Heparin abrogates peritoneal dialysate-induced reduction of perlecan synthesis and epithelial-to-mesenchymal transdifferentiation in human peritoneal mesothelial cells

Abstract

INTRODUCTION AND AIMS:

Injury to the mesothelium during chronic peritoneal dialysis (PD) results in the deterioration of the structural and functional properties of the peritoneal membrane. We have previously demonstrated that high dialysate glucose concentrations reduced perlecan synthesis in human peritoneal mesothelial cells (HPMC). Heparin is a negatively charged glycosaminoglycan (GAG) that is often used in PD to prevent fibrin formation. Heparin has been shown to improve the histologic and functional properties of the peritoneum, the mechanism of which remains to be defined. We therefore investigated the effects of heparin on cell proliferation, cell morphology, TGF-β1 secretion and perlecan synthesis in HPMC stimulated with spent dialysate solutions.

METHODS:

HPMC were isolated from omental specimens by enzymatic digestion. Confluent growth arrested HPMC were stimulated with spent non-infected or infected PD fluid for 24 h in the presence or absence of heparin (1 IU/ml). Cell morphology, proliferation, TGF-β1 secretion and assessment of bioactive TGF-β1 were determined by phase contrast microscopy, MTT assay, commercial TGF-β1 ELISA and the mink lung epithelial cell bioassay respectively. De novo synthesis of heparan sulfate GAG chains was investigated by metabolic labeling of HPMC with [35S]-sulfate (50μCi/ml) for 24h.

RESULTS:

Non-infected PD fluids induced epithelial-to-mesenchymal transdifferentiation in HPMC that was associated with a significant increase in cell proliferation, induction of TGF-β1 secretion and its bioactivation compared to medium alone (P<0.05 for all). These observations were more prominent in cells cultured with spent infected PD fluid. Exposure of HPMC to spent dialysate reduced perlecan core protein expression and its deposition in the extracellular matrix, mediated in part through increased TGF-β1 bioactivation. Metabolic labeling of HPMC with [35S]-sulfate showed that perlecan consisted solely of heparan sulfate (HS) GAG chains. Spent non infected and infected dialysate reduced de novo synthesis of perlecan HS GAG chains by 22.2% and 39.7% compared to control (P<0.01 for both), although the length and charge density were unaltered. Co-incubation of HPMC with heparin and spent dialysate significantly reduced HPMC activation, preserved cell morphology, decreased cell proliferation and TGF-β1 induction observed with dialysate alone. Furthermore, heparin significantly increased de novo synthesis of HS GAG chains and perlecan core protein.

CONCLUSIONS:

Our data demonstrate that heparin can improve the structural and functional integrity of the mesothelium by its ability to abrogate dialysate-induced HPMC activation and morphologic changes, down-regulate TGF-β1 secretion and increase perlecan synthesis.This abstract is presented as Free Communication on 25-May-09 during the session Optimizing peritoneal dialysis therapy.