l -Arginine Supplementation Protects Against Hepatic Ischemia–Reperfusion Lesions in Rabbits

L-Arginine

Supplementation Protects Against Hepatic Ischemia–Reperfusion Lesions in Rabbits M.O. Taha, M.J. Simões, M.A. Haddad, R.C. Capelato, N. Budny, A.H. Matsumoto, P.C.M. Soares, W.M. Santos, G.D. Armeato, C.M. Araki, J.S.M. Gomes, K.G. Magalhães, I.L.S. Tersariol, H.P. Monteiro, I. Oliveira, Jr, A. Jurkiewicz, and A. Caricati-Neto ABSTRACT We evaluated the effects of a substrate in the biosynthesis of nitric oxide (NO)-L-arginine (LARG)-on hepatic lesions caused by ischemia/reperfusion (I/R) injury in rabbit livers. Rabbits were pretreated with LARG (150 mg/kg IV) or saline solution 0.9% (SS) before the hepatic I/R procedure. The effects of LARG on hepatic injury were evaluated before and after I/R. The warm hepatic I/R procedure produced profound acute liver injury, as indicated by elevated values of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as well as a high apoptotic cell count. All changes were attenuated by treatment with LARG before the hepatic I/R procedure. These results suggested that LARG produced protective effects on hepatic I/R lesions. This protective effect of LARG was probably associated with blocking generation of superoxide anions during the hepatic I/R procedure. ASOCONSTRICTION and microcirculatory disturbances induced by superoxide-mediated endothelial cell dysfunction have been considered one of the major causes of liver injury associated with ischemia and subsequent reperfusion. Preischemic or postischemic administration of antioxidants, prostanoid-modulating drugs, adenosine-regulating agents, and antiendothelin antibodies, all of which act as vasodilators, have been shown to attenuate hepatic damage.1–3 Nitric oxide (NO), an endothelium-derived relaxing factor with various beneficial biologic actions, is a therapeutic candidate to protect the liver against noxious insults. The effects of NO on ischemia/reperfusion (I/R) injury of the liver have been conflicting. Some studies have demonstrated detrimental results with NO augmentation,4 whereas others5,6 observed no important improvements by these treatments. Although Shiraishi et al7 and Koeppel et al8 described beneficial properties of NO, their observations were confined to the effects on hepatic blood flow or liver enzyme release. In the present study, we tested our hypothesis that NO enhancement attenuates vascular dysfunction, lessens neutrophil-related tissue injury, and reduces I/R injury of the liver. For this purpose, we tested an NO precursor, 9 L-arginine (LARG), in rabbits undergoing ischemia for 30 minutes followed by reperfusion for 120 minutes. We evaluated the LARG effects on the hepatic lesions caused by I/R using the serum levels of aspartate aminotransferase (AST),

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alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as well as by the severity of liver lesions adjudged by histologic methods. METHODS Animals The experiments were performed using 3-month-old, male New Zealand rabbits, weighing 2.5 to 3.0 kg obtained from our animal care center. The day–night cycle was constant at 12 hours. The animals had free access to tap water and pelletted chow. The study design was approved by our Animal Ethics Committee. From the Departments of Surgery (M.O.T., M.A.H., J.S.M.G., K.G.M.), Morphology (M.J.S., I.O.Jr.), Biochemistry (H.P.M.), and Pharmacology (M.A.H., J.S.M.G., K.G.M., A.J., A.C.-N.), Federal University of São Paulo, Escola Paulista de Medicina; the Department of Medicine (R.C.C., N.B., A.H.M., P.C.M.S., W.M.S., G.D.A., C.M.A.), Federal University of Grande Dourados (UFGD), Grande-Dourados-MS, Brazil; and the University of Mogi das Cruzws (I.L.S.T.), São Paulo, Brazil. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnológico (CNPq), Brazil. Address reprint requests to Murched Omar Taha, MD, PhD, Department of Surgery, Federal University of São Paulo, Escola Paulista de Medicina, Rua Botucatu, 740, CEP 04023-900, São Paulo-SP, Brazil. E-mail: [email protected] © 2009 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 816 – 819 (2009)

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Fig 1. Histograms representing serum AST, ALT, and LDH levels before (basal) and after hepatic ischemia (I) and reperfusion (R) in rabbits treated with saline solution 0.9% (SS) or 150 mg/kg L-arginine (LARG). Data, expressed in U/L, represent mean values ⫾ SEM of 6 experiments. *Significantly different from SS group (P ⬍ .05). The protocol was prepared in accordance with the National Institutes of Health guidelines. Rabbits were pretreated with the substrate of NO biosynthesis LARG (150 mg/kg IV) at 10 minutes before the hepatic I/R procedure. LARG was obtained from Sigma Chemical Co. (St. Louis, Mo). Warm hepatic I/R procedures were performed as reported.1 In brief, rabbits were anesthetized with ketamine (60 mg/kg IP) and xylazine (20 mg/kg IP). Laparotomy via a middle incision exposed the liver lobes. After surgical exposure of the portal vein, rabbits were injected with heparin (100 U/kg) via tail vein to prevent the formation of blood clots during the ischemia period. The portal vein and hepatic artery were occluded for 60 minutes with a metallic clamp to induce hepatic ischemia. Thereafter, the clamp was removed to allow blood to flow through the liver again (reperfusion) for 120 minutes. The rabbits undergoing I/R were treated intravenously with either saline solution 0.9% (SS) or LARG 150 mg/kg. Blood samples were collected from the vena cava before and after I/R, before rapidly excising the liver. Portions of liver tissue fixed in 10% neutralized formalin for histologic evaluation or snap frozen in liquid nitrogen were maintained at ⫺80°C until homogenization for the various biochemical assays.

evaluation of liver injury. Photomicrographs were taken with a digital camera under a microscope.

Statistical Analysis Data expressed as mean values ⫾ SEM, were evaluated with ANOVA and the Newman–Keuls tests for multiple comparisons among groups. A P value less than .05 was considered significant.

RESULTS Effect of Treatment With LARG on Serum AST, ALT, and LDH Levels

As shown in Figure 1, the hepatic I/R procedure caused a marked increase in serum AST, ALT, and LDH levels above baseline levels (AST ⫽ 27 ⫾ 3 U/L; ALT ⫽ 34 ⫾ 4 U/L; LDH ⫽ 326 ⫾ 5 U/L; n ⫽ 12). Prior injection of LARG markedly decreased serum AST, ALT, and LDH levels compared with SS animals (P ⬍ .05). It seemed that LARG treatment tended to exert an improved protection, but the differences were not significant at either time point. These findings were consistent with the histologic observations shown in Figure 2.

Serum Assays Serum levels of AST, ALT, and LDH were analyzed using a commercially available diagnostic kit (Catachem Inc., Bridgeport, Conn), and expressed as units per liter.

Histologic Studies Fixed liver specimens were embedded in paraffin, sectioned at 4 ␮m thickness, and stained with hematoxylin and eosin for the

Effects of Treatment With LARG on Hepatic Lesions Caused by I/R

Figure 2 shows, led to marked lesions in the central lobule with significant inflammatory infiltration among the SS group undergoing hepatic I/R. A significant reduction in lesions was observed in the livers of animals receiving LARG compared with animals of the SS group 2. After

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Fig 2. Photomicrograph of the effects of I/R on hepatic tissue from rabbits treated with SS (left) or LARG 150 mg/kg (right). Note that present of apoptosis in hepatocytes of SS-treated rabbits (left), but not in the LARG-treated rabbits (right).

starting reperfusion, we visualized a profound number of apoptotic cells with nuclear staining among animals with the hepatic I/R procedure (Fig 2), which was prevented by LARG. DISCUSSION

This study showed that 30 minutes of hepatic warm ischemia followed by 120 minutes of reperfusion caused severe liver injury, resulting in increased serum levels of AST, ALT, and LDH and histologic liver lesions, including apoptosis. In contrast, enhanced generation of NO by LARG alleviated the injury. Pre- and postischemic administration of these agents allowed 100% survival of the animals. Histologic alterations were markedly attenuated by LARG treatment. NO is synthesized by NO synthases (NOS) through LARG to the L-citrulline pathway.10 NOS are classified as 2 types: calcium-dependent constitutive NOS and calciumindependent inducible NOS.11 Constitutive NOS located in vascular endothelial cells produce an appropriate amount of NO in response to circulatory environments.12 Inducible NOS synthesize NO excessively when stimulated by certain cytokines or endotoxin.13 NO has various biologic actions, such as vasodilation (vascular smooth muscle relaxation) and inhibition of platelet aggregation. The mechanism for both effects involves increased cyclic guanosine monophosphate after activation of soluble guanylate cyclase by NO.14,15 NO also has inhibitory effects on the endotheliumneutrophil interaction, partially through downregulation of the expression of adhesion molecules both on endothelium

and neutrophils16,17 and through production of cytokines by inflammatory cells.18 In the liver, NO has been reported to produce hepatic sinusoidal dilation, thereby improving the microcirculation to this organ by altering the morphofunctional activity of fat-storing (Ito) cells.19 Some studies have suggested that decreased NO production by injured endothelial cells after attack by reactive oxygen species is among the important mechanisms responsible for organ injury after I/R.20 In this work, we observed that elevated NO levels by treatment with the NO precursor LARG produced protective effects on hepatic lesions caused by I/R lesions in rabbits. Some studies have also demonstrated that portal injection of LARG (10 mg/kg) induced a temporary burst of intrahepatic NO after reperfusion, mitigating injury to rat or porcine livers subjected to 60 minutes of ischemia followed by reperfusion.7 In contrast, using an isolated rat liver perfusion model, Ma et al4 reported that inhibition of NO production by N␻-nitro-Larginine methyl ester hydrochloride (L-NAME) attenuated liver injury after ischemia. However, Jaeschke et al5 showed no substantial effects of L-NAME treatment on rat liver ischemic injury. The disparate results between our study and the other reports might be due to differences in animal species, models, and agents tested; the data from our preclinical study in rabbits revealed protective effects of NO against liver I/R injury. Although NO levels were not directly determined in this study, we believe that LARG administration augmented NO production by sinusoidal endothelial cells. Mammalian

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vascular endothelial cells produce NO, L-citrulline, and water from LARG and thereafter oxygen. Little information has been available as to the quantity of NO production by cells after exogenous LARG administration under normal conditions.21,22 It has been proposed, however, that once the cells are injured, they take up LARG preferentially23 and produce NO depending on LARG availability.24 The mechanisms by which NO provided hepatoprotective effects against I/R injury were not determined in our study, but several possibilities can be considered. For example, amelioration of microcirculatory disturbances could be produced by the vasodilatory effect of NO. NO has been shown to regulate hepatic sinusoidal circulation through morphofunctional influences on Ito cells,19 rather than through a direct effect on portal or arterial vascular smooth muscle cells. Because NO has been shown to modulate other vasoactive substances, such as endothelin25 and prostanoids,26 NO-driven changes among these vasoactive substances might contribute, at least in part, to the improved hepatic microcirculation after reperfusion. NO could also exert hepatoprotective effects by inhibiting polymorphonuclear cell adherence to endothelium, by scavenging superoxides and by increasing energy metabolism.27–29 Although the precise mechanisms are not understood, favorable energy metabolism undoubtedly contributed to the better survival among the treated animals. In conclusion, we demonstrated marked hepatoprotective effects of LARG against severe I/R injury in rabbit livers. This agent attenuated liver injury, rescuing all animals from a critical insult. Although further studies are required to elucidate the exact mechanisms by which LARG prevents ischemic liver injury, supplementation with LARG seems to be a possible clinical strategy because its does not cause adverse hypotensive effects.

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