Angiogenesis and Lymphangiogenesis in Peritoneal Dialysis

The ultrafiltration failure during peritoneal dialysis (PD) is related to inflammatory responses induced by bio-incompatible PD fluids, which may lead to deterioration of peritoneal membrane (PM) function. Mesothelial cells, lymphocytes, macrophages and other cell types present in the peritoneal cavity are stimulated to produce cytokines and growth factors that promote pathological processes. Due to these factors, blood and lymphatic vessels proliferate and could be responsible for hyperfiltration and PM failure type III and IV. Vessels proliferation may be related to fibrosis, being the cause and/or effect of the mesenchymal conversion of different cell types such as mesothelial (MMT), bone marrow-derived (fibrocytes) or endothelial (vascular- and lymph-endo-MT) cells. Lymphangiogenesis in PD is a poorly analysed process; however, its contribution to peritoneal function disorders has been recently recognized. VEGF production is associated with blood and lymphatic vessels proliferation, while specifically lymphangiogenesis is mainly regulated by VEGF-C and VEGF-D. Excessive lymphatic fluid drainage from the abdominal cavity may be related with macromolecule and isosmotic solutions reuptake and convective reabsorption of solutes that were cleared from plasma by diffusion. Some drugs have been shown to modulate tissue fibrosis, MMT, EndoMT, angiogenesis and lymphangiogenesis and could represent interesting therapeutic strategies to protect the PM

Pharmacological Preservation of Peritoneal Membrane in Peritoneal Dialysis

Peritoneal dialysis (PD) is an established renal replacement therapy for renal disease. It is based on the capacity of the peritoneum to act as a semipermeable membrane for the exchange of toxic solutes and water, which is called ultrafiltration capacity. Peritoneal membrane (PM) is lined by a monolayer of mesothelial cells (MCs), which lay on an extracellular matrix bed where other cell types and blood and lymphatic vessels can be found. Long-term exposure to hyperosmotic PD fluids (PDFs), peritonitis or hemoperitoneum causes peritoneal injury by the generation of an inflammatory state. Inflammatory cells and their mediators initiate a cascade of reactions promoting alterations in peritoneal cells, loss of MCs, fibrosis, vasculopathy, and angiogenesis, leading to ultrafiltration failure. Recent studies support that the so-called “mesothelial to mesenchymal transition” process of the MCs runs parallel to the anatomical and functional ridging of PM, which suggests that its inhibition might slow down or stop the PM damage. The fight against PM damage begins with the improvement in PDF biocompatibility. Complementary to this, an alternative approach to preserve the PM might be the use of pharmacological agents or molecular strategies. Here, we explain the existing research models for the development of new therapies and analyze several therapeutic options tested with them

T Helper 17/Regulatory T Cell Balance and Experimental Models of Peritoneal Dialysis-Induced Damage

Fibrosis is a general complication in many diseases. It is the main complication during peritoneal dialysis (PD) treatment, a therapy for renal failure disease. Local inflammation and mesothelial to mesenchymal transition (MMT) are well known key phenomena in peritoneal damage during PD. New data suggest that, in the peritoneal cavity, inflammatory changes may be regulated at least in part by a delicate balance between T helper 17 and regulatory T cells. This paper briefly reviews the implication of the Th17/Treg-axis in fibrotic diseases. Moreover, it compares current evidences described in PD animal experimental models, indicating a loss of Th17/Treg balance (Th17 predominance) leading to peritoneal damage during PD. In addition, considering the new clinical and animal experimental data, new therapeutic strategies to reduce the Th17 response and increase the regulatory T response are proposed. Thus, future goals should be to develop new clinical biomarkers to reverse this immune misbalance and reduce peritoneal fibrosis in PD.This work was supported in part by grants from Ministerio de Economia y competitividad SAF2010-21249 to Manuel López-Cabrera, Comunidad Autónoma de Madrid 2010-BMD2321 (FIBROTEAM) to Manuel Lopez Cabrera, and Fondo de Investigaciones Santitarias RETICS 06/0016 and PI 09/0064 to Rafael Selgas and FIS 12/01175 to Abelardo Aguilera Peralta. Georgios Liappas is fully supported from European Union, Seventh Framework Program “EuTRiPD,” under Grant Agreement PITN-GA-2011-287813. The authors would like to thank Juliette Siegfried and her team at ServingMed.com for editing the language of the paper.Peer Reviewe

Tamoxifen preserves the fibrinolytic capacity of TGF-β1-treated MCs.

<p>Omentum-derived MCs were treated or not with 1 ng/mL TGF-β1 during 48 hours, in the presence of different doses of Tamoxifen (0, 3 and 6 µM). <b>(A to C)</b> Stimulation of MCs with TGF-β1 inhibits the expression of the fibrinolytic factors uPA <b>(A)</b>, uPAR <b>(B)</b> and tPA <b>(C)</b>, and treatments with different doses of Tamoxifen restore the basal levels of these factors or increase their synthesis above basal levels. <b>(D)</b>. TGF-β1 treatment increases the expression of PAI-1, and its expression is not affected by Tamoxifen. The levels of these factors were measured in culture media supernatants by ELISA and results are depicted as nanograms per milligrams of total cellular proteins <b>(E)</b>. The PAI/tPA-ratio, an important marker of fibrinolytic capacity decline, increases in response to TGF-β1 and returns to basal levels when Tamoxifen is added at 6 µM. Box plots show the 25th and 75th percentiles, median, minimum and maximum values of five independent experiments. The symbols represent the statistical differences between the groups.</p

Tamoxifen blocks TGF-β1-induced MMT of MCs.

<p>Omentum-derived MCs were treated or not with 1 ng/mL TGF-β1 for 24 or 48 hours, in the presence of different doses of Tamoxifen (0, 3 and 6 µM). (<b>A</b>) Western blot analyses show that Tamoxifen treatment prevents TGF-β1-induced E-cadherin down-regulation as well as α-SMA, collagen I, fibronectin and MMP-2 up-regulation. A representative experiment is shown. (<b>B to F</b>) The experiments were repeated at least five times and results are depicted as means ± SE. The expressions of E-cadherin (<b>B</b>) and MMP-2 (<b>F</b>) were analyzed at 24 hours, whereas the expressions of α-SMA (<b>C</b>), collagen I (<b>D</b>) and fibronectin (<b>E</b>), were analyzed at 48 hours of treatments. (<b>G</b>) Analysis of the migration capacity in transwell units demonstrates that Tamoxifen (6 µM) reduces the TGF-β1-indued migratory capacity of MCs to basal levels. The experiments, made in triplicates, were repeated at least four times. Box plots represent the median, minimum and maximum values, as well as the 25th and 75th percentiles.</p

Tamoxifen reverts the MMT induced by TGF-β1 <i>in vitro</i>.

<p>Omentum-MCs were stimulated with TGF-β1 during 48 hours and then the cells were either left untreated, treated with TGF-β1 or treated with Tamoxifen (6 or 10 µM) during additional 48 hours. <b>(A)</b> Phase-contrast microscopy shows that Tamoxifen reverts partially the non-epithelioid morphology of omentum-derived MC treated with TGF-β1. <b>(B)</b> Quantitative RT-PCR analysis demonstrates that administration of Tamoxifen (6 µM) after TGF-β1 withdrawal restores partially E-cadherin expression. Bar graphic depicts the expression of E-cadherin-encoding mRNA in relative units (R.U.)</p

Parallelism between MMT, PM thickness and time on PD.

<p><b>(A)</b> Immunofluorescence microscopy images of parietal peritoneal sections stained for cytokeratin (green) and FSP-1 (red), with DAPI counterstaining, show accumulation of trans-differentiated mesothelial cells in the submesothelial space at 7, 15 and 30 days of PD mice. Progressive time-dependent increases of MMT and PM thickness is observed during PD fluid exposure. Representative slides are presented. Magnification ×200. <b>(B)</b> Quantification of the submesothelial MMT (cytokeratin/FSP-1 double positive cells per field) at different time points. <b>(C)</b> Quantification of peritoneal thickness ( µm) at different time points. Box Plots represent 25% and 75% percentiles, median, minimum and maximum values. Numbers above boxes depict means ± SE. Symbols show statistical differences between groups. <b>(D)</b> Correlation between both MMT and peritoneal thickness was determined by Spearman regression analysis.</p

Tamoxifen reverts partially the mesenchymal phenotype of effluent-derived MCs.

<p>Effluent-derived MCs with mesenchymal phenotype (as determined by non-epitheliod morphology, low expression of E-cadherin and up-regulated expression of mesenchymal markers) were treated with different doses of Tamoxifen (0, 3, 6, and 10 µM) and analyzed at 48 hours. Omentum-derived MCs were employed as control. (<b>A</b>) Western blot analyses show that Tamoxifen treatments do not re-induce E-cadherin expression but inhibit the expression of the mesenchymal molecules α-SMA, collagen I, fibronectin and MMP-2; being the effects of Tamoxifen more evident at high doses (6 and 10 µM). A representative experiment is shown. (<b>B to F</b>) The experiments were repeated with five different samples of effluent-derived MCs and results of the expression of E-cadherin <b>(B)</b>, α-SMA <b>(C)</b>, collagen I <b>(D)</b>, MMP-2 <b>(E)</b> and fibronectin <b>(F)</b> are depicted as means ± SE. Quantitative RT-PCR demonstrates that the expression of Snail mRNA is not inhibited by any dose of Tamoxifen tested <b>(G)</b>. Bars depict means ± SE of five independent experiments.</p