Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.
The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.
Department of Pediatrics and Adolescent Medicine and Zytoprotec GmbH, Vienna, Austria; and.
Rebecca
R
Herzog
Department of Nephrology and Medical Intensive Care, Charité University of Medicine Berlin, Berlin, Germany.
Thorsten O
TO
Bender
Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria;
Andreas
A
Vychytil
Zytoprotec GmbH, Vienna, Austria; and.
Katarzyna
K
Bialas
Department of Pediatrics and Adolescent Medicine and.
Christoph
C
Aufricht
Department of Pediatrics and Adolescent Medicine and Zytoprotec GmbH, Vienna, Austria; and klaus.kratochwill@meduniwien.ac.at.
Klaus
K
Kratochwill
D000117:Acetylglucosamine / Q000737:chemistry*; D002470:Cell Survival; D002478:Cells, Cultured; D015314:Dialysis Solutions / Q000737:chemistry* / Q000494:pharmacology; D004151:Dipeptides / Q000737:chemistry; D004848:Epithelium / Q000473:pathology*; D005947:Glucose / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006031:Glycosylation; D050884:HSP72 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D009852:Omentum / Q000166:cytology; D010530:Peritoneal Dialysis / Q000379:methods*; D010537:Peritoneum / Q000473:pathology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*
D015314:Dialysis Solutions; D004151:Dipeptides; D050884:HSP72 Heat-Shock Proteins; D011506:Proteins; D005973:Glutamine; D005947:Glucose; C054122:alanylglutamine; D000117:Acetylglucosamine