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Protective role of selectin ligand inhibition in a large animal model of kidney ischemia–reperfusion injury
http://www.kidney-international.org
original article
& 2006 International Society of Nephrology
Protective role of selectin ligand inhibition in a large animal model of kidney ischemia–reperfusion injury C Jayle1,2, S Milinkevitch1, F Favreau1, C Doucet1,2, JP Richer1,2, S Deretz2, G Mauco1, H Rabb3 and T Hauet1,2 1
INSERM E0324, Centre Hospitalier et Universitaire and Faculte de Medecine, Poitiers, France; 2INRA, Unite´ de Transplantation, Ge´ne´tique Expe´rimentale en Production Animale, Domaine du Magneraud, Surge`res, France and 3Division of Nephrology, John Hopkins University, Baltimore, Maryland, USA
Experiments in rodents have demonstrated an important role for selectins in kidney ischemia–reperfusion injury (IRI). However, the relevance of this in larger mammals, as well as the impact on long-term structure and function is unknown. We tested the hypothesis that small molecule selectin ligand inhibition attenuates IRI, cellular inflammation, and long-term effects on renal interstitial fibrosis. We used a porcine model of kidney IRI and used Texas Biotechnology Corporation (TBC)-1269, a selectin ligand inhibitor. Renal function, tissue inflammation, and tubulointerstitial fibrosis development were evaluated up to 16 weeks. Both warm and cold ischemia models were studied for relevance to native and transplant kidney injury. Pigs treated with TBC-1269 during 45 min of warm ischemia (WI) showed significantly increased glomerular filtration rate compared to control animals. In pigs with severe IRI (WI for 60 min), TBC-1269 treatment during IRI significantly increased renal recovery. Cellular inflammation was strongly reduced, particularly influx of CD4 cells. Quantitative measurement of fibrosis by picrosirius red staining showed strong reduction in TBC-1269-treated groups. TBC-1269 also reduced cold IRI, inflammation, and fibrosis in kidneys preserved for 24 h at 41C and autotransplanted. The selectin ligand inhibitor TBC-1269 provides a novel and effective approach to attenuate IRI in both warm and cold ischemia in large mammals, in both short and long terms. Selectin ligand inhibition is an attractive strategy for evaluation in human kidney IRI. Kidney International (2006) 69, 1749–1755. doi:10.1038/sj.ki.5000335; published online 19 April 2006 KEYWORDS: renal ischemia; reperfusion injury; selectin; T lymphocytes; fibrosis
Correspondence: T Hauet, Laboratoire de Biochimie et Toxicologie, Centre Hospitalier et Universitaire, Hopital Jean Bernard, La Miletrie, 86000 Poitiers, France. E-mail: [email protected] Received 19 August 2005; revised 15 October 2005; accepted 18 November 2005; published online 19 April 2006 Kidney International (2006) 69, 1749–1755
Ischemic acute renal failure remains a major clinical problem with high morbidity and mortality in native kidneys, as well as with worse short- and long-term allograft function in allografts. A number of studies have examined the role of inflammatory cells in the pathogenesis of post-ischemic injury (reviewed by Friedewald and Rabb1). Leukocyte rolling on the endothelium, which precedes firm adhesion via CD11/ CD18 and intercellular adhesion molecule (ICAM-1), is mediated by a class of adhesion molecules known as selectins, which consist of three different transmembrane receptors, E-, P-, and L-selectin.2 E- and L-selectin are involved at the stage of leukocytes rolling and tethering to the vascular endothelium. L-selectin is constitutively expressed on leukocytes, whereas E-selectin is expressed on only activated endothelium. P-selectin is expressed on platelets and activated endothelial cells, and appears to have some inducers different from those of E-selectin.3 Interstitial inflammation is associated with the development of tubulointerstitial fibrosis, characterized by excess accumulation of extracellular matrix in the renal interstitium.4 Renal fibrosis is generally regarded as the dark side of tissue repair mechanisms and the ultimate injury that leads to chronic nephropathy. Selectin blockade has been demonstrated to attenuate ischemia-reperfusion injury (IRI) in different models.5–8 However, blockade of one selectin can lead to compensatory inflammation conducted by the other selectins. A potent selectin antagonist, biosmanose Texas Biotechnology Corporation (TBC)-1269(1,6-Bis(3-(3-carboxymethylphenyl)-4(2-a-D-mannnopyranosylloxy)-phenyl)hexane) is a small molecule antagonist of E-, P-, and L-selectin binding to their ligand, sialyl Lewis X. The structure of TBC-1269 reveals two mannose and two carboxylic acid groups in a configuration to mimic the dimeric configuration of sialyl-Lewis (Figure 1). TBC-1269 was demonstrated to mitigate post-ischemic renal damage in a rodent model during the first 72 h after renal ischemia.9 A major concern in designing clinical trials is whether results in rodent models are translatable to man.10 We therefore conducted these studies in large mammals in an attempt to better set the stage for human studies. In addition, there is increasing concern that ischemic acute renal failure leads to long-term fibrotic changes.11,12 Furthermore, 1749
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C Jayle et al.: Protective effect of biosmanose in renal ischemia
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The outcomes after warm ischemia and reperfusion differed markedly between experimental groups. Two animals from group warm ischemia (WI) 60 died on post-operative days 9 and 10. Survival was 100% in control group (Cont), uninephrectomized group (Unif), WI 45, WI TBC 45, and WI TBC 60. Renal functional data are shown in Figure 2a and b. During the first week, Ccr was reduced in all experimental groups (WI 45 to WI 60) when compared to control and sham-operated groups (Figure 2a). However, Ccr was significantly increased in WI TBC 45 when compared to WI 45, between weeks 2 and 16, and in WI TBC 60, when compared to WI 60 particularly after day 7. In group WI 45, Ccr paralleled Cont and sham-operated groups but remained at lower level from Cont. Ccr remained at 60% of Cont and Unif value in group WI 60. TBC-1269 also provided effective kidney protection after 24 h cold preservation (Figure 2b). However, no change was noted when TBC-1269 was only added to the preservation solution without administering at reperfusion. Two animals were excluded in CI24 group because of renal artery thrombosis. In the warm ischemia model, proteinuria was detected and peaked at day 3 and decreased thereafter to day 7. Progressive proteinuria developed again after approximately 7–8 weeks (Figure 3a). A significant difference was detected between WI 45 and WI 60 groups, and TBC-1269-treated groups, particularly after 7–8 weeks. In the cold ischemia model, proteinuria was more pronounced during the first week when compared to the warm ischemia model (Figure 3b). TBC1269 also reduced proteinuria in cold-stored and autotransplanted groups. TBC-1269 when only added in the preservation solution had no influence in the outcome of proteinuria.
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RESULTS Effects of ischemic conditions and TBC-1269 on long-term renal function and survival
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Figure 1 | Structural diagram of TBC-1669.
findings in warm kidney IRI simulating native kidney injury may not be transferable to management of transplant patients who have a component of cold kidney IRI. The present study was designed to test the hypothesis that TBC-1269 reduced post-ischemic kidney damage in pig models of kidney IRI. The novel findings of this study include the following (1) TBC-1269 reduced kidney IRI in warm or cold ischemia in pigs; (2) the protective effect was associated with reduced CD4 and macrophage infiltration; and (3) long-term fibrosis was reduced by TBC-1269 treatment.
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Figure 2 | Influence of different ischemia models and addition of TBC-1269 on the creatine clearance (1a and b). Renal function was measured in the different experimental groups after different warm ischemia and compared to control group and uninephrectomized group. The values reported are the mean7s.d. *Po0.05, **Po0.05 WI 45 vs WI TB C45, WI 60 vs WI TBC 60.
TBC-1269 studied in animals submitted to the same Cont group conditions did not induce any change in the renal functions when compared to Cont group (Figure 3a and b). Effect of ischemic conditions and TBC-1269 on tissue injury of reperfused kidneys
Histological analysis of biopsy samples from warm-ischemia kidneys revealed differences in the tissue injury after 40 min of reperfusion and of the day-7 post-reperfusion. Kidneys from WI 45 and WI 60 had increased damage when compared to WI TBC 45 and particularly WI TBC 60 (Table 1). The degree of initial acute proximal tubule cell injury appears closely related to the duration of warm ischemia, particularly after 60 min of WI. In the cold preservation and transplantation model, TBC-1269 was also effective in reducing renal tubular injury when compared to controls. TBC-1269 added in the preservation solution alone without reperfusion was not protective when compared to TBC CI 24 group that received the agent during preservation and reperfusion. Effect of ischemic conditions and TBC-1269 on mononuclear cell infiltration in pig kidneys
Consistent with our previous studies in the pig model, the major inflammatory cell population detected by immunohistochemical long term studies after ischemia was CD4 þ T Kidney International (2006) 69, 1749–1755
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C Jayle et al.: Protective effect of biosmanose in renal ischemia
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Figure 3 | Influence of different ischemia models and administration of TBC-1269 on the urine protein excretion (2a and c). Renal function was measured in the different experimental groups after different warm ischemia time or 24-h preservation compared to TBC-1269-treated groups. The values reported are the mean7s.d. **Po0.01 and ***Po0.001 CI 24 vs TBC CI 24 and TBC CI 24A.
Table 1 | Degree of proximal tubule cell lesions and mitochondria damage after different ischemia conditions Warm ischemia Ischemic conditions Injury type time reperfusion WI 45 WI TBC 45 WI 60
WI TBC 60
Brush border integrity 30–45 min Day 7
3.570.2 3.470.2 2.670.21 3.770.2 3.670.2 3.070.11
Cell detachment 30–45 min Day 7
2.670.2 2.570.3 3.470.2 2.770,2* 1.570.2 1.470.2 2.7770.2 2.270.2*
Ischemic conditions Injury type time reperfusion Brush border integrity 30–45 min Day 7 Cell detachment 30–45 min Day 7
3.170.2* 3.570.2*
Cold ischemia CI24
TBCCI24
TBCCI24A
2.570.2 3.670.2 3.570.2# 3.570.2 4.270.2 4.070.1# #
2.470.2 1.970.2 2.070.2 1.770.2 1.370.2 1.270.2#
The histological lesions from early (30–45 min) and day 7 after warm ischemia were evaluated and given as a semiquantitative score (Materials and Methods). Brush border integrity and tubular necrosis in light microscopy studies. Values are means7s.d. for all parameters analyzed. *Po0.05 WI 90 vs WI 60, 1Po0.05 WI 60 vs WI 30 and WI 45, #Po0.05 TBC CI 24 and TBC CI 24A vs CI 24.
Kidney International (2006) 69, 1749–1755
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Figure 4 | Influence of different ischemia models and addition of TBC-1269 on CD4-positive cell infiltration in kidney tissue (3a and c). The number of positively stained cells per surface area were counted on biopsy samples from experimental groups. The values reported are the mean7s.d. from 8 to 12 separate determinations. # Po0.05, ##Po0.05 WI45 vs WITBC45 and 0Po0.05, 000Po0.05 WI60 vs WITBC60, **Po0.01 and ***Po0.001 CI24 vs TBCCI24 and TBCCI24A.
lymphocytes. No infiltrating cells were detected in Cont and Unif (data not shown). In contrast, the number of CD4 þ increased between weeks 1 and 2 after reperfusion in WI 45 and particularly WI 60 (Figure 4a). After week 2, CD4 þ cells plateaued up to 16 weeks following reperfusion in groups WI 45 and WI 60. In WI 60 and WI 45 groups, monocyte/ macrophage increased between weeks 1 and 2 and slightly decreased between weeks 2 and 4, then plateaued between weeks 4 and 16 (Figure 4b). Monocyte/macrophage number was significantly increased in WI 60 group when compared to WI 45 group. In WI kidneys treated with TBC-1269, CD4positive cell number was significantly reduced. Monocyte/ macrophage number was also different in WI TBC 45 and WI TBC 60 when compared to WI 45 and WI 60, respectively (Figure 5a). TBC-1269 was also efficient in cold ischemia model in reducing inflammatory cells (Figure 5b). TBC-1269 reduced CD4-positive cells and macrophages during the first week, and this beneficial effect remained detectable between weeks 4 and 16. Effect of ischemic conditions and TBC-1269 on interstitial fibrosis and tubular atrophy
Evolution of interstitial fibrosis is presented in Figure 4. In our hands, fibrosis is correlated with WI, particularly after 60 min (Figure 6, lower panel, a–d). Percentage of interstitial fibrosis area is also associated with progression of renal dysfunction particularly in WI 60 group. Picrosirius score was significantly different at weeks 7–8 between WI 45 and 1751
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C Jayle et al.: Protective effect of biosmanose in renal ischemia
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Figure 5 | Influence of different ischemia models and administration of TBC-1269 on monocyte/macrophages within kidney tissue (4a and b). The number of positively stained cells per surface area was counted on biopsy samples from experimental groups. The values reported are the mean7s.d. from 8 to 12 separate determinations. #Po0.05, ##Po0.05 WI 45 vs WI TBC 45 and 0Po0.05, 000 Po0.05 WI 60 vs WI TBC 60, **Po0.01 and ***Po0.001 CI 24 vs TBC CI 24, and TBC CI 24A.
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Figure 7 | Influence of cold ischemia on the onset of tubulointerstitial fibrosis and tubular atrophy score: the percentage of kidney areas displaying interstitial fibrosis stained with Picrosirius was measured over several weeks following cold ischemia within kidneys. *Po0.05 and **Po0.01 CI 24 vs TBC CI 24 and TBC CI 24A.
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WI 60 groups. These changes are accompanied by atrophy of the surrounding tubules (Figure 6, lower panel, e–h, upper panel). This difference in fibrosis increased at weeks 10–12 and more significantly by week 16. TBC-treated groups showed a significantly reduced fibrosis intensity particularly after 60 min of WI between weeks 8 to 16 (WI 60 vs WI TBC 60; Figure 6, lower panel) and weeks 10–12 and W 16 (WI 45 vs WI TBC 45; Figure 6). No difference was found at week 8 in WI 45 and WI TBC 45. Tubular atrophy scores were increased in WI 45 and WI 60 groups after week 8 when compared to TBC-1269-treated groups (Figure 6). In the cold ischemia model, TBC also reduced fibrosis (Figure 7, upper panel, a–c, lower panel) and tubular atrophy development (Figure 7, upper panel, d–f, lower panel).
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DISCUSSION 3 2.5 2
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Figure 6 | Influence of warm ischemia on the onset of tubulointerstitial fibrosis and tubular atrophy score: the percentage of kidney areas displaying interstitial fibrosis stained with Picrosirius was measured over several weeks following warm ischemia in kidneys. #Po0.05, ##Po0.01 WI45 vs WITBC45 and 0 Po0.05, 000Po0.05 WI60 vs WITBC60, *Po0.05 WI45 vs WITBC45, and WI60 vs WITBC60. 1752
IRI is the primary cause of acute renal failure in native kidney and can cause delayed graft function in transplanted kidneys. Ischemic injury to the kidney is associated with high morbidity and mortality and is a non-immunologic factor involved in chronic allograft nephropathy. In addition, recent report has demonstrated that patients with poor levels of early graft function are at an increased risk of death.13 The lack of suitable organs for transplantation from traditional sources has led clinicians to seek alternatives. Although related and unrelated live donation is being promoted as one option for increasing the donor pool, it is unlikely that this will in itself be able to completely bridge the gap. Non-heart beating and extended criteria organs, both typified by increased injury, can provide an alternative supply of organs, which should substantially increase the donor pool.14 Kidney International (2006) 69, 1749–1755
C Jayle et al.: Protective effect of biosmanose in renal ischemia
Consequently, therapeutics directed to improving the ability of the kidney to tolerate ischemic injury would have important implications. We found that selectin ligand binding with TBC-1269 significantly reduced post-ischemic renal dysfunction in pigs with different warm ischemia times, as well as in a cold ischemia model. Importantly, these findings on warm ischemia in a large mammal model confirm and extend earlier studies on rodents. Furthermore, we have evaluated a cold ischemia model as well, and demonstrated that administration in the perfusate is not adequate but that post-reperfusion dosing is crucial. Proteinuria occurred in the WI 45 and WI 60 groups, as well as CI 24 groups, and correlated with histologic injury and particularly tubulointersitital fibrosis. Proteinuria became prominent and progressive after week 8 in warm ischemia groups when compared to control and Unif. Interestingly, the protective effect of TBC-1269 was also prominent, long-term after warm ischemia. In cold preserved groups, TBC-1269-treated groups had reduced proteinuria and reduced fibrosis and tubular atrophy. We examined mononuclear leukocytes as potential mediators of late changes after IRI, and examined the impact of the selectin ligand blockade on these. TBC-1269 reduced monocyte/macrophage infiltration in WI TBC 45 and WI TBC 60 groups after weeks 6 and 8, respectively. We also evaluated the effects of TBC-1269 on CD4 þ cells infiltration, which increased with WI time. TBC-1269 reduced CD4 þ cell infiltration in both the WI model and the cold ischemia autotransplanted model. We cannot exclude the possibility that reduced macrophages and CD4 þ cells were a result of decreased injury, rather than the cause of decreased injury. Recent studies have demonstrated that the in vivo effects of TBC-1269 may be mediated through E-selectin but do not appear to involve leukocyte rolling. TBC-1269 was shown to inhibit rolling of a murine cell line (32DC13) on murine Pselectin in vitro and has pronounced anti-inflammatory activity in mice when given before induction of thioglycollate-induced peritonitis.15 In contrast, in this previous study, TBC-1269 failed to influence established leukocyte rolling in vivo at doses that match or exceed those with pronounced, anti-inflammatory activity. The authors hypothesized that TBC-1269 limits inflammation by a mechanism other than direct competition with cell-bound selectin ligands. An other recent study has demonstrated that TBC-1269 downregulated the expression of macrophage inflammatory protein (MIP)1a, MIP-1b, MIP-2, and kupffer cells, as well as expression of Akt, mitogen-activated protein kinases (p44/42), and ribosomal S6 kinase.16 Consequently, this selectin inhibition modulated the expression of Akt, mitogen-activated protein kinases (p44/42), and alterations in cellular signaling and chemokines. These alterations in cellular signaling and chemokine expression represent potential mechanisms or pathways of inflammatory response in ischemia-reperfusion. Mitogen-activated protein kinases have been the focus of a number of studies, as these compounds are involved in a Kidney International (2006) 69, 1749–1755
original article
number of important inflammatory cell signaling mechanisms.17 Recent studies have further elucidated the role of mitogen-activated protein kinases in the inflammatory response, as a result of trauma and/or IRI. Akt pathway is also involved in IRI and functional recovery of damaged organs. It was recently shown that biosmanose was the most advanced synthetic pan-selectin antagonist and that TBC1269 competitively inhibited, via steric/allosteric mechanisms, the binding of selectin function blocking antibody and was shown to bind all the three selectins to varying degrees.18,19 Previous studies have demonstrated that renal, ischemic injury results in permanent damage to peritubular capillaries and influence long-term function.20,21 Tubulo-interstitial fibrosis was significant in both warm and cold ischemia groups, long term. Picrosirius red-positive staining has been used for nearly 30 years and the use of computer-assisted quantification of fibrosis in the Sirius red-stained renal allograft biopsies appears to be a surrogate marker to predict long-term allograft function.22 In our model, Picrosirius red staining was improved by the selectin ligand inhibition. These data sets the stage for a clinical trial applying selectin ligand blockade to improve short- and long-term outcomes of kidney IRI in both the native kidney and allograft. We have noted some difference between warm ischemia and cold preservation, particularly for proteinuria and fibrosis development. Inflammatory cell kinetics were also specific for cold and warm ischemia. A recent study demonstrated that WI triggers injury primarily to proximal tubular cells, whereas cold ischemia damages glomerular podocytes and peritubular endothelial cells in addition to proximal tubules.23 These data support proteinuria output in cold ischemia group. As previously reported, these results suggest that organ preservation and cold ischemia could have pathophysiology distinct from warm IRI, particularly after 60 min of WI.24 The fact that TBC-1269 does not seem to have any beneficial impact when added to the preservation solution could be linked to the change of the drug effect at 41C. In addition, future studies are required to examine the molecular mechanisms of how selectin ligand blockade modulates kidney outcomes after IRI. MATERIALS AND METHODS Surgical procedures and experimental groups Large white male pigs, 30–35 kg, 3–4 month old (INRA, Le Magneraud, Surge`res, France) were prepared as previously described.25 All animals were fed with a standard diet (Socie´te´ Arrive, Saint Fulgent, France) and had free access to tap water. Rapid preoperative tranquilization was achieved by the rapid and atraumatic nasal administration of 0.2 mg/kg of midazolam (Laboratoire Roche, Neuilly-sur-Seine, France). The animals were then anesthetized with halothane (Laboratoire Belamont, Paris, France) and 100% oxygen. The surgical procedures were performed under sterile conditions. A 20-gauge plastic catheter (Becton Dickinson Vascular Access Inc., Sandy, UT, USA) was inserted into an ear vein. Atropine sulfate 10 mg/kg was given intravenously to reduce pharyngeal and tracheal secretion, and to 1753
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prevent post-intubation bradycardia. A midline abdominal incision was made to expose both kidneys. Warm ischemia was induced in the pigs by clamping the left renal pedicle for 45 min (WI 45, n ¼ 10) and 60 min (WI 60, n ¼ 10) with a vascular clamp followed by a right nephrectomy. Two other experimental groups were studied: WI TBC 45 (n ¼ 10) and WI TBC 60 (n ¼ 10). In these two experimental groups, TBC-1269 was administered 10 min before ischemia, during warm ischemia, and 1 h after reperfusion. TBC was given at a concentration of 25 mg/kg based on a previous dosing study.26 In the cold ischemia model, the left kidney was removed and cold-flushed with University of Wisconsin solution and coldstored at 41C for 24 h. The kidneys were re-implanted and the contralateral kidney was removed. Groups of animals were divided as follows: CI TBC 24, n ¼ 10 (TBC-1269 was administered 10 min before cold-flush and 1 h after reperfusion), CI TBC 24A (CI TBC 24 with TBC-1269 added in UW solution before cold-flush but not reperfusion, n ¼ 10), and CI 24, n ¼ 10. These experimental groups were compared to uninephrectomy group without kidney ischemia (Unif; n ¼ 8) and control group (Cont; n ¼ 6, age-matched group). TBC-1269 was also studied in control group condition (Cont þ TBC, n ¼ 6). All experiments were carried out in accordance with the Guidelines of the French Agricultural Office and the legislation governing animal studies. Any animal that died during the 16-week follow-up period was autopsied. All animals that died from causes other than renal failure, or developed renal artery, vein, or ureter problems were excluded. Functional parameters Endogenous creatinine clearance (Ccr, expressed in ml/min) and urinary protein were measured as previously described.25 Endogenous creatinine clearance is suggested to be a reliable indicator for renal excretory function in newborn and growing piglets.27,28 Pigs were placed in a cage to allow specific 24 h urine collections and plasma samples collection. Plasma and urinary creatinine (Cr) was measured enzymatically using an automatic analyzer (Hitachi, Paris, France) and Ccr was calculated using the following formula: urinary flow rate urinary Cr/plasma Cr. Urinary proteins was determined using a photometry method (Laboratoire Biorea, Talant, France). Histopathological studies After warm ischemia and 40 min of reperfusion, at day 1, day 3, day 5, day 7, day 14 and 4–5, 10–12, and 16 weeks after surgery, biopsy tissue samples from the deep cortex–outer medulla region were performed. Samples were fixed with Dubosq-Brasil and 10% formalin in 0.01 mmol/l phosphate buffer (pH 7.42) and embedded in paraffin. Conventional stains were applied (hematoxylin and eosin, periodic acid-Schiff). Different histological parameters (brush border integrity and intratubular detachment) were studied. Histological lesions were expressed in percent of kidney samples using a previously described semiquantitative scale24,25: (0) no abnormality; (1) mild lesions affecting less than 10% of kidney samples; (2) lesions affecting 10–25% of kidney samples; (3) lesions affecting 25–50% of kidney samples; (4) lesions affecting 50–75%; (5) lesions affecting more than 75% of kidney samples. Tubulointerstitial injury was defined as inflammatory cell infiltrates and interstitial fibrosis. To estimate the level of tubulointerstitial fibrosis, tissue sections were also labeled with Picrosirius for collagen identification (collagen I and III) as previously described.29,30 The amount of interstitial fibrosis was determined in Picrosirius-stained sections by a semiquantitative imaging technique.31 The percentage of Picrosirius-stained surface was determined on 10 different tissue 1754
C Jayle et al.: Protective effect of biosmanose in renal ischemia
sections viewed at 100 magnifications in each experimental condition and expressed as percent of the total surface area examined. Tubular atrophy was semiquantitatively scored on a scale of 0 to 5 þ by two pathologists blinded to the experimental conditions (0, normal; 1, small changes affecting 5–10%; 2, changes affecting 10–25% of specimen area; 3, changes affecting 25–50% of specimen area; 4, changes affecting 50–75% of specimen area; 5, changes affecting 75–100% of specimen area). Immunohistochemical studies Frozen and paraffin-embedded kidney biopsy sections (5 mm) from biopsies were processed for indirect immunohistochemistry using mouse monoclonal antibodies to detect macrophages (mouse antiporcine macrophage and monocyte, MCA1218, Serotec products, Oxford, UK) and CD4 þ cells (mouse antiporcine CD4 þ , MCA1749, Serotec products). Sections were deparaffined, rehydrated, and heated in a pressure cooker containing citrate buffer (pH 6) to boiling point for 2 min. The sections were then cooled, rinsed in phosphate-buffered saline, and processed for indirect immunohistochemistry as previously described20,24 by using the antibodies listed in Table 1. All sections were examined under blinded conditions and photographed. The number of MCA1218and CD4 þ -labeled cells per surface area (104/mm2) was counted on 10 different tissue sections for each of the experimental conditions as described.29 Statistical analysis Values are reported as the mean7s.d. from (n) experiments. Statistical significance was assessed using the unpaired t-test and the Mann–Whitney U-test or by one-way analysis of variance using the Bonferroni t-test or the Newman–Keuls test for comparison of two or more than two groups. A P-value o0.05 was considered significant. ACKNOWLEDGMENTS
This work was supported by grants from European Community (QLCK6-CT-2000-00064), from Re´gion Poitou Charentes and from Centre Hospitalier et Universitaire de Poitiers, Banque Tarneaud and an NIH R01 to HR. We thank Goujon JM and Barriere M for their precious criticisms and Lochon D, Ribardiere F, Challeroux C, Boutin JL, Quellard N, and Fernandez B for their technical assistance. We thank Kurt Berens (TBC) who provided us with TBC-1269. REFERENCES 1. 2. 3.
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Friedewald JJ, Rabb H. Inflammatory cells in ischemic renal failure. Kidney Int 2004; 66: 486–491. Lasky L. Selectins: interpreters of cell specific carbohydrates information during inflammation. Science 1992; 259: 964–969. Chamoun F, Burne M, O’Donnell M, Rabb H. Pathophysiologic role of selectins and their ligands in ischemia reperfusion injury. Front Biosc 2000; 5: 103–109. Eitner F, Floege J. Novel insights into renal fibrosis. Curr Opin Nephrol Hypertens 2003; 12: 227–252. Seekamp A, Till GO, Mulligan MS et al. Role of selectin in local and remote tissue injury following ischemia and reperfusion. Am J Pathol 1994; 144: 592–598. Springer TA. Traffic signals for lymphocytes recirculation and leukocytes emigration: the multistep paradigm. Cell 1994; 76: 301–314. Weyrich AS, Ma X, Lefer DJ et al. In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest 1993; 91: 2690–2692. Rabb H, Ramirez G, Saba SR et al. Renal ischemia–reperfusion injury in Lselectin deficient mice. Am J Physiol 1996; 271: F408–F413. Nemoto T, Burne MJ, Daniels F et al. Small molecule selectin ligand inhibition improves outcome in ischemic acute renal failure. Kidney Int 2001; 60: 2205–2214. Kidney International (2006) 69, 1749–1755
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Lieberthal W, Nigam SK. Acute renal failure II. Experimental models of acute renal failure: imperfect but indispensable. Am J Physiol Renal Physiol 2000; 278: F1–F12. Basile DP, Donohoe D, Roethe K, Osborn JL. Renal ischemic injury in permanent damage to peritubular capillaries and influence long-term function. Am J Physiol Renal Physiol 2001; 281: F887–F899. Burne-Taney MJ, Yokota N, Rabb H. Persistant renal and extra renal immune changes after severe ischemic injury. Kidney Int 2005; 67: 1002–1009. Ferna´ndez-Fresnedo G, Rodrigo E, Escallada R et al. Effect of early graft function on patient survival in renal transplantation. Transplant Proc 2003; 35: 1653–1654. Brook NR, Waller JR, Nicholson ML. Nonheart-beating kidney donation: current practice and future developments. Kidney Int 2003; 63: 1516–1529. Hicks AER, Abbitt KB, Dodd P et al. The anti-inflammatory effects of a selectin ligand mimetic TBC-1269, are not a result of competitive inhibition of leukocyte rolling in vivo. J Leukoc Biol 2005; 77: 59–66. Toledo-Pereyra LH, Lopez-Neblina F, Reuben JS et al. Selectin inhibition modulates Akt/MAPK signaling and chemokines expression after liver ischemia–reperfusion. J Invest Surg 2004; 17: 303–313. Lai EW, Toledo-Pereyra LH, Walsh J et al. The role of MAP kinase in trauma and ischemia–reperfusion. J Invest Surg 2004; 17: 45–53. Beauharnois ME, Lindquist KC, Marathe D et al. Affinity and kinetics of sialyl lewis-X and core-2 based oligosaccharides binding to L- and Pselectin. Biochemistry 2005; 44: 9507–9519. Aydt E, Wolf G. Development of synthetic pan-selectin antagonists: a new treatment strategy for chronic inflammation in asthma. Pathobiology 2003; 70: 297–301. Basile DP. Rarefaction of peritubular capillaries following ischemic acute renal failure: a potential factor predisposing to progressive nephropathy. Curr Opin Nephrol Hypertens 2004; 13: 1–7.
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& 2006 International Society of Nephrology
Protective role of selectin ligand inhibition in a large animal model of kidney ischemia–reperfusion injury C Jayle1,2, S Milinkevitch1, F Favreau1, C Doucet1,2, JP Richer1,2, S Deretz2, G Mauco1, H Rabb3 and T Hauet1,2 1
INSERM E0324, Centre Hospitalier et Universitaire and Faculte de Medecine, Poitiers, France; 2INRA, Unite´ de Transplantation, Ge´ne´tique Expe´rimentale en Production Animale, Domaine du Magneraud, Surge`res, France and 3Division of Nephrology, John Hopkins University, Baltimore, Maryland, USA
Experiments in rodents have demonstrated an important role for selectins in kidney ischemia–reperfusion injury (IRI). However, the relevance of this in larger mammals, as well as the impact on long-term structure and function is unknown. We tested the hypothesis that small molecule selectin ligand inhibition attenuates IRI, cellular inflammation, and long-term effects on renal interstitial fibrosis. We used a porcine model of kidney IRI and used Texas Biotechnology Corporation (TBC)-1269, a selectin ligand inhibitor. Renal function, tissue inflammation, and tubulointerstitial fibrosis development were evaluated up to 16 weeks. Both warm and cold ischemia models were studied for relevance to native and transplant kidney injury. Pigs treated with TBC-1269 during 45 min of warm ischemia (WI) showed significantly increased glomerular filtration rate compared to control animals. In pigs with severe IRI (WI for 60 min), TBC-1269 treatment during IRI significantly increased renal recovery. Cellular inflammation was strongly reduced, particularly influx of CD4 cells. Quantitative measurement of fibrosis by picrosirius red staining showed strong reduction in TBC-1269-treated groups. TBC-1269 also reduced cold IRI, inflammation, and fibrosis in kidneys preserved for 24 h at 41C and autotransplanted. The selectin ligand inhibitor TBC-1269 provides a novel and effective approach to attenuate IRI in both warm and cold ischemia in large mammals, in both short and long terms. Selectin ligand inhibition is an attractive strategy for evaluation in human kidney IRI. Kidney International (2006) 69, 1749–1755. doi:10.1038/sj.ki.5000335; published online 19 April 2006 KEYWORDS: renal ischemia; reperfusion injury; selectin; T lymphocytes; fibrosis
Correspondence: T Hauet, Laboratoire de Biochimie et Toxicologie, Centre Hospitalier et Universitaire, Hopital Jean Bernard, La Miletrie, 86000 Poitiers, France. E-mail: [email protected] Received 19 August 2005; revised 15 October 2005; accepted 18 November 2005; published online 19 April 2006 Kidney International (2006) 69, 1749–1755
Ischemic acute renal failure remains a major clinical problem with high morbidity and mortality in native kidneys, as well as with worse short- and long-term allograft function in allografts. A number of studies have examined the role of inflammatory cells in the pathogenesis of post-ischemic injury (reviewed by Friedewald and Rabb1). Leukocyte rolling on the endothelium, which precedes firm adhesion via CD11/ CD18 and intercellular adhesion molecule (ICAM-1), is mediated by a class of adhesion molecules known as selectins, which consist of three different transmembrane receptors, E-, P-, and L-selectin.2 E- and L-selectin are involved at the stage of leukocytes rolling and tethering to the vascular endothelium. L-selectin is constitutively expressed on leukocytes, whereas E-selectin is expressed on only activated endothelium. P-selectin is expressed on platelets and activated endothelial cells, and appears to have some inducers different from those of E-selectin.3 Interstitial inflammation is associated with the development of tubulointerstitial fibrosis, characterized by excess accumulation of extracellular matrix in the renal interstitium.4 Renal fibrosis is generally regarded as the dark side of tissue repair mechanisms and the ultimate injury that leads to chronic nephropathy. Selectin blockade has been demonstrated to attenuate ischemia-reperfusion injury (IRI) in different models.5–8 However, blockade of one selectin can lead to compensatory inflammation conducted by the other selectins. A potent selectin antagonist, biosmanose Texas Biotechnology Corporation (TBC)-1269(1,6-Bis(3-(3-carboxymethylphenyl)-4(2-a-D-mannnopyranosylloxy)-phenyl)hexane) is a small molecule antagonist of E-, P-, and L-selectin binding to their ligand, sialyl Lewis X. The structure of TBC-1269 reveals two mannose and two carboxylic acid groups in a configuration to mimic the dimeric configuration of sialyl-Lewis (Figure 1). TBC-1269 was demonstrated to mitigate post-ischemic renal damage in a rodent model during the first 72 h after renal ischemia.9 A major concern in designing clinical trials is whether results in rodent models are translatable to man.10 We therefore conducted these studies in large mammals in an attempt to better set the stage for human studies. In addition, there is increasing concern that ischemic acute renal failure leads to long-term fibrotic changes.11,12 Furthermore, 1749
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C Jayle et al.: Protective effect of biosmanose in renal ischemia
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The outcomes after warm ischemia and reperfusion differed markedly between experimental groups. Two animals from group warm ischemia (WI) 60 died on post-operative days 9 and 10. Survival was 100% in control group (Cont), uninephrectomized group (Unif), WI 45, WI TBC 45, and WI TBC 60. Renal functional data are shown in Figure 2a and b. During the first week, Ccr was reduced in all experimental groups (WI 45 to WI 60) when compared to control and sham-operated groups (Figure 2a). However, Ccr was significantly increased in WI TBC 45 when compared to WI 45, between weeks 2 and 16, and in WI TBC 60, when compared to WI 60 particularly after day 7. In group WI 45, Ccr paralleled Cont and sham-operated groups but remained at lower level from Cont. Ccr remained at 60% of Cont and Unif value in group WI 60. TBC-1269 also provided effective kidney protection after 24 h cold preservation (Figure 2b). However, no change was noted when TBC-1269 was only added to the preservation solution without administering at reperfusion. Two animals were excluded in CI24 group because of renal artery thrombosis. In the warm ischemia model, proteinuria was detected and peaked at day 3 and decreased thereafter to day 7. Progressive proteinuria developed again after approximately 7–8 weeks (Figure 3a). A significant difference was detected between WI 45 and WI 60 groups, and TBC-1269-treated groups, particularly after 7–8 weeks. In the cold ischemia model, proteinuria was more pronounced during the first week when compared to the warm ischemia model (Figure 3b). TBC1269 also reduced proteinuria in cold-stored and autotransplanted groups. TBC-1269 when only added in the preservation solution had no influence in the outcome of proteinuria.
*
*
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RESULTS Effects of ischemic conditions and TBC-1269 on long-term renal function and survival
**
*
Figure 1 | Structural diagram of TBC-1669.
findings in warm kidney IRI simulating native kidney injury may not be transferable to management of transplant patients who have a component of cold kidney IRI. The present study was designed to test the hypothesis that TBC-1269 reduced post-ischemic kidney damage in pig models of kidney IRI. The novel findings of this study include the following (1) TBC-1269 reduced kidney IRI in warm or cold ischemia in pigs; (2) the protective effect was associated with reduced CD4 and macrophage infiltration; and (3) long-term fibrosis was reduced by TBC-1269 treatment.
*
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Figure 2 | Influence of different ischemia models and addition of TBC-1269 on the creatine clearance (1a and b). Renal function was measured in the different experimental groups after different warm ischemia and compared to control group and uninephrectomized group. The values reported are the mean7s.d. *Po0.05, **Po0.05 WI 45 vs WI TB C45, WI 60 vs WI TBC 60.
TBC-1269 studied in animals submitted to the same Cont group conditions did not induce any change in the renal functions when compared to Cont group (Figure 3a and b). Effect of ischemic conditions and TBC-1269 on tissue injury of reperfused kidneys
Histological analysis of biopsy samples from warm-ischemia kidneys revealed differences in the tissue injury after 40 min of reperfusion and of the day-7 post-reperfusion. Kidneys from WI 45 and WI 60 had increased damage when compared to WI TBC 45 and particularly WI TBC 60 (Table 1). The degree of initial acute proximal tubule cell injury appears closely related to the duration of warm ischemia, particularly after 60 min of WI. In the cold preservation and transplantation model, TBC-1269 was also effective in reducing renal tubular injury when compared to controls. TBC-1269 added in the preservation solution alone without reperfusion was not protective when compared to TBC CI 24 group that received the agent during preservation and reperfusion. Effect of ischemic conditions and TBC-1269 on mononuclear cell infiltration in pig kidneys
Consistent with our previous studies in the pig model, the major inflammatory cell population detected by immunohistochemical long term studies after ischemia was CD4 þ T Kidney International (2006) 69, 1749–1755
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C Jayle et al.: Protective effect of biosmanose in renal ischemia
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Figure 3 | Influence of different ischemia models and administration of TBC-1269 on the urine protein excretion (2a and c). Renal function was measured in the different experimental groups after different warm ischemia time or 24-h preservation compared to TBC-1269-treated groups. The values reported are the mean7s.d. **Po0.01 and ***Po0.001 CI 24 vs TBC CI 24 and TBC CI 24A.
Table 1 | Degree of proximal tubule cell lesions and mitochondria damage after different ischemia conditions Warm ischemia Ischemic conditions Injury type time reperfusion WI 45 WI TBC 45 WI 60
WI TBC 60
Brush border integrity 30–45 min Day 7
3.570.2 3.470.2 2.670.21 3.770.2 3.670.2 3.070.11
Cell detachment 30–45 min Day 7
2.670.2 2.570.3 3.470.2 2.770,2* 1.570.2 1.470.2 2.7770.2 2.270.2*
Ischemic conditions Injury type time reperfusion Brush border integrity 30–45 min Day 7 Cell detachment 30–45 min Day 7
3.170.2* 3.570.2*
Cold ischemia CI24
TBCCI24
TBCCI24A
2.570.2 3.670.2 3.570.2# 3.570.2 4.270.2 4.070.1# #
2.470.2 1.970.2 2.070.2 1.770.2 1.370.2 1.270.2#
The histological lesions from early (30–45 min) and day 7 after warm ischemia were evaluated and given as a semiquantitative score (Materials and Methods). Brush border integrity and tubular necrosis in light microscopy studies. Values are means7s.d. for all parameters analyzed. *Po0.05 WI 90 vs WI 60, 1Po0.05 WI 60 vs WI 30 and WI 45, #Po0.05 TBC CI 24 and TBC CI 24A vs CI 24.
Kidney International (2006) 69, 1749–1755
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Figure 4 | Influence of different ischemia models and addition of TBC-1269 on CD4-positive cell infiltration in kidney tissue (3a and c). The number of positively stained cells per surface area were counted on biopsy samples from experimental groups. The values reported are the mean7s.d. from 8 to 12 separate determinations. # Po0.05, ##Po0.05 WI45 vs WITBC45 and 0Po0.05, 000Po0.05 WI60 vs WITBC60, **Po0.01 and ***Po0.001 CI24 vs TBCCI24 and TBCCI24A.
lymphocytes. No infiltrating cells were detected in Cont and Unif (data not shown). In contrast, the number of CD4 þ increased between weeks 1 and 2 after reperfusion in WI 45 and particularly WI 60 (Figure 4a). After week 2, CD4 þ cells plateaued up to 16 weeks following reperfusion in groups WI 45 and WI 60. In WI 60 and WI 45 groups, monocyte/ macrophage increased between weeks 1 and 2 and slightly decreased between weeks 2 and 4, then plateaued between weeks 4 and 16 (Figure 4b). Monocyte/macrophage number was significantly increased in WI 60 group when compared to WI 45 group. In WI kidneys treated with TBC-1269, CD4positive cell number was significantly reduced. Monocyte/ macrophage number was also different in WI TBC 45 and WI TBC 60 when compared to WI 45 and WI 60, respectively (Figure 5a). TBC-1269 was also efficient in cold ischemia model in reducing inflammatory cells (Figure 5b). TBC-1269 reduced CD4-positive cells and macrophages during the first week, and this beneficial effect remained detectable between weeks 4 and 16. Effect of ischemic conditions and TBC-1269 on interstitial fibrosis and tubular atrophy
Evolution of interstitial fibrosis is presented in Figure 4. In our hands, fibrosis is correlated with WI, particularly after 60 min (Figure 6, lower panel, a–d). Percentage of interstitial fibrosis area is also associated with progression of renal dysfunction particularly in WI 60 group. Picrosirius score was significantly different at weeks 7–8 between WI 45 and 1751
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Figure 5 | Influence of different ischemia models and administration of TBC-1269 on monocyte/macrophages within kidney tissue (4a and b). The number of positively stained cells per surface area was counted on biopsy samples from experimental groups. The values reported are the mean7s.d. from 8 to 12 separate determinations. #Po0.05, ##Po0.05 WI 45 vs WI TBC 45 and 0Po0.05, 000 Po0.05 WI 60 vs WI TBC 60, **Po0.01 and ***Po0.001 CI 24 vs TBC CI 24, and TBC CI 24A.
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Figure 7 | Influence of cold ischemia on the onset of tubulointerstitial fibrosis and tubular atrophy score: the percentage of kidney areas displaying interstitial fibrosis stained with Picrosirius was measured over several weeks following cold ischemia within kidneys. *Po0.05 and **Po0.01 CI 24 vs TBC CI 24 and TBC CI 24A.
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WI 60 groups. These changes are accompanied by atrophy of the surrounding tubules (Figure 6, lower panel, e–h, upper panel). This difference in fibrosis increased at weeks 10–12 and more significantly by week 16. TBC-treated groups showed a significantly reduced fibrosis intensity particularly after 60 min of WI between weeks 8 to 16 (WI 60 vs WI TBC 60; Figure 6, lower panel) and weeks 10–12 and W 16 (WI 45 vs WI TBC 45; Figure 6). No difference was found at week 8 in WI 45 and WI TBC 45. Tubular atrophy scores were increased in WI 45 and WI 60 groups after week 8 when compared to TBC-1269-treated groups (Figure 6). In the cold ischemia model, TBC also reduced fibrosis (Figure 7, upper panel, a–c, lower panel) and tubular atrophy development (Figure 7, upper panel, d–f, lower panel).
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Figure 6 | Influence of warm ischemia on the onset of tubulointerstitial fibrosis and tubular atrophy score: the percentage of kidney areas displaying interstitial fibrosis stained with Picrosirius was measured over several weeks following warm ischemia in kidneys. #Po0.05, ##Po0.01 WI45 vs WITBC45 and 0 Po0.05, 000Po0.05 WI60 vs WITBC60, *Po0.05 WI45 vs WITBC45, and WI60 vs WITBC60. 1752
IRI is the primary cause of acute renal failure in native kidney and can cause delayed graft function in transplanted kidneys. Ischemic injury to the kidney is associated with high morbidity and mortality and is a non-immunologic factor involved in chronic allograft nephropathy. In addition, recent report has demonstrated that patients with poor levels of early graft function are at an increased risk of death.13 The lack of suitable organs for transplantation from traditional sources has led clinicians to seek alternatives. Although related and unrelated live donation is being promoted as one option for increasing the donor pool, it is unlikely that this will in itself be able to completely bridge the gap. Non-heart beating and extended criteria organs, both typified by increased injury, can provide an alternative supply of organs, which should substantially increase the donor pool.14 Kidney International (2006) 69, 1749–1755
C Jayle et al.: Protective effect of biosmanose in renal ischemia
Consequently, therapeutics directed to improving the ability of the kidney to tolerate ischemic injury would have important implications. We found that selectin ligand binding with TBC-1269 significantly reduced post-ischemic renal dysfunction in pigs with different warm ischemia times, as well as in a cold ischemia model. Importantly, these findings on warm ischemia in a large mammal model confirm and extend earlier studies on rodents. Furthermore, we have evaluated a cold ischemia model as well, and demonstrated that administration in the perfusate is not adequate but that post-reperfusion dosing is crucial. Proteinuria occurred in the WI 45 and WI 60 groups, as well as CI 24 groups, and correlated with histologic injury and particularly tubulointersitital fibrosis. Proteinuria became prominent and progressive after week 8 in warm ischemia groups when compared to control and Unif. Interestingly, the protective effect of TBC-1269 was also prominent, long-term after warm ischemia. In cold preserved groups, TBC-1269-treated groups had reduced proteinuria and reduced fibrosis and tubular atrophy. We examined mononuclear leukocytes as potential mediators of late changes after IRI, and examined the impact of the selectin ligand blockade on these. TBC-1269 reduced monocyte/macrophage infiltration in WI TBC 45 and WI TBC 60 groups after weeks 6 and 8, respectively. We also evaluated the effects of TBC-1269 on CD4 þ cells infiltration, which increased with WI time. TBC-1269 reduced CD4 þ cell infiltration in both the WI model and the cold ischemia autotransplanted model. We cannot exclude the possibility that reduced macrophages and CD4 þ cells were a result of decreased injury, rather than the cause of decreased injury. Recent studies have demonstrated that the in vivo effects of TBC-1269 may be mediated through E-selectin but do not appear to involve leukocyte rolling. TBC-1269 was shown to inhibit rolling of a murine cell line (32DC13) on murine Pselectin in vitro and has pronounced anti-inflammatory activity in mice when given before induction of thioglycollate-induced peritonitis.15 In contrast, in this previous study, TBC-1269 failed to influence established leukocyte rolling in vivo at doses that match or exceed those with pronounced, anti-inflammatory activity. The authors hypothesized that TBC-1269 limits inflammation by a mechanism other than direct competition with cell-bound selectin ligands. An other recent study has demonstrated that TBC-1269 downregulated the expression of macrophage inflammatory protein (MIP)1a, MIP-1b, MIP-2, and kupffer cells, as well as expression of Akt, mitogen-activated protein kinases (p44/42), and ribosomal S6 kinase.16 Consequently, this selectin inhibition modulated the expression of Akt, mitogen-activated protein kinases (p44/42), and alterations in cellular signaling and chemokines. These alterations in cellular signaling and chemokine expression represent potential mechanisms or pathways of inflammatory response in ischemia-reperfusion. Mitogen-activated protein kinases have been the focus of a number of studies, as these compounds are involved in a Kidney International (2006) 69, 1749–1755
original article
number of important inflammatory cell signaling mechanisms.17 Recent studies have further elucidated the role of mitogen-activated protein kinases in the inflammatory response, as a result of trauma and/or IRI. Akt pathway is also involved in IRI and functional recovery of damaged organs. It was recently shown that biosmanose was the most advanced synthetic pan-selectin antagonist and that TBC1269 competitively inhibited, via steric/allosteric mechanisms, the binding of selectin function blocking antibody and was shown to bind all the three selectins to varying degrees.18,19 Previous studies have demonstrated that renal, ischemic injury results in permanent damage to peritubular capillaries and influence long-term function.20,21 Tubulo-interstitial fibrosis was significant in both warm and cold ischemia groups, long term. Picrosirius red-positive staining has been used for nearly 30 years and the use of computer-assisted quantification of fibrosis in the Sirius red-stained renal allograft biopsies appears to be a surrogate marker to predict long-term allograft function.22 In our model, Picrosirius red staining was improved by the selectin ligand inhibition. These data sets the stage for a clinical trial applying selectin ligand blockade to improve short- and long-term outcomes of kidney IRI in both the native kidney and allograft. We have noted some difference between warm ischemia and cold preservation, particularly for proteinuria and fibrosis development. Inflammatory cell kinetics were also specific for cold and warm ischemia. A recent study demonstrated that WI triggers injury primarily to proximal tubular cells, whereas cold ischemia damages glomerular podocytes and peritubular endothelial cells in addition to proximal tubules.23 These data support proteinuria output in cold ischemia group. As previously reported, these results suggest that organ preservation and cold ischemia could have pathophysiology distinct from warm IRI, particularly after 60 min of WI.24 The fact that TBC-1269 does not seem to have any beneficial impact when added to the preservation solution could be linked to the change of the drug effect at 41C. In addition, future studies are required to examine the molecular mechanisms of how selectin ligand blockade modulates kidney outcomes after IRI. MATERIALS AND METHODS Surgical procedures and experimental groups Large white male pigs, 30–35 kg, 3–4 month old (INRA, Le Magneraud, Surge`res, France) were prepared as previously described.25 All animals were fed with a standard diet (Socie´te´ Arrive, Saint Fulgent, France) and had free access to tap water. Rapid preoperative tranquilization was achieved by the rapid and atraumatic nasal administration of 0.2 mg/kg of midazolam (Laboratoire Roche, Neuilly-sur-Seine, France). The animals were then anesthetized with halothane (Laboratoire Belamont, Paris, France) and 100% oxygen. The surgical procedures were performed under sterile conditions. A 20-gauge plastic catheter (Becton Dickinson Vascular Access Inc., Sandy, UT, USA) was inserted into an ear vein. Atropine sulfate 10 mg/kg was given intravenously to reduce pharyngeal and tracheal secretion, and to 1753
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prevent post-intubation bradycardia. A midline abdominal incision was made to expose both kidneys. Warm ischemia was induced in the pigs by clamping the left renal pedicle for 45 min (WI 45, n ¼ 10) and 60 min (WI 60, n ¼ 10) with a vascular clamp followed by a right nephrectomy. Two other experimental groups were studied: WI TBC 45 (n ¼ 10) and WI TBC 60 (n ¼ 10). In these two experimental groups, TBC-1269 was administered 10 min before ischemia, during warm ischemia, and 1 h after reperfusion. TBC was given at a concentration of 25 mg/kg based on a previous dosing study.26 In the cold ischemia model, the left kidney was removed and cold-flushed with University of Wisconsin solution and coldstored at 41C for 24 h. The kidneys were re-implanted and the contralateral kidney was removed. Groups of animals were divided as follows: CI TBC 24, n ¼ 10 (TBC-1269 was administered 10 min before cold-flush and 1 h after reperfusion), CI TBC 24A (CI TBC 24 with TBC-1269 added in UW solution before cold-flush but not reperfusion, n ¼ 10), and CI 24, n ¼ 10. These experimental groups were compared to uninephrectomy group without kidney ischemia (Unif; n ¼ 8) and control group (Cont; n ¼ 6, age-matched group). TBC-1269 was also studied in control group condition (Cont þ TBC, n ¼ 6). All experiments were carried out in accordance with the Guidelines of the French Agricultural Office and the legislation governing animal studies. Any animal that died during the 16-week follow-up period was autopsied. All animals that died from causes other than renal failure, or developed renal artery, vein, or ureter problems were excluded. Functional parameters Endogenous creatinine clearance (Ccr, expressed in ml/min) and urinary protein were measured as previously described.25 Endogenous creatinine clearance is suggested to be a reliable indicator for renal excretory function in newborn and growing piglets.27,28 Pigs were placed in a cage to allow specific 24 h urine collections and plasma samples collection. Plasma and urinary creatinine (Cr) was measured enzymatically using an automatic analyzer (Hitachi, Paris, France) and Ccr was calculated using the following formula: urinary flow rate urinary Cr/plasma Cr. Urinary proteins was determined using a photometry method (Laboratoire Biorea, Talant, France). Histopathological studies After warm ischemia and 40 min of reperfusion, at day 1, day 3, day 5, day 7, day 14 and 4–5, 10–12, and 16 weeks after surgery, biopsy tissue samples from the deep cortex–outer medulla region were performed. Samples were fixed with Dubosq-Brasil and 10% formalin in 0.01 mmol/l phosphate buffer (pH 7.42) and embedded in paraffin. Conventional stains were applied (hematoxylin and eosin, periodic acid-Schiff). Different histological parameters (brush border integrity and intratubular detachment) were studied. Histological lesions were expressed in percent of kidney samples using a previously described semiquantitative scale24,25: (0) no abnormality; (1) mild lesions affecting less than 10% of kidney samples; (2) lesions affecting 10–25% of kidney samples; (3) lesions affecting 25–50% of kidney samples; (4) lesions affecting 50–75%; (5) lesions affecting more than 75% of kidney samples. Tubulointerstitial injury was defined as inflammatory cell infiltrates and interstitial fibrosis. To estimate the level of tubulointerstitial fibrosis, tissue sections were also labeled with Picrosirius for collagen identification (collagen I and III) as previously described.29,30 The amount of interstitial fibrosis was determined in Picrosirius-stained sections by a semiquantitative imaging technique.31 The percentage of Picrosirius-stained surface was determined on 10 different tissue 1754
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sections viewed at 100 magnifications in each experimental condition and expressed as percent of the total surface area examined. Tubular atrophy was semiquantitatively scored on a scale of 0 to 5 þ by two pathologists blinded to the experimental conditions (0, normal; 1, small changes affecting 5–10%; 2, changes affecting 10–25% of specimen area; 3, changes affecting 25–50% of specimen area; 4, changes affecting 50–75% of specimen area; 5, changes affecting 75–100% of specimen area). Immunohistochemical studies Frozen and paraffin-embedded kidney biopsy sections (5 mm) from biopsies were processed for indirect immunohistochemistry using mouse monoclonal antibodies to detect macrophages (mouse antiporcine macrophage and monocyte, MCA1218, Serotec products, Oxford, UK) and CD4 þ cells (mouse antiporcine CD4 þ , MCA1749, Serotec products). Sections were deparaffined, rehydrated, and heated in a pressure cooker containing citrate buffer (pH 6) to boiling point for 2 min. The sections were then cooled, rinsed in phosphate-buffered saline, and processed for indirect immunohistochemistry as previously described20,24 by using the antibodies listed in Table 1. All sections were examined under blinded conditions and photographed. The number of MCA1218and CD4 þ -labeled cells per surface area (104/mm2) was counted on 10 different tissue sections for each of the experimental conditions as described.29 Statistical analysis Values are reported as the mean7s.d. from (n) experiments. Statistical significance was assessed using the unpaired t-test and the Mann–Whitney U-test or by one-way analysis of variance using the Bonferroni t-test or the Newman–Keuls test for comparison of two or more than two groups. A P-value o0.05 was considered significant. ACKNOWLEDGMENTS
This work was supported by grants from European Community (QLCK6-CT-2000-00064), from Re´gion Poitou Charentes and from Centre Hospitalier et Universitaire de Poitiers, Banque Tarneaud and an NIH R01 to HR. We thank Goujon JM and Barriere M for their precious criticisms and Lochon D, Ribardiere F, Challeroux C, Boutin JL, Quellard N, and Fernandez B for their technical assistance. We thank Kurt Berens (TBC) who provided us with TBC-1269. REFERENCES 1. 2. 3.
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