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

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Abstract
Dialysate glucose has been implicated in the loss of peritoneal membrane function seen in long-term CAPD patients. 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-beta) antibody. Cell viability was measured by lactate dehydrogenase (LDH) release. Fibronectin (FN) and TGF-beta protein were measured by ELISA, and FN gene expression was measured by Northern analysis. Separately, the effects of recombinant TGF-beta(1) added to M199: dialysate at 5 mmol/l glucose were investigated. 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-beta 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-beta 0-1 ng/ml induced a dose-dependent increase in FN protein (280+/-45% increase at TGF-beta 1 ng/ml, P<0.0001), and FN mRNA levels (10.0+/-1.8-fold at TGF-beta 1 ng/ml). The increase in FN in response to 40 mmol/l glucose was significantly reduced by anti-TGF-beta antibody to levels not different from control (93.8+/-6.6%, P<0.05 vs no Ab). These data suggest that the pro-fibrotic effect of glucose dialysate on HPMC is mediated through stimulation of TGF-beta, which promotes FN gene expression and protein production.