Vascular endothelial growth factor (VEGF) is an important survival factor for endothelial cells in hypoxic environments. High glucose regulates certain aspects of VEGF expression in various cell types, including proximal tubular cells. Thus, ambient glucose levels may modulate the progression of chronic kidney disease, especially diabetic nephropathy. Immortalized rat proximal tubular cells (IRPTC) were cultured for 24 h under hypoxic conditions (1% O(2)), with or without high d-glucose (25 mM), or with or without high l-glucose (25 mM). Controls included culture in normoxic conditions and normal d-glucose (5.5 mM). VEGF mRNA expression was assessed by real-time quantitative PCR, and VEGF protein in the supernatant was assessed by ELISA. Hypoxia increased VEGF expression. This response was significantly blunted by high d-glucose (1.98 +/- 0.11- versus 2.65 +/- 0.27-fold increase for VEGF mRNA expression, 252.8 +/- 14.7 versus 324.0 +/- 11.5 pg/10(5) cells for VEGF protein; P < 0.05 both) but not by high l-glucose. It is interesting that hydrogen peroxide also blunted this response, whereas alpha-tocopherol restored the VEGF response to hypoxia in the presence of high d-glucose. For determination of involvement of the hypoxia-inducible factor (HIF)/hypoxia-responsible element (HRE) pathway, IRPTC that were stably transfected with HRE-luciferase were cultured under the previous conditions. High d-glucose also reduced luciferase activity under hypoxia, whereas alpha-tocopherol restored activity. In vivo experiments using streptozotocin-induced diabetic rats confirmed that hyperglycemia blunted HIF-HRE pathway activation. Insulin treatment restored activation of the HIF-HRE pathway in streptozotocin-induced diabetic rats. In conclusion, high glucose blunts VEGF response to hypoxia in IRPTC. This effect is mediated by the oxidative stress-regulated HIF-HRE pathway.