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Urinary trypsin inhibitor reduces inflammatory response in kidney induced by Lipopolysaccharide
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 4, 315–320. 2007 DOI: 10.1263/jbb.104.315
© 2007, The Society for Biotechnology, Japan
Urinary Trypsin Inhibitor Reduces Inflammatory Response in Kidney Induced by Lipopolysaccharide Masaaki Ueki,1* Satoshi Taie,1 Kousuke Chujo,1 Takehiko Asaga,1 Yasuyuki Iwanaga,1 Junichiro Ono,1 and Nobuhiro Maekawa1 Department of Anesthesiology & Emergency Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan1 Received 11 June 2007/Accepted 18 July 2007
Human urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used in Japan as a drug for patients with acute inflammatory disorders such as septic shock and pancreatitis. Lipopolysaccharide (LPS) triggers the sepsis syndrome by activating monocytes to produce proinflammatory cytokines, including tumor necrosis factor alpha (TNFα), which potently stimulate the activation of neutrophils. The inhibitory mechanism of UTI on the systemic inflammatory response induced by the intraperitoneal injection of LPS in the kidney is unclear. This study was undertaken to examine the inhibitory effects of UTI on renal injury associated with the systemic inflammatory response induced by LPS stimulation, with emphasis on systemic TNFα and the activation of neutrophils in rat kidney. The systemic inflammatory response syndrome was induced by LPS treatment. Serum and renal TNFα, renal cytokine-induced neutrophil chemoattractant-1 (CINC-1) and myeloperoxidase (MPO) levels, as well as renal function after LPS stimulation, were evaluated. UTI (50,000 U/kg) inhibited LPS-induced increases in the serum and renal tissue levels of TNFα, as well as the renal tissue levels of CINC-1 and MPO after LPS stimulation. UTI (50,000 U/kg) also inhibited the production of serum TNFα associated with the systemic inflammatory response syndrome induced by LPS stimulation, thereby attenuating neutrophil infiltration into renal tissues and subsequent neutrophil-mediated renal injury. These findings may have important implications in understanding the biologic functions of UTI. UTI may prove useful in protecting against acute renal injury associated with a systemic inflammatory response. [Key words: urinary trypsin inhibitor, lipopolysaccharide, neutrophil, acute renal dysfunction, cytokine-induced neutrophil chemoattractant-1 (CINC-1), tumor necrosis factor alpha]
hibits the enhanced production of proinflammatory molecules, such as TNFα (8), which are induced by LPS in vitro, the detailed mechanism underlying this inhibitory effect of UTI on systemic inflammatory response induced by LPS in the kidney remains to be elucidated. Therefore, in this study, we examined the inhibitory effects of UTI on renal injury associated with systemic inflammatory response induced by LPS stimulation, with emphasis on systemic TNFα and the activation of neutrophils in rat kidney.
Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria, is one of the major toxins that initiate the cascade of pathophysiological reactions called endotoxin shock (1). LPS triggers the sepsis syndrome by activating monocytes to produce proinflammatory cytokines, including tumor necrosis factor alpha (TNFα) (2). During sepsis, a broad array of humoral mediators including activated neutrophils are released into the systemic circulation (3). The overwhelming activation of neutrophils is known to elicit tissue damage by releasing inflammatory mediators such as neutrophil elastase and superoxide-derived free radicals (4, 5). Thus, the agents that inhibit the production of TNFα or the activation of neutrophils might prevent or attenuate tissue injury induced by LPS. Urinary trypsin inhibitor (UTI) is a multivalent Kunitztype serine protease inhibitor that is found in human urine and blood (6). UTI mainly inhibits proteases including α-chymotrypsin, plasmin, cathepsin G, and neutrophil elastase (7). UTI has been widely used as a renal protective drug in Japan for patients with septic shock and ischemia. Although UTI in-
MATERIALS AND METHODS UTI was a generous gift from Mochida Pharmaceutical (Tokyo). One unit of UTI is defined as the amount of UTI that inhibits the activity of 2 µg of trypsin and is the equivalent to 0.4 µg of UTI. LPS (Escherichia coli, serotype 0127:B8) was purchased from Sigma (St. Louis, MO, USA). Animal preparation and treatments All surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Kagawa University Faculty of Medicine. Rats were fasted for 12 h before surgery. Male Wistar rats weighing 220 to 270 g were obtained from Clea Japan (Osaka). The systemic inflammatory response syndrome was induced by
* Corresponding author. e-mail: [email protected] phone: +81-(0)87-891-2223 fax: +81-(0)87-891-2224 315
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LPS treatment. Animals were injected intraperitoneally with LPS (20 mg/kg body weight) dissolved in 1 ml of sterile saline. The experimental groups were divided into four. Sham groups Sham-operated control animals were injected with the same volume of the vehicle. UTI sham groups UTI sham animals (without LPS treatment) were injected intravenously with UTI (50,000 U/kg body weight). Yamaguchi et al. (9) reported that pretreatment with UTI at 50,000 U/kg but not 5000 U/kg significantly decreases serum CINC-1 levels in liver ischemia/perfusion. Thus, we decided that the present dose (50,000 U/kg) would have an optimal effect, which is necessary for inhibiting TNFα production associated with a systemic inflammatory response induced by the intraperitoneal injection of LPS. LPS groups LPS treatment group animals were injected with LPS. UTI + LPS groups UTI +LPS-treated animals were injected intravenously with UTI (50,000 U/kg) immediately after the injection of LPS. After the treatment, the rats were returned to their cages and provided with water ad libitum. Under anesthesia with sodium pentobarbital (50 mg/kg; Abbott Laboratories, North Chicago, IL, USA), a kidney was removed with infiltrating anesthesia of 1 ml lidocaine hydrochloride (AstraZeneca, Wilmington, DE, USA) at each specified time point (0–6 h). Enzyme immunoassays for serum TNFα, renal TNFα, cytokine-induced neutrophil chemoattractant-1 (CINC-1) and myeloperoxidase (MPO) levels TNFα was used as a proinflammatory cytokine. The concentrations of serum and renal TNFα were determined using an endogen ELISA kit (Pierce Biotechnology, Rockford, IL, USA). The renal CINC-1 serves as a chemotactic factor for the activation of neutrophils and chemotaxis to the injury site. The CINC-1 concentration in the rat kidney was determined by ELISA using a rat IL-8 assay kit (Panapharm Laboratories, Kumamoto). The renal MPO level was used as an indicator of neutrophil infiltration. The MPO level in the kidneys was determined using an ELISA kit (Oxis International, Foster City, CA, USA). At specified time points after LPS stimulation, the kidney was removed and homogenized with a homogenizer (Polytron PT10-35; Kinematika, Luzern, Switzerland) using 0.1 mol/l phosphate buffer (pH 7.4) containing 0.05% (weight/volume) sodium azide at 4°C. Homogenates were then sonicated for 20 s and centrifuged (2000×g for 10 min at 4°C). The supernatants were kept at −80°C until the measurement of the cytokine and chemokines concentrations. The concentrations of renal TNFα, CINC-1 and MPO in the supernatant were determined according to the manufacturer’s instructions and expressed as picograms per gram of tissue, picograms per gram of tissue, and nanogram per gram of tissue, respectively. Measurement of biochemical parameters At 6 h after reperfusion, a 3-ml blood sample was collected from the rats via the femoral artery. The samples were centrifuged (2000×g for 3 min) to separate the serum, and the plasma supernatant was stored at −80°C until the assays were performed. The blood urea nitrogen (BUN) and creatinine levels were measured (Special Reference Laboratories, Tokyo), and used as indicators of renal dysfunction. Histologic evaluation At 6 h after LPS stimulation, the kidney was removed, placed in formalin, and embedded in wax according to standard protocol. Five micrometer sections were cut and stained with hematoxylin and eosin for the light microscopy studies. A person who was blind to the experimental conditions counted the number of neutrophils per field under high-power (×400) and recorded the average of 10 fields for each sample (10). Statistical analysis All values shown in the text and figures are expressed as mean ±standard deviation (SD). Statistical comparisons were performed using the nonparametric Mann–Whitney U test for uncorrelated pairs. A p-value <0.05 was considered statistically significant.
RESULTS UTI decreases serum TNFα level after LPS stimulation The intraperitoneal injection of LPS triggered an inflammatory response characterized by an increase in serum TNFα level. As shown in Fig. 1a, the serum TNFα level in the LPS-treated rats increased markedly at 1 h after LPS stimulation and then gradually decreased. When UTI was administered at a dose of 50,000 U/kg, the serum TNFα level in the UTI+LPS-treated rats significantly decreased (p=0.004) in comparison with that in the LPS-treated rats at 1 h after LPS stimulation (Fig. 1b). UTI reduces expressions of renal TNFα, CINC-1 and myeloperoxidase after LPS stimulation To investigate the role of UTI in the expressions of proinflammatory cytokines and chemokines associated with the inflammatory response induced by LPS, we compared the renal levels of TNFα, CINC-1 and MPO after LPS stimulation. As shown in Fig. 2a, the renal TNFα level increased markedly at 3 h after LPS stimulation and then gradually decreased. The renal TNFα level in the UTI + LPS-treated rats significantly
FIG. 1. (a) Changes in serum TNFα concentration after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in serum TNFα concentration at 1 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean ± SD (n = 8).
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FIG. 2. (a) Changes in renal TNFα levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal TNFα levels at 3 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n =8).
FIG. 3. (a) Changes in renal CINC-1 levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal CINC-1 levels at 6 h after LPS stimulation. # p<0.05 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n = 8).
(p = 0.0017) decreased in comparison with that in the LPStreated rats at 3 h after LPS stimulation (Fig. 2b). As shown in Fig. 3a, the renal CINC-1 level increased markedly at 6 h after LPS stimulation. The renal CINC-1 level in the UTI + LPS-treated rats significantly (p = 0.0127) decreased in comparison with that in the LPS-treated rats at 6 h after LPS stimulation (Fig. 3b). As shown in Fig. 4a, the renal MPO level increased markedly at 6 h after LPS stimulation. The renal MPO level in the UTI+LPS-treated rats significantly (p =0.004) decreased in comparison with that in the LPS-treated rats at 6 h after LPS stimulation (Fig. 4b). UTI protects against LPS-induced acute renal injury The serum BUN and creatinine concentrations in the LPStreated rats before LPS stimulation were 19.1 ± 3.7 mg/dl and 0.27 ± 0.03 mg/dl, respectively. At 6 h after LPS stimulation, the serum BUN and creatinine concentrations in the LPS-treated rats were 37.0 ± 3.4 mg/dl and 0.54 ± 0.11 mg/dl, respectively. In the LPS-treated rats, the renal injury associated with a systemic inflammatory response induced by LPS injection was minimal. The serum BUN and creatinine con-
centrations in the UTI + LPS-treated rats were significantly (p = 0.0046, p= 0.0011) lower than those in the LPS-treated rats at 6 h after LPS stimulation (Table 1). Effects of UTI on renal histopathology induced by LPS stimulation Histopathological examination revealed mild neutrophil infiltration in the kidneys of the LPS-treated rats (Fig. 5). Furthermore, we performed morphometric analysis to quantitate the number of neutrophils in the kidney. We examined the effects of UTI on neutrophil infiltration in the kidney after LPS stimulation. Neutrophil infiltration in the UTI + LPS-treated rats was significantly (p < 0.0001) lower than that in the LPS-treated rats (Table 2). DISCUSSION In this study, we demonstrated that UTI inhibited the increases in serum and renal TNFα, renal CINC-1 and MPO levels after LPS stimulation and subsequent renal injury associated with a systemic inflammatory response induced by intraperitoneal injection of LPS. Sepsis is a leading cause of acute renal failure (11, 12),
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FIG. 4. (a) Changes in renal MPO levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal MPO levels at 6 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n =8).
but the pathogenesis of sepsis and the associated end-organ failure are highly complex, involving multiple inflammatory mediators. During sepsis, a broad array of humoral mediators including cytokines are released into the systemic circulation (3). Among the various cytokines, TNFα, which is released early into the circulation following LPS stimulation, has been shown to be a key early mediator of sepsis (13, 14). Another study has suggested that LPS causes damage to various tissues, including the kidney, by promoting tissue infiltration with neutrophils (15). LPS and other bacterial products are potent stimulators of polymorphonuclear leukocytes in vitro (16, 17). Activated neutrophils release various kinds of mediators, including proteases and oxygen radicals (18). A protease-antiprotease imbalance has been reported to be involved in a variety of inflammatory diseases (19). Indeed, neutrophil elastase and cathepsin G-deficient mice have been shown to be resistant to the lethal effects of LPS (20). In this study, we first demonstrated that UTI inhibited the production of both serum and renal TNFα in a rat LPS stim-
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FIG. 5. Histological findings in the groups. (a) Sham group; (b) LPS group; (c) UTI + LPS group. Histological examination of the kidney was performed at 6 h after LPS stimulation. Neutrophil infiltration is indicated by the arrow in panel b. Original magnification, ×200. Bars: 200 µm.
ulation model. Aosasa et al. (8) previously reported that UTI inhibited TNFα production in LPS-stimulated monocytes. In his study, UTI did not inhibit the TNFα mRNA expression or suppress the NFκB level. The precise mechanisms by which UTI inhibited LPS-stimulated TNFα production remain unclear at present. Molor-Erdene et al. (21) reported that UTI might inhibit the LPS-induced production of TNFα by inhibiting the activation of the human extracellular signal-regulated protein kinase 1/2 and early growth response factor-1 pathway in monocytes. TNFα can induce IL-8 gene expression in blood monocytes and other cells (22), leading to the secretion of IL-8 (23) and tissue damage via the activation and infiltration of neutrophils, and resulting in the release of lysozomal enzymes and superoxide anions by neutrophils (24). Maehara et al. (25) reported that UTI down regulated the IL-8 gene expression induced by LPS in HL-60 cells. Based on the biologic activity and peptide sequences of rat CINC-1, rat CINC-1 is a member of the IL-8 family (26). We measured
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TABLE 1. Renal function data BUN Creatinine (mg/dl) (mg/dl) Sham 21.5 ± 0.9 0.27 ± 0.02 UTI sham 19.8 ± 2.2 0.26 ± 0.04 0.54 ± 0.03a,b LPS 37.0 ± 3.4a,b UTI+ LPS 29.5 ± 5.9b,c,d 0.34 ± 0.06b,c,d BUN Blood urea nitrogen. Data are expressed as mean ± SD (n = 8). a p<0.01 compared with the sham group. b p<0.01 compared with the UTI sham group. c p<0.01 compared with the LPS group. d p<0.05 compared with the sham group. Group
TABLE 2. Neutrophils in kidney Average number of neutrophils/field Sham 0.22 ± 0.42 UTI sham 0.2 ± 0.4 LPS 1.9 ± 0.8a,b UTI+ LPS 1.0 ± 0.6a,b,c The number of neutrophils per field under high-power magnification was counted in kidney samples obtained 6 h after LPS stimulation. Data are expressed as mean ± SD (n = 4). a p<0.01 compared with the sham group. b p<0.01 compared with the UTI sham group. c p<0.01 compared with the LPS group. Group
the CINC-1 levels instead of the IL-8 levels in the rat inflammatory response model. In addition, in our study, UTI inhibited the production of renal CINC-1 in the rat LPS stimulation models. These experimental results have confirmed that UTI inhibits the LPS-induced IL-8 gene expression even in vivo. The MPO assay is widely used to quantify the number of neutrophils in a tissue and the MPO level serves as an index of inflammation because MPO is an enzyme that is released mainly from neutrophils (27). We used the MPO level as an index of the renal accumulation of activated neutrophils. Because rat CINC-1 activates the infiltration of neutrophils in tissue, the renal MPO level in the UTI + LPS-treated rats was significantly lower than that in the LPS-treated rats at 6 h after LPS stimulation. It is considered that the MPO level was inhibited because UTI depressed the CINC-1 level. UTI mainly inhibits proteases including α-chymotrypsin, plasmin, cathepsin G, and leukocyte elastase (7). UTI is synthesized from the inter-α-trypsin inhibitor through proteolytic cleavage by neutrophil elastase at the inflammation site (28). UTI participates as a component of the host defense mechanisms at the inflammation site. UTI has been widely used in Japan as a drug for patients with septic shock and pancreatitis. UTI had been used particularly for renal protection, but the molecular mechanism was unclear. Therefore, in this study, we examined the effects of UTI on renal injury associated with a systemic inflammatory response induced by LPS stimulation. In LPS-treated UTI-deficient mice, kidney damage, including neutrophil infiltration, was more prominent than in LPS-treated wild-type mice (29). These results suggest that UTI protects against organ damage associated with LPS possibly via the inhibition of neutrophil sequestration at the inflammation site.
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In this study, 50,000 U/kg of UTI was used. This dosage is 10 times higher than the therapeutic dosage of UTI used in the clinical setting. The UTI concentration is increased to 150 U/ml after the administration of 5000 U/kg of UTI (30). Molor-Erdene et al. (21) reported that although 100 U/ml of UTI (about 5000 U/kg of UTI) inhibited TNFα production in LPS-stimulated monocytes in vitro, 50,000 U/kg of UTI is needed to inhibit the lung TNFα production associated with the inflammatory response in rats administered with LPS. Yamaguchi et al. (9) reported that the pretreatment with UTI at 50,000 U/kg but not 5000 U/kg significantly decreased serum CINC-1 levels after liver ischemia/perfusion. Ito et al. (31) also reported that 50,000 U/kg of UTI inhibited the oleic acid-induced acute lung injury in rats via the inhibition of activated leukocytes. We chose the dosage of UTI according to the optimal dosage that was required to inhibit serum TNFα in their paper. We also previously reported that 50,000 U/kg of UTI improved energy metabolism during ischemia (32) and prevented hemorrhagic shock (33) in rat kidneys. Why such a large dose of UTI is necessary to inhibit LPS-induced cytokine and chemokine production in rats is unclear at present, but structural differences between human and rat UTIs might explain the decreased sensitivity of rats to human UTI (34). In this study, 50,000 U/kg of UTI inhibited the production of serum TNFα associated with a systemic inflammatory response syndrome induced by LPS stimulation, thereby attenuating neutrophil infiltration into renal tissues and subsequent neutrophil-mediated renal injury. However, further studies are required to evaluate the optimal dosage of UTI in cases of renal systemic inflammatory response induced by LPS stimulation. ACKNOWLEDGMENTS We are grateful to Mochida Pharmaceutical (Tokyo) for supplying the UTI used in this study.
REFERENCES 1. Michie, H. R., Manogue, K. R., Spriggs, D. R., Revhaug, A., O’Dwyer, S., Dinarello, C. A., Cerami, A., Wolff, S. M., and Wilmore, D. W.: Detection of circulating tumor necrosis factor after endotoxin administration. N. Engl. J. Med., 318, 1481–1486 (1988). 2. Parrillo, J. E.: Pathogenetic mechanisms of septic shock. N. Engl. J. Med., 328, 1471–1477 (1993). 3. De Vriese, A. S.: Prevention and treatment of acute renal failure in sepsis. J. Am. Soc. Nephrol., 14, 792–805 (2003). 4. Cohen, J.: The immunopathogenesis of sepsis. Nature, 420, 885–891 (2002). 5. Brown, K. A., Brain, S. D., Pearson, J. D., Edgeworth, J. D., Lewis, S. M., and Treacher, D. F.: Neutrophils in development of multiple organ failure in sepsis. Lancet, 368, 157–169 (2006). 6. Jonsson-Berling, B. M., Ohlsson, K., and Rosengren, M.: Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine. Biol. Chem. Hoppe-Seyler, 370, 1157–1161 (1989). 7. Morishita, H., Yamakawa, T., Matsusue, T., Kusuyama, T., Sameshima-Aruga, R., Hirose, J., Nii, A., Miura, T., Isaji, M., Horisawa-Nakano, R., and other 13 authors: Novel factor Xa and plasma kallikrein inhibitory-activities of
320
8.
9.
10.
11.
12.
13. 14.
15. 16.
17.
18. 19.
20. 21.
UEKI ET AL.
the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. Thromb. Res., 73, 193–204 (1994). Aosasa, S., Ono, S., Mochizuki, H., Tsujimoto, H., Ueno, C., and Matsumoto, A.: Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes. Shock, 15, 101–105 (2001). Yamaguchi, Y., Ohshiro, H., Nagao, Y., Odawara, K., Okabe, K., Hidaka, H., Ishihara, K., Uchino, S., Furuhashi, T., Yamada, S., Mori, K., and Ogawa, M.: Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion. J. Surg. Res., 94, 107– 115 (2000). Guo, R., Wnag, Y., Minto, A. W., Quigg, R. J., and Cunningham, P. N.: Acute renal failure in endotoxemia is dependent on caspase activation. J. Am. Soc. Nephrol., 15, 3093– 3102 (2004). Groeneveld, A. B., Tran, D. D., van der Meulen, J., Nauta, J. J., and Thijs, L. G.: Acute renal failure in the medical intensive care unit: predisposing, complicating factors and outcome. Nephron, 59, 602–610 (1991). Neveu, H., Kleinknecht, D., Brivet, F., Loirat, P., and Landais, P.: Prognostic factors in acute renal failure due to sepsis. Results of a prospective multicentre study. The French Study Group on Acute Renal Failure. Nephrol. Dial. Transplant., 11, 293–299 (1996). Beutler, B. A., Milsark, I. W., and Cerami, A.: Cachectin/ tumor necrosis factor: production, distribution, and metabolic fate in vivo. J. Immunol., 135, 3972–3977 (1985). Van Deventer, S. J., Buller, H. R., ten Cate, J. W., Aarden, L. A., Hack, C. E., and Sturk, A.: Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood, 76, 2520– 2526 (1990). Linas, S. L., Whittenburg, D., and Repine, J. E.: Role of neutrophil derived oxidants and elastase in lipopolysaccharidemediated renal injury. Kidney Int., 39, 618–623 (1991). Guthrie, L. A., McPhail, C., Henson, P. M., and Johnston, R. B., Jr.: Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme. J. Exp. Med., 160, 1656–1671 (1984). Smedly, L. A., Tonnesen, M. G., Sandhaus, R. A., Haslett, C., Guthrie, L. A., Johnston, R. B., Jr., Henson, P. M., and Worthen, G. S.: Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J. Clin. Invest., 77, 1233–1243 (1986). Weiss, S. J.: Tissue destruction by neutrophils. N. Engl. J. Med., 320, 365–376 (1989). Deng, X., Wang, X., Lasson, A., Sun, T. Z., Soltesz, V., and Andersson, R.: The involvement of multiple protease-antiprotease systems and gut origin sepsis in zymosan-associated endothelial barrier injury and multiple organ dysfunction in rats. Shock, 16, 298–303 (2001). Lehrer, R. I., Ganz, T., Selsted, M. E., Babior, B. M., and Curnutte, J. T.: Neutrophils and host defense. Ann. Intern. Med., 109, 127–142 (1988). Molor-Erdene, P., Okajima, K., Isobe, H., Uchiba, M., Harada, N., and Okabe, H.: Urinary trypsin inhibitor reduces LPS-induced hypotension by suppressing tumor necrosis factor-alpha production through inhibition of Egr-1 expression. Am. J. Physiol. Heart Circ. Physiol., 288, H1265–H1271 (2005).
J. BIOSCI. BIOENG.,
22. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W. E., Kung, H. F., Leonard, E. J., and Oppenheim, J. J.: Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med., 167, 1883–1893 (1988). 23. Strieter, R. M., Kunkel, S. L., Showell, H. J., Remick, D. G., Phan, S. H., Ward, P. A., and Marks, R. M.: Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta. Science, 243, 1467–1469 (1989). 24. Sekido, N., Mukaida, N., Harada, A., Nakanishi, I., Watanabe, Y., and Matsushima, K.: Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature, 365, 654–657 (1993). 25. Maehara, K., Kanayama, N., Halim, A., el Maradny, E., Oda, T., Fujita, M., and Terao, T.: Down-regulation of interleukin-8 gene expression in HL60 cell line by human Kunitztype trypsin inhibitor. Biochem. Biophys. Res. Commun., 206, 927–934 (1995). 26. Nakagawa, H., Komori, N., Shibata, F., Ikesue, A., Konishi, K., Fujioka, M., and Kato, H.: Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization. Biochem. J., 301, 545–550 (1994). 27. Krawisz, J. E., Sharon, P., and Stenson, W. F.: Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology, 87, 1344–1350 (1984). 28. Hirose, J., Ozawa, T., Miura, T., Isaji, M., Nagao, Y., Yamashiro, K., Nii, A., Kato, K., and Uemura, A.: Human neutrophil elastase degrades inter-alpha-trypsin inhibitor to liberate urinary trypsin inhibitor related proteins. Biol. Pharm. Bull., 21, 651–656 (1998). 29. Inoue, K., Takano, H., Shimada, A., Yanagisawa, R., Sakurai, M., Yoshino, S., Sato, H., and Yoshikawa, T.: Urinary trypsin inhibitor protects against systemic inflammation induced by lipopolysaccharide. Mol. Pharmacol., 67, 673– 680 (2005). 30. Sato, Y., Ishikawa, S., Otaki, A., Takahashi, T., Hasegawa, Y., Suzuki, M., Yamagishi, T., and Morishita, Y.: Induction of acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin. Inhibiting cytokines and postoperative organ injury. Jpn. J. Thorac. Cardiovasc. Surg., 48, 428– 434 (2000). 31. Ito, K., Mizutani, A., Kira, S., Mori, M., Iwasaka, H., and Noguchi, T.: Effect of Ulinastatin, a human urinary trypsin inhibitor, on the oleic acid-induced acute lung injury in rats via the inhibition of activated leukocytes. Injury, 36, 387–394 (2005). 32. Taie, S., Yokono, S., Ueki, M., and Ogli, K.: Effects of ulinastatin (urinary trypsin inhibitor) on ATP, intracellular pH, and intracellular sodium transients during ischemia and reperfusion in the rat kidney in vivo. J. Anesth., 15, 33–38 (2001). 33. Ueki, M., Yokono, S., and Ogli, K.: Effects of ulinastatin on rat renal energy metabolism and blood flow in hemorrhagic shock. J. Anesth., 9, 65–69 (1995). 34. Fries, E. and Blom, A. M.: Bikunin—not just a plasma proteinase inhibitor. Int. J. Biochem. Cell Biol., 32, 125–137 (2000).
© 2007, The Society for Biotechnology, Japan
Urinary Trypsin Inhibitor Reduces Inflammatory Response in Kidney Induced by Lipopolysaccharide Masaaki Ueki,1* Satoshi Taie,1 Kousuke Chujo,1 Takehiko Asaga,1 Yasuyuki Iwanaga,1 Junichiro Ono,1 and Nobuhiro Maekawa1 Department of Anesthesiology & Emergency Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan1 Received 11 June 2007/Accepted 18 July 2007
Human urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used in Japan as a drug for patients with acute inflammatory disorders such as septic shock and pancreatitis. Lipopolysaccharide (LPS) triggers the sepsis syndrome by activating monocytes to produce proinflammatory cytokines, including tumor necrosis factor alpha (TNFα), which potently stimulate the activation of neutrophils. The inhibitory mechanism of UTI on the systemic inflammatory response induced by the intraperitoneal injection of LPS in the kidney is unclear. This study was undertaken to examine the inhibitory effects of UTI on renal injury associated with the systemic inflammatory response induced by LPS stimulation, with emphasis on systemic TNFα and the activation of neutrophils in rat kidney. The systemic inflammatory response syndrome was induced by LPS treatment. Serum and renal TNFα, renal cytokine-induced neutrophil chemoattractant-1 (CINC-1) and myeloperoxidase (MPO) levels, as well as renal function after LPS stimulation, were evaluated. UTI (50,000 U/kg) inhibited LPS-induced increases in the serum and renal tissue levels of TNFα, as well as the renal tissue levels of CINC-1 and MPO after LPS stimulation. UTI (50,000 U/kg) also inhibited the production of serum TNFα associated with the systemic inflammatory response syndrome induced by LPS stimulation, thereby attenuating neutrophil infiltration into renal tissues and subsequent neutrophil-mediated renal injury. These findings may have important implications in understanding the biologic functions of UTI. UTI may prove useful in protecting against acute renal injury associated with a systemic inflammatory response. [Key words: urinary trypsin inhibitor, lipopolysaccharide, neutrophil, acute renal dysfunction, cytokine-induced neutrophil chemoattractant-1 (CINC-1), tumor necrosis factor alpha]
hibits the enhanced production of proinflammatory molecules, such as TNFα (8), which are induced by LPS in vitro, the detailed mechanism underlying this inhibitory effect of UTI on systemic inflammatory response induced by LPS in the kidney remains to be elucidated. Therefore, in this study, we examined the inhibitory effects of UTI on renal injury associated with systemic inflammatory response induced by LPS stimulation, with emphasis on systemic TNFα and the activation of neutrophils in rat kidney.
Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria, is one of the major toxins that initiate the cascade of pathophysiological reactions called endotoxin shock (1). LPS triggers the sepsis syndrome by activating monocytes to produce proinflammatory cytokines, including tumor necrosis factor alpha (TNFα) (2). During sepsis, a broad array of humoral mediators including activated neutrophils are released into the systemic circulation (3). The overwhelming activation of neutrophils is known to elicit tissue damage by releasing inflammatory mediators such as neutrophil elastase and superoxide-derived free radicals (4, 5). Thus, the agents that inhibit the production of TNFα or the activation of neutrophils might prevent or attenuate tissue injury induced by LPS. Urinary trypsin inhibitor (UTI) is a multivalent Kunitztype serine protease inhibitor that is found in human urine and blood (6). UTI mainly inhibits proteases including α-chymotrypsin, plasmin, cathepsin G, and neutrophil elastase (7). UTI has been widely used as a renal protective drug in Japan for patients with septic shock and ischemia. Although UTI in-
MATERIALS AND METHODS UTI was a generous gift from Mochida Pharmaceutical (Tokyo). One unit of UTI is defined as the amount of UTI that inhibits the activity of 2 µg of trypsin and is the equivalent to 0.4 µg of UTI. LPS (Escherichia coli, serotype 0127:B8) was purchased from Sigma (St. Louis, MO, USA). Animal preparation and treatments All surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Kagawa University Faculty of Medicine. Rats were fasted for 12 h before surgery. Male Wistar rats weighing 220 to 270 g were obtained from Clea Japan (Osaka). The systemic inflammatory response syndrome was induced by
* Corresponding author. e-mail: [email protected] phone: +81-(0)87-891-2223 fax: +81-(0)87-891-2224 315
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LPS treatment. Animals were injected intraperitoneally with LPS (20 mg/kg body weight) dissolved in 1 ml of sterile saline. The experimental groups were divided into four. Sham groups Sham-operated control animals were injected with the same volume of the vehicle. UTI sham groups UTI sham animals (without LPS treatment) were injected intravenously with UTI (50,000 U/kg body weight). Yamaguchi et al. (9) reported that pretreatment with UTI at 50,000 U/kg but not 5000 U/kg significantly decreases serum CINC-1 levels in liver ischemia/perfusion. Thus, we decided that the present dose (50,000 U/kg) would have an optimal effect, which is necessary for inhibiting TNFα production associated with a systemic inflammatory response induced by the intraperitoneal injection of LPS. LPS groups LPS treatment group animals were injected with LPS. UTI + LPS groups UTI +LPS-treated animals were injected intravenously with UTI (50,000 U/kg) immediately after the injection of LPS. After the treatment, the rats were returned to their cages and provided with water ad libitum. Under anesthesia with sodium pentobarbital (50 mg/kg; Abbott Laboratories, North Chicago, IL, USA), a kidney was removed with infiltrating anesthesia of 1 ml lidocaine hydrochloride (AstraZeneca, Wilmington, DE, USA) at each specified time point (0–6 h). Enzyme immunoassays for serum TNFα, renal TNFα, cytokine-induced neutrophil chemoattractant-1 (CINC-1) and myeloperoxidase (MPO) levels TNFα was used as a proinflammatory cytokine. The concentrations of serum and renal TNFα were determined using an endogen ELISA kit (Pierce Biotechnology, Rockford, IL, USA). The renal CINC-1 serves as a chemotactic factor for the activation of neutrophils and chemotaxis to the injury site. The CINC-1 concentration in the rat kidney was determined by ELISA using a rat IL-8 assay kit (Panapharm Laboratories, Kumamoto). The renal MPO level was used as an indicator of neutrophil infiltration. The MPO level in the kidneys was determined using an ELISA kit (Oxis International, Foster City, CA, USA). At specified time points after LPS stimulation, the kidney was removed and homogenized with a homogenizer (Polytron PT10-35; Kinematika, Luzern, Switzerland) using 0.1 mol/l phosphate buffer (pH 7.4) containing 0.05% (weight/volume) sodium azide at 4°C. Homogenates were then sonicated for 20 s and centrifuged (2000×g for 10 min at 4°C). The supernatants were kept at −80°C until the measurement of the cytokine and chemokines concentrations. The concentrations of renal TNFα, CINC-1 and MPO in the supernatant were determined according to the manufacturer’s instructions and expressed as picograms per gram of tissue, picograms per gram of tissue, and nanogram per gram of tissue, respectively. Measurement of biochemical parameters At 6 h after reperfusion, a 3-ml blood sample was collected from the rats via the femoral artery. The samples were centrifuged (2000×g for 3 min) to separate the serum, and the plasma supernatant was stored at −80°C until the assays were performed. The blood urea nitrogen (BUN) and creatinine levels were measured (Special Reference Laboratories, Tokyo), and used as indicators of renal dysfunction. Histologic evaluation At 6 h after LPS stimulation, the kidney was removed, placed in formalin, and embedded in wax according to standard protocol. Five micrometer sections were cut and stained with hematoxylin and eosin for the light microscopy studies. A person who was blind to the experimental conditions counted the number of neutrophils per field under high-power (×400) and recorded the average of 10 fields for each sample (10). Statistical analysis All values shown in the text and figures are expressed as mean ±standard deviation (SD). Statistical comparisons were performed using the nonparametric Mann–Whitney U test for uncorrelated pairs. A p-value <0.05 was considered statistically significant.
RESULTS UTI decreases serum TNFα level after LPS stimulation The intraperitoneal injection of LPS triggered an inflammatory response characterized by an increase in serum TNFα level. As shown in Fig. 1a, the serum TNFα level in the LPS-treated rats increased markedly at 1 h after LPS stimulation and then gradually decreased. When UTI was administered at a dose of 50,000 U/kg, the serum TNFα level in the UTI+LPS-treated rats significantly decreased (p=0.004) in comparison with that in the LPS-treated rats at 1 h after LPS stimulation (Fig. 1b). UTI reduces expressions of renal TNFα, CINC-1 and myeloperoxidase after LPS stimulation To investigate the role of UTI in the expressions of proinflammatory cytokines and chemokines associated with the inflammatory response induced by LPS, we compared the renal levels of TNFα, CINC-1 and MPO after LPS stimulation. As shown in Fig. 2a, the renal TNFα level increased markedly at 3 h after LPS stimulation and then gradually decreased. The renal TNFα level in the UTI + LPS-treated rats significantly
FIG. 1. (a) Changes in serum TNFα concentration after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in serum TNFα concentration at 1 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean ± SD (n = 8).
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FIG. 2. (a) Changes in renal TNFα levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal TNFα levels at 3 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n =8).
FIG. 3. (a) Changes in renal CINC-1 levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal CINC-1 levels at 6 h after LPS stimulation. # p<0.05 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n = 8).
(p = 0.0017) decreased in comparison with that in the LPStreated rats at 3 h after LPS stimulation (Fig. 2b). As shown in Fig. 3a, the renal CINC-1 level increased markedly at 6 h after LPS stimulation. The renal CINC-1 level in the UTI + LPS-treated rats significantly (p = 0.0127) decreased in comparison with that in the LPS-treated rats at 6 h after LPS stimulation (Fig. 3b). As shown in Fig. 4a, the renal MPO level increased markedly at 6 h after LPS stimulation. The renal MPO level in the UTI+LPS-treated rats significantly (p =0.004) decreased in comparison with that in the LPS-treated rats at 6 h after LPS stimulation (Fig. 4b). UTI protects against LPS-induced acute renal injury The serum BUN and creatinine concentrations in the LPStreated rats before LPS stimulation were 19.1 ± 3.7 mg/dl and 0.27 ± 0.03 mg/dl, respectively. At 6 h after LPS stimulation, the serum BUN and creatinine concentrations in the LPS-treated rats were 37.0 ± 3.4 mg/dl and 0.54 ± 0.11 mg/dl, respectively. In the LPS-treated rats, the renal injury associated with a systemic inflammatory response induced by LPS injection was minimal. The serum BUN and creatinine con-
centrations in the UTI + LPS-treated rats were significantly (p = 0.0046, p= 0.0011) lower than those in the LPS-treated rats at 6 h after LPS stimulation (Table 1). Effects of UTI on renal histopathology induced by LPS stimulation Histopathological examination revealed mild neutrophil infiltration in the kidneys of the LPS-treated rats (Fig. 5). Furthermore, we performed morphometric analysis to quantitate the number of neutrophils in the kidney. We examined the effects of UTI on neutrophil infiltration in the kidney after LPS stimulation. Neutrophil infiltration in the UTI + LPS-treated rats was significantly (p < 0.0001) lower than that in the LPS-treated rats (Table 2). DISCUSSION In this study, we demonstrated that UTI inhibited the increases in serum and renal TNFα, renal CINC-1 and MPO levels after LPS stimulation and subsequent renal injury associated with a systemic inflammatory response induced by intraperitoneal injection of LPS. Sepsis is a leading cause of acute renal failure (11, 12),
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FIG. 4. (a) Changes in renal MPO levels after LPS stimulation. Closed circles, LPS group; open circles, sham group. ** p<0.01 compared with the sham group. (b) Changes in renal MPO levels at 6 h after LPS stimulation. ## p<0.01 compared with the LPS group. ** p<0.01 compared with the sham group or UTI sham group. Data are expressed as mean± SD (n =8).
but the pathogenesis of sepsis and the associated end-organ failure are highly complex, involving multiple inflammatory mediators. During sepsis, a broad array of humoral mediators including cytokines are released into the systemic circulation (3). Among the various cytokines, TNFα, which is released early into the circulation following LPS stimulation, has been shown to be a key early mediator of sepsis (13, 14). Another study has suggested that LPS causes damage to various tissues, including the kidney, by promoting tissue infiltration with neutrophils (15). LPS and other bacterial products are potent stimulators of polymorphonuclear leukocytes in vitro (16, 17). Activated neutrophils release various kinds of mediators, including proteases and oxygen radicals (18). A protease-antiprotease imbalance has been reported to be involved in a variety of inflammatory diseases (19). Indeed, neutrophil elastase and cathepsin G-deficient mice have been shown to be resistant to the lethal effects of LPS (20). In this study, we first demonstrated that UTI inhibited the production of both serum and renal TNFα in a rat LPS stim-
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FIG. 5. Histological findings in the groups. (a) Sham group; (b) LPS group; (c) UTI + LPS group. Histological examination of the kidney was performed at 6 h after LPS stimulation. Neutrophil infiltration is indicated by the arrow in panel b. Original magnification, ×200. Bars: 200 µm.
ulation model. Aosasa et al. (8) previously reported that UTI inhibited TNFα production in LPS-stimulated monocytes. In his study, UTI did not inhibit the TNFα mRNA expression or suppress the NFκB level. The precise mechanisms by which UTI inhibited LPS-stimulated TNFα production remain unclear at present. Molor-Erdene et al. (21) reported that UTI might inhibit the LPS-induced production of TNFα by inhibiting the activation of the human extracellular signal-regulated protein kinase 1/2 and early growth response factor-1 pathway in monocytes. TNFα can induce IL-8 gene expression in blood monocytes and other cells (22), leading to the secretion of IL-8 (23) and tissue damage via the activation and infiltration of neutrophils, and resulting in the release of lysozomal enzymes and superoxide anions by neutrophils (24). Maehara et al. (25) reported that UTI down regulated the IL-8 gene expression induced by LPS in HL-60 cells. Based on the biologic activity and peptide sequences of rat CINC-1, rat CINC-1 is a member of the IL-8 family (26). We measured
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TABLE 1. Renal function data BUN Creatinine (mg/dl) (mg/dl) Sham 21.5 ± 0.9 0.27 ± 0.02 UTI sham 19.8 ± 2.2 0.26 ± 0.04 0.54 ± 0.03a,b LPS 37.0 ± 3.4a,b UTI+ LPS 29.5 ± 5.9b,c,d 0.34 ± 0.06b,c,d BUN Blood urea nitrogen. Data are expressed as mean ± SD (n = 8). a p<0.01 compared with the sham group. b p<0.01 compared with the UTI sham group. c p<0.01 compared with the LPS group. d p<0.05 compared with the sham group. Group
TABLE 2. Neutrophils in kidney Average number of neutrophils/field Sham 0.22 ± 0.42 UTI sham 0.2 ± 0.4 LPS 1.9 ± 0.8a,b UTI+ LPS 1.0 ± 0.6a,b,c The number of neutrophils per field under high-power magnification was counted in kidney samples obtained 6 h after LPS stimulation. Data are expressed as mean ± SD (n = 4). a p<0.01 compared with the sham group. b p<0.01 compared with the UTI sham group. c p<0.01 compared with the LPS group. Group
the CINC-1 levels instead of the IL-8 levels in the rat inflammatory response model. In addition, in our study, UTI inhibited the production of renal CINC-1 in the rat LPS stimulation models. These experimental results have confirmed that UTI inhibits the LPS-induced IL-8 gene expression even in vivo. The MPO assay is widely used to quantify the number of neutrophils in a tissue and the MPO level serves as an index of inflammation because MPO is an enzyme that is released mainly from neutrophils (27). We used the MPO level as an index of the renal accumulation of activated neutrophils. Because rat CINC-1 activates the infiltration of neutrophils in tissue, the renal MPO level in the UTI + LPS-treated rats was significantly lower than that in the LPS-treated rats at 6 h after LPS stimulation. It is considered that the MPO level was inhibited because UTI depressed the CINC-1 level. UTI mainly inhibits proteases including α-chymotrypsin, plasmin, cathepsin G, and leukocyte elastase (7). UTI is synthesized from the inter-α-trypsin inhibitor through proteolytic cleavage by neutrophil elastase at the inflammation site (28). UTI participates as a component of the host defense mechanisms at the inflammation site. UTI has been widely used in Japan as a drug for patients with septic shock and pancreatitis. UTI had been used particularly for renal protection, but the molecular mechanism was unclear. Therefore, in this study, we examined the effects of UTI on renal injury associated with a systemic inflammatory response induced by LPS stimulation. In LPS-treated UTI-deficient mice, kidney damage, including neutrophil infiltration, was more prominent than in LPS-treated wild-type mice (29). These results suggest that UTI protects against organ damage associated with LPS possibly via the inhibition of neutrophil sequestration at the inflammation site.
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In this study, 50,000 U/kg of UTI was used. This dosage is 10 times higher than the therapeutic dosage of UTI used in the clinical setting. The UTI concentration is increased to 150 U/ml after the administration of 5000 U/kg of UTI (30). Molor-Erdene et al. (21) reported that although 100 U/ml of UTI (about 5000 U/kg of UTI) inhibited TNFα production in LPS-stimulated monocytes in vitro, 50,000 U/kg of UTI is needed to inhibit the lung TNFα production associated with the inflammatory response in rats administered with LPS. Yamaguchi et al. (9) reported that the pretreatment with UTI at 50,000 U/kg but not 5000 U/kg significantly decreased serum CINC-1 levels after liver ischemia/perfusion. Ito et al. (31) also reported that 50,000 U/kg of UTI inhibited the oleic acid-induced acute lung injury in rats via the inhibition of activated leukocytes. We chose the dosage of UTI according to the optimal dosage that was required to inhibit serum TNFα in their paper. We also previously reported that 50,000 U/kg of UTI improved energy metabolism during ischemia (32) and prevented hemorrhagic shock (33) in rat kidneys. Why such a large dose of UTI is necessary to inhibit LPS-induced cytokine and chemokine production in rats is unclear at present, but structural differences between human and rat UTIs might explain the decreased sensitivity of rats to human UTI (34). In this study, 50,000 U/kg of UTI inhibited the production of serum TNFα associated with a systemic inflammatory response syndrome induced by LPS stimulation, thereby attenuating neutrophil infiltration into renal tissues and subsequent neutrophil-mediated renal injury. However, further studies are required to evaluate the optimal dosage of UTI in cases of renal systemic inflammatory response induced by LPS stimulation. ACKNOWLEDGMENTS We are grateful to Mochida Pharmaceutical (Tokyo) for supplying the UTI used in this study.
REFERENCES 1. Michie, H. R., Manogue, K. R., Spriggs, D. R., Revhaug, A., O’Dwyer, S., Dinarello, C. A., Cerami, A., Wolff, S. M., and Wilmore, D. W.: Detection of circulating tumor necrosis factor after endotoxin administration. N. Engl. J. Med., 318, 1481–1486 (1988). 2. Parrillo, J. E.: Pathogenetic mechanisms of septic shock. N. Engl. J. Med., 328, 1471–1477 (1993). 3. De Vriese, A. S.: Prevention and treatment of acute renal failure in sepsis. J. Am. Soc. Nephrol., 14, 792–805 (2003). 4. Cohen, J.: The immunopathogenesis of sepsis. Nature, 420, 885–891 (2002). 5. Brown, K. A., Brain, S. D., Pearson, J. D., Edgeworth, J. D., Lewis, S. M., and Treacher, D. F.: Neutrophils in development of multiple organ failure in sepsis. Lancet, 368, 157–169 (2006). 6. Jonsson-Berling, B. M., Ohlsson, K., and Rosengren, M.: Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine. Biol. Chem. Hoppe-Seyler, 370, 1157–1161 (1989). 7. Morishita, H., Yamakawa, T., Matsusue, T., Kusuyama, T., Sameshima-Aruga, R., Hirose, J., Nii, A., Miura, T., Isaji, M., Horisawa-Nakano, R., and other 13 authors: Novel factor Xa and plasma kallikrein inhibitory-activities of
320
8.
9.
10.
11.
12.
13. 14.
15. 16.
17.
18. 19.
20. 21.
UEKI ET AL.
the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. Thromb. Res., 73, 193–204 (1994). Aosasa, S., Ono, S., Mochizuki, H., Tsujimoto, H., Ueno, C., and Matsumoto, A.: Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes. Shock, 15, 101–105 (2001). Yamaguchi, Y., Ohshiro, H., Nagao, Y., Odawara, K., Okabe, K., Hidaka, H., Ishihara, K., Uchino, S., Furuhashi, T., Yamada, S., Mori, K., and Ogawa, M.: Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion. J. Surg. Res., 94, 107– 115 (2000). Guo, R., Wnag, Y., Minto, A. W., Quigg, R. J., and Cunningham, P. N.: Acute renal failure in endotoxemia is dependent on caspase activation. J. Am. Soc. Nephrol., 15, 3093– 3102 (2004). Groeneveld, A. B., Tran, D. D., van der Meulen, J., Nauta, J. J., and Thijs, L. G.: Acute renal failure in the medical intensive care unit: predisposing, complicating factors and outcome. Nephron, 59, 602–610 (1991). Neveu, H., Kleinknecht, D., Brivet, F., Loirat, P., and Landais, P.: Prognostic factors in acute renal failure due to sepsis. Results of a prospective multicentre study. The French Study Group on Acute Renal Failure. Nephrol. Dial. Transplant., 11, 293–299 (1996). Beutler, B. A., Milsark, I. W., and Cerami, A.: Cachectin/ tumor necrosis factor: production, distribution, and metabolic fate in vivo. J. Immunol., 135, 3972–3977 (1985). Van Deventer, S. J., Buller, H. R., ten Cate, J. W., Aarden, L. A., Hack, C. E., and Sturk, A.: Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood, 76, 2520– 2526 (1990). Linas, S. L., Whittenburg, D., and Repine, J. E.: Role of neutrophil derived oxidants and elastase in lipopolysaccharidemediated renal injury. Kidney Int., 39, 618–623 (1991). Guthrie, L. A., McPhail, C., Henson, P. M., and Johnston, R. B., Jr.: Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme. J. Exp. Med., 160, 1656–1671 (1984). Smedly, L. A., Tonnesen, M. G., Sandhaus, R. A., Haslett, C., Guthrie, L. A., Johnston, R. B., Jr., Henson, P. M., and Worthen, G. S.: Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J. Clin. Invest., 77, 1233–1243 (1986). Weiss, S. J.: Tissue destruction by neutrophils. N. Engl. J. Med., 320, 365–376 (1989). Deng, X., Wang, X., Lasson, A., Sun, T. Z., Soltesz, V., and Andersson, R.: The involvement of multiple protease-antiprotease systems and gut origin sepsis in zymosan-associated endothelial barrier injury and multiple organ dysfunction in rats. Shock, 16, 298–303 (2001). Lehrer, R. I., Ganz, T., Selsted, M. E., Babior, B. M., and Curnutte, J. T.: Neutrophils and host defense. Ann. Intern. Med., 109, 127–142 (1988). Molor-Erdene, P., Okajima, K., Isobe, H., Uchiba, M., Harada, N., and Okabe, H.: Urinary trypsin inhibitor reduces LPS-induced hypotension by suppressing tumor necrosis factor-alpha production through inhibition of Egr-1 expression. Am. J. Physiol. Heart Circ. Physiol., 288, H1265–H1271 (2005).
J. BIOSCI. BIOENG.,
22. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W. E., Kung, H. F., Leonard, E. J., and Oppenheim, J. J.: Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med., 167, 1883–1893 (1988). 23. Strieter, R. M., Kunkel, S. L., Showell, H. J., Remick, D. G., Phan, S. H., Ward, P. A., and Marks, R. M.: Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta. Science, 243, 1467–1469 (1989). 24. Sekido, N., Mukaida, N., Harada, A., Nakanishi, I., Watanabe, Y., and Matsushima, K.: Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature, 365, 654–657 (1993). 25. Maehara, K., Kanayama, N., Halim, A., el Maradny, E., Oda, T., Fujita, M., and Terao, T.: Down-regulation of interleukin-8 gene expression in HL60 cell line by human Kunitztype trypsin inhibitor. Biochem. Biophys. Res. Commun., 206, 927–934 (1995). 26. Nakagawa, H., Komori, N., Shibata, F., Ikesue, A., Konishi, K., Fujioka, M., and Kato, H.: Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization. Biochem. J., 301, 545–550 (1994). 27. Krawisz, J. E., Sharon, P., and Stenson, W. F.: Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology, 87, 1344–1350 (1984). 28. Hirose, J., Ozawa, T., Miura, T., Isaji, M., Nagao, Y., Yamashiro, K., Nii, A., Kato, K., and Uemura, A.: Human neutrophil elastase degrades inter-alpha-trypsin inhibitor to liberate urinary trypsin inhibitor related proteins. Biol. Pharm. Bull., 21, 651–656 (1998). 29. Inoue, K., Takano, H., Shimada, A., Yanagisawa, R., Sakurai, M., Yoshino, S., Sato, H., and Yoshikawa, T.: Urinary trypsin inhibitor protects against systemic inflammation induced by lipopolysaccharide. Mol. Pharmacol., 67, 673– 680 (2005). 30. Sato, Y., Ishikawa, S., Otaki, A., Takahashi, T., Hasegawa, Y., Suzuki, M., Yamagishi, T., and Morishita, Y.: Induction of acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin. Inhibiting cytokines and postoperative organ injury. Jpn. J. Thorac. Cardiovasc. Surg., 48, 428– 434 (2000). 31. Ito, K., Mizutani, A., Kira, S., Mori, M., Iwasaka, H., and Noguchi, T.: Effect of Ulinastatin, a human urinary trypsin inhibitor, on the oleic acid-induced acute lung injury in rats via the inhibition of activated leukocytes. Injury, 36, 387–394 (2005). 32. Taie, S., Yokono, S., Ueki, M., and Ogli, K.: Effects of ulinastatin (urinary trypsin inhibitor) on ATP, intracellular pH, and intracellular sodium transients during ischemia and reperfusion in the rat kidney in vivo. J. Anesth., 15, 33–38 (2001). 33. Ueki, M., Yokono, S., and Ogli, K.: Effects of ulinastatin on rat renal energy metabolism and blood flow in hemorrhagic shock. J. Anesth., 9, 65–69 (1995). 34. Fries, E. and Blom, A. M.: Bikunin—not just a plasma proteinase inhibitor. Int. J. Biochem. Cell Biol., 32, 125–137 (2000).