RESUMO
Altered nucleoside levels may be linked to pathogenic signaling through adenosine receptors. We hypothesized that adenosine dysregulation contributes to fibrosis in diabetic kidney disease. Our findings indicate that high glucose levels and experimental diabetes decreased uptake activity through the equilibrative nucleoside transporter 1 (ENT1) in proximal tubule cells. In addition, a correlation between increased plasma content of adenosine and a marker of renal fibrosis in diabetic rats was evidenced. At the cellular level, exposure of HK2 cells to high glucose, TGF-ß and the general adenosine receptor agonist NECA, induced the expression of profibrotic cell activation markers α-SMA and fibronectin. These effects can be avoided by using a selective antagonist of the adenosine A3 receptor subtype in vitro. Furthermore, induction of fibrosis marker α-SMA was prevented by the A3 receptor antagonist in diabetic rat kidneys. In conclusion, we evidenced the contribution of purinergic signaling to renal fibrosis in experimental diabetic nephropathy.
Assuntos
Adenosina/metabolismo , Nefropatias Diabéticas/metabolismo , Fibrose/metabolismo , Túbulos Renais/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Nefropatias Diabéticas/patologia , Células Epiteliais/metabolismo , Humanos , Túbulos Renais/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A(2B) receptor (A(2B)AR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A(2B)AR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A(2B)AR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Receptor A2B de Adenosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acetamidas/farmacologia , Adenosina/metabolismo , Adenosina/urina , Animais , Glicemia/metabolismo , Pressão Sanguínea/fisiologia , Peso Corporal/fisiologia , Diabetes Mellitus Experimental/urina , Nefropatias Diabéticas/urina , Histocitoquímica , Glomérulos Renais/química , Glomérulos Renais/metabolismo , Masculino , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
AIMS: Reduced expression of human equilibrative nucleoside transporter 1 (hENT1) results from nitric oxide (NO)-dependent reduced SLC29A1 transcriptional activity in human umbilical vein endothelial cells (HUVECs) from gestational diabetes. As expression of the transcription factor C/EBP homologous protein 10 (hCHOP, which forms heterodimers with C/EBPalpha transcription factor) is activated by NO and induced in diabetes mellitus, we hypothesize that hCHOP plays a role in the gestational diabetes-reduced hENT1 expression in HUVECs. METHODS AND RESULTS: HUVEC primary cultures from 42 normal and 42 gestational diabetic pregnancies were used for adenosine uptake assays. Real-time PCR (mRNA quantification), western blotting (protein abundance), and luciferase activity (SLC29A1 promoter activity) were used. hCHOP-C/EBPalpha activity was assayed by chromatin immunoprecipitation. Overlap extension mutagenesis was used to generate a mutated hCHOP-C/EBPalpha consensus site at the SLC29A1 promoter, and endothelial NO synthase (eNOS) siRNA recombinant adenovirus was used to knock down eNOS. hCHOP nuclear protein abundance and binding to DNA were higher in gestational diabetes, paralleled by reduced SLC29A1 promoter activity, hENT1 expression, and transport activity. These changes were blocked by hCHOP consensus sequence mutation (-1845G > T and -1844C > A), eNOS-siRNA-induced knockdown, and N(G)-nitro-L-arginine methyl ester (NOS inhibitor), and were mimicked by S-nitroso-N-acetyl-L, D-penicillamine (NO donor) in cells from normal pregnancies. hCHOP and C/EBPalpha overexpression mimicked gestational diabetes effects in cells from normal pregnancies, but did not alter SLC29A1 promoter activity or hENT1-adenosine transport in cells from gestational diabetes. CONCLUSION: The hCHOP-C/EBPalpha complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diabetes Gestacional , Células Endoteliais/fisiologia , Transportador Equilibrativo 1 de Nucleosídeo/genética , Óxido Nítrico/metabolismo , Fator de Transcrição CHOP/metabolismo , Adenosina/metabolismo , Arginina/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Células Cultivadas , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patologia , Diabetes Gestacional/fisiopatologia , Regulação para Baixo/fisiologia , Células Endoteliais/citologia , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Feminino , Humanos , Mutagênese/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Regiões Promotoras Genéticas/fisiologia , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/genética , Ativação Transcricional/fisiologia , Veias Umbilicais/citologiaRESUMO
AIMS: We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs. METHODS AND RESULTS: HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. CONCLUSION: hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.
Assuntos
Adenosina/metabolismo , Endotélio Vascular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/genética , Feminino , Humanos , Gravidez , Complicações na Gravidez/metabolismo , Regiões Promotoras Genéticas , Receptor do Fator de Crescimento Transformador beta Tipo II , Transcrição Gênica , VasodilataçãoRESUMO
High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial dysfunction.
Assuntos
Adenosina/metabolismo , Endotélio Vascular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/genética , Glucose/farmacologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Veias Umbilicais/metabolismo , Regiões 5' não Traduzidas/genética , Sequência de Bases , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Endotélio Vascular/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Dominantes , Humanos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Transcrição Gênica/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacosRESUMO
Human umbilical vein endothelial cells (HUVEC) from gestational diabetes exhibit reduced adenosine uptake and increased nitric oxide (NO) synthesis. Adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) is reduced by NO by unknown mechanisms in HUVEC. We examined whether gestational diabetes-reduced adenosine transport results from lower hENT1 gene (SLC29A1) expression. HUVEC from gestational diabetes exhibit reduced SLC29A1 promoter activity when transfected with pGL3-hENT1(-2154) compared with pGL3-hENT1(-1114) constructs, an effect blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), but unaltered by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). In cells from gestational diabetes transfected with pGL3-hENT1(-2154), L-NAME increased, but SNAP did not alter promoter activity and hENT1 expression. However, in cells from normal pregnancies L-NAME increased, but SNAP reduced promoter activity and hENT1 expression. Adenovirus-silenced eNOS expression increased hENT1 expression and activity in cells from normal or gestational diabetic pregnancies. Thus, reduced adenosine transport may result from downregulation of SLC29A1 expression by NO in HUVEC from gestational diabetes. These findings explain the accumulation of extracellular adenosine detected in cultures of HUVEC from gestational diabetes. In addition, fetal endothelial dysfunction could be involved in the abnormal fetal development and growth seen in gestational diabetes.
Assuntos
Diabetes Gestacional/metabolismo , Endotélio Vascular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Óxido Nítrico/farmacologia , Regiões Promotoras Genéticas , Adenosina/metabolismo , Adenoviridae/genética , Transporte Biológico , Estudos de Casos e Controles , Técnicas de Cultura de Células , Células Cultivadas , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Feminino , Genes Reporter , Humanos , Luciferases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Interferência de RNA , Veias Umbilicais/citologiaRESUMO
Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2 and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2) reduced maximal hENT1-adenosine transport velocity (V(max)) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N(omega)-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase [NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser(1177), but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44(mapk) phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44(mapk) may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.