Inhibition of STAT3 by S3I-201 suppress peritoneal fibroblast phenotype conversion and alleviate peritoneal fibrosis.

Peritoneal fibrosis is a common pathological response to long-term peritoneal dialysis (PD) and a major cause for PD discontinuation. Understanding the cellular and molecular mechanisms underlying the induction and progression of peritoneal fibrosis is of great interest. In our study, in vitro study revealed that signal transducer and activator of transcription 3 (STAT3) is a key factor in fibroblast activation and extracellular matrix (ECM) synthesis. Furthermore, STAT3 induced by IL-6 trans-signalling pathway mediate the fibroblasts of the peritoneal stroma contributed to peritoneal fibrosis. Inhibition of STAT3 exerts an antifibrotic effect by attenuating fibroblast activation and ECM production with an in vitro co-culture model. Moreover, STAT3 plays an important role in the peritoneal fibrosis in an animal model of peritoneal fibrosis developed in mice. Blocking STAT3 can reduce the peritoneal morphological changes induced by chlorhexidine gluconate. In conclusion, our findings suggested STAT3 signalling played an important role in peritoneal fibrosis. Therefore, blocking STAT3 might become a potential treatment strategy in peritoneal fibrosis.

LncRNA RPL29P2 promotes peritoneal fibrosis and impairs peritoneal transport function via miR-1184 in peritoneal dialysis.

Peritoneal dialysis (PD), hemodialysis and kidney transplantation are the three therapies to treat uremia. However, PD is discontinued for peritoneal membrane fibrosis (PMF) and loss of peritoneal transport function (PTF) due to damage from high concentrations of glucose in PD fluids (PDFs). The mechanism behind PMF is unclear, and there are no available biomarkers for the evaluation of PMF and PTF. Using microarray screening, we found that a new long noncoding RNA (lncRNA), RPL29P2, was upregulated in the PM (peritoneal membrane) of long-term PD patients, and its expression level was correlated with PMF severity and the PTF loss. In vitro and rat model assays suggested that lncRNA RPL29P2 targets miR-1184 and induces the expression of collagen type I alpha 1 chain (COL1A1). Silencing RPL29P2 in the PD rat model might suppress the HG-induced phenotypic transition of Human peritoneal mesothelial cells (HPMCs), alleviate HG-induced fibrosis and prevent the loss of PTF. Overall, our findings revealed that lncRNA RPL29P2, which targets miR-1184 and collagen, may represent a useful marker and therapeutic target of PMF in PD patients.

Matrix metalloproteinase-10 deficiency has protective effects against peritoneal inflammation and fibrosis via transcription factor NFκΒ pathway inhibition.

One of the most common causes of discontinued peritoneal dialysis is impaired peritoneal function. However, its molecular mechanisms remain unclear. Previously, by microarray analysis of mouse peritoneum, we showed that MMP (matrix metalloproteinase)-10 expression is significantly increased in mice with peritoneal fibrosis, but its function remains unknown. Chlorhexidine gluconate (CG) was intraperitoneally injected to wild-type and MMP-10 knockout mice to induce fibrosis to elucidate the role of MMP-10 on peritoneal injury. We also examined function of peritoneal macrophages and mesothelial cells obtained from wild-type and MMP-10 knockout mice, MMP-10-overexpressing macrophage-like RAW 264.7 cells and MeT-5A mesothelial cells, investigated MMP-10 expression on peritoneal biopsy specimens, and the association between serum proMMP-10 and peritoneal solute transfer rates determined by peritoneal equilibration test on patients. MMP-10 was expressed in cells positive for WT1, a mesothelial marker, and for MAC-2, a macrophage marker, in the thickened peritoneum of both mice and patients. Serum proMMP-10 levels were well correlated with peritoneal solute transfer rates. Peritoneal fibrosis, inflammation, and high peritoneal solute transfer rates induced by CG were all ameliorated by MMP-10 deletion, with reduction of CD31-positive vessels and VEGF-A-positive cells. Expression of inflammatory mediators and phosphorylation of NFκΒ subunit p65 at S536 were suppressed in both MMP-10 knockout macrophages and mesothelial cells in response to lipopolysaccharide stimulation. Overexpression of MMP-10 in RAW 264.7 and MeT-5A cells upregulated pro-inflammatory cytokines with phosphorylation of NFκΒ subunit p65. Thus, our results suggest that inflammatory responses induced by MMP-10 are mediated through the NFκΒ pathway, and that systemic deletion of MMP-10 ameliorates peritoneal inflammation and fibrosis caused by NFκΒ activation of peritoneal macrophages and mesothelial cells.

Knockdown of AK142426 suppresses M2 macrophage polarization and inflammation in peritoneal fibrosis via binding to c-Jun.

BACKGROUND: Peritoneal fibrosis is a common complication of peritoneal dialysis, which may lead to ultrafiltration failure and ultimately treatment discontinuation. LncRNAs participate in many biological processes during tumorigenesis. We investigated the role of AK142426 in peritoneal fibrosis. METHODS: The AK142426 level in peritoneal dialysis (PD) fluid was detected by quantitative real-time-PCR assay. The M2 macrophage distribution was determined by flow cytometry. The inflammatory cytokines of TNF-α and TGF-ß1 were measured by ELISA assay. The direct interaction between AK142426 and c-Jun was evaluated by RNA pull-down assay. In addition, the c-Jun and fibrosis related proteins were assessed by western blot analysis. RESULTS: The PD-induced peritoneal fibrosis mouse model was successfully established. More importantly, PD treatment induced M2 macrophage polarization and the inflammation in PD fluid, which might be associated with exosome transmission. Fortunately, AK142426 was observed to be upregulated in PD fluid. Mechanically, knockdown of AK142426 suppressed M2 macrophage polarization and inflammation. Furthermore, AK142426 could upregulate c-Jun through binding c-Jun protein. In rescue experiments, overexpression of c-Jun could partially abolish the inhibitory effect of sh-AK142426 on the activation of M2 macrophages and inflammation. Consistently, knockdown of AK142426 alleviated peritoneal fibrosis in vivo. CONCLUSIONS: This study demonstrated that knockdown of AK142426 suppressed M2 macrophage polarization and inflammation in peritoneal fibrosis via binding to c-Jun, suggesting that AK142426 might be a promising therapeutic target for patients of peritoneal fibrosis.

Galectin-1 promotes gastric cancer peritoneal metastasis through peritoneal fibrosis.

BACKGROUND: Peritoneal metastasis is one of the main causes of death in patients with gastric cancer (GC). Galectin-1 regulates various undesirable biological behaviors in GC and may be key in GC peritoneal metastasis. METHODS: In this study, we elucidated the regulatory role of galectin-1 in GC cell peritoneal metastasis. GC and peritoneal tissues underwent hematoxylin-eosin (HE), immunohistochemical (IHC), and Masson trichrome staining to analyze the difference in galectin-1 expression and peritoneal collagen deposition in different GC clinical stages. The regulatory role of galectin-1 in GC cell adhesion to mesenchymal cells and in collagen expression was determined using HMrSV5 human peritoneal mesothelial cells (HPMCs). Collagen and corresponding mRNA expression were detected with western blotting and reverse transcription PCR, respectively. The promoting effect of galectin-1 on GC peritoneal metastasis was verified in vivo. Collagen deposition and collagen I, collagen III, and fibronectin 1 (FN1) expression in the peritoneum of the animal models were detected by Masson trichrome and IHC staining. RESULTS: Galectin-1 and collagen deposition in the peritoneal tissues was correlated with GC clinical staging and were positively correlated. Galectin-1 enhanced the ability of GC cells to adhere to the HMrSV5 cells by promoting collagen I, collagen III, and FN1 expression. The in vivo experiments confirmed that galectin-1 promoted GC peritoneal metastasis by promoting peritoneal collagen deposition. CONCLUSION: Galectin-1-induced peritoneal fibrosis may create a favorable environment for GC cell peritoneal metastasis.

Novel Peritoneal Sclerosis Rat Model Developed by Administration of Bleomycin and Lansoprazole.

In our preliminary experiment, peritoneal sclerosis likely induced by peritoneal dialysis was unexpectedly observed in the livers of rats given bleomycin and lansoprazole. We examined whether this peritoneal thickening around the liver was time-dependently induced by administration of both drugs. Male Wistar rats were injected with bleomycin and/or lansoprazole for 2 or 4 weeks. The 3YB-1 cell line derived from rat fibroblasts was treated by bleomycin and/or lansoprazole for 24 h. The administration of both drugs together, but not individually, thickened the peritoneal tissue around the liver. There was accumulation of collagen fibers, macrophages, and eosinophils under mesothelial cells. Expressions of Col1a1, Mcp1 and Mcp3 genes were increased in the peritoneal tissue around the liver and in 3YB-1 cells by the administration of both drugs together, and Opn genes had increased expressions in this tissue and 3YB-1 cells. Mesothelial cells indicated immunoreactivity against both cytokeratin, a mesothelial cell marker, and αSMA, a fibroblast marker, around the livers of rats given both drugs. Administration of both drugs induced the migration of macrophages and eosinophils and induced fibrosis associated with the possible activation of fibroblasts and the possible promotion of the mesothelial-mesenchymal transition. This might become a novel model of peritoneal sclerosis for peritoneal dialysis.

The Transfer of the Hepatocyte Growth Factor Gene by Macrophages Ameliorates the Progression of Peritoneal Fibrosis in Mice.

Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-ß-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

PPARγ alleviates peritoneal fibrosis progression along with promoting GLUT1 expression and suppressing peritoneal mesothelial cell proliferation.

OBJECTIVE: Peritoneal fibrosis (PF) is commonly induced by bioincompatible dialysate exposure during peritoneal dialysis, but the underlying mechanisms remain elusive. This study aimed to investigate the roles of peroxisome proliferator-activated receptor gamma (PPARγ) in PF pathogenesis. METHODS: Rat and cellular PF models were established by high glucose dialysate and lipopolysaccharide treatments. Serum creatinine, urea nitrogen, and glucose contents were detected by ELISA. Histological evaluation was done through H&E and Masson staining. GLUT1, PPARγ, and other protein expression were measured by qRT-PCR, western blotting, and IHC. PPARγ and GLUT1 subcellular distribution were detected using confocal microscopy. Cell proliferation was assessed by MTT and Edu staining. RESULTS: Serum creatinine, urea nitrogen and glucose, and PPARγ and GLUT1 expression in rat PF model were reduced by PPARγ agonists Rosiglitazone or 15d-PGJ2 and elevated by antagonist GW9662. Rosiglitazone or 15d-PGJ2 repressed and GW9662 aggravated peritoneal fibrosis in rat PF model. PPARγ and GLUT1 were mainly localized in nucleus and cytosols of peritoneal mesothelial cells, respectively, which were reduced in cellular PF model, enhanced by Rosiglitazone or 15d-PGJ2, and repressed by GW9662. TGF-ß and a-SMA expression was elevated in cellular PF model, which was inhibited by Rosiglitazone or 15d-PGJ2 and promoted by GW9662. PPARγ silencing reduced GLUT1, elevated a-SMA and TGF-b expression, and promoted peritoneal mesothelial cell proliferation, which were oppositely changed by PPARγ overexpression. CONCLUSION: PPARγ inhibited high glucose-induced peritoneal fibrosis progression through elevating GLUT1 expression and repressing peritoneal mesothelial cell proliferation.

Activation of the RAS contributes to peritoneal fibrosis via dysregulation of low-density lipoprotein receptor.

Peritoneal dialysis (PD)-related peritoneal fibrosis (PF) is characterized by progressive extracellular matrix (ECM) accumulation in peritoneal mesothelial cells (PMCs) during long-term use of high glucose (HG)-based dialysates. Activation of the renin-angiotensin system (RAS) has been shown to be associated with PF. The aim of this study was to explore the underlying mechanism of the RAS in HG-induced PF. We treated C57BL/6 mice and a human PMC line with HG to induce a PF model and to stimulate ECM accumulation, respectively. RAS activity was blocked using valsartan or angiotensin II (ANGII) type 1 receptor siRNA. The major findings were as follows. First, mice in the HG group exhibited increased collagen deposition and expression of ECM proteins, including α-smooth muscle actin (α-SMA) and collagen type I in the peritoneum. Consistent with the in vivo data, HG upregulated α-SMA expression in human peritoneal mesothelial cells (HPMCs) in a time- and dose-dependent manner. Second, HG stimulation led to RAS activation in HPMCs, and inactivation of RAS decreased the expression of ECM proteins in vivo and in vitro, even during HG stimulation. Finally, RAS-mediated ECM production was associated with lipid accumulation in HPMCs and depended on the dysregulation of the low-density lipoprotein receptor (LDLr) pathway. HG-stimulated HPMCs showed increased coexpression of LDLr and α-SMA, whereas blockade of RAS activity reversed the effect. Furthermore, inhibition of LDLr signaling decreased α-SMA and collagen type I expression in HPMCs when treated with HG and ANG II. In conclusion, increased intracellular RAS activity impaired lipid homeostasis and induced ECM accumulation in HPMCs by disrupting the LDLr pathway, which contributed to PF.

Inflammatory macrophages switch to CCL17-expressing phenotype and promote peritoneal fibrosis.

Peritoneal fibrosis remains a problem in kidney failure patients treated with peritoneal dialysis. Severe peritoneal fibrosis with encapsulation or encapsulating peritoneal sclerosis is devastating and life-threatening. Although submesothelial fibroblasts as the major precursor of scar-producing myofibroblasts in animal models and M2 macrophage (Mϕ)-derived chemokines in peritoneal effluents of patients before diagnosis of encapsulating peritoneal sclerosis have been identified, attenuation of peritoneal fibrosis is an unmet medical need partly because the mechanism for cross talk between Mϕs and fibroblasts remains unclear. We use a sodium hypochlorite-induced mouse model akin to clinical encapsulated peritoneal sclerosis to study how the peritoneal Mϕs activate fibroblasts and fibrosis. Sodium hypochlorite induces the disappearance of CD11bhigh F4/80high resident Mϕs but accumulation of CD11bint F4/80int inflammatory Mϕs (InfMϕs) through recruiting blood monocytes and activating local cell proliferation. InfMϕs switch to express chemokine (C-C motif) ligand 17 (CCL17), CCL22, and arginase-1 from day 2 after hypochlorite injury. More than 75% of InfMϕs undergo genetic recombination by Csf1r-driven Cre recombinase, providing the possibility to reduce myofibroblasts and fibrosis by diphtheria toxin-induced Mϕ ablation from day 2 after injury. Furthermore, administration of antibody against CCL17 can reduce Mϕs, myofibroblasts, fibrosis, and improve peritoneal function after injury. Mechanistically, CCL17 stimulates migration and collagen production of submesothelial fibroblasts in culture. By breeding mice that are induced to express red fluorescent protein in Mϕs and green fluorescence protein (GFP) in Col1a1-expressing cells, we confirmed that Mϕs do not produce collagen in peritoneum before and after injury. However, small numbers of fibrocytes are found in fibrotic peritoneum of chimeric mice with bone marrow from Col1a1-GFP reporter mice, but they do not contribute to myofibroblasts. These data demonstrate that InfMϕs switch to pro-fibrotic phenotype and activate peritoneal fibroblasts through CCL17 after injury. CCL17 blockade in patients with peritoneal fibrosis may provide a novel therapy. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic or pharmacologic blockade of enhancer of zeste homolog 2 inhibits the progression of peritoneal fibrosis.

Dysregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the pathogenesis of many cancers. However, the role of EZH2 in peritoneal fibrosis remains unknown. We investigated EZH2 expression in peritoneal dialysis (PD) patients and assessed its role in peritoneal fibrosis in cultured human peritoneal mesothelial cells (HPMCs) and murine models of peritoneal fibrosis induced by chlorhexidine gluconate (CG) or high glucose peritoneal dialysis fluid (PDF) by using 3-deazaneplanocin A (3-DZNeP), and EZH2 conditional knockout mice. An abundance of EZH2 was detected in the peritoneum of patients with PD associated peritonitis and the dialysis effluent of long-term PD patients, which was positively correlated with expression of TGF-ß1, vascular endothelial growth factor, and IL-6. EZH2 was found highly expressed in the peritoneum of mice following injury by CG or PDF. In both mouse models, treatment with 3-DZNeP attenuated peritoneal fibrosis and inhibited activation of several profibrotic signaling pathways, including TGF-ß1/Smad3, Notch1, epidermal growth factor receptor and Src. EZH2 inhibition also inhibited STAT3 and nuclear factor-κB phosphorylation, and reduced lymphocyte and macrophage infiltration and angiogenesis in the injured peritoneum. 3-DZNeP effectively improved high glucose PDF-associated peritoneal dysfunction by decreasing the dialysate-to-plasma ratio of blood urea nitrogen and increasing the ratio of dialysate glucose at 2 h after PDF injection to initial dialysate glucose. Moreover, delayed administration of 3-DZNeP inhibited peritoneal fibrosis progression, reversed established peritoneal fibrosis and reduced expression of tissue inhibitor of metalloproteinase 2, and matrix metalloproteinase-2 and -9. Finally, EZH2-KO mice exhibited less peritoneal fibrosis than EZH2-WT mice. In HPMCs, treatment with EZH2 siRNA or 3-DZNeP suppressed TGF-ß1-induced upregulation of α-SMA and Collagen I and preserved E-cadherin. These results indicate that EZH2 is a key epigenetic regulator that promotes peritoneal fibrosis. Targeting EZH2 may have the potential to prevent and treat peritoneal fibrosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-145 promotes the epithelial-mesenchymal transition in peritoneal dialysis-associated fibrosis by suppressing fibroblast growth factor 10.

Peritoneal fibrosis is a common complication of long-term peritoneal dialysis (PD) and the principal cause of ultrafiltration failure during PD. The initial and reversible step in PD-associated peritoneal fibrosis is the epithelial-mesenchymal transition (EMT). Although the mechanisms in the EMT have been the focus of many studies, only limited information is currently available concerning microRNA (miRNA) regulation in peritoneal fibrosis. In this study, we aimed to characterize the roles of microRNA-145 (miR-145) and fibroblast growth factor 10 (FGF10) in peritoneal fibrosis. After inducing EMT with transforming growth factor-ß1 (TGF-ß1) in vitro, we found that miR-145 is significantly up-regulated, whereas FGF10 is markedly down-regulated, suggesting a close link between miR-145 and FGF10 in peritoneal fibrosis, further confirmed in luciferase reporter experiments. Furthermore, in human peritoneal mesothelial cells (i.e. HMrSV5 cells), miR-145 mimics induced EMT, whereas miR-145 inhibition suppressed EMT, and we also observed that miR-145 suppressed FGF10 expression. In vivo, we found that the exogenous delivery of an miR-145 expression plasmid both blocked FGF10 and intensified the EMT, whereas miR-145 inhibition promoted the expression of FGF10 and reversed the EMT. In conclusion, miR-145 promotes the EMT during the development of peritoneal fibrosis by suppressing FGF10 activity, suggesting that miR-145 represents a potential therapeutic target for managing peritoneal fibrosis.

Blockade of thrombospondin-1 ameliorates high glucose-induced peritoneal fibrosis through downregulation of TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is a profibrotic cytokine which induces mesothelial cell mesothelial-to-mesenchymal transition (MMT) and peritoneal fibrosis in patients receiving treatment of peritoneal dialysis. Because thrombospondin-1 (TSP-1) is able to activate latent TGF-ß1 in vivo, we investigated whether blockade of TSP-1 could modulate mesothelial cell MMT and ameliorate peritoneal fibrosis. METHODS: Human pleural mesothelial cells (Met-5A cells) were treated with TSP-1 and addition of TGF-ß1 neutralizing antibody to assess the effect of TSP-1 on MMT. Furthermore, TSP-1 blocking peptide Leu-Ser-Lys-Leu (LSKL) was applied to Met-5A cells treated with 4.25% d-glucose to determine its function in high glucose-induced MMT. Consequently, a uremic dialysate injection rat model was set up to confirm the results in vivo. RESULTS: Exposure of Met-5A cells to TSP-1 increased TGF-ß1 secretion, expression and bioactivity, triggered Smad3 phosphorylation, upregulated the expression of mesenchymal molecules including fibronectin, collagen type III, α-smooth muscle actin, Snail, and decreased calretinin expression. The effect was partially attenuated by TGF-ß1 neutralizing antibody. TSP-1 expression in Met-5A cells was increased by 4.25% d-glucose, followed by increased secretion and bioactivity of TGF-ß1, the onset of Smad3 phosphorylation and induction of MMT. LSKL significantly attenuated high glucose-mediated mesothelial cell MMT and ameliorated peritoneal fibrosis in uremic rats receiving dextrose dialysate injection. CONCLUSIONS: Taken together, these data demonstrated that TSP-1 contributes to mesothelial cell MMT by activating TGF-ß1/Smad3 signaling pathway and blockade of TSP-1 attenuates high glucose-mediated mesothelial cell MMT and peritoneal fibrosis.

Long noncoding RNA AK089579 inhibits epithelial-to-mesenchymal transition of peritoneal mesothelial cells by competitively binding to microRNA-296-3p via DOK2 in peritoneal fibrosis.

Peritoneal fibrosis (PF) represents a well-recognized complication associated with continuous ambulatory peritoneal dialysis therapy, characterized by a reversible epithelial-to-mesenchymal transition (EMT) at the early stage. The aim of the current study was to investigate the effects linked with the long noncoding RNA (lncRNA) AK089579 on the EMT of peritoneal mesothelial cells (PMCs) as well as the associated regulatory mechanisms of AK089579 downstream of tyrosine kinase 2 (DOK2) and microRNA-296-3p (miR-296-3p). Enrichment analysis, gene intersection association analysis, and a gene-gene intersection network were initially constructed to ascertain whether AK089579 regulated the expression of DOK2 through the mediation of miR-296-3p via the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in PF. After the PF mouse model had been constructed, the expression of the proteins associated with the JAK2/STAT3 signaling pathway and EMT and PMC migration and invasion were all determined accordingly. Based on the obtained results, AK089579 was determined to function as a competing endogenous RNA for miR-296-3p while acting to up-regulate the expression of DOK2, which is a target gene of miR-296-3p. AK089579 was detected to confer an inhibitory effect on the activation of the JAK2/STAT3 signaling pathway, whereby the migration and invasion of PMCs among the mice models were suppressed. Meanwhile, up-regulated miR-296-3p and down-regulated DOK2 produced contrasting effects when compared with the aforementioned findings. Treatment with wp10066, a JAK2/STAS3 signaling pathway inhibitor, was shown to reverse the effects exerted by up-regulated miR-296-3p. Taken together, the central findings of the current study present evidence highlighting the capability of the lncRNA AK089579 to bind competitively to miR-296-3p and indirectly enhance the expression of DOK2, which in turn suppresses the activation of the JAK2/STAT3 signaling pathway, whereby the EMT, migration, and invasion of PMCs was inhibited in PF.-Zhang, X. W., Wang, L., Ding, H. Long noncoding RNA AK089579 inhibits epithelial-to-mesenchymal transition of peritoneal mesothelial cells by competitively binding to microRNA-296-3p via DOK2 in peritoneal fibrosis.

MicroRNA-302c modulates peritoneal dialysis-associated fibrosis by targeting connective tissue growth factor.

Long-term peritoneal dialysis (PD) can lead to the induction of mesothelial/epithelial-mesenchymal transition (MMT/EMT) and fibrosis; these effects eventually result in ultrafiltration failure and the discontinuation of PD. MicroRNA-302c (miR-302c) is believed to be involved in regulating tumour cell growth and metastasis by suppressing MMT, but the effect of miR-302c on MMT in the context of PD is unknown. MiR-302c levels were measured in mesothelial cells isolated from the PD effluents of continuous ambulatory peritoneal dialysis patients. After miR-302c overexpression using lentivirus, human peritoneal mesothelial cell line (HMrSV5) and PD mouse peritoneum were treated with TGF-ß1 or high glucose peritoneal dialysate respectively. MiR-302c expression level and MMT-related factors alteration were observed. In addition, fibrosis of PD mouse peritoneum was alleviated by miR-302c overexpression. Furthermore, the expression of connective tissue growth factor (CTGF) was negatively related by miR-302c, and LV-miR-302c reversed the up-regulation of CTGF induced by TGF-ß1. These data suggest that there is a novel TGF-ß1/miR-302c/CTGF pathway that plays a significant role in the process of MMT and fibrosis during PD. MiR-302c might be a potential biomarker for peritoneal fibrosis and a novel therapeutic target for protection against peritoneal fibrosis in PD patients.

MicroRNA-21 contributes to high glucose-induced fibrosis in peritoneal mesothelial cells in rat models by activation of the Ras-MAPK signaling pathway via Sprouty-1.

Peritoneal fibrosis remains to be one of the most severe causes of failure in continuous peritoneal dialysis. The current study cultured peritoneal mesothelial cells in high glucose to stimulate the environment of peritoneal fibrosis model in rats, and investigate whether microRNA-21 (miR-21) targeting Sprouty-1 affects high glucose-induced fibrosis in peritoneal mesothelial cells via the rennin angiotensin system (Ras)-mitogen-activated protein kinase (MAPK) signaling pathway, as well as potential mechanisms. Peritoneal tissues in fibrosis rats were collected to extract peritoneal mesothelial cells, which, after in vitro culture, were transfected with a series of mimic or inhibitor of miR-21, or the small interfering RNA (siRNA) against Sprouty-1. Reverse-transcription quantitative polymerase chain reaction and western blot analyses were performed to determine the levels of related genes or proteins. MTT assay and flow cytometry were conducted to observe the cell viability and cell apoptosis of peritoneal mesothelial cells. Dual-luciferase reporter gene assay revealed that Sprouty-1 is the target gene of miR-21. Peritoneal fibrosis manifested with elevated miR-21, extracellular-signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), RAS and p38MAPK but reduced Sprouty-1. Cells transfected with miR-21 mimic exhibited decreased Sprouty-1 expressions, but increased levels of ERK, JNK, RAS, and p38MAPK. As for cellular process, miR-21 mimic or siRNA against Sprouty-1 exposure reduced cell viability, which resulted in more cells arrested at the G1 stage, and induced apoptosis. In contrast, miR-21 inhibitor exposure was observed to have induced effects on peritoneal mesothelial cells. These key findings provide evidence that miR-21 inhibits Sprouty-1 to promote the progression of fibrosis in peritoneal mesothelial cells by activating the Ras-MAPK signaling pathway.

Novel long non-coding RNA AV310809 promotes TGF-ß1 induced epithelial-mesenchymal transition of human peritoneal mesothelial cells via activation of the Wnt2/ß-catenin signaling pathway.

Peritoneal fibrosis (PF) is a crucial cause of the loss of peritoneal function in patients with peritoneal dialysis. To better understand the underlying mechanism of PF, we selected AV310809, which is one of the most highly upregulated lncRNA in fibrotic peritoneal tissue, for functional analysis. We used co-expression analysis to explore the potential relationship between AV310809 and coding genes. qPCR, WB and IF were applied to evaluate the expression and localization of AV310809, epithelial markers and proteins involved in the Wnt2/ß-catenin signaling pathway. The interaction between AV310809 and ß-catenin was examined using an RNA pulldown assay. The expression level of AV310809 was upregulated in fibrotic peritoneum and TGF-ß1 induced EMT in HPMCs. Ectopic overexpression of AV310809 promoted EMT and activated the Wnt2/ß-catenin signaling pathway. Furthermore, we demonstrated that AV310809 could interact with ß-catenin and blocking ß-catenin inhibited the augmentation of EMT by AV310809. These findings indicated that AV310809 promoted TGF-ß1-induced EMT in HPMCs through the activation of the Wnt2/ß-catenin signaling pathway, possibly by targeting ß-catenin. We suggest that AV310809 may be a new therapeutic target for the management of peritoneal dialysis-associated PF.

Functional molecules in mesothelial-to-mesenchymal transition revealed by transcriptome analyses.

Peritoneal fibrosis is a common complication of abdominal and pelvic surgery, and can also be triggered by peritoneal dialysis, resulting in treatment failure. In these settings, fibrosis is driven by activated myofibroblasts that are considered to be partly derived by mesothelial-to-mesenchymal transition (MMT). We hypothesized that, if the molecular signature of MMT could be better defined, these insights could be exploited to block this pathological cellular transition. Rat peritoneal mesothelial cells were purified by the use of an antibody against HBME1, a protein present on mesothelial cell microvilli, and streptavidin nanobead technology. After exposure of sorted cells to a well-known mediator of MMT, transforming growth factor (TGF)-ß1, RNA sequencing was undertaken to define the transcriptomes of mesothelial cells before and during early-phase MMT. MMT was associated with dysregulation of transcripts encoding molecules involved in insulin-like growth factor (IGF) and bone morphogenetic protein (BMP) signalling. The application of either recombinant BMP4 or IGF-binding protein 4 (IGFBP4) ameliorated TGF-ß1-induced MMT in culture, as judged from the retention of epithelial morphological and molecular phenotypes, and reduced migration. Furthermore, peritoneal tissue from peritoneal dialysis patients showed less prominent immunostaining than control tissue for IGFBP4 and BMP4 on the peritoneal surface. In a mouse model of TGF-ß1-induced peritoneal thickening, BMP4 immunostaining on the peritoneal surface was attenuated as compared with healthy controls. Finally, genetic lineage tracing of mesothelial cells was used in mice with peritoneal injury. In this model, administration of BMP4 ameliorated the injury-induced shape change and migration of mesothelial cells. Our findings demonstrate a distinctive MMT signature, and highlight the therapeutic potential for BMP4, and possibly IGFBP4, to reduce MMT. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Curcumin ameliorates peritoneal fibrosis via inhibition of transforming growth factor-activated kinase 1 (TAK1) pathway in a rat model of peritoneal dialysis.

BACKGROUND: Peritoneal fibrosis (PF) remains a serious complication of long-term peritoneal dialysis (PD). The goal of this study was to investigate the anti-fibrotic effects of curcumin on the PF response to PD and its' mechanism. METHODS: Male Sprague-Dawley rats were infused with 20 mL of 4.25% glucose-based standard PD fluid for 8 consecutive weeks to establish PF model and then divided into five groups: Control, received sham operation and 0.9% physiological saline; PD, received 4.25% standard PD fluid; Curcumin, PD rats injected intraperitoeally with curcumin for 8 weeks at doses of 10, 20 or 40 mg/kg. Masson's staining was performed to evaluate the extent of PF. Peritoneal Equilibration Test (PET) was conducted to assess ultrafiltration volume (UFV) and mass transfer of glucose (MTG), quantitative RT-PCR, and immunohistochemistry or western blotting were performed to measure the expression levels of inflammation and fibrosis-associated factors. We also detected the TGF-ß1 in peritoneal fluid by ELISA. RESULTS: Compared with the control group, the PD rats showed decreased UFV (2.54 ± 0.48 to 9.87 ± 0.78 mL, p < 0.05] and increased MTG (18.99 ± 0.86 to 10.85 ± 0.65 mmol/kg, p < 0.05) as well as obvious fibroproliferative response, with markedly increased peritoneal thickness (178.33 ± 4.42 to 25.26 ± 0.32um, p < 0.05) and higher expression of a-SMA, collagen I and TGF-ß1. Treatment with curcumin significantly increased UFV, reduced MTG and peritoneal thickness of PD rats. The elevated TGF-ß1 in peritoneal fluid of PD rats was significantly decreased by curcumin. It attenuated the increase in protein and mRNA of TGF-ß1, α-SMA and collagen I in peritoneum of PD rats. The mRNA expressions of TAK1, JNK and p38, as well as the protein expressions of p-TAK1, p-JNK and p-p38 in peritoneum of PD rats were reduced by curcumin. CONCLUSIONS: Present results demonstrate that curcumin showed a protective effect on PD-related PF and suggest an implication of TAK1, p38 and JNK pathway in mediating the benefical effects of curcumin.

Human umbilical cord mesenchymal stem cells facilitate the up-regulation of miR-153-3p, whereby attenuating MGO-induced peritoneal fibrosis in rats.

MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR-153-3p involved in anti-EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO-induced PF rats (Group MGO) and hUCMSCs-treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real-time PCR analyses were conducted in three groups. α-SMA, Snail1 and E-cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR-153-3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR-153-3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR-153-3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3'-untranslated region luciferase activity in RPMCs. These studies suggest that miR-153-3p is a critical molecule in anti-EMT effects of hUCMSCs in MGO-induced PF rats. MiR-153-3p might exert its beneficial effect through directly targeting Snai1.