High glucose contributes to the polarization of peritoneal macrophages to the M2 phenotype in vivo and in vitro.

Glucose is the primary osmotic medium used in most peritoneal dialysis (PD) solutions, and long­term exposure to high glucose is a major contributor to peritoneal fibrosis. Our previous study revealed that M2 macrophages participate in the development of PD­related fibrosis in a rat model. In the present study, the effects of high glucose on peritoneal macrophage polarization in vivo and in vitro were further evaluated. Continuous ambulatory PD (CAPD) patients with an overnight dwell of 1.5 or 2.5% glucose dialysate were recruited for this study. Overnight effluent samples from patients with CAPD (2,000 ml) were centrifuged to collect cells from the peritoneal cavity. J774A.1 cells (murine macrophages from ascites) were cultured in different concentrations of glucose. Macrophage phenotype markers were detected by flow cytometry. The levels of cytokines in PD effluent and the supernatant of murine macrophages were detected by enzyme­linked immunosorbent assays. The activity of arginase was determined by quantitative colorimetric analysis. In total, 107 CAPD subjects (92 patients using 1.5% glucose dialysate and 15 patients using 2.5% glucose dialysate) were recruited. The percentage of M1 macrophages (CD14­ and CCr7­positive cells) in the 1.5 and 2.5% glucose dialysate groups was 23.0±13.3 and 24.9±12.0%, respectively. The difference was not significant (P>0.05). The percentage of M2 macrophages (CD14­ and CD206­positive cells) in the 1.5% glucose dialysate group (36.2±11.4%) was significantly decreased compared to the 2.5% glucose dialysate group (43.2±7.4%) (P<0.05). Murine macrophages were cultured in a high­glucose in vitro environment, and the percentage of M1 macrophages in 138.8 mmol/l glucose medium significantly increased over time. The percentage of M2 macrophages increased in a glucose concentration­dependent and time­dependent manner. Arginase 1 in murine macrophages and the level of transforming growth factor ß1 in the supernatant increased in a glucose concentration­dependent manner. In conclusion, high glucose contributed to the polarization of peritoneal macrophages to the M2 phenotype, which may play an important role in the pathogenesis of PD­related fibrosis.

Parthenolide, an NF-κB inhibitor, alleviates peritoneal fibrosis by suppressing the TGF-ß/Smad pathway.

Transforming growth factor (TGF)-ß/Smad signalling plays a central role in the pathogenesis of peritoneal fibrosis related to peritoneal dialysis (PD). Parthenolide (PTL), a naturally occurring phytochemical, is isolated from the shoots of feverfew (Tanacetum parthenium) and displays analgesia, anti-inflammation and anticancer activities. In this study, we examined the therapeutic potential of PTL on PD-related peritoneal fibrosis induced by daily intraperitoneal injection of 4.25% dextrose-containing PD fluid (PDF) in vivo and TGF-ß1-induced epithelial-mesenchymal transition (EMT) in vitro. PTL was administered daily before PDF injection or after 14 days of PDF injection. Both PTL treatments showed a protective effect on peritoneal fibrosis and prevented peritoneal dysfunction. Similarly, PTL suppressed the expression of fibrotic markers (fibronectin and collagen I) and restored the expression of the epithelial marker (E-cadherin) in TGF-ß1-treated HMrSV5 cells. Furthermore, PTL inhibited TGF-ß1-induced Smad2 and Smad3 phosphorylation and nuclear translocation but did not influence Smad1/5/9 phosphorylation or activate other downstream signalling pathways of TGF-ß1, including AKT, extracellular signal-regulated kinase (ERK) or p38. In conclusion, PTL treatment may represent an effective and novel therapy for PD-associated peritoneal fibrosis by suppressing the TGF-ß/Smad pathway.

Adoptive transfer of DMSO-induced regulatory T cells exhibits a similar preventive effect compared to an in vivo DMSO treatment for chemical-induced experimental encapsulating peritoneal sclerosis in mice.

Encapsulating peritoneal sclerosis (EPS) is a severe complication of peritoneal dialysis (PD). This disease leads to intestinal obstruction with or without peritonitis. The imbalance between the populations of Th17 and regulatory T (Treg) cells (higher Th17 cells and lower Treg cells) is part of the pathogenesis of EPS formation. We demonstrated that dimethyl sulfoxide (DMSO) effectively inhibited autoimmune diabetes recurrence in the islet transplantation of NOD mice via the induction of the differentiation of Treg cells. In this study, we investigated the therapeutic potential of DMSO in the inhibition of EPS formation by a mouse model. Under DMSO treatment, the thickening of the parietal and visceral peritoneum was significantly reduced. The populations of CD4, CD8, and IFN-γ-producing CD4 and CD8 T cells were decreased. The populations of IL-4-producing CD4 T lymphocytes, IL-10-producing CD4 T lymphocytes, CD4 CD69 T lymphocytes and Treg lymphocytes were increased. The expression levels of the cytokines IFN-γ, IL-17a, TNF-α and IL-23, in ascites, were significantly decreased following the DMSO treatment. Furthermore, the differentiation of Treg cells was induced by DMSO from naïve CD4 T cells in vitro, and these cells were adoptively transferred into the EPS mice and significantly prevented EPS formation, exhibiting a comparable effect to the in vivo DMSO treatment. We also demonstrated that the differentiation of Treg cells by DMSO occurred via the activation of STAT5 by its epigenetic effect, without altering the PI3K-AKT-mTOR or Raf-ERK pathways. Our results demonstrated, for the first time, that in vivo DMSO treatment suppresses EPS formation in a mouse model. Furthermore, the adoptive transfer of Treg cells that were differentiated from naïve CD4 T cells by an in vitro DMSO treatment exhibited a similar effect to the in vivo DMSO treatment for the prevention of EPS formation.

Anti-C5a complementary peptide mitigates zymosan-induced severe peritonitis with fibrotic encapsulation in rats pretreated with methylglyoxal.

In a previous study of fungal peritoneal injury in peritoneal dialysis patients, complement (C)-dependent pathological changes were developed in zymosan (Zy)-induced peritonitis by peritoneal scraping. However, the injuries were limited to the parietal peritoneum and did not show any fibrous encapsulation of the visceral peritoneum, which differs from human encapsular peritoneal sclerosis (EPS). We investigated peritoneal injury in a rat model of Zy-induced peritonitis pretreated with methylglyoxal (MGO) instead of scraping (Zy/MGO peritonitis) to clarify the role of C in the process of fibrous encapsulation of the visceral peritoneum. Therapeutic effects of an anti-C5a complementary peptide, AcPepA, on peritonitis were also studied. In Zy/MGO peritonitis, peritoneal thickness, fibrin exudation, accumulation of inflammatory cells, and deposition of C3b and C5b-9 with loss of membrane C regulators were increased along the peritoneum until day 5. On day 14, fibrous encapsulation of the visceral peritoneum was observed, resembling human EPS. Peritoneal injuries and fibrous changes were significantly improved with AcPepA treatment, even when AcPepA was administered following injection of Zy in Zy/MGO peritonitis. The data show that C5a might play a role in the development of encapsulation-like changes in the visceral peritoneum in Zy/MGO peritonitis. AcPepA might have therapeutic effects in fungal infection-induced peritoneal injury by preventing subsequent development of peritoneal encapsulation.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

The peritoneum: healing, immunity, and diseases.

The peritoneum defines a confined microenvironment, which is stable under normal conditions, but is exposed to the damaging effect of infections, surgical injuries, and other neoplastic and non-neoplastic events. Its response to damage includes the recruitment, proliferation, and activation of a variety of haematopoietic and stromal cells. In physiological conditions, effective responses to injuries are organized; inflammatory triggers are eliminated; inflammation quickly abates; and the normal tissue architecture is restored. However, if inflammatory triggers are not cleared, fibrosis or scarring occurs and impaired tissue function ultimately leads to organ failure. Autoimmune serositis is characterized by the persistence of self-antigens and a relapsing clinical pattern. Peritoneal carcinomatosis and endometriosis are characterized by the persistence of cancer cells or ectopic endometrial cells in the peritoneal cavity. Some of the molecular signals orchestrating the recruitment of inflammatory cells in the peritoneum have been identified in the last few years. Alternative activation of peritoneal macrophages was shown to guide angiogenesis and fibrosis, and could represent a novel target for molecular intervention. This review summarizes current knowledge of the alterations to the immune response in the peritoneal environment, highlighting the ambiguous role played by persistently activated reparative macrophages in the pathogenesis of common human diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immune-Regulatory Molecule CD69 Controls Peritoneal Fibrosis.

Patients with ESRD undergoing peritoneal dialysis develop progressive peritoneal fibrosis, which may lead to technique failure. Recent data point to Th17-mediated inflammation as a key contributor in peritoneal damage. The leukocyte antigen CD69 modulates the setting and progression of autoimmune and inflammatory diseases by controlling the balance between Th17 and regulatory T cells (Tregs). However, the relevance of CD69 in tissue fibrosis remains largely unknown. Thus, we explored the role of CD69 in fibroproliferative responses using a mouse model of peritoneal fibrosis induced by dialysis fluid exposure under either normal or uremic status. We found that cd69-/- mice compared with wild-type (WT) mice showed enhanced fibrosis, mesothelial to mesenchymal transition, IL-17 production, and Th17 cell infiltration in response to dialysis fluid treatment. Uremia contributed partially to peritoneal inflammatory and fibrotic responses. Additionally, antibody-mediated CD69 blockade in WT mice mimicked the fibrotic response of cd69-/- mice. Finally, IL-17 blockade in cd69-/- mice decreased peritoneal fibrosis to the WT levels, and mixed bone marrow from cd69-/- and Rag2-/-γc-/- mice transplanted into WT mice reproduced the severity of the response to dialysis fluid observed in cd69-/- mice, showing that CD69 exerts its regulatory function within the lymphocyte compartment. Overall, our results indicate that CD69 controls tissue fibrosis by regulating Th17-mediated inflammation.

Peritoneal Dialysis Catheter Increases Leukocyte Recruitment in the Mouse Parietal Peritoneum Microcirculation and Causes Fibrosis.

UNLABELLED: ♦ BACKGROUND: The objective of this study was to examine the effects of a conventional dialysis solution and peritoneal catheter on leukocyte-endothelial cell interactions in the microcirculation of the parietal peritoneum in a subacute peritoneal dialysis (PD) mouse model. ♦ METHODS: An intraperitoneal (IP) catheter with a subcutaneous injection port was implanted into mice and, after a 2-week healing period, the animals were injected daily for 6 weeks with a 2.5% dextrose solution. Intravital microscopy (IVM) of the parietal peritoneum microcirculation was performed 4 hours after the last injection of the dialysis solution. Leukocyte-endothelial cell interactions were quantified and compared with catheterized controls without dialysis treatment and naïve mice. ♦ RESULTS: The number of rolling and extravascular leukocytes along with peritoneal fibrosis and neovascularization were significantly increased in the catheterized animals compared with naïve mice but did not significantly differ between the 2 groups of catheterized animals with sham injections or dialysis solution treatment. ♦ CONCLUSION: The peritoneal catheter implant increased leukocyte rolling and extravasation, peritoneal fibrosis and vascularization in the parietal peritoneum independently from the dialysis solution treatment.

New developments in peritoneal fibroblast biology: implications for inflammation and fibrosis in peritoneal dialysis.

Uraemia and long-term peritoneal dialysis (PD) can lead to fibrotic thickening of the peritoneal membrane, which may limit its dialytic function. Peritoneal fibrosis is associated with the appearance of myofibroblasts and expansion of extracellular matrix. The extent of contribution of resident peritoneal fibroblasts to these changes is a matter of debate. Recent studies point to a significant heterogeneity and complexity of the peritoneal fibroblast population. Here, we review recent developments in peritoneal fibroblast biology and summarize the current knowledge on the involvement of peritoneal fibroblasts in peritoneal inflammation and fibrosis.

Encapsulating peritoneal sclerosis is associated with T-cell activation.

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is an excessive fibrotic response of the peritoneum that may occur after long-term peritoneal dialysis (PD). The underlying pathophysiology is poorly understood, but involvement of peritoneal inflammatory T helper 1 cells may be pivotal. METHODS: Soluble interleukin-2 receptor alpha (sCD25) concentration was measured as a marker for T-cell activation in serum and ascites from EPS patients and various control patient groups. Peritoneal biopsies were stained for the presence of T cells, and T cells isolated from ascites of EPS patients were characterized in detail for differentiation status and cytokine expression. RESULTS: Serum sCD25 concentrations are significantly and specifically increased in EPS patients compared with haemodialysis, PD and predialysis patients. Peritoneal effluent of stable PD patients contains very low levels of sCD25, while sCD25 levels in ascites of EPS patients are high and indicative of local production. In the years preceding the diagnosis of EPS, the serum sCD25 concentrations increased while remaining at stable levels in control PD patients. The peritoneum and ascites of EPS patients showed a significant influx of T cells with relatively increased numbers of CD4(+) T cells. These T cells were fully differentiated and displayed a T helper 1 cell type with a pro-inflammatory cytokine profile. CONCLUSIONS: Increased serum sCD25 concentrations and peritoneal lymphocytosis in EPS patients indicate the involvement of activated T cells in the pathophysiology of excessive fibrosis.

Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production.

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4+ and CD8+ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8+ T cells showing a regulatory phenotype. The frequency of CD4+ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4+ and CD8+ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice.

The aldosterone receptor antagonist spironolactone prevents peritoneal inflammation and fibrosis.

Peritoneal fibrosis is a complication of patients with long-term continuous ambulatory peritoneal dialysis (CAPD). Reports have indicated that angiotensin (Ang) II may correlate with the development of peritoneal fibrosis. However, it is unknown whether aldosterone also has a role in the development of peritoneal inflammation and fibrosis. The aim of the present study was to clarify the role of aldosterone in peritoneal inflammation and fibrosis. A rat model of peritoneal inflammation and fibrosis was established by daily intraperitoneal injection of dialysates and lipopolysaccharide in a 4-day interval over a period of 7 days. The animals were randomly assigned to five groups as follows: control (C); peritoneal dialysis (PD); peritoneal dialysis-spironolactone (PD-S); peritoneal dialysis-cilazapril (PD-C); and peritoneal dialysis-spironolactone-cilazapril (PD-SC). After 30 days, the TGF-ß1 concentration in dialysates from all treatment groups was determined by ELISA. The histopathology of the parietal peritoneum was examined, and the expression of MCP-1, c-Jun, fibronectin (FN) and TGF-ß1 in the abdominal membrane was determined by immunohistochemistry. Mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) and CYP11B2 (aldosterone synthase) were analyzed by real time-PCR. Collagen deposition was significantly higher in PD compared with the other groups. The expression of MR, 11ß-HSD2 and CYP11B2 was significantly higher in PD compared with the other groups. Spironolactone and/or cilazapril treatment partially ablated the increase in monocyte chemoattractant protein (MCP)-1, p-c-Jun, transforming growth factor (TGF)-ß1, FN, MR, 11ß-HSD2 and CYP11B2. Furthermore, treatment with spironolactone and/or cilazapril also reduced the infiltration of CD-4- and ED-1-positive cells in rat peritoneal tissues after peritoneal fibrosis. Exogenous aldosterone may have a key role in the development of peritoneal inflammation and fibrosis. Spironolactone decreased peritoneal inflammation and fibrosis, which was associated with reduced secretion from peritoneal macrophages, inactivation of the c-Jun N-terminal kinase (JNK) pathway and subsequent downregulation of the expression of TGF-ß1.

Arginase activity is associated with fibrosis in experimental infection with Taenia crassiceps, but does not play a major role in resistance to infection.

Murine infection with Taenia crassiceps cysticerci is used as an experimental model for human and animal cysticercosis. In this infection parasites can be found associated with an inflammatory infiltrate enriched with macrophages. Experimental evidence exists supporting a role for either NO-producing classically activated (CAMΦ) or arginase- and CD301-expressing alternatively activated macrophages (AAMΦ) in T. crassiceps resistance. In both cell types, arginine is utilized as an important mediator in macrophage effector functions. To investigate whether there is an association between arginine availability, susceptibility to T. crassiceps and other parameters such as fibrosis, BALB/c mice were infected intraperitoneally with cysticerci and treated daily with the arginase inhibitor nor-NOHA or supplemented with l-arginine and followed for eight weeks. The numbers and developmental stages of parasites were evaluated as well as the presence of CD301+ AAMΦ, arginase activity and collagen deposition in the peritoneal membrane. Treatment with the arginase inhibitor or supplementation with l-arginine did not change the parasitic load or profile of the infection. However, the arginase inhibitor significantly decreased the deposition of collagen. These results suggest that arginase activity does not interfere with parasite control during experimental infection with T. crassiceps, but it is important for fibrosis in cysticercosis.

Inflammation, neoangiogenesis and fibrosis in peritoneal dialysis.

Peritoneal dialysis (PD) is a form of renal replacement therapy used in patients with end stage renal disease (ESRD). It is based on using the peritoneum as a semipermeable membrane through which ultrafiltration (UF) and diffusion occur. Despite several benefits, PD has long-term complications, including inflammation, neoangiogenesis and fibrosis. Several inflammatory molecules can be found in the dialysate of PD patients including: interleukins (IL), tumor necrosis factor α (TNF-α) and C-reactive protein (CRP). Angiogenesis results in increased effective surface area exchange. Consequently, the glucose-driven osmotic pressure of the peritoneal dialysis fluid (PDF) is significantly reduced leading to UF failure (UFF). Several factors are implicated in the development of peritoneal fibrosis (PF) in PD patients. The most important factor is the conventional bio-incompatible PD solution, which contains high concentration of glucose and glucose degradation products (GDP). Although there are several studies elucidating the mechanisms leading to UFF in PD patients, more studies needed to be developed in this area and more research is required to find mechanisms to delay or to minimize the occurrence of many deleterious changes in peritoneal membrane (PM) during PD.

Involvement of lymphocyte infiltration in the progression of mouse peritoneal fibrosis model.

Peritoneal fibrosis is a serious complication in patients with severe chronic kidney disease who are undergoing peritoneal dialysis (PD). One of the pathological characteristics of peritoneal fibrosis is the infiltration of macrophages in the thickened submesothelial compact zone. In addition, infiltration of lymphocytes, including T and B lymphocytes, is observed in the fibrotic peritoneum. However, the relationship between lymphocyte infiltration and progression of peritoneal fibrosis remains unclear. In this study, we investigated the role of lymphocytes in the development of peritoneal fibrosis induced by chlorhexidine gluconate (CG) by comparing the histological changes observed in severe combined immunodeficient (SCID) mice (largely lacking functional T and B lymphocytes) with those observed in wild-type (WT) mice. As expected, CG-injected WT mice showed a thickening of the submesothelial compact zone together with massive collagen deposition accompanied by increased numbers of infiltrating macrophages and T and B lymphocytes. In the peritoneum of SCID mice, the submesothelial compact zone was thicker and the number of macrophages and B lymphocytes was significantly higher than that observed in control immunodeficient and WT mice. In contrast, the number of T lymphocytes in the peritoneum of SCID mice was significantly lower than that in the peritoneum of WT mice. These results suggest that T and B lymphocytes modulate the process of peritoneal fibrosis via macrophage infiltration.

Post-transplantation encapsulating peritoneal sclerosis in a pediatric patient.

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is a serious complication of long-term peritoneal dialysis (PD), but only a few cases have been described in the pediatric patient population. There is no established medical treatment, and surgery has been reported with variable success. The number of reports of EPS being successfully treated with tamoxifen, based on its anti-fibrotic effects, are increasing. The role of sirolimus, an mTOR inhibitor with immunomodulatory and anti-proliferative properties, has been less well-defined. CASE-DIAGNOSIS/TREATMENT: A 17-year-old kidney transplant recipient, with a previous cumulative time on PD of 8 years and 3 months, developed severe bowel obstruction 8 months after undergoing a second kidney graft. Her immunosuppressive regimen consisted of tacrolimus, mycophenolate mofetil, and prednisolone. The patient underwent laparotomy, which revealed multiple thick leathery adhesions with an encapsulated small bowel. Enterolysis was performed, and total parenteral nutrition was commenced after surgery to provide an adequate food intake. Treatment with tamoxifen was initiated, but the patient developed significant liver toxicity 2 weeks later, and the drug was withdrawn. The immunosuppressive regimen was changed to an increased dose of prednisolone, and tacrolimus was replaced with sirolimos. At 20 months of follow-up, the patient remains symptom-free, with a functioning kidney transplant. CONCLUSION: Although EPS is a very rare condition in the pediatric population, it should be considered when a child or adolescent with a long-term history of PD presents with nonspecific gastrointestinal symptoms or with signs of bowel obstruction. There is an urgent need for alternative immunosuppressive protocols. The use of sirolimus in this group of patients remains controversial.