INTRODUCTION

During PD, dialysis fluid (called dialysate) is infused into the abdominal cavity through a specific surgery-placed catheter for a well defined period of time. The peritoneum acts as a membrane to allow excess fluids and waste products to pass from the bloodstream into the dialysate. Therefore, peritoneal membrane of PD patients, continuously in contact with bio-incompatible solution undergoes to significant histological/anatomical changes resulting in the progressive loss of its functions and unfavorable outcome (Liu J-2014 [1], Yung S-2011 [2]).

To avoid this condition, in the last ten years, researchers worldwide have started research programs to develop new generation PD solutions that are characterized by neutral pH and bicarbonate/lactate buffer formulations. However, although alternative osmotic agents such as amino acids and macromolecular solutions, including polypeptides and polyglucose (icodextrin) solutions, are now available, glucose is, at the moment, the most widely used osmotic agent in PD.

However, although relatively safe, effective, readily metabolized, and inexpensive, there is no doubt that long-term exposure of peritoneal tissues and cells to unphysiologic concentrations of glucose can adversely affect peritoneal membrane and mesothelial cells integrity (Sitter T-2005 [3]). The latter are biochemical active cells, disposed in monolayer in peritoneum, not only prevent friction and adhesions forming between adjacent parietal and visceral surfaces by synthesizing and secreting lubricants including glycosaminoglycans and surfactant, but also they are involved in several biological activities including serosal repair, secretion of inflammatory mediators, chemokines, growth factors, and extracellular matrix (ECM) components (Mutsaers SE-2002 [4],Strippoli R-2016 [5]).

Although morphologically they resemble epithelial cells and possess many epithelial characteristics (e.g., surface microvilli, apical/basal polarity, cytokeratins, and junctional complexes), if exposed to unphysiological stimuli they can undertake a morphological and functional changes  consistent with an epithelial-to-mesenchymal transition which has recently been termed mesothelial-to-mesenchymal transition (MMT) (Sandoval P-2016 [6]).

The ability of mesothelial cells to undergo MMT suggests that the mesothelium is a likely source of fibrogenic cells during serosal inflammation and tissue repair and therefore play important roles in peritoneal fibrosis and adhesion formation. Omental fibrosis may then induces dramatic clinical consequences with lost of the ultrafiltrative/dialytic capacity of this natural membrane and, in few cases, development of a life-threatening scleroting endocapuslatin peritonitis (SEP) (López-Cabrera M-2014 [7]).

We suppose that in the epithelial-to-mesenchymal transition, as in other cell lines, Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate (HS) chains and thus participates in extracellular remodeling, could have a central role ant it could represent a possible valuable new target for future pharmacological interventions.

Therefore, to achieve this objective, in this in vitro study, we employed several well standardized biomolecular strategies to analyze the contribution of HPSE in the glucose-induced MMT process and to evaluate whether its specific inhibition, by a recently available agent (SST0001), could control this pathological effects over-activated by high glucose concentrations.

METHODS

Cell culture and treatments

Rat peritoneal mesothelial cells (RPMC) were obtained using a standard trypsin/ethylenediaminetetraacetic acid (EDTA) digestion method from the peritoneal wall of male Wistar rats weighing.

Cells were cultured in complete medium to confluence, serum starved for 24 hours and then treated for 72 hours with high glucose (200mM). Subsequently cells were treated for 72 hours with or without SST0001 (200 microg/ml) (Fig.1A).

Gene expression analysis

Total RNA was extracted from cell monolayers using the “Trizol” reagent (Invitrogen), according to the manufacturer’s instruction. Yield and purity were checked using Nanodrop (EuroClone) and total RNA from each sample was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on an ABI-Prism 7500 using Power SYBR Green Master Mix 2X (Applied Biosystems).

Western Blotting

Cells were lysed in 50 mM Tris-HCl, pH 5.0, 150 mM NaCl,0.5% Triton X-100 with Complete Protease Inhibitor Mixture (Roche Applied Science). equal amounts of proteins were subjected to SDS-PAGE. Membranes were exposed to primary antibodies directed against GAPDH  and VEGF (Santa Cruz) overnight at 4 °C and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The signal was detected with SuperSignals West Pico Chemiluminescent substrate solution (Pierce) according to the manufacturer’s instructions and captured using Kodak Image Station 2000R.

Membranes were exposed to primary antibodies directed against GAPDH  and VEGF (Santa Cruz) overnight at 4 °C and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The signal was detected with SuperSignals West Pico Chemiluminescent substrate solution (Pierce) according to the manufacturer’s instructions and captured using Kodak Image Station 2000R.

Immunofluorescence

Mesothelial cells were seeded in 22-mm glass dishes and cultured to subconfluence and treated as previously described to analyze alpha-SMA, VIM, FN and E-CAD  and filamentous actin (F-actin). Cells were fixed in 4% paraformaldehyde and permeabilized. Cells  were incubated overnight at 4 °C with the specific primary antibodies in PBS with 1% BSA, then washed three times for 5 min with PBS before incubating them for 1 h at 37 °C with the secondary antibody. Cell nuclei were visualized by Hoechst 33258. Images were obtained with a confocal LeicaSP5 microscope.

Transepithelial resistance (TER) and permeability.

For this part of the study, we used the Millicell-ERS ohmmeter with electrodes (Millipore).

RESULTS

High glucose up-regulates heparanase in mesothelial cells

Gene expression level of HPSE in mesothelial cells was evaluated by real time PCR. After 72 hours of culture in high glucose (HG) medium in mesothelial cells HPSE expression was significantly increased (Fig. 1B).

HPSE inhibition prevents high glucose induced epithelial mesenchymal transition

Real time PCR and immunofluorescence analyses confirmed that HG induces EMT of mesothelial cells. (Fig. 2, 3). In particular, HG determined an up-regulation of all mesenchymal markers (alpha-SMA, VIM, FN, TGF-beta) and the neoangiogenetic factor VEGF (Fig. 4). Contarly, E-cadherin was down-regulated (Fig. 5).

Interestingly, the addition to cell culture of SST0001 reversed all the aforementioned biological/cellular effects.

HG induced also a modulation of cytoskeleton structure with a reduction of junctional actin and the increase of stress fibers (Fig.5). Image analysis showed a significant reduction of E-CAD and F-actin co-localization from 96% to 51% after HG treatment to mesothelial cells.

Mesothelial layer functionality

In order to evaluate whether the regulation of EMT by HPSE inhibition influenced also the mesothelial layer ultrafiltration function, mesothelial cells were grown to confluence on porous filters and then trans-epithelial resistance (TER) and the permeability to albumin were measured.

High glucose reduced TER of mesothelial cells monolayer and significantly increased albumin permeability (Fig. 6). The addiction of SST001 reported to basal value TER of mesothelial cells and significantly reduced the high glucose induced albumin permeability up-regulation (Fig. 6).

CONCLUSIONS

Our study demonstrates, for the first time, that HPSE inhibition was able to significantly reduce EMT and angiogenesis of mesothelial cells undergoing high glucose stress. This could definitely represent, if confirmed in in vivo studies, a valuable therapeutic tool to minimize fibrosis and to increase the dialytic time and technical efficiency.