Effect of high glucose on nitric oxide production and endothelial nitric oxide synthase protein expression in human glomerular endothelial cells.
BACKGROUND
Hyperglycemia directly contributes to the development of diabetic nephropathy. Nitric oxide (NO), a potent endothelium-derived vasodilator, has been suggested to participate in the regulation of renal blood flow, glomerular filtration rate, and mesangial matrix accumulation. Human vascular endothelial cells are known to exhibit functional heterogeneity, this prompted us to do the first study of NO bioavailability in human glomerular endothelial cells (HGECs), in response to high glucose exposure.


METHODS
NO release was examined by detecting nitrite generation by the Griess assay in HGECs exposed to control-level (5.5 mM) and high-level (15, 30 and 60 mM) glucose solutions at various time periods (24, 48 and 72 h) in the presence or absence of L-arginine (1 mM), or superoxide dismutase (SOD) (250 U/ml). In addition, we evaluated the effect of glucose on the expression of endothelial nitric oxide synthase (eNOS) in HGECs by Western blotting.


RESULTS
Final levels of nitrite generated in HGECs were reduced significantly, in a time- and concentration-dependent manner, after high glucose exposure. However, Western blot analysis revealed that eNOS protein expression was significantly upregulated at 12 h after exposure to high glucose concentrations (30 mM), reaching a peak at 48 h (twofold increase over baseline levels). The inhibitory effect of high glucose on NO production was restored by the addition of SOD. Addition of L-arginine (1 mM) to external media also reversed the inhibitory effect of high glucose on NO production of HGECs as well.


CONCLUSIONS
The present study demonstrated that high glucose increased eNOS protein expression, but decreased NO release finally. Decreased NO bioavailability seems to be associated with overproduction of superoxide and L-arginine deficiency. These findings provide an important clue in clarifying the molecular basis of the mechanisms by which elevated glucose leads to an imbalance between NO and superoxide, resulting in impaired endothelial function. In addition, restoration of NO function by both administration of L-arginine and adequate intake of antioxidants suggests a potential supportive treatment for patients with diabetic nephropathy.
Hoshiyama
Mari
M
Department of Cellular Physiology, Institute of Nephrology and Second Department of Internal Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
Li
Bing
B
Yao
Jian
J
Harada
Takashi
T
Morioka
Tetsuo
T
Oite
Takashi
T
D001120:Arginine / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005947:Glucose / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D020536:Enzyme Activators; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D005947:Glucose