Reperfusion Liver Injury Induces Down-Regulation of eNOS and Up-Regulation of iNOS in Lung Tissues

Hepatic

Reperfusion Liver Injury Induces Down-Regulation of eNOS and Up-Regulation of iNOS in Lung Tissues H.I. Lin, S.J. Chou, D. Wang, N.H. Feng, E. Feng, and C.F. Chen ABSTRACT Objectives. Acute lung injury and inflammation can occur after hepatic ischemia/ reperfusion (I/R). Little is known regarding the possible role of nitric oxide synthase expression in this complex type of lung injury. Methods. Real-time polymerase chain reactions and immunohistochemistry were used to assess the mRNA and protein expression of eNOS and iNOS in lung tissue after I/R challenge to the liver. Ischemia was induced by clamping the hepatic artery and portal vein for 40 minutes. After flow was restored, the liver was reperfused for 300 minutes. Blood samples were collected to assay three inflammatory parameters: tumor necrosis factor (TNF)-␣, hydroxyl radicals, and NO. Lung lavage samples were assayed for protein and myeloperoxidase. The expression of eNOS and iNOS in lung tissues (n ⫽ 3) was also evaluated after I/R challenge to the liver. The iNOS inhibitor aminoguanidine was also tested in this I/R model. Results. Reperfusion of the liver produced increased blood concentrations of TNF, hydroxyl radicals, and NO (P ⬍ .001; n ⫽ 8). Bronchial lavage fluids showed higher levels of protein and myeloperoxidase in the I/R than in the sham-treated group (P ⬍ .01). eNOS expression was down-regulated and iNOS expression up-regulated in I/R lung tissues (n ⫽ 3). The iNOS inhibitor aminoguanidine (10 mg/kg) significantly attenuated the lung injury. Conclusions. I/R injury to the liver induced lung injury involving systemic inflammatory responses and iNOS expression. Administration of aminoguanidine significantly attenuated the injury, suggesting that iNOS expression may play a critical role in lung injury induced by I/R of the liver.

From the Departments of Medicine (H.I.L.) and Surgery (S.J.C.), Catholic Cardinal Tien Hospital, Taipei, Taiwan; Department of Medicine (D.W.), College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan; Department of Internal Medicine, Kaohsiung Military General Hospital (N.H.F.), Kaohsiung, Taiwan; Pharmacology Specialist Program (E.F.), University of Toronto, Toronto, Ontario, Canada; and Division of Gastroenterology (C.F.C.),

Department of Internal Medicine, Cheng Hsin General Hospital; Department of Healthcare Information and Management, School of Health, Ming Chuan University, Taipei, Taiwan. Supported by grants from NSC 93-Cheng Hsin-13. Address reprint requests to Dr Chao-Fuh Chen, Department of Medicine, Cheng Hsin General Hospital, No. 45, Cheng Hsin Street, Peitou, Taipei, Taiwan. E-mail: [email protected]

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0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.012

Transplantation Proceedings, 38, 2203–2206 (2006)

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SCHEMIA AND REPERFUSION (I/R) injury to the liver remains an important clinical problem during shock, liver surgery, and transplantation. Kupffer cell activation,1 generation of xanthine oxidase-mediated oxygen radicals,2,3 and translocation of lipolysaccharide4 are often encountered in reperfusion-related injury to the liver or gut. Oxygen free radicals, nitric oxide (NO), cytokines, and activated white cells have been found to be involved in I/Rrelated liver injury.5 They may initiate a systemic inflammatory response that contributes to distant lung injury.6,7 Key pathophysiological events that occur in the pulmonary microvasculature take place at the interface of the alveolar capillary membrane, where sequestered inflammatory cells and mediators (both local and humoral) induce oxidative and nitrosative stress, involving iNOS expression and peroxynitrite production. The key cellular participants in this lung injury are polymorphonuclear neutrophils (PMN) and microvascular endothelial cells involving the chemoattractant CIN1.8 Adherence of the neutrophils to the endothelium as a result of elevated ICAM1 expression9 creates a microenvironment in which PMN-derived oxidants, XO and iNOS expression, NO burst, and peroxynitrite formation can cause injury to the alveolar capillary membrane with consequent pulmonary dysfunction. The purpose of this study was to characterize the role of eNOS and iNOS mRNA and protein expression in the lung dysfunction resulting from I/R of the liver. We also sought to ascertain whether the iNOS inhibitor aminoguanidine (AG) attenuated this lung injury. MATERIALS AND METHODS Preparation of Animals Male Sprague-Dawley rats (300 to 350 g, pathogen-free) were anesthetized with pentobarbital (50 mg/kg intraperitoneally). Via a midline laparotomy the liver hilum was exposed to reveal the common hepatic artery and portal vein. Ischemia was induced by clamping the common hepatic artery and portal vein for 40 minutes. Thereafter, blood flow was restored, and the liver was reperfused for 90 minutes. Blood samples were obtained immediately before ischemia and after reperfusion to measure changes in serum lactic dehydrogenase, tumor necrosis factor TNF-␣, hydroxyl radical, and NO.5,10

Measurement of TNF-␣ TNF-␣ concentrations in blood samples were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Endogen, Woburn, Mass, USA).

LIN, WANG, FENG ET AL injection into the chromatographic system (ENO-20, Eicom Nox Analyzer, Kyoto, Japan), the samples were deproteinized by ultrafiltration through membranes with a molecular mass cutoff of 3000. The method had a sensitivity of 30 pmol for both anions, as little as 0.05 to 0.1 mL sample volume was required, and linearity was observed at up to 60 nmol for each anion.

Measurement of Protein and Myloperoxydase in Lung Lavage Fluid Lungs were lavaged with 5 mL of saline (three times, with 2, 2, and 1 ml) at the end of the experiment. Lavage samples were centrifuged (1300 rpm) at room temperature for 10 minutes. The concentration of protein in the supernate was determined by measuring the change in absorbance at 630 nm after reaction with bromocresol green.12 The concentration of myeloperoxidase in the lavage fluid was measured according to the manufacturer’s instructions using the MPO-EIA ELISA kit (Bioxytech).

RNA Isolation and Real-Time Polymerase Chain Reaction Isolation of mRNA from mesenteric blood vessel and small intestine, as well as mRNA reverse-transcription to cDNA and real-time polymerase chain reaction were performed according to the methods we have previously described.13

Immunohistochemistry Mesenteric blood vessels and small intestinal tissues were dissected after I/R challenge for immunochemical analysis of the protein expression of eNOS and iNOS. Frozen liver sections (5 ␮m) from the I/R and sham groups were first incubated with blocking reagent, then with the appropriate dilution of primary antibody (mouse anti-rat eNOS or anti-rat iNOS monoclonal antibody at a titer of 1:50; Chemicon MAb, 13421, Temecular, Calif, USA), and finally with an anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibody at a titer of 1:100. Sections were labeled and developed with HRP substrate solution and counterstained with a hematoxylin stain kit (PS003, Gene Research Laboratory, Taiwan).10

Experimental Design Animals were randomly divided into three groups. In the AG group (n ⫽ 7), rats received AG (10 mg/kg; Sigma) by intravenous bolus injection 30 minutes prior to clamping of the common hepatic artery and portal vein. In the control group (n ⫽ 8), rats were given no treatment except saline before clamping of the common hepatic artery and portal vein. Rats in the sham-operated group (n ⫽ 7) were prepared in the same manner as the control group (n ⫽ 7), but their vessels were not clamp. After I/R challenge, the lung tissue was separated for NO isoforms mRNA and IHC analysis, from three rats in each group.

Data Analysis Measurement of Methylguanidine Because the formation of methylguanidine (MG) is an indicator of hydroxyl radical production in the blood, we measured MG levels by spectrofluorometry (Jusco 821-FP, Hachioji, Japan).11

Data were expressed as mean values ⫾ SEM. Comparisons between pre-I/R and post-I/R were performed with Student paired t tests. Comparisons between post-I/R and AG intervention group user independent t tests. Values of P ⬍ .05 were considered statistically significant.

Measurement of NO by High-Performance Liquid Chromatography

RESULTS

High-performance liquid chromatography was used to measure nitrite and nitrate anions derived from NO in plasma. Before

We demonstrated that I/R of the liver induces distant lung injury, as evidenced by increased bronchoalveolar lavage

REPERFUSION LIVER INJURY

Fig 1. Lavage protein and myeloperoxidase (MPO) concentrations in the sham-operated and I/R groups and the effect of iNOS inhibitor AG. *P ⬍ .05, **P ⬍ .01 significantly different between sham and I/R group or intervention with AG group. ⫹P ⬍ .05 significantly different between I/R and intervention with AG group.

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stress, and activated inflammatory cells sequestered in the lung. Similar results on iNOS and eNOS have been reported by Virlos and coworkers,15 who observed differential expression of pulmonary NO synthase isoforms after intestinal ischemia-reperfusion. Liu and coworkers16 have demonstrated that the eNOS inhibitor L-NAME can aggravate the reperfusion lung injury. iNOS expression could induce a substantial and sustained release of NO, which could further react with the superoxide released by the sequestered white cells to form peroxynitrite and hydroxyl radicals, causing damage to the alveolar-capillary membranes. Terada and coworkers2 have demonstrated that reperfusion of the liver or intestine induced xanthine oxidase-mediated production of oxygen radicals. Therefore, xanthine oxidase and oxidants from inflammatory cells or other pathways are apparently all involved in the lung injury induced by liver reperfusion. Expression of cytokines, such as TNF-␣, may induce increased ICAM 1 expression in the lung,9 causing more PMNs to adhere to the lung tissues, thereby aggravating the distant lung injury. We have now demonstrated that the cytokine TNF-␣, hydroxyl radicals, NO, and LDH all increase after the reperfusion injury to the liver (Fig 2). These data suggest that the ischemic liver is a potential source of the inflammatory mediators that are associated with hepatic I/R-induced pulmonary injury. We conclude that I/R injury to the liver can induce lung injury involving systemic inflammatory responses and iNOS expression. Administration of aminoguanidine may significantly attenuate this injury, suggesting that iNOS expression may play a critical role in the lung injury induced by I/R of the liver.

protein and myeloperoxidase concentration, which are indicators of pulmonary PMN infiltration and vascular permeability (Fig 1). We have now shown that the iNOS inhibitor aminoguanidine significantly attenuated the lung injury and sequestration of white cells (Fig 1). The lung injury may be related to the expression of iNOS mRNA and protein. Indeed, we observed a marked increase in the mRNA expression of iNOS (6.4 ⫾ 1.1-fold increase) in the I/R-challenged group when compared with sham-operated group. In contrast, eNOS expression was down-regulated after I/R. Immunohistochemical analysis of iNOS protein in lung tissues revealed a marked increase in the expression of iNOS (8.2 ⫾ 2.2-fold increase) but not eNOS in the I/R group, when compared with the sham-treated group. DISCUSSION

In this study, the ischemic liver model was induced by clamping both the hepatic artery and portal vein; therefore, not only were Kupffer cells activated in the liver1 but the enteric system was also subject to translocation of bacteria and endotoxins as a result of the I/R challenge.3 Lipopolysaccharide and bacteria are important proximal mediators of the remote organ injury associated with I/R of the liver,14 which leads to systemic inflammatory responses and an ensuing increase in cytokines, oxidative stress, nitrosative

Fig 2. MG, NO, and TNF levels in the sham-operated and I/R groups and the effect of iNOS inhibitor AG. ***P ⬍ .001 significantly different between sham and I/R group or intervention with AG group. ⫹P ⬍ .05; ⫹⫹P ⬍ .01 significantly different between post-I/R and intervention with AG.

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LIN, WANG, FENG ET AL 9. Meyer K, Brown MF, Zibari G, et al: ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion: a clue to the mechanism of multiple organ failure. J Pediatr Surg 33:350, 1998 10. Lin HI, Wang D, Leu FJ, et al: Ischemia and reperfusion of liver induces eNOS and iNOS expression: effect of a NO donor and NOS inhibitor. Chinese J Physiol 47:121, 2004 11. Nakamura K, Ienaga K, Yokozawa T, et al: Production of methyl guanidine from creatinine via cretol by active oxygen species. Nephron 58:42, 1991 12. Doumas BT: Determination of serum albumin. Standard Methods Clin Chem 7:175, 1972 13. Chen CF, Leu FJ, Chiang KF, et al: Reperfusion but ischemia induces the expression of inducible nitric oxide synthase in rat liver. Gastroenterol J Taiwan 22:1, 2005 14. Souza DG, Vieira AT, Soares AC, et al: The essential role of the intestinal microbiota in facilitating acute inflammatory responses. J Immunol 173:4137, 2004 15. Virlos IT, Inglott FS, Williamson RC, et al: Differential expression of pulmonary nitric oxide synthase isoforms after intestinal ischemia-reperfusion. Hepatogastroenterology 50:31, 2003 16. Liu P, Xu B, Hock CE: Inhibition of nitric oxide synthesis by L-name exacerbates acute lung injury induced by hepatic ischemiareperfusion. Shock 16:211, 2001