Luiz S. Aroeira

Epithelial to Mesenchymal Transition and Peritoneal Membrane Failure in Peritoneal Dialysis Patients: Pathologic Significance and Potential Therapeutic Interventions

Peritoneal dialysis (PD) is a form of renal replacement and is based on the use of the peritoneum as a semipermeable membrane across which ultrafiltration and diffusion take place. Nevertheless, continuous exposure to bioincompatible PD solutions and episodes of peritonitis or hemoperitoneum cause acute and chronic inflammation and injury to the peritoneal membrane, which progressively undergoes fibrosis and angiogenesis and, ultimately, ultrafiltration failure. The pathophysiologic mechanisms that are involved in peritoneal functional impairment have remained elusive. Resident fibroblasts and infiltrating inflammatory cells have been considered the main entities that are responsible for structural and functional alterations of the peritoneum. Recent findings, however, demonstrated that new fibroblastic cells may arise from local conversion of mesothelial cells (MC) by epithelial-to-mesenchymal transition (EMT) during the inflammatory and repair responses that are induced by PD and pointed to MC as protagonists of peritoneal membrane deterioration. Submesothelial myofibroblasts, which participate in inflammatory responses, extracellular matrix accumulation, and angiogenesis, can originate from activated resident fibroblasts and from MC through EMT. This heterogeneous origin of myofibroblasts reveals new pathogenic mechanisms and offers novel therapeutic possibilities. This article provides a comprehensive review of recent advances on understanding the mechanisms that are implicated in peritoneal structural alterations, which have allowed the identification of the EMT of MC as a potential therapeutic target of membrane failure.

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, α-smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and α-SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.

Epithelial-to-mesenchymal transition of mesothelial cells is an early event during peritoneal dialysis and is associated with high peritoneal transport.

Ultrafiltration (UF) failure is a consequence of long-term peritoneal dialysis (PD). Fibrosis, angiogenesis, and vasculopathy are causes of this functional disorder after 3-8 years on PD. Epithelial-to-mesenchymal transition (EMT) of mesothelial cell (MC) is a key process leading to peritoneal fibrosis with functional deterioration. Our purpose was to study the peritoneal anatomical changes during the first months on PD, and to correlate them with peritoneal functional parameters. We studied 35 stable PD patients for up to 2 years on PD, with a mean age of 45.3+/-14.5 years. Seventy-four percent of patients presented loss of the mesothelial layer, 46% fibrosis (>150 microm) and 17% in situ evidence of EMT (submesothelial cytokeratin staining), which increased over time. All patients with EMT showed myofibroblasts, while only 36% of patients without EMT had myofibroblasts. The number of peritoneal vessels did not vary when we compared different times on PD. Vasculopathy was present in 17% of the samples. Functional studies were used to define the peritoneal transport status. Patients in the highest quartile of mass transfer area coefficient of creatinine (Cr-MTAC) (>11.8 ml min(-1)) showed significantly higher EMT prevalence (P=0.016) but similar number of peritoneal vessels. In the multivariate analysis, the highest quartile of Cr-MTAC remained as an independent factor predicting the presence of EMT (odds ratio 12.4; confidence interval: 1.6-92; P=0.013) after adjusting for fibrosis (P=0.018). We concluded that, during the first 2 PD years, EMT of MCs is a frequent morphological change in the peritoneal membrane. High solute transport status is associated with its presence but not with increased number of peritoneal vessels.

CTGF Promotes Inflammatory Cell Infiltration of the Renal Interstitium by Activating NF-κB

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-kappaB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-kappaB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-gamma, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-kappaB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-kappaB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-kappaB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-kappaB pathway.

BMP-7 blocks mesenchymal conversion of mesothelial cells and prevents peritoneal damage induced by dialysis fluid exposure

BACKGROUND During peritoneal dialysis (PD), mesothelial cells (MC) undergo an epithelial-to-mesenchymal transition (EMT), and this process is associated with peritoneal membrane (PM) damage. Bone morphogenic protein-7 (BMP-7) antagonizes transforming growth factor (TGF)-beta1, modulates EMT and protects against fibrosis. Herein, we analysed the modulating role of BMP-7 on EMT of MC in vitro and its protective effects in a rat PD model. METHODS Epitheliod or non-epitheliod MC were analysed for the expression of BMP-7, TGF-beta1, activated Smads, epithelial cadherin (E-cadherin), collagen I, alpha smooth muscle cell actin (alpha-SMA) and vascular endothelial growth factor (VEGF) using standard procedures. Rats were daily instilled with PD fluid with or without BMP-7 during 5 weeks. Histological analyses were carried out in parietal peritoneum. Fibrosis was quantified with van Gieson or Massons trichrome staining. Vasculature, activated macrophages and invading MC were quantified by immunofluorescence analysis. Quantification of infiltrating leukocytes and MC density in liver imprints was performed by May-Grünwald-Giemsa staining. Hyaluronic acid levels were determined by ELISA. RESULTS MC constitutively expressed BMP-7, and its expression was downregulated during EMT. Treatment with recombinant BMP-7 resulted in blockade of TGF-beta1-induced EMT of MC. We provide evidence of a Smad-dependent mechanism for the blockade of EMT. Exposure of rat peritoneum to PD fluid resulted in inflammatory and regenerative responses, invasion of the compact zone by MC, fibrosis and angiogenesis. Administration of BMP-7 decreased the number of invading MC and reduced fibrosis and angiogenesis. In contrast, BMP-7 had no effect on inflammatory and regenerative responses, suggesting that these are EMT-independent, and probably upstream, processes. CONCLUSIONS Data point to a balance between BMP-7 and TGF-beta1 in the control of EMT and indicate that blockade of EMT may be a therapeutic approach to ameliorate peritoneal membrane damage during PD.

Alternative activation of macrophages in human peritoneum: implications for peritoneal fibrosis

BACKGROUND Depending on the cytokine microenvironment, macrophages (Mϕ) can adopt a proinflammatory (M1) or a profibrotic (M2) phenotype characterized by the expression of cell surface proteins such as CD206 and CD163 and soluble factors such as CC chemokine ligand 18 (CCL18). A key role for Mϕ in fibrosis has been observed in diverse organ settings. We studied the Mϕ population in a human model of peritoneal dialysis in which continuous stress due to dialysis fluids and recurrent peritonitis represent a risk for peritoneal membrane dysfunction reflected as ultrafiltration failure (UFF) and peritoneal fibrosis. METHODS We used flow cytometry and quantitative reverse transcription-polymerase chain reaction to analyse the phenotype of peritoneal effluent Mϕ and tested their ability to stimulate the proliferation of human fibroblasts. Mϕ from non-infected patients were compared with those from patients with active peritonitis. Cytokine production was evaluated by enzyme-linked immunosorbent assay (ELISA) in spent dialysates and cell culture supernatants. RESULTS CD206(+) and CD163(+) M2 were found within peritoneal effluents by flow cytometry analysis, with increased frequencies of CD163(+) cells during peritonitis (P = 0.003). TGFB1, MMP9 and CCL18 messenger RNA (mRNA) levels in peritoneal macrophages (pMϕ) were similar to those found in M2 cells differentiated in vitro. The ability of pMϕ to stimulate fibroblast proliferation correlated with CCL18 mRNA levels (r = 0.924, P = 0.016). CCL18 production by pMϕ was confirmed by immunostaining of cytospin samples and ELISA. Moreover, CCL18 effluent concentrations correlated with decreased peritoneal function, which was evaluated as dialysate to plasma ratio of creatinine (r = 0.724, P < 0.0001), and were significantly higher in patients with UFF (P = 0.0025) and in those who later developed sclerosing peritonitis (P = 0.024). CONCLUSIONS M2 may participate in human peritoneal fibrosis through the stimulation of fibroblast cell growth and CCL18 production as high concentrations of CCL18 are associated with functional deficiency and fibrosis of the peritoneal membrane.

Cyclooxygenase-2 Mediates Dialysate-Induced Alterations of the Peritoneal Membrane

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.

A Nanoconjugate Apaf-1 Inhibitor Protects Mesothelial Cells from Cytokine-Induced Injury

BACKGROUND Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization. METHODOLOGY Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice. CONCLUSION Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

IL-17A is a novel player in dialysis-induced peritoneal damage

The classical view of the immune system has changed by the discovery of novel T-helper (Th) subsets, including Th17 (IL-17A-producing cells). IL-17A participates in immune-mediated glomerulonephritis and more recently in inflammatory pathologies, including experimental renal injury. Peritoneal dialysis patients present chronic inflammation and Th1/Th2 imbalance, but the role of the Th17 response in peritoneal membrane damage has not been investigated. In peritoneal biopsies from dialyzed patients, IL-17A immunostaining was found mainly in inflammatory areas and was absent in the healthy peritoneum. IL-17A-expressing cells included lymphocytes (CD4+ and γδ), neutrophils, and mast cells. Elevated IL-17A effluent concentrations were found in long-term peritoneal dialysis patients. Studies in mice showed that repeated exposure to recombinant IL-17A caused peritoneal inflammation and fibrosis. Moreover, chronic exposure to dialysis fluids resulted in a peritoneal Th17 response, including elevated IL-17A gene and protein production, submesothelial cell infiltration of IL-17A-expressing cells, and upregulation of Th17 differentiation factors and cytokines. IL-17A neutralization diminished experimental peritoneal inflammation and fibrosis caused by chronic exposure to dialysis fluids in mice. Thus, IL-17A is a key player of peritoneum damage and it may be a good candidate for therapeutic intervention in peritoneal dialysis patients.

Influence of Bicarbonate/Low-GDP Peritoneal Dialysis Fluid (Bicavera) on In Vitro and Ex Vivo Epithelial-to-Mesenchymal Transition of Mesothelial Cells

♦ Background: Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions. ♦ Objectives: This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo. ♦ Methods: In vitro studies: Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium. Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs. ♦ Results: Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters. Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor β1. Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity. ♦ Conclusions: Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.