Journal: Clinical and Vaccine Immunology : CVI
Article Title: Glucose-Based Peritoneal Dialysis Fluids Downregulate Toll-Like Receptors and Trigger Hyporesponsiveness to Pathogen-Associated Molecular Patterns in Human Peritoneal Mesothelial Cells ▿
doi: 10.1128/CVI.00453-09
Figure Lengend Snippet: Glucose-based peritoneal dialysis solutions decrease TLR2 and TLR4 expression in human peritoneal mesothelial cells. Cells were treated with 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, and 7.5% icodextrin (Extraneal) for 6, 12, 24, 36, and 48 h for mRNA expression and for 24, 48, and 72 h for protein expression. Real-time PCR and immunoblot analyses were used to determine the TLR2 and TLR4 mRNA and protein expression levels. Incubation of cells with 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, and 7.5% icodextrin for 6 and 12 h did not influence TLR2 (A) and TLR4 (B) mRNA expression. However, at 24, 36, and 48 h, TLR2 and TLR4 mRNA expression in the glucose-based PD solution treatment groups was significantly downregulated compared to that in the control group (P < 0.01). (C) In addition, incubation of cells with 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, and 7.5% icodextrin for 24 h did not influence TLR2 and TLR4 protein expression. However, at 48 (D) and 72 (E) h, TLR2 and TLR4 protein expression in the glucose-based PD solution treatment groups was significantly downregulated compared to that in the control group (P < 0.05). Icodextrin-based PD solutions did not influence TLR2 and TLR4 expression. (F) Cells were analyzed at 48 h by immunofluorescence with anti-TLR2 and anti-TLR4 antibodies, and the intensity values of the cells were measured with LSM 510 confocal software. The positions of the nuclei are indicated by DAPI (4′,6-diamidino-2-phenylindole) fluorescence. Data are expressed as means ± SD for three individual experiments. D1.5%, D2.5%, D4.25%, and E7.5% represent 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, and 7.5% icodextrin, respectively. *, P < 0.05; **, P < 0.01 versus control.
Article Snippet: Rabbit anti-human TLR2 polyclonal antibody, rabbit anti-human TLR4 monoclonal antibody (MAb), rabbit anti-human polyclonal antibodies against p38 MAPK, phospho-p38 MAPK Thr180/Tyr182 , JNK, phospho-JNK Thr183/Tyr185 , p44/42 MAPK, NF-κB p65, and phospho-NF-κB p65 Ser536 , and mouse anti-human polyclonal antibodies against phospho-p44/42 MAPK Thr202/Tyr204 were purchased from Cell Signaling Technology Inc. (Danvers, MA).
Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Control, Immunofluorescence, Software, Fluorescence