The number of patients who are listed for renal transplantation has outgrown the number of donor organs, and this imbalance between supply and demand requires that available organs be used optimally, with minimization of immediate and delayed graft losses. Ischemia-reperfusion (I/R) injury leads to a delay of graft function, the incidence for which in cadaveric donor transplantation is approximately 25% (1,2). In addition to the impact on immediate graft function, prolonged cold ischemia contributes to late graft failure, probably as a result of postischemic fibrosis and arteriolosclerosis in the postischemic graft (3–5). In one large study of 27,096 kidney transplant recipients, delayed graft function without immunologic rejection was associated with a reduction of the graft half-life from 12.9 to 8 yr (6). Current immunosuppressive regimens can do very little to prevent such graft failure. However, if it were possible to protect the donor organ from acute I/R injury, then this could lead to reduced inflammatory injury and better early and late graft outcomes.
The pathogenesis of I/R injury is complex, and several mediators participate in the manifestations of injury. The complement system is a major contributory factor in several organ systems, including native and transplant kidneys (7–14). Complement-deficient and complement-depleted animals show a significant reduction of postischemic acute renal failure and late fibrosis (8,15–19). The complement cascade therefore makes a logical therapeutic target for the reduction of graft reperfusion damage.
A soluble form of complement receptor type 1 (CR1; CD35) and one of its derivatives (APT070) have shown promising effects as therapeutic complement inhibitors (16,20–22). CR1 is a natural membrane-bound complement regulator that consists of 30 short consensus-repeating subunits (SCR), with potent inhibitory activity of the classical and alternative pathways. It has specificity for C3b and C4b, with distinct sites for both proteins, a capacity for displacement of the catalytic subunits from the C3 or C5 convertases of both activating pathways, and co-factor function for the degradation of C3b and C4b by factor I (23,24). It has limited tissue distribution, being expressed primarily on erythrocytes and leukocytes (25). Recombinant soluble CR1 (250 kD) has been shown to be effective in allograft rejection (26).
On the basis of the findings that the first three SCR (SCR1–3) of CR1 at the N-terminal contain the relevant biologic activities of CR1 (24,27), a derivative known as APT070 has been generated. APT070 consists of three functional units: A membrane-associating peptide sequence, a membrane-inserting myristoyl group, and SCR1–3 from CR1 (28). The advantages of membrane-localizing CR1 over the whole soluble CR1 molecule include a much smaller size and membrane-binding properties that allow it to bind effectively to the cell membrane bilayer. These properties allow improved penetration into tissue, increased potency, and radically altered biodistribution in vivo.
Therapeutic studies using APT070 have proved effective in several experimental disorders that are associated with complement activation, such as vascular shock (29), rheumatoid arthritis (30), and acute myocardial infarction, as well as in renal transplantation (16). Previously, we developed a novel strategy to deliver APT070 into the donor kidney to inhibit complement activation at the injury site, without systemic complement depletion. Using a rat transplant I/R injury model, we have shown that treatment of donor kidneys with APT070 before transplantation reduced inflammatory injury and immunogenicity in donor kidneys, thereby improving graft performance in the short and long terms (16). However, acute renal failure in the previous model was induced by a short period of cold ischemia (30 min) and therefore was self-limiting, and it is unknown whether this treatment approach has the potential to prevent graft loss with more severe I/R damage. The use of “marginal” donor kidneys (e.g., grafts that have been exposed to extreme periods of cold ischemia before transplantation) could be one solution for the shortage of donor organs. If the donor organ could be protected from such tissue injury, then the number of kidneys that are suitable for transplantation could be increased.
In this study, we tested the hypothesis that APT070 can protect donor organs from initial injury caused by prolonged cold ischemia, thereby increasing the number of viable donor organs in a life-sustaining rat renal isograft model. In this model, there was no MHC mismatch between donor and recipient, which allowed us to study the graft outcome associated with cold ischemic time in the absence of an immune response. In addition, removal of both native kidneys at the time of transplantation enabled us to monitor the graft function immediately after transplantation. Preliminary experiments were performed to determine a cold ischemic time (CIT) that causes graft loss of approximately 50%. Using this predetermined CIT, we treated donor kidneys with APT070 or control agent before transplantation. After transplantation, we studied renal function, tissue injury, complement deposition, and graft survival.
Materials and Methods
Male DA rats (Harlan Olac, Bicester, UK) that weighed 215 to 230 g were used throughout the study period. Animals had free access to standard rat diet and water ad libitum. All experiments were carried out in accordance with the restrictions of a Home Office license for animal experimentation.
Construction of APT070
The plasmid pDB1081-1 was constructed to contain the gene coding for the N-terminal first three short consensus repeats of the long homologous repeat-A of human complement receptor type 1 (CR1), with an additional C-terminal cysteine codon (27). This protein sequence was transformed into expression host Escherichia coli BL21 (DE3). 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-2thiopyridyl)-Cys-carboxamide (APT542) was prepared by solid-phase synthesis and was termed myristoylated peptide (m-p). SCR1–3-cys then was coupled to the tag m-p and formed the membrane-localizing complement regulatory molecule m-pSCR1–3, also known as APT070, or Microcept (16,28). Analysis by SDS-PAGE showed the complement inhibitor had a molecular mass of 24 kD. The m-p moiety APT542 (lacking SCR1–3) was used as a control agent in this study.
Hemolytic Assay for APT070 Activity
The functional inhibition of complement activation by APT070 was assessed using a hemolytic assay as described previously (31). In brief, sensitized sheep red blood cells were resuspended in gelatin veronal–buffered saline at 109/ml. A total of 50 μl of sensitized red blood cells were incubated with a predetermined volume of rat serum, which caused maximal hemolysis and a titration of APT070 or control agent at 37°C in a 150-μl total volume. After 1 h, 100 μl of ice-cold gelatin veronal–buffered saline was added to each well, and the plate was centrifuged for 10 min. Supernatants were collected and transferred to a new 96-well plate. Absorbance was measured at 540 nm using a microplate reader. The percentage of lysis in the presence of APT070 was compared with that in the presence of control agent.
Rat Renal Transplantation
The technique for the life-sustaining renal transplant model in rat was adapted from a previously published method (32). The donor animal was anesthetized with gaseous enflurane and oxygen. A midline laparotomy was performed, and the left kidney and aorta were dissected. Ligatures were placed around the aorta cephalad and caudad to the renal artery. A Portex catheter (Smiths Medical, Kent, UK) was inserted into the aorta, and ligatures were secured. The kidney was perfused with 5 ml of ice-cold Soltran Kidney perfusion solution (Baxter, Berkshire, UK) with or without therapeutic agents for 5 min. The kidney then was excised and placed in ice-cold Soltran perfusion solution for the period of ischemia. In the recipient, microaneurysm clips (Codman, Berks, UK) were used to clamp the renal pedicle. The first native kidney was removed, and the prepared donor kidney was placed in the orthotopic position. End-to-end anastomosis of the renal artery, vein, and ureter was carried out using 10/0 nylon sutures (BEAR Medical Corporation, Chiba, Japan). A warm ischemia time of 60 min was used. At the end of the warm ischemic time, the microaneurysm clips were removed. The second recipient kidney then was removed. Surgical failure occurred in <10% of transplants performed.
The end point of graft survival was defined by death of the animal or serum creatinine >475 μmol/L, the point at which no animal survived in preliminary studies. To determine the effect of CIT on renal isograft survival, we varied the CIT (0.5 to 20 h) and identified the time associated with graft survival of 50% (which we called CIT∼50). The CIT∼50 was approximately 16 h in preliminary experiments. We used CIT of 16 h to compare the effect of APT070 and control substance to determine whether this would alter the number of surviving grafts.
Measurement of Serum Creatinine
Serum creatinine was measured by mass spectrometry and performed by hospital research services.
Assessment of Renal Tubular Damage
Formalin-fixed and paraffin-embedded sections (2 μm) were stained using the periodic acid-Schiff reaction. Sections then were viewed and analyzed in a blinded manner by two experienced investigators using LUCIA image analysis software (Jencons-PLS, Forest Row, UK). Two sections per animal were examined. The tubules with any of the following morphologic features were regarded as damaged tubules: Nonintact/denuded basement membrane, cytoplasmic degeneration/loss of polarity, nonuniform distribution of nuclei, individual epithelial necrosis, and narrowing of luminal space. The numbers of damaged tubules within 10 random fields in the corticomedullary junction were counted at a magnification of ×400 and expressed as a percentage of the total tubules per field.
Frozen sections (4 μm) were acetone fixed for 10 min. For C3 staining, goat anti-rat C3 (ICN Biomedicals, Hampshire, UK) was followed by FITC-conjugated donkey anti-goat Ig. For APT070 staining, mouse anti-human SCR1–3 (Inflazyme Pharmaceutical Ltd, Richmond, British Columbia, Canada) was followed by FITC-conjugated goat anti-mouse Ig. For C9 staining, rabbit anti-rat C9 (a gift from B.P. Morgan) was followed by FITC-conjugated donkey anti-rabbit Ig. The secondary antibodies were purchased from Jackson Laboratories (Bar Harbor, ME). Isotype-matched controls were also included. Sections were examined using LUCIA image analysis software.
APT070 Binding to Renal Tubular Epithelial Cells In Vitro
Binding of APT070 to rat renal tubular epithelial cells (NRK52E) was assessed by flow cytometric analysis. Briefly, cells were incubated with a titration of APT070 (0 to 100 μg/ml) or molar equivalent titrations of control agent at 4°C. After 1 h of incubation, cells were washed with PBS that contained 2% FCS. Washed cells then were stained with mouse anti-human SCR1–3, followed by FITC-conjugated goat anti-mouse Ig. The stained cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Oxford, UK).
All creatinine data were expressed as mean ± SEM. The means of two groups were compared by t test. Graft survival of the two treatment groups was compared by log-rank test. Differences were considered significant at P < 0.05.
CIT Influences Short-Term Graft Outcome
The design of our model differs in two important respects from previously published work with APT070. First, the CIT explored was much longer than in the previous study, with the intent of causing irreversible graft failure (in unprotected grafts) in a significant number of cases. Second, removal of both of the recipient native kidneys was performed simultaneously at the time of the transplant surgery so that measurement of graft function could begin immediately. We therefore started by determining the effect of CIT on complement-associated graft injury in our model. Donor kidneys were perfused with ice-cold Soltran kidney solution and stored at 4°C for various times (0.5 to 20 h). The kidneys that underwent cold ischemic insult then were transplanted into syngeneic recipients.
In the first of these experiments, at 24 h after transplantation, histopathologic changes in the graft that was exposed to short (3 h) and long (16 h) CIT were examined. As shown in Figure 1, the extent of tissue damage was considerably increased in the grafts that had undergone long (16 h) CIT compared with grafts that had undergone short (3 h) CIT (Figure 1). Quantification of tubular damage also showed that the extent of damage was related to the CIT, with a significant increase in the percentage of damaged tubules in the grafts that were exposed to long (16 h) CIT than those that were exposed to short (3 h) CIT (88 ± 3.8 [n = 4] versus 39% ± 12.3 [n = 4]; P < 0.01). These findings indicate that prolonged cold storage time increases the extent of tubular damage in our model. In addition to renal tubular damage, complement deposition was more apparent in the grafts that were exposed to long (16 h) CIT. Although weak positive staining for both C3 and C9 was observed in normal kidneys, staining was more intense in the grafts that were exposed to long (16 h) CIT and to a lesser extent in the grafts that were exposed to short (3 h) CIT compared with normal kidneys (Figure 2).
In a second experiment, renal function for animals that had received grafts that were exposed to 0.5, 3, 6, 9, 16, and 20 h of CIT and for control animals was monitored from 0 to 3 d posttransplantation. As shown in Figure 3A, the increase in serum creatinine paralleled the rise in CIT. Sham and unilateral nephrectomy control groups had almost normal creatinine values (<30 μmol/L). At the end point of this set of experiments (at 3 d posttransplantation), the surviving animals were killed and the extent of tubule damage in the grafts was assessed. As shown in Figure 3B, tubule damage correlated well with renal dysfunction. Together, these data demonstrate that the CIT is a determinant of the extent of renal tubule damage, complement deposition within the graft, and loss of graft function.
Verification of Intrarenal Delivery of APT070
Before performing in vivo experiments, we confirmed the functional activity of APT070 and their binding property in vitro using a hemolytic assay and renal tubular epithelial cell–binding assay. Incubation of red cells with APT070 effectively inhibited complement-mediated cell lysis in a dose-dependent manner. At the concentration of 1 μg/ml (38 nmol/L), APT070 inhibited 50% of cell lysis under the hemolytic conditions used (data not shown). In addition, binding studies showed that APT070 bound to tubular epithelial cells (NRK52E) in a dose-dependent manner (0 to 100 μg/ml; data not shown). Because a prolonged CIT was used in our model, we next determined whether APT070 could distribute and bind to donor kidneys efficiently under such severe conditions. As shown in Figure 4, at the end of the incubation of donor kidney with APT070, the binding of APT070 on renal tissue was detected clearly in donor kidneys that were exposed to 0.5 or 16 h of CIT using immunochemical staining (Figure 4, A through D). The staining was negative on the kidneys that were perfused with m-p control agent (Figure 4, E and F). In addition to the heavy staining observed in the glomeruli, positive staining was more specifically located at the basolateral surfaces of tubular epithelium and also observed at some peritubular endothelium. The pattern and the intensity of staining were comparable in donor kidneys that were exposed to 0.5 or 16 h of CIT. Therefore, our results indicate that, first, APT070 is able access the tubulointerstitial space and bind to cell membrane bilayer; second, the delivery and the binding of APT070 to donor kidneys are rapid and efficient; and, finally, the prolonged cold preservation period did not cause internalization of the complement inhibitor, at least at the end of the incubation period.
Pretreatment of Donor Kidney with APT070 Increases the Number of Usable Donor Organs
We next studied whether pretreatment of donor kidney with APT070 could increase the number of donor organs that remained viable after transplantation. Donor kidneys were perfused with either APT070 (200 μg) or m-p moiety control agent (16.7 μg, the equivalent molar concentration to APT070) and stored at 4°C for 16 h, as determined by preliminary experiments (see Materials and Methods). Pretreated kidneys then were transplanted into syngeneic recipients. Both native kidneys were removed at time of transplantation. Graft survival was monitored for 28 d. Graft loss was defined as death of the animal or serum creatinine >475 μmol/L. As shown in Figure 5, APT070-pretreated donor kidneys had a significantly increased graft survival rate compared with control-treated kidneys (63.6 [14 of 22] versus 26.3% [five of 19]; P = 0.03).
In the same group of recipients, the renal function of the graft was monitored daily until day 7 after transplantation and then weekly until day 28. The creatinine levels for the surviving animals (APT070 n = 14; control agent n = 5) are shown in Figure 6. Pretreatment of donor kidney with APT070 significantly improved renal function at 24 to 72 h posttransplantation compared with control treatment. These data together demonstrate that pretreatment of donor kidney with APT070 can protect the graft from the injury that is caused by prolonged cold ischemia, thereby improving the immediate posttransplantation survival rate.
Pretreatment of Donor Kidney with APT070 Attenuated Renal Tubular Damage and Complement Deposition
To determine whether pretreatment of donor kidney with APT070 could attenuate renal tubular damage and complement deposition within the grafts, we performed an additional set of kidney transplants. As in the above experiment, APT070 or control agent–pretreated kidneys (for 16 h of CIT) were transplanted into syngeneic recipients, but, in this experiment, the animals were killed after 10 or 24 h after transplantation. The histologic changes (10-h samples) and complement deposition (24-h samples) in the grafts were examined.
As shown in Figure 7, APT070 treatment resulted in less tubular damage than control treatment. Quantification of tubular damage showed that APT070 reduced the percentage of damage tubules by almost 35% compared with control treatment (57 ± 4.6 [APT070 n = 4] versus 91% ± 3.9 [control n = 4]; P < 0.01). In addition to the attenuated tubular damage, APT070 treatment reduced complement deposition in the grafts, with minimal C3 and C9 deposits on the basolateral surface of the tubules. In contrast, control agent–treated grafts demonstrated intense staining for both C3 and C9 (Figure 8). Together, these results suggest that pretreatment of donor organs with APT070 can inhibit complement activation at the injury site and prevent subsequent tubular damage.
Previous research in rodent models has identified complement as an important mediator of renal I/R damage. Such studies have shown that warm ischemia ranging from 25 to 58 min induces reversible acute tubular necrosis in native mouse kidney (8,18,33–35). Studies in rat kidney transplant models have indicated that a similar pattern of self-limiting acute renal failure follows exposure to cold ischemia of approximately 30 min (16). In both cases, the extent of postischemic acute renal failure was reduced substantially by preventing complement activation within the kidney. In clinical practice, however, renal transplantation often necessitates more prolonged periods of cold ischemia, with cold storage times as long as 24 to 30 h and a mean CIT of approximately 18 h (36). This work examines a more stringent, clinically relevant model of rodent ischemia to determine whether complement-based therapy can rescue grafts that otherwise would fail as a result of severe postischemic damage. We show, first, that the extent of complement activation and associated damage within the graft are proportional to the degree of cold ischemia (CIT), thereby providing a rational basis for complement inhibitory therapy. Second, our results establish that intervention to reduce intragraft complement activation can increase the number of viable transplants that remain functional after transplantation.
Membrane-localizing complement regulator APT070 was shown to be effective in preventing I/R injury in a proof-of-concept study that was characterized by mild renal failure with spontaneous recovery of function in untreated grafts (16). Our study applied a life-sustaining model with an extended CIT that was designed to simulate the conditions that might be represented in clinical practice. Our data showed that the treatment of donor kidney with APT070 increased the number of functional transplant organs by more than two-fold after initial injury caused by prolonged CIT (16 h). This therefore extends earlier studies in rodents that used a short ischemic time (30 min), suggesting that membrane-localizing complement regulator not only is capable of inhibiting complement activation within donor grafts under mildly ischemic conditions but also has a striking effect on complement inhibition in the extremes of reperfusion damage.
Using a mouse renal isograft model, we recently showed a close relationship between intrarenal C3 gene expression and the length of CIT. In addition, tissue damage and acute renal failure were less severe in C3-deficient grafts than in C3-sufficient grafts, regardless of whether the recipients were C3 positive or C3 negative. This suggests that local synthesis of C3, primarily identified in the tubular epithelium, was an essential determinant of complement-mediated reperfusion damage, whereas circulating C3 had a negligible effect (37). Our data showing increasing complement deposition as a result of prolonged ischemia is consistent with these findings that indicated that the extent of cold storage–induced damage is a function of complement deposition in the graft. Moreover, because local intragraft production of C3 plays a key role in acute complement-mediated reperfusion damage, this strengthens the rationale for targeting complement inhibitors to the graft. The complement inhibitory domains of CR1 (in APT070) contain C3b-binding sites that lead to dissociation and inactivation of C3b from the classical and alternative pathway convertases. Our strategy of “planting” APT070 at graft epithelium was effective in preventing local complement activation and widespread tubular damage at the extreme of reperfusion damage.
Most strategies to prevent complement-mediated I/R damage are based on systemic complement inhibition (15,19,38). Our strategy was to treat donor organ with membrane-localizing complement regulator by intragraft delivery. The strength of our strategy relied on locally targeting complement components at the area where complement is produced, is activated and deposited, and causes most damage, avoiding the need for systemic complement depletion.
Because the protein sequence of SCR1–3 that was used for constructing APT070 was derived from human CR1 gene coding sequence, there is the possibility that rats into which APT070-treated kidneys are transplanted could generate anti-human antibodies, potentially leading to inflammation within the kidney. Although our data do not exclude such a possibility, several factors suggest that the likelihood of autologous antibody-inducing injury is relatively small in our model. First, APT070 is a small molecule compared with CR1, thereby minimizing the potential for it to be immunogenic. Second, the intragraft delivery protocol used here restricted the amount of APT070 that entered the circulation. In support, we have observed that systemic complement activity in rats into which APT070-treated kidneys were transplanted is comparable to normal rats, suggesting that negligible amounts of APT070 were present in the recipient circulation (data not shown). Third, the majority of the membrane-localizing complement regulator that is bound to kidney tissue was internalized by 40 h after intragraft delivery of APT070 (data not shown). Therefore, the lack of surface-bound APT070 molecules on the tubular epithelium and negligible amounts of APT070 that are present in the circulation may elicit little or no immune response and may not serve as a target for circulating antibody. In addition, our data show that treatment had a net beneficial effect on donor kidney inflammation. It therefore is unlikely that rat anti-APT070 antibodies, if present, caused significant injury. Finally, Phase I clinical studies have shown that APT070 is well tolerated and does not generate an antibody response (39). Therefore, it is unlikely that antibody generation is relevant in a clinical context.
Our study shows that complement activation is an important mechanism contributing to postoperative graft loss as a consequence of prolonged cold storage damage. It also establishes the effectiveness of intragraft delivery of membrane-localizing complement regulator in a stringent model that is more close to a common situation in clinical transplantation, suggesting that this strategy could increase significantly the number of donor organs that remain functioning after transplantation. Therefore, the results that were generated from this study not only enhance our understanding of the mechanism by which prolonged cold ischemia reduces immediate graft survival but also provide essential information about the effectiveness of targeted complement inhibitors that could lead to more effective strategies for improving the use of donor organs.
This work was supported by the Wellcome Trust and the Medical Research Council of the UK.
Published online ahead of print. Publication date available at www.jasn.org.
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