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      Effect of High Glucose on Nitric Oxide Production and Endothelial Nitric Oxide Synthase Protein Expression in Human Glomerular Endothelial Cells

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          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 m M) and high-level (15, 30 and 60 m M) glucose solutions at various time periods (24, 48 and 72 h) in the presence or absence of L-arginine (1 m M), 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 m M), 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 m M) 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.

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          Most cited references 10

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          Vascular endothelial cells synthesize nitric oxide from L-arginine.

          Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue and for the inhibition of platelet aggregation and platelet adhesion attributed to endothelium-derived relaxing factor. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay, chemiluminescence or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
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            The role of oxidative stress in the onset and progression of diabetes and its complications: asummary of a Congress Series sponsored byUNESCO-MCBN, the American Diabetes Association and the German Diabetes Society

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              Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy.

              The overproduction of nitric oxide (NO) is reported in the diabetic kidney and considered to be involved in glomerular hyperfiltration. The precise mechanism of NO production in the diabetic kidney is, however, not known. In this report, we compare the localization of endothelial cell nitric oxide synthase (ecNOS) isoform expression in the kidney tissue of streptozotocin (STZ)-induced diabetic rats and 5/6 nephrectomized rats and clarify the pivotal role of ecNOS for the glomerular hyperfiltration in the early stages of diabetic nephropathy. In diabetic rats, the diameters of afferent arterioles, the glomerular volume, creatinine clearance, and urinary NO2/NO3 were increased after the induction of diabetes. Efferent arterioles were, however, not altered. Insulin or L-NAME treatment returned the diameters of afferent arterioles, glomerular volume, creatinine clearance, and urinary NO2/NO3 to normal. The expression of ecNOS in afferent arterioles and glomeruli of diabetic rats increased during the early stages of the disease, but was not altered in efferent arterioles. Treatment with either insulin or L-NAME decreased ecNOS expression in afferent arterioles and in glomeruli. In contrast, the ecNOS expression was upregulated in both afferent and efferent arterioles and in the glomeruli of 5/6 nephrectomized rats, where the dilatation of afferent and efferent arterioles and glomerular enlargement were observed. Treatment with L-NAME ameliorated the ecNOS expression and dilatation of arterioles. We conclude that enhanced NO synthesis by ecNOS in afferent arterioles and glomerular endothelial cells in response to the hyperglycaemic state could cause preferential dilatation of afferent arterioles, which ultimately induces glomerular enlargement and glomerular hyperfiltration.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                October 2003
                17 November 2004
                : 95
                : 2
                : e62-e68
                Department of Cellular Physiology, Institute of Nephrology and Second Department of Internal Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
                73673 Nephron Exp Nephrol 2003;95:e62–e68
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 6, References: 36, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/73673
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