Effect of high glucose on superoxide in human mesangial cells: role of angiotensin II.
OBJECTIVE
Reactive oxygen species, and especially superoxide (O2*),have been implicated in diabetic nephropathy. O2* accumulation in cells is dependent on O2* production (by NADH/NADPH oxidase) as well as scavenging by superoxide dismutase (SOD) activity. This study was designed to investigate the effects of high glucose (HG) on O2* accumulation and SOD activity in human mesangial cells (HMC) and to determine if these effects are mediated by angiotensin II (Ang II).


METHODS
HMC were incubated in media containing 10 mM glucose (control, C), 30 mM glucose (HG), 10 mM glucose + either 20 mM 2-deoxy-D-glucose (2-DG) or 20 mM mannitol (high mannitol, HM) (osmotic controls), or Ang II (10(-5) M). Ang II action was antagonized by employing 10(-4) M of Ang II receptor antagonists (losartan or irbesartan) or 10(-4) M of NADH/NADPH oxidase inhibitors [diphenyleneiodonium chloride (DPI) or apocynin]. Superoxide and total SOD activity were assayed using chemiluminescence of lucigenin.


RESULTS
Incubation of HMC in HG resulted in a 1.6-fold increase in Ang I (p < 0.05) and a 1.4-fold increase in Ang II levels (p < 0.05) in cell lysates. These changes were accompanied by a >2-fold increase in O2* accumulation (p < 0.01), which was inhibited by losartan and irbesartan. Exogenous Ang II increased net O2* accumulation by 2.7-fold (p < 0.01), which was normalized by losartan and irbesartan. DPI and apocynin blocked the HG and Ang II-induced increases in O2* (p < 0.01). HG but not exogenous Ang II inhibited total SOD activity by 30%, which was not affected by losartan.


CONCLUSIONS
High glucose increases O2* accumulation in HMC primarily by increasing its production via the Ang II-NADH/NADPH oxidase system.
Leehey
David J
DJ
Veterans Affairs Hospital, Hines, IL 60141, USA. dleehey@lumc.edu
Isreb
Majd A
MA
Marcic
Sonja
S
Singh
Ashok K
AK
Singh
Rekha
R
D000804:Angiotensin II / Q000502:physiology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D018929:Cell Culture Techniques; D003928:Diabetic Nephropathies / Q000503:physiopathology*; D005947:Glucose / Q000502:physiology*; D006801:Humans; D000077405:Irbesartan; D007678:Kidney Glomerulus / Q000166:cytology / Q000502:physiology*; D019808:Losartan / Q000494:pharmacology; D016877:Oxidants / Q000032:analysis*; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000032:analysis*; D013777:Tetrazoles / Q000494:pharmacology
D047228:Angiotensin II Type 1 Receptor Blockers; D001713:Biphenyl Compounds; D016877:Oxidants; D013777:Tetrazoles; D013481:Superoxides; D000804:Angiotensin II; D013482:Superoxide Dismutase; D005947:Glucose; D000077405:Irbesartan; D019808:Losartan