Single exposure of mesothelial cells to glucose degradation products (GDPs) yields early advanced glycation end-products (AGEs) and a proinflammatory response.
BACKGROUND
Fluids commonly used for peritoneal dialysis (PD) have a low pH and a high glucose content. Furthermore, heat sterilization of dialysis fluids degrades some of the glucose into glucose degradation products (GDPs), such as methylglyoxal (MGO) and 3-deoxyglucosone (3-DG). Mesothelial cells (MCs) form the first line in the peritoneal cavity and are constantly exposed to these nonphysiological conditions. Since MCs play an important role in the regulation of inflammatory responses in the peritoneal cavity, we studied the kinetics of MC uptake of highly purified GDP species, along with their effect on various cellular biological and immunological parameters.


METHODS
Methylglyoxal and 3-DG were purified and added to MC cultures. Complexing to medium components or uptake by MCs was analyzed over time by HPLC of the culture supernatant and by immunocytochemistry of MCs for MGO-modified proteins. Furthermore, MCs were exposed to a single dose of MGO or 3-DG and analyzed for apoptosis, proliferation by MTT assay, and [3H]-thymidine incorporation. Incorporation of [35S]-methionine was determined in order to analyze de novo protein synthesis. Expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), CD44, and vascular cell adhesion molecule-1 (VCAM-1) was analyzed by cell-bound ELISA. Effects of MGO and 3-DG on cytokine production were also analyzed.


RESULTS
Substitution of MGO and 3-DG in culture medium resulted in a spontaneous decrease in MGO over time, whereas 3-DG levels decreased minimally. The concentration of these GDPs was more reduced in the presence of MCs, indicating binding to and/or uptake by MCs of these GDPs. Mesothelial cells that had been cultured in the presence of MGO showed positive staining with a monoclonal that specifically recognizes MGO-modified proteins, demonstrating complexing to mesothelial cellular proteins. Cell-bound ELISA showed a two- to three-fold induction of expression of VCAM-1 by MGO and 3-DG; the expression of ICAM-1 and CD44 was not changed. Mesothelial cells showed a twofold increase in interleukin (IL)-6 and IL-8 production after exposure to 3-DG. Furthermore, incubation with MGO and 3-DG induced apoptosis and reduced the proliferation of cells, but did not influence protein synthesis.


CONCLUSIONS
In the current report we demonstrate that MCs take up MGO and 3-DG and form early advanced glycation end-products. Upon short exposure to a single GDP, MCs react with enhanced cytotoxic damage and a proinflammatory response, evidenced by increased VCAM-1 expression and elevated production of IL-6 and IL-8.
Welten
Angelique G A
AG
Department of Molecular Cell Biology, VU University Medical Center, Amsterdam, The Netherlands. aga.welten.cell@med.vu.nl
Schalkwijk
Casper G
CG
ter Wee
Piet M
PM
Meijer
Sybren
S
van den Born
Jacob
J
Beelen
Robert J H
RJ
D017209:Apoptosis; D002455:Cell Division / Q000187:drug effects; D002468:Cell Physiological Phenomena / Q000187:drug effects; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000008:administration & dosage* / Q000031:analogs & derivatives*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D007249:Inflammation / Q000139:chemically induced* / Q000276:immunology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D009852:Omentum / Q000187:drug effects / Q000276:immunology; D011765:Pyruvaldehyde / Q000008:administration & dosage*; D013997:Time Factors; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D017127:Glycation End Products, Advanced; D018960:Hyaluronan Receptors; D015850:Interleukin-6; D016209:Interleukin-8; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D011765:Pyruvaldehyde; D003847:Deoxyglucose; C016350:3-deoxyglucosone