Characterization of infiltrating macrophages in high glucose-induced peritoneal fibrosis in rats

Alternatively activated macrophages (M2 macrophages) are involved in tissue remodeling and fibrosis, but their effects on peritoneal fibrosis (PF) induced by high glucose peritoneal dialysate have yet to be fully established. In this study, PF was induced in male Sprague-Dawley rats by intraperitoneal injection with Lactate-G4.25% dialysate (20 ml/rat/day) for 4 weeks. Control rats were given an intraperitoneal injection with saline. Establishment of the PF model was verified by Masson's trichrome and H&E staining. M1 macrophages (co-localization of CD68 and CCr7) and M2 macrophages (co-localization of CD68 and CD206) was assayed by immunofluorescence and immunohistochemistry. The levels of dialysate cytokines driving macrophage differentiation (including IFN-γ, IL-2 and IL-4) were detected by ELISA. The expression of transforming growth factor (TGF)-β, p-Smad3, p-Smad2/3 and Smad7 in M2 macrophages (co-localization of CD68, CD206 and TGF-β, or p-Smad3, or p-Smad2/3, or Smad7) was measured by immunofluorescence. We found that PF rats had significantly thicker peritoneal membranes compared to control rats, indicating the successful establishment of our PF model. Compared to controls, PF rats had more peritoneal macrophages (CD68+ cells), more peritoneal M1 macrophages and a greater percentage of peritoneal M2 macrophages. PF rats also had significantly greater levels of dialysate cytokine IL-4, which promotes differentiation to M2 macrophages, higher expression levels of TGF-β in peritoneal M2 macrophages, upregulation of phosphorylated Smad3 and Smad2/3, and downregulation of Smad7 in peritoneal M2 macrophages. Our results indicate that M2 macrophages may play an important role in PF induced by high glucose, and that the cytokine environment in the abdominal cavities of PF rats promotes differentiation to M2 macrophages. The function of M2 macrophages in PF may be related to the TGF-β/Smad signaling pathways.