Epithelial-mesenchymal Transition In Human Peritoneal Mesothelial Cells Research Articles

The antioxidative effects of empagliflozin on high glucose‑induced epithelial-mesenchymal transition in peritoneal mesothelial cells via the Nrf2/HO-1 signaling

High glucose (HG)-induced epithelial-mesenchymal transition (EMT) and oxidative stress play an important role in peritoneal fibrosis, which could be regulated by the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. This study aimed to investigate whether empagliflozin could inhibit HG-induced EMT and oxidative stress via activating the Nrf2/HO-1 signaling pathway. We used HG-based peritoneal dialysis (PD) solution in rats and HG in human peritoneal mesothelial cells (HPMCs) to induce EMT in vivo and in vitro respectively. The peritoneal structure and function were evaluated by hematoxylin and eosin, Masson’s trichrome staining, and the peritoneal equilibrium test. Oxidative stress was measured by assay kits. EMT was analyzed using immunohistochemistry and western blot. The PD rats showed decreased ultrafiltration capacity and increased levels of oxidative stress. Histopathological analysis revealed markedly peritoneal thickening, excessive collagen deposition, increased expression of α-SMA, Collagen-I, and Fibronectin, and decreased expression of E‑cadherin. Empagliflozin significantly ameliorated the aforementioned changes. The protein expression levels of nuclear Nrf2 (N-Nrf2) and HO-1 increased in PD rats, which were further promoted by treatment with empagliflozin. In in vitro experiments, the EMT of HPMCs was induced with 60 mM glucose for 24 h and inhibited by empagliflozin. Empagliflozin suppressed oxidative stress and promoted the protein expression of N-Nrf2 and HO-1 in HG‑stimulated HPMCs, which was reversed by the Nrf2 inhibitor. In conclusion, empagliflozin exerted a protective effect against HG-induced EMT and suppressed oxidative stress in PMCs by activating the Nrf2/HO-1 signaling pathway.

Elabela Attenuates the TGF-β1-Induced Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells in Patients Receiving Peritoneal Dialysis.

Peritoneal fibrosis (PF), a common complication in patients receiving peritoneal dialysis (PD), is primarily caused by the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). PF is the main reason for patients on PD to withdraw from PD. Effective treatment is unavailable for this complication at present. Elabela (ELA) is a polypeptide hormone secreted by the vascular endothelium and kidney. Peptide hormones ELA and apelin (APLN) have various protective effects on the cardiovascular and urinary systems and have potential therapeutic effects on organ fibrosis. ELA and APLN are less studied in PD population. Here, we aimed to investigate the clinical significance of ELA in patients on PD and to evaluate the therapeutic effect of ELA on EMT of HPMCs. Compared with those in patients with stage 5 chronic kidney disease who are not on dialysis, serum ELA levels in patients on PD increased with the improvement of residual renal function at PD duration <36 months and decreased to pre-dialysis levels at PD duration ≥36 months, suggesting that dialysis duration is the main risk factor affecting serum ELA levels in patients on PD. In addition, serum APLN levels decreased in the early stage of PD and recovered to the pre-dialysis level with the prolongation of dialysis time. Notably, serum APLN levels were positively correlated with dialysis duration in patients undergoing PD. To establish the EMT model, we stimulated HPMCs using transforming growth factor-beta 1 (TGF-β1) in cell experiments performed in vitro. ELA-32 treatment reversed the TGF-β1-induced reduction in the expression of the epithelial cell marker and suppressed the expression of mesenchymal cell markers by inhibiting the phosphorylation of SMAD2/3, ERK1/2, and AKT. Therefore, our findings imply that ELA-32 can interfere with the EMT of HPMCs by inhibiting the activation of the TGF-β/SMAD2/3, ERK1/2, and AKT pathways, providing novel insights on the potential therapeutic use of ELA for treating PD-related PF.

Requirement of Histone Deacetylase 6 for Interleukin-6 Induced Epithelial-Mesenchymal Transition, Proliferation, and Migration of Peritoneal Mesothelial Cells.

Aims: Influenced by microenvironment, human peritoneal mesothelial cells (HPMCs) acquired fibrotic phenotype, which was identified as the protagonist for peritoneal fibrosis. In this study, we examined the role of histone deacetylase 6 (HDAC6) for interleukin-6 (IL-6) induced epithelial-mesenchymal transition (EMT), proliferation, and migration of HPMCs.Methods: The role of HDAC6 in IL-6-elicited EMT of HPMCs was tested by morphological observation of light microscope, immunoblotting, and immune-fluorescence assay; and the function of HDAC6 in proliferation and migration of HPMCs was examined by CCK-8 assay, wound healing experiment, and immunoblotting.Results: IL-6 stimulation significantly increased the expression of HDAC6. Treatment with tubastatin A (TA), a highly selective HDAC6 inhibitor, or silencing of HDAC6 with siRNA decreased the expression of HDAC6. Moreover, TA or HDAC6 siRNA suppressed IL-6-induced EMT, as evidenced by decreased expressions of α-SMA, Fibronectin, and collagen I and the preserved expression of E-cadherin in cultured HPMCs. Mechanistically, HDAC6 inhibition suppressed the expression of transforming growth factor β (TGFβ) receptor I (TGFβRI), phosphorylation of Smad3, secretion of connective tissue growth factor (CTGF), and transcription factor Snail. On the other hand, the pharmacological inhibition or genetic target of HDAC6 suppressed HPMCs proliferation, as evidenced by the decreased optical density of CCK-8 and the expressions of PCNA and Cyclin E. The migratory rate of HPMCs also decreased. Mechanistically, HDAC6 inhibition blocked the activation of JAK2 and STAT3.Conclusion: Our study illustrated that IL-6-induced HDAC6 not only regulated IL-6 itself downstream JAK2/STAT3 signaling but also co-activated the TGF-β/Smad3 signaling, leading to the change of the phenotype and mobility of HPMCs. HDAC6 could be a potential therapeutic target for the prevention and treatment of peritoneal fibrosis.

Nitro-oleic acid inhibits the high glucose-induced epithelial-mesenchymal transition in peritoneal mesothelial cells and attenuates peritoneal fibrosis.

As an electrophilic nitroalkene fatty acid, nitro-oleic acid (OA-NO2) exerts multiple biological effects that contribute to anti-inflammation, anti-oxidative stress, and antiapoptosis. However, little is known about the role of OA-NO2 in peritoneal fibrosis. Thus, in the present study, we examined the effects of OA-NO2 on the high glucose (HG)-induced epithelial-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs) and evaluated the morphological and immunohistochemical changes in a rat model of peritoneal dialysis-related peritoneal fibrosis. In in vitro experiments, we found that HG reduced the expression level of E-cadherin and increased Snail, N-cadherin, and α-smooth muscle actin expression levels in HPMCs. The above-mentioned changes were attenuated by pretreatment with OA-NO2. Additionally, OA-NO2 also inhibited HG-induced activation of the transforming growth factor-β1/Smad signaling pathway and NF-κB signaling pathway. Meanwhile, OA-NO2 inhibited HG-induced phosphorylation of Erk and JNK. The results from the in vivo experiments showed that OA-NO2 notably relieved peritoneal fibrosis by decreasing the thickness of the peritoneum; it also inhibited expression of transforming growth factor-β1, α-smooth muscle actin, N-cadherin, and vimentin and enhanced expression of E-cadherin in the peritoneum. Collectively, these results suggest that OA-NO2 inhibits the HG-induced epithelial-mesenchymal transition in HPMCs and attenuates peritoneal dialysis-related peritoneal fibrosis.

Arctigenin alleviates TGF-β1-induced epithelial-mesenchymal transition and PAI-1 expression via AMPK/NF-κB pathway in peritoneal mesothelial cells

Peritoneal fibrosis (PF) caused by long-term peritoneal dialysis is closely associated with the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). Moreover, the anti-fibrotic role of Arctigenin (Arc) has been reported in several fibrosis disorders. Therefore, the preventive effect of Arc on transforming growth factor-β1 (TGF-β1)-induced EMT and the underlying mechanisms in HPMCs was investigated in this study. Firstly, the PD model was established by TGF-β1 stimulation in cultured HPMCs in vitro, we found that TGF-β1 significantly increased the EMT markers (α-SMA, vimentin, and fibronectin) and plasminogen activator inhibitor type 1 (PAI-1) expressions, but decreased epithelial marker (E-cadherin). Co-treatment with Arc (10, 20, 40 μM) ameliorated TGF-β1-induced EMT in a dose-dependent manner, and the expression of PAI-1 was also inhibited by Arc, which was abrogated by restoration of PAI-1. Moreover, Arc enhanced the phosphorylated AMP-activated protein kinase (AMPK), but inhibited the phosphorylated IκBα level and nuclear translocation of nuclear factor κB (NF-κB) p65 in TGF-β1-induced HPMCs. ChIP and Luciferase reporter assays verified that the increased binding capacity of NF-κB to the promoter of PAI-1 induced by TGF-β1 was reversely attenuated by Arc in HPMCs. However, the effect of Arc on TGF-β1-induced NF-κB activation, PAI-1 expression and EMT in HPMCs was attenuated by AMPK agonist Compound C. In conclusion, these data demonstrated that Arc suppressed TGF-β1-induced EMT by activating the AMPK/NF-κB pathway to inhibit PAI-1 expression in HPMCs. Therefore, Arc might act as a potential therapeutic agent for PD treatment.

Zinc supplementation inhibits the high glucose‑induced EMT of peritoneal mesothelial cells by activating the Nrf2 antioxidant pathway.

The high glucose (HG)-induced epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) serves an important role in peritoneal fibrosis (PF) during peritoneal dialysis. Our previous study reported that zinc (Zn) supplementation prevented the HG-induced EMT of rat PMCs in vitro. In the present study, the role of Zn in HG-induced EMT was investigated in vivo using a rat model of PF. Additionally, the molecular mechanisms underlying HG-induced EMT were studied in human PMCs (HPMCs). In the rat model of PF, HG treatment increased the glucose transfer capacity and decreased the ultrafiltration volume. Histopathological analysis revealed peritoneal thickening, increased expression of vimentin and decreased expression of E-cadherin. ZnSO4 significantly ameliorated the aforementioned changes, whereas Zn inhibition by clioquinol significantly aggravated the effects of HG on rats. The effects of Zn on HPMCs was assessed using western blot analysis, Transwell assays and flow cytometry. It was revealed that Zn also significantly suppressed the extent of the EMT, and reduced reactive oxygen species production and the migratory ability of HG-induced HPMCs, whereas Zn inhibition by N',N',N',N'-tetrakis (2-pyridylmethyl) ethylenediamine significantly potentiated the HG-induced EMT of HPMCs. HG-stimulated HPMCs exhibited increased expression of nuclear factor-like 2 (Nrf2) in the nucleus, and total cellular NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase-1 (HO-1), the target proteins of the Nrf2 antioxidant pathway. Zn supplementation further promoted nuclear Nrf2 expression, and increased the expression of target proteins of the Nrf2 antioxidant pathway, whereas Zn depletion decreased nuclear Nrf2, NQO1 and HO-1 expression compared with the HG group. In conclusion, Zn supplementation was proposed to suppress the effects of HG on the EMT by stimulating the Nrf2 antioxidant pathway and subsequently reducing oxidative stress in PMCs.

Matrine suppresses lipopolysaccharide-induced fibrosis in human peritoneal mesothelial cells by inhibiting the epithelial-mesenchymal transition.

Background:Peritoneal fibrosis is the primary reason that patients with end-stage renal disease (ESRD) have to cease peritoneal dialysis. Peritonitis caused by Gram-negative bacteria such as Escherichia coli (E. coli) were on the rise. We had previously shown that matrine inhibited the formation of biofilm by E. coli. However, the role of matrine on the epithelial-mesenchymal transition (EMT) in peritoneal mesothelial cells under chronic inflammatory conditions is still unknown.Methods:We cultured human peritoneal mesothelial cells (HPMCs) with lipopolysaccharide (LPS) to induce an environment that mimicked peritonitis and investigated whether matrine could inhibit LPS-induced EMT in these cells. In addition, we investigated the change in expression levels of the miR-29b and miR-129-5p.Results:We found that 10 μg/ml of LPS induced EMT in HPMCs. Matrine inhibited LPS-induced EMT in HPMCs in a dose-dependent manner. We observed that treatment with matrine increased the expression of E-cadherin (F = 50.993, P < 0.01), and decreased the expression of alpha-smooth muscle actin (F = 32.913, P < 0.01). Furthermore, we found that LPS reduced the expression levels of miR-29b and miR-129-5P in HPMCs, while matrine promoted the expression levels of miR-29b and miR-129-5P.Conclusions:Matrine could inhibit LPS-induced EMT in HPMCs and reverse LPS inhibited expressions of miR-29 b and miR-129-5P in HPMCs, ultimately reduce peritoneal fibrosis. These findings provide a potential theoretical basis for using matrine in the prevention and treatment of peritoneal fibrosis.

AGEs induce epithelial to mesenchymal transformation of human peritoneal mesothelial cells via upregulation of STAT3.

Epithelial-mesenchymal transition (EMT) in peritoneum was induced during peritoneal dialysis (PD), which finally caused progressive fibrosis. However, the underlying mechanisms were not well elucidated. We established advanced glycation end-products (AGEs)-induced EMT model using primary human peritoneal mesothelial cells (HPMCs). The working concentration and time of AGEs were optimized. Then the expression and activation signal transducer and activator of transcription 3 (STAT3), a key factor of EMT in cancer, were detected. The regulation of STAT3 by miRNA was also explored. 50μg/ml AGEs treatment for 24h can successfully induce EMT in HPMCs. AGEs treatment upregulated and activated STAT3. miRNA-454, potentially targeting STAT3, was down-regulated in AGEs-treated HPMCs. Overexpression of miRNA-454 prevented AGEs- induced EMT in HPMCs. AGEs induce epithelial to mesenchymal transformation of Human peritoneal mesothelial cells via upregulation of STAT3.

Tanshinone IIA mitigates peritoneal fibrosis by inhibiting EMT via regulation of TGF-β/smad pathway

Purpose : To explore the effects of tanshinone IIA (T-IIA) on Dianeal-N PD-4 (PDF)-induced expression of fibrogenic cytokines in human peritoneal mesothelial cells (HPMCs), and to elucidate the mechanisms of action involved. Methods : Seven groups of HPMCs were used in the study: control group, PDF group, T-IIA group, LY364947 group, and 2 transforming growth factor-β (TGF-β) groups (TGF-β+ 50 μM T-IIA and TGF-β+ 100 μM T- IIA). The expression levels of mRNA and protein of TGF-β, smad2, smad7, α-smooth muscle actin(α-SMA), fibronectin, collagen І, E-cadherin, N-cadherin, matrix metalloprotein-2(MMP-2), and MMP-9 in the various groups were determined by reverse transcription-polymerase chain reaction (RTPCR) and Western blotting as appropriate. Results : The expressions of α-SMA, fibronectin, collagen І, TGF-β and smad2 were significantly upregulated in HPMCs by PDF treatment, but smad7 was down-regulated, relative to the control group (p < 0.01).These PDF-induced effects were reversed by T-IIA (p < 0.05). Inhibition of TGF-β/smad pathway by LY364947 treatment led to significant decrease in the expressions of fibrosis-related proteins, when compared with PDF group (p < 0.05). TGF-β treatment also produced numerous spindleshaped HPMCs characteristic of epithelial-mesenchymal transition (EMT). However, this morphological transition was alleviated, and the expression levels of EMT-related proteins were significantly downregulated by exposure to the two doses of T-IIA (p < 0.05). Conclusion : Tanshinone IIA inhibits EMT in HPMCs by regulating TGF-β/smad pathway, thus mitigating peritoneal fibrosis. Therefore, T-IIA has promising potential as a new drug for the treatment of peritoneal dialysis (PD)-induced fibrosis. Keywords : Peritoneal dialysis, Peritoneal fibrosis, Tanshinone IIA, Epithelial-mesenchymal transition

Small interfering RNA targeting ILK inhibits EMT in human peritoneal mesothelial cells through phosphorylation of GSK‑3β.

Emerging evidence has suggested that human peritoneal mesothelial cells (HPMCs) undergo epithelial‑mesenchymal transition (EMT) in peritoneal fibrosis. The molecular mechanisms underlying peritoneal fibrosis and the key molecules involved are not yet fully elucidated. In order to enhance the understanding of peritoneal fibrosis, the present study investigated the roles of integrin‑linked kinase (ILK) and glycogen synthase kinase3β (GSK‑3β) in high glucose‑induced phenotypic alterations of HPMCs. It was observed that HPMCs exhibited a cobblestone morphology under normal glucose conditions, whereas under high glucose conditions they had a spindle morphology. Additionally, under high glucose conditions it was found that E‑cadherin expression was decreased and vimentin expression was increased in HPMCs, suggesting HPMCs underwent EMT. ILK expression in high glucose conditions was also increased in a dose‑ and time‑dependent manner. The role of ILK in the induction of EMT in HPMCs was further investigated using small interfering RNA (siRNA). Following knockdown of ILK gene expression by siRNA, low vimentin expression as well as high E‑cadherin expression were observed, suggesting that EMT was inhibited. ILK‑knockdown also inhibited phosphorylation of GSK‑3β. These results indicate that ILK‑knockdown inhibits EMT of HPMCs through inhibition of GSK‑3β phosphorylation. These findings suggest that ILK may be used as a novel diagnostic and therapeutic target for HPMC fibrosis in the future.

Mechanisms of Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells During Peritoneal Dialysis

A growing body of evidence indicates that epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMC) may play an important role in the development and progression of peritoneal fibrosis during long-term peritoneal dialysis (PD) leading to failure of peritoneal membrane function. Here, we review our own observations and those of others on the mechanisms of EMT of HPMC and suggest potential therapeutic strategies to prevent EMT and peritoneal fibrosis during long-term PD. We found that high glucose and H2O2 as well as transforming growth factor-β1 (TGF-β1) induced EMT in HPMC and that high glucose-induced EMT was blocked not only by inhibition of TGF-β1 but also by antioxidants or inhibitors of mitogen-activated protein kinases (MAPK). Since MAPKs are downstream target molecules of reactive oxygen species (ROS), these data suggest that high glucose-induced generation of ROS and subsequent MAPK activation mediate high glucose-induced EMT in HPMC. We and others also observed that bone morphogenetic protein-7 (BMP-7) prevented EMT in HPMC. Glucose degradation products (GDP) were shown to play a role in inducing EMT. Involvement of a mammalian target of rapamycin (mTOR) in TGF-β1-induced EMT has also been proposed in cultured HPMC. A better understanding of the precise mechanisms involved in EMT of HPMC may provide new therapeutic strategies for inhibiting peritoneal fibrosis in long-term PD patients.