NDT Advance Access originally published online on August 25, 2006
Nephrology Dialysis Transplantation 2006 21(11):3082-3086; doi:10.1093/ndt/gfl451
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The Fischer–Lewis model of chronic allograft rejection—a summary
Renal Transplant Unit, Glasgow Western Infirmary, Glasgow, Scotland, UK
Correspondence and offprint requests to: Maria L. Marco, Renal Transplant Unit, Glasgow Western Infirmary, Dumbarton Road, Glasgow G11 GNT, Scotland, UK. Email: mlmarco2003{at}yahoo.co.uk
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Abstract |
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Abstract
Description of the Fischer-Lewis...Although the mechanisms involved in human allograft rejection differ in degree from those in animal allograft rejection, animal models are invaluable for the investigation of their cause and effect. A significant advantage of the animal model is that lesions, comparable with those seen in human allograft rejection, can be generated over a much shorter time scale. Chronic rejection in animal models seems to be driven principally by immune mechanisms, whereas a myriad of non-immune mechanisms are operating in human allograft rejection. Despite these limitations, well-established animal models allow investigators to manipulate allograft rejection and thereby make contributions to the understanding of its pathogenesis.
In addition, animal models provide the opportunity of identifying mechanisms whereby the rejection process might be arrested or reversed. Furthermore, such models have the potential of investigating the induction of tolerance by means that may be applicable to the human situation.
Chronic rejection has been studied using well-established and reproducible kidney allograft models in rats. In such models the histopathological changes observed in the rejecting renal allograft are similar to those occurring during chronic rejection in humans, and they provide a means of studying the underlying mechanisms involved in its pathogenesis.
Keywords: animal models of rejection; chronic allograft nephropathy; Fischer–Lewis model
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Description of the Fischer–Lewis model |
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In 1969, White et al. [1] described a reproducible animal model of chronic renal allograft rejection. Using two inbred strains of rat, differing at minor histocompatibility loci, Fischer kidneys were grafted orthotopically to Lewis recipients. Chronic rejection was characterized by a slow deterioration of renal function, progressive destruction of renal parenchyma and persistent alloantibody production until final rejection after 48 weeks. This model was initially designed to study the progress of chronic rejection and the associated pathological lesions. Having established the normal pattern of chronic renal allograft rejection, this model was subsequently used in experiments designed to sustain renal function and arrest the process of rejection with the use of immunosuppression or immunomodulation.
Four weeks post transplantation, biopsies showed mild focal mononuclear cell infiltration, occasional thickening of the glomerular basement membrane and minor proliferation of mesangial cells. At 8–12 weeks there was increased mononuclear cell infiltration of vessel walls with glomerular changes ranging from mild basement membrane thickening to significant glomerular damage, associated with proteinuria. In addition, there was also thickening of the tubular basement membrane and focal tubular necrosis. At 28 weeks, renal damage was more extensive with interstitial fibrosis, vascular intimal thickening and disruption of the internal elastic lamina accompanied by mononuclear cell infiltration. Despite these pathological changes, some grafts sustained adequate renal function at this stage while those with poor renal function showed more severe glomerular damage, fibrosis and scarring of the interstitium, tubular atrophy and moderate thickening of vessel walls.
White et al. [1] compared these pathological and functional observations with those obtained with Lewis isografts and with Lewis renal allografts in Fischer recipients. In addition Lewis recipients, previously sensitized by Fischer skin grafts, subsequently demonstrated acute accelerated rejection of both skin and renal allografts from Fischer donors. The Fischer–Lewis model has become the most intensively studied and widely accepted animal model of chronic renal allograft rejection because of its similarity to the progress of events following human renal transplantation.
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Introduction of ciclosporin in the Fischer–Lewis model |
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Diamond et al. [2], using the Fischer–Lewis model, gave a short course of ciclosporin (5 mg/kg intramuscular (IM) for 10 days) to prevent or reverse acute rejection episodes. Suppressing acute rejection allowed an analysis of the progression of the chronic rejection while the animals survived, despite declining renal function, for 12 months. In this experiment, chronic rejection changes, such as transplant glomerulopathy, were established 24 weeks post transplant. Diamond et al. [2] associated the progressive albuminuria and other functional abnormalities with the development of significant glomerulosclerosis and infiltration of macrophages and CD4+ lymphocytes in the tubular interstitium. This latter finding is similar to that which occurs in rejecting human renal allografts. Although this infiltration was not initially observed in isograft controls, subsequent immunohistochemical analysis revealed minimal macrophage and T-cell infiltration in the control animals.
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Cytokines and adhesion molecules in the Fischer–Lewis model |
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In a later study, using the same animal model, Hancock et al. [3] performed a more detailed analysis of this cellular infiltration in an attempt to identify the immunologically activated cells producing the cytokines responsible for mesangial proliferation, increased matrix production and fibrosis.
In this study, allografts showed an initial peak of IgM and IgG at 2–4 weeks post transplant, which decreased thereafter. Immunohistology showed deposition of IgM, IgG, C3 and fibrin in vessel walls and glomeruli. At 12 weeks, there was an interleukin-2 (IL-2)-activated macrophage infiltrate and CD4+ T cells were discovered in glomeruli and blood vessels.
There was also staining for cytokines tumour necrosis factor-
(TNF-), IL-1 and IL-6. In vitro binding studies showed increased binding of naïve host lymphocytes to allograft vs isografts, correlating with up-regulation of intercellular adhesion molecule-1 expression by graft endothelium. The authors suggest a 3-phase process in the development of chronic rejection: an antibody-mediated phase, a cellular response responsible for the release of cytokines and an up-regulation of adhesion molecules which induces the scarring-fibrosis process that leads to graft loss.
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Reversibility of chronic rejection in the Fischer–Lewis model |
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In 1994, Tullius et al. [4] used the same animal model to study the reversibility of chronic allograft rejection. Allografts, retransplanted to the recipient strain within 12 weeks of grafting, showed a reversal of the histological and immunological changes, whereas the changes associated with the later stages of chronic rejection, particularly fibrosis, were irreversible.
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Manipulation of the cellular immune response in the Fischer–Lewis model |
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Since the histological and immunological events associated with allograft rejection are alloantigen dependent, various attempts have been made to delay or prevent alloantigen recognition. Two essential steps are required for host T cells to recognize alloantigens: the T cell receptor must bind to the antigen presented by the antigen-presenting cell and the CD28 molecule of the T-cell must interact with the B7-1 (CD80) and B7-2 (CD86) molecules on the antigen-presenting cells. Agents that block the CD28-B7 co-stimulation pathway induce a state of antigen-specific unresponsiveness.
Azuma et al. [5] prevented the development of chronic rejection by blockade of the T-cell co-stimulatory pathway with a single dose of CTLA4Ig, a fusion protein that binds to B7 molecules on the surface of the antigen-presenting cells. Given on the second day after grafting, it prevented the development of proteinuria and the morphological evidence of transplant arteriosclerosis and focal and segmental glomerulosclerosis. Furthermore, administration of a single dose of CTLA4Ig 8 weeks after grafting significantly reduced the development of progressive proteinuria and attenuated mononuclear cell infiltration, glomerulosclerosis and intimal hyperplasia [5].
The CD28-B7 T-cell co-stimulatory pathway has also been blocked using a monoclonal antibody [6]. A single dose of a signalling anti-CD28 antibody at transplantation prolongs graft survival and preserves kidney morphology in the Fischer–Lewis model. Animals receiving only an initial short course of ciclosporin developed chronic rejection while those given anti-CD28 and ciclosporin had well-preserved graft morphology and increased recipient survival.
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Effect of different immunosuppressive drugs in the Fischer–Lewis model |
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Although immunosuppressive drugs are effective in treating acute rejection episodes in human recipients of renal transplants, none of the current agents are ideal in allowing morbidity-free graft survival and indeed, the nephrotoxic effect of calcineurin inhibitors may contribute to the development of chronic allograft nephropathy (CAN).
Chronic rejection in the Fischer–Lewis model is suppressed by mycophenolate mofetil (MMF) given either immediately or 8 weeks after grafting [7].
MMF acts by inhibiting de novo DNA synthesis, cell surface molecule expression and antibody production. Similarly, chronic rejection in this model is suppressed by daily administration of Gamma lactone, a specific macrophage inhibitor. After 32 weeks, macrophage infiltration of the graft was reduced, most of the glomeruli and arteries were histologically normal and there was no proteinuria. Withdrawal of the drug after 8 weeks was followed by progressive proteinuria and the appearance of histological changes characteristic of chronic rejection [8].
The macrolide antibiotic, rapamycin, reduces DNA synthesis and halts mitotic activity in vascular smooth muscle cells, thereby inhibiting their proliferation and migration. SDZ-RAD, a derivative of rapamycin with a more favourable pharmacokinetic profile, possesses potent immunosuppressive properties. Viklicky et al. [9] administered this drug to Lewis recipients of Fischer renal allografts and observed diminished macrophage and lymphocyte infiltrations and a significant reduction in the expression of TGF-ß and cell adhesion molecules such as ICAM-1 and VCAM-1 within the allograft.
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Effect of hypertension and anti-hypertensive treatment on structure and function in CAN |
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Schindler et al. [10] studied the effect of hypertension on Fischer renal allografts and on the expression of growth factors contributing to tissue damage. In hypertensive allograft recipients, major histocompatibility (MHC) expression was increased compared with that in normotensive recipients indicating that hypertension renders the allograft more immunogenic. Hypertensive allograft recipients also showed increased expression of such growth factors as platelet derived growth factor (PDGF), transforming growth factor (TGF)-ß, vascular endothelium growth factor (VEGF) and collagen IV that was not observed in hypertensive isograft recipients. Since hypertension in animal recipients of renal allografts increases the expression of MHC and various growth factors this may explain the acute rejection episodes experienced by hypertensive renal allograft patients.
The histopathological evolution of chronic allograft rejection, the effectiveness of ciclosporin, and the effects of anti-hypertensive drugs on graft structure and function were studied using the Fischer–Lewis model [11]. The five groups of recipients were either left untreated, received an 8-week course of ciclosporin, triple anti-hypertensive therapy, monotherapy with an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin blocker. The animals in the four treatment groups demonstrated adequate renal function and decreased proteinuria while the allografts in the untreated animals progressed to chronic rejection. Interestingly, ciclosporin prevented chronic rejection and did not induce hypertension. The anti-hypertensive drugs had a modulatory effect on graft glomerulosclerosis.
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Angiotensin antagonists in the Fischer–Lewis model |
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Angiotensin II increases glomerular capillary pressure, stimulates the synthesis of extracellular matrix and contributes to the overexpression of cytokines and cell adhesion molecules. Pharmacological blockade of angiotensin II in Lewis recipients of Fischer renal allografts reduces morphological damage to the kidney, diminishes the expression of cytokines and growth factors and ameliorates the progression of proteinuria [12,13].
Noris et al. [12] investigating the synergistic effect of MMF and Losartan in the Fischer–Lewis model, observed that administration of either agent alone provided only partial protection of renal function, whereas together the prevention of chronic injury and progressive dysfunction was much greater.
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Growth factors in the Fischer–Lewis model |
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TGF-ß1 mRNA transcript levels rise in acute and chronic renal transplant rejection, and immunohistochemical studies have located the TGF-ß1 protein predominantly within the glomeruli [14]. A further detailed study of TGF-ß1 expression after renal allografting confirmed a 2-fold increase in TGF-ß1 mRNA at 4 weeks, and a 5-fold increase at 52 weeks.
Similar increases in mRNA are reported for PAI-1, biglycan, decorin and type I collagen genes, which TGF-ß1 is known to promote in fibrotic models. Immunostaining for proteins induced by TGF-ß1 demonstrated the deposition of tenascin, extra domian A (EDA)-fibronectin and decorin, in vascular, interstitial and glomerular tissues of allografts from 4–8 weeks post transplantation which was not observed in isografts [15]. Currently, this model has not been used to demonstrate the effect of inhibiting or neutralizing TGF-ß1 expression.
Endothelin-A (ET-A), produced by endothelial cells, is a potent vasoconstrictor and induces muscle proliferation. The effect of endothelin on tissue cells is regulated by A and B receptors. Blockade of the ET-A receptors has a beneficial effect on the renal allograft with treated animals having improved graft survival and function accompanied by a significant reduction in the histopathological changes and the intensity of cellular infiltration compared with controls [16].
Another study by the same investigators demonstrated that blockade of both endothelin receptors had no effect on graft survival or function [17].
Hepatocyte growth factor (HGF) has been shown to protect against acute ischaemic injury and have a role in renal regeneration [18].
Allografts in the HGF-treated group of animals retained normal renal architecture in the absence of fibrosis while those in the control group developed CAN.
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Other drugs effective in preventing chronic rejection in the Fischer–Lewis model |
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Heparin and its derivatives have been shown to inhibit smooth muscle cell proliferation in vitro and in vivo, independent of their anti-coagulant properties [19]. The proposed mechanism of action is blockade of the G1-phase of the cell cycle, thus preventing smooth muscle proliferation and migration into the intima [20]. Furthermore, heparin and its derivatives affect matrix composition by increasing heparan sulphate and chondroitin sulphate, as well as decreasing elastin and collagen [20], which may in turn reduce the formation of mitogenic cytokines and growth factors [PDGF, fibroblast growth factor (FGF), epidermal growth factor (EGF)] associated with increased smooth muscle cell proliferation.
Low-molecular-weight heparin has been shown to prolong the survival of cardiac allografts in rats, and its prolonged administration reduces the development of the associated chronic vascular disease [21]. Using the Fischer–Lewis renal allograft model, Braun et al. [21] have also shown that both early and late administration of low-molecular-weight heparin significantly improved renal allograft survival.
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Lipid-lowering agents |
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Hyperlipidemia is very common in patients requiring renal transplants. ‘Statins’ are effective in lowering lipid levels and their use after kidney transplantation affords some protection against cardiovascular disease. Apart from their lipid-lowering effect, statins have additional properties beneficial to allograft recipients as shown by Ji et al. [22] using pravastatin after cardiac, liver and kidney allografts in rats. In the Fisher–Lewis renal allograft model, pravastatin reduced macrophage and T-cell graft infiltration, decreased intragraft expression of TGF-ß, caused an attenuation of the recipients anti-donor IgG alloantibodies and an up-regulation of the anti-apoptotic gene Bag-1 which has been implicated in T-cell activation-induced apoptosis [22].
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Infection effect in the Fischer–Lewis model |
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Infectioning accelerates allograft rejection. Lipopolysaccharide (LPS), a potent stimulator of macrophage activity, given as a single dose to Lewis recipients of Fischer renal allografts accelerated and intensified the histological changes associated with chronic rejection [23]. Macrophage infiltration of glomeruli was increased, proteinuria was progressive and at 16 weeks, the number of sclerosed glomeruli was double than that seen in controls. In addition, there was increased expression of cytokines, adhesion molecules and extracellular matrix proteins.
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Effect of nephron mass in the Fischer–Lewis model |
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In Lewis recipients of Fischer renal allografts, reduction of nephron mass by
50% induces glomerular hypertension and hyperfiltration [24]. |
Ischaemia-reperfusion and donor age contribution to CAN in the Fischer–Lewis model |
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Clinical experience with renal transplantation has shown that donor age and preservation injury are detrimental for long-term allograft survival [25]. In addition, prolonged ischaemia renders organs more immunogenic and increases the incidence of acute rejection episodes [26]. Tullius et al. [27] studied the relative contributions of donor age and ischaemia-reperfusion injury on renal allograft survival in the Fischer–Lewis model by using donors of different ages and varying the time period between harvesting and transplantation [27]. Age-related changes were monitored in control animals of similar donor age. The progression of CAN was accelerated in older kidneys and in kidneys exposed to longer periods of ischaemia. In control animals, renal morphology began to deteriorate after 18 months of age. The morphology of allografts from young donors after prolonged ischaemia was comparable with that of allografts from older donors exposed to moderate periods of ischaemia. Kidneys from donors <1 year old tolerated prolonged ischaemia well, whereas those from older donors were particularly sensitive to any period of ischaemia.
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Tolerance studies in the Fischer–Lewis model |
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Since previous sensitization usually accelerated rejection of subsequent allografts from the same donor strain this experimental device has been applied to the Fischer–Lewis animal model [28–30]. In one study [30], the first renal allograft was removed and replaced by a second renal allograft at 2, 4, 8 and 12 weeks after initial grafting. To determine the effect of immunosuppression on second renal allograft recipients, a control group was denied immunosuppression. Instead of the anticipated early rejection of these second renal allografts, donor-specific tolerance was induced, which was independent of both immunosuppression and the time interval between grafts. Fischer heart allografts in these second renal allograft recipients functioned indefinitely while third-party heart allografts were rejected in 2 weeks. The transfer of splenocytes from recipients of second renal allografts into native Lewis rats induced the indefinite function of subsequent donor-specific heart allografts. The findings of this tolerance study in the Fischer–Lewis model highlight the paramount importance of specific alloantigen-related effects for the initiation of chronic graft acceptance.
Conflict of interest statement. None declared.
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References |
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Accepted in revised form: 30. 6.06
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