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From the Department of Renal Medicine and Transplantation,* King’s College, University of London, London; and Adprotech Limted, Essex, United Kingdom
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Abstract |
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To address the difficulties of preventing I/R damage, we have treated donor organs with a novel, modified membrane-binding complement regulator. Our approach is designed to prevent complement activation specifically within a transplanted organ. The ligands for complement regulators are strictly the active fragments of complement components present only at the site of complement activation. It is now recognized that the kidney itself is a significant source of complement components.5 Recent experimental work has suggested that such local synthesis of complement has great influence on local tissue injury in the transplanted kidney6 and that local production in the kidney, for example of the pivotal component C3, which links the activation and terminal cascades of complement, is increased in response to I/R injury.7 In addition, ischemia/reperfusion injury and its contributory factors have a serious negative impact on acute rejection and the long-term results in clinical transplantation.8
We have modified a form of human complement receptor type 1 (CR1, CD35) as a means to reduce I/R injury in transplanted kidneys. Human CR1 is found on the surface of almost all peripheral blood cells, but has a limited distribution on other cell types. It is, however, a potent inhibitor of complement activation, having decay accelerating activity for the disassembly of the complexed C3 convertases of the classical and alternative activation pathways, and co-factor activity for Factor I, which inactivates C3b (Figure 1a)
.9
The active site in CR1 was initially reported to reside in the first four short consensus repeats (SCRs) in each long homologous repeat10
but is probably bounded by the first three domains—at least in long homologous repeat (LHR)-A.11
Each of the functions of CR1 have been identified in other complement control proteins, but the presence of multiple functions in CR1 makes it an ideal candidate for therapeutic tissue engineering.12
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.13,14
This was based on a linear array of membrane-binding units, each of a different specificity and assembled into a synthetic peptide termed MSWP114
based on the myristoyl electrostatic switch15
for membrane localization. The myristoylated peptide membrane-binding product of this process, termed m-pSCR1-3 (Figure 1b)
, is a soluble, extrinsically administered protein with membrane-binding properties capable of intrinsically regulating complement activation on cell surfaces and able to exert a local and persistent anti-inflammatory effect.16 Here we report the ability of the membrane-targeted complement regulator to bind to rat and human kidney, and to reduce antigen-independent and antigen-dependent injury of rat donor kidney in subsequent transplantation studies.
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Materials and Methods |
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The gene coding for the N-terminal SCR1-3 of LHR-A of CR1 with a codon for an additional C-terminal cysteine was constructed as described by Dodd and colleagues,17 and a plasmid, pDB1081–1 constructed as described in Mossakowska and colleagues11 for transformation into the expression host Escherichia coli BL21 (DE3). The protein product of the expression vector was purified and shown to inhibit antibody-sensitized red blood cell lysis by human serum, to inhibit C3a release, and to function as a co-factor for Factor I.17 The analysis of activity revealed that the 21-kd SCR1-3 fragment was active against both the classical and alternative pathways and appeared to be correctly folded.11,13,17
Membrane-Binding Reagents
The membrane-localizing thiol-reactive agent N-(myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-thiopyridyl)-Cys-carboxamide was prepared by solid-phase synthesis as described previously.11 The corresponding reduced (free cysteine) form lacking SCR1-3 and used as a control agent in this study was designated "m-p" (myristoylated peptide).
Construction of a Membrane-Binding Complement Regulatory Molecule, m-pSCR(1-3)
A plasmid encoding SCR1-3cys of CR1 was constructed and expressed in E. coli BL21 (DE3) as described by Mossakowska and Smith,12
purified by the method of Dodd and colleagues,17
coupled to the membrane-binding tag m-p, and isolated as described.13
The product had Mr 
24 kd by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a 50% inhibitory concentration in a classical pathway hemolytic assay of 0.15 nmol/L. using a commercially available antibody-sensitized sheep red blood cell preparation (Diamedix, Miami, FL) and a dilution of human serum of 1:400.11
Kidney Transplantation and Treatment with m-pSCR(1-3)
Animals were purchased from Harlan Olac (Bicester, UK) and maintained with standard rat chow and water ad libitum. All animal experiments were conducted within the restrictions of a Home Office License for animal experimentation. Anesthesia was performed with isoflurane administered through a vaporizer. The left donor kidney was dissected and the aorta ligated cephalad and caudad to the renal artery. A portex tube catheter (0.5-mm outside diameter) (Portex Ltd, Hythe, UK) was inserted between the ligatures and the kidney was perfused with 5 ml of Soltran Kidney Perfusion Solution (Baxter Health Care, UK) containing 200 µg of m-pSCR1-3 (40 µg/ml) throughout a period of 5 minutes. The kidney was then placed in ice-cold saline for 30 minutes. Orthotopic renal transplantation was performed using the method of Fabre and colleagues18 and microvascular clips releasing the blood flow were removed once 60 minutes of warm ischemia had elapsed. Syngeneic transplantation was performed using DA rats. The 7-day experiment used groups of six rats, and the 20-week experiment used groups of five and six control and treated rats, respectively. The allograft experiment with Fischer F344 (RT1lv1) rat donors and Lewis (RT1l) rat recipients used groups of six control and seven treated rats.
Analysis of Transplant Recipients
At regular intervals after transplantation blood samples were collected for analysis of blood urea nitrogen as previously described.19 Kidney transplants were harvested at various time intervals and portions of tissue were placed in 4% formal saline for standard histological analysis, or frozen in liquid nitrogen for subsequent immunohistochemical staining, using antibodies against C3d (DAKO, Glostrup, Denmark), C5b-9 (Quidel, CA), CD3 (Becton Dickinson, Abingdon, UK), and CD45 (Becton Dickinson, Abingdon, UK), and for morphometric image analysis as previously described.20
Analysis of m-pSCR(1-3) Binding to Rat Kidney Cells
Immortalized rat renal endothelial cells21 or primary cultured rat tubular epithelial cells7 were incubated with varying concentrations of m-pSCR1-3 in phosphate-buffered saline (PBS) for 40 minutes at 4°C. For immunocytochemistry cells were grown on glass coverslips and for flow cytometry cells were detached from plastic culture flasks and aliquots of 5 x 105 cells exposed to m-pSCR(1-3). For immunocytochemistry cells were washed twice in PBS and then fixed in cold acetone and stained using Cy3-labeled 3E10 [mouse anti-human SCR1-3 monoclonal antibody (mAb); provided by Adprotech Ltd.] for 30 minutes at 4°C, mounted in Fluoromount (Sigma), and examined using a fluorescence microscope.
For flow cytometry, cells were washed twice in PBS and then incubated with Cy3-labeled 3E10 for 30 minutes at 4°C, washed twice, and fixed in 1% paraformaldehyde in PBS. The samples were analyzed using a FACscan flow cytometer (Becton Dickinson).
Mixed Lymphocyte Reaction
Responder and stimulator lymphocytes were prepared as previously described.6 Cellular proliferation was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI). This is a colorimetric method in which methyl tetrazolium salt (MTS) is reduced into a soluble formazan product. The absorbance at 490 nm provides an index of the number of viable proliferating cells, which was determined at days 1, 3, 5, and 7 of the assay.
Perfusion of Human Tumor Nephrectomy Specimens
Human kidneys were obtained, with research ethics committee approval and patient consent, from patients undergoing nephrectomy for the removal of renal cell carcinoma in which there was a defined tumor mass recognized by preoperative imaging. Organs were placed on ice and perfused via the renal artery with 1 L of ice-cold Soltran solution with (n = 4) or without (n = 1) m-pSCR1-3 at 80 µg/ml. Tissue samples were taken at frequent time intervals (approximately every 2 hours) for 24 hours, and processed for histological analysis, for immunohistochemistry, and by enzyme-linked immunosorbent assay to determine the presence of m-pSCR(1-3).
Statistics
Student’s t-test or repeated measures analysis of variance was used where appropriate to determine significance between treatment groups; P < 0.05 was taken as significant.
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Results |
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We incubated cultured rat glomerular endothelial cells with m-pSCR1-3 and assessed binding by immunocytochemistry using a labeled mouse anti-human SCR1-3 mAb (3E10) (Figure 2, a and b)
. We found that m-pSCR(1-3), but not the untagged SCR1-3 bound in a dose-dependent manner (Figure 2, c and d)
. Binding of m-pSCR1-3 also occurred on cultured rat tubular epithelial cells (data not shown). In a hemolytic assay using antibody-sensitized sheep erythrocytes, the addition of the m-p membrane-binding tag to SCR1-3 produced a substantial (100-fold) increase in inhibitory activity compared to untagged SCR1-3 (unpublished data).13
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The routine flushing of donor kidneys with perfusion fluid before transplantation presents an ideal opportunity to introduce m-pSCR1-3 into donor organs. We used this approach in a rat model of syngeneic renal transplantation. We perfused the donor graft with ice-cold kidney perfusion solution containing either m-pSCR1-3 or a control substance, before exposing the donor tissue to 30 minutes of cold ischemia and 60 minutes of warm ischemia. This schedule was chosen because it produced histological and functional damage followed by partial recovery in untreated transplants.
The distribution of m-pSCR1-3 in the donor kidney is shown in Figure 3; a to c
. The pattern of staining was consistent with binding of m-pSCR1-3 to the glomerular capillary and peritubular capillary walls and the tubular epithelium at its basal surface. Histological analysis at 24 hours after transplantation showed reduced signs of acute tubular necrosis (tubular attenuation and cellular swelling, and associated neutrophil infiltration) in m-pSCR1-3-treated grafts (Figure 3, d and e)
. At 24 hours after transplantation tissue myeloperoxidase activity, taken here as a measure of neutrophil activity, was significantly reduced in m-pSCR1-3-treated grafts compared to control grafts (Figure 3f)
. Deposition of the complement split product C3d (not shown) and the membrane attack complex C5b-9 was absent or weak in m-pSCR1-3-treated grafts, whereas staining was more intense and widespread in control-treated grafts (Figure 4)
. These results indicate that m-pSCR1-3 treatment reduced the cleavage of C3 and the subsequent formation of C3d and C5b-9. The amount of tissue-bound m-pSCR1-3 detected by immunohistochemistry declined after 24 hours, so that on day 7 it was undetected in treated tissues.
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. Among control grafts, including those treated with the untagged complement regulator, SCR(1-3), there was no significant difference in function. Therefore both functional and structural improvement required the inhibitor to be bound to treated cell surfaces via the m-p moiety.
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We next investigated whether reducing the acute complement-mediated I/R injury could improve the long-term outcome of isografts. This is important because early graft injury may be a driver of late graft failure.22
In this experiment we followed m-pSCR1-3- or control-treated ischemic isografts for up to 20 weeks after transplantation. We found that m-pSCR1-3 treatment reduced the degree of functional (Figure 5b)
and histological graft injury throughout the period of study, such that by 20 weeks m-pSCR1-3-treated grafts showed only mild features of cellular swelling (Figure 5, c and e)
, whereas control grafts demonstrated widespread swelling and intimal proliferation of the arteriolar walls (Figure 5, d and f)
. Thus, transient initial treatment of the donor organ had a durable and beneficial effect.
m-pSCR(1-3) Treatment of Donor Kidney Reduces Allograft Rejection
To examine the effect of reduced I/R injury on the immunogenicity of the graft, we used the well-described Fischer F344 (RTlv1) to Lewis (RTll) renal allograft model.23
The donor and recipient differ by a single MHC class I allele, resulting in a mild and spontaneously resolving acute rejection episode that peaks between 10 and 14 days after transplantation. The protocol for inducing graft ischemia and treatment with complement inhibitor or control solution was the same as before. On day 14 after transplantation, histological damage (Figure 6
; a to d) and renal dysfunction (Figure 6e)
in allografts treated with m-pSCR1-3 were reduced. This protective effect of m-pSCR1-3 was evident throughout the period of acute rejection.
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In addition we examined the anti-donor T cell proliferative response of the recipient on day 14 after transplantation. The T-cell response in recipients of m-pSCR1-3-treated grafts was reduced in comparison with recipients of control grafts (Figure 6g)
, indicating that the treatment of donor organs with the tagged complement regulator led to modulation of recipient T-cell stimulation.
m-pSCR(1-3) Binds to ex Vivo Perfused Human Kidney
To assess the feasibility of treating human organs, we obtained human tumor nephrectomy specimens in which the whole kidney had been removed on a short length of renal artery. We then perfused the organ with a solution containing m-pSCR1-3 and examined the distribution of m-pSCR1-3 in the nontumor tissue. Immunohistochemical analysis showed that m-pSCR1-3 localized, as in the rat studies, to the glomerular and tubular structures of the kidney (Figure 7, a and b)
. The presence of m-pSCR1-3 in blocks of tissue was confirmed by enzyme-linked immunosorbent assay (data not shown). Although the level of inhibitor detected by enzyme-linked immunosorbent assay was variable, suggesting that perfusion or tissue binding was not equal throughout the kidney, the inhibitor was detected in all areas sampled.
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Discussion |
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I/R injury in clinical practice manifests most acutely as delayed graft function and may permanently decrease the function of transplanted organs. Increased morbidity and mortality associated with delayed graft function have been noted in clinical studies and represent a relationship between the condition of the engrafted tissue and the subsequent host response.25,26 In European studies delayed graft function affects as many as 20 to 30% of cadaveric renal transplants.27 Moreover, the increasing discrepancy between numbers of patients listed for transplantation and potential donors has necessitated the use of so-called suboptimal donors, ie, older than 50 years old and/or organs that have experienced more than 24 hours of cold ischemia. At present no specific agent is used to reduce the susceptibility of transplanted organs to I/R injury, the risks and implications of which increase significantly with suboptimal donor organs.27
Within minutes of I/R injury, activation and deposition of complement occurs, with subsequent up-regulation of cytokines and adhesion molecules28 that mediate the infiltration of leukocytes. Leukocyte accumulation and adhesion to microvascular endothelium in postischemic tissues is thought to be important through the release of toxic oxygen radicals and inflammatory cytokines.29 Postischemic complement activation is thought to cause direct endothelial and epithelial tissue damage30 and C5a-mediated neutrophil chemotaxis,31 which were reduced in the m-pSCR1-3-treated grafts in our study.
Soluble forms of naturally occurring complement regulators have been assessed in models of I/R injury.2-4 The soluble inhibitors currently available have been given systemically and have relatively short functional half-lives. This has necessitated the use of high doses that systemically abrogate the function of the complement system and that have the potential to lower host anti-microbial defenses. This is potentially undesirable for transplant recipients receiving other immunosuppressive drugs. Attempts have been made to prolong the half-life of such reagents, for example by linking complement regulators to albumin,32 or to sialyl Lewis-x moieties to target endothelial surfaces.33 The coupling of complement regulators to Ig domains has also been shown to prolong half-life,34 but not to protect the kidney from I/R injury.35 In our study, the untagged form of SCR(1-3) of CR1 similarly did not prevent I/R injury suggesting that the complement regulator must be bound to the target of complement activation at the site of tissue injury for it to be effective. Our therapeutic targeting strategy has overcome these difficulties.
We have addressed two issues in the targeting of therapeutic complement regulators. Firstly we have considered where in the complement cascade therapeutic intervention is effective. Secondly, we have determined how to target complement inhibition to those tissues that bear damage. By using CR1 activity as the basis for intervention, we focused our approach on the inactivation of C3b, because this is the pivotal bridging step between the activation and terminal pathways. Recent studies of I/R in C6-deficient mice have highlighted the importance of direct membrane damage caused by the membrane attack complex,36 while the upstream release of the fragments C3a and C5a, which have anaphylactic and chemotactic properties, contributes to local inflammation. The CR1-derived molecule used in our novel construct is a structurally minimal component, retaining the activities of CR1 needed for breakdown of complexed C3b within C3 convertases, and thus for its inhibitory effect on the generation of C3a, C5a, and membrane attack complex.11 The design of the membrane-binding tail exploits the membrane-inserting property of myristate, in combination with an electrostatic contribution to binding from a synthetic basic peptide sequence that interacts with negatively charged phospholipid headgroups, enabling binding to the cell surface. Combined with routine perfusion of the donor organ, this would permit organ-specific delivery and retention of m-pSCR(1-3). This two-site membrane interaction used in the targeting peptide exploits additive binding affinities of small molecules for spatially related components of cellular membranes14 and allows flexibility because the m-p tails can be modified to suit different applications. Site-directed regulators of complement activation have a wide variety of clinical applications beyond transplantation14,16 and this type of membrane localization strategy is also of wide applicability.
Our results show a significant antigen-independent component of injury late after transplantation that was preventable by modifying acute I/R injury. Additionally, there was an effect on the antigen-specific component of injury, suggested by the finding of reduced alloreactivity in the recipients of m-pSCR1-3-treated grafts. Thus the overall potential to improve graft function could reside in the benefit of reduced immunogenicity of the graft as well as reduced postischemic damage. A clinical corollary may be found in the results of live unrelated organ transplantation, in which ischemic times are minimized and consequently clinical outcomes are improved compared to cadaveric renal transplantation with equivalent degrees of MHC mismatch.37
Complement activation is possibly one of a number of antigen-independent mechanisms that enhance the recognition of foreign tissue by the recipient immune system. Current debate on the role of nonspecific injury in the stimulation of T-cell immunity would suggest that reduced inflammation could attenuate specific immunity.38 The results presented here support this concept, and suggest that intragraft inhibition of complement decreases the local inflammatory environment and reduces the formation of C3 fragments with immunoregulatory properties. Indeed, recent experiments using a mouse kidney allograft model found that donor organs with reduced capacity for the production of C3 elicit weaker alloimmunity,6 suggesting an important effect of local complement. Thus the transplanted kidney may be a particularly important target for therapeutic complement regulation.
To maximize the efficacy of therapeutic complement regulation, we introduced the complement regulator before complement activation, that is, before reperfusion of the transplant organ with recipient blood. By adding m-pSCR1-3 to the perfusion solution we were able to intervene in the pathogenesis of I/R injury. This method bypassed the inherent time delay for DNA transcription and protein translation required using gene transfection technologies to introduce site-specific protein expression. In organ transplantation this is particularly relevant because of the lack of notice clinicians receive before an organ transplant. The degree of protection we observed was achieved with a single application of a molecule that has been shown to be acceptable for use in humans.20
In conclusion, we have shown that it is possible to modify the biological properties of donor kidney by targeting a complement regulatory protein to potential sites of tissue injury, thereby reducing the late consequences of inflammatory and immunological injury. The approach described here in a rat model has the potential to be applied to clinical evaluation, possibly in conjunction with other emerging therapies for transplant I/R damage, such as superoxide dismutase mimetics39 and adhesion molecule blockade.40 Transient modification of the donor organ without the need for systemic complement inactivation might have a significant advantage for kidney transplant recipients.
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Acknowledgements |
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Footnotes |
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Supported by the Guy’s and St. Thomas’ Kidney Patients Association (to J. P.) and Adprotech Ltd.
J. R. P. and M. E. J. contributed equally to the study.
Accepted for publication June 25, 2003.
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compared with control-treated grafts perfused only with carrier solution 
(P = 0.03). MPO levels in SCR(1-3) 
- and m-p 
-treated grafts were not significantly different from controls. Sham-treated animals 
had a laparotomy 24 hours before MPO measurement (n = 4/group).
, SCR(1-3) lacking the m-p tail —
—
, or the m-p tag alone –
–
or in sham-operated rats undergoing nephrectomy without transplantation ...
.... Data are shown from day 3 because this was the point from which renal function was dependent on the transplanted kidney alone. Treatment with m-pSCR1-3 led to significantly improved renal function compared to any one of the control groups (P < 0.001; n = 6 animals/group). b: Blood urea nitrogen throughout 20 weeks after transplantation after perfusion of donor graft with perfusion solution alone -
