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

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 ?-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.

SUBMITTER: Nagasu H

PROVIDER: S-EPMC4874489 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

Nagasu Hajime H Satoh Minoru M Kiyokage Emi E Kidokoro Kengo K Toida Kazunori K Channon Keith M KM Kanwar Yashpal S YS Sasaki Tamaki T Kashihara Naoki N

Laboratory investigation; a journal of technical methods and pathology 20151109 1

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 ...[more]

PMID: 26552047

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Project description:BACKGROUND:Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS:We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS:Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS:These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

Project description:RationaleThe kynurenine (Kyn) pathway is the major route for tryptophan (Trp) metabolism in mammals. The Trp-Kyn pathway is reported to regulate several fundamental biological processes, including cell death.ObjectiveThe aim of this study was to elucidate the contributions and molecular mechanism of Trp-Kyn pathway to endothelial cell death.Methods and resultsEndogenous reactive oxygen species, endothelial cell apoptosis, and endothelium-dependent and endothelium-independent vasorelaxation were measured in aortas of wild-type mice or mice deficient for nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase subunits (p47(phox) or gp91(phox)) or indoleamine-pyrrole 2,3-dioxygenase 1 with or without angiotensin (Ang) II infusion. As expected, AngII increased plasma levels of Kyn- and 3-hydroxykynurenine-modified proteins in endothelial cells in vivo. Consistent with this, AngII markedly increased the expression of indoleamine-pyrrole 2,3-dioxygenase in parallel with increased expression of interferon-γ. Furthermore, in wild-type mice, AngII significantly increased oxidative stress, endothelial cell apoptosis, and endothelial dysfunction. These effects of AngII infusion were significantly suppressed in mice deficient for p47(phox), gp91(phox), or indoleamine-pyrrole 2,3-dioxygenase 1, suggesting that AngII-induced enhancement of Kynurenines via NAD(P)H oxidase-derived oxidants causes endothelial cell apoptosis and dysfunction in vivo. Furthermore, interferon-γ neutralization eliminates AngII-increased superoxide products and endothelial apoptosis by inhibiting AngII-induced Kynurenines generation, suggesting that AngII-activated Kyn pathway is interferon-γ-dependent. Mechanistically, we found that AngII-enhanced 3-hydroxykynurenine promoted the generation of NAD(P)H oxidase-mediated superoxide anions by increasing the translocation and membrane assembly of NAD(P)H oxidase subunits in endothelial cells, resulting in accelerated apoptosis and consequent endothelial dysfunction.ConclusionsKyn pathway activation accelerates apoptosis and dysfunction of the endothelium by upregulating NAD(P)H-derived superoxide.

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

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

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