Autophagy promotes fibrosis and apoptosis in the peritoneum during long-term peritoneal dialysis

Abstract

Long-term peritoneal dialysis is accompanied by functional and histopathological alterations in the peritoneal membrane. In the long process of peritoneal dialysis, high-glucose peritoneal dialysis solution (HGPDS) will aggravate the peritoneal fibrosis, leading to decreased effectiveness of peritoneal dialysis and ultrafiltration failure. In this study, we found that the coincidence of elevated TGF-β1 expression, autophagy, apoptosis and fibrosis in peritoneal membrane from patients with peritoneal dialysis. The peritoneal membranes from patients were performed with immunocytochemistry and transmission electron microscopy. Human peritoneal mesothelial cells were treated with 1.5%, 2.5% and 4.25% HGPDS for 24 hrs; Human peritoneal mesothelial cells pre-treated with TGF-β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. We further detected the production of TGF-β1, activation of TGF-β1/Smad2/3 signalling, induction of autophagy, EMT, fibrosis and apoptosis. We also explored whether autophagy inhibition by siRNA targeting Beclin 1 reduces EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. HGPDS increased TGF-β1 production, activated TGF-β1/Smad2/3 signalling and induced autophagy, fibrosis and apoptosis hallmarks in human peritoneal mesothelial cells; HGPDS-induced Beclin 1-dependent autophagy in human peritoneal mesothelial cells; Autophagy inhibition by siRNA Beclin 1 reduced EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. Taken all together, these studies are expected to open a new avenue in the understanding of peritoneal fibrosis, which may guide us to explore the compounds targeting autophagy and achieve the therapeutic improvement of PD.

Keywords: Beclin 1-dependent autophagy; apoptosis; fibrosis; high-glucose peritoneal dialysis solution; human peritoneal mesothelial cells.

Figure 1

Expression of TGF‐β1, autophagy and EMT hallmarks in peritoneal membrane (PM) from patients before and with PD. (A–D) The PM tissues of each patient before and with PD were randomly selected for immunohistochemistry (IHC) assay. Representative photomicrographs of TGF‐β1, N‐cadherin, MAP‐LC3 and Beclin 1 expression as determined by IHC staining in peritoneum samples of patients before PD and with PD. Image magnification: 100× (left); 200× (right).

Figure 2

Characteristic morphology of autophagy, fibrosis and apoptosis of peritoneal membrane from patients before and with PD under transmission electron microscope. Transmission electron microscopy images of the peritoneum tissues of each patient before and with PD. Tissues were fixed immediately in 1% glutaraldehyde, post‐fixed in 2% osmium tetroxide and embedded in Epon resin. Representative areas were picked for ultrathin sectioning. Transmission electron microscopy is performed using a Tecnai G2 Spirit Bio TWIN (FEI Co.) at 120 kV. N: nuclear; CF: Collagen fibre; White arrows, autophagosomes; Yellow arrows, apoptotic empty vacuoles. Scale bars: 1 μm, 500 nm (right) and 2 μm (left).

Figure 3

High‐glucose peritoneal dialysis solution (HGPDS) increased TGF‐β1 production, activated TGF‐β1/Smad2/3 signalling and induced autophagy, fibrosis and apoptosis hallmarks in human peritoneal mesothelial cells (HPMCs). (A) The levels of TGF‐β1 from Control, 1.5%, 2.5% and 4.25% HGPDS‐treated cells were determined by ELISA. Each bar represents the mean ± S.D. Statistical analysis was performed using Student's t‐test, *P < .05, **P < .01. (B and C) HGPDS induced expression of TGF‐β1 and activated TGF‐β1/Smad2/3 signalling in HPMCs. (D) Effect of HGPDS on MAP‐LC3 lipidation and Beclin 1 expression levels in cells is analysed by Western blotting. HPMCs were treated with 1.5%, 2.5%, and 4.25% HGPDS for 48 hrs. (E and F) HPMCs were treated with 1.5%, 2.5%, and 4.25% HGPDS for 48 hrs. Western blotting revealed that HGPDS induced expression of both fibrosis and apoptosis hallmarks. GAPDH was used as internal controls to ascertain equal loading.

Figure 4

HPGDS induced Beclin 1‐dependent autophagy in human peritoneal mesothelial cells (HPMCs). (A and B) Transmission electron microscopy images of HPMCs following high‐glucose peritoneal dialysis solution (HGPDS) treatment. The HPMCs were treated with 4.25% HGPDS or vehicle for 24 hrs. Cells were then fixed immediately in 1% glutaraldehyde and post‐fixed in 2% osmium tetroxide. The cell pellets or sections were embedded in Epon resin. Representative areas were picked for ultrathin sectioning. Transmission electron microscopy is performed using a Tecnai G2 Spirit Bio TWIN (FEI Co.) at 120 kV. (A) The HPMCs were treated with vehicle as control (a) and 4.25% HGPDS for 24 hrs (b). Scale bars: 2 μm. (B) Cells treated with vehicle as a control with normal nuclear and organelles. N: nuclear; M: mitochondrion; ER: endoplasmic reticulum. Scale bars: 500 nm (a). Cells are treated with 4.25% HGPDS for 24 hrs. N: nuclear; White arrows, representative autophagic structures; Yellow arrows, degradative autophagic vacuoles. Scale bars: 1 μm (b, c and d), 500 nm (e and f). (C) Western blotting analysis shows the effect of 4.25% HPGDS treatment of cells transfected with siRNA Beclin 1 or siRNA Ctrl on MAP‐LC3 lipidation and p62/SQSTM1 expression. (D) Western blotting analysis shows the effect of 4.25% HPGDS treatment of cells cotreated with TGF‐β1 (10 ng/ml) on MAP‐LC3 lipidation and p62/SQSTM1 expression. (E) and (F) HPMCs pre‐treated with TGF‐β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. Cells were then probed by SNLYSO sensor (an autolysosome florescent probe), and autophagic cells were analysed and quantitated by flowcytometer. Results from three independent experiments are shown as means ± S.D. **P < 0.01, *P < 0.05 versus untreated control group. ^## P < 0.01 versus 4.25% HGPDS‐treated group.

Figure 5

Autophagy inhibition reduced EMT and fibrosis in HPMCs. (A) Morphologic changes of cultured HPMCs (Magnification 100×). Human peritoneal mesothelial cells (HPMCs) transfected with siRNA Ctrl or siRNA Beclin 1 were treated with 2.5% high‐glucose peritoneal dialysis solution (HGPDS), 4.25% HGPDS for 24 hrs. Normal cells grew like characteristic cobblestone. Cells treated with HGPDS showed fibroblast morphology. Transfection with siRNA Beclin 1 attenuated such changes of cell morphology. (B) HPMCs were plated in 6‐well plates, and a cross scratch was wounded with a sterile micropipette tip. Cells transfected with siRNA Beclin 1 or vehicle were treated with 4.25% HGPDS for 24, 48, and 72 hrs after wounding. Then the migrating cells in denuded zone were assessed using an inverted microscope. Image magnification: 10×. (C) Western blotting analysis shows the effect of 4.25% HPGDS treatment of cells transfected with siRNA Beclin 1 or siRNA Ctrl on the protein levels of MMP‐2, MMP‐9, Fibronectin and Collagen Iα. (D) Effect of 4.25% HPGDS treatment of cells transfected with siRNA Beclin 1 or siRNA Ctrl on the RNA levels of MMP‐2 and MMP‐9. Melt curve analysis was employed at the end of each PCR to confirm the specificity of the PCR product. Data were analysed according to the comparative Ct method, and target genes expression of MMP‐2 and MMP‐9 was normalized to GAPDH expression levels in each sample. (E) Western blotting analysis shows the effect of 4.25% HPGDS treatment of cells transfected with siRNA Beclin 1 or siRNA Ctrl on the protein levels of epithelial‐mesenchymal transition markers. GAPDH was used as loading control. **P < 0.01, *P < 0.05 versus untreated control group

Figure 6

Autophagy inhibition rescued apoptosis in human peritoneal mesothelial cells. (A and B) Human peritoneal mesothelial cells pre‐treated with TGF‐β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% high‐glucose peritoneal dialysis solution (HGPDS) or vehicle for 24 hrs. The sum of early and late apoptotic cells ratio (%) was quantitated by flowcytometer analysis of Annexin V/PI. Results from three independent experiments are shown as means ± S.D. Results from three independent experiments are shown as means ± S.D. **P < 0.01, *P < 0.05 versus untreated control group. ^## P < 0.01, ^# P < 0.05 versus 4.25% HGPDS‐treated group. (C and D) Human peritoneal mesothelial cells transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. The apoptosis‐related protein such as Bax, Bcl‐2, Mcl‐1, PARP, caspase‐3 and caspase‐8 was assessed by Western blotting assay. GAPDH was used as loading control.