The role of peritoneal alternatively activated macrophages in the process of peritoneal fibrosis related to peritoneal dialysis

Abstract

It has been confirmed that alternatively activated macrophages (M2) participate in tissue remodeling and fibrosis occurrence, but the effect of M2 on peritoneal fibrosis related to peritoneal dialysis (PD) hasn't been elucidated. This study was therefore conducted to assess the association between M2 and peritoneal fibrosis related to PD. In this study, peritoneal fibrosis was induced by intraperitoneal (i.p.) injection of Lactate-4.25% dialysate (100 mL/kg) to C57BL/6J mice for 28 days, and liposome-encapsulated clodronate (LC, the specific scavenger of macrophages) was used to treat the peritoneal fibrosis mice model by i.p. injection at day 18 and day 21. All animals were sacrificed at day 29. Parietal peritonea were stained with Masson's trichrome, and the expression of type I collagen (Col-I), fibronectin, mannose receptor (CD206), transforming growth factor beta (TGF-β), chemokine receptor 7 (CCR7), chitinase 3-like 3 (Ym-1) and arginase-1 (Arg-1) was determined by Western blotting, immunofluorescence and quantitative real-time PCR. Our results revealed that peritoneal thickness, Col-I, fibronectin, CD206, TGF-β, Ym-1 and Arg-1 were upregulated in the peritoneal fibrosis mice model, and all of these indexes were downregulated in those treated with LC. Additionally, there was no difference in the level of CCR7 between the model and treatment group. Our study indicated that peritoneal M2 played an important role in the process of peritoneal fibrosis related to PD and might be a potential target for intervention therapy of peritoneal fibrosis.

Figure 1

Masson’s trichrome staining of parietal peritoneum slices. (A₁) Normal control (original magnification: ×200); (A₂) Normal control (original magnification: ×400); (B₁) Peritoneal fibrosis (PF) group (original magnification: ×200); (B₂) PF group (original magnification: ×400); (C) The thickness of the peritoneum (mean ± SD). The thickness of the peritoneum in the PF group was significantly thicker than that in the NC group. * p < 0.05, vs. NC group.

Figure 2

Macrophage polarization in PF mice. (A) The number of M1 didn’t change between normal control (NC) and PF mice. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CCR7; co-location signals were identified as M1; (B) The number of M2 in PF mice markedly increased. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CD206; co-location signals were identified as M2; (C) The cell count of M1 and M2 in the peritoneum of NC and PF mice.

Figure 3

Liposome-encapsulated clodronate (LC) depleted almost all the M2 in the peritoneum without an effect on M1. (A) The number of M1 didn’t change among the four groups. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CCR7; co-location signals were identified as M1; (B) The number of M2 significantly increased after injecting Lactate-4.25% dialysate; liposome-encapsulated PBS (LP) didn’t affect M2 infiltration, but LC depleted peritoneal M2 efficiently. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CD206; co-location signals were identified as M2; (C) TGF-β was expressed by M2, and LC inhibited the expression of TGF-β by depletion of M2. Blue corresponds to nuclear staining, green corresponds to F4/80, red corresponds to CD206 and yellow corresponds to TGF-β staining.

Figure 3

Liposome-encapsulated clodronate (LC) depleted almost all the M2 in the peritoneum without an effect on M1. (A) The number of M1 didn’t change among the four groups. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CCR7; co-location signals were identified as M1; (B) The number of M2 significantly increased after injecting Lactate-4.25% dialysate; liposome-encapsulated PBS (LP) didn’t affect M2 infiltration, but LC depleted peritoneal M2 efficiently. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CD206; co-location signals were identified as M2; (C) TGF-β was expressed by M2, and LC inhibited the expression of TGF-β by depletion of M2. Blue corresponds to nuclear staining, green corresponds to F4/80, red corresponds to CD206 and yellow corresponds to TGF-β staining.

Figure 4

Effect of LC on Lactate-4.25% dialysate-induced overexpression of CD206, TGF-β, Arg-1 and Ym-1. Values were expressed as the mean ± SD. (A) The overexpression of CD206 and TGF-β induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; (B) The relative protein level of CD206 normalized by GAPDH; (C) The relative protein level of TGF-β normalized by GAPDH; (D) The mRNA level of Arg-1 and Ym-1 were depressed by LC treatment. ^#p < 0.05, vs. NC and LC group.

Figure 4

Effect of LC on Lactate-4.25% dialysate-induced overexpression of CD206, TGF-β, Arg-1 and Ym-1. Values were expressed as the mean ± SD. (A) The overexpression of CD206 and TGF-β induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; (B) The relative protein level of CD206 normalized by GAPDH; (C) The relative protein level of TGF-β normalized by GAPDH; (D) The mRNA level of Arg-1 and Ym-1 were depressed by LC treatment. ^#p < 0.05, vs. NC and LC group.

Figure 5

Representative photomicrographs (original magnification: ×200) of Masson’s trichrome staining in the (A₁) NC group, (B₁) PF group, (C₁) LP group and (D₁) LC group. Representative photomicrographs (original magnification: ×400) of Masson’s trichrome staining in the (A₂) NC group, (B₂) PF group, (C₂) LP group and (D₂) LC group.

Figure 6

LC depressed Col-I deposition via depletion of peritoneal M2. Values were expressed as the mean ± SD. (A) The overexpression of Col-I induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; (B) The relative protein level of Col-I normalized by GAPDH; (C) The mRNA level of Col-I was depressed by LC treatment; (D) Immunofluorescence staining of Col-I in the four groups. Blue corresponds to nuclear staining; green corresponds to Col-I staining. ^#p < 0.05, vs. NC and LC group.

Figure 7

LC depressed both the protein and mRNA level of fibronectin via depletion of peritoneal M2. Values were expressed as the mean ± SD. (A) The overexpression of fibronectin induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; (B) The relative protein level of fibronectin normalized by GAPDH; (C) The mRNA level of fibronectin was depressed by LC treatment; (D) Immunofluorescence staining of fibronectin in the four groups. Blue corresponds to nuclear staining, and red corresponds to fibronectin staining. ^#p < 0.05 vs. NC and LC group.