Autophagy caused by oxidative stress promotes TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells

Epithelial-to-mesenchymal transition (EMT) is one of the main causes of peritoneal fibrosis. However, the pathophysiological mechanisms of EMT, specifically its relationship with autophagy, are still unknown. This study aimed to evaluate the role of autophagy in transforming growth factor-beta 1 (TGF-β1)-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-β1 (2 and 5 ng/mL) and changes in autophagy markers and the relationship between autophagy and EMT were evaluated. We also identified changes in EMT- and autophagy-related signaling pathways after autophagy and NADPH oxidase 4 (NOX4) inhibition. TGF-β1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, resulting in mitochondrial damage. Treatment with GKT137831 (20 μM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-β1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 μM) downregulated TGF-β1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (3-MA) (2 mM) or autophagy-related gene 5 (ATG5) gene silencing decreased the TGF-β1-induced EMT in HPMCs. The suppressor of mothers against decapentaplegic 2/3 (Smad2/3), autophagy-related phosphoinositide 3-kinase (PI3K) class III, and protein kinase B (Akt) pathways, and mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinase (ERK) and P38, were involved in TGF-β1-induced EMT. Autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Direct inhibition of autophagy and its indirect inhibition through the reduction of mitochondrial damage by upstream NOX4 inhibition reduced EMT in HPMCs. These results suggest that autophagy could serve as a therapeutic target for the prevention of peritoneal fibrosis in patients undergoing peritoneal dialysis.


INTRODUCTION
Peritoneal dialysis (PD) utilizes the peritoneal membrane to exchange uremic toxins and water, effectively treating patients with end-stage kidney disease.However, long-term exposure to PD solutions with high concentrations of glucose and glucose degradation products causes functional and morphological changes to the peritoneum, which results in ultrafiltration failure [1,2].Mesothelial cells are the primary cells of the peritoneum, and their phenotypic transition is a crucial mechanism underlying peritoneal fibrosis [3,4].Following the initiation of PD, human peritoneal mesothelial cells (HPMCs) progressively lose their epithelial phenotype and acquire myofibroblast-like characteristics through epithelial-tomesenchymal transition (EMT) [3].Therefore, there has been considerable research interest in the pathophysiological mechanisms of EMT and its downregulation.
Reactive oxygen species (ROS) are generated in various intraperitoneal cells, including HPMCs, macrophages, and mast cells [5], and ROS generation is a well-known cause of peritoneal fibrosis in HPMCs [6].High glucose concentrations increase the levels of transforming growth factor-beta 1 (TGF-β1), which upregulates cellular ROS through increased mitochondrial metabolism and NADPH oxidase (NOX) [7].Various cell signaling pathways are involved in ROS-induced EMT; however, the precise mechanisms have yet to be elucidated [8].
Autophagy is a protein degradation system that regulates cellular homeostasis [9,10].Cytoplasmic components, including various proteins and organelles, are sequestered into doublemembrane cytosolic vesicles called autophagosomes, which fuse with a lysosome for degradation and recycling [11,12].Autophagy regulates TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts [13] and induces mesothelial cell transformation [14].However, the role of autophagy and its relationship with oxidative stress in HPMCs remains uncertain.This study investigated the role of autophagy in the EMT of HPMCs caused by PD.This will help to identify the novel mechanisms underlying peritoneal dysfunction in PD patients and provide new therapeutic targets.

MATERIALS AND METHODS Primary culture of HPMCs
Omenta were obtained from patients who had undergone abdominal surgery.These patients provided informed consent to the use of their tissue in this study.HPMCs were isolated from the omenta using enzymatic disaggregation.For this, the cells were treated with trypsinethylenediaminetetraacetic acid 0.25% for 30 min in a water bath at 37 °C.The cultured HPMCs were seeded at 80% confluence and maintained in medium 199 (M199) supplemented with 20% fetal bovine serum (FBS) at 37 °C under 5% CO 2 conditions.The primary cultured cells used were between passages 2 and 5.

Cell viability assay
To study cell viability in the presence of 3-MA or GKT137831, cultured HPMCs were plated on 96-well plates and incubated with 1% FBScontaining M199 medium for 24 h.Subsequently, the cells were treated with 3-MA (1-10 mM) or GKT137831 (1-50 μM).Cell viability was analyzed using Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions.Absorbance was measured at 450 nm with a microplate reader (SPARK 10 M, Tecan, Durham, NC, USA).The results were expressed as percentages of the control value.

NOX4 inhibition
For the inhibition of NOX4, a NOX1/4 dual inhibitor, GKT137831, was used.HPMCs were preincubated with 20 µM GKT137831 for 2 h before stimulation with TGF-β1 (2 and 5 ng/mL) to prevent ROS formation and then incubated with TGF-β1 for a further 48 h.

Intracellular ROS measurement
The concentrations of intracellular ROS in the HPMCs were measured using a 2′,7′-dichlorofluorescin diacetate (DCF-DA)-Cellular ROS Assay Kit (Abcam, Cambridge, MA, USA), according to the manufacturer's instructions.Briefly, cells were plated in 96-well black plates (1.5 × 10 4 cells/well) and treated with TGF-β1 (2 and 5 ng/mL).After incubation for 24 h, 25 μM DCF-DA was added to each well and incubated for 45 min at 37 °C in dark conditions.Their fluorescence was measured using a microplate reader (SPARK 10 M, Tecan) at excitation and emission wavelengths of 485 and 535 nm, respectively.The fluorescence signal was expressed as a percentage of the control.

Hydrogen peroxide assay
Extracellular H 2 O 2 was measured using an Amplex Red Hydrogen Peroxide Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.Briefly, HPMCs were seeded in a 96-well black plate and treated with TGF-β1 (2 and 5 ng/mL).The H 2 O 2 released from the treated HPMCs reacted with the Amplex Red reagent, in which horseradish peroxidase produced the red fluorescent oxidation product, resorufin.The resorufin fluorescence was measured using a fluorescence microplate reader (SPARK 10 M, Tecan) at excitation and emission wavelengths of 560 and 590 nm, respectively.The H 2 O 2 concentrations were calculated using standard curves.

Transmission electron microscopy
To compare the autophagy levels of HPMCs incubated for 48 h with or without TGF-β1 (2 and 5 ng/mL), we used transmission electron microscopy (TEM).After overnight fixation with 2.5% glutaraldehyde at 4 °C, the treated HPMCs were washed with phosphate-buffered saline (PBS) and post-fixed in 1% osmium (VIII) oxide (OsO 4 ).The samples were then dehydrated in a series of graded ethanol solutions, embedded in Epon resin, and then cut into 60-80 nm-thick sections.These ultrathin sections were observed using a Hitachi HT7000 electron microscope (Tokyo, Japan).

Autophagy flux assay
A Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) was used to measure autophagy in HPMCs, according to the manufacturer's protocol.Hoechst and Cyto-ID Green (Enzo Life Sciences) detection reagents (1:500 final dilution) were added directly to cells in an 8-well chamber slide.The cells were observed using a Zeiss Axio microscope.For plate reader measurements, the Hoechst and green detection reagent (1:1000 final dilution) solutions were added to cells grown in 96-well plates.Their fluorescence was measured using a microplate reader (SPARK 10 M, Tecan) at the excitation and emission wavelengths of 480 and 530 nm, respectively.

RNA extraction and real-time quantitative RT-PCR
Total ribonucleic acid (RNA) was extracted from treated HPMCs using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions.The total RNA (1 µg) was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a PrimeScript cDNA Synthesis Kit (TaKaRa Shuzo Co. Ltd., Otsu, Japan).All primers for the quantitative reverse transcription PCR (qRT-PCR) (Suppl.Table 1) were designed using Primer Express v.1.5(Applied Biosystems, Foster City, CA, USA) software.The qRT-PCR was performed, in duplicate, on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems).All samples were analyzed using the comparative Ct method (2 −ΔΔCt ) for the relative quantification of gene expression and normalization with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression.

MitoSOX staining
MitoSOX Red selectively targets mitochondria and forms a stable fluorescent compound through superoxide oxidation.This was imaged using a green alter on a fluorescent microscope.For live-cell imaging, cells were stained with 5 mM MitoSOX™ Mitochondrial Superoxide Indicators (M36008, Invitrogen) for 10 min in an incubator, then washed twice with AM media.The live imaging was performed using an LSM 800 confocal microscope (Zeiss LSM 800 Laser Scanning Confocal Microscope SOP).

Mitochondrial oxygen consumption rate measurement
Cells were seeded on a Seahorse XF 96-well plate overnight for the measurement of their oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).Before measurement, the cells were washed and equilibrated for 1 h at 37 °C with XF base medium (102353-100; Agilent Technologies) supplemented with 1X GlutaMAX (35050; Gibco), 1 mM sodium pyruvate (S8636; Sigma-Aldrich), and 25 mM glucose (G7528; Sigma-Aldrich) (pH 7.4).A prehydrated sensor cartridge was loaded with the mitochondrial inhibitors to deliver a final concentration of 1 μM oligomycin (75351; Sigma-Aldrich), 1 μM carbonyl cyanide-ptrifluoromethoxy phenylhydrazone (FCCP) (C2920; Sigma-Aldrich), and 0.5 μM rotenone (R8875; Sigma-Aldrich) + 0.5 μM antimycin A (A8674; Sigma-Aldrich).This was placed on the XF 96-well plate, and the OCR was measured before and after the sequential injection of the mitochondrial inhibitors.For ECAR measurement, the cells were washed and the medium was replaced with glucose-or pyruvate-free XF base medium supplemented with 1 mM glutamine (G8540; Sigma-Aldrich) for 1 h before the assay.Subsequently, the cells were loaded with a sensor cartridge containing glucose, oligomycin (75351; Sigma-Aldrich), and 2-deoxyglucose (D6134; Sigma-Aldrich).These were injected at final concentrations of 10 mM, 2 μM, and 50 mM, respectively.The OCR and ECAR were measured using a Seahorse XFe 96 (Agilent Technologies) and analyzed using Wave 2.6.0 software after normalization to the total cell number.

Statistical analysis
Mann-Whitney U-tests were used to compare the mean values between two groups as the sample size was small.Statistical analyses were performed using SPSS v. 22.0 (IBM Corp., Armonk, NY, USA).P-values < 0.05 were considered statistically significant.

Ethics statement
The study protocol was reviewed and approved by the Institutional Review Board of the Kyungpook National University Hospital (KNUH-2016-04-002-001).The study was conducted in accordance with the principles of the 2013 revision of the Declaration of Helsinki.Written informed consent to specimen use and study publication was obtained from all patients who provided omentum specimens.

TGF-β1 induces EMT and ROS generation in HPMCs
Treatments with 2 and 5 ng/mL TGF-β1 decreased the messenger RNA (mRNA) expression of the epithelial cell marker, E-cadherin, and increased that of mesenchymal markers such as fibronectin and α-SMA (Fig. 1A).Consequently, the protein expression of E-cadherin decreased and that of fibronectin and α-SMA increased after treatment with TGF-β1.This indicated that TGF-β1 had induced EMT in the HPMCs (Fig. 1B, C).Similarly, the protein expression levels of phosphorylated Smad2 (p-Smad2), p-Smad2/ Smad2, phosphorylated Smad3 (p-Smad3), and p-Smad3/Smad3 were substantially increased (Fig. 1B, C).These results indicated that treatment with TGF-β1 induces fibrosis in HPMCs by upregulating the Smad2/3 signaling pathways.
The changes in oxidative stress markers and the NOX homologs, NOX1, 2, and 4, after treatment with TGF-β1 were also evaluated.An increase in NOX expression in TGF-β1-exposed HPMCs is one of the earliest indications of oxidative stress injury and EMT [15].Treatment with TGF-β1 upregulated NOX2 and NOX4 and downregulated NOX1 mRNA expression (Fig. 1D).Specifically, NOX4 mRNA was significantly higher after 2 and 5 ng/mL TGF-β1 treatments than in the control group.P22phox is a NOX membrane partner and is essential for NOX-induced ROS generation [16].The mRNA expression of P22phox was upregulated after treatment with 5 ng/mL TGF-β1 (Fig. 1D).Treatment with 2 and 5 ng/mL TGF-β1 increased ROS generation in HPMCs, as determined using intracellular ROS (DCF-DA) and H 2 O 2 assays (Fig. 1E, F).

TGF-β1 activates autophagy in HPMCs
TEM images confirmed an increase in the formation of autophagosomes in HPMCs after treatment with TGF-β1 (Fig. 2A).TGF-β1activated autophagy was also confirmed using autophagy flux assays (Fig. 2B).Autophagy flux increased after treatment with 2 and 5 ng/mL TGF-β1 to a similar degree to that observed in the positive control treated with 0.5 μM rapamycin (Fig. 2C).
Changes in individual markers of autophagy were identified.The mRNA expression and protein levels of the autophagy initiation marker Beclin 1 and autophagosome markers LC3B and ATG5 were significantly increased after treatment with TGF-β1 (Fig. 2D-F).Conversely, the mRNA expression and protein levels of p62, an autophagy regulator, decreased after TGF-β1 treatment.This indicated that TGF-β1 increased the degradation of p62.These results consistently indicate that autophagy activation increased after treatment with TGF-β1.

Autophagy inhibition downregulates TGF-β1-induced EMT in HPMCs
To confirm whether autophagy was associated with EMT, we identified changes in TGF-β1-induced EMT after autophagy inhibition.When cells were pretreated with 2 mM of the autophagy inhibitor 3-MA, the activation of autophagy flux was significantly lower than after treatment with TGF-β1 alone (2 and 5 ng/mL) (Fig. 3A).Cotreatment with 3-MA (2 mM) and TGF-β1 (2 and 5 ng/ mL) led to the downregulation of autophagy activation marker proteins, including Beclin 1, LC3B, and ATG5, and the upregulation of autophagy inactivation marker protein p62.This was confirmed by western blot analysis (Fig. 3B, C).The protein levels of fibrosis markers such as fibronectin and α-SMA were significantly lower in HPMCs cotreated with 3-MA and TGF-β1 than those treated with TGF-β1 alone.Immunofluorescence staining revealed that intracellular autophagy formation was reduced by treatment with 3-MA (Fig. 3D).Pretreatment with the autophagy inhibitor 3-MA (2 mM) resulted in substantially less autophagic flux activity than treatment with TGF-β1 alone (5 ng/mL) (Fig. 3D, upper phase).Additionally, fibrosis markers were increased by treatment with TGF-β1 alone (5 ng/mL), and decreased by autophagy inhibition (Fig. 3D).These results indicate that intracellular autophagy is part of the fibrosis induction mechanism.
We confirmed the effects of autophagy inhibition through ATG5 gene silencing, which codes for an essential protein in phagophore membrane extensions in autophagic vesicles.After ATG5 silencing in HPMCs, TGF-β1-induced protein levels of Beclin 1, LC3B, and ATG5 were downregulated, whereas those of p62 were upregulated, indicating the inactivation of autophagy (Suppl.Fig. S1).The TGF-β1-induced increase in mesenchymal marker protein levels, such as fibronectin and α-SMA, was reduced after the ATG5 gene was silenced.
These results confirmed that autophagy inhibition prevents TGF-β1-induced EMT and fibrosis in HPMCs, although the autophagy inhibition mechanisms are different.
These results indicated that NOX4 suppression inactivates TGF-β1-activated autophagy and that autophagy is a critical mechanism in HPMC fibrosis caused by NOX4-derived ROS.
We investigated cellular respiration in HPMCs by evaluating the OCR and ECAR.To determine the maximum oxidative capacity, we injected oligomycin into the culture wells followed by the unbinding agent FCCP.TGF-β1 (5 ng/mL) consistently raised the basal OCR after 48 h.The OCR reached its highest point in a dose-response pattern, and this increase was associated with the generation of adenosine triphosphate (ATP), an OCR sensitive to oligomycin, total oxidative capacity, and an OCR sensitive to FCCP.After 48 h exposure to TGF-β1 (5 ng/mL), the oxidative capacity of cells was higher than that of the control group (Fig. 5E, G).Treatment with GKT137831 (20 μM) reduced the TGF-β1-induced increase in oxidative capacity (Fig. 5E).This followed the same trend as the results obtained with the autophagy inhibitor 3-MA (2 mM) (Fig. 5G).GKT137831 (20 μM) reduced the abnormal respiration rate of mitochondria induced in control cells by TGF-β1 (5 ng/mL) and exhibited similar mitochondrial protection to that of direct autophagy inhibition with 3-MA (2 mM) (Fig. 5E, G).Additionally, using a Seahorse XF glycolysis stress test (Agilent Technologies), we found that TGF-β1-treated (5 ng/mL) HPMCs displayed a higher ECAR than control cells (Fig. 5F, H).A parameter analysis revealed significantly enhanced glycolysis and glycolytic capacity in the TGF-β1-induced (5 ng/mL) HPMCs.This suggests that TGF-β1-induced EMT progression causes more rapid glucose use via the glycolytic pathway in HPMCs.GKT137831 (20 μM) reduced the TGF-β1-stimulated glycolysis to a more normal level than 3-MA (2 mM) (Fig. 5F, H).
Autophagy inhibition suppresses TGF-β1-induced activation of the Smad2/3, PI3K/Akt, and MAPK signaling pathways We evaluated changes in the activation of the fibrosis-related Smad2/3, autophagy-related PI3K/Akt, and redox-sensitive MAPK pathways (ERK/P38/JNK) after autophagy inhibition to identify the signaling mechanisms underlying autophagy-mediated EMT.Autophagy was found to be inhibited by 3-MA and ATG5 gene silencing.
These changes in the downregulation of the signaling pathways were similar to those after 3-MA treatment and ATG5 gene silencing, both of which directly inhibit autophagy.These findings indicate that NOX4 activation triggers mitochondrial dysfunction and autophagy, which serve as the primary pathophysiological mechanisms behind EMT in HPMCs.

DISCUSSION
This study demonstrates the importance of autophagy in TGF-β1induced EMT in HPMCs.TGF-β1 treatment activates autophagy through increased NOX4-derived ROS generation, resulting in mitochondrial damage.Both direct inhibition of the autophagy pathway and indirect suppression of autophagy through NOX4 inhibition reduced TGF-β1-induced EMT in HPMCs.These findings suggest autophagy inhibition as a potential therapeutic strategy for the prevention of peritoneal fibrosis and the preservation of peritoneal membrane function in patients undergoing PD.
EMT is a complex process involving multiple biochemical changes.These allow epithelial cells to acquire mesenchymal-like phenotypes characterized by increased migratory capacity and invasiveness [1,17].TGF-β1 is generated by high levels of glucose and glucose degradation products in the PD fluid and has been identified as a prominent contributor to peritoneal EMT among patients undergoing PD, which culminates in the loss of peritoneal membrane function [6].Previous studies have shown that an increase in ROS via NOX activation by TGF-β1 is essential to the progression of peritoneal EMT [18,19].Nonetheless, the exact mechanism by which ROS triggers EMT has not previously been determined.Therefore, we elucidated the effect of autophagy activation on TGF-β1-induced EMT, particularly in the context of NOX4-derived ROS-induced EMT.
Autophagy is an intracellular degradation process that occurs in all mammalian cells and is essential to eliminate cellular waste ).The data are presented as mean ± standard error (SE).* P < 0.05 vs. control, *** P < 0.001 vs. control; # P < 0.05 vs. TGF-β1 2 ng/mL; ## P < 0.01 vs. TGF-β1 2 ng/mL; ### P < 0.001 vs. TGF-β1 2 ng/mL; + P < 0.05 vs. TGF-β1 5 ng/mL; ++ P < 0.01 vs. TGF-β1 5 ng/mL; and +++ P < 0.001 vs. TGF-β1 5 ng/mL.components such as cytoplasm, organelles, and damaged proteins [20,21].The stages of autophagy initiation, elongation, maturation, formation of the double-membrane autophagosome, fusion with lysosomes to form autolysosomes, and degradation [22,23].Autophagy can exert either protective or detrimental effects on cells.In pleural mesothelial cells, autophagy protects against EMT.A study has shown that impairment of autophagy by NOX4 increases EMT, and that NOX4 is induced by mycobacterial infection [19].In the present study, NOX4 expression was induced by TGF-β1 treatment and autophagy activation resulted in EMT in HPMCs.Following TGF-β1 treatment, the levels of autophagy initiation markers (Beclin 1) and autophagosome markers (LC3B and ATG5) increased, indicating heightened autophagy activity.To establish the link between autophagy and EMT, we inhibited autophagy using 3-MA and ATG5 gene silencing.The results demonstrated that the inhibition of autophagy decreases EMT, suggesting that autophagy is associated with and contributes to EMT.The differing results of our study and previous research on the role of autophagy in the EMT of mesothelial cells suggest that the role of autophagy may vary depending on the location and type of cell and the degree of cellular stimulation, even within the same mesothelial cell.
Interestingly, our findings suggest that activation of autophagy by TGF-β1 is indirectly induced by increased ROS within the mitochondria and mitochondrial damage.Two previous studies have reported that a high-glucose dialysis solution and the consequent production of TGF-β1 induces autophagy activation, promoting peritoneal fibrosis [2,24].However, these studies only identified the relationship between autophagy activation, fibrosis, and apoptosis.They did not reveal the relationships between autophagy and upstream pathways, such as NOX-ROS, mitochondrial damage, and changes in the related signaling pathways.Nor did they evaluate the effect of indirect autophagy inhibition through the inhibition of upstream pathways.Therefore, their interpretation of the inhibitory effects of autophagy was limited.Moreover, we confirmed the close relationship between NOX-ROS-induced mitochondrial damage and autophagy activation, confirming its effect on fibrosis by inhibiting the various pathways involved in each step.
NOX4 was first identified as a renal-specific NOX that is abundantly expressed in endothelial and vascular smooth muscle cells [25,26].NOX4 activation produces ROS, and NOX4-derived ROS plays a pivotal role in autophagy activation in various cells [27,28].The inhibition of NOX4 by GKT137831 decreased ROS generation and NOX4 expression.Subsequently, this reduced autophagy activation and the downregulation of EMT markers.Therefore, NOX4-derived ROS is vital to this process, connecting autophagy and EMT.We further identified a novel mechanism of EMT via mitochondrial damage and autophagy activation caused by NOX4-derived ROS generation.The PI3K class III signaling pathway is a key regulator of mitochondrial dysfunction [29] and mitochondrial apoptosis [30].Treatment with GKT137831 reduced TGF-β1-activated ROS and mitochondrial dysfunction through PI3K class III phosphorylation.This demonstrates that NOX4 significantly promotes oxidative stress and mitochondrial dysfunction in TGF-β1-induced fibrosis.
Our study explores the effect of autophagy inhibition on various signaling pathways, including Smad2/3, PI3K/Akt, and MAPK.Autophagy inhibition reduced the phosphorylation of Smad2/3, PI3K class III, Akt, and ERK, suggesting that autophagy plays a role in the activation of signaling pathways that contribute to EMT.We also investigated the effect of NOX4 inhibition on the signaling pathways.GKT137831 reduced Smad2/3, PI3K class III, Akt, and P38 phosphorylation, further supporting the role of NOX4 in activating these pathways and leading to EMT. Figure 8 summarizes these results.TGF-β1 activated NOX4 expression, and NOX4 generated ROS in HPMCs.We confirmed that mitochondrial damage and autophagy activation are mechanisms by which ROS induces EMT.Various signaling pathways are involved in TGF-β1-induced NOX4-ROS-mitochondrial damageautophagy-EMT in HPMCs.
This study had several limitations.First, only in vitro experiments were performed.Follow-up studies are required to confirm the association between autophagy and peritoneal fibrosis as well as the effect of autophagy inhibition.Second, we did not identify changes in the sub-pathways of the signaling pathways.Third, autophagy can have protective and damaging effects, and we were unable to compare the effects of gradual autophagy activation on fibrosis in detail.Therefore, further research is required.

Fig. 2
Fig. 2 TGF-β1 induced autophagy activation in HPMCs, which was confirmed using transmission electron microscopy (TEM), an autophagy flux assay, and western blotting analysis.A Representative TEM images of the autophagic morphology.Autophagosomes were frequently observed after TGF-β1 treatment (2 and 5 ng/mL) in HPMCs.The red boxed portion is shown at high magnification on the right.The red arrow indicates the autophagosomes.B TGF-β1-induced autophagy was evaluated by staining using a Cyto-ID Autophagy Detection Kit.Rapamycin (0.5 μM; Rap) was used as a positive control.The stained cells were observed and photographed under fluorescence microscopy (blue, nucleus/ Hoechst 33342; green, autophagosomes/ Cyto-ID).C The intensity of the Cyto-ID green was quantified using a plate reader.D TGF-β1 (2 and 5 ng/mL) increased the mRNA expression of Beclin 1, LC3B, and ATG5, and decreased the mRNA expression of p62.(E, F) TGF-β1 (2 and 5 ng/mL) increased the protein levels of Beclin 1, LC3B, and ATG5, and decreased the protein levels of p62.The results were calculated as values relative to the control.Data are presented as mean ± standard error (SE).n = 4 per group.* P < 0.05 vs. control; ** P < 0.01 vs. control; and *** P < 0.001 vs. control.

Fig. 8 A
Fig. 8 A summary of the results of the present study.In HPMCs, TGF-β1-induced NOX4 activation resulted in increased reactive oxygen species (ROS) generation, leading to mitochondrial damage.Increased ROS within the mitochondria activates the PI3K/Akt pathway, induces mitochondrial damage, and promotes the activation of autophagy.Autophagy activation promotes epithelial-to-mesenchymal transition (EMT) in HPMCs via the Smad2/3, PI3K/Akt, and ERK/P38 pathways.Both direct inhibition of autophagy by 3-MA or ATG5 gene silencing, and indirect inhibition of autophagy through NOX4 inhibition by GKT137831, ameliorated the TGF-β1-induced EMT in HPMCs.Upregulation is shown in red and downregulation in blue.