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Vol. 112, No. 2, 2009
Issue release date: May 2009
Nephron Exp Nephrol 2009;112:e59–e69
(DOI:10.1159/000213896)

Mizoribine Suppresses the Progression of Experimental Peritoneal Fibrosis in a Rat Model

Takahashi S.a · Taniguchi Y.c · Nakashima A.b · Arakawa T.a · Kawai T.a · Doi S.a · Ito T.d · Masaki T.b · Kohno N.a · Yorioka N.b
Departments of aMolecular and Internal Medicine and bAdvanced Nephrology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, cDivision of Clinical Pharmacotherapeutics, Department of Pharmaceutical Science, Hiroshima International University, Kure, and dSection of Nephrology, Department of Internal Medicine, Shimane University Faculty of Medicine, Shimane, Japan
email Corresponding Author

Abstract

Background/Aims: Peritoneal fibrosis is a serious complication of peritoneal dialysis (PD). It has been reported that administration of mizoribine, an effective immunosuppressant, ameliorated renal fibrosis in a rat model of unilateral ureteral obstruction. We therefore examined the effects of mizoribine in an experimental model of peritoneal fibrosis. Methods: 24 rats were given a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline. The rats were divided into three groups (n = 8 per group) that received either vehicle or mizoribine at a dose of 2 or 8 mg/kg once a day. 28 days after the start of the treatments the rats were sacrificed and peritoneal tissue samples collected. Macrophage infiltration (ED1), myofibroblast accumulation (α-smooth muscle actin (SMA)) and expression of type III collagen, transforming growth factor (TGF)-β and monocyte chemotactic protein-1 (MCP-1) were examined by immunohistochemistry. Results: Mizoribine significantly suppressed submesothelial zone thickening and reduced macrophage infiltration. Mizoribine also reduced collagen III+ area and decreased the number of α-SMA+, TGF-β+ and MCP-1+ cells. The magnitude of the changes observed was dose-dependent. Conclusion: The administration of mizoribine prevented the progression of peritoneal fibrosis in this rat model. Mizoribine may represent a novel therapy for peritoneal sclerosis in patients undergoing long-term PD.


 Outline


 goto top of outline Key Words

  • Macrophage
  • Mizoribine
  • Peritoneal dialysis
  • Peritoneal fibrosis
  • Experimental peritoneal fibrosis, rat model

 goto top of outline Abstract

Background/Aims: Peritoneal fibrosis is a serious complication of peritoneal dialysis (PD). It has been reported that administration of mizoribine, an effective immunosuppressant, ameliorated renal fibrosis in a rat model of unilateral ureteral obstruction. We therefore examined the effects of mizoribine in an experimental model of peritoneal fibrosis. Methods: 24 rats were given a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline. The rats were divided into three groups (n = 8 per group) that received either vehicle or mizoribine at a dose of 2 or 8 mg/kg once a day. 28 days after the start of the treatments the rats were sacrificed and peritoneal tissue samples collected. Macrophage infiltration (ED1), myofibroblast accumulation (α-smooth muscle actin (SMA)) and expression of type III collagen, transforming growth factor (TGF)-β and monocyte chemotactic protein-1 (MCP-1) were examined by immunohistochemistry. Results: Mizoribine significantly suppressed submesothelial zone thickening and reduced macrophage infiltration. Mizoribine also reduced collagen III+ area and decreased the number of α-SMA+, TGF-β+ and MCP-1+ cells. The magnitude of the changes observed was dose-dependent. Conclusion: The administration of mizoribine prevented the progression of peritoneal fibrosis in this rat model. Mizoribine may represent a novel therapy for peritoneal sclerosis in patients undergoing long-term PD.

Copyright © 2009 S. Karger AG, Basel


goto top of outline Introduction

Peritoneal dialysis (PD) has been used for more than two decades as an attractive treatment for end-stage kidney disease. However, long-term PD treatment is associated with histopathological alterations in the peritoneum [1]. Continuous exposure to bioincompatible dialysis fluids and repeated episodes of bacterial peritonitis play a major role in the alteration of peritoneal function and structure that occurs with time [2, 3]. The characteristic pathologic findings in the peritoneum of patients on long-term continuous ambulatory peritoneal dialysis (CAPD) therapy include marked peritoneal fibrosis with marked accumulation of collagen and loss of peritoneal mesothelial cells [4]. Indeed, a minority of patients may develop the serious complication of encapsulating peritoneal sclerosis (EPS) that is associated with a high mortality and characterized by severe progressive peritoneal fibrosis [5]. Therapeutic strategies for EPS are limited and include the appropriate use of steroids [6]. However, there is no experimental model for peritoneal fibrosis that is similar to the fibrosis that develops in patients on CAPD.

Suga et al. [7] developed an experimental model of peritoneal fibrosis in rats induced by the peritoneal injection of chlorhexidine gluconate (CG). CG-induced peritoneal fibrosis in rats is very similar to that seen in patients with EPS as many of the pathologic findings in the peritoneum of CAPD patients including the increased expression of type III collagen, α-smooth muscle actin (α-SMA) and macrophage infiltration were also observed in the peritoneum of animals injected with CG [8].

Mizoribine is an imidazole nucleoside isolated from Eupenicillium brefeldianum and is an orally administered immunosuppressive agent. Mizoribine inhibits the conversion of inosine 5′-nucleotide to guanosine 5′-nucleotide in the purine nucleotide biosynthetic pathway and has similar immunosuppressive effects upon both humoral and cellular immunity to mycophenolate mofetil [9,10,11]. The efficacy of this agent has been demonstrated in patients with diverse conditions including renal transplant recipients [12] and patients with rheumatoid arthritis [13], Sjögren’s syndrome [14], lupus nephritis [15] and primary nephrotic syndrome [16]. Moreover, the incidence of adverse effects including myelosuppression, hepatotoxicity and nephrotoxicity is lower with this drug than other immunosuppressive agents. Furthermore, recent studies have demonstrated that mizoribine improves renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) in the rat [17] in a dose-dependent manner [18].

In this study, we examined the therapeutic efficacy of mizoribine in a rat model of peritoneal fibrosis induced by the administration of CG.

 

goto top of outline Methods

goto top of outline Animals

52 Wistar rats weighing 190–200 g were obtained from Charles River Laboratories Japan (Yokosuka, Japan). The animals were housed in the animal facility of Hiroshima University with free access to food and water. The Institutional Animal Care and Use Committee at Hiroshima University (Hiroshima, Japan) approved all the animal protocols and the experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals.

goto top of outline Experimental Protocol

Peritoneal fibrosis was induced by the intraperitoneal injection of 0.1% CG in 15% ethanol dissolved in saline as described previously [19]. Briefly, rats received a daily intraperitoneal injection of 0.1% CG in 15% ethanol dissolved in 2 ml of saline for a period of 28 days. The intraperitoneal injection of CG was performed under anesthesia with isoflurane in order to ensure the accuracy of the injection. Intraperitoneal injections of CG were performed in the left portion of the abdomen, whereas the right portion of the peritoneum was processed for histological evaluation in order to avoid mechanical damage of the peritoneum caused by repeated injections confounding the findings.

The rats were divided into three groups that received either: (1) vehicle buffer (CG + vehicle group, n = 8), (2) mizoribine at 2 mg/kg body weight (b.w.) (CG + 2 mg mizoribine group, n = 8), (3) mizoribine at 8 mg/kg b.w. (CG + 8 mg mizoribine group, n = 8).

Mizoribine (Asahi Kasei Pharma Corp., Tokyo, Japan) was dissolved in 0.5 ml of distilled water and administered by daily oral gavage just before the CG injection. Control rats received a daily intraperitoneal injection of vehicle only (15% ethanol dissolved in 2 ml of saline) for 28 days (control group, n = 8). Rats were killed 28 days after starting CG injection and the parietal peritoneal tissues were carefully dissected prior to fixation in 10% formalin and embedding in paraffin.

goto top of outline Histological Analysis

Formalin-fixed, paraffin-embedded sections (4 μm) were stained with hematoxylin and eosin (HE) for light microscopic observation. Cross sections of the abdominal wall were examined and the thickness of the submesothelial collagenous zone above the abdominal muscle layer was defined as the peritoneal thickness [20]. The extent of peritoneal thickening was determined by analysis of digitized images using image analysis software NIS-Elements D (Nikon Corp., Tokyo, Japan). The image was transformed into a matrix of 1,280 × 960 pixels and viewed at ×100 magnification. We selected a width of 840 μm in the examined field under the microscope and measured the area of the submesothelial layer within this selected width. For each sample, eight such areas were selected and the average area of the submesothelial layer was determined.

goto top of outline Immunohistochemistry Analysis

Immunohistochemical analyses were performed using 4-μm tissue sections as described previously [21, 22]. The following primary antibodies were used: (1) mouse monoclonal anti-rat ED1 antibody as a macrophage marker (1:200 dilution, MCA341R; Serotec, Oxford, UK); (2) polyclonal rabbit anti-rat type III collagen antibody (1:500 dilution, AB757P; Chemicon International Inc., Temecula, Calif., USA); (3) mouse monoclonal anti-α-SMA (1:1,000 dilution, A2547; Sigma, St. Louis, Mo., USA); (4) polyclonal rabbit anti-mouse transforming growth factor (TGF-β1) antibody (1:1,000 dilution, sc-146; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA); (5) polyclonal rabbit anti-rat monocyte chemotactic protein-1 (MCP-1) antibody (1:250 dilution, FL-148; Santa Cruz Biotechnology, Inc.). Tissue sections were placed in 0.01 M citrate buffer (pH 6.0) and heated for 10 min in a microwave oven. This treatment was used for ED1, TGF-β1, and MCP-1 staining. Sections were blocked in 5% fetal calf serum, 5% bovine serum albumin and 10% normal goat serum in phosphate-buffered saline for 60 min and then incubated overnight at 4°C with primary antibody diluted in 10% normal goat serum and 5% normal rat serum. After washing, endogenous peroxidase activity was blocked by incubating tissue sections in 0.6% H2O2 in methanol for 20 min. For ED1 and α-SMA immunostaining, tissue sections were incubated with goat anti-mouse immunoglobulin-G (IgG) conjugated with horseradish peroxidase (HRP, P0447; Dako, Glostrup, Denmark, diluted 1/50) for 45 min at room temperature followed by a complex of HRP-conjugated mouse anti-HRP IgG (P0850; Dako, diluted 1/50) for 45 min at room temperature. For type III collagen and MCP-1 immunostaining, tissue sections were incubated with goat anti-rabbit IgG conjugated with HRP (P0448; Dako, diluted 1/50) for 45 min at room temperature followed by a complex of HRP-conjugated rabbit anti-HRP IgG (Z0113; Dako, diluted 1/50) for 45 min at room temperature. Immunostaining for TGF-β1 was conducted using the Vectastain ABC Elite reagent kit (Vector Laboratories, Burlingame, Calif., USA) according to the manufacturer’s protocol. Goat anti-rabbit IgG (1:250 dilution, 65-6140; Zymed, Carlsbad, Calif., USA) was used as a secondary antibody. Specific antibody binding was detected by color development following reaction with H2O2 and 3,3-diaminobenzidine tetrahydrochloride. In each peritoneal sample, the numbers of ED1-positive cells, α-SMA-positive cells, TGF-β-positive cells and MCP-1-positive cells were counted in 10 fields (×400 magnification). In order to assess the area positive for type III collagen immunostaining, the image files (1,280 × 960) at ×200 magnification were analyzed using Lumina Vision software (Mitani, Fukui, Japan). The positive area was shown as the mean of diaminobenzidine-positive pixel values obtained from five image files in each section.

Two color immunostaining was used to detect colocalization of ED1 and TGF-β. After staining for TGF-β and development with diaminobenzidine to give a brown color, the sections were placed in 0.01 M citrate buffer (pH 6.0) and heated for 10 min in a microwave oven. The sections were then blocked as described above, incubated overnight at 4°C with the monoclonal anti-ED1 antibody and incubated further with goat anti-mouse HRP for 45 min at room temperature. This was followed by incubation for 45 min at room temperature in a complex of HRP-conjugated mouse anti-HRP IgG and development with Vector SG to give a blue/gray color.

goto top of outline Pharmacokinetic Analysis

20 female Wistar rats weighing 190–200 g were used for the pharmacokinetic analysis. Rats were divided into two groups with 10 rats receiving mizoribine at 2 mg/kg b.w. by oral gavage (2 mg mizoribine group) and the remaining 10 rats receiving mizoribine at 8 mg/kg b.w. by oral gavage (8 mg mizoribine group). In each group, blood samples were collected from 5 rats at 0.5, 2 and 4 h after administering mizoribine, whilst the blood samples of the remaining 5 rats were collected 1, 3 and 6 h after administering mizoribine. To minimize the influence of blood loss upon the pharmacokinetic analysis, we collected blood three times per rat. The serum concentration of mizoribine was determined by high-performance liquid chromatography [23]. The simulation values of pharmacokinetics analysis were performed with the statistical software WinNonlin Version 5.2 (Pharsight Corp., Mountain View, Calif., USA) and fitted to the one-compartment model.

goto top of outline Statistical Analysis

Results are expressed as means ± SE for each group. Statistical analysis was performed with analysis of variance by Tukey’s post-hoc test. Data differences were deemed significant at p < 0.05.

 

goto top of outline Results

goto top of outline Morphologic Examination

Morphologic changes were assessed by HE staining. A monolayer of mesothelial cells was observed in normal rats covering the peritoneal surface without any thickening of the peritoneum (fig. 1a). The appearance of the peritoneum of control rats receiving intraperitoneal vehicle only (15% ethanol dissolved in saline) was similar to that of the normal rats (fig. 1b). The daily intraperitoneal injection of CG for 28 days resulted in marked thickening of the submesothelial compact zone associated with increased cellularity (fig. 1c). The administration of mizoribine suppressed both the thickness of the submesothelial zone and the increased cellularity induced by CG (fig. 1d, e). Morphological evaluation revealed a significant inhibitory effect of mizoribine on CG-induced peritoneal thickening with this anti-fibrotic effect being dose-dependent (control group 17.4 ± 1.7 × 103 μm2; CG + vehicle group 222.7 ± 8.5 × 103 μm2; CG + mizoribine 2 mg group 128.5 ± 4.1 × 103 μm2; CG + mizoribine 8 mg group 85.8 ± 3.3 × 103 μm2; fig. 1f).

FIG01
Fig. 1. Histological appearance of the peritoneum. Representative light microscopic appearance of peritoneal tissues (HE, ×100) in (a) normal rats, (b) 15% ethanol/saline-injected rats (control group C), (c) CG-injected rats treated with the vehicle (CG + vehicle group V), (d) CG-injected rats treated with mizoribine at a dose of 2 mg/kg (CG + 2 mg mizoribine group M2), and (e) CG-injected rats treated with mizoribine at a dose of 8 mg/kg (CG + 8 mg mizoribine group M8). (f) The increased thickness of the submesothelial compact zone in the CG-injected rats was diminished in a dose-dependent manner by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.01 vs. C, # p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Macrophage Infiltration

We examined the peritoneal expression of the macrophage marker ED1. ED1+ cells were rarely observed in the control group (1.4 ± 0.6 ED1+ cells/10 fields; fig. 2a). The number of ED1+ cells in the peritoneum was significantly increased in rats receiving injections of CG (491.1 ± 34.5 ED1+ cells/10 fields, p < 0.01; fig. 2b, e) compared with the control group. Daily treatment with 2 mg of mizoribine significantly suppressed macrophage infiltration of the peritoneum induced by CG injection compared with the group receiving CG and vehicle (189.8 ± 7.7 vs. 491.1 ± 34.5 ED1+ cells/10 fields, p < 0.01; fig. 2c, e). In addition, the number of ED1+ cells in the peritoneum was further decreased in rats treated with 8 mg of mizoribine per day compared to rats treated with 2 mg of mizoribine daily thereby indicating a dose-dependent effect (93.0 ± 8.4 vs. 189.8 ± 7.7 ED1+ cells/10 fields, p < 0.01; fig. 2d, e). The number of ED1+ cells per submesothelial area (mm2) was also analyzed and the results were similar to those expressed as the number of ED1+ cells per field with mizoribine exerting a significant anti-inflammatory and therapeutic effect (table 1). These data suggested that the marked reduction in the number of ED1+ macrophages was not merely a reflection of reduced submesothelial tissue volume. These results demonstrate that treatment with mizoribine significantly inhibits infiltration of the peritoneum by ED1+ macrophages following the administration of CG, with this action possibly being related to the potent immunosuppressive effect of mizoribine.

TAB01
Table 1. Morphometric analysis of immunohistochemical findings

FIG02
Fig. 2. Immunohistochemical analysis of ED1 expression. Representative light microscopic appearance of peritoneal tissues (×200) in (a) control group, (b) CG + vehicle group, (c) CG + 2 mg mizoribine group and (d) CG + 8 mg mizoribine group. (e) The increased number of ED1+ cells in rats injected with CG was decreased in a dose-dependent manner by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.01 vs. C, #p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Immunohistochemical Analysis of Type III Collagen

To examine the effects of mizoribine on peritoneal fibrosis induced by CG, we next examined the expression of type III collagen. Marked expression of type III collagen in the submesothelial zone was observed in rats receiving CG compared to the control group receiving vehicle alone (573.2 ± 28.6 vs. 6.6 ± 1.4 × 103 pixels, p < 0.01; fig. 3a, b, e). Daily treatment with 2 mg of mizoribine significantly reduced the peritoneal expression of type III collagen induced by CG compared to rats receiving CG alone (306.8 ± 15.5 vs. 573.2 ± 28.6 × 103 pixels, p < 0.01; fig. 3c, e). In addition, the expression of type III collagen in the submesothelial zone was further and significantly decreased in rats treated with 8 mg of mizoribine per day compared to rats treated with 2 mg of mizoribine per day thereby indicating a dose-dependent therapeutic and anti-fibrotic effect (181.7 ± 11.8 vs. 306.8 ± 15.5 ×103 pixels, p < 0.01; fig. 3d, e).

FIG03
Fig. 3. Immunohistochemical analysis of type III collagen expression. Representative light microscopic appearance of peritoneal tissues (×200) in (a) control group, (b) CG + vehicle group, (c) CG + 2 mg mizoribine group and (d) CG + 8 mg mizoribine group. (e) The increased number of type III collagen+ pixels in rats injected with CG was decreased in a dose-dependent manner by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.01 vs. C, # p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Immunohistochemical Analysis of α-SMA Expression

We also determined the number of α-SMA+ myofibroblasts in the peritoneum associated with peritoneal fibrosis. In the control group, the expression of α-SMA was only observed in vascular smooth muscle cells (6.9 ± 2.5 α-SMA+ cells/10 fields; fig. 4a). In rats administered CG, α-SMA expression was found in myofibroblasts in addition to vascular smooth muscle cells with numerous α-SMA+ myofibroblasts evident in the thickened peritoneal tissues (707.8 ± 36.1 α-SMA+ cells/10 fields; fig. 4b). Treatment with mizoribine significantly inhibited the increase in peritoneal α-SMA+ myofibroblast number induced by CG compared to rats administered CG alone (291.0 ± 24.1 vs. 707.8 ± 36.1 α-SMA+ cells/10 fields, p < 0.01; fig. 4c, e). The increase in the number of α-SMA+ cells in the peritoneum was further and significantly suppressed in rats treated with 8 mg mizoribine daily compared rats treated with 2 mg mizoribine (140.8 ± 15.5 vs. 291.0 ± 24.1 α-SMA+ cells/10 fields, p < 0.01; fig. 4d, e). The number of α-SMA+ cells per submesothelial area (mm2) was also determined and similar results were obtained (table 1) thereby indicating that the reduction in the number of α-SMA+ cells in mizoribine-treated rats was not merely a reflection of reduced submesothelial tissue volume.

FIG04
Fig. 4. Immunohistochemical analysis of α-SMA expression. Representative light microscopic appearance of peritoneal tissues (×200) in (a) control group, (b) CG + vehicle group, (c) CG + 2 mg mizoribine group and (d) CG + 8 mg mizoribine group. (e) The increased number of α-SMA+ cells in rats administered CG was dose-dependently decreased by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.01 vs. C, # p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Immunohistochemical Analysis of TGF-β

We next examined the peritoneal expression of the profibrotic growth factor TGF-β that is associated with peritoneal fibrosis. In the control group, TGF-β+ cells were rarely observed (6.0 ± 1.9 TGF-β+ cells/10 fields; fig. 5a) and immunoreactivity for TGF-β was observed in the submesothelial area and in the fibrotic layer. The number of peritoneal TGF-β+ cells was increased markedly in rats receiving CG alone compared to the control group (617 ± 27.6 vs. 6.0 ± 1.9 TGF-β+ cells/10 fields, p < 0.01; fig. 5b, e). Daily treatment with 2 mg of mizoribine significantly suppressed the increase in the number of peritoneal TGF-β+ cells compared to rats administered CG alone (249.4 ± 11.3 vs. 617 ± 27.6 TGF-β+ cells/10 fields, p < 0.01; fig. 5c, e). The number of peritoneal TGF-β+ cells was further reduced in rats treated with 8 mg mizoribine daily compared to rats receiving 2 mg of mizoribine (127.6 ± 7.6 vs. 249.4 ± 11.3 TGF-β+ cells/10 fields, p < 0.01; fig. 5d, e). The number of TGF-β+ cells per submesothelial area (mm2) was also determined and similar results were observed (table 1) suggesting that the reduction in the number of TGF-β+ cells was not merely a reflection of reduced submesothelial tissue volume.

FIG05
Fig. 5. Immunohistochemical analysis of TGF-β expression. Representative light microscopic appearance of peritoneal tissues (×200) in (a) control group, (b) CG + vehicle group, (c) CG + 2 mg mizoribine group and (d) CG + 8 mg mizoribine group. (e) The increased number of TGF-β+ cells in rats administered CG was dose-dependently decreased by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.01 vs. C, # p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Immunohistochemical Analysis of MCP-1

We next examined the peritoneal expression of MCP-1, a chemotactic factor which attracts monocytes. MCP-1+ cells were observed rarely in the control group (29.0 ± 11.3 MCP-1+ cells/10 fields; fig. 6a). The number of peritoneal MCP-1+ cells was increased markedly in rats receiving CG alone compared to the control group (1,338.4 ± 102.4 vs. 29.0 ± 11.3 MCP-1+ cells/10 fields, p < 0.01; fig. 6b, e). Daily treatment with 2 mg of mizoribine significantly suppressed the increase in the number of peritoneal MCP-1+ cells compared to rats administered CG alone (476.5 ± 50.4 vs. 1,338.4 ± 102.4 MCP-1+ cells/10 fields, p < 0.01; fig. 6c, e). The number of peritoneal MCP-1+ cells was further reduced in rats treated with 8 mg mizoribine daily compared to rats receiving 2 mg of mizoribine (228.3 ± 28.1 vs. 476.5 ± 50.4 MCP-1+ cells/10 fields, p < 0.01; fig. 6d, e). The number of MCP-1+ cells per submesothelial area (mm2) was also determined and similar results were observed (table 1) suggesting that the reduction in the number of MCP-1+ cells was not merely a reflection of reduced submesothelial tissue volume.

FIG06
Fig. 6. Immunohistochemical analysis of MCP-1 expression. Representative light microscopic appearance of peritoneal tissues (×200) in (a) control group, (b) CG + vehicle group, (c) CG + 2 mg mizoribine group and (d) CG + 8 mg mizoribine group. (e) The increased number of MCP-1+ cells in rats administered CG was dose-dependently decreased by treatment with mizoribine. Values are expressed as the mean ± SE. * p < 0.05 vs. C, ** p < 0.01 vs. C, # p < 0.01 vs. V, † p < 0.01 vs. M2, by Tukey’s post-hoc test.

goto top of outline Double Staining of ED1 and TGF-β

We also performed double staining of ED1 and TGF-β. The majority of ED1+ macrophages in the submesothelial compact zone showed immunoreactivity for TGF-β (fig. 7), demonstrating colocalization of ED1 and TGF-β.

FIG07
Fig. 7. Two-color immunohistochemistry of ED1 and TGF-β. Representative light microscopic appearance of peritoneal tissues (×400). The majority of ED1+ cells (blue/gray) in the submesothelial compact zone showed immunoreactivity for TGF-β (brown).

goto top of outline Pharmacokinetic Parameters

The change of serum concentration of mizoribine over time is shown in figure 8 and the pharmacokinetic profile of mizoribine was analyzed using this data (table 2). The time to maximum serum concentration (Tmax) of mizoribine was 1.04 h in the group treated with 2 mg mizoribine and 1.21 h in the 8 mg mizoribine group. The maximum serum concentration (Cmax) of mizoribine was 0.514 μg/ml in the group treated with 2 mg mizoribine and 2.214 μg/ml in the 8 mg mizoribine group. The elimination half-life (T1/2) of mizoribine was 1.42 h in the group treated with 2 mg mizoribine and 1.68 h in the 8 mg mizoribine group. The area under the serum concentration-time curve infinity (AUCinf) of mizoribine was 1.61 μg·h/ml in the group treated with 2 mg mizoribine and 8.46 μg·h/ml in the 8 mg mizoribine group.

TAB02
Table 2. Pharmacokinetic parameter (one-compartment model)

FIG08
Fig. 8. Time-related changes in serum mizoribine concentration.

 

goto top of outline Discussion

In the present study, we demonstrated that administration of mizoribine markedly reduced macrophage infiltration in the experimental rat model of peritoneal fibrosis induced by CG with this effect being dose-dependent. Moreover, treatment with mizoribine diminished collagen accumulation in the thickened submesothelial area. These findings indicate that mizoribine might be useful in preventing the progression of peritoneal fibrosis and implicate macrophage infiltration, at least in part, in the development of peritoneal fibrosis.

The inhibitory effect of mizoribine upon the number of ED1+ macrophages in the thickened submesothelial area may be mediated via the potent immunosuppressive action of mizoribine. Mizoribine inhibits both humoral and cellular immunity via the inhibition of de novo purine biosynthesis [9]. Sato et al. [18] reported that treatment with mizoribine significantly ameliorated tubulointerstitial fibrosis and infiltration by macrophages and T lymphocytes in rats with UUO. Kikuchi et al. [24] also showed mizoribine inhibited renal macrophage accumulation and prevented the progression of glomerulosclerosis and interstitial fibrosis in non-insulin-dependent diabetic rats. It was reported that macrophages can synthesize extracellular matrix protein such as collagen and fibronectin which may cause fibrosis [25, 26].In this regard, the infiltrating macrophages secrete important soluble factors such as TGF-β [27], and it is therefore possible that the decrease in submesothelial area caused by administration of mizoribine may be associated closely with the inhibitory effect upon macrophage infiltration.

Characteristic histological changes of peritoneal fibrosis such as thickening of the submesothelial area and the accumulation of type III collagen were significantly inhibited by treatment with mizoribine. The pivotal role of TGF-β1 in the development of peritoneal fibrosis results from the effect of TGF-β1 upon human peritoneal mesothelial cells including the induction of epithelial mesenchymal transition and the de novo synthesis of type III collagen [28, 29]. Immunohistochemical analysis indicated a significant inhibitory effect of mizoribine on the number of TGF-β+ cells in the peritoneum. Thus, the inhibitory effect of mizoribine on the accumulation of type III collagen induced by administration of CG may be partly mediated via its inhibitory effect upon TGF-β expression. Immunoreactivity for α-SMA and TGF-β1 was observed in the submesothelial area in rats administered CG. Treatment with mizoribine suppressed the increase in the number of peritoneal α-SMA+ cells in rats administered CG in parallel with its inhibitory effect on TGF-β expression and accumulation of extracellular matrix. The de novo synthesis of α-SMA by human peritoneal mesothelial cells after TGF-β1 stimulation has been reported [29], and it is possible that the therapeutic effect of mizoribine on peritoneal fibrosis may partly result from its regulatory role upon the conversion of peritoneal mesothelial cells to myofibroblasts.

The expression of MCP-1, a chemotactic factor which attracts monocytes, was inhibited significantly by treatment with mizoribine. Mizoribine inhibited upregulation of chemokines such as MCP-1, as well as suppressing the proliferation of macrophages. As macrophage accumulation in the thickened submesothelial area was suppressed, the production of TGF-β may also have been inhibited, resulting in prevention of peritoneal fibrosis. The colocalization of ED1 and TGF-β observed with double immunostaining supports this hypothesis.

For the analysis of immunohistochemical findings, we also determined the number of ED1+, α-SMA+, TGF-β+ and MCP-1+ cells per submesothelial area (mm2). The results were similar to the measurement of cell number per field, thereby demonstrating that the significant therapeutic effects of mizoribine did not merely reflect the differences in submesothelial thickening between experimental groups.

No adverse events were observed in this study despite the fact that the higher dose of mizoribine used was greater than the conventional dose of mizoribine used (up to 5 mg/kg b.w.). Stypinski et al. [30] reported that the daily administration of mizoribine up to 12 mg/kg b.w. caused no significant adverse events in healthy male volunteers except for a slight elevation in serum uric acid. Tanaka et al. [31] reported that a peak serum level of mizoribine of at least 2.5–3.0 μg/ml is necessary to achieve satisfactory clinical efficacy of the drug in the treatment of lupus nephritis. Pharmacokinetic analysis of the rats in this study indicated that the Cmax was lower than 2.5–3.0 μg/ml when the dose of mizoribine administered was 8 mg/kg b.w. Moreover, rats receiving 8 mg mizoribine daily exhibited a more marked amelioration in peritoneal fibrosis than rats receiving 2 mg mizoribine. Therefore, the administration of mizoribine at a dose of 8 mg/kg b.w. is optimal for preventing the progression of peritoneal fibrosis in rats.

In the present study, we induced peritoneal fibrosis by the intraperitoneal injection of CG. It is true that CG-induced peritoneal fibrosis does not fully replicate the peritoneal sclerosis or EPS observed in patients on long-term PD. However, there is not an ideal experimental model that simulates long-term PD, and CG-induced peritoneal fibrosis undoubtedly exhibits key features of peritoneal fibrosis.

In conclusion, the administration of mizoribine prevented the progression of peritoneal fibrosis in a rat model. Mizoribine is a potentially useful therapy for peritoneal sclerosis in patients undergoing long-term PD. As mizoribine has lower nephrotoxicity than other immunosuppressive agents, its benefit may be in preserving residual renal function of PD patients in comparison with other immunosuppressive agents.

 

goto top of outline Acknowledgement

The authors would like to thank Asahi-Kasei Pharm, Tokyo, for conducting the measurements of the blood levels of mizoribine.


 goto top of outline References
  1. Selgas R, Fernandez-Reyes MJ, Bosque E, Bajo MA, Borrego F, Jimenez C, Del Peso G, De Alvaro F: Functional longevity of the human peritoneum: how long is continuous peritoneal dialysis possible? Results of a prospective medium long-term study. Am J Kidney Dis 1994;23:64–73.
  2. Davies SJ, Bryan J, Phillips L, Russell GI: Longitudinal changes in peritoneal kinetics: the effects of peritoneal dialysis and peritonitis. Nephrol Dial Transplant 1996;11:498–506.
  3. Ito T, Yorioka N: Peritoneal damage by peritoneal dialysis solutions. Clin Exp Nephrol 2008;12:243–249.
  4. Williams JD, Craig KJ, Topley N, Von Ruhland C, Fallon M, Newman GR, Mackenzie RK, Williams GT: Morphologic changes in the peritoneal membrane of patients with renal disease. J Am Soc Nephrol 2002;13:470–479.
  5. Gandhi VC, Humayun HM, Ing TS, Daugirdas JT, Jablokow VR, Iwatsuki S, Geis WP, Hano JE: Sclerotic thickening of the peritoneal membrane in maintenance peritoneal dialysis patients. Arch Intern Med 1980;140:1201–1203.
  6. Kawanishi H, Moriishi M: Encapsulating peritoneal sclerosis: prevention and treatment. Perit Dial Int 2007;27(suppl 2):S289–S292.

    External Resources

  7. Suga H, Teraoka S, Ota K, Komemushi S, Furutani S, Yamauchi S, Margolin S: Preventive effect of pirfenidone against experimental sclerosing peritonitis on rats. Exp Toxicol Pathol 1995;47:287–291.
  8. Mishima Y, Miyazaki M, Abe K, Ozono Y, Shioshita K, Xia Z, Harada T, Taguchi T, Koji T, Kohno S: Enhanced expression of heat shock protein 47 in rat model of peritoneal fibrosis. Perit Dial Int 2003;23:14–22.
  9. Koyama H, Tsuji M: Genetic and biochemical studies on the activation and cytotoxic mechanism of bredinin, a potent inhibitor of purine biosynthesis in mammalian cells. Biochem Pharmacol 1983;32:3547–3553.
  10. Kusumi T, Tsuda M, Katsunuma T, Yamamura M: Dual inhibitory effect of bredinin. Cell Biochem Funct 1989;7:201–204.
  11. Ishikawa H: Mizoribine and mycophenolate mofetil. Curr Med Chem 1999;6:575–597.
  12. Akiyama T, Okazaki H, Takahashi K, Hasegawa A, Tanabe K, Uchida K, Takahara S, Toma H: Mizoribine in combination therapy with tacrolimus for living donor renal transplantation: analysis of a nationwide study in Japan. Transplant Proc 2005;37:843–845.
  13. Tanaka E, Inoue E, Kawaguchi Y, Tomatsu T, Yamanaka H, Hara M, Kamatani N: Acceptability and usefulness of mizoribine in the management of rheumatoid arthritis in methotrexate-refractory patients and elderly patients, based on analysis of data from a large-scale observational cohort study. Mod Rheumatol 2006;16:214–219.
  14. Nakayamada S, Saito K, Umehara H, Ogawa N, Sumida T, Ito S, Minota S, Nara H, Kondo H, Okada J, Mimori T, Yoshifuji H, Sano H, Hashimoto N, Sugai S, Tanaka Y: Efficacy and safety of mizoribine for the treatment of Sjögren’s syndrome: a multicenter open-label clinical trial. Mod Rheumatol 2007;17:464–469.
  15. Tanaka H, Tsugawa K, Tsuruga K, Suzuki K, Nakahata T, Ito E, Waga S: Mizoribine for the treatment of lupus nephritis in children and adolescents. Clin Nephrol 2004;62:412–417.
  16. Honda M: Nephrotic syndrome and mizoribine in children. Pediatr Int 2002;44:210–216.
  17. Sakai T, Kawamura T, Shirasawa T: Mizoribine improves renal tubulointerstitial fibrosis in unilateral ureteral obstruction-treated rat by inhibiting the infiltration of macrophages and the expression of α-smooth muscle actin. J Urol 1997;158:2316–2322.
  18. Sato N, Shiraiwa K, Kai K, Watanabe A, Ogawa S, Kobayashi Y, Yamagishi-Imai H, Utsunomiya Y, Mitarai T: Mizoribine ameliorates the tubulointerstitial fibrosis of obstructive nephropathy. Nephron 2001;89:177–185.
  19. Nishino T, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T, Ozono Y, Koji T, Kohno S: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats. Kidney Int 2003;64:887–896.
  20. Yoshio Y, Miyazaki M, Abe K, Nishino T, Furusu A, Mizuta Y, Harada T, Ozono Y, Koji T, Kohno S: TNP-470, an angiogenesis inhibitor, suppresses the progression of peritoneal fibrosis in mouse experimental model. Kidney Int 2004;66:1677–1685.
  21. Doi S, Masaki T, Arakawa T, Takahashi S, Kawai T, Nakashima A, Naito T, Kohno N, Yorioka N: Protective effects of peroxisome proliferator-activated receptor γ ligand on apoptosis and hepatocyte growth factor induction in renal ischemia-reperfusion injury. Transplantation 2007;84:207–213.
  22. Hirai T, Masaki T, Kuratsune M, Yorioka N, Kohno N: PDGF receptor tyrosine kinase inhibitor suppresses mesangial cell proliferation involving STAT3 activation. Clin Exp Immunol 2006;144:353–361.
  23. Hosotsubo H, Takahara S, Taenaka N: Simplified high-performance liquid chromatographic method for determination of mizoribine in human serum. J Chromatography 1988;432:340–345
  24. Kikuchi Y, Imakiire T, Yamada M, Saigusa T, Hyodo T, Hyodo N, Suzuki S, Miura S: Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Nephrol Dial Transplant 2005;20:1573–1581.
  25. Vaage J, Lindblad WJ: Production of collagen type I by mouse peritoneal macrophages. J Leukoc Biol 1990;48:274–280.
  26. Nathan CF: Secretory products of macrophages. J Clin Invest 1987;79:319–326.
  27. Diamond JR, Kees-Folts D, Ding G, Frye JE, Restrepo NC: Macrophages, monocyte chemoattractant peptide-1, and TGF-β1 in experimental hydronephrosis. Am J Physiol 1994;266:F926–F933.
  28. Margetts PJ, Bonniaud P, Liu L, Hoff CM, Holmes CJ, West-Mays JA, Kelly MM: Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol 2005;16:425–436.
  29. Yang AH, Chen JY, Lin JK: Myofibroblastic conversion of mesothelial cells. Kidney Int 2003;63:1530–1539.
  30. Stypinski D, Obaidi M, Combs M, Weber M, Stewart AJ, Ishikawa H: Safety, tolerability and pharmacokinetics of higher-dose mizoribine in healthy male volunteers. Br J Clin Pharmacol 2007;63:459–468.
  31. Tanaka H, Tsugawa K, Nakahata T, Kudo M, Suzuki K, Ito E: Implication of the peak serum level of mizoribine for control of the serum anti-dsDNA antibody titer in patients with lupus nephritis. Clin Nephrol 2005;63:417–422.

 goto top of outline Author Contacts

Noriaki Yorioka, MD, PhD
Department of Advanced Nephrology, Graduate School of Biomedical Sciences
Hiroshima University, 1-2-3 Kasumi Minami-ku
Hiroshima City 734-8551 (Japan)
Tel. +81 82 257 1506, Fax +81 82 257 1508, E-Mail nyorioka@hiroshima-u.ac.jp


 goto top of outline Article Information

Received: June 12, 2008
Accepted: December 1, 2008
Published online: April 23, 2009
Number of Print Pages : 11
Number of Figures : 8, Number of Tables : 2, Number of References : 31


 goto top of outline Publication Details

Nephron Experimental Nephrology

Vol. 112, No. 2, Year 2009 (Cover Date: May 2009)

Journal Editor: Hughes J. (Edinburgh)
ISSN: 1660-2129 (Print), eISSN: 1660-2129 (Online)

For additional information: http://www.karger.com/NEE


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Background/Aims: Peritoneal fibrosis is a serious complication of peritoneal dialysis (PD). It has been reported that administration of mizoribine, an effective immunosuppressant, ameliorated renal fibrosis in a rat model of unilateral ureteral obstruction. We therefore examined the effects of mizoribine in an experimental model of peritoneal fibrosis. Methods: 24 rats were given a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline. The rats were divided into three groups (n = 8 per group) that received either vehicle or mizoribine at a dose of 2 or 8 mg/kg once a day. 28 days after the start of the treatments the rats were sacrificed and peritoneal tissue samples collected. Macrophage infiltration (ED1), myofibroblast accumulation (α-smooth muscle actin (SMA)) and expression of type III collagen, transforming growth factor (TGF)-β and monocyte chemotactic protein-1 (MCP-1) were examined by immunohistochemistry. Results: Mizoribine significantly suppressed submesothelial zone thickening and reduced macrophage infiltration. Mizoribine also reduced collagen III+ area and decreased the number of α-SMA+, TGF-β+ and MCP-1+ cells. The magnitude of the changes observed was dose-dependent. Conclusion: The administration of mizoribine prevented the progression of peritoneal fibrosis in this rat model. Mizoribine may represent a novel therapy for peritoneal sclerosis in patients undergoing long-term PD.



 goto top of outline Author Contacts

Noriaki Yorioka, MD, PhD
Department of Advanced Nephrology, Graduate School of Biomedical Sciences
Hiroshima University, 1-2-3 Kasumi Minami-ku
Hiroshima City 734-8551 (Japan)
Tel. +81 82 257 1506, Fax +81 82 257 1508, E-Mail nyorioka@hiroshima-u.ac.jp


 goto top of outline Article Information

Received: June 12, 2008
Accepted: December 1, 2008
Published online: April 23, 2009
Number of Print Pages : 11
Number of Figures : 8, Number of Tables : 2, Number of References : 31


 goto top of outline Publication Details

Nephron Experimental Nephrology

Vol. 112, No. 2, Year 2009 (Cover Date: May 2009)

Journal Editor: Hughes J. (Edinburgh)
ISSN: 1660-2129 (Print), eISSN: 1660-2129 (Online)

For additional information: http://www.karger.com/NEE


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Selgas R, Fernandez-Reyes MJ, Bosque E, Bajo MA, Borrego F, Jimenez C, Del Peso G, De Alvaro F: Functional longevity of the human peritoneum: how long is continuous peritoneal dialysis possible? Results of a prospective medium long-term study. Am J Kidney Dis 1994;23:64–73.
  2. Davies SJ, Bryan J, Phillips L, Russell GI: Longitudinal changes in peritoneal kinetics: the effects of peritoneal dialysis and peritonitis. Nephrol Dial Transplant 1996;11:498–506.
  3. Ito T, Yorioka N: Peritoneal damage by peritoneal dialysis solutions. Clin Exp Nephrol 2008;12:243–249.
  4. Williams JD, Craig KJ, Topley N, Von Ruhland C, Fallon M, Newman GR, Mackenzie RK, Williams GT: Morphologic changes in the peritoneal membrane of patients with renal disease. J Am Soc Nephrol 2002;13:470–479.
  5. Gandhi VC, Humayun HM, Ing TS, Daugirdas JT, Jablokow VR, Iwatsuki S, Geis WP, Hano JE: Sclerotic thickening of the peritoneal membrane in maintenance peritoneal dialysis patients. Arch Intern Med 1980;140:1201–1203.
  6. Kawanishi H, Moriishi M: Encapsulating peritoneal sclerosis: prevention and treatment. Perit Dial Int 2007;27(suppl 2):S289–S292.

    External Resources

  7. Suga H, Teraoka S, Ota K, Komemushi S, Furutani S, Yamauchi S, Margolin S: Preventive effect of pirfenidone against experimental sclerosing peritonitis on rats. Exp Toxicol Pathol 1995;47:287–291.
  8. Mishima Y, Miyazaki M, Abe K, Ozono Y, Shioshita K, Xia Z, Harada T, Taguchi T, Koji T, Kohno S: Enhanced expression of heat shock protein 47 in rat model of peritoneal fibrosis. Perit Dial Int 2003;23:14–22.
  9. Koyama H, Tsuji M: Genetic and biochemical studies on the activation and cytotoxic mechanism of bredinin, a potent inhibitor of purine biosynthesis in mammalian cells. Biochem Pharmacol 1983;32:3547–3553.
  10. Kusumi T, Tsuda M, Katsunuma T, Yamamura M: Dual inhibitory effect of bredinin. Cell Biochem Funct 1989;7:201–204.
  11. Ishikawa H: Mizoribine and mycophenolate mofetil. Curr Med Chem 1999;6:575–597.
  12. Akiyama T, Okazaki H, Takahashi K, Hasegawa A, Tanabe K, Uchida K, Takahara S, Toma H: Mizoribine in combination therapy with tacrolimus for living donor renal transplantation: analysis of a nationwide study in Japan. Transplant Proc 2005;37:843–845.
  13. Tanaka E, Inoue E, Kawaguchi Y, Tomatsu T, Yamanaka H, Hara M, Kamatani N: Acceptability and usefulness of mizoribine in the management of rheumatoid arthritis in methotrexate-refractory patients and elderly patients, based on analysis of data from a large-scale observational cohort study. Mod Rheumatol 2006;16:214–219.
  14. Nakayamada S, Saito K, Umehara H, Ogawa N, Sumida T, Ito S, Minota S, Nara H, Kondo H, Okada J, Mimori T, Yoshifuji H, Sano H, Hashimoto N, Sugai S, Tanaka Y: Efficacy and safety of mizoribine for the treatment of Sjögren’s syndrome: a multicenter open-label clinical trial. Mod Rheumatol 2007;17:464–469.
  15. Tanaka H, Tsugawa K, Tsuruga K, Suzuki K, Nakahata T, Ito E, Waga S: Mizoribine for the treatment of lupus nephritis in children and adolescents. Clin Nephrol 2004;62:412–417.
  16. Honda M: Nephrotic syndrome and mizoribine in children. Pediatr Int 2002;44:210–216.
  17. Sakai T, Kawamura T, Shirasawa T: Mizoribine improves renal tubulointerstitial fibrosis in unilateral ureteral obstruction-treated rat by inhibiting the infiltration of macrophages and the expression of α-smooth muscle actin. J Urol 1997;158:2316–2322.
  18. Sato N, Shiraiwa K, Kai K, Watanabe A, Ogawa S, Kobayashi Y, Yamagishi-Imai H, Utsunomiya Y, Mitarai T: Mizoribine ameliorates the tubulointerstitial fibrosis of obstructive nephropathy. Nephron 2001;89:177–185.
  19. Nishino T, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T, Ozono Y, Koji T, Kohno S: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats. Kidney Int 2003;64:887–896.
  20. Yoshio Y, Miyazaki M, Abe K, Nishino T, Furusu A, Mizuta Y, Harada T, Ozono Y, Koji T, Kohno S: TNP-470, an angiogenesis inhibitor, suppresses the progression of peritoneal fibrosis in mouse experimental model. Kidney Int 2004;66:1677–1685.
  21. Doi S, Masaki T, Arakawa T, Takahashi S, Kawai T, Nakashima A, Naito T, Kohno N, Yorioka N: Protective effects of peroxisome proliferator-activated receptor γ ligand on apoptosis and hepatocyte growth factor induction in renal ischemia-reperfusion injury. Transplantation 2007;84:207–213.
  22. Hirai T, Masaki T, Kuratsune M, Yorioka N, Kohno N: PDGF receptor tyrosine kinase inhibitor suppresses mesangial cell proliferation involving STAT3 activation. Clin Exp Immunol 2006;144:353–361.
  23. Hosotsubo H, Takahara S, Taenaka N: Simplified high-performance liquid chromatographic method for determination of mizoribine in human serum. J Chromatography 1988;432:340–345
  24. Kikuchi Y, Imakiire T, Yamada M, Saigusa T, Hyodo T, Hyodo N, Suzuki S, Miura S: Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Nephrol Dial Transplant 2005;20:1573–1581.
  25. Vaage J, Lindblad WJ: Production of collagen type I by mouse peritoneal macrophages. J Leukoc Biol 1990;48:274–280.
  26. Nathan CF: Secretory products of macrophages. J Clin Invest 1987;79:319–326.
  27. Diamond JR, Kees-Folts D, Ding G, Frye JE, Restrepo NC: Macrophages, monocyte chemoattractant peptide-1, and TGF-β1 in experimental hydronephrosis. Am J Physiol 1994;266:F926–F933.
  28. Margetts PJ, Bonniaud P, Liu L, Hoff CM, Holmes CJ, West-Mays JA, Kelly MM: Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol 2005;16:425–436.
  29. Yang AH, Chen JY, Lin JK: Myofibroblastic conversion of mesothelial cells. Kidney Int 2003;63:1530–1539.
  30. Stypinski D, Obaidi M, Combs M, Weber M, Stewart AJ, Ishikawa H: Safety, tolerability and pharmacokinetics of higher-dose mizoribine in healthy male volunteers. Br J Clin Pharmacol 2007;63:459–468.
  31. Tanaka H, Tsugawa K, Nakahata T, Kudo M, Suzuki K, Ito E: Implication of the peak serum level of mizoribine for control of the serum anti-dsDNA antibody titer in patients with lupus nephritis. Clin Nephrol 2005;63:417–422.