Glutathione-Related Responses to Hyperglycemic and Hyperosmotic Stress in Cultured Human Retinal Pericytes and Endothelial Cells: An Exploratory Study.

Background/Objectives: Hyperglycemic and hyperosmotic stress may disturb glutathione-dependent antioxidant defenses in retinal vascular cells. This exploratory in vitro study evaluated glutathione-related biochemical endpoints in separate human retinal pericyte and endothelial-cell experiments and to identify candidate endpoints for independent validation. Methods: Passage-3 human retinal pericytes and endothelial cells were exposed for 24 h to five conditions: 5.5 mmol/L glucose (control), 25 or 50 mmol/L glucose, and 5.5 mmol/L glucose supplemented with 19.5 or 44.5 mmol/L mannitol. Glutathione peroxidase (GPx), glutathione reductase (GR), total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), the GSH/GSSG ratio, glutathione S-transferase (GST), and gamma-glutamyl transferase (GGT) were measured in a mixture of culture medium and cell lysate. Each condition comprised 10 technical replicate wells from one culture experiment; the cell types were examined in separate, unmatched runs. Results: In the pericyte experiment, GPx and GR decreased across several glucose and mannitol conditions, and GGT increased by 39-72%. In the separate endothelial experiment, GGT was the dominant response (eta squared = 0.763), increasing by 66-116%; GGT at 50 mmol/L glucose was 30% higher than the corresponding 44.5 mmol/L mannitol condition. Conclusions: The study identifies GGT as the principal candidate endpoint for biological replication and GPx and GR as additional candidates in pericytes. It does not support comparisons between cell types. All findings are preliminary because they derive from technical wells in single, unmatched experiments.