Peritoneal Membrane Histological Analysis

Parietal peritoneal tissues were biopsied in the standard manner and processed as reported previously [22 (link),23 (link)]. Parietal peritoneal membrane was obtained from the anterior abdominal wall when the PD catheter was removed at the cessation of PD. To avoid detachment of mesothelial cells, all procedures were carried out carefully. Peritoneal membrane tissues fixed with formalin were stained with hematoxylin and eosin (HE) and Masson’s trichrome [23 (link)–26 (link)] and were also stained with phosphotungstic acid hematoxylin (PTAH) reagent to detect fibrin formation, as described previously [27 (link)]. Immunostaining was performed on paraffin-embedded tissues as described previously [23 (link)–25 (link),28 (link)]. The antibodies used in these experiments are summarized in S1 Table. Briefly, 4-μm-thick sections of formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. Endogenous peroxidase activity was inhibited with 3% H₂O₂ in methanol. For antigen retrieval to detect CD31 and CD68, the slides were boiled in a solution of 0.04 M citrate and 0.12 M phosphate (pH 5.8) for 30 min at 98°C. After washing, nonspecific protein-binding sites were blocked with normal goat serum (Dako, Glostrup, Denmark). Then, sections were incubated with primary antibodies, mouse monoclonal antibodies against CD31 (JC/70A; Dako), CD68 (PGM1; Dako), and podoplanin (D2-40; Dako) overnight at 4°C. For advanced glycation end-products (AGEs), sections were incubated with mouse anti-AGE antibody (6D12; TransGenic, Kobe, Japan) for 60 min at room temperature. After washing with phosphate-buffered saline, sections were treated with a conjugate of polyclonal goat anti-mouse immunoglobulin (Ig) G antibodies and horseradish peroxidase-labeled polymer (Histofine^® Simple Stain; Nichirei, Tokyo, Japan) as the secondary reagent. Enzyme activity was detected by 3,3'-diaminobenzidine (Nichirei). For analysis of collagen volume fraction (collagen density), we applied the methods described by Morelle et al. [29 (link)]. Briefly, we stained peritoneal membrane tissues with a Picrosirius Red Stain kit (Polyscience, Warrington, PA), then observed the tissues under circularly polarized light microscopy (Zeiss Z1 image microscopy, Carl Zeiss, Oberkochen, Germany). Collagen volume fraction was assessed using ImageJ software version 1.5 (http://imagej.nih.gov/ij/) and was calculated as a percentage of the submesothelial area.