Lang JD Jr, Teng X, Chumley P, Crawford JH, Isbell TS, Chacko BK, Liu Y, Jhala N, Crowe DR, Smith AB, Cross RC, Frenette L, Kelley EE, Wilhite DW, Hall CR, Page GP, Fallon MB, Bynon JS, Eckhoff DE, Patel RP. Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation. J Clin Invest. 2007; 117:2583–2591. (Reprinted with permission).
Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.
The procedure of liver transplantation is inevitably accompanied by some degree of ischemia-reperfusion injury (IRI). IRI accounts for up to 10% of early graft failure and can contribute to the subsequent development of both acute and chronic rejection.1 Preexisting morbidity and successive insults including brain death, cold storage, warm ischemia and reperfusion result in cumulative damage to the cadaveric graft. Endogenous danger signals such as high-mobility group box 1 (HMGB1) combine with bacterial endotoxin translocated from the gut to signal through Toll-like receptors and activate an innate immune response, with production of proinflammatory cytokines and chemokines. Complement activation, endothelial damage, and upregulation of vascular adhesion molecules are accompanied by leucocyte infiltration, derangement of cellular metabolism, apoptosis and necrosis of parenchymal cells and further impairment of organ function.2
Studies of the role of nitric oxide (NO) in liver injury have yielded seemingly paradoxical results, with NO being found to either mediate or prevent liver damage.3 In part, these different outcomes depend upon the cellular source and amount of NO production. NO is generated within the liver by two of the three isoforms of NO synthase, constitutively expressed endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS). In general, low-level NO production by eNOS in the hepatic vasculature prevents endothelial dysfunction, platelet adhesion and neutrophil accumulation, maintains perfusion of the liver and protects against hepatic damage caused by lipopolysaccharide, cytokines or toxins including CCl4, paracetamol and ethanol.3 Conversely, upregulation of iNOS during conditions such as immune-mediated hepatitis, endotoxemia, hemorrhagic shock or IRI can result in the generation of large amounts of NO by multiple cell types including Kupffer cells and hepatocytes, and the potentiation of liver injury through enhanced production of TNF-α and IL-6.4, 5 Nevertheless, administration of pharmacological doses of NO donors is mostly associated with protection from IRI.3, 6
Inhaled nitric oxide (iNO) is a well-established agent in the therapy of neonatal hypoxemia and pulmonary hypertension.7 In this setting, iNO reduces pulmonary vascular resistance without appreciably affecting systemic hemodynamic parameters. The majority of the nitric oxide reaching the systemic circulation after inhalation is rapidly scavenged by binding to oxyhemoglobin, accounting for the lack of systemic vasodilatation. However, a proportion can form adducts with protein thiol groups, yielding compounds such as S-nitrosoalbumin and S-nitrosohemoglobin, or be oxidized in the blood to nitrite. These reactions allow NO to be carried to the periphery in a stable, bioavailable form, and released or regenerated there in response to the lower environmental oxygen tension. Documented extrapulmonary effects of iNO include inhibition of platelet and leucocyte adhesion, and experimental administration of iNO in animal models has resulted in protection against mesenteric ischemia and reduced infarct size with preservation of myocardial function after coronary occlusion.7
In this study by Lang and colleagues, recovery of liver function following orthotopic liver transplantation was more rapid in patients who received iNO intraoperatively than in those treated with placebo. No differences in inflammatory markers were detected between groups at one hour post-reperfusion, but hepatocyte apoptosis was attenuated in patients receiving iNO. NO can prevent apoptosis by a variety of mechanisms. NO-stimulated cGMP formation blocks caspase activation, whereas NO can directly inhibit caspase activity by s-nitrosylation of cysteine residues at the active site.3 NO activates mitochondrial ATP-sensitive potassium channels, stabilizing mitochondrial function and preventing release of cytochrome C.8, 9 Upregulation of Bcl-2, HSP70 and Hemoxygenase-1 may also contribute to NO-mediated cytoprotection.3 While the sampling time may have been too early to detect the full impact of iNO treatment on liver inflammation, it is also possible that the maximum benefit of NO therapy in reducing inflammation and liver damage in the peritransplant period would be achieved by commencing treatment earlier, either by treating the prospective organ donor with iNO, or by adding a source of NO to the preservation solution. As an example of this latter approach, addition of the NO donor glyceryl trinitrate to a commercial preservation solution significantly improved and extended cardiac preservation in a mouse model.10 iNO has recently been shown to up-regulate glucocorticoid receptors in lung, liver and kidney and to potentiate the anti-inflammatory effects of cortocosteroids in endotoxemia,11 suggesting that treatment with iNO or other NO sources could also synergise with steroids to reduce organ damage during transplantation. Further studies will be important to optimize the timing of delivery of these agents, and to determine whether their use can extend acceptable ischemia times or improve outcomes in the recipients of organs from marginal donors.