Protective effect of a novel and selective inhibitor of inducible nitric oxide synthase on experimental crescentic glomerulonephritis in WKY rats
Daisuke Ogawa1,
Kenichi Shikata1,,
Mitsuhiro Matsuda1,
Shinichi Okada1,
Hitomi Usui1,
Jun Wada1,
Naoyuki Taniguchi2 and
Hirofumi Makino1
1 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama and
2 Department of Biochemistry, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Osaka, Japan
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Abstract
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Background. Nitric oxide (NO) plays important roles in a variety of pathophysiological processes. It has been reported that inducible NO synthase (iNOS) is upregulated in the glomeruli of patients with glomerulonephritis, although there has been no direct evidence that NO generated by iNOS contributes to the progression of glomerulonephritis. ONO-1714, a novel cyclic amidine analog, is a selective inhibitor of iNOS. To elucidate the role of iNOS in the pathogenesis of experimental crescentic glomerulonephritis, we examined the effect of ONO-1714 given to rats with nephrotoxic serum (NTS) nephritis.
Methods. We induced NTS nephritis in WistarKyoto (WKY) rats. These rats were given ONO-1714 or physiological saline intraperitoneally for 14 days using an osmotic pump after intraperitoneal injection with NTS.
Results. Glomerular expression of iNOS and urinary excretion of NO metabolites (nitrite/nitrate) were increased in rats after injection of NTS. As compared with the control group, ONO-1714 significantly reduced proteinuria, crescent formation, glomerular infiltration of macrophages and urinary excretion of nitrite/nitrate.
Conclusion. The present results suggest that NO radicals generated by iNOS contribute to the progression of experimental crescentic glomerulonephritis in WKY rats. The selective iNOS inhibitor ONO-1714 may be beneficial for the treatment of crescentic glomerulonephritis.
Keywords: crescent; glomerulonephritis; inducible nitric oxide synthase; macrophage; nitric oxide; ONO-1714
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Introduction
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Nitric oxide (NO) is an important mediator in a variety of physiological and pathophysiological processes. Three isoforms of NO synthase (NOS), neuronal or brain NOS (nNOS, type I), inducible NOS (iNOS, type II), and endothelial NOS (eNOS, type III), which produce NO from the amino acid, L-arginine, have been identified. In the kidney, nNOS is expressed in the macula densa, and has been suggested to participate in the tubuloglomerular feedback system [1]. eNOS is constitutively expressed in endothelial cells. The expression of eNOS is modulated by shear stress, endothelial cell proliferation and cytokines [2]. The expression of iNOS is induced in various types of cells, including macrophages, in the inflammatory lesions. Although iNOS expression is hardly detected in the normal kidney, mesangial cells, glomerular epithelial cells and infiltrating leukocytes express iNOS in human glomerulonephritis [3,4]. Elevation of iNOS mRNA expression has also been demonstrated in glomeruli in experimental glomerulonephritis [5]. NO may be protective because it inhibits platelet aggregation and leukocyte adhesion to endothelial cells [6]. However, an excessive amount of NO radicals may accelerate the tissue injury in inflammatory lesions [7]. No direct evidence has been provided as to whether iNOS is involved in the process of glomerular injury or crescent formation in glomerulonephritis. ONO-1714, a novel cyclic amidine analog, is a selective inhibitor of iNOS. In the present study, to elucidate the role of iNOS in the disease processes of crescentic glomerulonephritis, we examined the preventive effects of ONO-1714 on experimental crescentic glomerulonephritis in WKY rats.
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Materials and methods
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Preparation of nephrotoxic serum (NTS)
The preparation of NTS was described previously [8]. Briefly, normal Wistar rat kidneys were fully perfused with physiological saline through a catheter placed in the aorta. Renal cortical tissue was removed, homogenized and diluted with physiological saline at
20% suspension. Two milliliters of renal cortical homogenate were emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI). This emulsion was injected subcutaneously into rabbits once a week for 1 month. Seven days after the last injection, the rabbits were bled from the inferior vena cava under anesthesia. The sera were decomplemented for 30 min at 56°C and absorbed with freshly harvested rat erythrocytes. Preliminary immunohistochemical experiments showed that intraperitoneal injection of 1.0 ml of NTS into WistarKyoto (WKY) rats of
140 g resulted in linear binding of rabbit IgG along the glomerular basement membrane (GBM).
ONO-1714
ONO-1714, (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo-[4.1.0] heptane hydrochloride, was synthesized by Ono Pharmaceutical Co., Ltd (Osaka, Japan) as described previously [9]. ONO-1714, a cyclic amidine analog, inhibits human iNOS with a Ki of 1.88 nM and rodent iNOS with similar potency in vitro [9]. The agent was dissolved in physiological saline immediately before use.
Experimental design
WKY rats (female, 140 g) were obtained from Charles River Japan (Atsugi, Kanagawa, Japan), and fed on standard rat chow and water ad libitum. At first, to evaluate the iNOS expression in the kidney, kidney samples were obtained from normal WKY rats (n=3) and WKY rats injected with 1.0 ml of NTS intraperitoneally after 14 days (n=3). The frozen sections were stained with anti-iNOS antibody. The method is described in detail in the Histopathology section.
Sixteen rats were randomly divided into two groups and injected with 1.0 ml of NTS intraperitoneally. One group (n=8) received continuous intraperitoneal administration of 0.17 µg/kg/min of ONO-1714 by an osmotic pump (ALZA Co., Palo Alto, CA) for 14 days. Another control group (n=8) received administration of physiological saline instead of ONO-1714. At days 1, 4, 8, 11 and 14, urine samples were collected for 24 h and urinary protein levels were measured by using the Pyrogallol red method. Creatinine clearance was measured on day 14. Urine samples of normal and NTS-treated WKY rats were also obtained on day 14 for nitrite and nitrate assay. Systemic blood pressure was measured by tail-cuff plethysmography (UR-5000; Ueda Seisakusho, Tokyo, Japan). At day 14, the rats were killed under ether anesthesia, and both kidneys were removed.
Histopathology
Light microscopy
The renal tissue was fixed in 10% formalin and embedded in paraffin. Paraffin sections (4 µm) were stained with periodic acid-Schiff's reagent. A glomerulus was considered to exhibit crescent formation when two or more layers of cells were observed in Bowman's space. The number of glomeruli with crescent formation was counted in 50 glomeruli per rat.
Immunofluorescence staining
iNOS expression was examined by the indirect immunofluorescence method as previously described [8]. Briefly, the frozen sections (4 µm) were fixed with cold acetone for 3 min and stained with mouse anti-iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 24 h at 4°C. As a negative control, the primary antibody was omitted from the reaction. Then, the sections were stained with fluorescein isothiocyanate-labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 min at room temperature, and observed using a fluorescence microscope (LSM 510; Carl Zeiss, Jena, Germany).
Immunoperoxidase staining
Leukocyte infiltration in the glomerulus was examined by immunoperoxidase staining using a Vectastain ABC Kit and Avidin Biotin Blocking Kit (Vector Laboratories, Burlingame, CA) as described previously [8]. In brief, the frozen sections (4 µm) were fixed with cold acetone for 3 min and non-specific protein binding was blocked by incubating with normal goat serum and avidin for 20 min. The sections were incubated with mouse anti-rat monocyte/macrophage (ED1; Serotec, Oxford, UK) in a solution containing biotin for 60 min at room temperature. Then, the sections were incubated with biotin-labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories) for 30 min at room temperature. Endogenous peroxidase activity was inhibited by incubating in methanol containing 0.3% H2O2 for 30 min. After that, the sections were incubated with ABC reagent (containing avidin and biotinated-HRP) for 30 min. Peroxidase activity was developed in 3,3-diaminobenzidine and hydrogen peroxide. The sections were then counterstained with Mayer's hematoxylin. The number of ED1-positive cells per glomerulus was counted.
Measurement of urinary excretion of nitrite/nitrate
Nitrite and nitrate (NOx), oxidized forms of NO, in urine samples on day 14 were determined using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI). Basically, the nitrate in the sample was reduced to nitrite with a nitrate reductase contained in the assay kit; nitrite levels were then determined spectrophotometrically as the total NOx concentration.
Statistical analysis
All values are expressed as the mean±SEM. Statistical analyses were performed using the paired Student's t-test and one-way ANOVA. Values of P<0.05 were considered statistically significant. Each section was evaluated by two independent observers with no prior knowledge of the experimental design.
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Results
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Expression of iNOS in the glomeruli
Based on the results of the indirect immunofluorescence study, there was little or no expression of iNOS in the normal glomeruli (Figure 1A). In contrast, in the glomeruli of the rats injected with NTS, the expression of iNOS was prominent on leukocytes and glomerular cells in addition to the crescent portion (Figure 1B). Negative controls showed no staining in the glomerulus both in normal (Figure 1C) and NTS-treated rats (Figure 1D).

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Fig. 1. Distribution of iNOS in the glomerulus of a normal rat (A) and an NTS-treated rat (B) on day 14. In the NTS-treated kidney, iNOS expression is exhibited in glomerular cells and infiltrated leukocytes. On the other hand, in the normal rat kidney, iNOS is expressed faintly in the glomerulus. Negative controls showed no staining in the glomerulus both in normal (C) and NTS-treated rats (D). Original magnification,x200.
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Metabolic data
Figure 2 shows urinary protein excretion on days 1, 4, 8, 11 and 14 in the control and ONO-1714 groups. Before day 8, there was no difference between the groups in urinary protein excretion. However, after day 11, the urinary protein excretion in the ONO-1714 group decreased significantly compared with that in the control group (day 11, 21.1±7.1 vs 43.0±25.2 mg/day; day14, 99.1±22.4 vs 135.3±13.3 mg/day). No significant difference in systemic blood pressure and creatinine clearance was noted between ONO-1714-treated and control rats (Table 1).

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Fig. 2. Twenty four hour urinary protein excretion on days 1, 4, 8, 11 and 14 in the control group (open squares) and the ONO-1714 group (closed squares). Before day 8, there was no difference between the groups in urinary protein excretion; however, after day 11, the urinary protein excretion in the ONO-1714 group increased less significantly compared with that in the control group (mean±SEM; *P <0.05, **P <0.01).
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Renal histopathology
By light microscopy, the glomeruli in the normal rats showed no crescent formation. In the control group, severe leukocyte infiltration, mesangial matrix expansion, necrotizing lesions and cellular crescent formation were observed on day 14 (Figure 3A). On the other hand, in the ONO-1714 group, histological changes in the kidneys were mild (Figure 3B). The frequency of crescent formation was significantly suppressed in the ONO-1714 group compared with the control group (25.1±2.0 vs 35.4±2.7%).

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Fig. 3. Glomeruli of the control group (A) and the ONO-1714 group (B) on day 14. The light micrograph shows mononuclear cell infiltration, exadative lesions and crescent formation in the glomeruli of the control group. In contrast, the histological changes are mild in the ONO-1714 group. Original magnification,x200. Periodic Schiff staining.
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In the immunoperoxidase study, the control group exhibited a massive influx of macrophages into the glomeruli (Figure 4A). In contrast, in the ONO-1714 group, infiltration of macrophages was significantly decreased (Figure 4B). The number of macrophages in the glomeruli was significantly lower in the ONO-1714 group than in the control group (18.1±0.8 vs 16.0±1.5/glomerulus).

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Fig. 4. Intraglomerular macrophages in the control group (A) and the ONO-1714 group (B) on day 14. The control group shows a massive influx of macrophages into the glomerulus (A). In contrast, in the ONO-1714 group, infiltration of macrophages is significantly blunted (B). Original magnification,x200.
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Urinary excretion of nitrite/nitrate
In control rats, urinary excretion of nitrite/nitrate was significantly elevated on day 14 compared with that in normal rats. In ONO-1714-treated rats, urinary nitrite/nitrate levels were significantly reduced on day 14 compared with those in control rats (Figure 5).

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Fig. 5. Urinary excretion of nitrite/nitrate in normal (open squares), control (closed squares) and ONO-1714-treated rats (shaded squares) on day 14. In the ONO-1714 group, urinary excretion of nitrite/nitrate was significantly reduced compared with that in control rats (mean±SEM; *P <0.05, **P <0.01).
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Discussion
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WKY rats are known to be more susceptible to anti-GBM antibody than other strains, and develop progressive proteinuria and crescentic glomerulonephritis [10]. Kawasaki et al. [10] reported that this model showed the upregulation of intercellular adhesion molecule-1 (ICAM-1) in the glomeruli and a high influx of leukocytes mainly composed of macrophages and CD8-positive T lymphocytes, both of which play a central role in the progression of crescentic glomerulonephritis. Our previous study also demonstrated that the stable prostacyclin analog, beraprost, prevents glomerular crescent formation and proteinuria by reduction of both glomerular expression of ICAM-1 and macrophage infiltration [8]. These studies indicate that macrophages play a critical role in the development of crescentic glomerulonephritis.
There have been many reports suggesting the involvement of NO in the induction and progression of several types of experimental glomerulonephritis [5,11,12]. Macrophages express iNOS in several models of experimental glomerulonephritis, including immune complex glomerulonephritis [11], anti-Thy-1 glomerulonephritis [5] and crescentic glomerulonephritis [12]. It is well known that macrophages can generate high amounts of NO by iNOS for a prolonged period of time after stimulation by endotoxin or cytokines. It has been reported that L-NMMA, a non-selective NOS inhibitor, prevented mesangial cell lysis in rat Thy-1 nephritis [13]. In line with these findings, NO radicals may play important roles in the pathophysiology of glomerulonephritis, although it is still unclear whether NO radicals are protective or harmful in this disease [14].
ONO-1714, a novel cyclic amidine analog, is a potent inhibitor of iNOS. In fact, ONO-1714 was shown to have a 10-fold greater inhibitory effect on human iNOS than on human eNOS in vitro [9]. When the inhibitory activity of ONO-1714 on iNOS was compared with the activity of other NOS inhibitors in vivo, ONO-1714 was found to be 451-fold more potent than L-NMMA and >20 000-fold more potent than aminoguanidine [9]. The effects of ONO-1714 on the arginine pathway and the arginine transporter have not been examined. ONO-1714 has also shown anti-inflammatory effects in septic lung injury [15] and in dextran sulfate sodium colitis [16], although the effects of this agent on renal disease have not been tested.
The present study is the first report that a selective inhibitor of iNOS prevents glomerulonephritis. This agent significantly reduced urinary excretion of NO metabolites (nitrite/nitrate), proteinuria, the accumulation of macrophages in the glomeruli and crescent formation in experimental crescentic glomerulonephritis in WKY rats. No adverse effects, such as elevation of blood pressure, were seen during the course of this experiment. These results suggest that the decrease in the amount of NO radicals generated by iNOS resulted in the improvement in glomerular damage. High levels of NO radicals have been reported to inactivate iron-containing enzymes in the mitochondria, resulting in cytotoxicity [17]. We also reported that NO and its derivatives directly inactivate glutathione peroxidase, resulting in an increase in intracellular peroxides that are responsible for cellular damage [18]. Peroxinitrite produced by reaction of NO with superoxide anion mediates lipid peroxidation and nitration of tyrosine residues, leading to dysfunction of proteins [12,19]. The above mechanisms may participate in the development of crescentic glomerulonephritis. The present results strongly suggest that NO radicals contribute to the development of crescentic glomerulonephritis.
Cattel et al. [20] reported that proteinuria and glomerular macrophage infiltration was not attenuated in iNOS-deficient mice with anti-GBM glomerulonephritis; however, this might be due to either differences in the experimental animal or differences in the observed period. iNOS-deficient mice were killed 6 days after NTS injection in their experiment, while WKY rats were killed 14 days after NTS injection in our experiment. In our study, there was no difference between the control and ONO-1714 group in urinary protein excretion before day 8, the same as their results. However, on days 11 and 14, the urinary protein excretion in the ONO-1714 group decreased significantly compared with that in the control group (Figure 2).
In conclusion, our results suggest that a selective iNOS inhibitor, ONO-1714, ameliorates crescentic glomerulonephritis in WKY rats. These results provide the first direct evidence that a selective iNOS inhibitor may be beneficial for the treatment of crescentic glomerulonephritis.
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Acknowledgments
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A portion of this study was supported by a Grant-in-Aid for Scientific Research (C) (11671036 to K.S.) from the Ministry of Education, Science, Culture, Sports and Technology of Japan. We thank Ms Atsuko Yuasa (Okayama Central Hospital, Okayama, Japan) for excellent technical assistance.
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Notes
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Correspondence and offprint requests to: Kenichi Shikata, MD, PhD, Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.Email: shikata{at}md.okayama-u.ac.jp 
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Received for publication: 9.11.01
Accepted in revised form: 19. 6.02