ZnSO₄ (Xinhua Pure Chemical Industries), 4.25% dextrose PD fluid (Baxter Healthcare Ltd.), Zn chelator N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN; Sigma-Aldrich; Merck KGaA) and clioquinol (CQ; Sigma-Aldrich; Merck KGaA) were used during the study. Monoclonal antibodies against E-cadherin (cat. no. 3195), vimentin (cat. no. 5741), NAD(P)H quinone dehydrogenase 1 [cat. no. 3187, (NQO-1)], heme oxygenase-1 [cat. no. 5853, (HO-1)], Nrf-2 (cat. no. 12721), GAPDH (cat. no. 5174) and Lamin B (cat. no. 13435) were purchased from Cell Signaling Technology, Inc. A Pierce ECL detection kit was obtained from Pierce (Thermo Fisher Scientific, Inc.). All reagents used were of analytical grade.

A total of 40 male Sprague-Dawley rats (200–250 g, 8–10 weeks) were obtained from the Center of Experimental Animals of China Medical University (Shenyang, China). Rats were housed in controlled conditions, at a temperature of 20–25°C with 40–70% humidity under a 12:12-h light/dark cycle, with access to food and water ad libitum.

Rats were randomly allocated into four groups with 10 rats in each group: The negative control (NC) group, which were treated daily via intraperitoneal administration of 100 ml/kg of 0.9% saline for 4 weeks; the HG group, which received daily intraperitoneal administration of 100 ml/kg of 4.25% dextrose PD fluid for 4 weeks; the HG + Zn group, which received daily intraperitoneal administration of 3 mg/kg ZnSO₄ and 100 ml/kg of 4.25% dextrose PD fluid for 4 weeks; the HG + CQ group, which were treated daily via intraperitoneal administration of 20 mg/kg CQ and 100 ml/kg of 4.25% dextrose PD fluid for 4 weeks. Prior to sacrifice, 100 ml/kg of 4.25% dextrose PD fluid was intraperitoneally administered to the rats. At 2 h later, the rats were anaesthetized, the peritonea were opened, and all the fluid contents were immediately collected to determine the volume (ml). The ultrafiltration volume (UV) was calculated using the following formula: Drainage volume-initial injection volume. The glucose concentration in the dialysate was determined using a glucose assay kit (cat. no. ab65333, Abcam) to assess the transfer capacity of glucose, according to the manufacture's protocols. In addition, the visceral and parietal peritonea were kept for subsequent examination.

All animal experiments in the present study were approved by the Institutional Animal Ethics Committee of China Medical University. All animal procedures were performed according to the Animal Care Institutional Guidelines for experimental animals.

For pathological examination, the parietal peritoneum was fixed in 4% paraformaldehyde for 12 h at room temperature. Paraffin-embedded sections (4 µm) were prepared and stained with hematoxylin (room temperature for ١٥ min) and eosin (room temperature for 3 min; H&E) and/or Masson's trichrome (room temperature for 10 min); 5 random visual fields from each section were imaged under a light microscope (magnification, ×10 and 20) and selected for measuring the thickness of the peritoneum. Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) was used to analyze the images. ‘Peritoneal thickness’, which indicated the extent of fibrosis, was defined as the thickness of the submesothelial zone measured from the inner surface of the abdominal muscle to the mesothelium (21). The average of five random visual fields was used to quantify the thickness of each section.

For immunohistochemical staining, paraffin-embedded sections were heated at 60°C for 10 min, washed with xylene and rehydrated in a descending alcohol series. The deparaffinized sections were blocked with 3% hydrogen peroxide (room temperature for 10 min) and non-immune goat serum (KIT-9710; Fuzhou Maixin Biotech Co., Ltd.; room temperature for 20 min). Then, tissues were incubated with primary antibodies against E-cadherin (1:200; Cell Signaling Technology, Inc.) and vimentin (1:200; Cell Signaling Technology, Inc.) at 4°C overnight following antigen retrieval in boiling sodium citrate buffer (10 mM, pH 6.0) for 5 min. After washing with phosphate-buffered saline (PBS), tissue sections were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (KIT-9710; Fuzhou Maixin Biotech Co., Ltd.) at 37°C for 30 min. Reaction products were visualized by incubation with 3,3′-diaminobenzidine at 37°C for 30 min and then counterstained with hematoxylin at room temperature for 3 min. As a negative control, tissue samples were subject to the same staining procedures with no incubation with primary antibodies. A minimum of five fields were randomly selected in each section and images were acquired by a fluorescence microscope (Nikon Corporation) at a magnification of ×20. Image-Pro Plus was used to calculate the number of cells positive for E-cadherin and vimentin in each field. According to the proportion of positive cells, the following standard was used: 0, negative; 1, 1–20%; 2, 21–50%; 3, 51–80%; 4, 81–100%.

HMrSV5, the HPMC line, was provided by Professor Huimian Xu of The First Affiliated Hospital of China Medical University (Shenyang, China). Cells were isolated from human omentum by enzymatic disaggregation and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) (22). All cells were incubated at 37°C in a 5% CO₂. atmosphere. Confluent cells presented as a monolayer with characteristic cobblestone-like appearance. The following groups were allocated: The NC group (HPMCs without any treatment); the HG group [HPMCs treated with HG (126 mM)]; the HG + Zn group [HPMCs co-treated with HG (126 mM) and ZnSO₄ (10 µM)]; the HG + TPEN group [HPMCs co-treated with HG (١٢٦ mM) and TPEN (1 µM)]; and the Zn group [HPMCs treated with ZnSO₄ (10 µM) alone]. Cultures were incubated for ٤٨ h.

Cell migration ability was assessed using a Transwell assay with 24-well Transwell chambers (Corning, Inc.) with 8-µm pores. A cell suspension containing 5×10⁵ cells/ml was seeded into the upper chamber in serum-free medium. The medium in the lower chamber contained 10% FBS. Cells were untreated or pre-treated with ZnSO₄ (10 µM) or TPEN (١ µM) in the presence or absence of HG (١٢٦ mM). Following incubation at 37°C for 24 h, a cotton-tipped swab was used to carefully remove the cells that had not migrated through the pores; the cells that had migrated onto the lower surface were stained with 0.1% crystal violet for 5 min at room temperature. Five random visual fields in each section were analyzed under a light microscope (magnification, ×20) and cells were counted. Three independent experiments were performed.

An ROS assay was performed using an ROS assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocols. Briefly, HPMCs were treated as aforementioned. Then, 5×10⁶ cells were incubated with 10 µmol/l dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) probes at 37°C for 30 min. Following washing with PBS three times, DCFH-DA was deacetylated by a intracellular nonspecific esterase to 2,7-dichlorofluorescein (DCF), a fluorescent compound. The fluorescence spectrum of DCF which is excited at 488 nm and emitted at 525 nm is very similar to FITC. So FITC can be used to detect DCF fluorescence using a flow cytometer (BD FACSCaliber; BD Biosciences). Results were analyzed using Cell Quest software version 5.1 (BD Biosciences).

For western blotting, tissues and HPMCs were lysed in RIPA buffer (Sigma-Aldrich; Merck KGaA) with protease inhibitors (Sigma-Aldrich; Merck KGaA). Supernatants containing total proteins were harvested to detect HO-1, NQO1, E-cadherin, vimentin and GAPDH. Nuclear proteins of the HPMCs were extracted using a nuclear protein extraction kit (Sigma-Aldrich; Merck KGaA) to detect Lamin B and nuclear factor-like 2 (Nrf2). Protein (30 µg) determined by BCA assay (Beyotime Institute of Biotechnology) was separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in Tris-Buffered saline containing 0.1% Tween-20 for 1 h at room temperature, and then incubated with specific primary antibodies (1:1,000; Cell Signaling Technology, Inc.) against HO-1, NQO, GAPDH, E-cadherin, vimentin, Nrf2 and Lamin B at 4°C overnight. Blots were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. E030120-02; 1:2,000; EarthOx Life Sciences) for 1 h at room temperature. Proteins were visualized using chemiluminescence and clarity western ECL substrate (Bio-Rad Laboratories, Inc.). Densitometric analysis of the western blots was performed using ImageJ v1.48 software (National Institutes of Health).

SPSS (Version 17.0; SPSS, Inc.) was used to analyze statistics. All data were presented as the mean ± standard deviation, and all experiments were performed at least three times. After statistical significance was established by one-way ANOVA, post hoc multiple comparisons were performed using Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1, the rats in the HG group exhibited significantly reduced UV values and an increased glucose transfer capacity compared with the NC. Zn supplementation significantly reduced the glucose transfer capacity and increased the UV compared with HG treatment alone, whereas CQ treatment significantly increased the glucose transfer capacity, but decreased the UV.

It was observed via H&E staining that inflammatory cells infiltrated the interstitium, fibers were exposed, mesothelial cells became round and cylindrical, and cells shed in the HG group (Fig. 2A and B). Furthermore, the HG + CQ group presented a further elevated inflammatory response and significantly thicker peritoneum compared with the HG group. Masson's trichrome staining also demonstrated that HG treatment significantly increased the thickness of the peritoneal collagen fibers compared with the NC group (Fig. 2C and D). This thickening may increase the permeability of peritoneal membrane and the absorption of glucose, which could reduce the osmotic pressure of peritoneal permeation and affect ultrafiltration. Immunohistochemical staining revealed significantly increased expression of vimentin, and decreased expression of E-cadherin in the HG group, compared with the NC group (Fig. 3). Zn supplementation significantly ameliorated these pathological alterations, whereas CQ aggravated the effects of HG on the PMCs (Figs. 2 and 3).

Concentrations and durations of treatment of HG, Zn and TPEN were established according to our previous studies (20,23,24). Following exposure of HPMCs to HG for 48 h, E-cadherin expression decreased, whereas the expression of vimentin was significantly upregulated compared with the NC group (Fig. 4). Co-treatment with Zn (10 µM) for ٤٨ h in HPMCs resulted in a significant alleviation of HG-induced EMT-associated alterations, whereas co-treatment with the Zn chelator TPEN (1 µM) significantly aggravated HG-induced changes in expression, consistent with the results of the in vivo experiments.

The roles of Zn in the migration of HPMCs were investigated by using a Transwell cell migration assay; cells exhibit increased migration following EMT (25,26). HG stimulation for 24 h significantly increased the migration of HPMCs compared with the NC group (Fig. 5). Co-treatment with ZnSO₄ significantly reversed the HG-stimulated increase in migration, whereas co-treatment with the Zn chelator TPEN further promoted the migration of HPMCs (Fig. 5).

The effects of Zn on the intracellular generation of ROS were also determined, as ROS generation may promote the onset of EMT. HG significantly increased the levels of ROS in HPMCs compared with the NC group (Fig. 6). Co-treatment with ZnSO₄ significantly inhibited the HG-stimulated increase in ROS levels, whereas co-treatment with the Zn chelator TPEN further increased ROS levels in HPMCs (Fig. 6).

The Nrf2 antioxidant pathway has been reported to be involved in regulating ROS generation and the EMT (27,28). Therefore, the effects of Zn on the activity of the Nrf2 antioxidant pathway in HPMCs was determined via western blotting. As presented in Fig. 7, the expression levels of nuclear Nrf2 and target proteins of the Nrf2 antioxidant pathway, including NQO1 and HO-1, were upregulated in HG-induced HPMCs compared with the NC group. Co-treatment with TPEN (1 µM) notably inhibited the expression of these proteins, whereas co-treatment with ZnSO₄ (10 µM) significantly promoted the expression of nuclear Nrf٢, NQO١ and HO-١ compared with the HG group. The group treated with Zn alone presented no significant alterations in expression compared with the NC group.