SAHA Suppresses Peritoneal Fibrosis in Mice

Abstract

Objective: Long-term peritoneal dialysis causes peritoneal fibrosis in submesothelial areas. However, the mechanism of peritoneal fibrosis is unclear. Epigenetics is the mechanism to induce heritable changes without any changes in DNA sequences. Among epigenetic modifications, histone acetylation leads to the transcriptional activation of genes. Recent studies indicate that histone acetylation is involved in the progression of fibrosis. Therefore, we examined the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on the progression of peritoneal fibrosis in mice.

Methods: Peritoneal fibrosis was induced by the injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. SAHA, or a dimethylsulfoxide and saline vehicle, was administered subcutaneously every day from the start of the CG injections for 3 weeks. Morphologic peritoneal changes were assessed by Masson's trichrome staining, and fibrosis-associated factors were assessed by immunohistochemistry.

Results: In CG-injected mice, a marked thickening of the submesothelial compact zone was observed. In contrast, the administration of SAHA suppressed the progression of submesothelial thickening and type III collagen accumulation in CG-injected mice. The numbers of fibroblast-specific protein-1-positive cells and α-smooth muscle actin α-positive cells were significantly decreased in the CG + SAHA group compared to that of the CG group. The level of histone acetylation was reduced in the peritoneum of the CG group, whereas it was increased in the CG + SAHA group.

Conclusions: Our results indicate that SAHA can suppress peritoneal thickening and fibrosis in mice through up-regulation of histone acetylation. These results suggest that SAHA may have therapeutic potential for treating peritoneal fibrosis.

Keywords: SAHA; Suberoylanilide hydroxamic acid; chlorhexidine gluconate; histone deacetylase inhibitor; peritoneal fibrosis.

Figure 1 —

The result of Masson’s trichrome staining of peritoneal tissues. (A) In normal mice, the monolayer of mesothelial cells covered the entire surface of the peritoneum. (B) In the control group, peritoneal tissues were almost normal without thickening of the submesothelial zone. (C) The peritoneal tissues of the mice in the CG group showed marked thickening of the submesothelial compact zone and presence of numerous cells. (D) SAHA significantly prevented the progression of submesothelial thickening. (A-D), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (E) Bar graph showing the thickness of the submesothelial compact zone. Data are expressed as mean±SEM. * represents p<0.05; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean.

Figure 2 —

The result of the immunohistochemical analysis for type III collagen. (A) In the control group, type III collagen expression in the submesothelial compact zone was equivalent to that of normal mice. (B) In the CG group, type III collagen was diffusely expressed in the submesothelial compact zone. (C) Type III collagen expression was clearly decreased in the CG+SAHA group. (D) The peritoneal tissue of CG+SAHA group was incubated with normal IgG instead of type III collagen antibody as a negative control. (A-D), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (E) Bar graph showing the positive areas for type III collagen. Data are expressed as mean±SEM; * represents p<0.05; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean.

Figure 3 —

The result of the immunohistochemical analysis for FSP-1 and αSMA. (A) The number of FSP-1-positive cells increased markedly in the CG group. (B) The expression of FSP-1 was inhibited in the CG+SAHA group. (C) The peritoneal tissue of CG+SAHA group was incubated with normal IgG instead of FSP-1 antibody as a negative control. (A-C), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (D) Bar graph showing the number of FSP-1-positive cells. Data are expressed as mean±SEM. (E) In the CG group, the number of αSMA–positive cells was increased. (F) The number of αSMA-positive cells was reduced in the CG+SAHA group. (G) The peritoneal tissue of CG+SAHA group was incubated with normal IgG instead of αSMA antibody as a negative control. (E–G), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (H) Bar graph showing the number of αSMA–positive cells. Data are expressed as mean±SEM. * represents p<0.05; FSP-1 = fibroblast-specific protein-1; <alpha>SMA = <alpha>-smooth muscle actin; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean.

Figure 4 —

The results of immunohistochemical analysis for phosphorylated Smad2/3 and TGF-β dependent profibrotic genes expression. (A) In the CG group, a number of phosphorylated-Smad2/3-positive cells were observed in the thickened peritoneal compact zone. (B) These numbers were significantly decreased in the CG+SAHA group. (C) The peritoneal tissue of CG+SAHA group was incubated with normal IgG instead of phosphorylated Smad2/3 antibody as a negative control. (A–C), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (D) Bar graph showing the number of phosphorylated-Smad2/3–positive cells. Data are expressed as mean±SEM. (E) COLIα1 expression levels were measured by quantitative RT-PCR relative to β-actin controls. Values are relative to the control group±SEM. (F) Fibronectin expression levels were measured by quantitative RT-PCR relative to β-actin controls. Values are relative to the control group±SEM. (G) CTGF expression levels were measured by quantitative RT-PCR relative to β-actin controls. Values are relative to the control group±SEM. * represents p<0.05; TGF = transforming growth factor; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean; CTGF = connective tissue growth factor; RT-PCR = real time PCR.

Figure 5 —

The result of the immunofluorescence staining for H3K9 acetylation. (A) The control group showed low levels of H3K9 acetylation in the cells of the peritoneum. (B) In the CG group, moderate levels of H3K9 acetylation were evident by the thickened submesothelial compact zone. (C) In the CG+SAHA group, the level of H3K9 acetylation tended to be higher than that in the CG group. (D) The peritoneal tissue of the CG+SAHA group was incubated with normal IgG instead of H3K9 acetylation antibody as a negative control. (A–D), magnification 200×; bars indicate the thickness of the submesothelial compact zone. CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid.

Figure 6 —

The result of the immunofluorescence staining for BMP-7. (A) In the control group, very few BMP-7–positive cells were shown in the peritoneum. (B) The CG group showed some BMP-7–positive cells in the submesothelial compact zone. (C) The BMP-7–positive cells were increased in the CG+SAHA group. (D) The peritoneal tissue of the CG+SAHA group was incubated with normal IgG instead of BMP-7 antibody as a negative control. (A–D), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (E) Bar graph showing the BMP-7–positive cells. Data are expressed as mean±SEM. (F) BMP-7 expression levels were measured by quantitative real-time RT-PCR relative to β-actin controls. Values are relative to the control group±SEM. * represents p<0.05; BMP-7 = bone morphogenetic protein 7; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean; RT-PCR = real time PCR.

Figure 7 —

The result of the immunohistochemical analysis for F4/80 and CD31. (A) In the CG group, a number of F4/80-positive cells were observed in thickened peritoneal compact zone. (B) These numbers were significantly decreased in the CG+SAHA group. (C) The peritoneal tissue of CG+SAHA group was incubated with normal IgG instead of F4/80 antibody as a negative control. (A–C), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (D) Bar graph showing the number of F4/80–positive cells. Data are expressed as mean±SEM. (E) The number of CD31–positive vessels increased markedly in the CG group. (F) The number of CD31–positive vessels was reduced in the CG+SAHA group. (G) The peritoneal tissue of the CG+SAHA group was incubated with normal IgG instead of CD31 antibody as a negative control. (E-G), magnification 200×; bars indicate the thickness of the submesothelial compact zone. (H) Bar graph showing the number of CD31-positive vessels. Data are expressed as mean±SEM. * represents p<0.05; CG = chlorhexidine gluconate; SAHA = suberoylanilide hydroxamic acid; SEM = standard error of the mean.