Role of reactive oxygen species in high concentration glucose-induced growth inhibition of human peritoneal mesothelial cells

Introduction

Peritoneal dialysis (PD) is an effective life-sustaining treatment for patients with end-stage renal disease. The peritoneum may become dysfunctional as a dialysis organ over time because of progressive membrane injury. As a result of prolonged exposure to bioincompatible PD fluid (PDF) that contains high levels of glucose and degradation products of glucose, low pH, and high osmolality, peritonitis develops (1). Human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal membrane injury (2). Hypertrophy and arrest of the cell cycle induced by high concentration glucose (HG) in renal mesangial cells in the early stage of diabetic nephropathy, which might be one of the factors of the initiation or progression of the disease (3). A recent study demonstrated that HG inhibits the growth of HPMCs (4). Another study showed that HG causes hypertrophy, senescence, cell cycle arrest, apoptosis, and necrosis, epithelial-to-mesenchymal transition (EMT), peritoneal angiogenesis, which are detrimental to continuous ambulatory PD (CAPD) patients (5,6). Regarding the mechanism of HG cytotoxicity, the oxidative stress state induced by reactive oxygen species (ROS) has been the subject of increasing interest for CAPD patients (7). There is not only increased generation of ROS by mesothelial cells but also by peritoneal phagocytes. ROS can damage HPMCs, but the mechanism of damage remains unclear. ROS can inhibit cell growth and induce cell cycle arrest in other types of cells, such as mesangial and proximal tubular cells (3). Belonging to the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p21^Waf1 and p27^kip1 are negative regulators of the cell cycle. Some effects of HG have been associated with increased expression of p21^Waf1 (8). Additionally, mammalian cells produce hydrogen peroxide (H₂O₂), in low concentrations, H₂O₂ plays an important role in cell proliferation, immunity, and metabolism. However, higher non-physiologic concentrations of H₂O₂ can cause oxidative stress. Stress can lead to cell death if it is not alleviated (9). H₂O₂ is involved in various processes including inducing hypertrophic and senescent phenotypes in some cell types, arresting the cell cycle, and increasing the expression of p21^Waf1 and p27^Kip1 (10-12). Presently, there is no report on the reciprocal effects of these factors and the role of ROS in HG-induced changes in the cell cycle and growth of HPMCs. Therefore, we designed this preliminary study to explore these correlations. We present the following article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-4352/rc).

Methods

Cell culture

A HPMC line, HMrSV5 (kindly provided by Prof. Pierre Ronco, Hospital Tenon, Paris, France), was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco, NY, USA), 20 mmol/L hydroxyethyl piperazine ethane sulfonic acid (HEPES; Gibco, NY, USA), and 2 mmol/L L-glutamine at 37 ℃ in a humidified atmosphere with 5% CO₂.

Research design

The study was divided into 2 parts—part A and part B—to observe the effects of HG and H₂O₂ purchased from Nanjing Chemical Reagent (Nanjing, China) on HPMCs, respectively. All the test solutions used DMEM for all stimulations.

Part A comprised 8 groups: group a1 with 0.1% glucose in DMEM (G₀) as the control group, group a2 with 1.35% glucose (1.35G), group a3 with 3.86% glucose (3.86G), group a4 with 3.86% mannitol (MCE, Shanghai, China) (3.86M), group a5 with pyruvate 35 mmol/L in DMEM (G₀ + P), group a6 with 3.86% glucose and pyruvate (MCE) 35 mmol/L (3.86G + P), group a7 with catalase 500 U/L (G₀ + C), and group a8 with 3.86% glucose and catalase 500 U/L in DMEM (3.86G + C). Part B consisted of 6 groups: group b1 with H₂O₂ 0.5 mmol/L in DMEM (G₀ + 0.5H), group b2 with H₂O₂ 0.1 mmol/L (G₀ + 0.1H), group b3 with H₂O₂ 0.5 mmol/L and 3.86% glucose (3.86G + 0.5H), group b4 with H₂O₂ 0.1 mmol/L and 3.86% glucose (3.86G + 0.1H), group b5 with H₂O₂ 0.5 mmol/L, 3.86% glucose, and pyruvate 35 mmol/L (3.86G + 0.5H + P), group b6 with 0.1% glucose (G₀, control group). Unless noted otherwise, the stimulation time was 24 and 12 h in part A and B, respectively. Each experiment was performed with 3 independent replicates.

Morphological observation

The HPMCs were harvested from culture flasks by trypsin digestion and centrifugation as described previously，and inoculated into a 24-well culture plate at 6.0×10⁴ cells/well and grown at 37 ℃ in a 5% CO₂ incubator. When the cells reached to logarithmic phase, incubated with 0.1% glucose, 3.86% glucose, and 0.5 mmol/L H₂O₂. Then, the morphological changes were observed under a phase-contrast microscope 24 h post-stimulation.

Flow cytometric analysis

Cells of groups a1–a6 and b1–b6 were assessed by flow cytometry 12–24 h post-incubation. Subsequently, the cells were washed with phosphate-buffered saline (PBS), trypsinized, fixed with 70% ethanol at 4 ℃ overnight, and stained with propidium iodide (PI) for 30 min. Then, 10⁶ cells/mL in each group were harvested and analyzed on a FACS Epics XL Flow Cytometer (Coulter) to elucidate cell cycle distribution and calculate the proportion of cells in the G₁ phase.

Cell proliferation studies with 3H-thymidine incorporation

The proliferation of the HPMCs was estimated indirectly based on the incorporation of ³H-thymidine into growing cells that were seeded in a 96-well plate at a density of 10³ cells/well. After 48 h, the cells were exposed to the test solutions (groups a1–a8 and b1–b6, i.e., 14 groups) for 24 h. Subsequently, ³H-thymidine (1 Ci/well, procured from Chinese Science Academy, Shanghai Institute of Nuclear Research) was added for an additional 6-h incubation period. Then, the test solutions were discharged, and the cells were trypsinized and collected on a filter paper using a semi-automatic cell collector (type of DYQIII). The filter papers were placed into the tube, and the scintillation reagent was added to count the samples in a beta counter (Pharmacia).

Reverse-transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from HPMCs in the a1–a6 and b1–b6 groups using TRIzol as stated by the protocol. An equivalent of 2 µg of RNA was reversed transcribed into complementary DNA (cDNA) using M-MLV reverse transcriptase (Gibco, NY, USA). PCR amplification was performed in a total volume of 50 µL using the following protocol: 94 ℃ for 5 min, 25 cycles of 94 ℃ for 1 min, 65 ℃ (p21) for 30 s, 58 ℃ (GAPDH) for 30 s, 72 ℃ for 1 min, and finally, 72 ℃ for 7 min. The PCR products were separated by 2% agarose gel electrophoresis with 0.5 µg/mL ethidium bromide and photographed. Bands were scanned using a densitometer (Gensnap), and band density was compared to that of the housekeeping gene. The sequences of the amplification primers were as follows: p21^Waf1 (sense: 5'-CAGGGGACAGCAGAGGAAGA-3', antisense 5'-GGGCGGCCAGGGTATGTAC-3'), p27^Kip1 (sense: 5'-ATGTCAAACGTGCGAGTGTC-3', antisense: 5'-TCTGTAGTAGAACTCGGGCAA-3'), GAPDH (sense: 5'-CCACCCATGGCAAATTCCATGGCA-3', antisense: 5'-TCTAGAGGGCAGGTCAGGTCCACC-3').

Immunocytochemistry (ICC)

HPMCs were grown on 1×1 cm² slides and stimulated with various test solutions (groups a1–a8 and b1–b6) for 12–24 h. Then, the stimulations were removed, and the cell layer was rinsed with PBS. Subsequently, the cells were fixed with freshly prepared 4% paraformaldehyde at room temperature for 20 min. Then, the cells were rehydrated with PBS and incubated with a primary antibody (mouse anti-human p21^Waf1 or rabbit anti-human p27^Kip1) for 4 ℃ overnight (1:100 dilution, Santa Cruz, CA, USA). After washing with PBS, the cells were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (37 ℃ for 2 h), and the immune reaction was detected by 3,3'-diaminobenzidine (DAB). The nuclei were counterstained with hematine. Computer software was used for semiquantitative analysis to calculate the proportion of positive cells.

Western blot

Groups a1, a3, a6, and b3 were stimulated in a 90-mm dish for 24 h. Then total cellular proteins extracted from the HPMCs, quantified, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). After electrophoresis and transfer (nitrocellulose membrane, Bio-Rad, Amresco), the polyvinylidene fluoride (PVDF) membranes were blocked in 5% milk prior and probed with mouse monoclonal antibody to human p21^Waf1 and rabbit anti-human p27^Kip1 (Santa Cruz, CA, USA). This was followed by incubation with the secondary antibody, namely HRP-conjugated goat anti-mouse IgG (Yeasen, Shanghai, China). Finally, the membranes were developed using enhanced chemiluminescence (ECL) blotting substrate (Amersham, USA) and exposed to X-ray film. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. The immunoreactive band intensity was measured on an image analysis system (Genesnap).

Statistical analysis

Data are shown as the mean ± standard error of the mean (SEM). Each experiment was repeated at least 3 times. Student’s t-test or one-way analysis of variance (ANOVA) were used for statistical analysis using SPSS 10 software (IBM Corporation, Armonk, NY, USA). P<0.05 was considered statistically significant.

Results

Morphological observations

Morphological changes were observed after 24-h stimulation. The HPMCs in group G₀ (control group) showed a typical polygonal and cobblestone monolayer appearance when grown to confluency. In the 3.86G group, the nucleolus was obvious, with the appearance of poly-nuclei and hypertrophy of the cell. In the 0.5 mmol/L H₂O₂ group, the changes were similar to that in the HG group.

Effects of various stimulations on cell growth (proliferation)

No significant difference in cell growth was observed among group a1 (G₀), a5 (G₀ + P), and a7 (G₀ + C), and the count per minute (CPM) was higher than that in group a2 (1.35G) or a3 (3.86G). No difference was detected between the cell numbers in group a6 (3.86G + P) and a8 (3.86G + C), but both had significantly higher cell numbers than group a3 (Figure 1A).

Figure 1 ³H-thymidine incorporation analysis. (A) Effect of glucose and antioxidants on HPMCs proliferation. *, a2 vs. a1, a2 vs. a3, P<0.05; ^#, a3 vs. a5, a3 vs. a7, P<0.05. a1: with 0.1% glucose in DMEM (G₀), as the control group; a2: with 1.35% glucose (1.35G); a3: with 3.86% glucose (3.86G); a4: with 3.86% mannitol (3.86M); a5: with pyruvate 35 mmol/L in DMEM (G₀ + P); a6: with 3.86% glucose and pyruvate 35 mmol/L (3.86G + P); a7: with catalase 500 U/L (G₀ + C); a8: with 3.86% glucose and catalase 500 U/L in DMEM (3.86G + C). (B) Effect of glucose, H₂O₂, and the antioxidant on the proliferation of HPMCs. *, b1 vs. b6, b1 vs. b3, P<0.05; ^#, b5 vs. b3, P<0.05. b1: with H₂O₂ 0.5 mmol/L in DMEM (G₀ + 0.5H); b2: with H₂O₂ 0.1 mmol/L (G₀ + 0.1H); b3: with H₂O₂ 0.5 mmol/L and 3.86% glucose (3.86G + 0.5H); b4: with H₂O₂ 0.1 mmol/L and 3.86% glucose (3.86G + 0.1H); b5: with H₂O₂ 0.5 mmol/L, 3.86% glucose, and pyruvate 35 mmol/L (3.86G + 0.5H + P); b6: with 0.1% glucose (G₀), as the control group. CPM, count per minute; HPMC, human peritoneal mesothelial cell; DMEM, Dulbecco’s modified Eagle’s medium; H₂O₂, hydrogen peroxide.

A high concentration of H₂O₂ (0.5 mmol/L) inhibited the proliferation of HPMCs, which was not demonstrated with the 0.1 mmol/L dose (b2 vs. b6, P>0.05). This effect was accentuated by HG conditions (b1 vs. b3, P<0.05), while antioxidant treatment attenuated this effect (b3 vs. b5, P<0.05) (Figure 1B).

Effects of various stimulations on the cell cycle

HG conditions elevated the proportion of G₁ phase cells (shown as percent in parentheses) in a dose-dependent manner. Compared to the a2 (77.1±7.40) or a3 (82.3±6.20) groups, the a1 group (68.2±42.5) (control) differed significantly (P<0.05). However, no significant difference was detected between the proportion in high osmolality a4 (70.1±5.21) and a1 (control) (P>0.05). Without HG, pyruvate did not change the proportion of G1 phase significantly [a5 (72.2±6.22) vs. a1] (P>0.05). After adding the antioxidant, the proportion in the a6 group (72.2±5.32) was decreased compared to the a3 group (82.3±6.20) (P<0.05).

Moreover, the proportion of G₁ phase cells was increased, and HG enhanced this effect_. [comparing group b1 (75.2±5.35) with b6 (65.3±7.21), b3 (86.2±7.10) with b1, and b4 (80.2±8.12) with b2 (71.1±4.02), P<0.05]. However, after adding the antioxidants, the proportion was decreased when comparing the b5 group (72.2±4.22) to the b3 group (P<0.05).

Effects of various stimulations on p21^Waf1 expression

The expression of p21^Waf1 mRNA in group a3 was maximal. Following stimulation of HPMCs with HG and H₂O₂ (Figure 2A-2D), p21^Waf1 mRNA expression was elevated compared with the control in a dose-dependent manner (a3 vs. a2, a2 vs. a1; b2 vs. b6, P<0.05). After adding the antioxidant in the HG- (a3 vs. a6) or H₂O₂-containing medium (b3 vs. b5), the expression was significantly reduced in both. Moreover, no statistically significant difference was detected in p21^Waf1 gene expression among groups a4, a5, and control (Figure 2C). On the other hand, HG enhanced the effect of H₂O₂ (b4 vs. b2), as shown in Figure 2D (P<0.05).

Figure 2 Effects of various stimulations on p21^Waf1. (A,C) Effect of glucose, mannitol, and pyruvate on the expression of p21^Waf1 mRNA in HPMCs. Lane 1: a3 (3.86G); lane 2: a2 (1.38G); lane 3: a6 (3.86G + P); lane 4: a1 (G₀); lane 5: a4 (3.86M); lane 6: a5 (G₀ + P); lane 7: marker. *, a2 vs. a1, P<0.05; ^#, a3 vs. a2, P<0.05; ^Δ, a3 vs. a6, P<0.05. (B,D) The effect of glucose, H₂O₂, and pyruvate on the expression of p21^Waf1 mRNA in HPMCs. Lane 1: b1 (G₀ + 0.5H); lane 2: b2 (G₀ + 0.1); lane 3: b3 (3.86G + 0.5H); lane 4: b4 (3.86G + 0.1H); lane 5: b5 (3.86G + 0.5H + P); lane 6: b6 (G₀); lane 7: marker. *, b2 vs. b6, P<0.05; ^#, b3 vs. b5, P<0.05; ^Δ, b4 vs. b2, P<0.05. (E-G) ICC analysis. (E) Effect of glucose and antioxidants on the expression of p21^Waf1 protein. *, a2 vs. a1, P<0.05; ^#, a3 vs. a2, P<0.05; ^Δ, a6 vs. a3, P<0.05. (F) Effect of glucose, H₂O₂, and pyruvate (antioxidant) on the expression of p21^Waf1 protein. *, b1 vs. b2, P<0.05; ^#, b1 vs. b6, P<0.05; ^Δ, b3 vs. b1, P<0.05; ^☆, b3 vs. b5, P<0.05. (G) ICC analysis of the expression of p21^Waf1 protein with glucose, H₂O₂, and pyruvate (antioxidant) in HPMCs (×20) (DAB staining). a, a3 (3.86G); b, a2 (1.35G); c, a6 (3.86G + P); d, a1 (G₀); e, b3 (3.86G + 0.5H); f, b5 (3.86G + 0.5H + P); g, b1 (G₀ + 0.5H); h, b6 (G₀). (H) Western blot analysis of p21^Waf1 protein expression in the HPMCs of each group. Lane 1: G₀ (a1); lane 2: 3.86G (a3); lane 3: 3.86G + 0.5H (b3); lane 4: 3.86G + P (a6). a1: with 0.1% glucose in DMEM (G₀), as the control group; a2: with 1.35% glucose (1.35G); a3: with 3.86% glucose (3.86G). a4: with 3.86% mannitol (3.86M); a5: with pyruvate 35 mmol/L in DMEM (G₀ + P); a6: with 3.86% glucose and pyruvate 35 mmol/L (3.86G + P); a7: with catalase 500 U/L (G₀ + C); a8: with 3.86% glucose and catalase 500 U/L in DMEM (3.86G + C). b1: with H₂O₂ 0.5 mmol/L in DMEM (G₀ + 0.5H); b2: with H₂O₂ 0.1 mmol/L (G₀ + 0.1H); b3: with H₂O₂ 0.5 mmol/L and 3.86% glucose (3.86G + 0.5H); b4: with H₂O₂ 0.1 mmol/L and 3.86% glucose (3.86G + 0.1H); b5: with H₂O₂ 0.5 mmol/L, 3.86% glucose, and pyruvate 35 mmol/L (3.86G + 0.5H + P); b6: with 0.1% glucose (G₀), as the control group. HPMCs, human peritoneal mesothelial cells; H₂O₂, hydrogen peroxide; ICC, immunocytochemistry; DAB, 3,3'-diaminobenzidine; DMEM, Dulbecco’s modified Eagle’s medium.

The expression of p21^Waf1 protein in the HG group (a3) was maximal, followed by the a2, a6, a8, a1, a4, a5, and a7 groups (Figure 2E). As a response to HG stimulation, p21^Waf1 protein expression was higher in HPMCs than in controls in a dose-dependent manner (a3 vs. a2, a2 vs. a1, P<0.05). This effect was reduced significantly by the antioxidant (a6 vs. a3, P<0.05). No difference was observed between p21 expression in the high osmolality (a4) group and the control group (Figure 2E). The expression of p21^Waf1 protein in the HG group (b3) was the strongest, followed by the b4, b5, b1, b2, and b6 groups. We found that exogenous H₂O₂ elevated the expression of p21 (b1 vs. b6, b1 vs. b2, P<0.05), which was enhanced by HG (b3 vs. b1), and the antioxidant counteracted the effect of exogenous H₂O₂ (b3 vs. b5, P<0.05) (Figure 2F). The representative ICC images for p21 are shown in Figure 2G.

The results of Western blot were similar to that of ICC. The expression between the HG group (a3) and the HG plus H₂O₂ group (b3) did not differ significantly, and both groups had stronger expression than the control group (a1). The expression (a6) was reduced after the addition of antioxidants but remained higher than the control (P<0.05) (Figure 2H).

Effects of various stimulations on p27^Kip1 expression

The representative results of RT-PCR for p27 are shown in Figure 3A,3B. Following stimulation of HPMCs with HG (Figure 3A) and H₂O₂ (Figure 3B), no significant differences were observed in p27 gene expression (Figure 3A,3B) (P>0.05).

Figure 3 Effects of various stimulations on p27^Kip1. (A) Effect of glucose, mannitol, and pyruvate on the expression of p27^Kip1 mRNA in HPMCs. No significant differences were detected in p27 gene expression (P>0.05). Lane 1: a3 (3.86G); lane 2: a2 (1.38G); lane 3: a6 (3.86G + P); lane 4: a1 (G₀); lane 5: a4 (3.86M); lane 6: a5 (G₀ + P); lane 7: marker. (B) Effect of glucose, H₂O₂, and pyruvate on the expression of p27^Kip1 mRNA in HPMCs. No significant differences were detected in p27 gene expression (P>0.05). Lane 1: b1 (G₀ + 0.5H); lane 2: b2 (G₀ + 0.1H); lane 3: b3 (3.86G + 0.5H); lane 4: b4 (3.86G + 0.1H); lane 5: b5 (3.86G + 0.5H + P); lane 6: b6 (G₀); lane 7: marker. (C-E) ICC analysis. (C) Effect of glucose and antioxidants on the expression of p27^Kip1 protein. *, a1 vs. a2, a1 vs. a3, P<0.05. (D) Effect of glucose, H₂O₂, and antioxidants on the expression of p27^Kip1 protein. *, b6 vs. b1, P<0.05. (E) ICC analysis of the expression of p27^Kip1 protein with glucose, H₂O₂, and pyruvate (antioxidant) in HPMCs (×20) (DAB staining). a, a3 (3.86G); b, a6 (3.86G + P); c, b1 (G₀ + 0.5H); d, a1 (G₀). (F) Western blot analysis of p27^Kip1 protein expression in HPMCs in each group. Lane 1: G₀ (a1); lane 2: 3.86G (a3); lane 3: 3.86G + 0.5H (b3); lane 4: 3.86G + P (a6). a1: with 0.1% glucose in DMEM (G₀), as the control group; a2: with 1.35% glucose (1.35G); a3: with 3.86% glucose (3.86G). a4: with 3.86% mannitol (3.86M); a5: with pyruvate 35 mmol/L in DMEM (G₀ + P); a6: with 3.86% glucose and pyruvate 35 mmol/L (3.86G + P); a7: with catalase 500 U/L (G₀ + C); a8: with 3.86% glucose and catalase 500 U/L in DMEM (3.86G + C). b1: with H₂O₂ 0.5 mmol/L in DMEM (G₀ + 0.5H); b2: with H₂O₂ 0.1 mmol/L (G₀ + 0.1H); b3: with H₂O₂ 0.5 mmol/L and 3.86% glucose (3.86G + 0.5H); b4: with H₂O₂ 0.1 mmol/L and 3.86% glucose (3.86G + 0.1H); b5: with H₂O₂ 0.5 mmol/L, 3.86% glucose, and pyruvate 35 mmol/L (3.86G + 0.5H + P); b6: with 0.1% glucose (G₀), as the control group. HPMCs, human peritoneal mesothelial cells; H₂O₂, hydrogen peroxide; ICC, immunocytochemistry; DAB, 3,3'-diaminobenzidine; DMEM, Dulbecco’s modified Eagle’s medium.

Following the stimulation of HPMCs with HG, the expression of p27^Kip1 protein in the a3, a6, a8, and a2 groups was similar, with approximately 100% positive expression and no significant differences. Moreover, p27 expression in the a1, a4, a5, and a7 groups was also similar, approximately 75% positive, which was significantly different when compared to that of the a3, a6, a8, a2 groups (P<0.05). This indicated that HG increased p27 expression. However, a significant change was observed in p27 expression after pyruvate addition. Also, no difference was observed in p27 expression between the high osmolality group (a4) and the control group (Figure 3C). Both exogenous H₂O₂ and H₂O₂ plus glucose elevated the expression of p27. The positive cell percent of all 5 groups with HG or H₂O₂ was nearly 100% compared to that of the b6 group (75%), which reached statistical significance (Figure 3D). The representative ICC images for p27 are shown in Figure 3E. The results of Western blot were similar to p21^Waf1 (Figures 2H,3F).

Discussion

In our study, we preliminarily demonstrated: (I) the effect of ROS and HG on the growth (proliferation) of HPMCs and the role of p21 and p27 in this process; (II) the role of ROS in HG-induced changes in cell growth in HPMCs.

Effects of HG and exogenous H₂O₂ on the growth and cell cycle of HPMCs

This study suggested that HG inhibits cell proliferation, as described previously (4). We also found that exogenous H₂O₂ inhibits the proliferation of HPMCs in a dose-dependent manner. The low concentration of H₂O₂ did not have an obvious effect, which is supported by similar study on cultured human tracheal epithelium and renal proximal tubule cells (3). On the other hand, we also observed that the addition of H₂O₂ in HG medium enhanced the cytotoxicity of H₂O₂. These results suggest that peritonitis or long-term dialysis might cause injury to CAPD patients from the increased generation of H₂O₂, which might be harmful in the presence of HG.

We speculated that these effects are related to the changes in HG- or exogenous H₂O₂-induced cell cycle. In this study, we found that both HG and exogenous H₂O₂ can induce cell cycle arrest of HPMCs at the G₁ phase, but not with HG-induced high osmolality. This finding was supported by Zhang et al. (10) who demonstrated that H₂O₂ induced the premature senescence of human diploid fibroblasts by increasing p21 gene expression and arresting the cell cycle at the G₁ phase. Therefore, based on the current results, we can deduce that HG inhibits the growth of HPMCs by arresting cell cycle progression.

Effects of HG and exogenous H₂O₂ on the expression of p21^Waf1 and p27^Kip1 in HPMCs

In diabetes research, p21 and p27 have been associated with the cytotoxicity of HG. We found that both HG and exogenous H₂O₂ increased the expression of p21^Waf1, which was supported by Lin et al. (8) in a diabetic nephropathy study and Kim et al. (11) in human fibroblast cells. For p27^Kip1, protein expression was increased by either HG or exogenous H₂O₂, but neither of them influenced the gene expression of p27^Kip1. Elson et al. (13) demonstrated that in most situations, the gene expression of p27 is consistent in the cell cycle, indicating that the alteration of p27 occurs at the protein level, corresponding to our results. In a study on diabetic nephropathy, Aitken et al. (14) demonstrated that HG inhibits cell proliferation (cell growth) in a dose-dependent manner, which is associated with the increased expression of p27^Kip1. Thus, we inferred that both HG and exogenous H₂O₂ can inhibit the growth of HPMCs by arresting cell cycle progression via increased expression of p21 and p27.

Effects of endogenous ROS on HG-induced growth inhibition and the expression of p21^Waf1 and p27^Kip1 in HPMCs

HG-induced growth inhibition and cell cycle arrest in HPMCs are attributed to a complex mechanism. Recently, the role of ROS in PD has been under intense focus. ROS are now being accepted as signaling molecules which activate protein kinases, transcription factors, and gene expression (15). Horiuchi et al. (16) found that cultured HPMCs can produce H₂O₂. Moreover, mesothelial cells in culture significantly increased the generation of H₂O₂ when exposed to HG. Recently, a study measured ROS generation by HPMCs cultured under HG using a ROS-sensitive fluorescence probe and confocal microscope (15). On the other hand, peritoneal infection or long-term, continuous stimulation by the biologically incompatible peritoneal dialysate is likely to produce ROS in the peritoneal cavity, which might harm HPMCs and CAPD patients.

In this study, HG-induced p21^Waf1 expression was downregulated at both the gene and protein level after the addition of antioxidants to the HG media. Moreover, G₁ phase arrest and inhibited cell growth were attenuated, indicating that the antioxidants (pyruvate and catalase) counteracted the effect of endogenous ROS. Shostak et al. reported that pyruvate could improve the peritoneal dialysate-induced inhibition of proliferation in HPMCs (17). These results were supported by other similar study (18). We also found a specific scavenger for H₂O₂, namely catalase, which can improve the inhibition of cell growth. This finding suggests endogenous H₂O₂ is a vital part of ROS, which is linked to HG-induced cell growth inhibition. Although a study has reported the ROS-increased expression of p27^Kip1 and HG-increased expression of p27^Kip1, the effect of endogenous ROS is not certain. At least, in our preliminary study, we could not demonstrate any significant influence of endogenous ROS on the expression of p27, as shown by ICC results in our previous study (19). Thus, we infer that in our methods, the incubation time of HG with HPMCs is limited, which might restrict the effect of ROS. Therefore, we could not detect the effect of endogenous H₂O₂ on p27 expression with ICC, which is different from CAPD in vivo. Also, the HPMCs were in contact with HG for a prolonged period. Western blotting presented a different positive result, indicating a certain effect of endogenous ROS on p27 expression. These discrepancies in the results may be due to the high sensitivity of Western blots compared to ICC in quantitative analysis. Thus, it may be inferred that HG-dependent cell damage is a function of both the reduced production of scavengers and the increased generation of ROS by mesothelial cells.

Interestingly, one study suggested that HG increases the expression of p21 and p27, mediated by TGF-β1 (20). These results indicate that chronic exposure to elevated glucose might result in TGF-β1-mediated accelerated senescence of HPMCs in vitro. Jin et al. demonstrated that the role of HG is related to ROS (21), but to the best of our knowledge, the role of ROS in p21 and p27 expression in HPMCs has not yet been studied. Our preliminary results suggested that HG inhibits cell growth by increasing ROS-mediated p21 and p27 expression.

Nevertheless, the present study has some limitations. We could not directly measure H₂O₂ concentration in HG medium. Furthermore, the significance of direct measurement is with little meaning since the effect of H₂O₂ could be influenced by other factors, such as cell types and the microenvironment. Our previous study found that high glucose increased the expression of monocyte chemoattractant protein-l (MCP-1) and interlukin-8 (IL-8), and endogenous ROS could induce the expression of IL-8 (22). Many researches have confirmed that the classical signaling pathway involved about p21^Waf1 is p21^WAF1/CIP1, and the classical signaling pathway involved in p27^Kip1 is mitogen-activated extracellular signal-regulated kinase-extracellular regulated protein kinases (MEK-ERK) signaling (23,24). But the mechanisms of HG-induced changes in the cell cycle and cell proliferation in HPMCs have not yet been clarified, although ROS may play a role. Thus, the signal transduction pathways through which ROS affects the cell cycle and proliferation need to be investigated further.

Acknowledgments

We thank Prof. Yijie Wu, Department of Endocrinology, Shanghai General Hospital, for revising the English manuscript; Prof. Shanlin Liu, Department of Biochemistry, Shanghai Medical College, Fudan University, China, for his kind suggestions in this study; and Dr. Kaitlyn Reasoner for her kind help in revising and polishing the English writing of this article.

Funding: This study was supported by the National Natural Science Foundation of China (No. 81800676).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-4352/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-4352/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-4352/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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(English Language Editor: C. Betlazar-Maseh)