Deletion of p38 MAPK in macrophages ameliorates peritoneal fibrosis and inflammation in peritoneal dialysis

Abstract

One of the most common causes of peritoneal dialysis withdrawal is ultrafiltration failure which is characterized by peritoneal membrane thickening and fibrosis. Although previous studies have demonstrated the inhibitory effect of p38 MAPK inhibitors on peritoneal fibrosis in mice, it was unclear which specific cells contribute to peritoneal fibrosis. To investigate the role of p38 MAPK in peritoneal fibrosis more precisely, we examined the expression of p38 MAPK in human peritoneum and generated systemic inducible p38 MAPK knockout mice and macrophage-specific p38 MAPK knockout mice. Furthermore, the response to lipopolysaccharide (LPS) was assessed in p38 MAPK-knocked down RAW 264.7 cells to further explore the role of p38 MAPK in macrophages. We found that phosphorylated p38 MAPK levels were increased in the thickened peritoneum of both human and mice. Both chlorhexidine gluconate (CG)-treated systemic inducible and macrophage-specific p38 MAPK knockout mice ameliorated peritoneal thickening, mRNA expression related to inflammation and fibrosis, and the number of αSMA- and MAC-2-positive cells in the peritoneum compared to CG control mice. Reduction of p38 MAPK in RAW 264.7 cells suppressed inflammatory mRNA expression induced by LPS. These findings suggest that p38 MAPK in macrophages plays a critical role in peritoneal inflammation and thickening.

Keywords: Fibrosis; Macrophage; Peritoneal dialysis; p38 MAPK.

Fig. 1

Phosphorylation of p38 MAPK in human and mouse peritoneum and generation of systemic p38 MAPK knockout mice. (a) Immunohistochemistry of phosphorylated p38 MAPK in the human peritoneum at the time of catheter insertion (beginning) in a 63-year-old male nephrosclerosis patient (left panel) and a 64-year-old male chronic glomerulonephritis patient (right panel), and at the time of catheter removal (withdrawal) after 11 years of peritoneal dialysis in a 76-year-old female IgA nephropathy patient (left panel) and a 69-year-old female IgA nephropathy patient (right panel). (b) Schema of the experimental protocol. Male ROSA26-CreER^T2; p38αfl/fl mice or control [p38αfl/fl, Cre (−)] mice were treated with 0.2 mg/gBW (body weight) tamoxifen orally for 5 days at 8–9 weeks of age. Systemic p38 KO or control mice at 13–15 weeks of age were treated with intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) at 0.01ml/gBW dissolved in 15% ethanol and 85% phosphate-buffered saline (PBS), or PBS alone, three times a week for 3 weeks. (c) Expression of peritoneal Mapk14 mRNA in control or systemic p38 MAPK knockout mice with PBS or CG. Ctrl PBS; n = 9, systemic p38 KO PBS; n = 9, Ctrl CG; n = 11, systemic p38 KO CG; n = 11. (d) Double immunofluorescence staining for phosphorylated p38 MAPK (p-p38) and F4/80 in control and systemic p38 MAPK knockout mice in PBS- or CG-treated mouse peritoneum. Triangles indicate double positive cells. High magnification images of immunofluorescence staining for p-p38, F4/80, and DAPI in control and systemic p38 MAPK knockout mice in CG-treated mouse peritoneum. Ctrl; control mice, Systemic p38 KO; systemic p38 MAPK knockout mice. LPF; Low power field, HPF; High power field, Scale bars represent 100 µm. Values are expressed as means ± SD. *P < 0.05, **P < 0.01.

Fig. 2

Systemic deletion of p38 MAPK ameliorated peritoneal thickening and fibrosis. (a) Representative images of Masson’s trichrome staining of the peritoneum after 3 weeks of PBS or CG injection in control and systemic p38 MAPK KO mice. Double-headed arrows indicate the submesothelial zone. (b) Mean peritoneal membrane thickness of the submesothelial zone. (c) Peritoneal mRNA expression levels of Il1b, Il6, Emr1, Tlr4, Col1a1, Acta2, Fn1, and Ctgf in control and systemic p38 MAPK KO mice treated with PBS or CG three times for 3 weeks. Ctrl; control mice, Systemic p38 KO; systemic p38 MAPK knockout mice. Scale bars represent 100 µm. Values are expressed as means ± SD. *P < 0.05, **P < 0.01.

Fig. 3

Immunohistochemical staining of αSMA and MAC-2 in control and systemic p38 MAPK KO mice. (a) Immunohistochemical staining of αSMA in the peritoneum of control and systemic p38 KO mice treated with PBS or CG, shown in photomicrographs at low and high magnification. (b) The number of αSMA-positive cells in the peritoneum. (c) Immunohistochemical staining of MAC-2 in the peritoneum, shown in photomicrographs at low and high magnification. (d) The number of MAC-2-positive cells in the peritoneum. LPF; Low power field, HPF; High power field, Scale bars represent 100 µm. Ctrl; control mice, Systemic p38 KO; systemic p38 MAPK knockout mice. Ctrl PBS; n = 9, systemic p38 KO PBS; n = 9, Ctrl CG; n = 11, systemic p38 KO CG; n = 11. Values are expressed as means ± SD. **P < 0.01.

Fig. 4

Generation of macrophage-specific p38 MAPK knockout mice. (a) Macrophage-specific p38 MAPK knockout mice or control mice at 13–15 weeks of age were treated by intraperitoneal injection of CG or PBS three times a week for 3 weeks. (b) Expression of peritoneal Mapk14 mRNA in control or macrophage-specific p38 knockout mice with PBS or CG. Ctrl PBS; n = 6, p38^ΔMΦ KO PBS; n = 6, Ctrl CG; n = 11, p38^ΔMΦKO CG; n = 11. (c) Double immunofluorescence staining for p-p38 and F4/80 in control and macrophage-specific p38 MAPK knockout mice in PBS- or CG-treated mouse peritoneum. Triangles indicate double positive cells. High magnification of immunofluorescence staining for p-p38, F4/80 and DAPI in control and macrophage-specific p38 MAPK knockout mice in CG-treated mouse peritoneum. Ctrl; control mice, p38^ΔMΦ KO; macrophage-specific p38 MAPK knockout mice. Scale bars represent 100 µm. Values are expressed as means ± SD. n.s.; not significant, **P < 0.01.

Fig. 5

Macrophage-specific deletion of p38 MAPK also ameliorated peritoneal thickening and fibrosis. (a) Representative images of Masson’s trichrome staining of the peritoneum after 3 weeks of PBS or CG injection in control and macrophage-specific p38 MAPK knockout mice. Double-headed arrows indicate the submesothelial zone. (b) Mean peritoneal membrane thickness of the submesothelial zone. (c) Peritoneal mRNA expression levels of Il1b, Il6, Emr1, Tlr4, Col1a1, Acta2, Fn1, and Ctgf in control and macrophage-specific p38 MAPK knockout mice treated with PBS or CG three times per week for 3 weeks. Ctrl; control mice, p38^ΔMΦ KO; macrophage-specific p38 MAPK knockout mice. Scale bars represent 100 µm. Values are expressed as means ± SD. *P < 0.05, **P < 0.01.

Fig. 6

Immunohistochemical staining of αSMA and MAC-2 in control and macrophage-specific p38 MAPK knockout mice. (a) Immunohistochemical staining of αSMA in the peritoneum of control and macrophage-specific p38 MAPK knockout mice treated with PBS or CG, shown in photomicrographs at low and high magnification. (b) The number of αSMA-positive cells in the peritoneum. (c) Immunohistochemical staining of MAC-2 in the peritoneum, shown in photomicrographs at low and high magnification. (d) The number of MAC-2-positive cells in the peritoneum. LPF; Low power field, HPF; High power field, Scale bars represent 100 µm. Ctrl; control mice, p38^ΔMΦ KO; macrophage-specific p38 MAPK knockout mice. Ctrl PBS; n = 6, p38^ΔMΦ KO PBS; n = 6, Ctrl CG; n = 11, p38^ΔMΦKO CG; n = 11. Values are expressed as means ± SD. **P < 0.01.

Fig. 7

Knockdown of p38 MAPK suppressed mRNA expression of inflammatory cytokines induced by lipopolysaccharide in RAW 264.7 cells. (a) The cell culture protocol for transfection siRNA targeting Mapk14 (si-Mapk14) or negative control (si-NC) into RAW 264.7 cells. (b,c) Western blot analyses of phosphorylated p38 MAPK, total p38 MAPK, and β-actin in RAW 264.7 cells with or without p38 MAPK knockdown. Original blots/gels are presented in Supplementary Fig. S5. (d) Double immunofluorescence staining for phosphorylated p38 MAPK (p-p38) and F4/80 in RAW 264.7 cells. Scale bars represent 400 μm. (e) Expression of Il1b, Il6, Ccl2, and Il10 mRNA in RAW 264.7 cells with or without p38 MAPK knockdown. Values are expressed as means ± SD. **P < 0.01.