Treatment with CO-RMs during cold storage improves renal function at reperfusion
2006; Elsevier BV; Volume: 69; Issue: 2 Linguagem: Inglês
10.1038/sj.ki.5000016
ISSN1523-1755
AutoresAshraf Sandouka, Barry Fuller, B. E. MANN, C.J. Green, Roberta Foresti, Roberto Motterlini,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoLow concentrations of carbon monoxide (CO) can protect tissues against ischemia–reperfusion (I–R) injury. We have recently identified a novel class of compounds, CO-releasing molecules (CO-RMs), which exert important pharmacological activities by carrying and delivering CO to biological systems. Here, we examined the possible beneficial effects of CO liberated from CO-RMs on the damage inflicted by cold storage and I–R in isolated perfused kidneys. Hemodynamic and biochemical parameters as well as mitochondrial respiration were measured in isolated perfused rabbit kidneys that were previously flushed with CO-RMs and stored at 4°C for 24 h. Two water-soluble CO-RMs were tested: (1) sodium boranocarbonate (CORM-A1), a boron-containing carbonate that releases CO at a slow rate, and (2) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a transition metal carbonyl that liberates CO very rapidly in solution. Kidneys flushed with Celsior solution supplemented with CO-RMs (50 μM) and stored at 4°C for 24 h displayed at reperfusion a significantly higher perfusion flow rate (PFR), glomerular filtration rate, and sodium and glucose reabsorption rates compared to control kidneys flushed with Celsior solution alone. Addition of 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, prevented the increase in PFR mediated by CO-RMs. The respiratory control index from kidney mitochondria treated with CO-RMs was also markedly increased. Notably, renal protection was lost when kidneys were flushed with Celsior containing an inactive compound (iCO-RM), which had been deliberately depleted of CO. CO-RMs are effective therapeutic agents that deliver CO during kidney cold preservation and can be used to ameliorate vascular activity, energy metabolism and renal function at reperfusion. Low concentrations of carbon monoxide (CO) can protect tissues against ischemia–reperfusion (I–R) injury. We have recently identified a novel class of compounds, CO-releasing molecules (CO-RMs), which exert important pharmacological activities by carrying and delivering CO to biological systems. Here, we examined the possible beneficial effects of CO liberated from CO-RMs on the damage inflicted by cold storage and I–R in isolated perfused kidneys. Hemodynamic and biochemical parameters as well as mitochondrial respiration were measured in isolated perfused rabbit kidneys that were previously flushed with CO-RMs and stored at 4°C for 24 h. Two water-soluble CO-RMs were tested: (1) sodium boranocarbonate (CORM-A1), a boron-containing carbonate that releases CO at a slow rate, and (2) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a transition metal carbonyl that liberates CO very rapidly in solution. Kidneys flushed with Celsior solution supplemented with CO-RMs (50 μM) and stored at 4°C for 24 h displayed at reperfusion a significantly higher perfusion flow rate (PFR), glomerular filtration rate, and sodium and glucose reabsorption rates compared to control kidneys flushed with Celsior solution alone. Addition of 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, prevented the increase in PFR mediated by CO-RMs. The respiratory control index from kidney mitochondria treated with CO-RMs was also markedly increased. Notably, renal protection was lost when kidneys were flushed with Celsior containing an inactive compound (iCO-RM), which had been deliberately depleted of CO. CO-RMs are effective therapeutic agents that deliver CO during kidney cold preservation and can be used to ameliorate vascular activity, energy metabolism and renal function at reperfusion. Kidney transplantation is the treatment of choice for end-stage renal disease.1.Salahudeen A.K. Cold ischemic injury of transplanted kidneys: new insights from experimental studies.Am J Physiol Renal Physiol. 2004; 287: F181-F187Crossref PubMed Scopus (142) Google Scholar Most kidneys prior to transplantation are exposed to a period of cold storage (CS), which can limit but not completely avoid tissue injury and graft dysfunction in transplanted patients.2.Perico N. Cattaneo D. Sayegh M.H. et al.Delayed graft function in kidney transplantation.Lancet. 2004; 364: 1814-1827Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar Therefore, new strategies for mitigating cold ischemic damage and novel therapies for successful kidney transplantations are required. CS procedures are widely used for preserving cadaveric kidneys prior to transplant; these techniques involve intravascular flushing of the isolated organ using a hypothermic solution followed by storage at low temperatures for the time required to transfer the graft to the surgery unit. In addition to the injury imposed by CS, kidneys are subjected to further damage at reperfusion when warm oxygenated blood (37°C) is reintroduced into the transplanted graft. The pathophysiological consequences of CS followed by warm reperfusion involve cellular edema and the generation of reactive oxygen species, which trigger acute inflammatory responses and promote apoptosis once the graft is transplanted.3.Burns A.T. Davies D.R. McLaren A.J. et al.Apoptosis in ischemia/reperfusion injury of human renal allografts.Transplantation. 1998; 66: 872-876Crossref PubMed Scopus (134) Google Scholar, 4.McLaren A.J. Friend P.J. Trends in organ preservation.Transplant Int. 2003; 16: 701-708Crossref PubMed Scopus (85) Google Scholar Mitochondria are a key contributor to cell survival, as mitochondrial respiration and oxidative phosphorylation are essential for keeping the adenosine 5′ triphosphate demands and restoring cellular energy after adenosine 5′ triphosphate depletion caused by ischemia.5.Jassem W. Fuggle S.V. Rela M. et al.The role of mitochondria in ischemia/reperfusion injury.Transplantation. 2002; 73: 493-499Crossref PubMed Scopus (201) Google Scholar Several approaches have been used to counteract the damaging mechanisms of CS-mediated injury and consequently ameliorate renal function at transplantation. Perhaps one of the most surprising and unforeseen strategies involves the use of carbon monoxide (CO), which has recently attracted attention as a fundamental cell signalling mediator and cytoprotective agent against apoptosis and ischemia–reperfusion (I–R) injury.6.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 (179) Google Scholar Low levels of CO are produced endogenously in mammalian tissues by heme oxygenase (HO), the first and rate-limiting step in heme catabolism.7.Maines M.D. The heme oxygenase system: a regulator of second messenger gases.Annu Rev Pharmacol Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2201) Google Scholar, 8.Foresti R. Motterlini R. The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis.Free Radical Res. 1999; 31: 459-475Crossref PubMed Scopus (240) Google Scholar Different types of HO enzymes have been characterized, including constitutive (HO-2) and inducible (HO-1) isoforms. HO-1 is a stress protein that possesses the peculiar feature of being finely upregulated by stimuli or pathological events that trigger oxidative and nitrosative stress.9.Motterlini R. Green C.J. Foresti R. et al.Regulation of heme oxygenase-1 by redox signals involving nitric oxide.Antiox Redox Signal. 2002; 4: 615-624Crossref PubMed Scopus (142) Google Scholar, 10.Foresti R. Green C.J. Motterlini R. et al.Generation of bile pigments by heme oxygenase: a refined cellular stratagem in response to stressful insults.Biochem Soc Symp. 2004; 71: 177-192Crossref PubMed Scopus (69) Google Scholar The induction of HO-1 and the consequent increase in endogenous CO production play important roles in vasorelaxation,11.Sammut I.A. Foresti R. Clark J.E. et al.Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1.Br J Pharmacol. 1998; 125: 1437-1444Crossref PubMed Scopus (213) Google Scholar, 12.Motterlini R. Gonzales A. Foresti R. et al.Heme oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertensive responses in vivo.Circ Res. 1998; 83: 568-577Crossref PubMed Scopus (246) Google Scholar inhibition of cell proliferation,13.Morita T. Mitsialis S.A. Koike H. et al.Carbon monoxide controls the proliferation of hypoxic vascular smooth muscle cells.J Biol Chem. 1997; 272: 32804-32809Crossref PubMed Scopus (294) Google Scholar, 14.Durante W. Schafer A.I. Carbon monoxide and vascular cell function [Review].Int J Mol Med. 1998; 2: 255-262PubMed Google Scholar blockade of apoptotic pathways,15.Zhang X. Shan P. Otterbein L.E. et al.Carbon monoxide inhibition of apoptosis during ischemia–reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3.J Biol Chem. 2003; 278: 1248-1258Crossref PubMed Scopus (248) Google Scholar suppression of inflammation,16.Otterbein L.E. Bach F.H. Alam J. et al.Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.Nat Med. 2000; 6: 422-428Crossref PubMed Scopus (1824) Google Scholar protection against organ rejection,17.Otterbein L.E. Zuckerbraun B.S. Haga M. et al.Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury.Nat Med. 2003; 9: 183-190Crossref PubMed Scopus (441) Google Scholar and I–R injury.18.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 (201) Google Scholar In essence, both the use of potent HO-1 inducers and administration of low doses of CO gas have been employed to evaluate and sustain a therapeutic role of CO in I–R injury in the kidney.18.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 (201) Google Scholar, 19.Sikorski E.M. Hock T. Hill-Kapturczak N. et al.The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury.Am J Physiol Renal Physiol. 2004; 286: F425-F441Crossref PubMed Scopus (211) Google Scholar, 20.Matsumoto M. Makino Y. Tanaka T. et al.Induction of renoprotective gene expression by cobalt ameliorates ischemic injury of the kidney in rats.J Am Soc Nephrol. 2003; 14: 1825-1832Crossref PubMed Scopus (219) Google Scholar, 21.Nath K.A. Balla G. Vercellotti G.M. et al.Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.J Clin Invest. 1992; 90: 267-270Crossref PubMed Scopus (601) Google Scholar, 22.Agarwal A. Kim Y. Matas A.J. et al.Gas-generating systems in acute renal allograft rejection in the rat.Transplantation. 1996; 61: 93-98Crossref PubMed Scopus (64) Google Scholar As a novel approach to deliver CO, our group has recently identified a class of compounds, termed CO-releasing molecules (CO-RMs), which are able to transport and release CO both in vivo and in vitro in a controllable manner under physiological conditions.23.Motterlini R. Clark J.E. Foresti R. et al.Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities.Circ Res. 2002; 90: E17-E24Crossref PubMed Google Scholar, 24.Motterlini R. Mann B.E. Johnson T.R. et al.Bioactivity and pharmacological actions of carbon monoxide-releasing molecules.Curr Pharm Des. 2003; 9: 2525-2539Crossref PubMed Scopus (224) Google Scholar Different types of CO-RMs have been characterized and their biological activities assessed. Two water-soluble CO-RMs have recently shown promising results in our studies: tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a metal carbonyl complex that rapidly liberates CO in physiological buffers,24.Motterlini R. Mann B.E. Johnson T.R. et al.Bioactivity and pharmacological actions of carbon monoxide-releasing molecules.Curr Pharm Des. 2003; 9: 2525-2539Crossref PubMed Scopus (224) Google Scholar, 25.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 and sodium boranocarbonate (CORM-A1), a newly identified generator of CO that does not contain a transition metal and liberates CO at a much slower rate under physiological conditions.26.Motterlini R. Sawle P. Bains S. et al.CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.FASEB J. 2005; 19: 284-286PubMed Google Scholar The specific effect of CO liberated from CO-RMs in modulating important physiological effects has been confirmed by the parallel use of specific inactive compounds (iCO-RMs), which do not liberate CO in the cellular environment.23.Motterlini R. Clark J.E. Foresti R. et al.Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities.Circ Res. 2002; 90: E17-E24Crossref PubMed Google Scholar, 25.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, 26.Motterlini R. Sawle P. Bains S. et al.CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.FASEB J. 2005; 19: 284-286PubMed Google Scholar Previous data from our own laboratory and results provided by collaborators support a critical role for CO-RMs in vasorelaxation,23.Motterlini R. Clark J.E. Foresti R. et al.Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities.Circ Res. 2002; 90: E17-E24Crossref PubMed Google Scholar, 26.Motterlini R. Sawle P. Bains S. et al.CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.FASEB J. 2005; 19: 284-286PubMed Google Scholar, 27.Foresti R. Hammad J. Clark J.E. et al.Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.Br J Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (263) Google Scholar suppression of inflammation,28.Sawle P. Foresti R. Mann B.E. et al.Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages.Br J Pharmacol. 2005; 145: 800-810Crossref PubMed Scopus (351) Google Scholar protection against hypoxia–reoxygenation, and oxidative stress as well as mitigation of I–R injury,25.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 allograft rejection,25.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 and myocardial infarction.29.Guo Y. Stein A.B. Wu W.J. et al.Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo.Am J Physiol Heart Circ Physiol. 2004; 286: H1649-H1653Crossref PubMed Scopus (202) Google Scholar, 30.Stein A.B. Guo Y. Tan W. et al.Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction.J Mol Cell Cardiol. 2005; 38: 127-134Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Thus, the hypothesis that low concentrations of CO released from CO-RMs could protect against cold ischemia-mediated injury and be used as a novel therapeutic strategy for organ preservation and transplantation is tantalizing. This study was designed to assess how cold preservation of kidneys in the presence of CO-RMs could affect renal biochemical and physiological functions at reperfusion using an ex vivo isolated kidney preparation. To decide upon the effective dose range to be used in the cold preservation protocols, the effect of 0, 10, 25 and 50 μM CORM-A1 was examined over a 45 min perfusion time on the isolated perfused kidney (Figure 1). Addition of 50 μM CORM-A1 significantly increased perfusion flow rate (PFR) compared to controls (Figure 1a). On the other hand, there was no detectable increase in PFR with 10 and 25 μM CORM-A1; notably, iCORM-A1 (50 μM), which does not release CO, was also totally ineffective (Figure 1b). CORM-A1, but not iCORM-A1, also significantly increased urine flow rates (UFR) (P<0.05) in a time- and concentration-dependent manner when compared to the control group (Figure 1c and d). Similarly, perfusion of isolated kidneys in the presence of CORM-A1 resulted in a marked concentration- and time-dependent increase in glomerular filtration rate (GFR), but no significant changes were observed with the negative control, iCORM-A1 (Figure 1f and e). Interestingly, another water-soluble CO carrier (CORM-3) did not cause any change in the renal hemodynamics of freshly isolated kidneys (data not shown). The rate and amount of CO liberated from CO-RMs were measured in the solutions used for kidney perfusion as well as organ preservation. We found that the rate of CO release by CORM-A1 in Krebs solution at 37°C directly correlates with the concentrations used (Figure 2a). Specifically, the calculated rates of CO release were 6.16±0.12, 14.50±0.66, and 30.16±1.33 nmol/h for 10, 25, and 50 μM CORM-A1, respectively. Predictably, iCORM-A1 did not release any detectable CO in the Krebs solution at 37°C (Figure 2a). Given the fact that 50 μM CO-RMs was chosen as the ideal concentration for preserving kidneys at low temperatures, we measured the kinetics of CO release from CORM-A1 and CORM-3 in Celsior solution at 4°C. As observed with Krebs solution, we found that the addition of iCO-RMs to deoxymyoglobin dissolved in Celsior solution at 4°C did not produce any detectable carbonmonoxy myoglobin over a 24 h period (data not shown). Addition of CORM-3 (50 μM) increased carbonmonoxy myoglobin formation over time, reaching a maximal level after 4 h; the rate of CO release in the first 2 h was calculated as 14.0±1.0 nmol/h (see Figure 2b). Interestingly, and in contrast to CORM-3, the increase in carbonmonoxy myoglobin after the addition of 50 μM CORM-A1 to the cold Celsior solution was slower and the calculated rate of CO release was only 1.39±0.05 nmol/h (see Figure 2b). Kidneys flushed in the presence of CORM-A1 and CORM-3 prior to 24 h CS in Celsior solution (CS+CO-RMs) produced a significantly higher (P<0.05) PFR on the isolated system at reperfusion (Figure 3a and b). In addition, the use of both CO-RMs during the flushing procedure of kidneys prior to CS significantly increased UFR (Figure 4a and b) and GFR (Figure 5a and b) (P<0.05). Interestingly, all the renal parameters evaluated (PFR, UFR, and GFR) were not affected when kidneys were flushed with Celsior solution supplemented with the negative controls (iCO-RMs), indicating that CO liberated from CO-RMs was directly involved in the improved renal function at reperfusion.Figure 4Effect of CO-RMs on urine flow rate in isolated kidneys after cold storage. Rabbit kidneys were initially flushed with Celsior solution alone or supplemented with (a) 50 μM CORM-A1 or (b) CORM-3 and then subjected to a 24 h cold storage (CS) (see Materials and Methods for details). Urine flow rate was then measured in isolated kidneys over 90 min after a period of initial equilibration. iCORM-A1 and iCORM-3, which do not release CO, were used as negative controls. Each line represents the mean±s.e.m. of n=6 for each group. *P<0.05 vs CS group.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of CO-RMs on glomerular filtration rate (GFR) in isolated kidneys after cold storage. Rabbit kidneys were initially flushed with Celsior solution alone or supplemented with (a) 50 μM CORM-A1 or (b) CORM-3 and then subjected to a 24 h cold storage (CS) (see Materials and Methods for details). GFR was then measured in isolated kidneys over 90 min after a period of initial equilibration. iCORM-A1 and iCORM-3, which do not release CO, were used as negative controls. Each line represents the mean±s.e.m. of n=6 for each group. *P<0.05 vs CS group.View Large Image Figure ViewerDownload (PPT) As mitochondrial function contributes significantly to the outcome of I–R injury in organ transplantation,5.Jassem W. Fuggle S.V. Rela M. et al.The role of mitochondria in ischemia/reperfusion injury.Transplantation. 2002; 73: 493-499Crossref PubMed Scopus (201) Google Scholar we assessed the influence of CO liberated from CO-RMs during CS on mitochondrial oxygen consumption and viability (Figure 6). Cold preservation (24 h) followed by 2 h reperfusion resulted in a significant decrease in the renal mitochondria respiratory control index (RCI) from 4 (control) to 2.5. Remarkably, the use of CO-RMs during CS resulted in a statistically significant (P<0.05) improvement of RCI values (RCI=4.6 with CORM-A1; RCI=4.5 with CORM-3). Figure 7 shows the effect of 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase,27.Foresti R. Hammad J. Clark J.E. et al.Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.Br J Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (263) Google Scholar on PFR in freshly isolated kidneys perfused with CORM-A1 (Figure 7a) and in kidneys subjected to CS+CORM-A1 and reperfusion (Figure 7b). In both conditions, the effect of CORM-A1 treatment on PFR was completely abolished by the presence of ODQ (30 μM). PFR was not affected at reperfusion when kidneys were flushed with Celsior containing ODQ alone (data not shown). A similar decrease in PFR by ODQ was observed in kidneys stored in the presence of CORM-3 (data not shown). As shown in Table 1, glucose (RGLU) and Na+ (RNa) reabsorption were found to be significantly increased in kidneys that were previously flushed with CORM-A1. Both RGLU and RNa levels were unchanged in kidneys treated with iCORM-A1. Notably, both iCORM-3 and iCORM-3 did not significantly affect RGLU and RNa levels (Table 1). The urinary release of gamma-glutamyltransferase (GGT), a marker of tubular injury, was increased at reperfusion after CS and treatment with CORM-A1 or CORM-3 (but not iCO-RMs) reduced these values by more than two-fold. Urinary nitrite was significantly reduced when CORM-3 was used, but was unchanged after treatment with CORM-A1.Table 1Effect of CO-RMs on tubular function and other biochemical parameters at reperfusion after cold storage (CS)%RGLU%RNaGGT (U/l)Nitrite (μM)% Protein leakageCS54.8±6.316.4±2.818.0±9.23.8±0.57.0±0.2CS+CORM-A1 (50 μM)72.7±3.9*29.3±1.9*8.7±2.13.4±0.47.7±0.3CS+iCORM-A1 (50 μM)54.3±7.018.1±2.830.0±12.83.4±0.67.6±1.1CS+CORM-3 (50 μM)69.8±6.925.3±2.86.4±2.72.2±0.4*7.3±0.5CS+iCORM-3 (50 μM)71.3±5.026.0±3.415.8±5.55.8±0.58.0±0.6RGLU=glucose reabsorption; RNa=sodium reabsorption; GGT=gamma-glutamyltransferase. [2]Isolated rabbit kidneys were flushed with Celsior solution (4°C) alone or Celsior supplemented with CORM-A1 or CORM-3. Kidneys were then stored at 4°C for 24 h and then reperfused ex vivo as discussed in the Materials and Methods section.*P<0.05 vs CS. Open table in a new tab RGLU=glucose reabsorption; RNa=sodium reabsorption; GGT=gamma-glutamyltransferase. [2]Isolated rabbit kidneys were flushed with Celsior solution (4°C) alone or Celsior supplemented with CORM-A1 or CORM-3. Kidneys were then stored at 4°C for 24 h and then reperfused ex vivo as discussed in the Materials and Methods section. *P<0.05 vs CS. CO-RMs are a novel class of bioactive agents that have been recently identified to substantiate the important biological function of CO in mammals.23.Motterlini R. Clark J.E. Foresti R. et al.Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities.Circ Res. 2002; 90: E17-E24Crossref PubMed Google Scholar, 24.Motterlini R. Mann B.E. Johnson T.R. et al.Bioactivity and pharmacological actions of carbon monoxide-releasing molecules.Curr Pharm Des. 2003; 9: 2525-2539Crossref PubMed Scopus (224) Google Scholar, 25.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 We have proposed that their pharmacological properties could be exploited for the therapeutic delivery of CO in the treatments of a variety of pathophysiological states that affect the cardiovascular and immune systems.25.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, 27.Foresti R. Hammad J. Clark J.E. et al.Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.Br J Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (263) Google Scholar, 28.Sawle P. Foresti R. Mann B.E. et al.Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages.Br J Pharmacol. 2005; 145: 800-810Crossref PubMed Scopus (351) Google Scholar, 31.Johnson T.R. Mann B.E. Clark J.E. et al.Metal carbonyls: a new class of pharmaceuticals?.Angew Chem Int Ed Engl. 2003; 42: 3722-3729Crossref PubMed Scopus (232) Google Scholar Here, we show that CO liberated from water-soluble CO-RMs exerts significant beneficial effects on renal vascular function of fresh isolated kidneys as well as kidneys stored at low temperatures (4°C) in Celsior solution, a clinical strategy commonly used to preserve organs for transplantation. The fact that CO-RMs were used only during the flushing procedure prior to CS indicates the feasibility of utilizing these CO carriers as adjuvants of preservation solutions to greatly limit the damage of donor organs. Impairment of renal blood flow during I–R plays a significant role in the exacerbation of tissue injury in a number of kidney diseases.32.Regan M.C. Young L.S. Geraghty J. et al.Regional renal blood flow in normal and disease states.Urol Res. 1995; 23: 1-10Crossref PubMed Scopus (33) Google Scholar It has been shown that a persistent reduction in renal blood flow can be attributed to a decreased GFR observed in renal allografts following an ischemic event.33.Alejandro V. Scandling Jr, J.D. Sibley R.K. et al.Mechanisms of filtration failure during postischemic injury of the human kidney. A study of the reperfused renal allograft.J Clin Invest. 1995; 95: 820-831Crossref PubMed Google Scholar In addition, intense vasoconstriction and endothelial damage are prominent features of CS-mediated injury.34.Wilhelm S.M. Simonson M.S. Robinson A.V. et al.Cold ischemia induces endothelin gene upregulation in the preserved kidney.J Surg Res. 1999; 85: 101-108Abstract Full Text PDF PubMed Scopus (20) Google Scholar In the present study, we initially found that freshly isolated kidneys perfused with CORM-A1, a boron-containing CO generator, resulted in a significant increase in PFR. Moreover, PFR markedly increased in reperfused isolated kidneys previously flushed with either CORM-A1 or CORM-3 and subjected to a 24 h CS procedure. The fact that iCO-RMs were ineffective clearly demonstrates that CO is directly responsible for the observed pharmacological effects. Since the perfusion pressure in our isolated model was kept constant (100 mmHg), we conclude that the increase in PFR by CO-RMs is mainly due to a decrease in vascular resistance. We also found that the beneficial vascular effects mediated by CORMs were lost in the presence of ODQ, a guanylate cyclase inhibitor. These results are supported by data showing that endogenous CO promotes renal vasodilatation in chronically hypoxic rats35.O'Donaughy T.L. Walker B.R. et al.Renal vasodilatory influence of endogenous carbon monoxide in chronically hypoxic rats.Am J Physiol Heart Circ Physiol. 2000; 279: H2908-H2915PubMed Google Scholar and are consistent with previous studies from our laboratory demonstrating that both CORM-3 and CORM-A1 exert vasorelaxation of precontracted aortas as well as systemic hypotension through stimulation of the cyclic 3′, 5′ guanosine monophosphate pathway.26.Motterlini R. Sawle P. Bains S. et al.CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.FASEB J. 2005; 19: 284-286PubMed Google Scholar, 27.Foresti R. Hammad J. Clark J.E. et al.Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.Br J Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (263) Google Scholar We cannot exclude a priori that other mechanisms, particularly activation of K+ channels36.Kaide J.I. Zhang F. Wei Y. et al.Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors.J Clin Invest. 2001; 107: 1163-1171Crossref PubMed Scopus (170) Google Scholar and modulation of the vasoconstrictor endothelin,37.Pollock D.M. Endothelin antagonists in the treatment of renal failure.Curr Opin Invest Drugs. 2001; 2: 513-520PubMed Google Scholar could participate in CO-mediated renal vasorelaxation as these two pathways have been shown to be potential targets for CO-RMs in cardiac and smooth muscle cells.25.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, 38.Stanford S.J. Walters M.J. Mitchell J.A. et al.Carbon monoxide inhibits endothelin-1 release by human pulmonary artery smooth muscle cells.Eur J Pharmacol. 2004; 486: 349-352Crossref PubMed Scopus (29) Google Scholar Thus, the present
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