MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF-ß1/Smad2/3 signaling pathway.

BACKGROUND: Peritoneal fibrosis (PF) is the main complication of peritoneal dialysis (PD) and the most common cause of cessation from PD. There is still no effective therapeutic approach to reserve PF. We aimed to investigate the role of miR-132-3p and underlying potential mechanisms in PF. METHODS: A total of 18 Sprague-Dawley (SD) rats were divided randomly into three groups (n = 6): (i)Control group (ii)PF group (iii)PF+Losartan group; Rats in the PF group and PF+Losartan group received daily intraperitoneal injections of 3 mg/kg chlorhexidine for 14 days, and rats in the PF+Losartan group simultaneously received daily intraperitoneal injections of 2 mg/kg losartan for 14 days. The control group was injected with saline in the same volume. Met-5A cells were treated for 24h with TGF-ß1 dissolved in recombinant buffered saline at a concentration of 10 ng/ml, meanwhile, PBS solution as a negative control. The human peritoneal solution was collected for the detection of miR-132-3p. RESULTS: In vivo, SD rats were infused with chlorhexidine to establish PF model, and we found that miR-132-3p significantly decreased and the expressions of transforming growth factor-ß1 (TGF-ß1), and Smad2/3 were up-regulated in PF. In vitro, miR-132-3p mimics suppressed TGF-ß1/Smad2/3 activity, whereas miR-132-3p inhibition activated the pathway. In human peritoneal solution, we found that the expression of miR-132-3p decreased in a time-dependent model and its effect became more pronounced with longer PD duration. CONCLUSION: MiR-132-3p ameliorated PF by suppressing TGF-ß1/Smad2/3 activity, suggesting that miR-132-3p represented a potential therapeutic approach for PF.

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.

High glucose dialysate-induced peritoneal fibrosis: Pathophysiology, underlying mechanisms and potential therapeutic strategies.

Peritoneal dialysis is an efficient renal replacement therapy for patients with end-stage kidney disease. However, continuous exposure of the peritoneal membrane to dialysate frequently leads to peritoneal fibrosis, which alters the function of the peritoneal membrane and results in withdrawal from peritoneal dialysis in patients. Among others, high glucose dialysate is considered as a predisposing factor for peritoneal fibrosis in patients on peritoneal dialysis. Glucose-induced inflammation, metabolism disturbance, activation of the renin-angiotensin-aldosterone system, angiogenesis and noninflammation-induced reactive oxygen species are implicated in the pathogenesis of high glucose dialysate-induced peritoneal fibrosis. Specifically, high glucose causes chronic inflammation and recurrent peritonitis, which could cause migration and polarization of inflammatory cells, as well as release of cytokines and fibrosis. High glucose also interferes with lipid metabolism and glycolysis by activating the sterol-regulatory element-binding protein-2/cleavage-activating protein pathway and increasing hypoxia inducible factor-1α expression, leading to angiogenesis and peritoneal fibrosis. Activation of the renin-angiotensin-aldosterone system and Ras-mitogen activated protein kinase signaling pathway is another contributing factor in high glucose dialysate-induced fibrosis. Ultimately, activation of the transforming growth factor-ß1/Smad pathway is involved in mesothelial-mesenchymal transition or epithelial-mesenchymal transition, which leads to the development of fibrosis. Although possible intervention strategies for peritoneal dialysate-induced fibrosis by targeting the transforming growth factor-ß1/Smad pathway have occasionally been proposed, lack of laboratory evidence renders clinical decision-making difficult. We therefore aim to revisit the upstream pathways of transforming growth factor-beta1/Smad and propose potential therapeutic targets for high glucose-induced peritoneal fibrosis.

Exosomal lnc-CDHR derived from human umbilical cord mesenchymal stem cells attenuates peritoneal epithelial-mesenchymal transition through AKT/FOXO pathway.

OBJECTIVE: Chronic stimulation of peritoneal dialysis (PD) fluid leads to the epithelial-mesenchymal transformation (EMT) of mesothelial cells, peritoneal fibrosis (PF), and ultimately ultrafiltration failure. Some studies have proposed that mesenchymal stem cells (MSCs) can alleviate PF. This study aimed to investigate whether the exosomes from human umbilical cord MSCs (hUMSCs) could alleviate peritoneal EMT. METHODS: Human peritoneal mesothelial cell line (HMrSV5) were treated with high glucose (HG) for 48 hours to induce the peritoneal EMT model. An inverted fluorescence microscope was used to observe the internalization of exosomes derived from hUMSCs (hUMSC-Exos). Western blot and real-time PCR were used to evaluate the expression of α-SMA, Vimentin, E-cadherin, PTEN, and AKT/FOXO3a. The relationships of lncRNA CDHR and miR-3149, miR-3149 and PTEN were detected by dual luciferase reporter gene assay. RESULTS: Compared with HG-induced HMrSV5, E-cadherin and PTEN levels significantly increased whereas α-SMA and Vimentin levels significantly decreased after treatment of hUMSC-CM and hUMSC-Exos (P < 0.05). An inverted fluorescence microscope showed HMrSV5 can absorb exosomes to alleviate EMT. Furthermore, exosomes extracted from lnc-CDHR siRNA-transfected hUMSCs can't ameliorate HMrSV5 EMT. Moreover, both CDHR overexpressed and miR-3149 inhibitor in HG-induced HMrSV5 alleviated the expression of α-SMA, and Vimentin, and increased the expression of E-cadherin and PTEN, and AKT/FOXO3a. A rescue experiment showed that CDHR overexpressed expression was repressed by miR-3149 in the HG-induced peritoneal EMT model. CONCLUSIONS: Exosomal lnc-CDHR derived from hUMSCs may competitively bind to miR-3149 to regulate suppression on target PTEN genes and alleviate EMT of HMrSV5 through AKT/FOXO pathway.

Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model.

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified. We investigated transglutaminase 2 (TG2) as a possible novel therapeutic target for peritoneal injury. TG2 and fibrosis, inflammation, and angiogenesis were investigated in a chlorhexidine gluconate (CG)-induced model of peritoneal inflammation and fibrosis, representing a noninfectious model of PD-related peritonitis. Transforming growth factor (TGF)-ß type I receptor (TGFßR-I) inhibitor and TG2-knockout mice were used for TGF-ß and TG2 inhibition studies, respectively. Double immunostaining was performed to identify cells expressing TG2 and endothelial-mesenchymal transition (EndMT). In the rat CG model of peritoneal fibrosis, in situ TG2 activity and protein expression increased during the development of peritoneal fibrosis, as well as increases in peritoneal thickness and numbers of blood vessels and macrophages. TGFßR-I inhibitor suppressed TG2 activity and protein expression, as well as peritoneal fibrosis and angiogenesis. TGF-ß1 expression, peritoneal fibrosis, and angiogenesis were suppressed in TG2-knockout mice. TG2 activity was detected by α-smooth muscle actin-positive myofibroblasts, CD31-positive endothelial cells, and ED-1-positive macrophages. CD31-positive endothelial cells in the CG model were α-smooth muscle actin-positive, vimentin-positive, and vascular endothelial-cadherin-negative, suggesting EndMT. In the CG model, EndMT was suppressed in TG2-knockout mice. TG2 was involved in the interactive regulation of TGF-ß. As inhibition of TG2 reduced peritoneal fibrosis, angiogenesis, and inflammation associated with TGF-ß and vascular endothelial growth factor-A suppression, TG2 may provide a new therapeutic target for ameliorating peritoneal injuries in PD.

TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. AIM OF STUDY: The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. MATERIALS AND METHODS: The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P‒T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experimental verification. RESULTS: TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mesentery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/STAT signaling pathway, such as JAK2 and STAT3. The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-ß1-stimulated HMrSV5 cells, E-cadherin expression levels were dramatically reduced whereas Vimentin, p-JAK2, α-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-ß1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. CONCLUSION: ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Activated protein C inhibits mesothelial-to-mesenchymal transition in experimental peritoneal fibrosis.

BACKGROUND: In addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) in endothelial cells. OBJECTIVES: To investigate the protective effect of APC in a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model. METHODS: Peritoneal fibrosis was induced in wild-type as well as EPCR- and PAR1-deficient mice via daily injection of CG (0.2 mL of 0.1% CG in 15% ethanol and 85% saline) for 21 days with or without concomitant injection of recombinant human APC derivatives (50 µg/kg of bodyweight). The expression of proinflammatory cytokines and profibrotic markers as well as collagen deposition were analyzed using established methods. RESULTS: CG significantly upregulated the expression of transforming growth factor-ß1 in peritoneal tissues, which culminated in the deposition of excessive extracellular matrix proteins, thickening of the peritoneal membrane, and mesothelial-to-mesenchymal transition in damaged tissues. APC potently inhibited CG-induced peritoneal fibrosis and downregulated the expression of proinflammatory cytokines, collagen deposition, Smad3 phosphorylation, and markers of mesothelial-to-mesenchymal transition (α-smooth muscle actin, vimentin, and N-cadherin). APC also inhibited transforming growth factor-ß1-mediated upregulation of α-smooth muscle actin, Smad3, and fibronectin in human primary mesothelial cells. Employing signaling-selective and anticoagulant-selective variants of APC and mutant mice deficient for either EPCR or PAR1, we demonstrated that the EPCR-dependent signaling function of APC through PAR1 activation was primarily responsible for its antifibrotic activity in the CG-induced peritoneal fibrosis model. CONCLUSION: APC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.

Pharmacologic Inhibition of Histone Deacetylase 6 Prevents the Progression of Chlorhexidine Gluconate-Induced Peritoneal Fibrosis by Blockade of M2 Macrophage Polarization.

Peritoneal fibrosis contributes to ultrafiltration failure in peritoneal dialysis (PD) patients and thus restricts the wide application of PD in clinic. Recently we have demonstrated that histone deacetylase 6 (HDAC6) is critically implicated in high glucose peritoneal dialysis fluid (HG-PDF) induced peritoneal fibrosis, however, the precise mechanisms of HDAC6 in peritoneal fibrosis have not been elucidated. Here, we focused on the role and mechanisms of HDAC6 in chlorhexidine gluconate (CG) induced peritoneal fibrosis and discussed the mechanisms involved. We found Tubastatin A (TA), a selective inhibitor of HDAC6, significantly prevented the progression of peritoneal fibrosis, as characterized by reduction of epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) protein deposition. Inhibition of HDAC6 remarkably suppressed the expression of matrix metalloproteinases-2 (MMP2) and MMP-9. Administration of TA also increased the expression of acetylation Histone H3 and acetylation α-tubulin. Moreover, our results revealed that blockade of HDAC6 inhibited alternatively M2 macrophages polarization by suppressing the activation of TGF-ß/Smad3, PI3K/AKT, and STAT3, STAT6 pathways. To give a better understanding of the mechanisms, we further established two cell injured models in Raw264.7 cells by using IL-4 and HG-PDF. Our in vitro experiments illustrated that both IL-4 and HG-PDF could induce M2 macrophage polarization, as demonstrated by upregulation of CD163 and Arginase-1. Inhibition of HDAC6 by TA significantly abrogated M2 macrophage polarization dose-dependently by suppressing TGF-ß/Smad, IL4/STAT6, and PI3K/AKT signaling pathways. Collectively, our study revealed that blockade of HDAC6 by TA could suppress the progression of CG-induced peritoneal fibrosis by blockade of M2 macrophage polarization. Thus, HDAC6 may be a promising target in peritoneal fibrosis treatment.

Use of paclitaxel carried in solid lipid nanoparticles to prevent peritoneal fibrosis in rats.

BACKGROUND: Progressive fibrous thickening of peritoneal membrane (PM) is a major complication of long-term peritoneal dialysis. TGF-ß/SMAD pathway activation, inflammation and neoangiogenesis have an important role in PM changes induced by peritoneal dialysis. Here, we investigated the effects of paclitaxel (PTX) carried in lipid core nanoparticles (LDE) on the development of peritoneal fibrosis (PF) in rats. METHODS: To induce PF, 21 male Wistar rats (300-350g) were injected with chlorhexidine gluconate for 15 consecutive days and randomly assigned to three groups: 1)PF, n = 5: no treatment; 2)LDE, n = 8: treated with LDE only, 3/3 days during 15 days; 3)LDE-PTX, n = 8: treated with PTX (4mg/kg) associated with LDE, 3/3 days during 15 days. A Control group without PF induction (n = 5) was designed, received saline solution, 3/3 days. Peritoneum function tests were performed, and anterior abdominal wall samples of the PM were collected for analyses of peritoneal thickness, immunohistochemitry, and gene expression. RESULTS: LDE-PTX treatment preserved the membrane function, maintaining the ultrafiltration rate and mass transfer of glucose at normal levels. LDE-PTX also prevented PM thickening induced by chlorhexidine gluconate injections. LDE-PTX treatment reduced the number of myofibroblasts infiltrating PM and inhibited the cell proliferation. Gene expression of fibronectin, FSP-1, VEGF, TGF-ß, and SMAD3 were reduced by LDE-PTX. CONCLUSIONS: LDE-PTX was effective to prevent development of PF and preserve the PM filtration capacity in this rat model, with clear-cut actions on pro-fibrotic mechanisms. Thus, LDE-PTX can be candidate for future clinical trials as adjuvant to peritoneal dialysis to prevent PF development, since this preparation is devoid of toxicity as shown previously.

A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage.

Peritoneal fibrosis is an important complication of peritoneal dialysis (PD). To investigate and address this problem, an appropriate animal model of PD is required. The present protocol establishes a chlorhexidine gluconate (CG) induced peritoneal fibrosis model that mimics the condition of a patient with PD. Peritoneal fibrosis was induced by intraperitoneal injection of 0.1% of CG in 15% ethanol for 3 weeks (administered every other day), for a total of nine times in male C57BL/6 mice. Peritoneal functional tests were then performed on day 22. After the mice were sacrificed, the parietal peritoneum of the abdominal wall and the visceral peritoneum of the liver were harvested. They were thicker and more fibrotic when analyzed microscopically after Masson's trichrome staining. The ultrafiltration rate decreased, and glucose mass transport indicated a CG-induced increase in peritoneal permeability. The PD model thus established may have applications in improving PD technology, dialysis efficacy, and prolonging patient survival.

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.

Astragalus Total Saponins Ameliorate Peritoneal Fibrosis by Promoting Mitochondrial Synthesis and Inhibiting Apoptosis.

Peritoneal fibrosis (PF) is a disease caused by prolonged exposure of the peritoneum to high levels of dialysis fluid. Astragalus total saponins (ATS) is a phytochemical naturally occurring in Radix Astragali that has anti-inflammatory and anti-oxidant properties. In this study, we constructed an in vivo model of PF using 4.25% glucose-containing administered intraperitoneally to rats and incubated peritoneal mesothelial cells (PMCs) with 4.25% glucose-containing peritoneal dialysis fluid to construct an in vitro model of PF. Furthermore, siRNA of PGC-1[Formula: see text] was used to inhibit the expression of PGC-1[Formula: see text] to further investigate the mechanism of the protective effect of ATS on PF. In both in vivo and in vitro models, ATS treatment showed a protective effect against PF, with ATS reducing the thickness of peritoneal tissues in PF rats, increasing the viability of PMCs, increasing the mitochondrial membrane potential and reducing apoptosis ratio. ATS treatment also reduced the expressions of peritoneal fibrosis markers (Smad2, p-Smad2 and [Formula: see text]-SMA) and apoptosis markers (Caspase3, cleaved-Caspase3 and Bax) and restored the expressions of mitochondrial synthesis proteins (PGC-1[Formula: see text], NRF1 and TFAM) in ATS-treated peritoneal tissues or PMCs. Furthermore, in the presence of PGC-1[Formula: see text] inhibition, the protective effect of ATS on PF was blocked. In conclusion, ATS treatment may be an effective therapeutic agent to inhibit high glucose-induced in peritoneal fibrosis through PGC-1[Formula: see text]-mediated apoptosis.

Increased expression of epimorphin in a peritoneal fibrosis mouse model.

BACKGROUND: Long-term peritoneal dialysis results in functional and histopathological alterations of the peritoneal membrane, leading to peritoneal fibrosis (PF). The mechanism of PF has not been fully elucidated, and at present there is no effective therapy for PF. Epimorphin is a mesenchymal protein that not only regulates morphogenesis in organ development but is implicated in tissue repair. However, the role of epimorphin in PF has not yet been clarified. METHODS: PF was induced in C57/Bl6 mice by intraperitoneal injection of chlorhexidine gluconate (CG-injected mice) three times a week for 3 weeks. The parietal peritoneum was subsequently dissected and assessed by Masson's trichrome staining, and epimorphin expression was analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, epimorphin-positive regions were analysed by multiple immunofluorescence staining using fibrosis-associated markers. In addition, normal rat fibroblast cells (NRK-49F) were treated with transforming growth factor-ß (TGF-ß) in the presence or absence of epimorphin. The expression of fibrosis-associated markers was assessed by real-time RT-PCR. RESULTS: In CG-injected mice, Masson's trichrome staining showed marked thickening of the submesothelial compact zone. Weak epimorphin expression was observed in the narrow submesothelial compact zone beneath the mesothelial cells in control mice; however, epimorphin expression was stronger in the submesothelial compact zone in CG-injected mice. Epimorphin expression was observed mainly in α-smooth muscle actin (α-SMA)-positive myofibroblasts. Epimorphin suppressed the TGF-ß-induced upregulation of α-SMA and platelet-derived growth factor receptor-ß in cultured cells. CONCLUSIONS: Our results suggest that epimorphin may be a therapeutic target for fibrotic diseases of the peritoneum.

Mitochonic acid-5 ameliorates chlorhexidine gluconate-induced peritoneal fibrosis in mice.

Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, attributable to inflammation and mitochondrial dysfunction. Mitochonic acid-5 (MA-5), an indole-3-acetic acid derivative, improves mitochondrial dysfunction and has therapeutic potential against various diseases including kidney diseases. However, whether MA-5 is effective against peritoneal fibrosis remains unclear. Therefore, we investigated the effect of MA-5 using a peritoneal fibrosis mouse model. Peritoneal fibrosis was induced in C57BL/6 mice via intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks. MA-5 was administered daily by oral gavage. The mice were divided into control, MA-5, CG, and CG + MA-5 groups. Following treatment, immunohistochemical analyses were performed. Fibrotic thickening of the parietal peritoneum induced by CG was substantially attenuated by MA-5. The number of α-smooth muscle actin-positive myofibroblasts, transforming growth factor ß-positive cells, F4/80-positive macrophages, monocyte chemotactic protein 1-positive cells, and 4-hydroxy-2-nonenal-positive cells was considerably decreased. In addition, reduced ATP5a1-positive and uncoupling protein 2-positive cells in the CG group were notably increased by MA-5. MA-5 may ameliorate peritoneal fibrosis by suppressing macrophage infiltration and oxidative stress, thus restoring mitochondrial function. Overall, MA-5 has therapeutic potential against peritoneal fibrosis.

Effects of TGF-ß1 Receptor Inhibitor GW788388 on the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells.

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

Hepatocyte growth factor ameliorates methylglyoxal-induced peritoneal inflammation and fibrosis in mouse model.

BACKGROUND: Peritoneal dialysis (PD) is essential for patients with end-stage renal disease. Peritoneal fibrosis (PF) is a complex inflammatory, fibrogenic process. No effective treatments are available to prevent these processes. Hepatocyte growth factor (HGF) possesses anti-inflammatory and anti-fibrotic properties. The aim of this study was to analyze whether HGF suppresses MGO-induced peritoneal inflammation and fibrosis in a mouse model. METHODS: PF was induced by intraperitoneal (IP) injections of MGO for 14 days. C57/BL/6 mice were divided into three groups: Sham group (only vehicle); Sham + MGO group (PF induced by MGO); and HGF + MGO group (PF mice treated with recombinant human-HGF). PF was assessed from tissue samples by Masson's trichrome staining. Inflammation and fibrosis-associated factors were assessed by immunohistochemistry and quantitative real-time PCR. RESULTS: MGO-injected mice showed significant thickening of the submesothelial compact zone with PF. Treatment with HGF significantly reduced PM thickness and suppressed the expression of collagen I and III and α-SMA. Expression of profibrotic and proinflammatory cytokines (TGF-ß, TNF-α, IL-1ß) was reduced by HGF treatment. The number of macrophages, and M1 and M2 macrophage-related markers, such as CD86, CD206, and CD163, was reduced in HGF + MGO mice. CONCLUSION: HGF attenuates MGO-induced PF in mice. Furthermore, HGF treatment reduces myofibroblast and macrophage infiltration, and attenuates the upregulated expression of proinflammatory and profibrotic genes in peritoneal tissues. HGF might be an effective approach to prevent the development of PF in patients undergoing PD.

Mesenchymal stem cells cultured in serum-free medium ameliorate experimental peritoneal fibrosis.

BACKGROUND: Mesenchymal stem cells (MSCs) provide potential treatments for peritoneal fibrosis. However, MSCs cultured in media containing serum bring risks of infection and other problems. In this study, we compared the effect of human MSCs in serum-free medium (SF-MSCs) on peritoneal fibrosis with that of MSCs cultured in medium containing 10% fetal bovine serum (10%MSCs). METHODS: Peritoneal fibrosis was induced by intraperitoneally injecting 0.1% chlorhexidine gluconate (CG). SF-MSCs or 10%MSCs were intraperitoneally administered 30 min after the CG injection. Ten days after the CG and MSC injections, we performed histological analyses and peritoneal equilibrium testing. In the in vitro experiments, we used transforming growth factor (TGF)-ß1-stimulated human peritoneal mesothelial cells incubated in conditioned medium from MSCs to examine whether the SF-MSCs showed enhanced ability to produce antifibrotic humoral factors. RESULTS: Histological staining showed that the SF-MSCs significantly suppressed CG-induced cell accumulation and thickening compared with that of the 10%MSCs. Additionally, the SF-MSCs significantly inhibited mesenchymal cell expression, extracellular matrix protein deposition and inflammatory cell infiltration. Peritoneal equilibration testing showed that compared with administering 10%MSCs, administering SF-MSCs significantly reduced the functional impairments of the peritoneal membrane. The in vitro experiments showed that although the conditioned medium from MSCs suppressed TGF-ß1 signaling, the suppression did not significantly differ between the SF-MSCs and 10%MSCs. CONCLUSIONS: Serum-free culture conditions can enhance the antifibrotic abilities of MSCs by suppressing inflammation. Administering ex vivo expanded SF-MSCs may be a potential therapy for preventing peritoneal fibrotic progression.

An inhibitor of Krüppel-like factor 5 suppresses peritoneal fibrosis in mice.

BACK GROUND: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. RESULTS: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor ß-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. CONCLUSIONS: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.

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.

Trehalose ameliorates peritoneal fibrosis by promoting Snail degradation and inhibiting mesothelial-to-mesenchymal transition in mesothelial cells.

Peritoneal fibrosis (PF) is a severe complication of peritoneal dialysis, but there are few effective therapies for it. Recent studies have revealed a new biological function of trehalose as an autophagy inducer. Thus far, there are few reports regarding the therapeutic effects of trehalose on fibrotic diseases. Therefore, we examined whether trehalose has anti-fibrotic effects on PF. PF was induced by intraperitoneal injection of chlorhexidine gluconate (CG). CG challenges induced the increase of peritoneal thickness, ColIα1 mRNA expression and hydroxyproline content, all of which were significantly attenuated by trehalose. In addition, CG challenges induced a marked peritoneal accumulation of α-SMA+ myofibroblasts that was reduced by trehalose. The number of Wt1+ α-SMA+ cells in the peritoneum increased following CG challenges, suggesting that a part of α-SMA+ myofibroblasts were derived from peritoneal mesothelial cells (PMCs). The number of Wt1+ α-SMA+ cells was also suppressed by trehalose. Additionally, trehalose attenuated the increase of α-SMA and ColIα1 mRNA expression induced by TGF-ß1 through Snail protein degradation, which was dependent on autophagy in PMCs. These results suggest that trehalose might be a novel therapeutic agent for PF through the induction of autophagy and the suppression of mesothelial-to-mesenchymal transition in PMCs.