Improvement of endothelial nitric oxide synthase activity retards the progression of diabetic nephropathy in db/db mice

Division of Nephrology, Department of Medicine, George M. O'Brien Kidney and Urologic Diseases Center, Vanderbilt University School of Medicine, Nashville Veterans Affairs Hospital, Nashville, Tennessee, USA.
Kidney International (Impact Factor: 8.56). 07/2012; 82(11). DOI: 10.1038/ki.2012.248
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Impaired endothelial nitric oxide synthase (eNOS) activity may be involved in the pathogenesis of diabetic nephropathy. To test this, we used the type 2 diabetic db/db mouse (BKS background) model and found impaired eNOS dimerization and phosphorylation along with moderate glomerular mesangial expansion and increased glomerular basement membrane (GBM) thickness at 34 weeks of age. Cultured murine glomerular endothelial cells exposed to high glucose had similar alterations in eNOS dimerization and phosphorylation. Treatment with sepiapterin, a stable precursor of the eNOS cofactor tetrahydrobiopterin, or the nitric oxide precursor L-arginine corrected changes in eNOS dimerization and phosphorylation, corrected permeability defects, and reduced apoptosis. Sepiapterin or L-arginine, administered to db/db mice from weeks 26 to 34, did not significantly alter hyperfiltration or affect mesangial expansion, but reduced albuminuria and GBM thickness, and decreased urinary isoprostane and nitrotyrosine excretion (markers of oxidative stress). Although there was no change in glomerular eNOS monomer expression, both sepiapterin and L-arginine partially reversed the defect in eNOS dimerization and phosphorylation. Hence, our results support an important role for eNOS dysfunction in diabetes and suggest that sepiapterin supplementation might have therapeutic potential in diabetic nephropathy.Kidney International advance online publication, 11 July 2012; doi:10.1038/ki.2012.248.

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    • "These observations suggest that glomerular hemodynamic effects also promote formation of glomerular nodular lesions in diabetic pigs. Similarly, diabetic nephropathy was accelerated in eNOS-knockout mice, attenuated by improvement of eNOS activity in db/db mice [32], [33], and antihypertensive therapy alone significantly suppressed the development of nodular lesions and mesangiolysis in diabetic eNOS-knockout mice [34]. Based on these reports and our current findings, our results suggest the involvement of glomerular hypertension in nodular lesion formation in diabetes. "
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    ABSTRACT: Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α) P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3) gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI) with advanced glycation end-product (AGE) and Nε-carboxymethyl-lysine (CML) deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β) was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the somewhat acute and constitutive formation of nodules in this mammalian model might provide information facilitating identification of the principal mechanism underlying diabetic nodular sclerosis.
    PLoS ONE 03/2014; 9(3):e92219. DOI:10.1371/journal.pone.0092219 · 3.23 Impact Factor
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    • "For some experiments, sepiapterin (30 mg / kg) was administered orally by gavage, one day before exposure to hypoxia and every other day during hypoxia, at each chamber opening. Consequently, the dose of sepiapterin was adapted from previous studies [20,21]. "
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    ABSTRACT: Tetrahydrobiopterin (BH4), which fosters the formation of and stabilizes endothelial NO synthase (eNOS) as an active dimer, tightly regulates eNOS coupling / uncoupling. Moreover, studies conducted in genetically-modified models demonstrate that BH4 pulmonary deficiency is a key determinant in the pathogenesis of pulmonary hypertension. The present study thus investigates biopterin metabolism and eNOS expression, as well as the effect of sepiapterin (a precursor of BH4) and eNOS gene deletion, in a mice model of hypoxic pulmonary hypertension. In lungs, chronic hypoxia increased BH4 levels and eNOS expression, without modifying dihydrobiopterin (BH2, the oxidation product of BH4) levels, GTP cyclohydrolase-1 or dihydrofolate reductase expression (two key enzymes regulating BH4 availability). In intrapulmonary arteries, chronic hypoxia also increased expression of eNOS, but did not induce destabilisation of eNOS dimers into monomers. In hypoxic mice, sepiapterin prevented increase in right ventricular systolic pressure and right ventricular hypertrophy, whereas it modified neither remodelling nor alteration in vasomotor responses (hyper-responsiveness to phenylephrine, decrease in endothelium-dependent relaxation to acetylcholine) in intrapulmonary arteries. Finally, deletion of eNOS gene partially prevented hypoxia-induced increase in right ventricular systolic pressure, right ventricular hypertrophy and remodelling of intrapulmonary arteries. Collectively, these data demonstrate the absence of BH4/BH2 changes and eNOS dimer destabilisation, which may induce eNOS uncoupling during hypoxia-induced pulmonary hypertension. Thus, even though eNOS gene deletion and sepiapterin treatment exert protective effects on hypoxia-induced pulmonary vascular remodelling, increase on right ventricular pressure and / or right ventricular hypertrophy, these effects appear unrelated to biopterin-dependent eNOS uncoupling within pulmonary vasculature of hypoxic wild-type mice.
    PLoS ONE 11/2013; 8(11):e82594. DOI:10.1371/journal.pone.0082594 · 3.23 Impact Factor
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    • "reacts with eNOS-derived NO to generate ONOO- which oxidizes BH4 to BH2. BH4 reduction has been implicated in the pathogenesis of a variety of conditions such as hyperphenylalaninemia [44], diabetes [45], ischemia-reperfusion injury [46], hypertension [47], Alzheimer disease and Parkinson’s disease [48], and many of these pathological conditions were shown to be ameliorated by exogenous BH4 [44-47] or sepiapterin [49-51] supplementation. BH4 is critical for the maintenance of eNOS dimers and is functionally related to S-glutathionylation, a powerful regulator of eNOS uncoupling [52]. "
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    ABSTRACT: Increased levels of the sugar metabolite methylglyoxal (MG) in vivo were shown to participate in the pathophysiology of vascular complications in diabetes. Alterations of endothelial nitric oxide synthase (eNOS) activity by hypophosphorylation of the enzyme and enhanced monomerization are found in the diabetic milieu, and the regulation of this still remains undefined. Using various pharmacological approaches, we elucidate putative mechanisms by which MG modulates eNOS-associated functions of MG-stimulated superoxide (O2[bullet]-) production, phosphorylation status and eNOS uncoupling in EA.hy926 human endothelial cells. In cultured EA.hy926 endothelial cells, the effects of MG treatment, tetrahydrobiopterin (BH4; 100 muM) and sepiapterin (20 muM) supplementation, NOS inhibition by NG-nitro-L-arginine methyl ester (L-NAME; 50 muM), and inhibition of peroxynitrite (ONOO-) formation (300 muM Tempol plus 50 muM L-NAME) on eNOS dimer/monomer ratios, Ser-1177 eNOS phosphorylation and 3-nitrotyrosine (3NT) abundance were quantified using immunoblotting. O2[bullet]--dependent fluorescence was determined using a commercially available kit and tissue biopterin levels were measured by fluorometric HPLC analysis. In EA.hy926 cells, MG treatment significantly enhanced O2[bullet]- generation and 3NT expression and reduced Ser-1177 eNOS phosphorylation, eNOS dimer/monomer ratio and cellular biopterin levels indicative of eNOS uncoupling. These effects were significantly mitigated by administration of BH4, sepiapterin and suppression of ONOO- formation. L-NAME treatment significantly blunted eNOS-derived O2[bullet]- generation but did not modify eNOS phosphorylation or monomerization. MG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with O2[bullet]- generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.
    Cardiovascular Diabetology 09/2013; 12(1):134. DOI:10.1186/1475-2840-12-134 · 4.02 Impact Factor
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