Protective effect of Cyclo(His-Pro) on peritoneal fibrosis through regulation of HDAC3 expression.

Peritoneal dialysis is a common treatment for end-stage renal disease, but complications often force its discontinuation. Preventive treatments for peritoneal inflammation and fibrosis are currently lacking. Cyclo(His-Pro) (CHP), a naturally occurring cyclic dipeptide, has demonstrated protective effects in various fibrotic diseases, yet its potential role in peritoneal fibrosis (PF) remains uncertain. In a mouse model of induced PF, CHP was administered, and quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry was employed to identify PF-related protein signaling pathways. The results were further validated using human primary cultured mesothelial cells. This analysis revealed the involvement of histone deacetylase 3 (HDAC3) in the PF signaling pathway. CHP administration effectively mitigated PF in both peritoneal tissue and human primary cultured mesothelial cells, concurrently regulating fibrosis-related markers and HDAC3 expression. Moreover, CHP enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) while suppressing forkhead box protein M1 (FOXM1), known to inhibit Nrf2 transcription through its interaction with HDAC3. CHP also displayed an impact on spleen myeloid-derived suppressor cells, suggesting an immunomodulatory effect. Notably, CHP improved mitochondrial function in peritoneal tissue, resulting in increased mitochondrial membrane potential and adenosine triphosphate production. This study suggests that CHP can significantly prevent PF in peritoneal dialysis patients by modulating HDAC3 expression and associated signaling pathways, reducing fibrosis and inflammation markers, and improving mitochondrial function.

Selective therapeutic efficacy of tyrosine kinase inhibitor sorafenib on the restoration of methylglyoxal-induced peritoneal fibrosis.

Peritoneal fibrosis, a common complication observed in long-term peritoneal dialysis patients, can gradually lead to ultrafiltration failure and the development of encapsulating peritoneal sclerosis. Although mechanisms of peritoneal fibrosis have been proposed, effective therapeutic options are unsatisfactory. Recently, several tyrosine kinase inhibitors have proven to be anti-fibrosis in rodent models. To assess the potential therapeutic effects of tyrosine kinase inhibitors on peritoneal fibrosis in the larger animal model, a novel porcine model of peritoneal fibrosis induced by 40 mM methylglyoxal in 2.5 % dialysate was established, and two different doses (20 mg/kg and 30 mg/kg) of sorafenib were given orally to evaluate their therapeutic efficacy in this study. Our results showed that sorafenib effectively reduced adhesions between peritoneal organs and significantly diminished the thickening of both the parietal and visceral peritoneum. Angiogenesis, vascular endothelial growth factor A production, myofibroblast infiltration, and decreased endothelial glycocalyx resulting from dialysate and methylglyoxal stimulations were also alleviated with sorafenib. However, therapeutic efficacy in ameliorating loss of mesothelial cells, restoring decreased ultrafiltration volume, and improving elevated small solutes transport rates was limited. In conclusion, this study demonstrated that sorafenib could potentially be used for peritoneal fibrosis treatment, but applying sorafenib alone might not be sufficient to fully rescue methylglyoxal-induced peritoneal defects.

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.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis.

PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

The disruptor of telomeric silencing 1-like (DOT1L) promotes peritoneal fibrosis through the upregulation and activation of protein tyrosine kinases.

The disruptor of telomeric silencing 1-like (DOT1L), a specific histone methyltransferase that catalyzed methylation of histone H3 on lysine 79, was associated with the pathogenesis of many diseases, but its role in peritoneal fibrosis remained unexplored. Here, we examined the role of DOT1L in the expression and activation of protein tyrosine kinases and development of peritoneal fibrosis. We found that a significant rise of DOT1L expression in the fibrotic peritoneum tissues from long-term PD patients and mice. Inhibition of DOT1L significantly attenuated the profibrotic phenotypic differentiation of mesothelial cells and macrophages, and alleviated peritoneal fibrosis. Mechanistically, RNA sequencing and proteomic analysis indicated that DOT1L was mainly involved in the processes of protein tyrosine kinase binding and extracellular matrix structural constituent in the peritoneum. Chromatin immunoprecipitation (ChIP) showed that intranuclear DOT1L guided H3K79me2 to upregulate EGFR in mesothelial cells and JAK3 in macrophages. Immunoprecipitation and immunofluorescence showed that extranuclear DOT1L could interact with EGFR and JAK3, and maintain the activated signaling pathways. In summary, DOT1L promoted the expression and activation of tyrosine kinases (EGFR in mesothelial cells and JAK3 in macrophages), promoting cells differentiate into profibrotic phenotype and thus peritoneal fibrosis. We provide the novel mechanism of dialysis-related peritoneal fibrosis (PF) and the new targets for clinical drug development. DOT1L inhibitor had the PF therapeutic potential.

2-Deoxy-glucose ameliorates the peritoneal mesothelial and endothelial barrier function perturbation occurring due to Peritoneal Dialysis fluids exposure.

Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (RTM), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by RTM, dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier.

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.

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.

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.

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.

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.

Histone deacetylase 8 inhibition prevents the progression of peritoneal fibrosis by counteracting the epithelial-mesenchymal transition and blockade of M2 macrophage polarization.

Background: Peritoneal dialysis (PD) is an effective replacement therapy for end-stage renal disease patients. However, long-term exposure to peritoneal dialysate will lead to the development of peritoneal fibrosis. Epigenetics has been shown to play an important role in peritoneal fibrosis, but the role of histone deacetylases 8 (HDAC8) in peritoneal fibrosis have not been elucidated. In this research, we focused on the role and mechanisms of HDAC8 in peritoneal fibrosis and discussed the mechanisms involved. Methods: We examined the expression of HDAC8 in the peritoneum and dialysis effluent of continuous PD patients. Then we assessed the role and mechanism of HDAC8 in peritoneal fibrosis progression in mouse model of peritoneal fibrosis induced by high glucose peritoneal dialysis fluid by using PCI-34051. In vitro, TGF-ß1 or IL-4 were used to stimulate human peritoneal mesothelial cells (HPMCs) or RAW264.7 cells to establish two cell injury models to further explore the role and mechanism of HDAC8 in epithelial-mesenchymal transition (EMT) and macrophage polarization. Results: We found that HDAC8 expressed highly in the peritoneum from patients with PD-related peritonitis. We further revealed that the level of HDAC8 in the dialysate increased over time, and HDAC8 was positively correlated with TGF-ß1 and vascular endothelial growth factor (VEGF), and negatively correlated with cancer antigen 125. In mouse model of peritoneal fibrosis induced by high glucose dialysate, administration of PCI-34051 (a selective HDAC8 inhibitor) significantly prevented the progression of peritoneal fibrosis. Treatment with PCI-34051 blocked the phosphorylation of epidermal growth factor receptor (EGFR) and the activation of its downstream signaling pathways ERK1/2 and STAT3/HIF-1α. Inhibition of HDAC8 also reduced apoptosis. In vitro, HDAC8 silencing with PCI-34051 or siRNA inhibited TGF-ß1-induced EMT and apoptosis in HPMCs. In addition, continuous high glucose dialysate or IL-4 stimulation induced M2 macrophage polarization. Blockade of HDAC8 reduced M2 macrophage polarization by inhibiting the activation of STAT6 and PI3K/Akt signaling pathways. Conclusions: We demonstrated that HDAC8 promoted the EMT of HPMCs via EGFR/ERK1/2/STAT3/HIF-1α, induced M2 macrophage polarization via STAT6 and PI3K/Akt signaling pathways, and ultimately accelerated the process of peritoneal fibrosis.

Effects of sitagliptin on peritoneal membrane: The potential role of mesothelial cell tight junction proteins.

BACKGROUND: The roles of tight junction (TJ) proteins in peritoneal membrane transport and peritoneal dialysis (PD) require further characterisation. Dipeptidyl peptidase-4 is expressed in mesothelial cells, and its activity may affect peritoneal membrane function and morphology. METHODS: Human peritoneal mesothelial cells (HPMCs) were isolated and cultured from omentum obtained during abdominal surgery, and paracellular transport functions were evaluated by measuring transmesothelial electrical resistance (TMER) and dextran flux. Sprague-Dawley rats were infused daily with 4.25% peritoneal dialysate with and without sitagliptin administration for 8 weeks. At the end of this period, rat peritoneal mesothelial cells (RPMCs) were isolated to evaluate TJ protein expression. RESULTS: In HPMCs, the protein expression of claudin-1, claudin-15, occludin and E-cadherin was decreased by TGF-ß treatment but reversed by sitagliptin co-treatment. TMER was decreased by TGF-ß treatment but improved by sitagliptin co-treatment. Consistent with this, dextran flux was increased by TGF-ß treatment and reversed by sitagliptin co-treatment. In the animal experiment, sitagliptin-treated rats had a lower D2/D0 glucose ratio and a higher D2/P2 creatinine ratio than PD controls during the peritoneal equilibration test. Protein expression of claudin-1, claudin-15 and E-cadherin decreased in RPMCs from PD controls but was not affected in those from sitagliptin-treated rats. Peritoneal fibrosis was induced in PD controls but ameliorated in sitagliptin-treated rats. CONCLUSION: The expression of TJ proteins including claudin-1 and claudin-15 was associated with transport function both in HPMCs and in a rat model of PD. Sitagliptin prevents peritoneal fibrosis in PD and can potentially restore peritoneal mesothelial cell TJ proteins.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Alleviate Peritoneal Dialysis-Associated Peritoneal Injury.

Peritoneal fibrosis is a critical sequela that limits the application of peritoneal dialysis (PD). This study explored the role and mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) in preventing PD-associated peritoneal injury. C57BL/6 mice were randomized into three groups: a control (saline), peritoneal injury [2.5% glucose peritoneal dialysate + lipopolysaccharide (LPS)], and peritoneal injury + exosome group. After 6 weeks, mice were dissected, and the parietal peritoneum was collected. The level of peritoneal structural and functional damage was assessed. Additionally, transcriptome analysis of the peritoneum and miRNA sequencing on BMSC-Exos were performed. The parietal peritoneum had significantly thickened, and peritoneal function was impaired in the peritoneal injury group. Peritoneal structural and functional damage was significantly reduced after exosome treatment, while peritoneal inflammation, fibrosis, angiogenesis, and mesothelial damage significantly increased. Transcriptomic analysis showed that the BMSC-Exos affected the cell cycle process, cell differentiation, and inflammatory response regulation. Significant pathways in the exosome group were enriched by inflammation, immune response, and cell differentiation, which constitute a molecular network that regulates the peritoneal protective mechanism. Additionally, inflammatory factors (TNF-α, IL-1ß), fibrosis markers (α-SMA, collagen-III, fibronectin), profibrotic cytokines (TGF-ß1), and angiogenesis-related factor (VEGF) were downregulated at the mRNA and protein levels through BMSC-Exos treatment. BMSC-Exos treatment can prevent peritoneal injury by inhibiting peritoneal fibrosis, inflammation, and angiogenesis, showing a multitarget regulatory effect. Therefore, BMSC-Exos therapy might be a new therapeutic strategy for treating peritoneal injury.

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.

Inhibition of EZH2 suppresses peritoneal angiogenesis by targeting a VEGFR2/ERK1/2/HIF-1α-dependent signaling pathway.

The catalytic subunit of polycomb repressive complex 2 (PRC2), enhancer of zeste homolog 2 (EZH2), has been reported to be involved in angiogenesis in some tumors and autoimmune diseases. However, the mechanisms by which EZH2 regulates peritoneal angiogenesis remain unclear. We detected the expression of EZH2 in clinical samples and the peritoneal tissue of a mouse peritoneal fibrosis model induced by chlorhexidine gluconate (CG). In addition, we further investigated the mechanisms by which inhibition of EZH2 by 3-deazaneplanocin A (3-DZNeP) alleviated the CG-induced peritoneal fibrosis mouse model in vivo and 3-DZNeP or EZH2 siRNA treatment in cultured human peritoneal mesothelial cells (HPMCs) and human umbilical vein endothelial cells (HUVECs). The expression of EZH2 in the peritoneum of long-term peritoneal dialysis (PD) patients and the CG-induced peritoneal fibrosis mouse model was remarkably increased and this was positively associated with higher expression of vascular markers (CD31, CD34, VEGF, p-VEGFR2). Peritoneal injection of 3-DZNeP attenuated angiogenesis in the peritoneum of CG-injured mice; improved peritoneal membrane function; and decreased phosphorylation of STAT3, ERK1/2, and activation of Wnt1/ß-catenin. In in vitro experiments, we demonstrated that inhibition of EZH2 by 3-DZNeP or EZH2 siRNA decreased tube formation and the migratory ability of HUVECs via two pathways: the Wnt1/ß-catenin pathway and the IL-6/STAT3 pathway. Suppression of the Wnt1/ß-catenin pathway and the IL-6/STAT3 pathway subsequently reduced VEGF production in HPMCs. Using specific inhibitors of VEGFR2, ERK1/2, and HIF-1α, we found that a VEGFR2/ERK1/2/HIF-1α axis existed and contributed to angiogenesis in vitro. Moreover, phosphorylation of VEGFR2 and activation of the ERK1/2 pathway and HIF-1α in HUVECs could be suppressed by inhibition of EZH2. Taken together, the results of this study suggest that EZH2 may be a novel target for preventing peritoneal angiogenesis in PD patients. © 2022 The Pathological Society of Great Britain and Ireland.

Role of IGF-1R in epithelial-mesenchymal transdifferentiation of human peritoneal mesothelial cells.

BACKGROUND: Peritoneal fibrosis (PF) is caused by epithelial-mesenchymal transdifferentiation (EMT) in the peritoneum under high glucose (HG) conditions. The study aimed to explored the role of Insulin-like growth factor 1 receptor (IGF-1R) in the regulation of EMT in human peritoneal mesothelial cells (HPMCs). METHODS: We used HG peritoneal dialysis fluid (PDF) to induce in vivo PF in mice, and treated HPMCs with HG in vitro to stimulate EMT. RESULTS: In the mice, the higher the glucose concentration in the dialysate, the more obvious the peritoneal tissue thickening and the more that collagen was deposited. The in vitro study indicated that the expression of IGF-1R, α-SMA, vimentin was upregulated, while the expression of occludin, ZO-1, and E-cadherin was downregulated in HPMCs under HG and IGF-1R overexpression conditions. Conversely, the expression of IGF-1R, α-SMA, and vimentin was downregulated, while the expression of occludin, ZO-1, and E-cadherin was upregulated in IGF-1R-underexpressed HPMCs under HG conditions. The cell migration abilities were increased, while the cell adhesion abilities were reduced in HPMCs under HG and IGF-1R overexpression conditions. In contrast, cell migration abilities were reduced, while cell adhesion abilities were increased in IGF-1Runderexpressed HPMCs under HG conditions. CONCLUSIONS: Targeting at IGF-1R may provide novel insights into the prevention and treatment of PF.

Human Amnion-Derived Mesenchymal Stromal Cells: A New Potential Treatment for Carbapenem-Resistant Enterobacterales in Decompensated Cirrhosis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. METHODS: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. RESULTS: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. CONCLUSION: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.

Macrophages regulates the transition of pericyte to peritoneal fibrosis through the GSDMD/IL-1ß axis.

BACKGROUND: End stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. METHODS: Histological examination was conducted using Hematoxylin and eosin (HE) staining and Masson's trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1ß concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). RESULTS: Mice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1ß production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD-/- alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD-/- attenuated interleukin-1beta (IL-1ß) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. CONCLUSION: These results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1ß axis, which provides a new therapeutic target.

Blocking core fucosylation of epidermal growth factor (EGF) receptor prevents peritoneal fibrosis progression.

OBJECTIVE: Peritoneal fibrosis (PF) ultimately causes ultrafiltration failure and peritoneal dialysis (PD) termination, but there are few effective therapies for it. Core fucosylation, which is catalyzed by α1,6-fucosyltransferase (Fut8) in mammals, may play a crucial role in PF development. This study aims to assess the effects of inhibiting core fucosylation of epidermal growth factor (EGF) receptor on PF rats. METHODS: PF rats (established by 4.25% glucose dialysate) were treated with either an adenovirus-Fut8 short hairpin RNA (Fut8shRNA) or adenovirus-control. Masson's staining and net ultrafiltration were performed at week six. Fut8 level and core fucosylation of EGF receptor and collagen I in the peritoneal membrane were assessed, and EGF signaling was detected, including signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) and their phosphorylation. Monocyte chemoattractant protein-1 (MCP-1) in peritoneal effluent was examined. RESULTS: Fut8 was upregulated in PF rats but decreased after Fut8shRNA treatment. EGF and EGF receptor expression was upregulated in PF rats, while core fucosylation of EGF receptor decreased after Fut8shRNA treatment. Masson's staining results showed an increase in peritoneal thickness in PF rats but a decrease after Fut8shRNA treatment. Fut8shRNA treatment increased net ultrafiltration, reduced the expression of collagen I and MCP-1 compared to PF rats. Fut8shRNA treatment suppressed phosphorylation of STAT3 and NF-κB in the peritoneal membrane of PF rats. CONCLUSIONS: Fut8shRNA treatment ameliorated the fibrotic changes in PF rats. A potential mechanism may be that Fut8shRNA treatment inactivated EGF signaling pathway by suppressing the phosphorylation of STAT3 and NF-κB.