J Am Soc Nephrol 10: 264–270, 1999 The B4 Antagonist ONO-4057 Inhibits Nephrotoxic Serum Nephritis in WKY Rats

SATORU SUZUKI,* TAKESHI KURODA,† JUN-ICHIROU KAZAMA,† NAOFUMI IMAI,† HIDEKI KIMURA,* MASAAKI ARAKAWA,† and FUMITAKE GEJYO* *Department of Clinical and Laboratory Medicine, Fukui Medical University, Fukui, Japan; and †Department of Medicine (II), Niigata University School of Medicine, Niigata, Japan.

Abstract. To evaluate the role of (LTB4) in ONO-4057 treatment significantly reduced proteinuria and he- glomerulonephritis, this study was conducted to examine maturia, suppressed the glomerular accumulation of mono- whether ONO-4057, an LTB4 receptor antagonist, moderated cytes/macrophages, and reduced the formation of crescentic nephritis caused by the injection of nephrotoxic serum (NTS) glomeruli in a dose-dependent manner. These results suggest into Wistar-Kyoto rats. Rats were given intraperitoneal injec- that LTB4 is responsible for the crescentic formations and tions of ONO-4057 or phosphate-buffered saline 24 h before renal dysfunction associated with NTS nephritis. The LTB4 the injection of NTS. These rats subsequently received equal receptor antagonist ONO-4057 may thus be beneficial in the doses of ONO-4057 or phosphate-buffered saline 3 h and 1, 2, treatment of crescentic glomerulonephritis. 3, 4, 5, and 6 d later. Compared with the control groups,

The nephrotoxic serum (NTS) nephritis, which is produced by monocyte chemotaxis, margination, degranulation, and the gen- the administration of a heterologous antibody against glomer- eration of active oxygen species (18). The administration of LTB4 ular basement membrane (GBM), is a well established exper- to rats with mild NTS GN increases the glomerular infiltration of imental model of glomerular immune injury resulting in glo- PMN and enhances their adhesion to mesangial cells (10,19). merulonephritis (GN) (1). The glomerular lesions induced by However, no direct evidence has been provided as to whether NTS vary depending on the species. The administration of LTB4 influences the infiltration and activation of monocytes/ small doses of NTS to the Wistar-Kyoto (WKY) rat causes the macrophages or the crescentic formation in GN. Hence, we at- formation of a crescentic GN (2). In this rat model, it has been tempted to evaluate the involvement of LTB4 in crescentic GN in reported that the infiltration of T lymphocytes and monocytes/ WKY rats by administering a specific LTB4 receptor antagonist, macrophages, along with a few polymorphonuclear leukocytes ONO-4057 (5-[2-(2-carboxyethel)-3-{6-(para-methoxyphenyl)- (PMN), is the most prominent pathologic finding. The mono- 5E-hexenyl}oxyphenoxy] valeric acid), which inhibited human cytes/macrophages as well as CD8-positive T lymphocytes are neutrophil aggregation, chemotaxis or degranulation induced by putatively involved in both the development of glomerular LTB4, and the LTB4-induced rise in cytosolic free calcium (20– lesions and crescentic formation (3). However, the specific 22), to these animals. involvement of monocytes/macrophages in the development of glomerular lesions and crescentic formation is not known. Materials and Methods Lipoxygenase products, particularly (LT), are po- Animals tentially important modulators of inflammation in various forms Eight-week-old inbred male WKY rats were obtained from Charles of acute GN (4–10). The synthesis of glomerular LTB4 synthesis River Japan, Inc. (Atsugi, Kanagawa, Japan). The rats were housed is markedly enhanced early in the course of several forms of individually in metabolic cages to obtain urine samples. They were glomerular immune injury, including NTS nephritis (10–14). fed standard rat chow and given free access to water throughout the Activated PMN, monocytes, and probably resident glomerular study. macrophages each generate LTB4 through the sequential actions of 5-lipoxygenase and LTA4 hydrolase on and Rabbit Anti-Rat GBM Antiserum (NTS) LTA4 (8,15–17). Notably, LTB4 is a stimulus for PMN and Rat GBM that was obtained from perfused renal cortices (13) was digested with trypsin (Sigma Chemical Co., St. Louis, MO) for3hat 37°C. After heating the digest at 60°C for 30 min, the mixture was ϫ Received March 11, 1998. Accepted August 24, 1998. centrifuged at 76,000 g for 1 h and the supernatant was lyophilized. Correspondence to Dr. Satoru Suzuki, Department of Clinical and Laboratory For the production of NTS, two white rabbits were subcutaneously Medicine, Fukui Medical University, Matsuoka, Fukui, 910-1193 Japan. injected with the lyophilized sample that was emulsified in complete Phone: 0776-61-3111; Fax: 0776-61-8120; E-mail: [email protected] Freund’s adjuvant. Subsequent injections were conducted 2, 4, and 6 1046-6673/1002-0264$03.00/0 wk after the initial immunization. Rabbits were bled in the seventh Journal of the American Society of Nephrology week and the serum was collected. Sera exhibiting a nephrotoxic Copyright © 1999 by the American Society of Nephrology potency, as demonstrated by inducing proteinuria in rats after the J Am Soc Nephrol 10: 264–270, 1999 ONO-4057 in Nephrotoxic Serum Nephritis 265

intraperitoneal injections of PBS. Twenty-four hours later, all rats were injected with 0.2 ml of NTS through the tail vein. The rats were then administered the same doses of ONO-4057 or PBS, intraperito- neally, 3 h after the NTS injection and again 1, 2, 3, 4, 5, and 6 d later. Five rats in each group were killed by axillar bleeding at 1, 2, 3, 5, or 7 d after the injection of NTS.

Histologic Examination At the time of sacrifice, one kidney was excised from each rat and divided into three parts for examination by light, electron, and immu- nofluorescence microscopy. Light Microscopy. The renal tissue was fixed in buffered for- malin and embedded in paraffin for light microscopic examination. Sections 2- to 3-␮m-thick were then individually stained with hema- toxylin-eosin, periodic acid-Schiff, periodic acid-silver methenamine (PAM), Masson-trichrome, and PAM-Masson. Upon examination, the percentage of crescentic glomeruli per 100 glomeruli of each rat was calculated. Immunofluorescence Microscopy. Each kidney tissue speci- men was immediately embedded in OCT medium (Miles Laborato- ries, Elkhart, IN) and frozen in a mixture of acetone and dry ice. Frozen sections (2 to 3 ␮m in thickness) were cut in a cryostat and Figure 1. Urinary protein excretion in Wistar-Kyoto (WKY) rats stored at Ϫ80°C until use. The cryostat sections were cut serially, treated with ONO-4057 plus nephrotoxic serum (NTS). Rats were rinsed in PBS for 15 min, and fixed in absolute acetone for 10 min. administered 75 mg/kg ONO-4057 (■), 300 mg/kg ONO-4057 (}), or FITC-conjugated rabbit anti-rat IgG, FITC-conjugated goat anti-rat PBS (F), intraperitoneally, and injected 24 h later with 0.2 ml of NTS. C3, FITC-conjugated goat anti-rat fibrinogen, and FITC-conjugated They were administered the same dose of ONO-4057 or PBS intra- goat anti-rabbit immunoglobulins (all obtained from Cappel, peritoneally 3 h after NTS injection and again at 1, 2, 3, 4, 5, and 6 d Malverne, PA) were used for direct immunofluorescence. The pres- ଙ later. Data are mean Ϯ SD. E, untreated rats. P Ͻ 0.001 versus ence of rat monocytes/macrophages was examined immunohisto- ଙଙ ଙଙଙ control; P Ͻ 0.001 versus 75 mg/kg ONO-4057; P Ͻ 0.05 chemically on frozen tissue specimens by using the monoclonal versus 75 mg/kg ONO-4057. antibodies MRCOX-41 (Biomedicals AG, Rheinstrasse, Switzerland) (23). The number of monocytes/macrophages per 20 glomeruli of each rat was calculated. intravenous injection of 0.2 ml of antiserum, were pooled. The pooled Electron Microscopy. The renal specimens for electron micros- antiserum was heat-inactivated at 56°C for 30 min, and stored at copy were fixed in 2.5% glutaraldehyde, followed by osmium tetrox- Ϫ20°C until used. ide and embedded in Epon 812. Ultrathin sections stained with tannic acid and lead citrate were examined. ONO-4057 ONO-4057 was supplied by Ono Pharmaceutical Co. (Osaka, Ja- pan). It was dissolved in phosphate-buffered saline (PBS), pH 7.4, Urinalysis immediately before use. The amount of protein excreted in the urine per 24 h was deter- mined by using the Bio-Rad protein assay (Bio-Rad Laboratories, Experimental Design Richmond, CA). Occult blood was measured by using N-Multistix Rats (n ϭ 25 in each group) first received intraperitoneal doses of SG-L (Bayer-Sankyo Co., Tokyo, Japan). The amount of occult blood either 75 or 300 mg/kg ONO-4057. The control group received present was graded as negative or positive.

Table 1. Hematuria in WKY rats treated with ONO-4057 plus NTSa

Days after NTS Injection

ONO-4057 13 5 7

n Neg. Pos. n Neg. Pos. n Neg. Pos. n Neg. Pos.

0 25 25 0 (0%) 15 12 3 (20%) 10 6 4 (40%) 5 0 5 (100%) 75 mg/kg 25 25 0 (0%) 15 15 0 (0%) 10 10 0 (0%) 5 4 1 (20%)b 300 mg/kg 25 25 0 (0%) 15 15 0 (0%) 10 10 0 (0%) 5 5 0 (0%)c

a Experimental procedures are described in Materials and Methods. n ϭ number of rats. WKY, Wistar-Kyoto; NTS, nephrotoxic serum; Neg., negative; Pos., positive. b P Ͻ 0.05 versus control. c P Ͻ 0.01 versus control. 266 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 264–270, 1999

Statistical Analyses Data are expressed as mean Ϯ SD. Differences among data sets were assessed by performing t test, ␹2 test, and Fisher exact test for four-fold tables, where appropriate. A level of P Ͻ 0.05 was accepted as statistically significant.

Results Effect of ONO-4057 on Proteinuria The amount of urinary protein excretion increased markedly after day 5 in the control- and ONO-4057-treated groups (Fig- ure 1). The urinary protein levels reached 62 Ϯ 9 mg/d on day 7 in the control group. By comparison, this increase was significantly suppressed in the rats given ONO-4057 in a dose-dependent manner. On day 7, a dose of 75 mg/kg yielded 13 Ϯ 3 mg/d urinary protein, whereas a dose of 300 mg/kg yielded a urinary protein level of 3 Ϯ 3 mg/d (P Ͻ 0.001).

Effect of ONO-4057 on Hematuria As shown in Table 1, hematuria was frequently noted in rats given PBS. This hematuria was significantly suppressed on days 3, 5, and 7 in all groups receiving ONO-4057.

Histology and Immunohistochemistry In the control group, glomerular lesions were induced by the intravenous injection of 0.2 ml of NTS on day 3; these lesions were characterized by endocapillary hypercellularity. In this group of rats, mesangial proliferation, severe necrotizing le- sions, and marked crescentic formation were observed on day 5 and thereafter. These alterations, however, were not observed in the ONO-4057-treated groups (Figure 2). Our immunoflu- orescence study showed that rabbit IgG intensely stained the capillary walls of both control- and ONO-4057-treated rats in a linear pattern throughout the time course of the experiment (Figure 3). Although there was a gradual decrease in the intensity of rabbit IgG staining throughout the experiment in each group, these changes were not statistically significant. Rat IgG and C3 antigens were also detected, but the staining intensity of these markers was similarly faint in groups. A small number of PMN were noted in the glomerular capillaries Figure 2. Light micrographs showing glomeruli of the control group of both control- and ONO-4057-treated rats on days 1 and 3. (A) and the 300 mg/kg ONO-4057 group (B) on day 7. The control The glomerular accumulation of monocytes/macrophages in- group shows mononuclear cell proliferation, exudative lesion, and creased gradually in the control group (Figure 4). This increase crescentic formation. The ONO-4057 group shows a slight increase in mononuclear cells. Magnification, ϫ400. Periodic acid-Schiff stain. was significantly suppressed in all of the groups given ONO- 4057, and occurred in a dose-dependent manner (Figure 5).

Effect of ONO-4057 on Crescentic Formation oped crescentic GN, which leads to glomerulosclerosis with As shown in Figure 6, the frequency of crescentic glomeruli progressive renal dysfunction, when given a small dose of formation was significantly and dose-dependently suppressed NTS. In this study, we observed that the administration of in the ONO-4057 groups compared with the control group. The ONO-4057, an LTB4 receptor antagonist (22), significantly suppression of crescentic glomeruli formation was first ob- reduced crescentic formation and proteinuria in a dose-depen- served on day 5 and was most pronounced on day 7. dent manner. These findings suggest that LTB4 may govern the course of glomerular injury in NTS nephritis in WKY rats. Discussion Kawasaki et al. (3) reported that crescentic GN in WKY rats The glomerular injury induced by NTS varies with the was characterized by the early infiltration of CD8-positive cells species, the age and gender of the recipient, the quantity and in glomeruli; those authors suspected that the increased sus- type of NTS used, and the duration of the disease consisting of ceptibility of WKY rats to NTS may be related to a CD8- the heterologous and autologous phases (24). WKY rats devel- positive cell-related injury. The previous study showed that a J Am Soc Nephrol 10: 264–270, 1999 ONO-4057 in Nephrotoxic Serum Nephritis 267

Figure 4. Immunofluorescence micrograph showing glomerular accu- mulation of monocyte/macrophage (the control group, at day 3). Magnification, ϫ400.

ONO-4057 does not cross-react with MCP-1 receptor (M. Odani, personal communication). These findings suggest that in WKY rats, the mechanism governing glomerular lesion formation Figure 3. Immunofluorescence micrograph showing glomerular dep- induced by NTS may vary according to the amount of NTS osition of rabbit IgG. Rabbit IgG is distributed in a linear pattern along administered, and that CD8-positive cells or monocytes/mac- glomerular basement membrane in both the control group (A) and the rophages may participate in glomerular lesion formation. 300 mg/kg ONO-4057 group (B) on day 1. Magnification, ϫ400. The semiquantitative evaluation of immunoglobulin deposi- tion revealed no significant difference in the deposition of rabbit immunoglobulins, rat IgG, and rat C3 between glomeruli small number of PMN accumulated in the glomerular capillar- from rats in any group. These data suggest that ONO-4057 ies on days 1 and 3 and that the glomerular accumulation of does not influence the glomerular deposition of immunoglobu- monocytes/macrophages increased gradually through days 3, 5, lins by NTS administration. and 7. Fujinaka et al. (25) also reported the infiltration of PMN Goldman et al. (28) reported that PMN express separate into the glomerular capillaries at 1 h and 1 and 3 d after NTS high- and low-affinity receptors for LTB4. Through the high- injection in cases of crescentic GN of WKY rats. Wada et al. affinity receptors, LTB4 acts as the most potent chemotactic (26) reported that the decrease of the number of monocytes/ substance for PMN and also increases PMN aggregation and macrophages in glomeruli of WKY rats caused by administer- adhesion to the endothelium (18). Through the low-affinity ing specific polyclonal neutralizing antimonocyte chemotactic receptors, LTB4 acts as a calcium ionophore (29), leading to and activating factor/monocyte chemoattractant protein-1 PMN activation (30,31). Concerning the monocytes, it has (MCAF/MCP-1) prevented crescentic formation, thereby de- been reported that LTB4 provoked rapid monocyte-mesangial creasing the excreted amounts of protein to normal levels on cell adhesion at nanomolar concentrations by interacting with days 3 and 6. Furthermore, our present study shows that the monocytes (7). In addition, the recruitment of monocytes might decreased number of monocytes/macrophages in glomeruli of be modulated by LTB4 (32), and LTB4 could direct monocyte- WKY rats achieved by administering ONO-4057 prevented mediated events since it is a potent and selective agonist for crescentic formation and decreased the excreted amounts of peripheral blood monocyte function (33). Furthermore, LTB4 protein. Although Yokomizo et al. (27) recently reported the receptors have been identified on monocytes (34) and macro- cloning of the complementary DNA encoding a cell surface phages (17). This study showed that the number of monocytes/ LTB4 receptor and revealed that an LTB4 receptor is a member macrophages in the glomeruli was decreased by administering of the chemokine superfamily receptors, it has been shown that ONO-4057. These findings suggest that ONO-4057 inhibits 268 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 264–270, 1999

Figure 5. Intraglomerular accumulation of monocyte/macrophage in WKY rats treated with ONO-4057 plus NTS. Data are expressed as the number of stained cells per glomerular cross section. Procedure as in Figure 1. ᮀ, control group; z, the 75 mg/kg ONO-4057 group; ■, 300 mg/kg ONO-4057 group. ଙ P Ͻ 0.001 versus control; ଝ P Ͻ 0.001 versus 75 mg/kg ONO-4057.

crescentic formation caused by the accumulation of mono- cytes/macrophages. Recently, Werb and Gordon (35) showed that stimulated macrophages secreted elastase. It has been reported that human monocytes and macrophages have elastolytic activity, which is mainly caused by cell surface-related PMN elastase (36,37). It has also been reported that elastase degrades heparan sulfate proteoglycan (HSPG) within the subendothelial matrix in vitro, suggesting that a proteolytic cleavage of HSPG may be in- volved in proteinuria (38,39), and that the interruption of the GBM, probably caused in part by lysosomal enzymes such as elastase, may cause hematuria (40). The present study shows that the LTB4 receptor antagonist ONO-4057 significantly reduces proteinuria, hematuria, and the accumulation of mono- cytes/macrophages in rats in a dose-dependent manner. On the basis of the above results, our findings suggest that decreased amounts of elastase, due to the reduced number of monocytes/ macrophages in glomeruli of WKY rats given ONO-4057, may reduce the magnitude of glomerular damage. However, our study showed that the administration of ONO- 4057 did not completely prevent glomerular lesions or renal dysfunction. Conversely, Wada et al. (26) reported that anti- MCAF/MCP-1 antibodies decreased the number of macro- phages in glomeruli, and prevented crescentic formation and proteinuria. Kawasaki et al. (3) reported that CD8-positive Figure 6. Crescentic formation in renal sections of WKY rats treated cells also play a key role in crescentic formation. These results with ONO-4057 plus NTS. Procedure and symbols as in Figure 1. J Am Soc Nephrol 10: 264–270, 1999 ONO-4057 in Nephrotoxic Serum Nephritis 269 suggest that several factors may be important in modulating A4 hydrolase from glomerular mesangial cells using polymerase crescentic formation in WKY rats. chain reaction (PCR). FEBS Lett 299: 273–277, 1992 We conclude that LTB4 or its receptor is partly responsible 15. Brady HR, Papayianni A, Serhan CN: Leukocyte adhesion pro- for the crescentic formation and renal dysfunction mediated by motes biosynthesis of lipoxygenase products by transcellular NTS nephritis in WKY rats. These results provide good evi- routes. Kidney Int 45: S90–S97, 1994 16. Williams JD, Czop JK, Austen KF: Release of leukotrienes by dence that an LTB4 receptor antagonist may prove beneficial human monocytes on stimulation of their phagocytic receptor for in the treatment of crescentic GN. particulate activators. J Immunol 132: 3034–3040, 1984 17. Badr KF: Glomerulonephritis: Roles for lipoxygenase pathway Acknowledgments in pathophysiology and therapy. Curr Opin Nephrol Hypertens 6: 111–118, 1997 The authors are grateful to Hideki Maegawa and Masaki Mori for 18. Samuelsson B, Dahlen S-E, Lindgren JA, Rouzer CA, Serhan technical assistance. CN: Leukotrienes and lipoxins: Structures, biosynthesis, and biological effects. Science 237: 1171–1176, 1987 19. Brady HR, Denton MD, Brenner BM, Serhan CN: Neutrophil References adhesion to glomerular mesangial cells: Regulation by lipoxy- 1. Unanue ER, Dixon FJ: Experimental glomerulonephritis: Immu- genase-derived eicosanoids. Adv Exp Med Biol 314: 347–359, nological events and pathogenetic mechanisms. Adv Immunol 6: 1991 1–90, 1967 20. Kishikawa K, Tateishi N, Maruyama T, Seo R, Toda M, Miy- 2. Granados R, Mendrick DL, Rennke HG: Antibody-induced cres- amoto T: ONO-4057, a novel, orally active leukotriene B4 an- cent formation in WKY rats: Potential role of antibody-depen- tagonist: Effects on LTB4-induced neutrophil functions. Prosta- dent cell cytotoxicity (ADCC) in vivo. Kidney Int 37: 414, 1990 glandins 44: 261–275, 1992 3. Kawasaki K, Yaoita E, Yamamoto T, Kihara I: Depletion of 21. Kishikawa K, Nakao S, Matsumoto S, Kondo K, Hamanaka N: CD8-positive cells in nephrotoxic serum nephritis of WKY rats. Estimation of antagonistic activity of ONO-4057 against leuko- Kidney Int 41: 1517–1526, 1992 triene B4 in humans. Adv Prostaglandin Thromboxane Leukot 4. Badr KF, Schreiner GF, Wasserman M, Ishikawa I: Preservation Res 23: 279–281, 1995 of glomerular capillary ultrafiltration coefficient during rat neph- 22. Kishikawa K, Matsunaga N, Maruyama T, Seo R, Toda M, rotoxic serum nephritis by a specific leukotriene D4 receptor Miyamoto T, Kawasaki A: ONO-LB-457: A novel and orally antagonist. J Clin Invest 81: 1702–1709, 1988 active leukotriene B4 receptor antagonist. Adv Prostaglandin

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