Effects of glucose dialysate on extracellular matrix production by human peritoneal mesothelial cells (HPMC): The role of TGF-β

Abstract

Background. Dialysate glucose has been implicated in the loss of peritoneal membrane function seen in long-term CAPD patients. Methods. In order to investigate this in vitro, human peritoneal mesothelial cells (HPMC) were cultured in a 50 : 50 mix of dialysis solution and M199 for 12 h. The dialysate was laboratory manufactured and designed to be identical in composition to PD4 (LAB). The final glucose concentration ranged between 5 and 40 mmol/l. Experiments were conducted in the presence and absence of an anti-transforming growth factor-beta (TGF-β) antibody. Cell viability was measured by lactate dehydrogenase (LDH) release. Fibronectin (FN) and TGF-β protein were measured by ELISA, and FN gene expression was measured by Northern analysis. Separately, the effects of recombinant TGF-β1 added to M199: dialysate at 5 mmol/l glucose were investigated. Results. Forty millimoles per litre D-glucose LAB caused a decrease in cell viability, as evidenced by an increase in LDH release (6.0±1.3 vs 2.6±0.7%). This effect was dependent on osmolality. Forty millimoles per litre D-glucose LAB stimulated a 15.4±4.6% increase in FN, a 46.5±18.3% increase in TGF-β protein (both P < 0.05), and 1.4±0.09-fold increase in FN mRNA compared with 5 mmol/l D-glucose LAB. Exogenous TGF-β 0-1 ng/ml induced a dose-dependent increase in FN protein (280±45% increase at TGF-β 1 ng/ml, P < 0.0001), and FN mRNA levels (10.0±1.8-fold at TGF-β 1 ng/ml). The increase in FN in response to 40 mmol/l glucose was significantly reduced by anti-TGF-β antibody to levels not different from control (93.8±6.6%, P < 0.05 vs no Ab). Conclusions. These data suggest that the pro-fibrotic effect of glucose dialysate on HPMC is mediated through stimulation of TGF-β, which promotes FN gene expression and protein production.