Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts
2008; Elsevier BV; Volume: 74; Issue: 8 Linguagem: Inglês
10.1038/ki.2008.342
ISSN1523-1755
AutoresAtsunori Nakao, Gaetano Faleo, Hiroko Shimizu, Kiichi Nakahira, Junichi Kohmoto, Ryujiro Sugimoto, Augustine M.K. Choi, Kenneth R. McCurry, Toru Takahashi, Noriko Murase,
Tópico(s)Neonatal Health and Biochemistry
ResumoRenal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 μm). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury. Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 μm). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury. Currently, kidney transplantation (KTx) is widely practiced around the world and has been accepted as the preferred strategy of renal replacement therapy for end-stage renal disease. However, ischemia/reperfusion (I/R) injury due to cold storage and warm reoxygenation, obligatory to KTx procedure, remains unfavorable factor in KTx, contributing to delayed graft function and subsequent chronic graft deterioration.1.Gueler F. Gwinner W. Schwarz A. et al.Long-term effects of acute ischemia and reperfusion injury.Kidney Int. 2004; 66: 523-527Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,2.Daemen M.A. de Vries B. Buurman W.A. Apoptosis and inflammation in renal reperfusion injury.Transplantation. 2002; 73: 1693-1700Crossref PubMed Scopus (174) Google Scholar Recent evidences have implicated that cytochrome P450 (CYP), a large group of heme proteins abundantly present in the kidney, play critical roles in the renal I/R injury process.3.Paller M.S. Jacob H.S. Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney.Proc Natl Acad Sci USA. 1994; 91: 7002-7006Crossref PubMed Scopus (114) Google Scholar,4.Takahashi T. Morita K. Akagi R. et al.Protective role of heme oxygenase-1 in renal ischemia.Antioxid Redox Signal. 2004; 6: 867-877Crossref PubMed Scopus (51) Google Scholar,5.Tamura Y. Imaoka S. Gemba M. et al.Effects of ischemia-reperfusion on individual cytochrome P450 isoforms in the rat kidney.Life Sci. 1997; 60: 143-149Crossref PubMed Scopus (22) Google Scholar CYPs are susceptible to oxidant stress and substantially damaged during renal I/R injury by reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide. Damaged CYPs are prone to degrade and release free heme/iron.5.Tamura Y. Imaoka S. 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Free heme toxicity and its detoxification systems in human.Toxicol Lett. 2005; 157: 175-188Crossref PubMed Scopus (531) Google Scholar In addition, intracellular free heme can be a major source of iron, which participates in the generation of more detrimental hydroxyl radicals from hydrogen peroxide via Fenton reaction, or becomes highly reactive iron–oxygen complexes such as ferryl or perferryl ion.13.Paller M.S. Hedlund B.E. Role of iron in postischemic renal injury in the rat.Kidney Int. 1988; 34: 474-480Abstract Full Text PDF PubMed Scopus (166) Google Scholar,14.Baliga R. Zhang Z. Baliga M. et al.Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity.Kidney Int. 1998; 54: 1562-1569Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Thus, CYP degradation and increased intracellular free heme due to renal I/R injury could play a significant role in the renal graft damage. Accordingly, the strategies to inhibit CYP degradation and subsequent heme/iron release will have a substantial effect in ameliorating I/R injury and improve renal transplant outcomes. Carbon monoxide (CO) possesses a strong affinity to heme moiety of heme proteins15.Blomberg L.M. Blomberg M.R. Siegbahn P.E. A theoretical study on the binding of O2, NO and CO to heme proteins.J Inorg Biochem. 2005; 99: 949-958Crossref PubMed Scopus (85) Google Scholar,16.Koley A.P. Buters J.T. Robinson R.C. et al.CO binding kinetics of human cytochrome P450 3A4. Specific interaction of substrates with kinetically distinguishable conformers.J Biol Chem. 1995; 270: 5014-5018Crossref PubMed Scopus (88) Google Scholar and could alter the biological behaviors of CYPs,17.Ball E.G. Strittmatter C.F. Cooper O. The reaction of cytochrome oxidase with carbon monoxide.J Biol Chem. 1951; 193: 635-647Abstract Full Text PDF PubMed Google Scholar,18.Miller M.A. Hales C.A. Role of cytochrome P-450 in alveolar hypoxic pulmonary vasoconstriction in dogs.J Clin Invest. 1979; 64: 666-673Crossref PubMed Scopus (30) Google Scholar as the binding of nitric oxide inhibits the biological action of CYP4A219.Sun C.W. Alonso-Galicia M. Taheri M.R. et al.Nitric oxide-20-hydroxyeicosatetraenoic acid interaction in the regulation of K+ channel activity and vascular tone in renal arterioles.Circ Res. 1998; 83: 1069-1079Crossref PubMed Scopus (152) Google Scholar and CYP omega-hydroxylase.20.Oyekan A.O. Youseff T. Fulton D. et al.Renal cytochrome P450 omega-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride.J Clin Invest. 1999; 104: 1131-1137Crossref PubMed Scopus (99) Google Scholar We hypothesized that the binding of CO to renal CYP would stabilize CYP, and prevent CYP degradation and detrimental heme/iron release in renal grafts, leading to potent protection of kidney grafts from I/R injury. We have tested our hypothesis in this study using the orthotopic rat KTx model with 24 h cold preservation. CO was mixed into UW preservation solution and ex vivo delivered to excised kidney grafts during cold preservation and before transplantation. Our data provide a novel mechanism of the protective effects of ex vivo organ-targeted CO delivery against transplant-induced I/R injury. CO solubility in UW solution elevated to 40.6±1.6 μmol/l after CO supplementation from 0.7±0.6 μmol/l in untreated UW solution.21.Nakao A. Toyokawa H. Tsung A. et al.Ex vivo application of carbon monoxide in university of wisconsin solution to prevent intestinal cold ischemia/reperfusion injury.Am J Transplant. 2006; 6: 2243-2255Crossref PubMed Scopus (80) Google Scholar When kidney grafts were perfused with and stored in CO-bubbled UW, tissue CO contents at the end of 24 h cold preservation elevated to 23.3±4.2 pmol/mg tissue, whereas the grafts stored in control UW showed 1.43 pmol/mg tissue. These results demonstrate that CO gas can be effectively delivered to kidney grafts during cold storage before transplantation using ex vivo organ-targeted CO delivery protocol. However CO levels of the grafts stored in both control UW and UW with CO 3 h after reperfusion were basal levels, indicating that CO quickly disappeared from the grafts within 3 h after reperfusion (Figure 1a). Total CYP contents in the microsomal fraction of the grafts were determined at the end of cold storage and 3 h after KTx. Graft CYP contents were maintained throughout the cold storage period and the levels at the end of cold storage were similar to those of naive kidneys. Total CYP contents, however, were significantly decreased in the kidney grafts preserved in control UW at 3 h after KTx. On the other hand, the kidney grafts stored in UW with CO maintained a nearly normal concentration of CYP at the same time point (Figure 1b). These data indicate that ex vivo organ-targeted CO delivery during cold storage prevents CYP breakdown during I/R process. As degraded CYP during I/R injury possibly become the source of detrimental intracellular free heme, microsomal heme contents in the kidney grafts were assessed 3 h after reperfusion. Microsomal heme content elevated significantly in the grafts stored in control UW. Ex vivo organ-targeted CO delivery protocol significantly decreased intracellular free heme concentration (Figure 1c). Although heme plays an essential role in the body as a component of heme proteins, intracellular free heme is injurious. Therefore, heme concentration in the cell is tightly controlled mostly via heme biosynthesis rate-limiting enzyme, 5-aminolevulinate synthase (ALAS-1).22.Handschin C. Lin J. Rhee J. et al.Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha.Cell. 2005; 122: 505-515Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar,23.May B.K. Dogra S.C. Sadlon T.J. et al.Molecular regulation of heme biosynthesis in higher vertebrates.Prog Nucleic Acid Res Mol Biol. 1995; 51: 1-51Crossref PubMed Scopus (118) Google Scholar We therefore examined sequential changes of graft mRNA levels for ALAS-1 using northern blot analysis. In the control UW group, ALAS-1 mRNA levels in the kidney grafts were markedly suppressed at 3 h after reperfusion, and gradually increased to the normal level by 6 h (Figure 2a). The decrease of ALAS-1 mRNA levels in the control UW group at 3 h was significantly inhibited in UW with the CO group (Figure 2b). These results indicate that increased microsomal heme levels in I/R injury in the control UW group coincidently downregulate heme biosynthesis due to negative feedback mechanism, whereas CO in the UW group maintains normal heme biosynthesis. In addition, these results also reveal that increased intracellular heme contents during renal I/R injury are not caused by accelerated heme biosynthesis, but heme release from degraded heme proteins such as CYP. Further, the inhibition of heme elevation with CO is not due to ALAS-1 downregulation. Conversely, we assessed the activation of heme-degrading enzyme heme oxygenase-1 (HO-1), which plays a primary role to eliminate toxic free heme and to protect cells from heme-induced oxidative stress.12.Kumar S. Bandyopadhyay U. Free heme toxicity and its detoxification systems in human.Toxicol Lett. 2005; 157: 175-188Crossref PubMed Scopus (531) Google Scholar HO-1 mRNA levels in the kidney grafts rapidly elevated after reperfusion and peaked at 3 h in the control UW group. CO significantly inhibited intragraft HO-1 mRNA upregulation. Cold storage in control UW or UW containing CO did not alter graft HO-1 mRNA expression (Figure 3a). In correlation with mRNA levels, HO-1 protein levels gradually increased after reperfusion and were markedly increased 6 h after reperfusion (Figure 3b). CO in UW significantly reduced HO-1 protein levels in the graft 6 h after reperfusion (Figure 3c). These data reveal that an increase of intracellular heme in renal I/R injury triggers the induction of heme catalytic enzyme, HO-1.24.Tenhunen R. Marver H.S. Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc Natl Acad Sci USA. 1968; 61: 748-755Crossref PubMed Scopus (1425) Google Scholar,25.Shibahara S. Yoshida T. Kikuchi G. Mechanism of increase of heme oxygenase activity induced by hemin in cultured pig alveolar macrophages.Arch Biochem Biophys. 1979; 197: 607-617Crossref PubMed Scopus (47) Google Scholar The magnitude of the graft oxidative injury was determined by measuring tissue malondialdehyde (MDA) level. MDA is the most abundant byproduct of lipid peroxidation caused by ROS generated during I/R injury. Graft MDA levels did not change during cold storage and were maintained at the basal level. However, MDA levels in the grafts stored in control UW significantly elevated and peaked at 3 h after reperfusion. Supplementation of CO into UW resulted in a significant reduction of MDA levels in the grafts 3 h after reperfusion, suggesting that CO given during cold storage reduced injurious ROS and inhibited cellular lipid peroxidation associated with I/R injury (Figure 4a). Both CYP-derived free heme and catalytic iron-derived ROS, generated during I/R injury, may participate in direct oxidative damage of graft parenchymal cells. Sequential expression of inflammatory mediators including cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and early growth response-1 (Egr-1) were examined. Graft mRNA levels for these mediators rapidly elevated and peaked at 3 h after KTx. CO in UW effectively downregulated I/R-induced activation of inflammatory mediators in the kidney grafts (Figure 4b). Prolonged cold storage of the grafts in control UW for 24 h resulted in tubular atrophy, cellular infiltration, and mild glomerulitis at 28 days after KTx. These histopathological changes were less severe in the grafts stored in UW with CO (Figure 5a). Glomerulosclerosis or vasculitis was not seen in the grafts in either group. A number of ED1+ infiltrating macrophages in control grafts was increased to 55.2±16.2 cells per high power field (HPF) compared to 29.7±11.8 cells per HPF in naive kidneys. The grafts stored in CO-treated UW showed significantly reduced number of ED1+ infiltrating macrophages (Figure 5b). Cold I/R injury resulted in progressive deterioration of renal graft function, and creatinine clearance (CCr) dramatically decreased to 0.28±0.11 ml/min at 28 days after KTx, which was less than 12% of that seen in normal animals. Furthermore, whereas normal animals excreted small amounts of urine protein (0.2 mg per 24 h), the grafts in control UW showed significant proteinuria (155.3±41.7 mg per 24 h) at 28 days after KTx. When kidney grafts were preserved for 24 h in 5% CO-bubbled UW, graft functions at 28 days after KTx were significantly improved (CCr: 0.64±0.26 ml/min; proteinuria: 96.7±25.4 mg per 24 h) compared to those of control UW (Figure 6a and b). Mean body weight gains at 28 days after KTx in the recipients of control UW group and CO-treated UW group were -3.42 and 9.01%, respectively (Figure 6c). In correlation to improved renal functions, ex vivo organ-targeted CO delivery significantly enhanced recipient survival to a median of >100 days, compared to 51 days of control UW group (Figure 6d). The recipients in both control UW and UW with CO groups showed high levels of serum creatinine (7.9–9.0 mg per 100 ml), potassium (6.5–8.9 mEq/l), and urea nitrogen (481–789 mg per 100 ml) at their terminal phase. At autopsy, these recipients did not have any surgical complication or other abnormality. Increasing evidences have demonstrated that inhalation of CO at low concentrations (20–500 p.p.m.) prevents I/R injury in various experimental organ transplantation models.26.Nakao A. Choi A.M. Murase N. Protective effect of carbon monoxide in transplantation.J Cell Mol Med. 2006; 10: 650-671Crossref PubMed Scopus (97) Google Scholar,27.Neto J.S. Nakao A. Kimizuka K. et al.Protection of transplant-induced renal ischemia-reperfusion injury with carbon monoxide.Am J Physiol Renal Physiol. 2004; 287: F979-F989Crossref PubMed Scopus (168) Google Scholar,28.Akamatsu Y. Haga M. Tyagi S. et al.Heme oxygenase-1-derived carbon monoxide protects hearts from transplant-associated ischemia reperfusion injury.FASEB J. 2004; 18: 771-772Crossref PubMed Scopus (169) Google Scholar,29.Kohmoto J. Nakao A. Kaizu T. et al.Low-dose carbon monoxide inhalation prevents ischemia/reperfusion injury of transplanted rat lung grafts.Surgery. 2006; 140: 179-185Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Although inhaled CO could be used for the therapy, there are considerable concerns to apply CO as in vivo treatment. To minimize the concerns for the possible adverse effects associating with in vivo CO inhalation, we have recently developed the ex vivo organ-targeted CO delivery method by perfusing and storing excised grafts with CO-bubbled preservation solution before transplantation. Using the intestinal transplantation model, we showed that ex vivo delivered CO could ameliorate intestinal I/R injury following prolonged cold ischemia.21.Nakao A. Toyokawa H. Tsung A. et al.Ex vivo application of carbon monoxide in university of wisconsin solution to prevent intestinal cold ischemia/reperfusion injury.Am J Transplant. 2006; 6: 2243-2255Crossref PubMed Scopus (80) Google Scholar In the current study using the kidney transplant model, we confirmed that gaseous CO in UW preservation solution could be effectively delivered to excised kidney grafts, prevent renal I/R injury, and improve renal graft function and survival. The beneficial effects of ex vivo delivered CO associated with the inhibition of CYP degradation and heme release in kidney grafts and amelioration of oxidative injuries. In the bloodless ex vivo condition specifically occurring in a transplant setting, there is no CO binding to hemoglobin that is typically seen with inhaled CO in the living animal. Therefore, CO could bind more efficiently to target molecules, most likely heme proteins, without forming carboxyhemoglobin. Thus, ex vivo organ-targeted CO delivery method might involve mechanisms of action different from those of the inhalation delivery method. As CYPs are known to play critical roles in I/R injury associated with renal transplantation,30.Orrenius S. Ellin A. Jakobsson S.V. et al.The cytochrome P-450-containing mono-oxygenase system of rat kidney cortex microsomes.Drug Metab Dispos. 1973; 1: 350-357PubMed Google Scholar,31.Carroll M.A. Balazy M. Huang D.D. et al.Cytochrome P450-derived renal HETEs: storage and release.Kidney Int. 1997; 51: 1696-1702Abstract Full Text PDF PubMed Scopus (90) Google Scholar we tested our hypothesis in this study that CO binding to heme moiety of CYP, in advance, can stabilize and protect CYP from degradation caused by oxidative attack initiated by reperfusion. As the binding activity of CO may not be CYP specific, CO also may bind to various heme proteins in the kidney grafts. However, as the kidney is richly endowed with CYP particularly in the proximal tubules, CO in UW could effectively bind to CYP during cold storage of the kidney grafts. In fact, the study demonstrated that excised kidney grafts exposed to CO during the cold preservation period could preserve CYP, decrease free heme release, and maintain normal intracellular heme concentration and metabolism. In contrast, in untreated grafts, intracellular heme metabolism was shifted towards heme catabolism due to a large increase of intracellular heme concentration. Prevention of CYP degradation could be the result mediated by other mechanisms. As kidney grafts stored in UW with CO showed the downregulation of inflammatory mediators such as TNFα and IL-6, the inhibition of proinflammatory responses could be a direct effect of CO. Thus preventing oxidative stress and the preservation of the CYP content could be an indirect effect of CO on CYP. However, previous reports using CYP inhibitors such as cimetidine or piperonyl butoxide show that CYP degradation and detrimental heme/iron increase from the organ during I/R injury are inhibited by these drugs due to their binding to heme and CYP stabilization.32.Caballero F. Gerez E. Batlle A. et al.Interaction of cimetidine with P450 in a mouse model of hepatocarcinogenesis initiation.Br J Cancer. 2002; 86: 630-635Crossref PubMed Scopus (5) Google Scholar,33.Fisher C.W. Mayer R.T. Characterization of house fly microsomal mixed function oxidases: inhibition by juvenile hormone i and piperonyl butoxide.Toxicology. 1982; 24: 15-31Crossref PubMed Scopus (8) Google Scholar It is also known that hemoglobin becomes more stable and resistant against oxidative stress when oxygen binds to hemoglobin and forms oxyhemoglobin.34.Shikama K. Nature of the FeO2 bonding in myoglobin and hemoglobin: a new molecular paradigm.Prog Biophys Mol Biol. 2006; 91: 83-162Crossref PubMed Scopus (68) Google Scholar Although further studies will be necessary to confirm the actual binding site of CO, it is tempting to conceive that CO-binding to heme moiety of renal CYP could prevent CYP degradation and block heme/iron release during kidney I/R injury. Another possible action of CO on CYP might be an inhibition of CYP's enzymatic activities.35.Estabrook R.W. Franklin M.R. Hildebrandt A.G. Factors influencing the inhibitory effect of carbon monoxide on cytochrome P-450-catalyzed mixed function oxidation reactions.Ann NY Acad Sci. 1970; 174: 218-232Crossref PubMed Scopus (71) Google Scholar CYPs act as a monooxygenase and can generate detrimental ROS including hydrogen peroxide and superoxide anion during the nicotinamide adenine dinucleotide phosphate-dependent electron transfer.36.Nordblom G.D. Coon M.J. Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.Arch Biochem Biophys. 1977; 180: 343-347Crossref PubMed Scopus (147) Google Scholar In addition, CYPs contribute to the metabolization of arachidonic acid and generation of vasoconstrictor eicosanoids, which are known to elicit vasoconstriction of the graft vasculature during I/R injury.37.Schwartzman M. Ferreri N.R. Carroll M.A. et al.Renal cytochrome P450-related arachidonate metabolite inhibits (Na++K+) ATPase.Nature. 1985; 314: 620-622Crossref PubMed Scopus (234) Google Scholar,38.Fleming I. Cytochrome p450 and vascular homeostasis.Circ Res. 2001; 89: 753-762Crossref PubMed Scopus (314) Google Scholar Thus, an inhibition of CYP enzymatic activities by CO may lead to reduced ROS generation and improved graft microcirculation. However, considering that significant amount of CYP is degraded during I/R injury, the inhibitions of these CYP enzymatic activities by CO are unlikely to play major roles in I/R injury process.9.Bysani G.K. Kennedy T.P. Ky N. et al.Role of cytochrome P-450 in reperfusion injury of the rabbit lung.J Clin Invest. 1990; 86: 1434-1441Crossref PubMed Scopus (67) Google Scholar It needs to be determined if optimum concentrations of CO in UW preservation solution can be more effective for kidney or other organ transplantation. Using 5% gaseous CO in this study, CO solubility reached 40.6 μmol/l, which appeared to be effective for KTx experiments. This CO concentration was chosen based on the previous studies using CO-releasing molecules.39.Clark J.E. Naughton P. Shurey S. et al.Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule.Circ Res. 2003; 93: e2-e8Crossref PubMed Google Scholar,40.Sandouka A. Fuller B.J. Mann B.E. et al.Treatment with CO-RMs during cold storage improves renal function at reperfusion.Kidney Int. 2006; 69: 239-247Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar These reports showed that cardiomyocytes and renal vascular system were protected against oxidative stress at CO concentrations of 10–50 μmol/l in the perfusate containing CO-releasing molecules. Future studies are warranted to determine if a higher concentration of CO may bring additional protections against renal I/R injury. In conclusion, our results demonstrated that renal CYPs were degraded during I/R injury, followed by an increase of intracellular free heme and oxidative injury of the kidney grafts. The perfusion with and storage in the CO-supplemented UW solution prevented CYP degradation and ameliorated transplant-induced renal graft I/R injury. Ex vivo organ-targeted CO delivery is an innovative, attractive, and simple strategy, which can be applicable in clinical practice. Further, ex vivo CO delivery provides a unique setting to analyze the functional mechanisms of CO's protective effects in the whole organ system. UW solution (Viaspan, Du Pont, Wilmington, DE, USA) was vigorously bubbled for 5 min at 4 °C with compressed 5% CO gas mixed in air (PRAXAIR, Danbury, CT, USA) under sterile conditions in the hume hood. The solubility of CO was determined using TRI lyzer (Taiyo, Osaka, Japan). Tissue CO content was measured using a method as previously described.41.Sunderman Jr, F.W. Downs J.R. Reid M.C. et al.Gas-chromatographic assay for heme oxygenase activity.Clin Chem. 1982; 28: 2026-2032PubMed Google Scholar Briefly, 1 ml of homogenized kidney tissue and 3 ml of phthalate solution (pH 4.01) was transferred into the vacuum tube and 0.5 ml of potassium ferricyanide (Sigma, St Louis, MO, USA) was added. The solution was mixed vigorously to release CO from the tissue homogenate at room temperature. Gas phase (1 ml) was taken into 10 ml vacuum tube, and released CO was measured by TRI lyzer. Results were expressed as CO (pmol) per mg tissue weight. Inbred male LEW (RT.1l) rats weighing 200–250 g were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN, USA). Animals were maintained in laminar flow cages in the animal facility at the University of Pittsburgh with a standard diet and water supplied ad libitum. All procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Pittsburgh and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals. Orthotopic KTx was performed using a previously described technique.27.Neto J.S. Nakao A. Kimizuka K. et al.Protection of transplant-induced renal ischemia-reperfusion injury with carbon monoxide.Am J Physiol Renal Physiol. 2004; 287: F979-F989Crossref PubMed Scopus (168) Google Scholar,42.Fisher B.L.S. Microvascular surgical techniques in research, with special reference to renal transplantation in the rat.Surgery. 1965; 58: 904-914Google Scholar,43.Nakao A. Neto J.S. Kanno S. et al.Protection against ischemia/reperfusion injury in cardiac and renal transplantation with carbon monoxide, biliverdin and both.Am J Transplant. 2005; 5: 282-291Crossref PubMed Scopus (190) Google Scholar In short, after intravenous heparinization (300 U), the donor left kidney was removed with the left renal artery and a short aortic segment, and the left renal vein with a patch of vena cava. The excised graft was flushed with and preserved in UW or CO-bubbled UW at 4 °C for 24 h. After recipient's left nephrectomy, the kidney graft was orthotopically transplanted into syngeneic recipient by end-to-side microvascular anastomoses between graft aorta and recipient infrarenal abdominal aorta, and between graft renal vein and recipient infrarenal vena cava with 10-0 suture. End-to-end ureteral anastomosis was performed using 10-0 suture. Recipient animals were given 20 mg prophylactic cefamandole nafate immediately after surgery and for 3 days postoperatively. The remaining right native kidney was removed 10 days after KTx for the experiments assessing renal functi
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