RESUMO
Intracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1-Atg7-Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-γ (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.
Assuntos
Autofagia , Macrófagos Alveolares/metabolismo , Pseudomonas aeruginosa/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/ultraestrutura , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Fagossomos/ultraestruturaRESUMO
BACKGROUND: We previously demonstrated that cellular and extracellular components of the blood-urine barrier in renal glomeruli are susceptible to damage in OVE transgenic mice, a valuable model of human diabetic nephropathy that expresses profound albuminuria. METHODS: To test our hypothesis that glomerular filtration barrier damage in OVE mice may be the result of oxidative insult to podocytes, 150-day-old bi-transgenic OVENmt diabetic mice that overexpress the antioxidant metallothionein specifically in podocytes were examined by enzyme-linked immunosorbent assay for albuminuria mitigation and by unbiased transmission electron microscopy (TEM) stereometry for protection from chronic structural diabetic complications. RESULTS: Although blood glucose and HbA(1c) levels were indistinguishable in OVE and OVENmt animals, albuminuria was significantly reduced (average >7-fold) in OVENmt mice through 8 months of age. Interestingly, the Nmt transgene provided significant glomerular protection against diabetic nephropathic complications outside of the podocyte. Glomerular filtration barrier damage was reduced in OVENmt mice, including significantly increased area occupied by endothelial luminal fenestrations (~13%), significantly reduced glomerular basement membrane (GBM) thickening (~17%) and significantly less podocyte effacement (~18%). In addition, OVENmt mice exhibited significantly reduced glomerular volume (~50%), fewer glomerular endothelial cells (~33%), fewer mesangial cells (~57%) and fewer total glomerular cells (~40%). CONCLUSIONS: These results provide evidence of oxidative damage to podocytes induces primary diabetic nephropathic features including severe and sustained albuminuria, specific glomerular filtration barrier damage and alterations in glomerular endothelial and mesangial cell number. Importantly, these diabetic complications are significantly mitigated by podocyte targeted metallothionein overexpression.
Assuntos
Albuminúria/prevenção & controle , Nefropatias Diabéticas/prevenção & controle , Barreira de Filtração Glomerular/patologia , Metalotioneína/biossíntese , Podócitos/metabolismo , Animais , Nefropatias Diabéticas/fisiopatologia , Membrana Basal Glomerular/fisiopatologia , Barreira de Filtração Glomerular/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Metalotioneína/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Podócitos/patologiaRESUMO
BACKGROUND/AIMS: Glomerular lesions in diabetic nephropathy (DN) have been studied in numerous murine diabetic models, but the critical feature of aging is often absent. Since histopathology indicates that in mice, DN glomerular lesions may just begin to develop at about 5 months of age, we utilized the long-lived OVE26 transgenic diabetic model for stereometric analyses of DN glomerulopathic aging. METHODS: Albuminuria was determined by ELISA, and transmission electron microscopy stereometry was utilized exclusively to demonstrate changes in glomerular cell density and number, and alterations in the glomerular filtration barrier in OVE26 mice at 60, 150, and 450 days of age. RESULTS: Compared to age-matched controls, albuminuria in diabetic mice is significant at 60 days. At 150 days, glomerular volume and mesangial, endothelial and total cell numbers, and podocyte effacement are significantly increased, while podocyte, endothelial, and total cell density are significantly decreased. Endothelial fenestrations are decreased, and glomerular basement membrane thickness is increased. At 450 days, stereometric alterations are exacerbated. CONCLUSION: Our data indicate that in OVE26 mice, albuminuria precedes morphological glomerular lesions and could be due to early-onset hyperglycemia. Moreover, in this model, most DN glomerulopathic lesions occur relatively late in life, and it is possible that they may result from prolonged hyperglycemia-induced oxidative stress.
Assuntos
Envelhecimento/patologia , Albuminúria/urina , Nefropatias Diabéticas/patologia , Glomérulos Renais/patologia , Fatores Etários , Análise de Variância , Animais , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Estatísticas não ParamétricasRESUMO
The diabetic-prone BioBreeding Wistar (BB/DP) rat is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 90% of the animals (BB/DPh) are hyperglycemic by 90-120 days of age, while the remaining ~10% (BB/DPn) and diabetes-resistant rats (BB/DR) are normoglycemic for life. The transmission electron microscope data from this study demonstrate expected significant age- and diabetes-related increases in retinal capillary basement membrane (RCBM) widths in (BB/DPh) rats relative to BB/DR animals. However, the data show, for the first time, an unexpected significant RCBM thickening in (BB/DPn) rats compared to BB/DR animals at 6 months and 1 year post-onset of hyperglycemia.
Assuntos
Membrana Basal/ultraestrutura , Glicemia/metabolismo , Capilares/ultraestrutura , Diabetes Mellitus Tipo 1/patologia , Retinopatia Diabética/patologia , Vasos Retinianos/ultraestrutura , Fatores Etários , Animais , Peso Corporal , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Retinopatia Diabética/sangue , Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Endogâmicos BBRESUMO
Diabetes-induced oxidative stress is one of the major contributors to dysfunction of endothelial progenitor cells (EPCs) and impaired endothelial regeneration. Thus, we tested whether increasing antioxidant protein metallothionein (MT) in EPCs promotes angiogenesis in a hind limb ischemia (HLI) model in endothelial MT transgenic (JTMT) mice with high-fat diet- and streptozocin-induced diabetes. Compared with littermate wild-type (WT) diabetic mice, JTMT diabetic mice had improved blood flow recovery and angiogenesis after HLI. Similarly, transplantation of JTMT bone marrow-derived mononuclear cells (BM-MNCs) stimulated greater blood flow recovery in db/db mice with HLI than did WT BM-MNCs. The improved recovery was associated with augmented EPC mobilization and angiogenic function. Further, cultured EPCs from patients with diabetes exhibited decreased MT expression, increased cell apoptosis, and impaired tube formation, while cultured JTMT EPCs had enhanced cell survival, migration, and tube formation in hypoxic/hyperglycemic conditions compared with WT EPCs. Mechanistically, MT overexpression enhanced hypoxia-inducible factor 1α (HIF-1α), stromal cell-derived factor (SDF-1), and vascular endothelial growth factor (VEGF) expression and reduced oxidative stress in ischemic tissues. MT's pro-EPC effects were abrogated by siRNA knockdown of HIF-1α without affecting its antioxidant action. These results indicate that endothelial MT overexpression is sufficient to protect against diabetes-induced impairment of angiogenesis by promoting EPC function, most likely through upregulation of HIF-1α/SDF-1/VEGF signaling and reducing oxidative stress.
Assuntos
Quimiocina CXCL12/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/prevenção & controle , Células Progenitoras Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metalotioneína/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Animais , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Quimiocina CXCL12/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Membro Posterior/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isquemia/genética , Isquemia/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Metalotioneína/genética , Camundongos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Podocytes are critical components of the selective filtration barrier of the glomerulus and are susceptible to oxidative damage. For investigation of the role of oxidative stress and podocyte damage in diabetic nephropathy, transgenic mice that overexpress the antioxidant protein metallothionein (MT) specifically in podocytes (Nmt mice) were produced. MT expression was increased six- and 18-fold in glomeruli of two independent lines of Nmt mice, and podocyte-specific overexpression was confirmed. Glomerular morphology and urinary albumin excretion were normal in Nmt mice. OVE26 transgenic mice, a previously reported model of diabetic nephropathy, were crossed with Nmt mice to determine whether an antioxidant transgene targeted to podocytes could reduce diabetic nephropathy. Double-transgenic OVE26Nmt mice developed diabetes similar to OVE26 mice, but MT overexpression reduced podocyte damage, indicated by more podocytes, less glomerular cell death, and higher density of podocyte foot processes. In addition, expansion of glomerular and mesangial volume were significantly less in OVE26Nmt mice compared with OVE26 mice. Four-month-old OVE26Nmt mice had a 70 to 90% reduction in 24-h albumin excretion, but this protection does not seem to be permanent. These results provide evidence for the role of oxidative damage to the podocyte in diabetic mice and show that protection of the podocyte can reduce or delay primary features of diabetic nephropathy.
Assuntos
Antioxidantes/metabolismo , Nefropatias Diabéticas/prevenção & controle , Metalotioneína/genética , Metalotioneína/metabolismo , Podócitos/metabolismo , Albuminúria/prevenção & controle , Animais , Apoptose , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Expressão Gênica , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Podócitos/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: Antioxidant therapy has shown some promise in critical care medicine in which glutathione depletion and heart failure are often seen in critically ill patients. This study was designed to examine the impact of glutathione depletion and the free radical scavenger, metallothionein (MT), on cardiac function. DESIGN: Friend virus B and MT transgenic mice were given the glutathione synthase inhibitor buthionine sulfoximine (buthionine sulfoximine [BSO], 30 mmol/L) in drinking water for 2 wks. MEASUREMENTS: Echocardiographic and cardiomyocyte functions were evaluated, including myocardial geometry, fraction shortening, peak shortening, time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), intracellular Ca2+ rise, sarcoplasmic reticulum Ca2+ release, and intracellular Ca2+ decay rate. Sacro (endo)plasmic reticulum Ca2+-ATPase function was evaluated by 45Ca uptake. Highly reactive oxygen species, caspase-3, and aconitase activity were detected by fluorescent probe and colorimetric assays. MAIN RESULT: BSO elicited lipid peroxidation, protein carbonyl formation, mitochondrial damage, and apoptosis. BSO also reduced wall thickness, enhanced end systolic diameter, depressed fraction shortening, peak shortening, +/-dL/dt, sarcoplasmic reticulum Ca2+ release, 45Ca uptake, and intracellular Ca2+ decay, leading to prolonged TR90. BSO-induced mitochondrial loss and myofilament aberration. MT transgene itself had little effect on myocardial mechanics and ultrastructure. However, it alleviated BSO-induced myocardial functional, morphologic, and carbonyl changes. Western blot analysis showed reduced expression of sacro (endo)plasmic reticulum Ca2+-ATPase2a, Bcl-2 and phosphorylated GSK-3beta, enhanced calreticulin, Bax, p53, myosin heavy chain-beta isozyme switch, and IkappaB phosphorylation in FVB-BSO mice, all of which with the exception of p53 were nullified by MT. CONCLUSION: Our findings suggest a pathologic role of glutathione depletion in cardiac dysfunction and the therapeutic potential of antioxidants.
Assuntos
Butionina Sulfoximina/farmacologia , Cardiomiopatias/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glutationa/deficiência , Metalotioneína/uso terapêutico , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/etiologia , Glutationa Sintase/antagonistas & inibidores , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , UltrassonografiaRESUMO
We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age-matched OVE diabetic mice to produce bi-transgenic OVE-JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE-JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day-old OVE-JTMT mice, blood glucose and HbA1c were indistinguishable from age-matched OVE mice. However, endothelial-specific MT overexpression in OVE-JTMT mice mitigated several DN complications including significantly increased non-fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE-JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560-576, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Nefropatias Diabéticas/metabolismo , Células Endoteliais/metabolismo , Glomérulos Renais/metabolismo , Metalotioneína/metabolismo , Podócitos/metabolismo , Animais , Glicemia/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Glomérulos Renais/patologia , Metalotioneína/genética , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Podócitos/patologiaRESUMO
BACKGROUND: Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. METHODS: To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. RESULTS: High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. CONCLUSIONS: Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.
Assuntos
Gorduras na Dieta/efeitos adversos , Fatores de Transcrição Forkhead/metabolismo , Miócitos Cardíacos/fisiologia , Obesidade/fisiopatologia , Aconitato Hidratase/metabolismo , Animais , Apoptose/fisiologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Ceramidas/metabolismo , Proteína Forkhead Box O3 , Técnicas In Vitro , Masculino , Óxido Nítrico Sintase/metabolismo , Obesidade/etiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Sirtuína 1 , Sirtuínas/metabolismo , Triglicerídeos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Regulação para CimaRESUMO
The effect of diabetes in vivo has not been examined on isolated podocytes. To achieve this, GFP was expressed constitutively in podocytes of PGFP transgenic mice which were bred to OVE mice to produce diabetic OVE-GFP mice. Viewing GFP fluorescence, foot processes of OVE-GFP podocytes were visually and measurably effaced, which did not occur with less severe STZ diabetes. Over 300,000 podocytes were purified from each PGFP mouse but only 49,000 podocytes per diabetic OVE-GFP mouse. The low yield from OVE-GFP mice appeared to be due to more fragile state of most OVE-GFP diabetic podocytes which did not survive the isolation process. Diabetic podocytes that were isolated had high levels of the lipid peroxidation product 4-HNE and they were more sensitive to death due to oxidative stress. Gene array analysis of OVE-GFP podocytes showed strong diabetes induction of genes involved in inflammation. Four CXC chemokines were induced at least 3-fold and the chemokine CXCL1 was shown for the first time to be specifically induced in podocytes by OVE, dbdb and STZ diabetes.
Assuntos
Expressão Gênica , Podócitos/ultraestrutura , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Proteínas de Fluorescência Verde/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Podócitos/metabolismoRESUMO
Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.
Assuntos
Cardiomiopatias/prevenção & controle , Angiopatias Diabéticas/prevenção & controle , Regulação da Expressão Gênica/fisiologia , Metalotioneína/genética , Contração Miocárdica/fisiologia , Actinas/genética , Animais , Antioxidantes/metabolismo , Fator Natriurético Atrial/genética , Sequência de Bases , Glicemia/metabolismo , Northern Blotting , Cardiomiopatias/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Dissulfeto de Glutationa/metabolismo , Insulina/sangue , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Transcrição Gênica , Triglicerídeos/sangueRESUMO
Many diabetic patients suffer from a cardiomyopathy that cannot be explained by poor coronary perfusion. Reactive oxygen species (ROS) have been proposed to contribute to this cardiomyopathy. Consistent with this we found evidence for induction of the antioxidant genes for catalase in diabetic OVE26 hearts. To determine whether increased antioxidant protection could reduce diabetic cardiomyopathy, we assessed cardiac morphology and contractility, Ca(2+) handling, malondialdehyde (MDA)-modified proteins, and ROS levels in individual cardiomyocytes isolated from control hearts, OVE26 diabetic hearts, and diabetic hearts overexpressing the antioxidant protein catalase. Diabetic hearts showed damaged mitochondria and myofibrils, reduced myocyte contractility, slowed intracellular Ca(2+) decay, and increased MDA-modified proteins compared with control myocytes. Overexpressing catalase preserved normal cardiac morphology, prevented the contractile defects, and reduced MDA protein modification but did not reverse the slowed Ca(2+) decay induced by diabetes. Additionally, high glucose promoted significantly increased generation of ROS in diabetic cardiomyocytes. Chronic overexpression of catalase or acute in vitro treatment with rotenone, an inhibitor of mitochondrial complex I, or thenoyltrifluoroacetone, an inhibitor of mitochondrial complex II, eliminated excess ROS production in diabetic cardiomyocytes. The structural damage to diabetic mitochondria and the efficacy of mitochondrial inhibitors in reducing ROS suggest that mitochondria are a source of oxidative damage in diabetic cardiomyocytes. We also found that catalase overexpression protected cardiomyocyte contractility in the agouti model of type 2 diabetes. These data show that both type 1 and type 2 diabetes induce damage at the level of individual myocytes, and that this damage occurs through mechanisms utilizing ROS.
Assuntos
Catalase/metabolismo , Citoproteção , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Miócitos Cardíacos , Animais , Cálcio/metabolismo , Catalase/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Expressão Gênica , Membranas Intracelulares/metabolismo , Malondialdeído/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miocárdio/enzimologia , Miocárdio/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
OVE26 mice are a transgenic model of severe early-onset type 1 diabetes. These mice develop diabetes within the first weeks of life and can survive well over a year with no insulin treatment, and they maintain near normal body weight. To determine whether OVE26 mice provide a valuable model of chronic diabetic nephropathy (DN), OVE26 diabetic mice were compared with their nondiabetic littermates for functional and structural characteristics of DN. OVE26 mice exhibited pronounced polyuria and significant albuminuria by 2 months of age (305 microg/24 h in OVE26 vs. 20 microg/24 h in controls). Albumin excretion rate increased progressively with age and exceeded 15,000 microg/24 h at 9 months of age. The profound loss of albumin led to hypoalbuminemia in some diabetic animals. Albuminuria coincided with an elevation in blood pressure as measured by tail cuff. The glomerular filtration rate (GFR) in OVE26 mice measured using fluorescein isothiocynate inulin clearance demonstrated that GFR increased significantly from 2 to 3 months of age and then decreased significantly from 5 to 9 months. GFR in 9-month-old diabetic mice was significantly lower than that of 9-month-old control mice. The decline in GFR coincided with a significant increase in renal vascular resistance. Structural studies showed an almost twofold increase in kidney weight between 2 and 5 months. Diabetic mice also showed progressively enlarged glomeruli and expanded mesangium with diffuse and nodular expansion of mesangial matrix. Tubulointerstitial fibrosis was also observed in these mice. Glomerular basement membrane was thickened in OVE26 mice. In summary, OVE26 mice demonstrate that most of the characteristics of human DN can be produced by chronic hyperglycemia in a murine model. This model will be useful for improved understanding and treatment of DN.
Assuntos
Nefropatias Diabéticas/fisiopatologia , Envelhecimento , Animais , Glicemia/metabolismo , Peso Corporal , Nefropatias Diabéticas/patologia , Taxa de Filtração Glomerular , Humanos , Rim/anatomia & histologia , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Circulação Renal , Resistência VascularRESUMO
The diabetic-prone BioBreeding Wistar rat (BB/DP) is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 80-90% of the animals are hyperglycemic (BB/DP(h)) by 90-120 days of age while those that do not become diabetic in adolescence (BB/DP(n)) remain normoglycemic for life. Likewise, rats in the diabetes-resistant (BB/DR) strain are normoglycemic. Although renal morphological studies have been carried out in this model, ultrastructural observations of age- and diabetes-related extracellular matrix (ECM) changes, including glomerular basement membrane (GBM) morphometry, are not available. Moreover, possible renal changes in the relatively uncommon BB/DP(n) control animals have not been reported. The current electron microscopic study was carried out to investigate temporal changes in detergent-treated acellular ECM in BB/DP(h) rats at 2 weeks, 3 months, 6 months, and 1 year postonset of moderate hyperglycemia. Age-matched BB/DR and BB/DP(n) control animals were also examined. Our data demonstrate age- and diabetes-related alterations in mesangial matrix distributions and GBM widths and show for the first time significant increases in GBM thickening in both hyperglycemic (BB/DP(h)) and normoglycemic (BB/DP(n)) rats when compared to age-matched BB/DR controls. Surprisingly, the rate of increase is greatest in BB/DP(n) animals. Although the pathogenesis of diabetic basement membrane disease is not completely understood, GBM thickening is widely regarded as a morphological consequence of hyperglycemia. However, data in the current investigation show that ECM alterations, including significantly increased GBM thickness, may occur in genetically diabetic animals in the absence of hyperglycemia.
Assuntos
Membrana Basal/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hiperglicemia/metabolismo , Glomérulos Renais/metabolismo , Animais , Membrana Basal/patologia , Glicemia , Modelos Animais de Doenças , Matriz Extracelular , Hiperglicemia/patologia , Rim/patologia , Glomérulos Renais/patologia , Ratos , Ratos Endogâmicos BB , Ratos Sprague-DawleyRESUMO
Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues.
Assuntos
Membrana Basal/ultraestrutura , Capilares/ultraestrutura , Diabetes Mellitus Experimental/patologia , Animais , Membrana Basal/metabolismo , Peso Corporal , Capilares/metabolismo , Hemoglobinas/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica , Tamanho do ÓrgãoRESUMO
Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ~3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury.
Assuntos
Membrana Basal/metabolismo , Capilares/metabolismo , Vasos Coronários/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Metalotioneína/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Membrana Basal/ultraestrutura , Glicemia/metabolismo , Capilares/ultraestrutura , Vasos Coronários/ultraestrutura , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Genótipo , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Metalotioneína/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Type 1 and Type 2 diabetic patients are at high risk of developing diabetic nephropathy (DN). Renal functional decline is gradual and there is high variability between patients, though the reason for the variability is unknown. Enough diabetic patients progress to end stage renal disease to make diabetes the leading cause of renal failure. The first symptoms of DN do not appear for years or decades after the onset of diabetes. During and after the asymptomatic period structural changes develop in the diabetic kidney. Typically, but not always, the first symptom of DN is albuminuria. Loss of renal filtration rate develops later. This review examines the structural abnormalities of diabetic kidneys that are associated with and possibly the basis for advancing albuminuria and declining GFR. Mouse models of diabetes and genetic manipulations of these models have become central to research into mechanisms underlying DN. This article also looks at the value of these mouse models to understanding human DN as well as potential pitfalls in translating the mouse results to humans.
Assuntos
Diabetes Mellitus/patologia , Nefropatias Diabéticas/patologia , Rim/patologia , Albuminúria , Animais , Diabetes Mellitus/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Taxa de Filtração Glomerular , Glucose/toxicidade , Humanos , Rim/fisiopatologia , CamundongosRESUMO
One key mechanism for endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O(2)(*-) rather than NO because of deficient eNOS cofactor tetrahydrobiopterin (BH4). This study was designed to examine the effect of BH4 deficiency on cardiac morphology and function, as well as the impact of metallothionein (MT) on BH4 deficiency-induced abnormalities, if any. Friend virus B (FVB) and cardiac-specific MT transgenic mice were exposed to 2,4-diamino-6-hydroxy-pyrimidine (DAHP; 10 mmol/L, 3 weeks), an inhibitor of the BH4 synthetic enzyme GTP cyclohydrolase I. DAHP reduced plasma BH4 levels by 85% and elevated blood pressure in both FVB and MT mice. Echocardiography found decreased fractional shortening and increased end-systolic diameter in DAHP-treated FVB mice. Cardiomyocytes from DAHP-treated FVB mice displayed enhanced O(2)(*-) production, contractile and intracellular Ca(2+) defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, reduced intracellular Ca(2+) rise, and clearance. DAHP triggered mitochondrial swelling/myocardial filament aberrations and mitochondrial O(2)(*-) accumulation, assessed by transmission electron microscopy and MitoSOX Red fluorescence, respectively. DAHP also promoted the N(G)-nitro-l-arginine methyl ester-inhibitable O(2)(*-) production and eNOS phosphorylation at Thr497. Although MT had little effect on cardiac mechanics and ultrastructure, it attenuated DAHP-induced defects in cardiac function, morphology, O(2)(*-) production, and eNOS phosphorylation (Thr497). The DAHP-induced cardiomyocyte mechanical responses were alleviated by in vitro BH4 treatment. DAHP inhibited mitochondrial biogenesis, mitochondrial uncoupling protein 2, and chaperone heat shock protein 90, and all but uncoupling protein 2 were rescued by MT. Our data suggest a role for BH4 deficiency in cardiac dysfunction and the therapeutic potential of antioxidants against eNOS uncoupling in the heart.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/metabolismo , Metalotioneína/metabolismo , Mitocôndrias Cardíacas/metabolismo , Óxido Nítrico/biossíntese , Açúcares Ácidos/farmacologia , Análise de Variância , Animais , Biopterinas/sangue , Biopterinas/metabolismo , Western Blotting , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ecocardiografia , Masculino , Metalotioneína/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Consumo de Oxigênio/fisiologia , Probabilidade , RNA/análise , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
The Br/+ mutant mouse displays decreased embryological expression of the homeobox transcription factor Six2, resulting in hertitable renal hypoplasia. The purpose of this study was to characterize the renal physiological consequences of embryonic haploinsuffiency of Six2 by analyzing renal morphology and function in the adult Br heterozygous mutant. Adult Br/+ kidneys weighed 50% less than those from wild-type mice and displayed glomerulopathy. Stereological analysis of renal glomeruli showed that Br/+ kidneys had an average of 88% fewer glomeruli than +/+ kidneys, whereas individual glomeruli in Br/+ mice maintained an average volume increase of 180% compared with normal nephrons. Immunostaining revealed increased levels of endothelin-1 (ET-1), endothelin receptors A (ET(A)) and B (ET(B)), and Na-K-ATPase were present in the dilated renal tubules of mutant mice. Physiological features of chronic renal failure (CRF) including elevated mean arterial pressure, increased plasma creatinine, and dilute urine excretion were measured in Br/+ mutant mice. Electron microscopy of the Br/+ glomeruli revealed pathological alterations such as hypercellularity, extracellular matrix accumulation, and a thick irregular glomerular basement membrane. These results indicate that adult Br/+ mice suffer from CRF associated with reduced nephron number and renal hypoplasia, as well as glomerulopathy. Defects are associated with embryological deficiencies of Six2, suggesting that proper levels of this protein during nephrogenesis are critical for normal glomerular development and adult renal function.
Assuntos
Proteínas de Homeodomínio/genética , Hipertensão Renal/fisiopatologia , Falência Renal Crônica/fisiopatologia , Néfrons/anormalidades , Néfrons/fisiologia , Fatores de Transcrição/genética , Animais , Regulação para Baixo/fisiologia , Endotelina-1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipertensão Renal/genética , Hipertensão Renal/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Glomérulos Renais/anormalidades , Glomérulos Renais/fisiologia , Glomérulos Renais/ultraestrutura , Túbulos Renais/anormalidades , Túbulos Renais/fisiologia , Túbulos Renais/ultraestrutura , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Microscopia Eletrônica , Néfrons/ultraestrutura , Receptores de Endotelina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Recent studies show that podocyte nuclear density (N(V)) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. N(V) and N are generally reduced, and it has been hypothesized that these morphological changes may relate to the loss of glomerular permselectivity in diabetic nephropathy (DN). In the current study, OVE26 transgenic diabetic mice and age-matched (FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion and renal cortical tissues were prepared for morphometric analyses. ImageJ software and point counting analyses were carried out on light and transmission electron micrographs to determine glomerular volume (V(G)), N(V), and N. As expected, mean V(G) in OVE26 mice increased substantially ( approximately 134%) over the course of the study and was significantly increased over FVB mice at all ages. At 60 days, N(V) and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, N(V) was significantly reduced in diabetics but not N. In 450-day-old OVE26 animals, however, N(V) and N were both significantly decreased ( approximately 231% and approximately 99%, respectively) relative to age-matched FVB mice. These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood-urine barrier in the later stages of diabetic renal decompensation.