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

Cheng H, Wang H, Fan X, Paueksakon P, Harris RC - Kidney Int. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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 37232, USA.

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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High glucose increased bovine serum albumin (BSA) permeability and apoptosis in glomerular endothelial cells (GEnCs); these effects were inhibited by sepiapterin or L-arginine. (a) High glucose (HG), but not mannitol, increased permeability in GEnCs, indicated by BSA clearance at 5–40 min. *P<0.05 HG versus normal medium (NG) or osmolality control (mannitol at same concentration). (b) Sepiapterin (Sep) and L-arginine (L-arg) attenuated HG increases in permeability, *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation. (c) Effect of HG on apoptosis in GEnCs. Representative images of terminal transferase dUTP nick-end labeling (TUNEL) assay from four independent experiments. (d) Sep and L-arg prevented HG-induced apoptosis. *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation.
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fig1: High glucose increased bovine serum albumin (BSA) permeability and apoptosis in glomerular endothelial cells (GEnCs); these effects were inhibited by sepiapterin or L-arginine. (a) High glucose (HG), but not mannitol, increased permeability in GEnCs, indicated by BSA clearance at 5–40 min. *P<0.05 HG versus normal medium (NG) or osmolality control (mannitol at same concentration). (b) Sepiapterin (Sep) and L-arginine (L-arg) attenuated HG increases in permeability, *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation. (c) Effect of HG on apoptosis in GEnCs. Representative images of terminal transferase dUTP nick-end labeling (TUNEL) assay from four independent experiments. (d) Sep and L-arg prevented HG-induced apoptosis. *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation.

Mentions: Pilot studies indicated that incubation of cultured mouse GEnCs with HG for 48 h demonstrated significant alterations. At that time, GEnCs in HG increased bovine serum albumin (BSA) clearance compared with normal glucose (NG) medium or mannitol (9.9±0.6% in NG and 11.0±1.0% in mannitol vs. 40.8±1.7% in HG after 40 min, n=5, P<0.05) (Figure 1a) without significantly altering proliferation (data not shown). In addition, HG induced profound apoptosis in GEnCs (1±0.2% in NG vs. 7.9±0.4% in HG, n=4, P<0.05) (Figure 1b and c). Incubation in the high-glucose medium decreased eNOS dimerization (Figure 2a) and phosphorylation at Ser1179 (Figure 2b) without changing eNOS monomer expression or phosphorylation at Thr479, whereas incubation with mannitol as an osmolar control did not have the same effects (Supplementary Figure S1a and b online). HG also decreased NOS activity, indicated by decreased levels of the stable end products of NO (nitrate/nitrite) (to 0.53±0.07 fold of NG. n=4, P<0.05 compared with NG or mannitol control) (Figure 2c and Supplementary Figure S1c online).


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

Cheng H, Wang H, Fan X, Paueksakon P, Harris RC - Kidney Int. (2012)

High glucose increased bovine serum albumin (BSA) permeability and apoptosis in glomerular endothelial cells (GEnCs); these effects were inhibited by sepiapterin or L-arginine. (a) High glucose (HG), but not mannitol, increased permeability in GEnCs, indicated by BSA clearance at 5–40 min. *P<0.05 HG versus normal medium (NG) or osmolality control (mannitol at same concentration). (b) Sepiapterin (Sep) and L-arginine (L-arg) attenuated HG increases in permeability, *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation. (c) Effect of HG on apoptosis in GEnCs. Representative images of terminal transferase dUTP nick-end labeling (TUNEL) assay from four independent experiments. (d) Sep and L-arg prevented HG-induced apoptosis. *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473143&req=5

fig1: High glucose increased bovine serum albumin (BSA) permeability and apoptosis in glomerular endothelial cells (GEnCs); these effects were inhibited by sepiapterin or L-arginine. (a) High glucose (HG), but not mannitol, increased permeability in GEnCs, indicated by BSA clearance at 5–40 min. *P<0.05 HG versus normal medium (NG) or osmolality control (mannitol at same concentration). (b) Sepiapterin (Sep) and L-arginine (L-arg) attenuated HG increases in permeability, *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation. (c) Effect of HG on apoptosis in GEnCs. Representative images of terminal transferase dUTP nick-end labeling (TUNEL) assay from four independent experiments. (d) Sep and L-arg prevented HG-induced apoptosis. *P<0.05 HG versus normal medium or HG with Sep or L-arg coincubation.
Mentions: Pilot studies indicated that incubation of cultured mouse GEnCs with HG for 48 h demonstrated significant alterations. At that time, GEnCs in HG increased bovine serum albumin (BSA) clearance compared with normal glucose (NG) medium or mannitol (9.9±0.6% in NG and 11.0±1.0% in mannitol vs. 40.8±1.7% in HG after 40 min, n=5, P<0.05) (Figure 1a) without significantly altering proliferation (data not shown). In addition, HG induced profound apoptosis in GEnCs (1±0.2% in NG vs. 7.9±0.4% in HG, n=4, P<0.05) (Figure 1b and c). Incubation in the high-glucose medium decreased eNOS dimerization (Figure 2a) and phosphorylation at Ser1179 (Figure 2b) without changing eNOS monomer expression or phosphorylation at Thr479, whereas incubation with mannitol as an osmolar control did not have the same effects (Supplementary Figure S1a and b online). HG also decreased NOS activity, indicated by decreased levels of the stable end products of NO (nitrate/nitrite) (to 0.53±0.07 fold of NG. n=4, P<0.05 compared with NG or mannitol control) (Figure 2c and Supplementary Figure S1c online).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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 37232, USA.

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.

Show MeSH
Related in: MedlinePlus