Fig 1.
G9a expression is upregulated in MGO-injected mice and human Peritoneal Dialysis (PD) effluents.
(A) Immunohistochemical staining shows typical G9a expression in peritoneal tissues of control mice and methylglyoxal (MGO)-injected mice (original magnification, ×200). (B) Graph indicating the number of G9a-positive cells in mice with or without peritoneal injection of MGO (n = 5 for both groups). (C) Representative western blot analysis showing the levels of G9a expression in nonadherent cells of human PD effluents. Data are expressed as the mean ± SE. Statistical analysis was performed by the Student’s t-test. *P < 0.05.
Fig 2.
BIX01294 suppresses peritoneal thickening and cell density in MGO-injected mice.
(A) Masson’s trichrome staining shows the typical thickness of peritoneal tissue in control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates quantification of the peritoneal thickness in the three groups of mice. (C) Hematoxylin-eosin staining shows typical cellularity of peritoneal tissue in control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (D) Graph indicates quantification of cell density in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 mice per group.
Fig 3.
BIX01294 attenuates α-Smooth Muscle Actin (α-SMA) expression in mice with peritoneal fibrosis.
(A) Immunohistochemical staining shows typical α-SMA expression in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates the number of α-SMA-positive cells in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 mice per group.
Fig 4.
BIX01294 reduces collagen I and III expression in mice with peritoneal fibrosis.
(A) Immunohistochemical staining shows typical collagen I expression in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates the number of collagen I-positive pixels in the three groups of mice. (C) Immunohistochemical staining shows typical collagen III expression in the peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (D) Graph indicates the number of collagen III-positive pixels in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 mice per group.
Fig 5.
BIX01294 inhibits monocyte/macrophage infiltration, TGF-β1, and H3K9me1 in mice with peritoneal fibrosis.
(A) Immunohistochemical staining shows typical CD68 expression in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates the number of CD68-positive cells in the three groups of mice. (C) Immunohistochemical staining shows typical TGF-β1 expression in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (D) Graph indicates the number of TGF-β1-positive cells in the three groups of mice. (E) Two-color immunohistochemical staining shows that most CD68 cells (brown) are immunoreactive for TGF-β1 (blue-gray) (arrows). (F) TGF-β1 protein levels in mouse PD effluent were quantified by ELISA. (G) Immunohistochemical staining shows typical H3K9me1 levels in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (H) Graph indicates the number of H3K9me1-positive cells in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis were performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 mice per group.
Fig 6.
BIX01294 improves functional impairments of the peritoneal membrane in mice with peritoneal fibrosis.
Graph shows (A) the dialysate-to-plasma (D/P) ratio of blood urea nitrogen (BUN) and (B) peritoneal absorption of glucose from the dialysate (D/D0) in control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 during the 10-minute dwell of dialysate (4.25% Dianeal). Data are expressed as the mean ± SE. Statistical analysis were performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 mice per group.
Fig 7.
G9a expression is regulated by TGF-β1 in HPMCs, and BIX01294 represses TGF-β1-induced fibrotic changes.
(A) Representative western blot analysis showing the levels of G9a protein expression in TGF-β1 (5 ng/mL)-stimulated HPMCs at various time points. Quantification is shown in the lower panel. (B) Representative western blot analysis of the expression of α-SMA (C) zonula occludens-1 (ZO-1), and (D) secreted fibronectin of HPMCs. Quantification is shown in the lower panel. (E) Representative western blot analysis showing the levels of H3K9me1 in TGF-β1-stimulated in HPMCs. Quantification is shown in the lower panel. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *P < 0.05, n = 5 samples per group.