RESUMEN
Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-beta and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of (125)I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab's to RAGE or to TGF-beta. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-beta expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.
Asunto(s)
Nefropatías Diabéticas/etiología , Productos Finales de Glicación Avanzada/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Receptores Inmunológicos/fisiología , Actinas/análisis , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/fisiología , Fibroblastos/fisiología , Productos Finales de Glicación Avanzada/metabolismo , Túbulos Renales Proximales/citología , Ratas , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada , Factor de Crecimiento Transformador beta/análisisRESUMEN
AIMS/HYPOTHESIS: ALT-946, an inhibitor of advanced glycation with a minimal inhibitory effect on nitric oxide synthase, was compared with aminoguanidine in experimental diabetic nephropathy. METHODS: In vitro and in vivo assays were used to assess the ability of ALT-946 to inhibit AGE-protein cross-link formation. Diabetic animals were randomly allocated into groups receiving aminoguanidine for 32 weeks, ALT-946 or vehicle (untreated). As a delayed intervention protocol, an additional diabetic group was treated with ALT-946 from week 16 to week 32 of the study. Non-diabetic rats were studied concurrently. Systolic blood pressure, body weight, plasma glucose, glycated haemoglobin and urinary albumin excretion were measured serially. Accumulation of advanced-glycation end products in the kidney was assessed by immunohistochemistry. RESULTS: The ALT-946 inhibitor was more potent than aminoguanidine in inhibiting AGE-protein cross-linking both in vitro and in vivo. Increased albuminuria observed in diabetic rats was attenuated in all three treatment groups. We found no difference in body weight, blood pressure or glycaemic control with any of the treatments. The untreated diabetic group had a twofold increase in glomerular staining for advanced-glycation end products compared with the diabetic groups which received treatment. CONCLUSION/INTERPRETATION: ALT-946 is a potent inhibitor of advanced renal glycation end-product accumulation and reproduces the renoprotective effects of aminoguanidine. Therefore, ALT-946 should be considered as a treatment for preventing or retarding diabetic nephropathy.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/prevención & control , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Animales , Reactivos de Enlaces Cruzados , Diabetes Mellitus Experimental/metabolismo , Inhibidores Enzimáticos , Tasa de Filtración Glomerular , Productos Finales de Glicación Avanzada/análisis , Guanidinas/uso terapéutico , Humanos , Inmunohistoquímica , Riñón/química , Glomérulos Renales/química , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Albúmina Sérica BovinaRESUMEN
AIMS/HYPOTHESIS: Advanced glycation is postulated to have a pivotal role in mediating diabetic vascular complications. The emergence of thiazolium compounds such as N-phenacylthiazolium bromide which cleave preformed advanced glycation end products (AGEs) has allowed us to explore the effects of these agents on the vascular AGE accumulation and hypertrophy associated with diabetes. METHODS: Control and streptozotocin diabetic rats were selected at random for no treatment or treatment with N-phenacylthiazolium bromide (10 mg/kg intraperitoneally) and followed for 3 weeks. In a separate study, intervention with N-phenacylthiazolium bromide was delayed until after 3 weeks of diabetes and then given for 3 weeks (total of 6 weeks). RESULTS: Diabetes was associated with increased mesenteric vascular advanced glycation end products, as assessed by radioimmunoassay and immunohistochemistry. This increase in vascular AGE accumulation was prevented by N-phenacylthiazolium bromide treatment. Diabetes-associated mesenteric vascular hypertrophy was attenuated by treatment with N-phenacylthiazolium bromide only if given from the time of induction of diabetes. CONCLUSION/INTERPRETATION: Cross-link breakers seem to be effective in preventing or reversing accumulation of advanced glycation end-products in blood vessels and have the potential to play a part in the treatment of diabetic vascular complications.
Asunto(s)
Vasos Sanguíneos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Tiazoles/farmacología , Animales , Presión Sanguínea , Vasos Sanguíneos/química , Hemoglobina Glucada/análisis , Productos Finales de Glicación Avanzada/análisis , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-Dawley , Circulación Esplácnica , Tiazoles/administración & dosificaciónRESUMEN
AIMS/HYPOTHESIS: Previous studies in our laboratory have shown that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature and that this process can be attenuated by the inhibition of advanced glycation with aminoguanidine. Since aminoguanidine can also act as an inhibitor of nitric oxide synthase, the effect of a novel inhibitor of advanced glycation end-products, formation that does not inhibit nitric oxide synthase, known as 2,3 diaminophenazine (2,3 DAP) was evaluated. METHODS: Initially, in vitro assessment of the ability of 2,3 diaminophenazine to inhibit formation of advanced glycation products was performed. Subsequently, in vivo studies evaluating 2,3 diaminophenazine and aminoguanidine were carried out. Animals were followed for 3 weeks after induction of diabetes and randomised to no treatment, aminoguanidine or 2,3 diaminophenazine. Mesenteric vessels were weighed and advanced glycation end-products were measured by radioimmunoassay in vessel and kidney homogenates. In addition, these products were assessed in mesenteric vessels by immunohistochemistry. RESULTS: When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight. Treatment of diabetic rats with aminoguanidine or 2,3 diaminophenazine resulted in attenuation of vascular hypertrophy. Both aminoguanidine and 2,3 diaminophenazine reduced the formation of advanced glycation end-products as measured by radioimmunoassay and as assessed immunohistochemically in these vessels. This reduction was also observed in the kidney. CONCLUSION/INTERPRETATION: These data support the concept that the effects of aminoguanidine in reducing diabetes associated vascular hypertrophy are via inhibition of advanced glycation end-products dependent pathways.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Angiopatías Diabéticas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Fenazinas/farmacología , Animales , Diabetes Mellitus Experimental/metabolismo , Inducción Enzimática , Hipertrofia/prevención & control , Masculino , Arterias Mesentéricas/patología , Venas Mesentéricas/patología , Ratones , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Conejos , Radioinmunoensayo , Ratas , Ratas Sprague-DawleyRESUMEN
Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs are increased in experimental diabetes and treatment with the inhibitor of advanced glycation end products, aminoguanidine, has been shown to attenuate the level of these products in tissues undergoing complications. Recently, an AGE-binding protein has been isolated from bovine lung endothelial cells and termed the receptor for advanced glycated end products (RAGE). The present study sought to determine the distribution of AGE and RAGE in tissues susceptible to the long-term complications of diabetes including the kidney, eye, nerve, arteries as well as in a tissue resistant to such complications, the lung. Using polyclonal antisera both AGE and RAGE were found to co-localize in the renal glomerulus. AGE staining was clearly increased with age and was further increased by diabetes. Aminoguanidine treatment reduced AGE accumulation in the kidney. Co-localisation of AGE and RAGE was demonstrated in the inner plexiform layer and the inner limiting membrane of the retina and in nerve bundles from mesenteric arteries. In the aorta, both AGE and RAGE were found in the intima, media and adventitia. Medial staining was increased in diabetes and was reduced by aminoguanidine treatment. A similar pattern was observed for RAGE in the aorta. In the lung, RAGE was found widely distributed throughout the lung whereas the distribution of AGE staining was more limited, primarily localising to macrophages. The co-localisation of AGEs and RAGE in sites of diabetic microvascular injury suggests that this ligand-receptor interaction may represent an important mechanism in the genesis of diabetic complications.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Angiopatías Diabéticas/fisiopatología , Productos Finales de Glicación Avanzada/metabolismo , Microcirculación/fisiopatología , Receptores Inmunológicos/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Glucemia/metabolismo , Peso Corporal , Bovinos , Diabetes Mellitus Experimental/patología , Angiopatías Diabéticas/patología , Susceptibilidad a Enfermedades , Productos Finales de Glicación Avanzada/análisis , Guanidinas/farmacología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Arterias Mesentéricas/inervación , Microcirculación/patología , Ratas , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/análisis , Retina/metabolismo , Retina/patología , Albúmina Sérica Bovina/metabolismoRESUMEN
Advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic kidney. Aminoguanidine, an inhibitor of advanced glycation, has been shown to attenuate the development of AGEs as well as the progression of renal disease in experimental diabetes. However, the precise mechanisms through which aminoguanidine acts remain to be elucidated since it is also able to act as an inhibitor of nitric oxide synthase (NOS). This study has therefore compared the effects of aminoguanidine with the effects of two other inhibitors of NOS, L-NAME and methylguanidine, on the development of experimental diabetic nephropathy. Diabetic rats were randomised to receive no treatment, aminoguanidine (1 g/l in drinking water), L-NAME (5 mg/l in drinking water) or methylguanidine (1 g/l in drinking water). Diabetic rats had increased levels of albuminuria and urinary nitrite/nitrate excretion when compared to control rats. Renal AGEs measured by fluorescence as well as by a carboxymethyllysine reactive radioimmunoassay, were elevated in diabetic rats. No changes in inducible NOS (iNOS) protein expression were detected in experimental diabetes nor did aminoguanidine affect iNOS expression. Aminoguanidine did not affect blood glucose or HbA1c but it did prevent increases in albuminuria, urinary nitrites/nitrates and renal AGE levels as measured by fluorescence and radioimmunoassay. L-NAME and methylguanidine did not retard the development of albuminuria, nor did they prevent increases in renal AGE levels, as assessed by fluorescence. However, these treatments did prevent increases in AGEs, as measured by radioimmunoassay. This study indicates that the renoprotective effect of aminoguanidine in experimental diabetes cannot be reproduced by L-NAME or methylguanidine. It is likely that the effect of aminoguanidine is mediated predominantly by decreased AGE formation rather than via NOS inhibition. It also raises the possibility that inhibition of fluorescent AGE formation may be more renoprotective than inhibition of the formation of carboxymethyllysine-containing AGEs.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/orina , Inhibidores Enzimáticos/farmacología , Productos Finales de Glicación Avanzada/metabolismo , Glomérulos Renales/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Administración Oral , Albuminuria/metabolismo , Albuminuria/orina , Animales , Estudios de Cohortes , Diabetes Mellitus Experimental/inmunología , Inhibidores Enzimáticos/administración & dosificación , Productos Finales de Glicación Avanzada/inmunología , Productos Finales de Glicación Avanzada/orina , Guanidinas/administración & dosificación , Guanidinas/farmacología , Inmunohistoquímica , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Masculino , Metilguanidina/administración & dosificación , Metilguanidina/farmacología , NG-Nitroarginina Metil Éster/administración & dosificación , NG-Nitroarginina Metil Éster/farmacología , Nitratos/orina , Nitritos/orina , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Ultrastructural examinations have shown myofibroblastoid differentiation in sarcomatoid/desmoplastic mesotheliomas, but immunohistochemical expression of muscle actins seldom has been documented. We examined 10 sarcomatoid, 12 epithelial, and five biphasic mesotheliomas immunohistochemically for the expression of muscle-specific actin (MSA) and smooth muscle actin (SMA) and compared it with that in 12 specimens of lung cancer. All of the sarcomatoid mesotheliomas were found to be positive for both MSA and SMA. The epithelial cells in nine epithelial and two biphasic mesotheliomas were positive for MSA, but SMA was only positive in one epithelial mesothelioma. Conversely, the lung cancers were negative for both MSA and SMA in the epithelial cells, except for one specimen that was weakly positive for MSA. The stromal cells in both the epithelial mesotheliomas and lung cancers were negative for cytokeratin but were positive for MSA and SMA, whereas the sarcomatoid and biphasic mesothelioma spindle cells were positive for all three antibodies. We concluded that sarcomatoid mesothelioma was positive for MSA and SMA, which is in support of its myofibroblastic differentiation, and that positivity for MSA in some epithelial mesotheliomas might be of diagnostic value in differentiation from lung cancers.