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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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Sepiapterin (Sep) and L-arginine (L-arg) attenuated renal injury in db/db mice. (a) The upper panel indicates glomerular mesangial expansion in db/db mice at 34 weeks by Periodic Acid Schiff staining, Bars: 20 μm; the lower panel indicates glomerular basement membrane (GBM) thickness and foot process effacement (red arrow) in db/db mice with or without treatment with Sep/L-arg, bars: 2 μm. (b) GBM thickness. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (c) Sep or L-arg decreased albuminuria in db/db mice at 34 weeks of age. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (d) Sep or L-arg had no effect on hyperfiltration seen in db/db mice, determined by fluorescein isothiocyanate-inulin clearance. *P<0.05, BKS versus db/db mice regardless of treatments. (e) Sep or L-arg decreased elevations in urine isoprostane excretion in db/db mice. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (f) Increased nitrotyrosine accumulation in db/db mice was attenuated by Sep or L-arg treatment. Representative photo from three independent experiments. GFR, glomerular filtration rate.
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fig3: Sepiapterin (Sep) and L-arginine (L-arg) attenuated renal injury in db/db mice. (a) The upper panel indicates glomerular mesangial expansion in db/db mice at 34 weeks by Periodic Acid Schiff staining, Bars: 20 μm; the lower panel indicates glomerular basement membrane (GBM) thickness and foot process effacement (red arrow) in db/db mice with or without treatment with Sep/L-arg, bars: 2 μm. (b) GBM thickness. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (c) Sep or L-arg decreased albuminuria in db/db mice at 34 weeks of age. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (d) Sep or L-arg had no effect on hyperfiltration seen in db/db mice, determined by fluorescein isothiocyanate-inulin clearance. *P<0.05, BKS versus db/db mice regardless of treatments. (e) Sep or L-arg decreased elevations in urine isoprostane excretion in db/db mice. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (f) Increased nitrotyrosine accumulation in db/db mice was attenuated by Sep or L-arg treatment. Representative photo from three independent experiments. GFR, glomerular filtration rate.

Mentions: db/db mice are a well-established type 2 diabetic model, but some strains are relatively resistant to diabetic renal injury despite the significant hyperglycemia.15, 16db/db mice on the BKS background have been shown to develop hyperglycemia and diabetic renal injury.17, 18 We found that at 34 weeks, db/db (BKS) mice had developed glomerulopathy characterized by mesangial matrix accumulation (Figure 3a), foot process effacement, and increased glomerular basement membrane (GBM) thickness (154±6 in BKS control vs. 335±23 nm in untreated db/db mice) (Figure 3a and b) and had significant albuminuria (alb/cre: 18±3 in BKS control vs. 759±281 in untreated db/db, n=8, P<0.05) (Figure 3c) and hyperfiltration (glomerular filtration rate: 206±33 μl/min in BKS control; 403±36 in untreated db/db, n=6–12, P<0.05) (Figure 3d). Furthermore, urine F2-isoprostanes, a marker of oxidative stress, increased in the diabetic mice (5.7±0.9 in db/db mice vs. 1.4±0.1 ng 8-iso-PGF2α/mg Cr in BKS control, n=6–11, P<0.05) (Figure 3e). Increased oxidative stress in db/db mice was also confirmed by the accumulation of nitrotyrosine, another superoxide marker (Figure 3f).


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)

Sepiapterin (Sep) and L-arginine (L-arg) attenuated renal injury in db/db mice. (a) The upper panel indicates glomerular mesangial expansion in db/db mice at 34 weeks by Periodic Acid Schiff staining, Bars: 20 μm; the lower panel indicates glomerular basement membrane (GBM) thickness and foot process effacement (red arrow) in db/db mice with or without treatment with Sep/L-arg, bars: 2 μm. (b) GBM thickness. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (c) Sep or L-arg decreased albuminuria in db/db mice at 34 weeks of age. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (d) Sep or L-arg had no effect on hyperfiltration seen in db/db mice, determined by fluorescein isothiocyanate-inulin clearance. *P<0.05, BKS versus db/db mice regardless of treatments. (e) Sep or L-arg decreased elevations in urine isoprostane excretion in db/db mice. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (f) Increased nitrotyrosine accumulation in db/db mice was attenuated by Sep or L-arg treatment. Representative photo from three independent experiments. GFR, glomerular filtration rate.
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fig3: Sepiapterin (Sep) and L-arginine (L-arg) attenuated renal injury in db/db mice. (a) The upper panel indicates glomerular mesangial expansion in db/db mice at 34 weeks by Periodic Acid Schiff staining, Bars: 20 μm; the lower panel indicates glomerular basement membrane (GBM) thickness and foot process effacement (red arrow) in db/db mice with or without treatment with Sep/L-arg, bars: 2 μm. (b) GBM thickness. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (c) Sep or L-arg decreased albuminuria in db/db mice at 34 weeks of age. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (d) Sep or L-arg had no effect on hyperfiltration seen in db/db mice, determined by fluorescein isothiocyanate-inulin clearance. *P<0.05, BKS versus db/db mice regardless of treatments. (e) Sep or L-arg decreased elevations in urine isoprostane excretion in db/db mice. *P<0.05, compared with wild-type BKS; #P<0.05, versus each treatment. (f) Increased nitrotyrosine accumulation in db/db mice was attenuated by Sep or L-arg treatment. Representative photo from three independent experiments. GFR, glomerular filtration rate.
Mentions: db/db mice are a well-established type 2 diabetic model, but some strains are relatively resistant to diabetic renal injury despite the significant hyperglycemia.15, 16db/db mice on the BKS background have been shown to develop hyperglycemia and diabetic renal injury.17, 18 We found that at 34 weeks, db/db (BKS) mice had developed glomerulopathy characterized by mesangial matrix accumulation (Figure 3a), foot process effacement, and increased glomerular basement membrane (GBM) thickness (154±6 in BKS control vs. 335±23 nm in untreated db/db mice) (Figure 3a and b) and had significant albuminuria (alb/cre: 18±3 in BKS control vs. 759±281 in untreated db/db, n=8, P<0.05) (Figure 3c) and hyperfiltration (glomerular filtration rate: 206±33 μl/min in BKS control; 403±36 in untreated db/db, n=6–12, P<0.05) (Figure 3d). Furthermore, urine F2-isoprostanes, a marker of oxidative stress, increased in the diabetic mice (5.7±0.9 in db/db mice vs. 1.4±0.1 ng 8-iso-PGF2α/mg Cr in BKS control, n=6–11, P<0.05) (Figure 3e). Increased oxidative stress in db/db mice was also confirmed by the accumulation of nitrotyrosine, another superoxide marker (Figure 3f).

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