec- (Grade 2.0 � 0.6), remaining elevated at day 3 (GradeSOD did not prevent the development of these chronic histological 2.0 � 0.7) and declining by 24 weeks post-transplantchanges. *P � 0.01 vs. 1 hour CIT.

(Grade 1.7 � 0.8). Preservation with lec-SOD signifi-cantly attenuated induction of MHC Class I antigens atday 1 post-transplant (Grade 1.3 � 0.5; P � 0.037; Fig.

sing the kidney to 18 hours of cold ischemia. Preservation 5). MHC Class II antigens were not significantly up-of kidneys with lec-SOD significantly reduced the level regulated by 18 hours of cold ischemia, nevertheless max-of CD45� infiltration on day 1 (3.9 � 1.1% vs. 2.7 � imal expression was seen in all kidneys at 24 weeks,0.3%; P � 0.013), but not on day 3 or at 24 weeks irrespective of cold storage time and preservation withpost-transplantation (Fig. 4). To identify the leukocyte lec-SOD (Fig. 5).subpopulations that may be involved in contributing to Low levels of ICAM-1 expression were detected onthe response, staining for CD3� T lymphocytes and day 1 following transplantation. Significantly increasedED1� macrophages was performed. The pattern of levels of ICAM-1 were detected on day 3 in kidneys

stored for 18 hours compared with kidneys stored forED1� macrophage infiltration observed at day 1 and day


Fig. 3. Early infiltration of HIS48� granulo-cytes following cold ischemia and reperfusionis attenuated by preservation with lec-SOD.Granulocytes were assessed by morphometricpoint counting following indirect immunoper-oxidase staining of kidneys harvested one andthree days post-transplant with a granulocyte-specific monoclonal antibody, HIS48. A sig-nificant increase in granulocytes was detectedon day 1 in kidneys stored for 18 hours, re-maining at high levels on day 3 post-transplant(P � 0.01). Preservation with lec-SOD for 18hours significantly inhibited the granulocyteinfiltration 1 day after transplantation (P �0.017). Each time point is representative ofthe mean � SD for 4 to 7 animals. Symbolsare: ( ) no lec-SOD; (�) lec-SOD; *P � 0.01vs. preservation for one hour with no SOD atthe same time point; §P � 0.017 vs. preserva-tion for 18 hours with lec-SOD at the sametime point.

one hour (2.6 � 0.6 vs. 1.5 � 0.6; P � 0.01; Fig. 5). At plantation elicit a non-specific inflammatory response in24 weeks, elevated ICAM-1 expression was detected in the immediate post-transplant period, accelerating theall kidneys and this was significantly exacerbated by long development of renal allograft failure and enhancingcold ischemia. Preservation with lec-SOD did not influ- apoptotic cell death and alloimmune responses. Preser-ence expression of ICAM-1 at any time point (Fig. 5). vation with lec-SOD inhibited the development of post-

reperfusion inflammatory responses and delayed the on-Apoptosis set of chronic renal allograft failure induced by pro-

At day 1 post-transplantation, low levels of tubular longed cold ischemia, but had no apparent effect on latercell apoptosis were detected in kidneys stored for one alloimmune events. This study provides evidence to showhour and 18 hours (Fig. 6). However, by day 3 post- that production of reactive oxygen species at reperfusiontransplant, a significant increase in the level of tubular plays an important role in the pathogenesis of chroniccell apoptosis was observed in kidneys that were stored allograft failure and that preservation with lec-SOD mayfor 18 hours compared with kidneys stored for one hour be a useful therapeutic agent for protecting against the(1.61 � 0.39 vs. 0.44 � 0.08, respectively, P � 0.001). initial post-transplant damage.Preservation of kidneys with lec-SOD significantly re- In this rat model of chronic renal allograft failure, aduced the tubular apoptosis observed at day 3 (0.81 � progressive increase in proteinuria up to 24 weeks post-0.37 vs. 1.61 � 0.39; P � 0.0032) and at 24 weeks (0.67 � transplantation was detected in kidneys exposed to 180.41 vs. 1.21 � 0.57; P � 0.01) compared with allografts hours of cold ischemia compared with those preservedstored with no SOD for 18 hours (Fig. 6). for only one hour. The long-term damage to renal allo-

The results from this study suggest that following 18 grafts exposed to prolonged cold ischemia was preventedhours of cold ischemia, an initial inflammatory response by preservation of kidneys with lec-SOD. This progres-mediated by macrophages and granulocytes was evident sive elevation in proteinuria arising from cold ischemicwithin the first few days post-transplantation, but after 24 damage is similar to data reported from a rat kidneyweeks, T lymphocytes were the predominant leukocyte model of in situ cold ischemia, where proteinuria alsopopulation. Preservation with lec-SOD significantly in- developed 16 weeks after the initial ischemia/reperfusionhibited the initial inflammatory responses generated by injury [24, 25]. It may be surprising that deterioratingcold ischemia, but did not appear to be protective against renal function was not observed in the renal allograftssubsequent alloimmune responses that were exacerbated

with only one hour cold ischemia (subjected to weakby the initial ischemic injury. However, preservation with

alloimmune responses) as has been previously describedlec-SOD did appear to inhibit the induction of apoptotic[38], but this may be due to optimal preservation ofcell death by cold ischemia in the later post-transplantkidneys with Marshall’s solution in our study.period.

In this Fisher to Lewis rat renal allograft model, it waspossible to evaluate the effects of the weak alloimmune

DISCUSSION response in kidneys with a minimal one-hour ischemiatime and assess the additional effects of prolonged coldThe results in this current study show that the addi-

tional effects of cold ischemia on allogeneic renal trans- ischemia. In renal allografts with minimal ischemia time,


Fig. 4. Variations in leukocyte subpopula-tions in renal allografts exposed to long coldischemia during the early and late post-trans-plantation period. Total CD45� leukocytes,CD3� lymphocytes and ED1� macrophageswere assessed by morphometric point count-ing following indirect immunoperoxidase stain-ing of kidneys harvested 1 day, 3 days and 24weeks post-transplant. At day 1 and day 3post-transplantation, the increase in totalCD45� leukocyte infiltration correlated witha similar increase in macrophage and granulo-cytes induced by prolonged cold ischemia. By24 weeks post-transplantation, the number ofmacrophages had declined, whereas the levelof CD3� T lymphocyte infiltration had in-creased in renal allografts and further exacer-bated by 18 hours of cold ischemia. This wasnot 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; (�) lec-SOD; *P � 0.01 versuspreservation for 1 hour with no SOD at thesame time point; §P � 0.013 versus 18 hoursof preservation with lec-SOD at the same timepoint.

there was no evidence of an inflammatory response in these inflammatory events are likely to result from theeffects of cold ischemia and reperfusion injury. Indeed,the first three days following transplantation. In contrast,

in renal allografts exposed to 18 hours of cold ischemia, preservation with lec-SOD significantly inhibited the in-filtration of granulocytes, induction of MHC Class I anti-an inflammatory response was detected in the early

phase after transplantation (day 1 and day 3), mediated gens at day 1 post-transplantation and the developmentof apoptotic cell death in the early post-transplantationby granulocytes and macrophages, with up-regulated ex-

pression of MHC Class I antigens and ICAM-1. Further- period.The granulocyte infiltration detected at days 1 and 3more, in kidneys exposed to long cold ischemia, an in-

crease in apoptotic cell death was detected. Therefore, post-transplant is characteristic of an early inflammatory


Fig. 5. Variation in expression of major his-tocompatible complex (MHC) antigens andintercellular adhesion molecule-1 (ICAM-1)in renal allografts exposed to long cold isch-emia. MHC Class I, Class II and ICAM-1 ex-pression was assessed semiquantitatively fol-lowing indirect immunoperoxidase staining ofkidneys harvested 1 day, 3 days and 24 weekspost-transplant. A significant increase in ClassI expression was detected on day 1 in kidneysstored for 18 hours (P � 0.01) that was signifi-cantly attenuated by preservation with lec-SOD (P � 0.037). Expression of MHC ClassII antigens was not enhanced by cold ischemicinjury, but maximal levels were detected in allrenal allografts at 24 weeks post-transplant.ICAM-1 expression was minimal at day 1, butincreased by day 3 in renal allografts exposedto 18 hours of cold ischemia. At 24 weekspost-transplant, ICAM-1 levels were elevatedin all kidneys and this was exacerbated by coldischemia. Preservation with lec-SOD did notprevent up-regulated ICAM-1 expression inkidneys exposed to the long cold ischemia.Each time point is representative of themean � SD for 4 to 7 animals. Symbols are:( ) no lec-SOD; (�) lec-SOD; *P � 0.01 vs.preservation for 1 hour with no SOD at thesame time point; §P � 0.037 vs. preservationfor 18 hours with lec-SOD at the same timepoint; ¶P � 0.01 vs. preservation for 1 hourwith no SOD at day 1.

response generated as a result of oxygen free radical affinity to endothelial cells under cold hypoxic conditionsand remains on the cell surface up to three days followingdamage following ischemia/reperfusion [41–47]. Reac-

tive oxygen species are generated by endothelial cells reperfusion [37]. It is likely that direct antioxidant activ-ity by bound lec-SOD at the endothelial surface of ves-following hypoxia/reoxygenation [48, 49], resulting in in-

creased expression of adhesion molecules facilitating sels in the kidneys, prevented damage caused by an allo-independent ischemia/reperfusion injury.binding and activation of neutrophils [37, 50–56]. Experi-

mental models of ischemia and reperfusion have demon- At 24 weeks following transplantation, histologicalchanges associated with the diagnosis of chronic renalstrated that organ function was markedly improved and

neutrophil infiltration significantly inhibited following allograft failure were evident in renal allografts that wereexposed to long cold ischemia, but were not observedtreatment with exogenously administered SOD or trans-

fection with human SOD cDNA [27–35, 57, 58]. We have in those allografts with short cold ischemia times. Theinflammatory response involving granulocytes and mac-previously demonstrated that lec-SOD binds with high


Fig. 6. Preservation with lec-SOD inhibitedthe late development of apoptosis in renalallografts exposed to long periods of cold isch-emia. Apoptosis of tubular cells was assessedfollowing TUNEL staining using the Apopto-sis Index scoring system. Prolonged cold isch-emia did not increase the level of apoptosisobserved at day 1 post-transplant. However,the number of apoptotic cells increased sig-nificantly at 3 days and 24 weeks post-trans-plantation in kidneys stored for 18-hours coldischemia. Preservation of kidneys with lec-SOD significantly inhibited the developmentof apoptosis at 3 days and 24 weeks in kidneysexposed to long cold ischemia (P � 0.01).Symbols are: ( ) no lec-SOD; (�) lec-SOD;*P � 0.01 vs. 18 hours preservation with lec-SOD at the same time point.

rophages was absent in all kidneys at 24 weeks post- allograft failure. Preservation with lec-SOD for 18 hourssignificantly inhibited the early inflammatory responsetransplantation. However, an increase in CD3� T lym-

phocytes was detected in kidneys with minimal ischemia but not long-term alloimmune responses or histologicalchanges associated with chronic allograft failure. How-presumably as a result of alloimmune responses, and this

was significantly enhanced in kidneys that were exposed ever, chronic renal allograft dysfunction was preventedin allografts preserved with lec-SOD and was associatedto 18 hours of cold ischemia. Similarly, ICAM-1 expres-

sion was elevated by alloimmune responses at 24 weeks with a reduction in the level of apoptosis detected at 24weeks. Taken together, these results suggest that theand was enhanced by cold ischemic injury. The harmful

effects of cold ischemia also resulted in significantly inflammatory response generated by ischemia/reperfu-sion injury is mediated by reactive oxygen species. If thehigher levels of apoptotic cell death that was inhibited

by preservation with lec-SOD. Although preservation harmful effects of reactive oxygen species and reperfu-sion injury can be attenuated, then this may prevent thewith lec-SOD for 18 hours significantly improved long-

term allograft function and reduced apoptotic cell death, onset of apoptosis within the graft and thus preserverenal function. Lec-SOD may be a suitable agent for pro-no reduction in cellular infiltration, adhesion molecules

or MHC antigen expression were observed at 24 weeks tection of renal allografts against initial ischemia/reper-fusion injury with potentially beneficial effects on chroniccompared with untreated renal allografts.

The induction of apoptosis as a result of ischemia/ renal allograft dysfunction.reperfusion injury has been previously reported in anumber of experimental models [45, 59–62]. In clinical ACKNOWLEDGMENTSrenal allografts, correlations have been observed be- This work was supported by the National Kidney Research Fund,

UK and the Seikagaku Corporation, Japan. The results from this studytween delayed graft function and apoptosis followingwere accepted for oral presentation and presented at the 2000 XVIIIreperfusion [63]. Analysis of post-reperfusion biopsiesInternational Congress of the Transplantation Society, Rome, Italy.

from cadaver and living-related donor kidneys demon- A.J.M. was in receipt of a Hanson Trust Research Fellowship.strated a high number of apoptotic cells predominantly

Reprint requests to Dr. Susan V. Fuggle, Nuffield Department ofin cadaver renal allografts with long cold ischemia timesSurgery, University of Oxford, John Radcliffe Hospital, Headington,

[20]. The reduction in apoptotic cells following preserva- Oxford, OX3 9DU, United Kingdom.E-mail: [email protected] with lec-SOD may reflect the reduction in the avail-

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