Figure 1.
Paricalcitol reduced peritoneal membrane fibrosis, inflammation and ultrafiltration failure in mice exposed to PDF.
A) Paraffin sections of the peritoneal membrane from the 3 groups were stained with Masson's trichrome. B) Thickening of the peritoneal membrane was determined by morphometric analysis. C) Peritoneal permeability was determined by net ultrafiltration. D) The presence of inflammatory and mesothelial cells was determined by the expression of CD45 (green) and cytokeratin (red), respectively, in frozen sections of peritoneal membrane representative of each group. A green arrow indicates hematopoietic cells. A red arrow indicates mesothelial cells. E) The angiogenesis was determined by the expression of CD31 (green). Cytokeratin-positive cells are stained in red. The color balance was equally adjusted in immunofluorescence using Photoshop V10 for Mac (Abobe Systems Incorporated, US). n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. *P<.05; ***P<.001.
Figure 2.
Paricalcitol changed the peritoneal T cell population in mice instilled with PDF.
A) The paricalcitol group tends to increase the number of nucleated cells at the peritoneal cavity compared with the PDF group. B) The number of B cells was similar in the PDF and paricalcitol groups. C) There is no significant difference between the number of macrophages in the PDF and paricalcitol groups. D) Paricalcitol increased the numbers of CD4+ T cells compared with the PDF group. E) The treatment with paricalcitol increased CD8+ T cells in the peritoneal cavity. Statistical significance was determined using the Mann-Whitney test. ***P<.001; n≥5 in each group.
Figure 3.
Paricalcitol's enhancement of T cell numbers is inversely correlated with peritoneal thickness.
The increased levels of CD4+ (A) and CD8+ (B) T cells are strongly correlated with lower peritoneal fibrosis. n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. A correlation analysis was performed using Spearman's nonparametric test.
Figure 4.
Paricalcitol induced the recruitment of regulatory CD8+ T cells into the peritoneal cavity.
Paricalcitol (dashed box) tended to increase the frequency of CD4+ T cells expressing Foxp-3 (A), but not CTLA-4 (B) or membrane TGF-β (C). Paricalcitol increased the frequency of CD8+ T cells expressing Foxp-3 (A), CTLA-4 (B) and membrane TGF-β (C) compared with the PDF-group (white box). n≥9 in each group. Paricalcitol treated group had higher number of CD4+ CD8+ T cells expressing Foxp-3 than PDF group (D). Statistical significance was determined using the Mann-Whitney test. *P<.05; **P<.01; ***P<.001.
Figure 5.
Chemokine concentrations in the peritoneal cavity were not affected by paricalcitol treatment.
Concentrations of RANTES (A), MIP-1α (B) and MIP-1β (C) were determined in the peritoneal washing fluid. We observed no difference between the paricalcitol and the PDF group. n≥5 in each group.
Figure 6.
Paricalcitol induced the regulation of IL-17 production and affected peritoneal fibrosis outcomes.
A) Peritoneal concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α were not affected by paricalcitol treatment. B) Paricalcitol induced the suppression of IL-17 production in the peritoneal membrane. C) The IL-17 concentrations strongly correlate with peritoneal fibrosis. n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. **P<.01. A correlation analysis was performed using Spearman's nonparametric test.
Figure 7.
CD4+ and CD8+ T cells from paricalcitol-treated mice regulate IL-17 production.
CD4+- and CD8+-enriched T cell populations from mice treated with PDF and paricalcitol were stained with PKH67 and co-cultured with CD3+ T cells from PDF-treated mice. A control group of CD3+ T enriched cells from PDF-treated mice was also included. The cells were stimulated with CD3 and CD28, and after 2 days, brefeldin A was added for the last 12 h of culture. The cells were stained with anti-CD4 and anti-IL-17 antibodies and analyzed by flow cytometry. The PKH67negative CD4+ T cells were selected, and the frequency of IL17+ cells is shown in the figure. Statistical significance was determined using the Mann-Whitney test. **P<.01.