Fig 1.
Linagliptin suppresses peritoneal cell density and thickening in methylglyoxal (MGO)-injected mice.
(a, b) Representative light microscopy of peritoneal tissues on day 21 (a: hematoxylin-eosin staining; b: Masson’s trichrome staining; original magnification: ×200) in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. (c, d) The thickness of the submesothelial compact zone increased along with its cellularity until day 21 in mice treated with saline, whereas cell density and zone thickening were suppressed in mice treated with linagliptin. Bars indicate the compact zone area. Statistical analyses were performed using analysis of variance (ANOVA) followed by Tukey’s post-hoc test. *P < 0.05.
Fig 2.
Linagliptin inhibits α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) expression in mice with peritoneal fibrosis.
Immunohistochemical analyses of (a) α-SMA and (b) FSP-1 expression in peritoneal tissues on day 21 (original magnification: ×200) in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. (c, d) Accumulation of α-SMA+ and FSP-1+ cells was observed on day 21 in MGO-injected mice treated with saline, whereas the numbers of α-SMA-and FSP-1-expressing cells were significantly lower in MGO-injected mice treated with linagliptin. Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test. *P < 0.05.
Fig 3.
Linagliptin reduces type I and III collagen expression in mice with peritoneal fibrosis.
Immunohistochemical analyses of (a) type I collagen and (b) type III collagen expression in peritoneal tissues on day 21 (original magnification: ×200) in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. The numbers of type I and III collagen pixels were increased on day 21 in MGO-injected mice treated with saline but significantly decreased in MGO-injected mice treated with linagliptin (c, d). Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test. *P < 0.05.
Fig 4.
Linagliptin attenuates monocyte/macrophage infiltration and transforming growth factor-β1 (TGF-β1) expression in mice with peritoneal fibrosis.
(a) Immunohistochemical analysis of F4/80 expression in peritoneal tissue on day 21 in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. (b) Immunohistochemical analysis of TGF-β1 expression in peritoneal tissues on day 21 in MGO-injected mice treated with saline and MGO-injected mice treated with linagliptin. (c) The number of F4/80-expressing cells increased until day 21 in MGO-injected mice treated with saline, but significantly decreased by day 21 in MGO-injected mice treated with linagliptin. (d) The number of TGF-β1-expressing cells increased until day 21 in MGO-injected mice treated with saline, but significantly decreased by day 21 in MGO-injected mice treated with linagliptin. Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test. *P < 0.05 (original magnification in a and b: ×400).
Fig 5.
Linagliptin increases plasma GLP-1 (7–36) amide, improves peritoneal function, and decreases TGF-β1 levels in peritoneal fluid.
(a) Plasma GLP-1 (7–36) amide levels were analyzed in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. The plasma GLP-1 (7–36) amide level was significantly higher in MGO-injected mice treated with linagliptin than in MGO-injected mice treated with saline or in control mice. (b) Plasma glucose levels were measured in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. The plasma glucose level was significantly higher in MGO-injected mice treated with or without linagliptin than in control mice. Plasma glucose levels did not differ in MGO-injected mice treated with linagliptin or saline. (c) Peritoneal absorption of glucose from the dialysate (D/D0) was assessed in the three treatment groups during a 15-minute instillation of dialysate (4.25% Dianeal) at 100 mL/kg body weight. D/D0 values were significantly lower in MGO-injected mice treated with saline than in control mice, but were significantly increased in MGO-injected mice treated with linagliptin. (d) TGF-β1 levels in peritoneal fluid were assessed by ELISA and found to be significantly lower in MGO-injected mice treated with linagliptin than in MGO-injected mice treated with saline. Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test (a–c) or the Student’s t-test (d). *P < 0.05.
Fig 6.
GLP-1R is expressed in infiltrated inflammatory cells and MGO-induced GLP-1 expression is suppressed by linagliptin.
(a) Expression of GLP-1R was examined by western blotting. GLP-1R was expressed in the monocyte/macrophage cell line THP-1, but not in HPMCs. (b) Immunohistochemical analysis of GLP-1R expression in peritoneal tissue on day 21 in control mice, MGO-injected mice treated with saline, and MGO-injected mice treated with linagliptin. (c) The number of GLP-1R-expressing cells was increased until day 21 in MGO-injected mice treated with saline, but it was significantly decreased by day 21 in MGO-injected mice treated with linagliptin. Statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test. *P < 0.05. (original magnification in b: ×200).