Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice - PubMed (original) (raw)

Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

Hajime Nagasu et al. Lab Invest. 2016 Jan.

Abstract

Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes.

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Figures

Figure 1

Figure 1

Analysis of urinary albumin excretion of wild-type mice (WT, open circles), endothelial dominant Nox2 transgenic mice (NOX2TG; open squares), heterozygous Akita mice (_Ins2_Akita/Ins2+, black solid circles), and heterozygous Akita mice (Ins2_Akita/Ins2+) expressing a Nox2 transgene (NOX2TG-Akita, black solid squares), 10 mice per group. *P<0.05 versus WT group; †_P<0.05 versus Akita group.

Figure 2

Figure 2

Glomerular superoxide production and morphological changes in glomeruli of mice from various strains at 6 weeks of age. (a) Reactive oxygen species (ROS) detected by staining with 2′,7′-dichlorofluorescein (DCF) in isolated glomeruli. Magnification × 400. (b) Intensity of DCF staining relative to WT, 10 mice per group. Twenty glomeruli from each mouse were evaluated to calculate the mean intensity. *P<0.05 versus WT group; †P<0.05 versus Akita group. (c) Periodic acid-Schiff (PAS) staining images of glomeruli. Magnification × 400. (d) Glomerular matrix score, 10 mice per group. Twenty glomeruli from each mouse were evaluated to calculate the mean score was calculated. *P<0.05 versus WT group; †P<0.05 versus Akita group. NOX2TG, endothelial dominant Nox2 transgenic mice; NOX2TG-Akita, heterozygous Akita mice (_Ins2_Akita/Ins2+) expressing a Nox2 transgene; WT, wild-type mice.

Figure 3

Figure 3

Evaluation of glomerular endothelial injury and morphological changes in mice from various groups at 6 weeks of age. (a) Immunohistochemical analysis of plasmalemmal vesicle-1 (PV-1), a marker of glomerular capillary remodeling. Magnification × 400. (b) PV-1 staining score, 10 mice per group. Ten glomeruli from each mouse were evaluated to calculate the mean score. *P<0.05 versus WT group; †P<0.05 versus Akita group. (c) Assessment of glycocalyx of the endothelial surface layer detected by using tomato lectin staining. Magnification × 400. (d) Lectin-positive areas compared with WT, 10 mice per group. Ten glomeruli from each mouse were evaluated to calculate the mean area. *P<0.05 versus WT group; †P<0.05 versus Akita group. (e) Scanning electron micrographs of activated and swollen endothelial cells (arrows) of NOX2TG-Akita mice. Note the partial occlusion and disappearance of fenestrae (arrowheads). Scale bar = 1.0 _μ_m. (f) Transmission electron micrographs of glomerular capillary endothelia. Note the partial disappearance of fenestrae in NOX2TG-Akita mice (arrowheads). Scale bar = 1.0 _μ_m. ND, not detected; NOX2TG, endothelial dominant Nox2 transgenic mice; NOX2TG-Akita, heterozygous Akita mice (_Ins2_Akita/Ins2+) expressing a Nox2 transgene; WT, wild type.

Figure 4

Figure 4

Analysis of glomerular filtration in mice 6 weeks of age. Representative series of images showing filtration of macromolecules in the glomeruli of the indicated mouse group. Green color indicates the localization of 70-kDa fluorescein isothiocyanate (FITC)-labeled dextran. Magnification × 200.

Figure 5

Figure 5

Analyses of mesangial lesions in various groups of mice at 6 weeks of age. (a) Immunohistochemical analysis of _α_-smooth muscle actin (_α_-SMA) expression in glomeruli. Magnification × 400. (b) α_-SMA staining score, 10 mice per group. Ten glomeruli from each mouse were evaluated to calculate the mean score. *P<0.05 versus WT group; †_P<0.05 versus Akita group. (c) Electron micrographs of representative glomeruli from Akita and NOX2TG-Akita mice showing notable expansion of the mesangium in the latter group (arrows). Swollen endothelial cells are indicated by the arrowheads. Scale bar = 10 _μ_m. NOX2TG, endothelial dominant Nox2 transgenic mice; NOX2TG-Akita, heterozygous Akita mice (_Ins2_Akita/Ins2+) expressing a Nox2 transgene; WT, wild type.

Figure 6

Figure 6

Analysis of podocyte injury and morphological changes in mice at 6 weeks of age. (a) Immunohistochemical analysis of desmin expression in glomeruli, which is tremendously increased in NOX2TG-Akita mice group. Magnification × 400. (b) Desmin staining score, 10 mice per group. Ten glomeruli from each mouse were evaluated to calculate the mean score. *P<0.05 versus WT group; †P<0.05 versus Akita group. (c) Electron micrographs depicting ultrastructural changes in podocytes of Akita and NOX2TG-Akita mice. Numerous microvilli-like structures in the podocytes are readily seen (arrows). Scale bar = 5.0 _μ_m. NOX2TG, endothelial dominant Nox2 transgenic mice; NOX2TG-Akita, heterozygous Akita mice (_Ins2_Akita/Ins2+) expressing a Nox2 transgene; WT, wild type.

Figure 7

Figure 7

Analysis of the glomerular glycocalyx and urinary albumin excretion in mice NOX2TG-Akita treated with the NOX2 inhibitor gp91TAT. (a) Glycocalyx was detected using tomato lectin. Magnification × 400. (b) Relative lectin-positive area to WT. Treatment with gp91TAT led to the recovery of the lectin-stained area, 10 mice per group. Ten glomeruli from each mouse were evaluated to calculate the mean area. (c) Urinary albumin excretion. Cre, urinary creatinine. *P<0.05 versus WT group; †P<0.05. NOX2TG, endothelial dominant Nox2 transgenic mice; NOX2TG-Akita, heterozygous Akita mice (_Ins2_Akita/Ins2+) expressing a Nox2 transgene; WT, wild type.

References

    1. Alebiosu CO, Ayodele OE. The global burden of chronic kidney disease and the way forward. Ethn Dis. 2005;15:418–423. - PubMed
    1. Krolewski AS, Warram JH, Rand LI, et al. Epidemiologic approach to the etiology of type I diabetes mellitus and its complications. N Engl J Med. 1987;317:1390–1398. - PubMed
    1. Maeda S, Kobayashi MA, Araki S, et al. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes. PLoS Genet. 2010;6:e1000842. - PMC - PubMed
    1. Hamada Y, Miyata S, Nii-Kono T, et al. Overexpression of thioredoxin1 in transgenic mice suppresses development of diabetic nephropathy. Nephrol Dial Transplant. 2007;22:1547–1557. - PubMed
    1. Raj DS, Choudhury D, Welbourne TC, et al. Advanced glycation end products: a Nephrologist's perspective. Am J Kidney Dis. 2000;35:365–380. - PubMed

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