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


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-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).


CONCLUSIONS
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.
Medcalf
J F
JF
Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK.
Walls
J
J
Pawluczyk
I Z
IZ
Harris
K P
KP
D000906:Antibodies / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015314:Dialysis Solutions / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000008:administration & dosage* / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009994:Osmolar Concentration; D010537:Peritoneum / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000378:metabolism / Q000502:physiology*
D000906:Antibodies; D015314:Dialysis Solutions; D005353:Fibronectins; D016212:Transforming Growth Factor beta; D007770:L-Lactate Dehydrogenase; D005947:Glucose