RESUMEN
[Figure: see text].
Asunto(s)
Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Colesterol/metabolismo , Proteínas de Choque Térmico/administración & dosificación , Chaperonas Moleculares/administración & dosificación , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Vacunas Sintéticas/administración & dosificación , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos/sangre , Aorta/enzimología , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/inmunología , Aterosclerosis/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Inmunogenicidad Vacunal , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Persona de Mediana Edad , Chaperonas Moleculares/inmunología , Chaperonas Moleculares/metabolismo , Placa Aterosclerótica , Receptores de LDL/genética , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.
Asunto(s)
Apolipoproteína B-100/sangre , Apolipoproteínas E/sangre , Aterosclerosis/patología , Lipoproteínas LDL/sangre , Metaloproteinasa 14 de la Matriz/metabolismo , Receptores de LDL/metabolismo , Animales , Apolipoproteínas E/genética , Línea Celular Tumoral , Ésteres del Colesterol/metabolismo , Dependovirus/genética , Femenino , Células HEK293 , Células Hep G2 , Humanos , Masculino , Metaloproteinasa 14 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDL receptor (LDLR) degradation, increasing plasma levels of LDL cholesterol and the risk of cardiovascular disease. We have previously shown that, in addition to the epidermal growth factor precursor homology repeat-A of LDLR, at least three ligand-binding repeats (LRs) of LDLR are required for PCSK9-promoted LDLR degradation. However, how exactly the LRs contribute to PCSK9's action on the receptor is not completely understood. Here, we found that substitution of Asp at position 172 in the linker between the LR4 and LR5 of full-length LDLR with Asn (D172N) reduced PCSK9 binding at pH 7.4 (mimic cell surface), but not at pH 6.0 (mimic endosomal environment). On the other hand, mutation of Asp at position 203 in the LR5 of full-length LDLR to Asn (D203N) significantly reduced PCSK9 binding at both pH 7.4 and pH 6.0. D203N also significantly reduced the ability of LDLR to mediate cellular LDL uptake, whereas D172N had no detectable effect. These findings indicate that amino acid residues in the LRs of LDLR play an important role in PCSK9 binding to the receptor.
Asunto(s)
Proproteína Convertasa 9/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Secuencias Repetitivas de Aminoácido , Células HEK293 , Humanos , Ligandos , Lipoproteínas LDL/metabolismo , Mutación , Unión Proteica , Receptores de LDL/genéticaRESUMEN
Recently, we demonstrated that heat shock protein (HSP)-27 is protective against the development of experimental atherosclerosis, reducing plaque cholesterol content by more than 30%. Moreover, elevated HSP-27 levels are predictive of relative freedom from clinical cardiovascular events. HSP-27 signaling occurs via the activation of NF-κB, which induces a marked up-regulation in expression of granulocyte-monocyte colony-stimulating factor (GM-CSF), a cytokine that is known to alter ABC transporters involved in reverse cholesterol transport (RCT). Therefore, we hypothesized that HSP-27-derived GM-CSF has a potent role in impeding plaque formation by promoting macrophage RCT and sought to better characterize this pathway. Treatment of THP-1 cells, RAW-Blue cells, and primary macrophages with recombinant HSP-27 resulted in NF-κB activation via TLR-4 and was inhibited by various pharmacologic blockers of this pathway. Moreover, HSP-27-induced upregulation of GM-CSF expression was dependent on TLR-4 signaling. Recombinant (r)HSP-27 treatment of ApoE-/- female (but not male) mice for 4 wk yielded reductions in plaque area and cholesterol clefts of 33 and 47%, respectively, with no effect on GM-CSF-/-ApoE-/- mice. With 12 wk of rHSP-27 treatment, both female and male mice showed reductions in plaque burden (55 and 42%, respectively) and a 60% reduction in necrotic core area but no treatment effect in GM-CSF-/-ApoE-/- mice. In vitro functional studies revealed that HSP-27 enhanced the expression of ABCA1 and ABCG1, as well as facilitated cholesterol efflux in vitro by â¼10%. These novel findings establish a paradigm for HSP-27-mediated RCT and set the stage for the development of HSP-27 atheroprotective therapeutics.-Pulakazhi Venu, V. K., Adijiang, A., Seibert, T., Chen, Y.-X., Shi, C., Batulan, Z., O'Brien, E. R. Heat shock protein 27-derived atheroprotection involves reverse cholesterol transport that is dependent on GM-CSF to maintain ABCA1 and ABCG1 expression in ApoE-/- mice.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Apolipoproteínas E/metabolismo , Aterosclerosis/prevención & control , Colesterol/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Animales , Apolipoproteínas E/genética , Línea Celular , Regulación de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteínas de Choque Térmico HSP27/genética , Humanos , Macrófagos , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Low-density lipoprotein receptor (LDLR) catalyzes the uptake of LDL-cholesterol by liver and peripheral organs. The function of the LDLR is antagonized by pro-protein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR at the plasma membrane inducing LDLR degradation. Here, we report that matrix metalloproteinase-2 (MMP-2) interacts with and cleaves PCSK9, as evidenced by proteomic, chemical cross-linkage, blue native-PAGE and domain-specific antibodies Western blot analyses. Furthermore, MMP-2 overexpression renders Hepa1-c1c7 cells resistant to PCSK9-induced LDLR degradation. The data suggest that pathological MMP-2 overexpression may protect the LDLR from PCSK-9-induced degradation.
Asunto(s)
Metaloproteinasa 2 de la Matriz/fisiología , Proproteína Convertasas/fisiología , Receptores de LDL/metabolismo , Serina Endopeptidasas/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Ratones , Datos de Secuencia Molecular , Proproteína Convertasa 9 , ProteolisisRESUMEN
Proprotein convertase subtilisin kexin-like 9 (PCSK9) promotes the degradation of low density lipoprotein receptor (LDLR) and plays an important role in regulating plasma LDL-cholesterol levels. We have shown that the epidermal growth factor precursor homology domain A (EGF-A) of the LDLR is critical for PCSK9 binding at the cell surface (pH 7.4). Here, we further characterized the role of EGF-A in binding of PCSK9 to the LDLR. We found that PCSK9 efficiently bound to the LDLR but not to other LDLR family members. Replacement of EGF-A in the very low density lipoprotein receptor (VLDLR) with EGF-A of the LDLR promoted the degradation of the mutant VLDLR induced by PCSK9. Furthermore, we found that PCSK9 bound to recombinant EGF-A in a pH-dependent manner with stronger binding at pH 6.0. We also identified amino acid residues in EGF-A of the LDLR important for PCSK9 binding. Mutations G293H, D299V, L318D, and L318H reduced PCSK9 binding to the LDLR at neutral pH without effect at pH 6.0, while mutations R329P and E332G reduced PCSK9 binding at both pH values. Thus, our findings reveal that EGF-A of the LDLR is critical for PCSK9 binding at the cell surface (neutral pH) and at the acidic endosomal environment (pH 6.0), but different determinants contribute to efficient PCSK9 binding in different pH environments.
Asunto(s)
Factor de Crecimiento Epidérmico/química , Proproteína Convertasas/metabolismo , Precursores de Proteínas/química , Receptores de LDL/química , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Secuencia Conservada , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Proproteína Convertasa 9 , Unión Proteica , Estructura Terciaria de Proteína , Proteolisis , Receptores de LDL/genéticaRESUMEN
The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naive T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in 'lipotoxic danger signals' such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the re-establishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT.
Asunto(s)
Envejecimiento/inmunología , Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Timo/crecimiento & desarrollo , Timo/inmunología , Envejecimiento/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Caspasa 1/metabolismo , Microambiente Celular , Senescencia Celular/efectos de los fármacos , Senescencia Celular/inmunología , Ceramidas/metabolismo , Colesterol/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/patología , Eliminación de Gen , Trasplante de Células Madre Hematopoyéticas , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lípidos/toxicidad , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Tamaño de los Órganos/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Timocitos/efectos de los fármacos , Timocitos/inmunología , Timocitos/patología , Timo/enzimología , Timo/patologíaRESUMEN
We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated ß-galactosidase activity and expression of p53, transforming growth factor (TGF)-ß1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, ß-galactosidase, TGF-ß1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.
Asunto(s)
Senescencia Celular/fisiología , Indicán/metabolismo , FN-kappa B/metabolismo , Actinas/metabolismo , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/metabolismo , Animales , Benzotiazoles/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Masculino , FN-kappa B/antagonistas & inhibidores , Regiones Promotoras Genéticas , Pirrolidinas/farmacología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Sesquiterpenos/farmacología , Tiocarbamatos/farmacología , Tolueno/análogos & derivados , Tolueno/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Clinical evidence that the blockade of IL-1ß in type-2 diabetic patients improves glycemia is indicative of an autoinflammatory mechanism that may trigger adiposity-driven pancreatic damage. IL-1ß is a key contributor to the obesity-induced inflammation and subsequent insulin resistance, pancreatic ß-cell dysfunction, and the onset of type 2 diabetes. Our previous studies demonstrated that the ceramides activate the Nod-like receptor family, pyrin domain containing 3 (Nlrp3) inflammasome to cause the generation of mature IL-1ß and ablation of the Nlrp3 inflammasome in diet-induced obesity improves insulin signaling. However, it remains unclear whether the posttranslational processing of active IL-1ß in pancreas is regulated by the NLRP3 inflammasome or whether the alternate mechanisms play a dominant role in chronic obesity-induced pancreatic ß-cell exhaustion. Here we show that loss of ASC, a critical adaptor required for the assembly of the NLRP3 and absent in melanoma 2 inflammasome substantially improves the insulin action. Surprisingly, despite lower insulin resistance in the chronically obese NLRP3 and ASC knockout mice, the insulin levels were substantially higher when the inflammasome pathway was eliminated. The obesity-induced increase in maturation of pancreatic IL-1ß and pancreatic islet fibrosis was dependent on the NLRP3 inflammasome activation. Furthermore, elimination of NLRP3 inflammasome protected the pancreatic ß-cells from cell death caused by long-term high-fat feeding during obesity with significant increase in the size of the islets of Langerhans. Collectively, this study provides direct in vivo evidence that activation of the NLRP3 inflammasome in diet-induced obesity is a critical trigger in causing pancreatic damage and is an important mechanism of progression toward type 2 diabetes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Inflamasomas/metabolismo , Obesidad/metabolismo , Páncreas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Fibrosis , Inflamasomas/genética , Inflamación/metabolismo , Inflamación/patología , Insulina/sangre , Resistencia a la Insulina/fisiología , Interleucina-1beta/metabolismo , Leptina/sangre , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Obesidad/genética , Obesidad/patología , Páncreas/patologíaRESUMEN
BACKGROUND/AIM: Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease (CKD). The present study aimed to examine whether renal expression of Klotho is regulated by indoxyl sulfate, a uremic toxin, using rat kidneys and human proximal tubular cells (HK-2). METHODS: The effect of indoxyl sulfate on renal expression of Klotho was examined using (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). The effects of indoxyl sulfate, inhibitors of nuclear factor-κB (NF-κB) and an antioxidant on the expression of Klotho in HK-2 cells were examined. RESULTS: DH+IS and DN+IS rats showed decreased expression of Klotho mRNA in the kidneys as compared with DH and DN rats, respectively. Indoxyl sulfate suppressed the expression of Klotho mRNA and protein in HK-2 cells, whereas an antioxidant, N-acetylcysteine, and NF-κB inhibitors, pyrrolidine dithiocarbamate and isohelenin, alleviated these effects. CONCLUSIONS: Indoxyl sulfate downregulates Klotho expression in kidneys through production of reactive oxygen species and activation of NF-κB in proximal tubular cells. Indoxyl sulfate may be involved in reduced renal expression of Klotho in CKD.
Asunto(s)
Regulación de la Expresión Génica , Glucuronidasa/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno , Acetilcisteína/metabolismo , Animales , Antioxidantes/metabolismo , Regulación hacia Abajo , Humanos , Indicán/metabolismo , Proteínas Klotho , Pirrolidinas/química , Ratas , Ratas Endogámicas Dahl , Sesquiterpenos/química , Tiocarbamatos/químicaRESUMEN
BACKGROUND: Administration of indoxyl sulfate, a uremic toxin, promotes progression of chronic kidney disease in rats affected by the disease. Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease. This study aimed to clarify whether indoxyl sulfate could reduce klotho expression and contribute to cell senescence in the kidneys of hypertensive rats. METHODS: The rats used for this study were segregated in to the following 4 groups: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their kidneys were excised for histological and immunohistochemical analysis for klotho, senescence-associated ß-galactosidase, p16(INK4a), p21(WAF1/CIP1), p53, and retinoblastoma protein (Rb). RESULTS: DH + IS rats showed decreased expression of klotho, increased expression of senescence-associated ß-galactosidase, p16(INK4a), p21(WAF1/CIP1), p53, and Rb in renal tubular cells, and increased tubulointerstitial fibrosis and mesangial expansion as compared with DH rats. Further, DN + IS rats showed decreased expression of klotho as compared with DN rats. CONCLUSION: Administration of indoxyl sulfate to hypertensive rats reduced renal expression of klotho and promoted cell senescence with expression of senescence-related proteins, such as p16(INK4a), p21(WAF1/CIP1), p53, and Rb, which was accompanied by renal fibrosis.
Asunto(s)
Senescencia Celular , Glucuronidasa/metabolismo , Hipertensión/metabolismo , Hipertensión/patología , Indicán/metabolismo , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Riñón/metabolismo , Riñón/patología , Proteínas Klotho , Ratas , Ratas Endogámicas Dahl , Proteína de Retinoblastoma/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Various uremic toxins accumulate in patients with chronic renal failure (CRF) and one of them is indoxyl sulfate, which accelerates the progression of CRF through unknown mechanisms. The present study investigates how indoxyl sulfate promotes CRF using the proximal tubular cell line HK-2 and CRF rats. Indoxyl sulfate inhibited serum-induced cell proliferation and promoted the activation of senescence-associated ß-galactosidase, a marker of cellular senescence, and the expression of α-smooth muscle actin (α-SMA), a marker of fibrosis, through inducing p53 expression and phosphorylation. Pifithrin-α, p-nitro, a p53 inhibitor, blocked these effects. Indoxyl sulfate evoked reactive oxygen species (ROS), and the antioxidant N-acetylcysteine inhibited indoxyl sulfate-induced p53 expression and phosphorylation, as well as indoxyl sulfate-induced α-SMA expression. We previously demonstrated that although cellular senescence and fibrosis are detectable in the kidneys of CRF rats, the oral adsorbent AST-120 repressed these effects. Here, we found that ß-galactosidase, p53 and α-SMA were expressed and colocalized in the renal tubules of CRF rats, whereas AST-120 decreased the expression of these genes. Taken together, these findings indicate that indoxyl sulfate induces the expression and phosphorylation of p53 though ROS production, thus inhibiting cell proliferation and promoting cellular senescence and renal fibrosis.
Asunto(s)
Línea Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Indicán/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/fisiología , Túbulos Renales Proximales/fisiopatología , Proteína p53 Supresora de Tumor/metabolismo , Acetilcisteína/farmacología , Animales , Benzotiazoles/farmacología , Proliferación Celular/efectos de los fármacos , Senescencia Celular/fisiología , Fibrosis/metabolismo , Humanos , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/fisiopatología , Túbulos Renales Proximales/citología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). After 32weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated beta-galactosidase (SA-beta-gal), and senescence-related proteins such as p16(INK4a), p21(WAF1/CIP1), p53 and retinoblastoma protein (Rb). Both DH and DH+IS rats showed significantly higher systolic blood pressure than DN and DN+IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN+IS and DH+IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-beta-gal, p16(INK4a), p21(WAF1/CIP1), p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH+IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-beta-gal, p16(INK4a), p21(WAF1/CIP1), p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.
Asunto(s)
Aorta/patología , Enfermedades de la Aorta/patología , Calcinosis/patología , Senescencia Celular , Hipertensión/patología , Indicán/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Enfermedades de la Aorta/inducido químicamente , Enfermedades de la Aorta/metabolismo , Calcinosis/inducido químicamente , Calcinosis/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Hipertensión/metabolismo , Indicán/toxicidad , Ratas , Ratas Endogámicas Dahl , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Uremia/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND/AIM: Klotho, an anti-aging gene, is primarily expressed in the kidney, and its renal expression is decreased in chronic renal failure (CRF). We determined if administration of an oral sorbent, AST-120, increases the expression of Klotho, and inhibits cell senescence in the kidney of CRF rats. METHODS: CRF rats were produced by 4/5-nephrectomy. AST-120 was administered to the CRF rats at a dose of 4 g/kg with powder chow for 16 weeks, whereas powder chow alone was administered to control CRF rats. The expression of Klotho and cell senescence (senescence-associated beta-galactosidase: SA-beta-gal) was detected by immunohistochemistry. RESULTS: The expression of Klotho was significantly decreased in the kidney of CRF rats as compared with normal rats. AST-120-treated CRF rats showed significantly increased renal expression of Klotho as compared with CRF rats. The expression of SA-beta-gal was significantly increased in the kidney of CRF rats as compared with normal rats. AST-120-treated CRF rats showed significantly decreased expression of SA-beta-gal in the kidney as compared with CRF rats. AST-120 significantly decreased serum and urine levels of indoxyl sulfate. CONCLUSIONS: AST-120 increased Klotho expression, and inhibited cell senescence in the kidney of CRF rats, probably by alleviating indoxyl sulfate overload on the kidney.
Asunto(s)
Carbono/farmacología , Glucuronidasa/biosíntesis , Óxidos/farmacología , Uremia/patología , Animales , Senescencia Celular , Inmunohistoquímica/métodos , Indicán/química , Riñón/metabolismo , Proteínas Klotho , Masculino , Mutación , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/tratamiento farmacológico , Factores de Tiempo , Uremia/metabolismoRESUMEN
BACKGROUND: Stage 5 chronic kidney disease (CKD) is associated with enhanced aortic calcification. The aim of this study was to determine if the administration of indoxyl sulphate (IS), a uraemic toxin, stimulates the progression of aortic calcification. METHODS: The rat groups consisted of (i) Dahl salt-resistant normotensive rats (DR) with intake of 0.3% salt, (ii) Dahl salt-sensitive hypertensive rats (DS) with intake of 2.0% salt and (iii) Dahl salt-sensitive hypertensive IS-administered rats (DS-IS) with intake of 2.0% salt and 200 mg/kg of IS in water. After 30 weeks, their aortic and kidney tissues were excised for histological and immunohistochemical analyses. RESULTS: Severe vascular calcification was observed by von Kossa staining in the arcuate aorta of all the DS-IS rats, but hardly in DS or DR rats. Immunohistochemistry demonstrated that osteopontin, core binding factor 1 (Cbfal), alkaline phosphatase (ALP), osteocalcin, IS and organic anion transporter (OAT) 3 were colocalized in the cells embedded in the aortic calcification area of DS-IS rats. Wall thickness was significantly increased in arcuate, thoracic and abdominal aortas of DS-IS rats compared with DS and DR rats. DS-IS rats showed significantly increased extent of glomerular hypertrophy, mesangial expansion, Masson's trichrome-positive tubulointerstitial area and glomerular and tubulointerstitial expression of transforming growth factor-ssl as compared with DS and DR rats. CONCLUSIONS: IS induced aortic calcification with expression of osteoblast-specific proteins and aortic wall thickening. IS is not only a nephrotoxin but also a vascular toxin, and may contribute to the progression of aortic calcification in stage 5 CKD patients.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Calcinosis/metabolismo , Calcinosis/patología , Osteoblastos/metabolismo , Análisis de Varianza , Animales , Enfermedades de la Aorta/inducido químicamente , Presión Sanguínea/efectos de los fármacos , Calcinosis/inducido químicamente , Factores de Unión al Sitio Principal/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunohistoquímica , Indicán , Glomérulos Renales/patología , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Osteopontina/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas Dahl , Cloruro de Sodio DietéticoRESUMEN
BACKGROUND: We have demonstrated that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, inhibits formation of advanced glycation end-products (AGEs) by trapping 3-deoxyglucosone. The present study aimed to clarify if PLP could exert beneficial effects on nephropathy in diabetic rats. METHODS: Streptozotocin (STZ)-induced diabetic rats were treated by oral administration of PLP or pyridoxamine (PM), another active form of vitamin B6, at a dose of 600 mg/kg/day for 16 weeks. AGEs [imidazolone, N(epsilon)-(carboxymethyl)lysine (CML) and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG)], transforming growth factor-beta1 (TGF-beta1), type 1 collagen and fibronectin were detected in the kidneys using immunohistochemistry. Gene expression of TGF-beta1 and receptor for AGEs (RAGEs) in the kidneys was determined using real-time quantitative polymerase chain reaction. RESULTS: Administration of PLP significantly inhibited albuminuria, glomerular hypertrophy, mesangial expansion, and interstitial fibrosis as compared with diabetic rats. PLP markedly inhibited accumulation of AGEs such as imidazolone, CML and CEdG, a DNA-linked AGE, in glomeruli. PLP significantly inhibited expression of TGF-beta1, type 1 collagen, fibronectin and RAGE in the kidneys. PLP was superior to PM in inhibiting accumulation of AGEs, expression of TGF-beta1, type 1 collagen, and fibronectin, and the development of diabetic nephropathy. CONCLUSIONS: PLP prevented progression of nephropathy in STZ-induced diabetic rats by inhibiting formation of AGEs. PLP is considered a promising active form of vitamin B6 for the treatment of AGE-linked disorders such as diabetic nephropathy.