RESUMO
NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in retinal endothelial cells (ECs) and is upregulated under hyperglycemic and hypoxic conditions. However, the role of endothelial Nox4 upregulation in long-term retinal blood vessel damage in diabetic retinopathy (DR) remains undefined. Here, we attempted to address this question using humanized EC-specific Nox4 transgenic (hNox4EC-Tg) and EC-specific Nox4 knockout (Nox4EC-KO) mouse models. Our results show that hNox4EC-Tg mice at age of 10-12 months exhibited increased tortuosity of retinal blood vessels, focal vascular leakage, and acellular capillary formation. In vitro study revealed enhanced apoptosis in brain microvascular ECs derived from hNox4EC-Tg mice, concomitant with increased mitochondrial ROS, elevated lipid peroxidation, decreased mitochondrial membrane potential, and reduced mitochondrial respiratory function. In contrast, EC-specific deletion of Nox4 decreased mitochondrial ROS generation, alleviated mitochondrial damage, reduced EC apoptosis, and protected the retina from acellular capillary formation and vascular hyperpermeability in a streptozotocin-induced diabetes mouse model. These findings suggest that sustained upregulation of Nox4 in the endothelium contributes to retinal vascular pathology in diabetes, at least in part, through impairing mitochondrial function. Normalization of Nox4 expression in ECs may provide a new approach for prevention of vascular injury in DR.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Retinopatia Diabética , Camundongos , Animais , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Células Endoteliais/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Retinopatia Diabética/metabolismo , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismoRESUMO
NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC-KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.
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Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Deleção de Genes , NADPH Oxidase 4/metabolismo , Neovascularização Fisiológica , Neovascularização Retiniana/enzimologia , Vasos Retinianos/crescimento & desenvolvimento , Animais , Camundongos , Camundongos Knockout , NADPH Oxidase 4/genética , Neovascularização Retiniana/genéticaRESUMO
OBJECTIVE: Glomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. METHODS: We studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. RESULTS: We demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3' untranslated region (3'-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3'-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. CONCLUSIONS: We established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.
Assuntos
Nefropatias Diabéticas/genética , Proteína Semelhante a ELAV 1/metabolismo , NADPH Oxidase 4/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/terapia , Proteína Semelhante a ELAV 1/genética , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/genética , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanism by which TSC2 inactivation or deficiency contributes to the pathology of tuberous sclerosis complex (TSC) is not fully clear. We show that renal angiomyolipomas from TSC patients and kidney cortex from Tsc2+/- mice exhibit elevated levels of reactive oxygen species (ROS). Downregulation of tuberin (protein encoded by TSC2 gene) in renal proximal tubular epithelial cells significantly increased ROS concomitant with enhanced Nox4. Similarly, we found elevated levels of Nox4 in the renal cortex of Tsc2+/- mice and in the renal angiomyolipomas from TSC patients. Tuberin deficiency is associated with activation of mTORC1. Rapamycin, shRNAs targeting raptor, or inhibition of S6 kinase significantly inhibited the expression of Nox4, resulting in attenuation of production of ROS in tuberin-downregulated proximal tubular epithelial cells. In contrast, activation of mTORC1 increased Nox4 and ROS. These results indicate that Nox4 may be a potential target for tuberin-deficiency-derived diseases. Using a xenograft model from tuberin-null tubular cells in nude mice, both anti-sense Nox4 and GKT137831, a specific inhibitor of Nox1/4, significantly inhibited the tumor growth. Thus, our results demonstrate the presence of an antagonistic relationship between tuberin and Nox4 to drive oncogenesis in the tuberin deficiency syndrome and identify Nox4 as a target to develop a therapy for TSC.
Assuntos
Angiomiolipoma/patologia , Nefropatias/patologia , Rim/patologia , NADPH Oxidase 4/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Angiomiolipoma/complicações , Angiomiolipoma/metabolismo , Animais , Estudos de Casos e Controles , Humanos , Rim/metabolismo , Nefropatias/complicações , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Nus , NADPH Oxidase 4/genética , Espécies Reativas de Oxigênio/metabolismo , Síndrome , Esclerose Tuberosa/complicações , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary-like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-ß cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.
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Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica/fisiologia , Animais , Becaplermina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with â¼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage-dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.
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Diabetes Mellitus Tipo 2/genética , Fator de Crescimento Insulin-Like II/metabolismo , Sítios de Splice de RNA/genética , Tecido Adiposo , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Variação Genética , Genótipo , Humanos , Fator de Crescimento Insulin-Like II/genética , Fígado , Americanos Mexicanos/genética , México , Isoformas de Proteínas , Células-Tronco , População BrancaRESUMO
OBJECTIVE: Type 2 diabetes (T2D) is the primary case of chronic kidney disease (CKD). Inflammation is associated with metabolic dysregulation in patients with T2D and CKD. Tryptophan (TRP) metabolism may have relevance to the CKD outcomes and associated symptoms. We investigated the relationships of TRP metabolism with inflammatory markers in patients with T2D and CKD. METHODS: Data were collected from a well-characterized cohort of type 2 diabetic individuals with all stages of CKD, including patients on hemodialysis. Key TRP metabolites (kynurenine [KYN], kynurenic acid [KYNA], and quinolinic acid [QA]), proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]), and C-reactive protein were measured in plasma. The KYN/TRP ratio was utilized as a surrogate marker for indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity. RESULTS: There was a significant inverse association between circulating TRP level and stages of CKD (P< 0.0001). Downstream bioactive TRP metabolites KYN, KYNA, and QA were positively and robustly correlated with the severity of kidney disease (P < 0.0001). In multiple linear regression, neither TNF-α nor IL-6 was independently related to KYN/TRP ratio after adjusting for estimated glomerular filtration rate (eGFR). Only TNF-α was independently related to KYN after taking into account the effect of eGFR. CONCLUSIONS: Chronic kidney disease secondary to T2D may be associated with accumulation of toxic TRP metabolites due to both inflammation and impaired kidney function. Future longitudinal studies to determine whether the accumulation of KYN directly contributes to CKD progression and associated symptoms in patients with T2D are warranted.
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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
BACKGROUND/AIM: Toll-like receptor 4 (TLR4) is one of the regulators of the innate immune response. Genetic variations in TLR4 have been associated with inflammatory diseases, including type 2 diabetes. However, to our knowledge, there are no reports on the role of variations in TLR4 in chronic kidney disease susceptibility. The objective of this study is to determine whether the genetic variants in TLR4 are associated with the estimated glomerular filtration rate (eGFR), a measure of renal function. METHODS: To evaluate the association between TLR4 variants and eGFR, we used data obtained from 434 Mexican American participants from the San Antonio Family Diabetes/Gallbladder Study. GFR was estimated using the Modification of Diet in Renal Disease formula. The Asp(299)Gly (rs4986790) and Thr(399)Ile (rs4986791) variants of TLR4 were genotyped using the TaqMan assay. Association analyses between genotypes and eGFR were performed using the measured genotype approach. RESULTS: Of the two genetic markers examined for association, only the Asp(299)Gly variant of TLR4 exhibited a nominally significant association with eGFR (p = 0.025) after accounting for the covariate effects of age and sex terms, diabetes, duration of diabetes, systolic blood pressure, body mass index, and antihypertensive treatment. Carriers of Gly299 had significantly decreased eGFR values. Although, the Thr(399)Ile variant failed to exhibit a statistically significant association with eGFR, the carriers of Ile399, however, showed a trend towards decrease in eGFR. CONCLUSION: We show for the first time that Asp(299)Gly variants of TLR4 are associated with decrease in renal function in Mexican Americans.
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AIM: Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. We explored the hypothesis that mammalian target of rapamycin complex 2 (mTORC2) mediates podocyte injury in diabetes. RESULTS: High glucose (HG)-induced podocyte injury reflected by alterations in the slit diaphragm protein podocin and podocyte depletion/apoptosis. This was paralleled by activation of the Rictor/mTORC2/Akt pathway. HG also increased the levels of Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using small interfering RNA (siRNA)-targeting Rictor in vitro decreased HG-induced Nox1 and Nox4, NADPH oxidase activity, restored podocin levels, and reduced podocyte depletion/apoptosis. Inhibition of mTORC2 had no effect on mammalian target of rapamycin complex 1 (mTORC1) activation, described by our group to be increased in diabetes, suggesting that the mTORC2 activation by HG could mediate podocyte injury independently of mTORC1. In isolated glomeruli of OVE26 mice, there was a similar activation of the Rictor/mTORC2/Akt signaling pathway with increase in Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using antisense oligonucleotides targeting Rictor restored podocin levels, reduced podocyte depletion/apoptosis, and attenuated glomerular injury and albuminuria. INNOVATION: Our data provide evidence for a novel function of mTORC2 in NADPH oxidase-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. CONCLUSION: mTORC2 and/or NADPH oxidase inhibition may represent a therapeutic modality for diabetic kidney disease. Antioxid. Redox Signal. 25, 703-719.
Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Glucose/farmacologia , Complexos Multiproteicos/metabolismo , NADPH Oxidases/genética , Podócitos/citologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Proteínas de Transporte/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Membrana/metabolismo , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Reactive oxygen species (ROS) are an important endogenous source of DNA damage and oxidative stress in all cell types. Deficiency in tuberin resulted in increased oxidative DNA damage in renal cells. In this study, the role of tuberin in the regulating of ROS and NADPH oxidases was investigated. Formation of ROS and activity of NADPH oxidases were significantly higher in mouse embryonic fibroblasts and in primary culture of rat renal proximal tubular epithelial tuberin-deficient cells compared to wild-type cells. In addition, expression of NADPH oxidase (Nox)1, Nox2, and Nox4 (Nox isoforms) was higher in mouse embryonic fibroblasts and renal proximal tubular epithelial tuberin-deficient cells compared to wild-type cells. Furthermore, activity levels of NADPH oxidases and protein expression of all Nox isoforms were higher in the renal cortex of rat deficient in tuberin. However, treatment of tuberin-deficient cells with rapamycin showed significant decrease in protein expression of all Nox. Significant increase in protein kinase C ßII expression was detected in tuberin-deficient cells, whereas inhibition of protein kinase C ßII by bisindolylmaleimide I resulted in decreased protein expression of all Nox isoforms. In addition, treatment of mice deficient in tuberin with rapamycin resulted in significant decrease in all Nox protein expression. Moreover, protein and mRNA expression of all Nox were highly expressed in tumor kidney tissue of patients with tuberous sclerosis complex compared to control kidney tissue of normal subjects. These data provide the first evidence that tuberin plays a novel role in regulating ROS generation, NADPH oxidase activity, and Nox expression that may potentially be involved in development of kidney tumor in patients with tuberous sclerosis complex.
Assuntos
Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Córtex Renal/enzimologia , Córtex Renal/metabolismo , Masculino , Camundongos , NADPH Oxidase 1 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiênciaRESUMO
BACKGROUND: The presence of population structure in a sample may confound the search for important genetic loci associated with disease. Our four samples in the Family Investigation of Nephropathy and Diabetes (FIND), European Americans, Mexican Americans, African Americans, and American Indians are part of a genome- wide association study in which population structure might be particularly important. We therefore decided to study in detail one component of this, individual genetic ancestry (IGA). From SNPs present on the Affymetrix 6.0 Human SNP array, we identified 3 sets of ancestry informative markers (AIMs), each maximized for the information in one the three contrasts among ancestral populations: Europeans (HAPMAP, CEU), Africans (HAPMAP, YRI and LWK), and Native Americans (full heritage Pima Indians). We estimate IGA and present an algorithm for their standard errors, compare IGA to principal components, emphasize the importance of balancing information in the ancestry informative markers (AIMs), and test the association of IGA with diabetic nephropathy in the combined sample. RESULTS: A fixed parental allele maximum likelihood algorithm was applied to the FIND to estimate IGA in four samples: 869 American Indians; 1385 African Americans; 1451 Mexican Americans; and 826 European Americans. When the information in the AIMs is unbalanced, the estimates are incorrect with large error. Individual genetic admixture is highly correlated with principle components for capturing population structure. It takes ~700 SNPs to reduce the average standard error of individual admixture below 0.01. When the samples are combined, the resulting population structure creates associations between IGA and diabetic nephropathy. CONCLUSIONS: The identified set of AIMs, which include American Indian parental allele frequencies, may be particularly useful for estimating genetic admixture in populations from the Americas. Failure to balance information in maximum likelihood, poly-ancestry models creates biased estimates of individual admixture with large error. This also occurs when estimating IGA using the Bayesian clustering method as implemented in the program STRUCTURE. Odds ratios for the associations of IGA with disease are consistent with what is known about the incidence and prevalence of diabetic nephropathy in these populations.
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Negro ou Afro-Americano/genética , Nefropatias Diabéticas/genética , Indígenas Norte-Americanos/genética , Americanos Mexicanos/genética , Polimorfismo de Nucleotídeo Único , População Branca/genética , Algoritmos , Mapeamento Cromossômico , Nefropatias Diabéticas/etnologia , Marcadores Genéticos/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Funções Verossimilhança , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Componente Principal , Estados Unidos/etnologiaRESUMO
Diabetic kidney disease (DKD) is the most common etiology of chronic kidney disease (CKD) in the industrialized world and accounts for much of the excess mortality in patients with diabetes mellitus. Approximately 45% of U.S. patients with incident end-stage kidney disease (ESKD) have DKD. Independent of glycemic control, DKD aggregates in families and has higher incidence rates in African, Mexican, and American Indian ancestral groups relative to European populations. The Family Investigation of Nephropathy and Diabetes (FIND) performed a genome-wide association study (GWAS) contrasting 6,197 unrelated individuals with advanced DKD with healthy and diabetic individuals lacking nephropathy of European American, African American, Mexican American, or American Indian ancestry. A large-scale replication and trans-ethnic meta-analysis included 7,539 additional European American, African American and American Indian DKD cases and non-nephropathy controls. Within ethnic group meta-analysis of discovery GWAS and replication set results identified genome-wide significant evidence for association between DKD and rs12523822 on chromosome 6q25.2 in American Indians (P = 5.74x10-9). The strongest signal of association in the trans-ethnic meta-analysis was with a SNP in strong linkage disequilibrium with rs12523822 (rs955333; P = 1.31x10-8), with directionally consistent results across ethnic groups. These 6q25.2 SNPs are located between the SCAF8 and CNKSR3 genes, a region with DKD relevant changes in gene expression and an eQTL with IPCEF1, a gene co-translated with CNKSR3. Several other SNPs demonstrated suggestive evidence of association with DKD, within and across populations. These data identify a novel DKD susceptibility locus with consistent directions of effect across diverse ancestral groups and provide insight into the genetic architecture of DKD.
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Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/genética , Negro ou Afro-Americano/genética , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etnologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hispânico ou Latino/genética , Humanos , Indígenas Norte-Americanos/genética , Proteínas de Ligação a RNA/genética , Estados Unidos , População Branca/genéticaRESUMO
Unilateral ureteral obstruction causes important tubulo-interstitial fibrosis in the kidney. Metformin reduces fibrosis in mice with diabetic nephropathy. We examined the effects of metformin in a mouse model of unilateral ureteral obstruction (UUO). Expression of inflammation and fibrosis markers was studied by immunohistochemistry, immunoblot and quantitative real-time polymerase chain reaction. Seven days after UUO, kidneys presented dilated tubules, expansion of the tubulo-interstitial compartment, and significant infiltration of inflammatory cells. Macrophage infiltration and inflammation markers expression were increased in obstructed kidneys and reduced by metformin. Metformin reduced expression of extracellular matrix proteins and profibrotic factor TGFß in obstructed kidneys, measured by immunohistochemistry. Interstitial fibroblast activation was evident in obstructed kidneys and ameliorated by metformin. UUO did not affect adenosine monophosphate-activated kinase (AMPK) activity, but metformin activated AMPK. Our results show that metformin prevents or slows down the onset of renal inflammation and fibrosis in mice with UUO, an effect that could be mediated by activation of AMPK.
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Anti-Inflamatórios/farmacologia , Metformina/farmacologia , Nefrite Intersticial/prevenção & controle , Obstrução Ureteral/tratamento farmacológico , Adenilato Quinase/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Fibrose , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/imunologia , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Nefrite Intersticial/etiologia , Obstrução Ureteral/complicaçõesRESUMO
PURPOSE: To employ high-resolution manganese-enhanced MRI (MEMRI) to study abnormal calcium activity in different cell layers in streptozotocin-induced diabetic rat retinas, and to determine whether MEMRI detects changes at earlier time points than previously reported. METHODS: Sprague-Dawley rats were studied 14 days (n = 8) and 30 days (n = 5) after streptozotocin (STZ) or vehicle (n = 7) injection. Manganese-enhanced MRI at 20 × 20 × 700 µm, in which contrast is based on manganese as a calcium analogue and an MRI contrast agent, was obtained in light and dark adaptation of the retina in the same animals in which one eye was covered and the fellow eye was not. The MEMRI activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. RESULTS: Manganese-enhanced MRI showed three layers, corresponding to the inner retina, outer retina, and the choroid. In normal animals, the outer retina showed higher MEMRI activity in dark compared to light; the inner retina displayed lower activity in dark compared to light; and the choroid showed no difference in activity. Manganese-enhanced MRI activity changed as early as 14 days after hyperglycemia and decreased with duration of hyperglycemia in the outer retina in dark relative to light adaptation. The choroid also had altered MEMRI activity at 14 days, which returned to normal by 30 days. No differences in MEMRI activity were detected in the inner retina. CONCLUSIONS: Manganese-enhanced MRI detects progressive reduction in calcium activity with duration of hyperglycemia in the outer retina as early as 14 days after hyperglycemia, earlier than any other time point reported in the literature.
Assuntos
Adaptação Ocular/fisiologia , Meios de Contraste , Adaptação à Escuridão/fisiologia , Imageamento por Ressonância Magnética/métodos , Compostos de Manganês , Retina/fisiologia , Animais , Diabetes Mellitus Experimental , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
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
Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H2O2 decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H2O2 in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H2O2. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina , Glicoproteínas de Membrana/fisiologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , NADPH Oxidases/fisiologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Regulação para Baixo , Perfilação da Expressão Gênica , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Masculino , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , NADPH Oxidase 2 , Estresse Oxidativo/efeitos dos fármacos , Palmitatos/farmacologia , Fosforilação , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Edulcorantes/farmacologiaRESUMO
BACKGROUND: The estimated glomerular filtration rate (eGFR) is a well-known measure of kidney function and is commonly used for the diagnosis and management of patients with chronic kidney disease. The inter-individual variation in eGFR has significant genetic component. However, the identification of underlying genetic susceptibility variants has been challenging. In an attempt to identify and characterize susceptibility genetic variant(s) we previously identified the strongest evidence for linkage of eGFR occurring on chromosome 9q21 in the Mexican American participants of San Antonio Family Heart Study (SAFHS). The objective of the present study was to examine whether the common genetic variants in Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2), a positional candidate gene on 9q21, contribute to variation in eGFR. RESULTS: Twelve tagging single nucleotide polymorphisms (SNPs) across the NTRK2 gene region were selected (r2 ≥ 0.80, minor allele frequency of ≥ 0.05) from the Hapmap database. SNPs were genotyped by TaqMan assay in the 848 Mexican American subjects participated in the SAFHS. Association analysis between the genotypes and eGFR (estimated by the Modification of Diet in Renal Disease equation) were performed by measured genotype approach as implemented in the program SOLAR. Of the 12 common genetic variants examined, the rs1036915 (located in 3'UTR) and rs1187274 (located in intron-14), present in perfect linkage disequilibrium, exhibited an association (P = 0.017) with eGFR after accounting for the effects of age, sex, diabetes, diabetes duration, systolic blood pressure and blood pressure medication. The carriers of minor allele of rs1036915 (G; 38%) had increased eGFR (104 ± 25 ml/min/1.73 m(2)) in comparison to the carriers of major allele A (98 ± 25 ml/min/1.73 m(2)). CONCLUSION: Together, our results suggest for the first time that the genetic variants in NTRK2 may regulate eGFR.
Assuntos
Predisposição Genética para Doença/epidemiologia , Taxa de Filtração Glomerular , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Receptor trkB/genética , Adulto , Feminino , Marcadores Genéticos , Predisposição Genética para Doença/genética , Humanos , Masculino , Americanos Mexicanos , Pessoa de Meia-Idade , Receptor trkB/metabolismo , Texas/epidemiologiaRESUMO
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
BACKGROUND: NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived reactive oxygen species in the myocardium. We investigated the role of Nox4 and Nox4-associated signaling pathways in the development of cardiac remodeling. METHODS AND RESULTS: Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: (1) control mice, littermates that are negative for hNox4 transgene but Cre positive; (2) c-hNox4 Tg mice; (3) angiotensin II (AngII)-infused control mice; and (4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited an ≈10-fold increase in Nox4 protein expression and an 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII infusion to control mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least in part by enhancing Nox4 expression. Moreover, hNox4 transgene and AngII infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed the Akt-mTOR and NFκB signaling pathways, and attenuated cardiac remodeling. CONCLUSIONS: Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling.