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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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Related in: MedlinePlus

Sepapterin (Sep) and L-arginine (L-arg) reversed high glucose–induced endothelial nitric oxide synthase (eNOS) impairment in glomerular endothelial cells (GEnCs). (a) High glucose did not alter eNOS expression, but decreased eNOS dimerization in GEnCs, which could be prevented by Sep or L-arg. Representative photo from three independent experiments. (b) High glucose decreased eNOS phosphorylation at Ser1179, without significant change at Thr479. Sep or L-arg coincubation reversed phosphorylation at Ser1179. Representative photo from three independent experiments. (c) High glucose reduced nitrate/nitrite production by GEnCs, which was corrected by supplementation with Sep or L-arg. *P<0.05 compared with normal medium or high glucose with Sep or L-arg. HG, high glucose; NG, normal glucose.
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fig2: Sepapterin (Sep) and L-arginine (L-arg) reversed high glucose–induced endothelial nitric oxide synthase (eNOS) impairment in glomerular endothelial cells (GEnCs). (a) High glucose did not alter eNOS expression, but decreased eNOS dimerization in GEnCs, which could be prevented by Sep or L-arg. Representative photo from three independent experiments. (b) High glucose decreased eNOS phosphorylation at Ser1179, without significant change at Thr479. Sep or L-arg coincubation reversed phosphorylation at Ser1179. Representative photo from three independent experiments. (c) High glucose reduced nitrate/nitrite production by GEnCs, which was corrected by supplementation with Sep or L-arg. *P<0.05 compared with normal medium or high glucose with Sep or L-arg. HG, high glucose; NG, normal glucose.

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)

Sepapterin (Sep) and L-arginine (L-arg) reversed high glucose–induced endothelial nitric oxide synthase (eNOS) impairment in glomerular endothelial cells (GEnCs). (a) High glucose did not alter eNOS expression, but decreased eNOS dimerization in GEnCs, which could be prevented by Sep or L-arg. Representative photo from three independent experiments. (b) High glucose decreased eNOS phosphorylation at Ser1179, without significant change at Thr479. Sep or L-arg coincubation reversed phosphorylation at Ser1179. Representative photo from three independent experiments. (c) High glucose reduced nitrate/nitrite production by GEnCs, which was corrected by supplementation with Sep or L-arg. *P<0.05 compared with normal medium or high glucose with Sep or L-arg. HG, high glucose; NG, normal glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Sepapterin (Sep) and L-arginine (L-arg) reversed high glucose–induced endothelial nitric oxide synthase (eNOS) impairment in glomerular endothelial cells (GEnCs). (a) High glucose did not alter eNOS expression, but decreased eNOS dimerization in GEnCs, which could be prevented by Sep or L-arg. Representative photo from three independent experiments. (b) High glucose decreased eNOS phosphorylation at Ser1179, without significant change at Thr479. Sep or L-arg coincubation reversed phosphorylation at Ser1179. Representative photo from three independent experiments. (c) High glucose reduced nitrate/nitrite production by GEnCs, which was corrected by supplementation with Sep or L-arg. *P<0.05 compared with normal medium or high glucose with Sep or L-arg. HG, high glucose; NG, normal glucose.
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