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

Contribution of lactate buffer, glucose and glucose degradation products to peritoneal injury in vivo

BACKGROUND: Long-term peritoneal dialysis (PD) is associated with the development of functional and structural alterations of the peritoneal membrane. In this study, we investigated the contribution of low pH lactate buffer, high glucose concentration and glucose degradation products to peritoneal injury in a rat peritoneal exposure model. METHODS: Rats received daily 10 ml of either heat-sterilized (3.86% glucose, pH 5.2, n = 8) or filter-sterilized PD fluid (3.86% glucose, pH 5.2, n = 8), or lactate buffer (pH 5.2, n = 8) via a mini vascular access port during a 10 week period. Untreated rats served as controls. RESULTS: The low pH lactate buffer instillation induced pronounced morphological changes including the induction of angiogenesis in various peritoneal tissues and mild damage to the mesothelial cell layer covering the peritoneum. It also evoked a cellular response characterized by an increased mesothelial cell density on the liver, the induction of milky spots and accumulation of omental mast cells in the omentum, and significant changes in the composition of peritoneal leukocytes. The addition of glucose to low pH lactate buffer (filter-sterilized PD fluid) strengthened most, but not all of the responses described above and induced a fibrogenic response. In addition to glucose and low pH lactate buffer, the presence of glucose degradation products (heat-sterilized PD fluid) significantly induced an additional omental milky spot response (P < 0.03) and caused profound mesothelial damage. The vessel density in the omentum and the mesentery was significantly correlated to both the number of tissue mast cells and the hyaluronan content in the peritoneal lavage, which might suggest a role for mast cells and hyaluronan in the induction of angiogenesis. CONCLUSIONS: Instillations of low pH lactate buffer, a high glucose concentration and glucose degradation products contribute differently and often cumulatively to peritoneal injury in vivo

Altered Gene Expression in Staphylococcus aureus upon Interaction with Human Endothelial Cells

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus