RESUMEN
In chronic kidney disease, elevated levels of circulating uremic toxins are associated with a variety of symptoms and organ dysfunction. Indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are microbiota-derived metabolites and representative uremic toxins. We have previously shown that the oral adsorbent AST-120 profoundly reduced pCS compared to IS in adenine-induced renal failure in mice. However, the mechanisms of the different attenuation effects of AST-120 between IS and pCS are unclear. To clarify the difference of AST-120 on IS and pCS, we investigated the levels of fecal indole and p-cresol, the respective precursors of IS and pCS, and examined the influence on the gut microbiota. Although fecal indole was detected in all groups analyzed, fecal p-cresol was not detected in AST-120 treatment groups. In genus level, a total of 23 organisms were significantly changed by renal failure or AST-120 treatment. Especially, AST-120 reduced the abundance of Erysipelotrichaceae uncultured and Clostridium sensu stricto 1, which have a gene involved in p-cresol production. Our findings suggest that, in addition to the adsorption of the uremic toxin precursors, AST-120 affects the abundance of some gut microbiota in normal and renal failure conditions, thereby explaining the different attenuation effects on IS and pCS.
Asunto(s)
Carbono/administración & dosificación , Carbono/farmacología , Cresoles/metabolismo , Heces/química , Microbioma Gastrointestinal/efectos de los fármacos , Indoles/metabolismo , Óxidos/administración & dosificación , Óxidos/farmacología , Administración Oral , Adsorción , Animales , Bacterias/efectos de los fármacos , Fallo Renal Crónico/microbiología , Fallo Renal Crónico/patología , Masculino , Ratones Endogámicos C57BLRESUMEN
Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC). Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs). Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 µM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22. Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.
Asunto(s)
Indicán/toxicidad , Miocitos del Músculo Liso/patología , Receptores Notch/metabolismo , Calcificación Vascular/patología , Animales , Aorta/citología , Aorta/patología , Calcio/análisis , Línea Celular , Dipéptidos/farmacología , Técnicas de Silenciamiento del Gen , Indicán/administración & dosificación , Miocitos del Músculo Liso/efectos de los fármacos , Ratas , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Calcificación Vascular/inducido químicamente , Calcificación Vascular/diagnósticoRESUMEN
The function of aryl hydrocarbon receptor repressor (AHRR) in the kidney is unclear. The present study investigated associations between AHRR Pro189Ala polymorphism and estimated glomerular filtration rates (eGFR), serum creatinine, and hemoglobin levels in 2775 Japanese adults without diabetes. In addition, we examined whether AHRR expression levels in the kidney of control and chronic kidney disease (CKD) rats were changed. Multiple linear regression analyses showed that carriers of the Ala allele had increased eGFR and lower concentrations of serum creatinine and hemoglobin (p < 0.05). Immunohistochemical analysis showed that the expression of AHRR was upregulated in the kidneys of rats with CKD. These findings suggest that AHRR plays distinct roles in kidney functions and hemoglobin values. The effects of the AHRR polymorphism might be intensified in the kidneys of patients with CKD.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Riñón/metabolismo , Polimorfismo Genético , Insuficiencia Renal Crónica/genética , Proteínas Represoras/genética , Adulto , Alelos , Sustitución de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Creatinina/sangre , Femenino , Expresión Génica , Tasa de Filtración Glomerular , Hemoglobinas/metabolismo , Humanos , Riñón/fisiopatología , Modelos Lineales , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Proteínas Represoras/metabolismoRESUMEN
Indoxyl sulfate (IS) induces fibrosis and inflammation in kidneys via oxidative stress through the induction of transforming growth factor-ß1 (TGF-ß1) and monocyte chemotactic protein-1 (MCP-1). Furthermore, IS is a potent endogenous agonist for aryl hydrocarbon receptor (AHR), which regulates the transcription of genes such as cytochrome P450 (CYP) 1A1. Indole-3-propionic acid (IPA) is an antioxidant and has been reported to be neuroprotective. We determined whether IPA suppresses IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in proximal tubular cells. The effects of IS on the expression of AHR, CYP1A1, TGF-ß1, and MCP-1 were studied using normotensive rats and hypertensive rats. The effects of IPA on IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 were studied using proximal tubular cells (HK-2). Furthermore, the effects of IPA on IS-induced expression and phosphorylation of signal transducer and activator of transcription 3 (Stat3) were studied in HK-2 cells. Administration of IS induced the expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in the tubular cells of rat kidneys. IPA significantly suppressed IS-induced mRNA and protein expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in HK-2 cells. IPA suppressed the IS-induced expression and phosphorylation of Stat3 in HK-2 cells. Furthermore, knockdown of Stat3 inhibited the IS-induced mRNA and protein expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in HK-2 cells. In conclusion, IPA suppressed the IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 through suppression of Stat3 in proximal tubular cells. Thus, IPA suppresses IS-induced expression of fibrotic and inflammatory genes in proximal tubular cells.
Asunto(s)
Indicán/toxicidad , Inflamación/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Propionatos/uso terapéutico , Animales , Western Blotting , Línea Celular , Quimiocina CCL2/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Humanos , Inmunohistoquímica , Inflamación/genética , Ratas , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
We have recently found that indoxyl sulfate induces prorenin expression in proximal tubular cells. The present study aimed to determine whether nuclear factor-κB (NF-κB) p65, signal transducer and activator of transcription 3 (Stat3), and reactive oxygen species are involved in indoxyl sulfate-induced prorenin expression in cultured human proximal tubular cells (HK-2 cells). Effects of indoxyl sulfate on prorenin expression were determined using HK-2 cells with small interfering RNAs (siRNAs) specific to NF-κB p65 and Stat3, N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. Indoxyl sulfate increased prorenin expression in HK-2 cells. siRNAs specific to NF-κB p65 and Stat3 inhibited indoxyl sulfate-induced prorenin expression. Both N-acetylcysteine and diphenyleneiodonium suppressed indoxyl sulfate-induced prorenin expression. Indoxyl sulfate upregulates the expression of prorenin via NF-κB p65, Stat3, and reactive oxygen species in proximal tubular cells.
Asunto(s)
Indicán/farmacología , Túbulos Renales Proximales/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Renina/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Cultivadas , Humanos , Túbulos Renales Proximales/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Renina/efectos de los fármacos , Factor de Transcripción STAT3/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Indoxyl sulfate (IS), a uremic toxin, has cardiovascular as well as uremic toxicity. We evaluated the prognostic value of blood IS level for long-term outcome. METHODS AND RESULTS: This study followed 311 patients with coronary artery disease. Plasma IS level and estimated glomerular filtration rate (eGFR) were determined. The endpoint was a major adverse cardiac event (MACE). Median follow-up was 759 days. IS was significantly higher in patients with MACE than in those without (P<0.001). Patients were divided according to quartiles (Q) of plasma IS level (Q1, Q2, Q3, and Q4). On Kaplan-Meier analysis a significantly lower MACE-free rate was obtained for Q4 compared with the other quartiles (P<0.001). In patients with eGFR ≥90, 89-60, 59-30, 29-15, and <15 ml·min(-1)·1.73 m(-2), the percentage of patients in Q4 was 0%, 13%, 29%, 100%, and 100%, respectively. In patients with eGFR 89-60 ml·min(-1)·1.73 m(-2), there was no significant difference in MACE-free rate between Q4 and the other quartiles; in patients with eGFR 59-30 ml·min(-1)·1.73 m(-2), a significantly lower MACE-free rate was obtained for Q4 compared with the other quartiles (P=0.832 and P=0.015, respectively). CONCLUSIONS: Plasma IS level is a significant predictor of MACE, especially in patients with eGFR 59-30 ml·min(-1)·1.73 m(-2).
Asunto(s)
Enfermedad de la Arteria Coronaria , Tasa de Filtración Glomerular , Indicán/sangre , Anciano , Anciano de 80 o más Años , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/mortalidad , Enfermedad de la Arteria Coronaria/fisiopatología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Tasa de Supervivencia , Factores de TiempoRESUMEN
BACKGROUND/AIMS: Interleukin-6 (IL-6) is one of the inflammation biomarkers with highest predictive value for outcome in chronic kidney disease (CKD) patients. The present study aimed to determine the effects of indoxyl sulfate (IS) on IL-6 expression in vascular cells. METHODS: IS was administered to normo- and hypertensive rats. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were incubated with or without IS. RESULTS: Immunohistochemistry revealed that IS-administered rats showed increased expression of IL-6 in the aortic tissues. IS increased IL-6 expression in HUVECs and HASMCs in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3) using small interfering RNA (siRNA) inhibited IS-induced expression of IL-6 in HUVECs and HASMCs. IS induced activation of aryl hydrocarbon receptor (AhR) and nuclear factor-κB (NF-κB) subunit p65 in HUVECs and HASMCs. Both AhR siRNA and p65 siRNA inhibited IS-induced expression of IL-6. AhR siRNA inhibited IS-induced phosphorylation and nuclear translocation of p65 without change in total p65 level. However, p65 siRNA did not inhibit IS-induced nuclear translocation of AhR. Thus, AhR is responsible for IS-induced p65 signaling transduction. CONCLUSION: IS induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3/AhR/NF-κB pathway.
Asunto(s)
Endotelio Vascular/metabolismo , Indicán/farmacología , Interleucina-6/metabolismo , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Independiente/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Independiente/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Endogámicas Dahl , Transducción de Señal/fisiología , Factores de Tiempo , eIF-2 Quinasa/metabolismoRESUMEN
OBJECTIVE: Nrf2 is a transcription factor that regulates the expression of antioxidant genes. This study aimed to investigate the association of Nrf2 gene single nucleotide polymorphisms (SNPs), rs35652124 (-653A/G) and rs6721961 (-617C/A), with laboratory data and mortality in hemodialysis (HD) patients. METHODS: Blood samples were obtained from 216 HD patients (119 males and 97 females; 60 diabetics and 156 non-diabetics) with mean age of 60.3±13.3 (SD) years, and mean HD duration of 9.10±8.28 years. Genotyping was performed using polymerase chain reaction with confronting two-pair primers (PCR-CTPP) assay. RESULTS: As for rs35652124, diastolic blood pressure (BP) was significantly high in total AA carriers. ß2-microglobulin was significantly low in male AA carriers. Systolic BP, diastolic BP and albumin were significantly high in female AA carriers. As for 6721961, systolic BP and diastolic BP were significantly high in female AA carriers. Cox proportional hazard analysis adjusted for age, HD duration, diabetes and Kt/V demonstrated that rs35652124 AA carriers showed higher cardiovascular mortality than (GG+GA) carriers. CONCLUSION: Nrf2 SNPs were associated with BP in Japanese HD patients. More notably, rs35652124 was associated with cardiovascular mortality in these patients.
Asunto(s)
Presión Sanguínea/genética , Enfermedades Cardiovasculares/genética , Factor 2 Relacionado con NF-E2/genética , Diálisis Renal/efectos adversos , Anciano , Enfermedades Cardiovasculares/mortalidad , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) is a transcription factor that regulates the expression of antioxidant genes by activating Nrf2-antioxidant response element (ARE) pathway. This study aimed to investigate association of Nrf2 gene single nucleotide polymorphisms (SNPs), rs35652124 (A --> G) and rs6721961 (C --> A), with various laboratory data in 464 health evaluation examinees. The genotyping of these SNPs was performed using polymerase chain reaction with confronting two-pair primers (PCR-CTPP) assay. The genotype frequencies of rs35652124 SNP were 21.1% for AA, 44.0% for AG, and 34.9% for GG. The frequency of A allele was 0.431. In male subjects, cholinesterase was significantly high, and HDL cholesterol was significantly low in (AG+GG) carriers. In female subjects, diastolic blood pressure (BP) was significantly low in (AG+GG) carriers. The genotype frequencies of rs6721961 SNP were 55.2% for CC, 34.7% for CA, and 10.1% for AA. The frequency of A allele was 0.275. In male subjects, systolic BP, diastolic BP and cholinesterase were significantly low, and iron was significantly high in (CA+AA) carriers. In female subjects, cholinesterase was significantly high in (CA+AA) carriers, and diastolic BP was significantly high in AA carriers. In conclusion, Nrf2 polymorphisms are associated with BP in Japanese.
Asunto(s)
Pueblo Asiatico/genética , Presión Sanguínea/genética , Factor 2 Relacionado con NF-E2/genética , Polimorfismo de Nucleótido Simple , Adulto , Biomarcadores/sangre , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Japón , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.
Asunto(s)
Compuestos Azo , Neoplasias Colorrectales , Pirazoles , Insuficiencia Renal Crónica , Humanos , Indicán/farmacología , Indicán/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores ErbB/genética , Indoles , Proliferación CelularRESUMEN
In chronic kidney disease (CKD), indoxyl sulfate, a uremic toxin, accumulates in serum, and the expression of angiotensinogen (AGT) is upregulated in renal proximal tubular cells. The present study aimed to determine the relationship between indoxyl sulfate and the upregulation of AGT expression in proximal tubular cells. Indoxyl sulfate induced expression of AGT in rat renal cortex and in cultured human proximal tubular cells (HK-2). In proximal tubular cells, indoxyl sulfate induced phosphorylation of cAMP response element-binding protein (CREB) on Ser-133, and small interfering RNA (siRNA) specific to CREB inhibited indoxyl sulfate-induced AGT expression. Our previous study demonstrated that indoxyl sulfate activated nuclear factor-κB (NF-κB) through reactive oxygen species (ROS) production. NF-κB inhibitors (pyrrolidine dithiocarbamate and isohelenin), NF-κB p65 siRNA, an antioxidant [N-acetylcysteine (NAC)], and a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor [diphenyleneiodonium (DPI)] suppressed indoxyl sulfate-induced AGT expression. Both NAC and DPI suppressed indoxyl sulfate-induced expression of NF-κB p65 and CREB. CREB siRNA suppressed indoxyl sulfate-induced NF-κB p65 expression, whereas both NF-κB inhibitors and NF-κB p65 siRNA prevented indoxyl sulfate-induced CREB expression. Furthermore, we focused on the expression of NADPH oxidase 4 (NOX4), because indoxyl sulfate induced NOX4 expression in vascular smooth muscle cells and vascular endothelial cells. Indoxyl sulfate induced the expression of NOX4 in proximal tubular cells, which was suppressed by NAC, DPI, NF-κB inhibitors, NF-κB p65 siRNA, and CREB siRNA. Taken together, CREB, NF-κB, and NOX4 coordinately upregulate indoxyl sulfate-induced AGT expression in proximal tubular cells.
Asunto(s)
Angiotensinógeno/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Indicán/farmacología , Túbulos Renales Proximales/citología , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Angiotensinógeno/genética , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación de la Expresión Génica/fisiología , Humanos , Corteza Renal/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/genética , FN-kappa B/genética , Ratas , Especies Reactivas de Oxígeno , Regulación hacia ArribaRESUMEN
BACKGROUND/AIM: Indoxyl sulfate-induced activation of nuclear factor (NF)-ĸB promotes transforming growth factor (TGF)-ß1 in human proximal tubular cells (HK-2 cells). The present study aimed to elucidate the cross talk among indoxyl sulfate, p53 and TGF-ß1-Smad3 signaling in proximal tubular cells. METHODS: The effects of indoxyl sulfate on the expression of TGF-ß1, Smad3, and α-smooth muscle actin (α-SMA) were determined using HK-2 cells. As for in vivo experiments the following animals were used: Dahl salt-resistant normotensive rats (DN) and indoxyl sulfate-administered Dahl salt-resistant normotensive rats (DN+IS). RESULTS: Both indoxyl sulfate and nutlin-3, a specific p53 inducer, stimulated TGF-ß1 expression, which was suppressed by pifithrin-α, p-nitro, a p53 inhibitor. Further, indoxyl sulfate stimulated TGF-ß1-induced expression of α-SMA by enhancing Smad3 expression and TGF-ß1-induced Smad3 phosphorylation. Indoxyl sulfate induced phosphorylation of extracellular signal-regulated kinase (ERK). U0126, an inhibitor of ERK pathway, prevented indoxyl sulfate-induced upregulation of Smad3 expression. Immunohistochemistry demonstrated that TGF-ß1 and Smad3 were localized in renal tubular cells, and that indoxyl sulfate increased the TGF-ß1 and Smad3-positive area in the kidney. CONCLUSION: Indoxyl sulfate stimulates p53-induced TGF-ß1 expression and TGF-ß1-induced α-SMA expression in proximal tubular cells. Indoxyl sulfate-induced Smad3 accelerates TGF-ß1-induced α-SMA expression through ERK activation. Thus, indoxyl sulfate enhances p53-TGF-ß1-Smad3 pathway in proximal tubular cells.
Asunto(s)
Actinas/metabolismo , Indicán/farmacología , Túbulos Renales Proximales/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Actinas/genética , Análisis de Varianza , Animales , Benzotiazoles/farmacología , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Imidazoles/farmacología , Túbulos Renales Proximales/citología , Masculino , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Dahl , Proteína smad3/genética , Tolueno/análogos & derivados , Tolueno/farmacología , Factor de Crecimiento Transformador beta1/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). A wide variation in individual response to EPO, however, is often observed, causing EPO resistance. EPO exhibits not only hematopoietic but also extra-hematopoietic functions such as endothelial effects. Indoxyl sulfate, a uremic toxin, is involved in endothelial dysfunction, and consequently, the pathogenesis of CKD-associated cardiovascular disease. The aim of the present study was to determine the effect of indoxyl sulfate on the extra-hematopoietic functions of EPO in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: HUVECs were incubated with or without indoxyl sulfate or an Akt inhibitor, and then stimulated with or without EPO. Indoxyl sulfate suppressed EPO-induced survival/proliferation, anti-apoptosis function, phosphorylation of endothelial nitric oxide synthase, and the expression of thrombospondin-1, an erythroid-stimulating factor, in HUVECs. Although EPO induced phosphorylation of both Akt and extracellular signal-regulated kinases (ERK) in HUVECs, indoxyl sulfate suppressed phosphorylation of Akt but not ERK. An Akt kinase inhibitor or Akt small interfering RNA suppressed all the EPO-induced cellular effects in HUVECs. As a site of action of indoxyl sulfate on EPO signaling, indoxyl sulfate attenuated EPO-induced tyrosine phosphorylation of EPO receptor (EPOR) in HUVECs. CONCLUSIONS: Indoxyl sulfate negatively regulates the EPOR-Akt pathway in endothelial cells, and might contribute to EPO resistance and endothelial dysfunction in patients with CKD.
Asunto(s)
Eritropoyetina/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Indicán/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Receptores de Eritropoyetina/efectos de los fármacos , Receptores de Eritropoyetina/metabolismo , Transducción de Señal/efectos de los fármacos , Trombospondina 1/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Serum indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic kidney disease (CKD). The aim of this study was to determine whether serum IS is associated with hemodynamic parameters or cardiac events in patients with nonischemic dilated cardiomyopathy (DCM). METHODS AND RESULTS: The 76 patients with DCM had their serum IS and plasma brain natriuretic peptide (BNP) levels measured, and underwent echocardiographic examination. Mean (± standard deviation) left ventricular ejection fraction (LVEF) and BNP levels in the patients were 32.5 ± 10.7% and 204 ± 219 pg/ml, respectively. Patients were divided into 2 groups, low IS (<0.9 µg/ml) and high IS (≥ 0.9 µg/ml), based on the median value of serum IS. Although there were no significant differences in LVEF and BNP between the groups, E/e' was significantly greater in the high IS group than in the low IS group. Furthermore, E/e' was an independent determinant of serum IS level. The risk of a cardiac event was significantly higher in the high IS group than in the low IS group (P=0.014). Moreover, serum IS was a significant predictor of cardiac events even after adjustment for BNP. CONCLUSIONS: Cardiac dysfunction is associated with the serum IS level, which might serve as a new prognostic marker in DCM patients with normal renal function or mild to moderate CKD.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/mortalidad , Indicán/sangre , Uremia/metabolismo , Uremia/mortalidad , Adulto , Anciano , Biomarcadores/sangre , Cardiomiopatía Dilatada/diagnóstico por imagen , Progresión de la Enfermedad , Femenino , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Valor Predictivo de las Pruebas , Pronóstico , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/mortalidad , Factores de Riesgo , Volumen Sistólico , UltrasonografíaRESUMEN
Accumulating evidence suggests that protein-bound uraemic toxins play an important role in uraemic complications, especially in cardiovascular disease. Notably, protein-bound uraemic toxins such as indoxyl sulphate, p-cresyl sulphate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) have emerged as important targets of therapeutic removal. Indoxyl sulphate stimulates reactive oxygen species production in human umbilical vein endothelial cells (HUVEC) most intensely, followed by CMPF. Indoxyl sulphate and CMPF inhibit cell growth of HUVEC. Haemodialysis (HD) even with a high-flux membrane cannot efficiently remove the protein-bound uraemic toxins because of their high albumin-binding property. Especially, indoxyl sulphate, p-cresyl sulphate, and CMPF showed high protein-binding ratios (more than 95%) and low reduction rates by HD (less than 35%). Removal of indoxyl sulphate and p-cresyl sulphate can be improved to some extent by increasing the diffusion of the free forms with super-flux membrane HD, increasing the dialyzer mass transfer area coefficient and dialysate flow, haemodiafiltration, daily HD, and addition of a sorbent to dialysate. However, CMPF is more strongly bound to albumin (with a binding ratio of 99-100%) than indoxyl sulphate and p-cresyl sulphate, and cannot be removed at all by conventional HD. Uraemic toxins strongly or covalently bound to albumin such that CMPF can be removed by protein-leaking HD. Protein-leaking HD with a polymethylmethacrylate membrane BK-F dialyzer can reduce serum levels of CMPF with improvement of anaemia as well as reduce plasma levels of homocysteine, pentosidine, and inflammatory cytokines.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Cresoles/sangre , Furanos , Indicán/sangre , Membranas Artificiales , Propionatos , Ésteres del Ácido Sulfúrico/sangre , Uremia/sangre , Uremia/terapia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Especies Reactivas de Oxígeno/sangre , Diálisis Renal/instrumentación , Diálisis Renal/métodosRESUMEN
BACKGROUND: Indoxyl sulfate, a uremic toxin, is accumulated in the serum of chronic kidney disease (CKD) patients, accelerating the progression of CKD. In CKD rat kidney, the expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its related genes are downregulated. AST-120, an oral sorbent, reduces serum indoxyl sulfate and slows the progression of CKD. The present study aimed to determine whether indoxyl sulfate downregulates Nrf2 expression in human proximal tubular cells and rat kidneys and whether AST-120 upregulates Nrf2 expression in CKD rat kidneys. METHODS: Effects of indoxyl sulfate on expression of Nrf2 were determined using HK-2 cells as human proximal tubular cells and the following animals: (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). Further, AST-120 was administered to subtotally nephrectomized CKD rats to determine its effect on the expression of Nrf2. RESULTS: Indoxyl sulfate downregulated Nrf2 expression in HK-2 cells. The indoxyl sulfate-induced downregulation of Nrf2 expression was alleviated by an inhibitor of nuclear factor-κB (NF-κB) (pyrrolidine dithiocarbamate) and small interfering RNA specific to NF-κB p65. DN+IS, DH, and DH+IS rats showed decreased renal expression of Nrf2 and its downstream target genes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and increased renal expression of 8-hydroxydeoxyguanosine (8-OHdG), a marker of reactive oxygen species (ROS), compared with DN. Thus, indoxyl sulfate, as well as hypertension, downregulated renal expression of Nrf2 in rats. AST-120 upregulated renal expression of Nrf2, HO-1 and NQO1 and suppressed renal expression of 8-OHdG compared with control CKD rats. CONCLUSIONS: Indoxyl sulfate downregulates renal expression of Nrf2 through activation of NF-κB, followed by downregulation of HO-1 and NQO1 and increased production of ROS. Further, AST-120 upregulates renal expression of Nrf2 in CKD rats by removing serum indoxyl sulfate, followed by upregulation of HO-1 and NQO1 and decreased production of ROS.
Asunto(s)
Indicán/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Uremia/metabolismo , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated ß-galactosidase (SA-ß-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-ß-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-ß-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.
Asunto(s)
Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Indicán/fisiología , Músculo Liso Vascular/fisiología , Proteínas Nucleares/biosíntesis , Estrés Oxidativo/fisiología , Precursores de Proteínas/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Células Cultivadas , Humanos , Lamina Tipo A , Músculo Liso Vascular/citología , Distribución Aleatoria , Ratas , Ratas Endogámicas DahlRESUMEN
Mass spectrometry (MS) has been successfully applied for the identification and quantification of uremic toxins and uremia-associated modified proteins. This review focuses on the recent progress in the MS analysis of uremic toxins. Uremic toxins include low-molecular weight solutes, protein-bound low-molecular weight solutes, and middle molecules (peptides and proteins). Based on MS analysis of these uremic toxins, the pathogenesis of the uremic symptoms will be elucidated to prevent and manage the symptoms. Notably, protein-bound uremic toxins such as indoxyl sulfate, p-cresyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid have emerged as important targets of therapeutic removal. Hemodialysis even with a high-flux membrane cannot efficiently remove the protein-bound uremic toxins because of their high albumin-binding property. The accumulation of these protein-bound uremic toxins in the blood of dialysis patients might play an important role in the development of uremic complications such as cardiovascular disease. Indoxyl sulfate is the most promising protein-bound uremic toxin as a biomarker of progress in chronic kidney disease. Novel dialysis techniques or membranes should be developed to efficiently remove these protein-bound uremic toxins for the prevention and management of uremic complications.
Asunto(s)
Espectrometría de Masas/métodos , Toxinas Biológicas/metabolismo , Uremia/metabolismo , Secuencia de Aminoácidos , Animales , Furanos/análisis , Furanos/metabolismo , Humanos , Indicán/análisis , Indicán/metabolismo , Datos de Secuencia Molecular , Propionatos/análisis , Propionatos/metabolismo , Proteínas/análisis , Proteínas/metabolismo , Toxinas Biológicas/análisisRESUMEN
BACKGROUND/AIM: Increased phosphorylation (activation) of signal transducer and activator of transcription 3 (Stat3) on tyrosine 705 leads to renal fibrosis. Indoxyl sulfate, a uremic toxin, induces renal fibrosis through expression of transforming growth factor-ß(1) (TGF-ß(1)) in proximal tubular cells. The present study aimed to determine whether Stat3 is involved in indoxyl sulfate-induced dysfunction of proximal tubular cells. METHODS: Localization of phosphorylated Stat3 in the kidneys of normal, subtotally nephrectomized, and AST-120-treated subtotally nephrectomized rats was examined by immunohistochemistry. The effect of indoxyl sulfate on phosphorylation of Stat3 and the role of Stat3 on indoxyl sulfate-induced cellular effects were examined using human proximal tubular cells (HK-2 cells). RESULTS: Subtotally nephrectomized rats showed increased immunostaining of phosphorylated Stat3 in the renal tubules compared with normal rats. Administration of AST-120, which reduces serum level of indoxyl sulfate, to subtotally nephrectomized rats reduced the immunostaining of phosphorylated Stat3 in the renal tubules. Indoxyl sulfate induced phosphorylation of Stat3 in HK-2 cells. Stat3 small interfering RNA suppressed indoxyl sulfate-induced expression of an inflammation marker gene (monocyte chemotactic protein-1), fibrosis marker genes (TGF-ß(1), α-smooth muscle actin) and a subunit of nuclear factor-ĸB (p65), and attenuated a cellular senescence marker, senescence-associated ß-galactosidase activity. CONCLUSIONS: Stat3 is involved in indoxyl sulfate-induced inflammatory and fibrotic gene expression and cellular senescence in proximal tubular cells.
Asunto(s)
Lesión Renal Aguda/genética , Lesión Renal Aguda/inmunología , Senescencia Celular/fisiología , Indicán/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Lesión Renal Aguda/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis/genética , Fibrosis/inmunología , Fibrosis/patología , Expresión Génica/inmunología , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/inmunología , Masculino , Nefrectomía , Fosforilación/fisiología , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismo , Uremia/genética , Uremia/inmunología , Uremia/patologíaRESUMEN
Cardiovascular disease (CVD) is prevalent in patients with chronic kidney disease (CKD). In hemodialysis (HD) patients, some protein-bound uremic toxins are considered to be associated with CVD. However, it is not yet known which uremic toxins are important in terms of endothelial toxicity. Serum samples were obtained from 45 HD patients before and after HD. Total and free serum concentrations of indoxyl sulfate, indoxyl glucuronide, indoleacetic acid, p-cresyl sulfate, p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide, phenylacetic acid, phenylacetyl glutamine, hippuric acid, 4-ethylphenyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) were simultaneously measured by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS). The effects of these solutes at their pre-HD mean and maximum serum concentrations on reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVEC) were measured with a ROS probe. Serum levels of 11 of the solutes (all except 4-ethylphenyl sulfate) were significantly increased in HD patients compared to healthy subjects. All 12 solutes showed changes in their protein-binding ratios. In particular, indoxyl sulfate, p-cresyl sulfate, CMPF, and 4-ethylphenyl sulfate showed high protein-binding ratios (>95 %) and low reduction rates by HD (<35 %). Indoxyl sulfate at its mean and maximum pre-HD serum concentrations-even with 4 % albumin-stimulated ROS production in HUVEC most intensely, followed by CMPF. In conclusion, the serum levels of 11 protein-bound uremic toxins were increased in HD patients. Indoxyl sulfate, p-cresyl sulfate, and CMPF could not be removed efficiently by HD due to their high protein-binding ratios. Indoxyl sulfate most intensely induced endothelial ROS production, followed by CMPF.