RESUMO
High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H2S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H2S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H2S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H2S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N(ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H2S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H2S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.
Assuntos
Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Sulfeto de Hidrogênio/farmacologia , Túbulos Renais Proximais/enzimologia , NADPH Oxidases/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/terapia , Células Epiteliais/patologia , Proteínas da Matriz Extracelular/metabolismo , Túbulos Renais Proximais/patologia , Camundongos , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacosRESUMO
Systemic fibrosis can be induced in humans with gadolinium-based contrast, and cumulative doses correlate with severity. Bone marrow-derived fibrocytes accumulate in the dermis. Whether target organs liberate chemokines to recruit these fibrocytes or whether fibrocytes are stimulated to home to the affected tissue is unknown. Transgenic (tagged) donor rats were treated with gadolinium-based contrast. Bone marrow was obtained from diseased animals and age-matched controls. Rats with subtotal nephrectomies were lethally irradiated and underwent salvage transplantation with either the contrast-naïve or contrast-exposed bone marrow. Groups were randomly assigned to control or contrast treatment. Contrast treatment led to dermal fibrosis, and this was exacerbated in recipients of contrast-exposed marrow. Fibronectin, C-C chemokine receptors (CCRs)2 and 7, and oxidative stress were all increased in skin from contrast-treated animals-all parameters more severe in recipients of contrast-treated animals. The respective ligands, monocyte chemoattractant protein and C-C motif ligand 19, were both elevated in skin from contrast-treated animals. Coadministration of gadolinium-based contrast and a CCR2 inhibitor reduced the severity of skin disease as well as dermal cellularity. The functional role of chemokines in the effects of gadolinium-based contrast was further confirmed in in situ coculture studies using neutralizing CCR2 antibodies. These data implicate dermal liberation of specific chemokines in the recruitment of circulating bone marrow-derived cells. The disease is augmented by bone marrow exposure to contrast, which explains why multiple exposures correlate with severity.-Drel, V. R., Tan, C., Barnes, J. L., Gorin, Y., Lee, D.-Y., Wagner, B. Centrality of bone marrow in the severity of gadolinium-based contrast-induced systemic fibrosis.
Assuntos
Medula Óssea/efeitos dos fármacos , Meios de Contraste/efeitos adversos , Gadolínio DTPA/efeitos adversos , Dermopatia Fibrosante Nefrogênica/induzido quimicamente , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Transplante de Medula Óssea , Meios de Contraste/administração & dosagem , Meios de Contraste/farmacologia , Feminino , Gadolínio DTPA/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Masculino , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Dermopatia Fibrosante Nefrogênica/patologia , Distribuição Aleatória , Ratos , Espécies Reativas de Oxigênio , Receptores CCR2/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismoRESUMO
Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase ß. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carbolinas/química , Glucose/química , Sulfeto de Hidrogênio/química , Óxido Nítrico/química , Podócitos/metabolismo , Transdução de Sinais , Animais , Cálcio/química , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Rim/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Inibidores da Fosfodiesterase 5/química , Fosforilação , Podócitos/citologia , Polirribossomos/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo , TadalafilaRESUMO
Systemic fibrosis from gadolinium-based magnetic resonance imaging contrast is a scourge for the afflicted. Although gadolinium-associated systemic fibrosis is a rare condition, the threat of litigation has vastly altered clinical practice. Most theories concerning the etiology of the fibrosis are grounded in case reports rather than experiment. This has led to the widely accepted conjecture that the relative affinity of certain contrast agents for the gadolinium ion inversely correlates with the risk of succumbing to the disease. How gadolinium-containing contrast agents trigger widespread and site-specific systemic fibrosis and how chronicity is maintained are largely unknown. This review highlights experimentally-derived information from our laboratory and others that pertain to our understanding of the pathophysiology of gadolinium-associated systemic fibrosis.
Assuntos
Meios de Contraste/efeitos adversos , Fibrose/induzido quimicamente , Gadolínio/efeitos adversos , Imageamento por Ressonância Magnética/efeitos adversos , Fibrose/epidemiologia , Fibrose/patologia , HumanosRESUMO
Transforming growth factor (TGF)-ß contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-ß exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-ß1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. TGF-ß1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-ß1 functions. Conversely, inhibition of AMPK with adenine 9-ß-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-ß1 and enhanced basal and TGF-ß1-induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-ß1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-ß transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-ß exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-ß. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Caderinas/metabolismo , Fibronectinas/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Camundongos , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Pirazolonas , Piridonas , Espécies Reativas de Oxigênio/metabolismoRESUMO
Platelet-derived growth factor BB and its receptor (PDGFRß) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.
Assuntos
Proliferação de Células , Retroalimentação Fisiológica/fisiologia , Células Mesangiais/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Proteína Oncogênica v-akt/biossíntese , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Retroalimentação Fisiológica/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , RatosRESUMO
Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.
Assuntos
Angiotensina II/farmacologia , Fibronectinas/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Disponibilidade Biológica , Fibrose , Inativação Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/enzimologia , Células Mesangiais/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Modelos Biológicos , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Ratos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor ß is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Quinases da Família src/metabolismo , Animais , Becaplermina , Movimento Celular , Células Cultivadas , Cromonas/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Morfolinas/farmacologia , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxirredução , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases da Família src/genéticaRESUMO
The small G proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory have shown that NADPH oxidase Nox4-derived ROS are involved in transforming growth factor (TGF)-ß1-induced rat kidney myofibroblast differentiation assessed by the acquisition of an α-smooth muscle actin (α-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link among Rac1 and RhoA and Nox4-dependent ROS generation in TGF-ß1-induced kidney myofibroblast activation. TGF-ß1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant α-SMA and Fn-EIIIA expression. RhoA but not Rac1 was involved in TGF-ß1 induction of Nox4 signaling of kidney myofibroblast activation. TGF-ß1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering RNA and ROCK using Y-27632 significantly reduced TGF-ß1-induced stimulation of Nox4 protein, NADPH oxidase activity, and α-SMA and Fn-EIIIA expression. Treatment with diphenyleneiodonium, an inhibitor of NADPH oxidase, did not decrease RhoA activation but inhibited TGF-ß1-induced α-SMA and Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase (DNA-directed) δ-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Rim/enzimologia , Miofibroblastos/enzimologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Movimento Celular , Ativação Enzimática , Fibronectinas/metabolismo , Fibrose , Rim/patologia , Miofibroblastos/patologia , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Oxirredução , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Ratos , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression, and 150% the number of apoptotic cells, effects that were completely blocked by 10 µM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out that was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.
Assuntos
Antioxidantes/uso terapêutico , Nefropatias Diabéticas/prevenção & controle , Glucose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Silimarina/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/fisiopatologia , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/biossíntese , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Silibina , Superóxidos/metabolismoRESUMO
Matrix protein accumulation is a prominent feature of diabetic nephropathy that contributes to renal fibrosis and decline in renal function. The pathogenic mechanisms of matrix accumulation are incompletely characterized. We investigated if the matrix metalloprotease a disintegrin and metalloprotease1 7 (ADAM17), known to cleave growth factors and cytokines, is activated in the kidney cortex of OVE26 type 1 diabetic mice and the potential mechanisms by which ADAM17 mediates extracellular matrix accumulation. Protein expression and activity of ADAM17 were increased in OVE26 kidney cortex. Using a pharmacological inhibitor to ADAM17, TMI-005, we determined that ADAM17 activation results in increased type IV collagen, Nox4, and NADPH oxidase activity in the kidney cortex of diabetic mice. In cultured mouse proximal tubular epithelial cells (MCTs), high glucose increases ADAM17 activity, Nox4 and fibronectin expression, cellular collagen content, and NADPH oxidase activity. These effects of glucose were inhibited when cells were pretreated with TMI-005 and/or transfected with small interfering ADAM17. Collectively, these data indicate a novel mechanism whereby hyperglycemia in diabetes increases extracellular matrix protein expression in the kidney cortex through activation of ADAM17 and enhanced oxidative stress through Nox enzyme activation. Additionally, our study is the first to provide evidence that Nox4 is downstream of ADAM17.
Assuntos
Proteínas ADAM/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Córtex Renal/metabolismo , NADPH Oxidases/metabolismo , Proteína ADAM17 , Animais , Western Blotting , Peso Corporal/fisiologia , Colágeno/metabolismo , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Imunofluorescência , Glucose/metabolismo , Glucose/farmacologia , Técnicas Imunoenzimáticas , Córtex Renal/enzimologia , Masculino , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Tamanho do Órgão/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
Watanabe et al. report that Nox4 NADPH oxidase catalytic moiety and the subunit p22(phox) mediate the increase in oxidative stress and human tubular epithelial cell injury induced by p-cresyl sulfate, a protein-bound uremic toxin. These findings could be instrumental for the design of novel therapeutic intervention utilizing small-molecule inhibitors specifically targeting Nox oxidases to prevent or slow down the progression of chronic kidney disease and the associated disorders due to uremic toxicity.
Assuntos
Cresóis/toxicidade , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Insuficiência Renal Crônica/induzido quimicamente , Ésteres do Ácido Sulfúrico/toxicidade , Animais , Humanos , MasculinoRESUMO
Nephrogenic systemic fibrosis (NSF) is associated with gadolinium-based magnetic resonance imaging (MRI) contrast exposure in the setting of acute or chronic renal compromise. It has been proposed that circulating fibrocytes mediate the disease. A study was conducted to determine whether bone marrow-derived fibroblast precursors are involved in contributing to organ fibrosis in MRI contrast-treated rodents with renal insufficiency. Rats status post 5/6 nephrectomy underwent bone marrow transplant from human placental alkaline phosphatase (hPAP)-expressing donors. After engraftment, animals were treated with gadolinium-based MRI contrast (2.5 mmol/kg IP), during weekdays for 4 weeks, or an equivalent volume of normal saline. Dermal cellularity in the contrast-treated group was fourfold that of control. Skin cells from the contrast-treated group demonstrated greater hPAP expression with co-expression of pro-collagen I and α-smooth muscle actin-positive stress fibers. Donor and host cells expressed CD34. Dihydroethidium staining of skin was greater in the contrast-treated animals, indicating oxidative stress. This was abrogated when the animals were co-administered the superoxide dismutase mimetic tempol. In conclusion, a bone marrow-derived cell population is increased in the dermis of MRI contrast-treated rodents. The cell markers are consistent with fibrocytes mediating the disease. These changes correlate with oxidative stress and expression of Nox4, suggestive of a novel therapeutic target. Elucidation of the mechanisms of MRI contrast-induced fibrosis may aid in discovering therapies to this devastating disease.
Assuntos
Medula Óssea/patologia , Fibroblastos/patologia , Fígado/patologia , Miocárdio/patologia , Dermopatia Fibrosante Nefrogênica/patologia , Estresse Oxidativo , Pele/patologia , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Colágeno Tipo I/metabolismo , Meios de Contraste/efeitos adversos , Derme/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fator XIIIa/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose , Humanos , Fígado/metabolismo , Imageamento por Ressonância Magnética , Nefrectomia , Dermopatia Fibrosante Nefrogênica/metabolismo , Tamanho do Órgão , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Pele/metabolismo , Dobras CutâneasRESUMO
Oxidative stress has been linked to the pathogenesis of the major complications of diabetes in the kidney, the heart, the eye or the vasculature. NADPH oxidases of the Nox family are a major source of ROS (reactive oxygen species) and are critical mediators of redox signalling in cells from different organs afflicted by the diabetic milieu. In the present review, we provide an overview of the current knowledge related to the understanding of the role of Nox in the processes that control cell injury induced by hyperglycaemia and other predominant factors enhanced in diabetes, including the renin-angiotensin system, TGF-ß (transforming growth factor-ß) and AGEs (advanced glycation end-products). These observations support a critical role for Nox homologues in diabetic complications and indicate that NADPH oxidases are an important therapeutic target. Therefore the design and development of small-molecule inhibitors that selectively block Nox oxidases appears to be a reasonable approach to prevent or retard the complications of diabetes in target organs. The bioefficacy of these agents in experimental animal models is also discussed in the present review.
Assuntos
Complicações do Diabetes/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Complicações do Diabetes/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/prevenção & controle , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , Espécies Reativas de Oxigênio/antagonistas & inibidores , Sistema Renina-Angiotensina/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoRESUMO
Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.
Assuntos
Fibronectinas/biossíntese , Fator de Crescimento Insulin-Like I/fisiologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais/metabolismo , NADPH Oxidases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/fisiologia , Animais , Linhagem Celular Transformada , Células Cultivadas , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Humanos , Túbulos Renais Proximais/fisiologia , Camundongos , NADPH Oxidase 4 , Fosforilação/fisiologia , Biossíntese de Proteínas/fisiologia , Ratos , Transcrição Gênica/fisiologiaRESUMO
Oxidative stress contributes to diabetic cardiomyopathy. This study explored the role of the NADPH oxidase Nox4 as a source of reactive oxygen species (ROS) involved in the development of diabetic cardiomyopathy. Phosphorothioated antisense (AS) or sense (S) oligonucleotides for Nox4 were administered for 2 wk to rats made diabetic by streptozotocin. NADPH oxidase activity, ROS generation, and the expression of Nox4, but Nox1 or Nox2, were increased in left ventricular tissue of the diabetic rats. Expression of molecular markers of hypertrophy and myofibrosis including fibronectin, collagen, α-smooth muscle actin, and ß-myosin heavy chain were also increased. These parameters were attenuated by the administration of AS but not S Nox4. Moreover, the impairment of contractility observed in diabetic rats was prevented in AS- but not S-treated animals. Exposure of cultured cardiac myocytes to 25 mM glucose [high glucose (HG)] increased NADPH oxidase activity, the expression of Nox4, and molecular markers of cardiac injury. These effects of HG were prevented in cells infected with adenoviral vector containing a dominant negative form of Nox4. This study provides strong evidence that Nox4 is an important source of ROS in the left ventricle and that Nox4-derived ROS contribute to cardiomyopathy at early stages of type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Actinas/biossíntese , Animais , Células Cultivadas , Colágeno/biossíntese , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicações , Cardiomiopatias Diabéticas/patologia , Fibronectinas/biossíntese , Glucose/farmacologia , Humanos , Masculino , Glicoproteínas de Membrana/biossíntese , NADH NADPH Oxirredutases/biossíntese , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Miosinas Ventriculares/biossínteseRESUMO
BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , NADPH Oxidases/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Camundongos , NADPH Oxidase 4 , RNA Mensageiro/genéticaRESUMO
Oxidative stress is implicated in human diseases. Some of the oxidative pathways are harbored in the mitochondria. NAD(P)H oxidases have been identified not only in phagocytic but also in somatic cells. Nox4 is the most ubiquitous of these oxidases and is a major source of reactive oxygen species (ROS) in many cell types and in kidney tissue of diabetic animals. We generated specific Nox4 antibodies, and found that Nox4 localizes to mitochondria. (i) Immunoblot analysis in cultured mesangial cells and kidney cortex revealed that Nox4 is present in crude mitochondria, in mitochondria-enriched heavy fractions, and in purified mitochondria; (ii) immunofluorescence confocal microscopy also revealed that Nox4 localizes with the mitochondrial marker Mitotracker; and (iii) the mitochondrial localization prediction program MitoProt indicated that the probability score for Nox4 is identical to mitochondrial protein cytochrome c oxidase subunit IV. We also show that in purified mitochondria, siRNA-mediated knockdown of Nox4 significantly reduces NADPH oxidase activity in pure mitochondria and blocks glucose-induced mitochondrial superoxide generation. In a rat model of diabetes, mitochondrial Nox4 expression is increased in kidney cortex. Our data provide evidence that a functional Nox4 is present and regulated in mitochondria, indicating the existence of a previously undescribed source of ROS in this organelle.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Animais , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica , Imunoprecipitação , Córtex Renal/citologia , Córtex Renal/metabolismo , Masculino , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Microscopia Confocal , NADPH Oxidase 4 , NADPH Oxidases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The present study was performed to investigate the underlying mechanism, particularly the roles of reactive oxygen species (ROS) and protein kinase C (PKC), in the diabetes-induced canonical transient receptor potential 6 (TRPC6) downregulation. We found that high glucose (HG) significantly reduced TRPC6 protein expression in cultured mesangial cells (MCs). TRPC6 protein was also significantly reduced in the glomeruli but not in the heart or aorta isolated from streptozotocin-induced diabetic rats. In the cultured MCs, H(2)O(2) suppressed TRPC6 protein expression in a dose- and time-dependent manner, which emulated the HG effect. Catalase as well as superoxide dismutase were able to prevent the inhibitory effect of HG on TRPC6. The antioxidant effect observed in cultured cells was also observed in diabetic rats treated with tempol for 2 wk, which exhibited a preservation of TRPC6 in the glomeruli. Specific knockdown of Nox4, a component of NADPH oxidase, increased TRPC6 protein expression. Furthermore, the PKC activator phorbol 12-myristate 13-acetate (PMA), but not its analog 4α-phorbol 12, 13-didecanoate (4α-PDD), suppressed TRPC6 expression, and this PMA effect was not affected by catalase. Moreover, Gö6976, but not LY333531, attenuated the negative effect of HG on TRPC6 expression. Gö6976 also inhibited H(2)O(2) effect on TRPC6. Furthermore, either knockdown of TRPC6 or HG treatment significantly decreased ANG II-stimulated MC contraction, and the HG-impaired MC contraction was rescued by overexpression of TRPC6. These results suggest that hyperglycemia in diabetes downregulated TRPC6 protein expression in MCs through a NADPH oxidase Nox4-ROS-PKC pathway, proving a mechanism for impaired MC contraction in diabetes.