Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose

Background. Peritoneal dialysis causes damage to peritoneal mesothelial cel ls primarily because dialysis fluids have a high glucose concentration. Thi s study examined the abnormalities of gap junctional intercellular communic ation (GJIC) in human peritoneal mesothelial cells (HPMCs) exposed to relat ively high levels of glucose. Also, ability of hexamethylene bisacetamide ( HMBA) to up-regulate GJIC in HPMCs exposed to high levels of glucose was me asured. Methods. An assay that monitors the recovery of fluorescence after photoble aching was used to measure GJIC in primary cultured HPMCs. The cells were e xposed to a low (10 mmol/L) or high (50 or 90 mmol/L) glucose level for a t otal of six days, and some cells were also incubated with or without HMBA ( I or 6 mmol/L) from day 4. The effects of incubation in these various envir onments on expression of the connexin 43 (Cx43) gene were investigated by t he reverse transcription-polymerase chain reaction (to detect Cx43 mRNA) or by immunofluorescence and Western blotting (to detect Cx43 protein). To ev aluate the influence of protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) on GJIC, specific inhibitors were added to cultures in a hig h glucose medium. Results. Gap junctional intercellular communication was inhibited in a conc entration- and time-dependent manner when cells were exposed to high glucos e. The addition of 6 mmol/L HMBA to cultures significantly enhanced GJIC de spite the presence of a high glucose concentration. High glucose also down- regulated Cx43 mRNA and protein expression, with the dose-dependent decreas e of Cx43 protein at gap junctions paralleled by a decrease in the phosphor ylation of this protein. As expected, treatment of cells with 6 mmol/L HMBA increased both Cx43 mRNA and protein levels despite exposure to high gluco se. The addition of PKC or MAPK inhibitors to high glucose cultures did not restore GJIC, and there was no significant change of Cx43 phosphorylation in the presence of these inhibitors. Conclusions. High glucose down-regulates GJIC in human peritoneal mesotheli al cells. It also decreases the levels of both Cx43 mRNA and Cx43 protein, with the latter becoming hypophosphorylated. HMBA appears to reverse all of these changes. These results are consistent with our hypothesis that HMBA protects HPMCs from the adverse effects of high glucose by reversing variou s processes that would otherwise lead to harmful loss of GJIC.