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

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          Abstract

          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.

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          Mechanisms of Increased Vascular Superoxide Production in Human Diabetes Mellitus: Role of NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase

          Background — Increased superoxide production contributes to reduced vascular nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of diabetes. We characterized the sources and mechanisms underlying vascular superoxide production in human blood vessels from diabetic patients with coronary artery disease compared with nondiabetic patients. Methods and Results — Vascular superoxide production was quantified in both saphenous veins and internal mammary arteries from 45 diabetic and 45 matched nondiabetic patients undergoing coronary artery bypass surgery. NAD(P)H-dependent oxidases were important sources of vascular superoxide in both diabetic and nondiabetic patients, but both the activity of this enzyme system and the levels of NAD(P)H oxidase protein subunits (p22phox, p67phox, and p47phox) were significantly increased in diabetic veins and arteries. In nondiabetic vessels, endothelial NO synthase produced NO that scavenged superoxide. However, in diabetic vessels, the endothelium was an additional net source of superoxide production because of dysfunctional endothelial NO synthase that was corrected by intracellular tetrahydrobiopterin supplementation. Furthermore, increased superoxide production in diabetes was abrogated by the protein kinase C inhibitor chelerythrine. Conclusions — These observations suggest important roles for NAD(P)H oxidases, endothelial NO synthase uncoupling, and protein kinase C signaling in mediating increased vascular superoxide production and endothelial dysfunction in human diabetes mellitus.
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            A new method for large scale isolation of kidney glomeruli from mice.

            Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5- micro m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 +/- 4699 (mean +/- SD, n = 14) with a purity of 97.5 +/- 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.
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              Endothelial nitric oxide synthase uncoupling impairs endothelial progenitor cell mobilization and function in diabetes.

              Uncoupling of the endothelial nitric oxide synthase (eNOS) resulting in superoxide anion (O(2)(-)) formation instead of nitric oxide (NO) causes diabetic endothelial dysfunction. eNOS regulates mobilization and function of endothelial progenitor cells (EPCs), key regulators of vascular repair. We postulate a role of eNOS uncoupling for reduced number and function of EPC in diabetes. EPC levels in diabetic patients were significantly reduced compared with those of control subjects. EPCs from diabetic patients produced excessive O(2)(-) and showed impaired migratory capacity compared with nondiabetic control subjects. NOS inhibition with N(G)-nitro-l-arginine attenuated O(2)(-) production and normalized functional capacity of EPCs from diabetic patients. Glucose-mediated EPC dysfunction was protein kinase C dependent, associated with reduced intracellular BH(4) (tetrahydrobiopterin) concentrations, and reversible after exogenous BH(4) treatment. Activation of NADPH oxidases played an additional but minor role in glucose-mediated EPC dysfunction. In rats with streptozotocin-induced diabetes, circulating EPCs were reduced to 39 +/- 5% of controls and associated with uncoupled eNOS in bone marrow. Our results identify uncoupling of eNOS in diabetic bone marrow, glucose-treated EPCs, and EPCs from diabetic patients resulting in eNOS-mediated O(2)(-) production. Subsequent reduction of EPC levels and impairment of EPC function likely contributes to the pathogenesis of vascular disease in diabetes.
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                Author and article information

                Journal
                Kidney Int
                Kidney Int
                Kidney International
                Nature Publishing Group
                0085-2538
                1523-1755
                December 2012
                11 July 2012
                : 82
                : 11
                : 1176-1183
                Affiliations
                [1 ]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
                [2 ]Department of Pathology, Microbiology and Immunology, George M. O'Brien Kidney and Urologic Diseases Center, Vanderbilt University School of Medicine, Nashville Veterans Affairs Hospital , Nashville, Tennessee, USA
                Author notes
                [* ]Division of Nephrology, Department of Medicine, C3121 MCN, George M. O'Brien Kidney and Urologic Diseases Center, Vanderbilt University School of Medicine, Nashville Veterans Affairs Hospital , Nashville, Tennessee 37232, USA. E-mail: ray.harris@ 123456vanderbilt.edu
                Article
                ki2012248
                10.1038/ki.2012.248
                3473143
                22785174
                6c27f39e-a03c-47b9-a102-cdb6a332956c
                Copyright © 2012 International Society of Nephrology

                This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                History
                : 07 February 2012
                : 20 April 2012
                : 22 May 2012
                Categories
                Original Article

                Nephrology
                db/db mice,diabetic nephropathy,enos,glomerular endothelial cells,sepiapterin
                Nephrology
                db/db mice, diabetic nephropathy, enos, glomerular endothelial cells, sepiapterin

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