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  • Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells.

Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells.

The Journal of endocrinology (2004-12-14)
Tusty-Jiuan Hsieh, Pierre Fustier, Chih-Chang Wei, Shao-Ling Zhang, Janos G Filep, Shiow-Shiu Tang, Julie R Ingelfinger, I George Fantus, Pavel Hamet, John S D Chan
ABSTRACT

We reported previously that insulin inhibits the stimulatory effect of high glucose on the expression of angiotensinogen (ANG) gene in both rat immortalized renal proximal tubular cells (IRPTCs) and non-diabetic rat renal proximal tubular cells (RPTCs), but has no effect in diabetic rat RPTCs. In the present study we investigated whether hyperglycaemia-induced resistance to the insulin-induced inhibition of expression of the ANG gene is mediated via the generation of reactive oxygen species (ROS) in RPTCs. Rat IRPTCs were cultured for 2 weeks in high-glucose (25 mM) or normal-glucose (5 mM) medium plus angiotensin II (Ang II) with or without a superoxide scavenger (tiron), or inhibitors of: NADPH oxidase (diphenylene iodinium, DPI), Ang II type 1 and 2 receptors (losartan and PD123319), angiotensin-converting enzyme (perindopril), protein kinase C (GF 109203X), or glutamine:fructose-6-phosphate amino-transferase (azaserine). Cellular generation of ROS, and ANG and renin mRNA levels were assessed by lucigenin assay and specific reverse transcriptase-PCR respectively. Phosphorylation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) was evaluated by western blotting. Prolonged exposure of IRPTCs to high concentrations of glucose or Ang II evoked generation of ROS and resistance to the insulin-induced inhibition of expression of the ANG gene and of p44/42 MAPK phosphorylation. Co-incubation of IRPTCs with tiron, DPI, losartan, PD123319, perindopril, GF 109203X or azaserine prevented ROS generation, restoring the inhibitory action of insulin on ANG gene expression and on p44/42 MAPK phosphorylation. In conclusion, our studies demonstrate that blockade of both ROS generation and activation of the intrarenal renin-angiotensin system improves the inhibitory action of insulin on ANG gene expression in IRPTCs in conditions of high glucose.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Azaserine, Hybri-Max, γ-irradiated, 50x, lyophilized powder, BioXtra, suitable for hybridoma
Sigma-Aldrich
Azaserine, ≥98% (TLC)