title: Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells.
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.
authors.affiliation: Research Centre, Centre hospitalier de l'Université de Montréal (CHUM), Hôtel-Dieu, 3850 Saint Urbain Street, Montreal, Quebec H2W 1T8, Canada.
authors.forename: Tusty-Jiuan
authors.initials: TJ
authors.lastname: Hsieh
authors.forename: Pierre
authors.initials: P
authors.lastname: Fustier
authors.forename: Chih-Chang
authors.initials: CC
authors.lastname: Wei
authors.forename: Shao-Ling
authors.initials: SL
authors.lastname: Zhang
authors.forename: Janos G
authors.initials: JG
authors.lastname: Filep
authors.forename: Shiow-Shiu
authors.initials: SS
authors.lastname: Tang
authors.forename: Julie R
authors.initials: JR
authors.lastname: Ingelfinger
authors.forename: I George
authors.initials: IG
authors.lastname: Fantus
authors.forename: Pavel
authors.initials: P
authors.lastname: Hamet
authors.forename: John S D
authors.initials: JS
authors.lastname: Chan
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000808:Angiotensinogen / Q000235:genetics*; D000818:Animals; D001377:Azaserine / Q000494:pharmacology; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D019808:Losartan / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D020913:Perindopril / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012083:Renin / Q000235:genetics
chemical_list: D047228:Angiotensin II Type 1 Receptor Blockers; D000806:Angiotensin-Converting Enzyme Inhibitors; D007093:Imidazoles; D007202:Indicators and Reagents; D007211:Indoles; D007328:Insulin; D008301:Maleimides; D011725:Pyridines; D017382:Reactive Oxygen Species; D000808:Angiotensinogen; D000804:Angiotensin II; C073402:PD 123319; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D001377:Azaserine; D011493:Protein Kinase C; D012083:Renin; D005947:Glucose; D019808:Losartan; C070515:bisindolylmaleimide I; D020913:Perindopril