RESUMO
Chronic kidney disease (CKD) results from the development of fibrosis, ultimately leading to end-stage renal disease (ESRD). Although human bone marrow-derived mesenchymal stem cells (MSCs) can accelerate renal repair following acute injury, the establishment of fibrosis during CKD may affect their potential to influence regeneration capacity. Here we tested the novel combination of MSCs with the antifibrotic serelaxin to repair and protect the kidney 7 d post-unilateral ureteral obstruction (UUO), when fibrosis is established. Male C57BL6 mice were sham-operated or UUO-inured (n = 4-6) and received vehicle, MSCs (1 × 10(6)), serelaxin (0.5 mg/kg per d), or the combination of both. In vivo tracing studies with luciferin/enhanced green fluorescent protein (eGFP)-tagged MSCs showed specific localization in the obstructed kidney where they remained for 36 h. Combination therapy conferred significant protection from UUO-induced fibrosis, as indicated by hydroxyproline analysis (P < 0.001 vs. vehicle, P < 0.05 vs. MSC or serelaxin alone). This was accompanied by preserved structural architecture, decreased tubular epithelial injury (P < 0.01 vs. MSCs alone), macrophage infiltration, and myofibroblast localization in the kidney (both P < 0.01 vs. vehicle). Combination therapy also stimulated matrix metalloproteinase (MMP)-2 activity over either treatment alone (P < 0.05 vs. either treatment alone). These results suggest that the presence of an antifibrotic in conjunction with MSCs ameliorates established kidney fibrosis and augments tissue repair to a greater extent than either treatment alone.
Assuntos
Fibrose/fisiopatologia , Falência Renal Crônica/fisiopatologia , Rim/fisiopatologia , Células-Tronco Mesenquimais/citologia , Relaxina/uso terapêutico , Insuficiência Renal Crônica/terapia , Animais , Diferenciação Celular , Proliferação de Células , Colágeno/metabolismo , Gelatinases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/lesões , Rim/metabolismo , Macrófagos/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Proteínas Recombinantes/uso terapêutico , Regeneração , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cinnamoylanthranilates including tranilast have been identified as promising antifibrotics that can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. Structure-activity relationships aimed at improving potency and metabolic stability have led to the discovery of FT061. This compound, which bears a bis-difluoromethoxy catechol, attenuates TGF-ß-stimulated production of collagen in cultured renal mesangial cells (approx 50% at 3 µM). When dosed orally at 20mg/kg to male Sprague Dawley rats, FT061 exhibited a high bioavailability (73%), Cmax of 200 µM and Tmax of 150 min, and a half-life of 5.4h. FT061 reduced albuminuria when orally dosed in rats at 200 mg kg/day in a late intervention study of a rat model of progressive diabetic nephropathy.
Assuntos
Albuminúria/tratamento farmacológico , Antifibrinolíticos/uso terapêutico , Ácidos Cafeicos/química , ortoaminobenzoatos/química , Administração Oral , Albuminúria/complicações , Albuminúria/metabolismo , Animais , Antifibrinolíticos/química , Antifibrinolíticos/farmacocinética , Ácidos Cafeicos/farmacocinética , Ácidos Cafeicos/uso terapêutico , Células Cultivadas , Colágeno/metabolismo , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Meia-Vida , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , ortoaminobenzoatos/farmacocinética , ortoaminobenzoatos/farmacologia , ortoaminobenzoatos/uso terapêuticoRESUMO
Endothelial nitric oxide synthase (eNOS) deficiency may contribute to the pathogenesis of diabetic nephropathy in both experimental models and humans, but the underlying mechanism is not fully understood. Here, we studied two common sequelae of endothelial dysfunction in diabetes: glomerular capillary growth and effects on neighboring podocytes. Streptozotocin-induced diabetes increased glomerular capillary volume in both C57BL/6 and eNOS(-/-) mice. Inhibiting the vascular endothelial growth factor receptor attenuated albuminuria in diabetic C57BL/6 mice but not in diabetic eNOS(-/-) mice, even though it inhibited glomerular capillary enlargement in both. In eNOS(-/-) mice, an acute podocytopathy and heavy albuminuria occurred as early as 2 weeks after inducing diabetes, but treatment with either captopril or losartan prevented these effects. In vitro, serum derived from diabetic eNOS(-/-) mice augmented actin filament rearrangement in cultured podocytes. Furthermore, conditioned medium derived from eNOS(-/-) glomerular endothelial cells exposed to both high glucose and angiotensin II activated podocyte RhoA. Taken together, these results suggest that the combined effects of eNOS deficiency and hyperglycemia contribute to podocyte injury, highlighting the importance of communication between endothelial cells and podocytes in diabetes. Identifying mediators of this communication may lead to the future development of therapies targeting endothelial dysfunction in albuminuric individuals with diabetes.
Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Óxido Nítrico Sintase Tipo III/deficiência , Podócitos/metabolismo , Podócitos/patologia , Albuminúria/etiologia , Albuminúria/prevenção & controle , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Capilares/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Podócitos/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
BACKGROUND: Diabetic nephropathy is the leading cause of kidney failure in the developed world. Tranilast has been reported to not only act as an anti-inflammatory and anti-fibrotic compound, but it also exerts anti-oxidative stress effects in diabetic nephropathy. Thioredoxin-interacting protein (Txnip) is the endogenous inhibitor of the anti-oxidant thioredoxin and is highly up-regulated in diabetic nephropathy, leading to oxidative stress and fibrosis. In this study, we aimed to investigate whether tranilast exerts its anti-oxidant properties through the inhibition of Txnip. METHODS: Heterozygous Ren-2 rats were rendered diabetic with streptozotocin. Another group of rats were injected with citrate buffer alone and treated as non-diabetic controls. After 6 weeks of diabetes, diabetic rats were divided into two groups: one group gavaged with tranilast at 200 mg/kg/day and another group with vehicle. RESULTS: Diabetic rats had a significant increase in albuminuria, tubulointerstitial fibrosis, peritubular collagen IV accumulation, reactive oxygen species (ROS) and macrophage infiltration (all P < 0.05). These changes were associated with an increase in Txnip mRNA and protein expression in the tubules and glomeruli of diabetic kidney. Treatment with tranilast for 4 weeks significantly attenuated Txnip up-regulation in diabetic rats and this was associated with a reduction in ROS, fibrosis and macrophage infiltration (all P < 0.05). CONCLUSIONS: This is the first study to demonstrate that tranilast not only has anti-inflammatory and anti-fibrotic effects as previously reported but also attenuates the up-regulation of Txnip and oxidative stress in diabetic nephropathy.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte/metabolismo , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Estresse Oxidativo/efeitos dos fármacos , ortoaminobenzoatos/farmacologia , Albuminúria/etiologia , Animais , Antioxidantes/farmacologia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/patologia , Feminino , Fibrose/etiologia , Fibrose/patologia , Técnicas Imunoenzimáticas , Hibridização In Situ , Luminescência , Macrófagos/metabolismo , Nefrite Intersticial/etiologia , Nefrite Intersticial/patologia , RNA Mensageiro/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
AIM: Early renal enlargement may predict the future development of nephropathy in patients with diabetes. The epidermal growth factor (EGF)-EGF receptor (EGFR) system plays a pivotal role in mediating renal hypertrophy, where it may act to regulate cell growth and proliferation and also to mediate the actions of angiotensin II through transactivation of the EGFR. In the present study we sought to investigate the effects of long-term inhibition of the EGFR tyrosine kinase in an experimental model of diabetes that is characterized by angiotensin II dependent hypertension. METHODS: Female heterozygous streptozotocin-diabetic TGR(mRen-2)27 rats were treated with the EGFR inhibitor PKI 166 by daily oral dosing for 16 weeks. RESULTS: Treatment of TGR(mRen-2)27 rats with PKI 166 attenuated the increase in kidney size, glomerular hypertrophy and albuminuria that occurred with diabetes. The reduction in albuminuria, with EGFR inhibition in diabetic TGR(mRen-2)27 rats, was associated with preservation of the number of glomerular cells staining positively for the podocyte nuclear marker, WT1. Immunostaining for WT1 inversely correlated with glomerular volume in diabetic rats. In contrast to agents that block the renin-angiotensin system (RAS), EGFR inhibition had no effect on either the quantity of mesangial matrix or the magnitude of tubular injury in diabetic animals. CONCLUSION: These observations indicate that inhibition of the tyrosine kinase activity of the EGFR attenuates kidney and glomerular enlargement in association with podocyte preservation and reduction in albuminuria in diabetes. Accordingly, targeting the EGF-EGFR pathway may represent a therapeutic strategy for patients who continue to progress despite RAS-blockade.
Assuntos
Albuminúria/prevenção & controle , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Receptores ErbB/antagonistas & inibidores , Rim/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/patologia , Albuminúria/fisiopatologia , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Receptores ErbB/metabolismo , Feminino , Hipertrofia , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Camundongos , Podócitos/metabolismo , Podócitos/patologia , Ratos , Ratos Transgênicos , Renina/genética , Renina/metabolismo , Proteínas WT1/metabolismoRESUMO
AIM: Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the ß isoform of this enzyme has been examined. However, PKC-ß is also increased in various forms of human glomerulonephritis, including IgA nephropathy. Accordingly, we sought to examine the effects of PKC-ß inhibition in the Thy1.1 model of mesangial proliferative glomerulonephritis. METHODS: Following administration of monoclonal OX-7, anti-rat Thy-1.1 antibody, Male Wistar rats were randomized to receive either the PKC-ß inhibitor, ruboxistaurin (10 mg/kg per day in chow) or vehicle. Animals were then examined 6 days later. RESULTS: PKC-ß inhibition was associated with reductions in mesangial cellularity and extracellular matrix deposition. Proteinuria was, however, unaffected. In vitro, PKC-ß inhibition showed modest, dose-dependent reductions in mesangial cell (3) H-thymidine and (3) H-proline incorporations, indices of cell proliferation and collagen synthesis, respectively. CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by PKC-ß inhibition suggests the potential clinical utility of this approach as a therapeutic strategy in non-diabetic glomerular disease.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Indóis/farmacologia , Maleimidas/farmacologia , Células Mesangiais/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Anticorpos Monoclonais , Linhagem Celular , Colágeno/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Glomerulonefrite Membranoproliferativa/enzimologia , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/patologia , Imunoglobulina G , Masculino , Células Mesangiais/enzimologia , Células Mesangiais/imunologia , Células Mesangiais/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteinúria/tratamento farmacológico , Proteinúria/enzimologia , Ratos , Ratos Wistar , Antígenos Thy-1/imunologiaRESUMO
Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Tiorredoxinas/fisiologia , Animais , Linhagem Celular , Nefropatias Diabéticas/genética , Cães , Feminino , Rim/fisiologia , Túbulos Renais Coletores/fisiologia , Túbulos Renais Proximais/fisiologia , RNA Mensageiro/genética , Ratos , Valores de Referência , Tiorredoxinas/genéticaRESUMO
BACKGROUND: Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the beta isoform of this enzyme is in advanced clinical development. However, PKC-beta is also increased in various forms of human glomerulonephritis with several potentially nephrotoxic factors, other than high glucose, resulting in PKC-beta activation. Accordingly, we sought to examine the effects of PKC-beta inhibition in a non-diabetic model of progressive kidney disease. METHODS: Subtotally nephrectomized (STNx) rats were randomly assigned to receive either the selective PKC-beta inhibitor, ruboxistaurin or vehicle. In addition to functional and structural parameters, gene expression of the podocyte slit-pore diaphragm protein, nephrin, was also assessed. RESULTS: STNx animals developed hypertension, proteinuria and reduced glomerular filtration rate (GFR) in association with marked glomerulosclerosis and tubulointerstitial fibrosis. Glomerular nephrin expression was also reduced. Without affecting blood pressure, ruboxistaurin treatment attenuated the impairment in GFR and reduced the extent of both glomerulosclerosis and tubulointerstitial fibrosis in STNx rats. In contrast, neither proteinuria nor the reduction in nephrin expression was improved by ruboxistaurin. CONCLUSIONS: These findings indicate firstly that PKC-beta inhibition may provide a new therapeutic strategy in non-diabetic kidney disease and secondly that improvement in GFR is not inextricably linked to reduction in proteinuria.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Indóis/uso terapêutico , Nefropatias/tratamento farmacológico , Nefropatias/enzimologia , Maleimidas/uso terapêutico , Proteína Quinase C/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , Proteínas de Membrana/genética , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Proteína Quinase C beta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Tranilast is an anti-inflammatory drug in use for asthma and atopic dermatitis. In studies over the last decade it has been revealed that tranilast can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. We report a structure-activity study aimed at optimizing the antifibrotic activity of tranilast. A series of cinnamoyl anthranilates were prepared and assessed for their ability to prevent TGF-beta-stimulated production of collagen in cultured renal mesangial cells. We reveal derivatives with improved potency and reduced cellular toxicity relative to tranilast. 3-Methoxy-4-propargyloxycinnamoyl anthranilate reduces albuminuria in a rat model of progressive diabetes, and thus has potential as an innovative treatment for diabetic nephropathy.
Assuntos
Ácidos Cafeicos/química , Cinamatos/química , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Rim/efeitos dos fármacos , Rim/patologia , ortoaminobenzoatos/química , Animais , Ácidos Cafeicos/farmacologia , Ácidos Cafeicos/uso terapêutico , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Colágeno/antagonistas & inibidores , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Fibrose , Ratos , Ratos Sprague-Dawley , ortoaminobenzoatos/farmacologia , ortoaminobenzoatos/uso terapêuticoRESUMO
OBJECTIVE: The pathological accumulation of extracellular matrix is a characteristic feature of diabetic cardiomyopathy that is directly related to a loss of function. Tranilast (n-[3,4-anthranilic acid), used for the treatment of fibrotic skin diseases, has also been shown to inhibit transforming growth factor-beta (TGF-beta)-induced matrix production in kidney epithelial cells. METHODS: To investigate the effects of tranilast in the diabetic heart, we examined its effects in cultured cardiac fibroblasts and then assessed its effects in (mRen-2)27 diabetic rats with established disease (8 weeks after streptozotocin). RESULTS: In vitro studies demonstrated a 58% reduction in TGF-beta1-induced 3[H]-hydroxyproline incorporation with tranilast 30 microM (p<0.01). At 16 weeks, diabetes in the Ren-2 rat was associated with increased cardiac fibrosis and evidence of TGF-beta1 activation, as measured by the abundance of phosphorylated Smad2. Despite persistent hyperglycaemia and hypertension, tranilast attenuated cardiac fibrosis by 37% (p<0.05) in association with reduction in phospho-Smad2 (p<0.01). CONCLUSION: These findings indicate that tranilast has antifibrotic actions in the Ren-2 model of experimental diabetic cardiac disease by mechanisms that might attributable to reduced TGF-beta activity.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Matriz Extracelular/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , ortoaminobenzoatos/uso terapêutico , Animais , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibrose , Coração/efeitos dos fármacos , Miocárdio/patologia , Fosforilação , Ratos , Proteína Smad2 , Transativadores/metabolismo , Fator de Crescimento Transformador beta1 , ortoaminobenzoatos/farmacologiaRESUMO
Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of the antioxidant thioredoxin, and a critical agent in the in vivo regulation of glucose. The well-described induction of TXNIP by high glucose may represent an important pathogenic trigger of complications arising in the diabetic environment, with sustained overexpression of TXNIP triggering the increased production of reactive oxygen species and collagen, both major contributors to the development of diabetic nephropathy (DN). To examine a possible therapeutic role for targeted TXNIP inhibition in DN, transgenic (mRen-2)27 rats were rendered diabetic with streptozotocin and then treated with 20 µM TXNIP deoxyribozyme (DNAzyme) delivered continuously over 12 weeks by an implanted osmotic mini-pump. Renal injury was measured using biochemical parameters of kidney function along with histological markers of damage. Catalytic activity of TXNIP DNAzyme was determined by TXNIP gene and peptide expression in the rat kidneys. TXNIP DNAzyme localization was demonstrated with a fluorescent-labelled TXNIP DNAzyme. A panel of markers was used to assess the extent of oxidative stress and renal fibrosis including superoxide level, nitrotyrosine staining, TGF-ß1, NLRP3 and collagen IV expression. Fluorescent-labelled TXNIP DNAzyme was localized to tubulo-epithelial cells, but was not identified in glomeruli or endothelial cells. Elevated renal cortical TXNIP gene and protein expression seen in kidneys of DN animals were significantly attenuated by TXNIP DNAzyme (p < 0.05). Downstream markers of TXNIP activity, particularly oxidative stress, inflammasome signalling, tubulo-interstitial fibrosis and collagen deposition, were also attenuated in the tubulo-interstitium of DN rats treated with TXNIP DNAzyme. Consistent with the identified site of action of the DNAzyme, the effects of the TXNIP inhibition were limited to the tubulo-interstitial compartment. This study supports the role of TXNIP as an important mediator of progressive tubulo-interstitial fibrosis in DN, and also supports the notion of TXNIP inhibition as a potential new therapeutic target for DN.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Animais , Glicemia/metabolismo , Proteínas de Transporte/metabolismo , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Progressão da Doença , Feminino , Fibrose , Rim/patologia , Testes de Função Renal , Glomérulos Renais/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
BACKGROUND: The high level of expression of vascular endothelial growth factor (VEGF) in normal podocyte foot processes suggests that VEGF has an important role in maintaining normal glomerular function. While altered VEGF expression occurs in many glomerular diseases, a direct role for VEGF in the pathogenesis of proteinuria has not been demonstrated. METHODS: Expression of VEGF and its receptors (VEGFR-1 and VEGFR-2) was examined in passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), by immunohistochemistry, in situ hybridization, Northern and Western blotting. Inhibition of VEGF in the PAN model was performed by administration of a blocking antibody. RESULTS: In both models, glomeruli showed upregulation of VEGF and VEGF receptors compared to control animals. VEGF mRNA was increased most significantly (5-fold) at day 5 after induction of PHN, prior to the onset of proteinuria, with persistent upregulation (3-fold) at day 21. Increased VEGF mRNA was also seen in PAN, but it was less marked. In situ hybridization and immunohistochemistry localized VEGF predominantly to podocytes. Increased expression of VEGFR-1 and VEGFR-2 protein was seen in glomerular endothelial cells of PHN and PAN rats by immunohistochemistry, as was VEGFR-2 mRNA by in situ hybridization. Upregulation of VEGFR-1 by endothelial cells was more striking in the PAN model than PHN. Administration of a blocking antibody to rats with PAN did not affect proteinuria, creatinine clearance or sodium excretion. CONCLUSION: The expression of VEGF and its receptors is significantly increased in the PHN and PAN rat models of proteinuria suggesting a role for VEGF in the disease process. VEGF may have an important role in promoting glomerular repair in a variety of glomerular diseases.
Assuntos
Modelos Animais de Doenças , Proteinúria/metabolismo , Proteinúria/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Linhagem Celular , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomerulonefrite/urina , Imuno-Histoquímica/métodos , Glomérulos Renais/química , Glomérulos Renais/patologia , Masculino , Nefrose/metabolismo , Nefrose/patologia , Nefrose/urina , Puromicina Aminonucleosídeo/toxicidade , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
BACKGROUND: Pathological deposition of extracellular matrix in the non-infarct zone (NIZ) of the ventricle post myocardial infarction (MI) is a key contributor to cardiac remodeling and heart failure. FT011, a novel antifibrotic compound, was evaluated for its efficacy in neonatal cardiac fibroblasts (NCF) and in an experimental MI model. METHODS AND RESULTS: Collagen synthesis in NCF was determined by (3)H-proline incorporation following stimulation with TGF-ß or angiotensin II (Ang II). FT011 inhibited collagen synthesis to both agents in a dose dependent manner. In vivo, Sprague Dawley rats underwent left anterior descending coronary artery ligation or sham surgery and were randomized one week later to receive either FT011 (200mg/kg/day) or vehicle for a further 4 weeks. Echocardiography and cardiac catheterization were performed, and tissues were collected for histological analysis of collagen, myocyte hypertrophy, interstitial macrophage accumulation and Smad2 phosphorylation. mRNA expression of collagens I and III and TGF-ß was measured using in situ hybridization and RT-PCR, respectively. FT011 treatment was associated with improved cardiac function (increased ejection fraction, fraction shortening and preload recruitable stroke work) and myocardial remodeling (reduced left ventricular diameter and volume at both end diastolic and systolic) compared with vehicle treatment. FT011 significantly reduced collagen matrix deposition, myocyte hypertrophy and interstitial macrophage infiltration, and mRNA expression of collagens I and III in NIZ compared with vehicle treatment. CONCLUSION: Anti-fibrotic therapy with FT011 in MI rats attenuated fibrosis and preserved systolic function.
Assuntos
Antifibrinolíticos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Ácidos Cafeicos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Remodelação Ventricular/efeitos dos fármacos , ortoaminobenzoatos/uso terapêutico , Animais , Animais Recém-Nascidos , Antifibrinolíticos/farmacologia , Pressão Sanguínea/fisiologia , Ácidos Cafeicos/farmacologia , Colágeno/antagonistas & inibidores , Colágeno/biossíntese , Masculino , Infarto do Miocárdio/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Remodelação Ventricular/fisiologia , ortoaminobenzoatos/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-ß or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD. EXPERIMENTAL APPROACH: Accordingly, we studied the effects of a recently described, small molecule anti-fibrotic drug, 3-methoxy-4-propargyloxycinnamoyl anthranilate (FT011, Fibrotech Therapeutics, Australia), which should have these effects. KEY RESULTS: In the in vitro setting, FT011 inhibited both TGF-ß1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. Consistent with these in vitro actions, when studied in a robust model of non-diabetic kidney disease, the 5/6 nephrectomised rat, FT011 attenuated the decline in GFR, proteinuria and glomerulosclerosis (p<0.05 for all). Similarly, in the streptozotocin-diabetic Ren-2 rat, a model of advanced diabetic nephropathy, FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. CONCLUSIONS AND IMPLICATIONS: Together these studies suggest that broadly antagonising growth factor actions, including those of TGF-ß1 and PDGF-BB, has the potential to protect the kidney from progressive injury in both the diabetic and non-diabetic settings.
Assuntos
Ácidos Cafeicos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , ortoaminobenzoatos/uso terapêutico , Albuminúria/complicações , Albuminúria/tratamento farmacológico , Animais , Becaplermina , Ácidos Cafeicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Feminino , Macrófagos/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Osteopontina/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-sis/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/patologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , ortoaminobenzoatos/farmacologiaRESUMO
AIMS: Cardiac remodelling in diabetes includes pathological accumulation of extracellular matrix and myocyte hypertrophy that contribute to heart dysfunction. Attenuation of remodelling represents a potential therapeutic target. We tested this hypothesis using a new anti-fibrotic drug, FT011 (Fibrotech Therapeutics Pty Ltd), on diabetic Ren-2 rats, a model which replicates many of the structural and functional manifestations of diabetic cardiomyopathy in humans. METHODS AND RESULTS: Homozygous Ren-2 rats were randomized to receive streptozotocin or vehicle then further randomized to FT011 (200 mg/kg/day) or vehicle treatment for 6 weeks. Prior to tissue collection, cardiac function was assessed via echocardiography and cardiac catheterization. Total collagen deposition and cardiomyocyte hypertrophy were assessed by picrosirius red and haematoxylin and eosin staining, respectively. Macrophage interstitial infiltration and type I and III collagen were quantitated by immunostaining. Without affecting blood pressure or hyperglycaemia, treatment of diabetic rats with FT011 significantly attenuated interstitial fibrosis (total collagen, 5.09 ±1.28 vs, 2.42 ±0.43%/area; type I collagen, 4.09 ±1.16 vs. 1.42 ±0.38%/area; type III collagen, 1.52 ±0.33 vs. 0.71 ±0.14 %/area; P < 0.05), cardiomyocyte hypertrophy (882 ±38 vs. 659 ±28 µm(2); P < 0.05), and interstitial macrophage influx (66 ±5.3 vs, 44 ±7.9 number/section; P < 0.05). Cardiac myopathic dilatation was normalized, as evidenced by reduced left ventricular inner diameter at diastole (0.642 ±0.016 vs. 0.577 ±0.024 cm), increased ejection fraction (75 ±1.1 vs. 83 ±1.2%) and preload recruitable stroke work relationship (44 ±6.7 vs. 77 ±6.3 slope-mmHg; P < 0.05), and reduced end-diastolic pressure-volume relationship (0.059 ±0.011 vs. 0.02 ±0.003 slope-mmHg/µL; P < 0.05). CONCLUSIONS: A direct anti-fibrotic agent, FT011, attenuates cardiac remodelling and dysfunction in experimental diabetic cardiomyopathy. This represents a novel therapy for the treatment of diabetic cardiomyopathy associated with cardiac fibrosis and hypertrophy.
Assuntos
Antifibrinolíticos/uso terapêutico , Ácidos Cafeicos/uso terapêutico , Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Miocárdio/patologia , Miócitos Cardíacos/patologia , ortoaminobenzoatos/uso terapêutico , Animais , Cateterismo Cardíaco , Cardiomegalia/etiologia , Cardiomegalia/patologia , Doença Crônica , Colágeno/metabolismo , Fibrose/tratamento farmacológico , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/metabolismo , Imuno-Histoquímica , Masculino , Ratos , Ratos Endogâmicos , Ratos Sprague-Dawley , EstreptozocinaRESUMO
BACKGROUND: Heart failure is a common cause of morbidity and mortality in diabetic patients that frequently manifests in the absence of impaired left ventricular systolic function. In contrast to the strong evidence base for the treatment of systolic heart failure, the treatment of heart failure with preserved left ventricular function is uncertain, and therapeutic targets beyond blockade of the renin-angiotensin-aldosterone and beta-adrenergic systems are being sought. One such target is the beta-isoform of protein kinase C (PKC), implicated in both the complications of diabetes and in cardiac dysfunction in the nondiabetic setting. METHODS AND RESULTS: Using a hemodynamically validated rodent model of diabetic diastolic heart failure, the (mRen-2)27 transgenic rat, we sought to determine whether selective inhibition of PKC-beta would preserve cardiac function and reduce structural injury. Diabetic rats were randomized to receive either vehicle or the PKC-beta inhibitor, ruboxistaurin (20 mg/kg per d) and followed for 6 weeks. Compared with untreated animals, ruboxistaurin-treated diabetic rats demonstrated preserved systolic and diastolic function, as measured by the slope of preload recruitable stroke work relationship (P<0.05) and the slope of the end-diastolic pressure volume relationship (P<0.01). Collagen I deposition and cardiomyocyte hypertrophy were both reduced in diabetic animals treated with ruboxistaurin (P<0.01), as was phosphorylated-Smad2, an index of transforming growth factor-beta activity (P<0.01 for all, versus untreated diabetic rats). CONCLUSIONS: PKC-ss inhibition attenuated diastolic dysfunction, myocyte hypertrophy, and collagen deposition and preserved cardiac contractility. PKC-beta inhibition may represent a novel therapeutic strategy for the prevention of diabetes-associated cardiac dysfunction.
Assuntos
Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Diabetes Mellitus Experimental/complicações , Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Indóis/farmacologia , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cateterismo Cardíaco , Cardiomiopatias/diagnóstico , Cardiomiopatias/patologia , Colágeno/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diástole , Matriz Extracelular/metabolismo , Expressão Gênica , Homozigoto , Hipertrofia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Estresse Oxidativo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Ratos , Ratos Transgênicos , Renina/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteína Smad2/metabolismoRESUMO
The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H(+)-ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment-specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H(+)-ATPase, the ion exchanger that secretes H(+) ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal-regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H(+)-ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H(+)-ATPase, suggests that the (P)RR may function primarily in distal nephron H(+) transport, recently noted to be, at least in part, an angiotensin II-dependent phenomenon.
Assuntos
Transporte Biológico/fisiologia , Túbulos Renais Coletores/citologia , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Túbulos Renais Coletores/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Fosforilação , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Simportadores de Sódio-Bicarbonato/metabolismo , Receptor de Pró-ReninaRESUMO
Diastolic dysfunction is an increasingly recognized complication of diabetes that develops in relatively young patients as a result of diabetic cardiomyopathy (DCM). With recent advances in echocardiographic technology now permitting the reliable assessment of diastolic function in the rat, we examined cardiac function and structure in diabetic rodents and assessed the effects of intervening with tranilast, an antifibrotic compound that has been shown to attenuate the actions of transforming growth factor-beta (TGF-beta) in cardiac fibroblasts. We also sought to examine the mechanism whereby tranilast inhibits the actions of TGF-beta. Six-week-old heterozygous (mRen-2)27 rats were randomized to receive either streptozotocin or citrate buffer and then further randomized to receive either tranilast (400 mg x kg(-1) x day(-1) by twice daily gavage) or vehicle for another 8 wk. Cell signaling was examined in neonatal cardiac fibroblasts. After 8 wk, diabetic rats showed evidence of impaired diastolic function with reduced early-to-late atrial wave ratio and prolonged deceleration time in association with fibrosis, apoptosis, and hypertrophy (all P < 0.05). Treatment with tranilast prevented the development of diastolic dysfunction and the histopathological features of DCM. While tranilast did not affect Smad phosphorylation, it significantly attenuated TGF-beta-induced p44/42 mitogen-activated protein kinase phosphorylation.
Assuntos
Cardiomiopatias/patologia , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/patologia , Modelos Animais de Doenças , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/prevenção & controle , ortoaminobenzoatos/administração & dosagem , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/metabolismo , Citocinas/metabolismo , Complicações do Diabetes/induzido quimicamente , Complicações do Diabetes/metabolismo , Diástole/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Ratos , Estreptozocina , Resultado do Tratamento , Ultrassonografia , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/metabolismoRESUMO
Ruboxistaurin is an inhibitor of the beta isoform of protein kinase C (PKC-beta) that reduces the actions of vascular endothelial growth factor (VEGF) and attenuates the progression of diabetic retinopathy. In the glomerulus VEGF is constitutively expressed where it likely has a role in maintaining endothelial cell integrity, particularly in disease states. Given its potential use in diabetic nephropathy, we sought to determine the effects of PKC-beta inhibition on VEGF and glomerular endothelial cells in experimental diabetic nephropathy. Studies were conducted in (mRen-2)27 rat, a transgenic rodent with hypertension and an enhanced renin-angiotensin system that following induction of diabetes with streptozotocin develops many of the features of diabetic nephropathy. Moreover, to mimic the clinical context, the effects of PKC-beta inhibition were examined both with and without concomitant angiotensin-converting enzyme (ACE) inhibitor therapy. Diabetic Ren-2 rats were randomized to receive either vehicle, the ACE inhibitor, perindopril (0.2 mg/l in drinking water), ruboxistaurin (10 mg.kg(-1).day(-1), admixed in chow), or their combination and studied for 12 wk. Diabetic Ren-2 rats displayed glomerular endothelial cell loss in association with overexpression of VEGF mRNA. Both cell loss and VEGF overexpression were attenuated by the administration of either perindopril or ruboxistaurin, as single agent treatments with their combination providing additional, incremental improvements, reducing these manifestations of injury down to levels seen in nondiabetic, normotensive, nontransgenic animals. Combination therapy was also associated with additional improvements in albuminuria and glomerulosclerosis.
Assuntos
Nefropatias Diabéticas/metabolismo , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Glomérulos Renais/metabolismo , Proteína Quinase C/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Animais Geneticamente Modificados , Autorradiografia , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Hibridização In Situ , Glomérulos Renais/efeitos dos fármacos , Proteína Quinase C beta , Ratos , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Inhibiting the actions of VEGF is a new therapeutic paradigm in cancer management with antiangiogenic therapy also under intensive investigation in a range of nonmalignant diseases characterized by pathological angiogenesis. However, the effects of VEGF inhibition on organs that constitutively express it in adulthood, such as the kidney, are mostly unknown. Accordingly, we examined the effect of VEGF inhibition on renal structure and function under physiological conditions and in the setting of the common renal stressors: hypertension and activation of the renin-angiotensin system. When compared with normotensive Sprague-Dawley (SD) rats, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats that overexpress renin with spontaneously hypertensive rat (SHR) kidneys showing VEGF expression levels that were intermediate between them. Administration of either an orally active inhibitor of the type 2 VEGF receptor (VEGFR-2) tyrosine kinase or a VEGF neutralizing antibody to TGR(mRen-2)27 rats resulted in loss of glomerular endothelial cells and transformation to a malignant hypertensive phenotype with severe glomerulosclerosis. VEGFR-2 kinase inhibition treatment was well tolerated in SDs and SHRs; although even in these animals there was detectable endothelial cell loss and rise in albuminuria. Mild mesangial expansion was also noted in hypertensive SHR, but not in SD rats. These studies illustrate: (i) VEGF has a role in the maintenance of glomerular endothelial integrity under physiological circumstances, (ii) glomerular VEGF is increased in response to hypertension and activation of the renin-angiotensin system, and (iii) VEGF signaling plays a protective role in the setting of these renal stressors.