RESUMEN
Renal fibrosis is a chronic pathological process that seriously endangers human health. However, the current therapeutic options for this disease are extremely limited. Previous studies have shown that signaling factors such as JAK2/STAT3, Smad3, and Myd88 play a regulatory role in renal fibrosis, and ß-elemene is a plant-derived sesquiterpenoid organic compound that has been shown to have anti-inflammatory, anti-cancer, and immunomodulatory effects. In the present study, the anti-fibrotic effect of ß-elemene was demonstrated by in vivo and in vitro experiments. It was shown that ß-elemene inhibited the synthesis of extracellular matrix-related proteins in unilateral ureteral obstruction mice, and TGF-ß stimulated rat interstitial fibroblast cells, including α-smooth muscle actin, vimentin, and connective tissue growth factor, etc. Further experiments showed that ß-elemene reduced the expression levels of the above-mentioned fibrosis-related proteins by blocking the phosphorylation of JAK2/STAT3, Smad3, and the expression or up-regulation of MyD88. Notably, knockdown of MyD88 attenuated the phosphorylation levels of STAT3 and Smad3 in TGF-ß stimulated NRK49F cell, which may be a novel molecular mechanism by which ß-elemene affects renal interstitial fibrosis. In conclusion, this study elucidated the anti-interstitial fibrosis effect of ß-elemene, which provides a new direction for future research and development of drugs related to chronic kidney disease.
Asunto(s)
Factor 88 de Diferenciación Mieloide , Insuficiencia Renal Crónica , Factor de Transcripción STAT3 , Sesquiterpenos , Proteína smad3 , Obstrucción Ureteral , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Fibrosis , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Ratas , Insuficiencia Renal Crónica/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Sesquiterpenos/farmacología , Proteína smad3/antagonistas & inhibidores , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/metabolismoRESUMEN
Renal fibrosis is the common pathway of chronic kidney disease progression. The nuclear receptor farnesoid X receptor [FXR, NR1H4 (nuclear receptor subfamily 1 group member 4)], a multifunctional transcription factor, plays a pivotal role in protecting against fibrosis. However, the mechanisms underlying these antifibrotic actions of FXR in kidney disease are largely unknown. Here, we show that agonist GW4064-mediated FXR activation inhibits the activity of the nonreceptor tyrosine kinase Src (proto-oncogene tyrosine-protein kinase), which is critical for regulation of yes-associated protein (YAP) phosphorylation and nuclear localization in renal fibrosis. Activation of FXR suppressed renal fibrosis and Tyr416-Src phosphorylation in TGF-ß-treated human renal proximal tubule epithelial (HK2) cells. Moreover, GW4064 treatment in HK2 cells increased Ser127 phosphorylation, cytosolic accumulation of YAP, and interaction of the hippo core kinases (Ste20-like kinase 1, large tumor suppressor kinase 1, and salvador homolog 1). Inhibition of Src using PP2 (Src kinase inhibitor) prevented renal fibrosis and increased Ser127 phosphorylation and cytosolic accumulation of YAP. The expression of fibrosis markers, inflammatory genes, and YAP target genes was increased in the kidneys of FXR knockout mice compared with those of wild-type mice. In addition, GW4064 or WAY-362450 (turofexorate isopropyl) treatment protected against unilateral ureteral obstruction-induced renal fibrosis. Collectively, our data support the novel conclusion that Src-mediated crosstalk between FXR and YAP protects against renal fibrosis, making this pathway a possible therapeutic target for chronic kidney disease.-Kim, D.-H., Choi, H.-I., Park, J. S., Kim, C. S., Bae, E. H., Ma, S. K., Kim, S. W. Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibrosis/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Isoxazoles/farmacología , Riñón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proto-Oncogenes Mas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Señalizadoras YAPRESUMEN
Receptor tyrosine kinases (RTKs) play important roles in the pathogenic processes of kidney fibrosis. However, the pathophysiological roles of recepteur d'origine nantais (RON), one of the receptor tyrosine kinases, have not yet been defined. We investigated whether the activation or sequence-specific small interfering RNA (siRNA) suppression of RON could regulate epithelial mesenchymal transition (EMT) and the expression of pro-fibrotic markers, and its underlying molecular mechanisms. Stable cell lines and transient transfection for RON and the transfected cells of siRNA for RON were developed to investigate the molecular mechanisms in human kidney proximal tubular epithelial (HK-2) and interstitial fibroblasts (NRK49F) cells. RON overexpression induced EMT and increased expression of fibrosis-related proteins such as N-cadherin, vimentin, transforming growth factor-ß (TGFß), αSMA, and fibronectin in HK-2 and NRK49F cells. RON overexpression increased various RTKs and the phosphorylation of Src (Y416) and Smad, while inhibition of RON by siRNA attenuated the expression of EMT- and fibrosis-related proteins and decreased RTKs such as insulin-like growth factor receptor (IGFR), fibroblast growth factor receptor 1 (FGFR1), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR), as well as the phosphorylation of Src and Smad pathways. siRNA silencing of Src also attenuated the expression of IGFR, FGFR1, VEGFR, and PDGFR. Inhibition of RON can exert an anti-fibrotic effect by the inhibition of EMT and other RTKs through control of Src and Smad pathways in HK-2 and NRK49F cells.
Asunto(s)
Transición Epitelial-Mesenquimal , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Familia-src Quinasas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Familia-src Quinasas/genéticaRESUMEN
TGF-ß/Smad signaling is a major pathway in progressive fibrotic processes, and further studies on the molecular mechanisms of TGF-ß/Smad signaling are still needed for their therapeutic targeting. Recently, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) was shown to improve renal fibrosis, making it an attractive target for chronic kidney diseases (CKDs). Here, we show the mechanism by which PGC-1α regulates the TGF-ß/Smad signaling pathway using HK-2 cell lines stably overexpressing empty vector (mock cells) or human PGC1α (PGC1α cells). Stable PGC-1α overexpression negatively regulated the expression of TGF-ß-induced epithelial-mesenchymal transition (EMT) markers (fibronectin, E-cadherin, vimentin, and α-SMA) and EMT-related transcription factors (Snail and Slug) compared to mock cells, inhibiting fibrotic progression. Interestingly, among molecules upstream of Smad2/3 activation, the gene expression of only TGFßRI, but not TGFßRII, was downregulated in PGC-1α cells. In addition, the downregulation of TGFßRI by PGC-1α was associated with the upregulation of let-7b/c, miRNA for which the 3' untranslated region (UTR) of TGFßRI contains a binding site. In conclusion, PGC-1α suppresses TGF-ß/Smad signaling activation via targeting TGFßRI downregulation by let-7b/c upregulation.
Asunto(s)
MicroARNs/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Insuficiencia Renal Crónica/genética , Sitios de Unión/genética , Línea Celular , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación de la Expresión Génica/genética , Humanos , Peroxisomas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Insuficiencia Renal Crónica/patología , Transducción de Señal , Proteínas Smad/genética , Factores de Transcripción de la Familia Snail/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic ß-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.
Asunto(s)
Lesión Renal Aguda/metabolismo , Apoptosis/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Animales , Apoptosis/genética , Biomarcadores , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Inmunohistoquímica , Túbulos Renales Proximales/patología , Masculino , Ratas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismoRESUMEN
BACKGROUND: Kidney is one of the major target organs in sepsis, while effective prevention of septic acute kidney injury has not yet been established. α-Lipoic acid (LA) has been known to exert beneficial effects against lipopolysaccharide (LPS)-induced damages in various organs such as heart, lung, and liver. We investigated the protective effect of LA on LPS-induced kidney injury. METHODS: Two groups of rats were treated with LPS (20 mg/kg, i.p.), one of which being co-treated with LA (50 mg/kg), while the control group was treated with vehicle alone. Human renal proximal tubular epithelial cells (HK-2 cells) were cultured with or without LPS (10 µg/ml) in the presence or absence of LA (100 µg/ml) for 3 h prior to LPS treatment. RESULTS: Serum creatinine level was increased in LPS-treated rats, which was attenuated by LA co-treatment. LPS treatment induced cleaved caspase-3 expression in the kidney, which was counteracted by LA. Terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells increased in the kidneys of LPS-treated rats compared with controls, which was counteracted by LA treatment. Protein expression of inducible nitric oxide synthase and cyclooxygenase-2 detected by immunoblotting and/or immunohistochemical staining, along with mRNA levels of pro-inflammatory cytokines detected by real-time polymerase chain reaction, was increased in the kidney with LPS administration, which was ameliorated with LA treatment. LA also protected LPS-induced tubular dysfunction, preserving type 3 Na(+)/H(+) exchanger and aquaporin 2 expressions in the kidney. Suppression of LPS-induced expression of cleaved caspase-3 by LA was also observed in HK-2 cells. Increased protein expression of phospho-extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases by LPS treatment was attenuated by LA pretreatment, while p38 was not affected by either LPS or LA treatment. MitoTracker Red demonstrated LA prevented LPS-induced increment of mitochondrial oxidative stress, where concurrent 4',6-diamidino-2-phenylindole staining also revealed marked fragmentation and condensation of nuclei in HK-2 cells treated with LPS, which was prevented by LA. CONCLUSION: LA treatment attenuates LPS-induced kidney injury, such as renal tubular dysfunction, by suppression of apoptosis, and inflammation.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Antioxidantes/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Ácido Tióctico/farmacología , Lesión Renal Aguda/patología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/biosíntesis , Línea Celular , Creatinina/sangre , Humanos , Canales Iónicos/efectos de los fármacos , Pruebas de Función Renal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , RatasAsunto(s)
Hematuria/etiología , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/genética , Defectos Congénitos del Transporte Tubular Renal/complicaciones , Defectos Congénitos del Transporte Tubular Renal/genética , Cálculos Urinarios/complicaciones , Cálculos Urinarios/genética , Adulto , Femenino , Humanos , Mutación , LinajeRESUMEN
Ferroptosis is a cell death mechanism characterized by intracellular iron accumulation and lipid peroxidation. Effects of uremic toxins on ferroptosis in the kidney are not well understood. We investigated whether protein-bound uremic toxins induce ferroptosis, resulting in cell death, using the bilateral ureteral obstruction (BUO) mouse model and kidney cells. In BUO mice, we observed elevated lipid peroxidation, increased iron concentration, and decreased glutathione peroxidase 4 (GPX4) expression. Levels of transferrin receptor 1 and system Xc-, which are involved in iron transport and storage, were also elevated, while those of ferritin heavy and light chains (FHC and FLC) were reduced. Treatment of HK-2 and NRK49F kidney cells with CMPF decreased GSH levels and the expression of GPX4, FHC, and FLC, and increased levels of ROS, lipid peroxidation, and intracellular iron concentration. CMPF-induced and erastin-induced decreases in GPX4 levels and increases in Bax and cytochrome C levels were counteracted by ferrostatin-1 pretreatment. However, GPX4 mRNA levels, protein abundance, or promoter activity were not restored by Z-VAD-FMK, a multi-caspase inhibitor. These results suggest that ferroptosis induced by CMPF treatment induces apoptosis, and inhibition of ferroptosis reduces apoptosis, suggesting that ferroptosis plays a role in triggering cell death by apoptosis.
Asunto(s)
Ferroptosis , Animales , Ratones , Tóxinas Urémicas , Apoptosis , Muerte Celular , Peroxidación de Lípido , Hierro/metabolismo , Riñón/metabolismoRESUMEN
The prevalence of kidney diseases has been increasing worldwide due to the aging population and has results in an increased socioeconomic burden as well as increased morbidity and mortality. A deep understanding of the mechanisms underlying the physiological regulation of the kidney and the pathogenesis of related diseases can help identify potential therapeutic targets. The farnesoid X receptor (FXR, NR1H4) is a primary nuclear bile acid receptor that transcriptionally regulates bile acid homeostasis as well as glucose and lipid metabolism in multiple tissues. The roles of FXR in tissues other than hepatic and intestinal tissues are poorly understood. In studies over the past decade, FXR has been demonstrated to have a protective effect against kidney diseases through its anti-inflammatory and antifibrotic effects; it also plays roles in glucose and lipid metabolism in the kidney. In this review, we discuss the physiological role of FXR in the kidney and its pathophysiological roles in various kidney diseases, including acute kidney injury and chronic kidney diseases, diabetic nephropathy, and kidney fibrosis. Therefore, the regulatory mechanisms involving nuclear receptors, such as FXR, in the physiology and pathophysiology of the kidney and the development of agonists and antagonists for modulating FXR expression and activation should be elucidated to identify therapeutic targets for the treatment of kidney diseases.
Asunto(s)
Nefropatías Diabéticas , Riñón , Anciano , Humanos , Ácidos y Sales Biliares/metabolismo , Nefropatías Diabéticas/metabolismo , Glucosa/metabolismo , Riñón/patología , Hígado/metabolismoRESUMEN
Peroxiredoxin II (Prdx II, a typical 2-Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II-/- mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation-sensitive proteins in Prdx II-/- mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano-UPLC-MSE shotgun proteomics. We found oxidation-sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II-/- mice, and abnormal RBCs of Prdx II-/- mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress-induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation-sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox-imbalanced diseases.
Asunto(s)
Cisteína/metabolismo , Eritrocitos/metabolismo , Peroxirredoxinas/genética , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Eritrocitos/enzimología , Técnicas de Inactivación de Genes , Homeostasis , Yodoacetamida/química , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Peroxirredoxinas/metabolismo , Mapas de Interacción de Proteínas , Proteoma/químicaRESUMEN
The side effects of cisplatin, a widely used chemotherapeutic agent, include nephrotoxicity. Previous studies have reported that cisplatin induces ferroptosis and lipid peroxide accumulation. Ferroptosis, a type of regulated cell death, is characterized by iron-dependent lipid peroxidation. Although previous studies have examined the regulation of ferroptosis in acute kidney injury (AKI), the regulatory mechanism of ferroptosis has not been elucidated. Here, the ability of activated farnesoid X receptor (FXR) to attenuate cisplatin-induced AKI through the regulation of ferroptosis was examined. FXR deficiency exhibited more ferroptosis responses, such as increase in lipid peroxidation, iron content and heme oxygenase 1 protein, and a decrease in glutathione/glutathione disulfide ratio and glutathione peroxidase 4 levels in HK2 cells and mice. Increased blood urea nitrogen, serum creatinine, and ferroptotic responses in the cisplatin-induced AKI mouse model were mitigated upon treatment with the FXR agonist GW4064 but were exacerbated in FXR knockout mice. RNA sequencing analysis revealed that ferroptosis-associated genes were novel targets of FXR. FXR agonist upregulated the expression of lipid and glutathione metabolism-related genes and downregulated cell death-related genes. Additionally, chromatin immunoprecipitation assays, using mice renal tissues, revealed that agonist-activated FXR could bind to its known target genes (Slc51a, Slc51b, Osgin1, and Mafg) and ferroptosis-related genes (Aifm2, Ggt6, and Gsta4). Furthermore, activated FXR-dependent MAFG, a transcriptional repressor, could bind to Hmox1, Nqo1, and Tf in the renal tissues of FXR agonist-treated mice. These findings indicate that activated FXR regulates the transcription of ferroptosis-related genes and protects against cisplatin-induced AKI.
Asunto(s)
Lesión Renal Aguda , Ferroptosis , Receptores Citoplasmáticos y Nucleares , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Animales , Cisplatino/efectos adversos , Ferroptosis/genética , Glutatión , Humanos , Hierro , Ratones , Ratones Endogámicos C57BL , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3ß inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFß-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFß, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFß, and α-SMA increased in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFß treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFß treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.
Asunto(s)
Indoles/farmacología , Enfermedades Renales/tratamiento farmacológico , Túbulos Renales Proximales/efectos de los fármacos , Oximas/farmacología , Animales , Línea Celular , Factor de Crecimiento del Tejido Conjuntivo/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/genética , Inhibidores Enzimáticos/farmacología , Fibrosis , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Enfermedades Renales/patología , Túbulos Renales Proximales/patología , Masculino , Inhibidor 1 de Activador Plasminogénico/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/efectos de los fármacos , Factor de Transcripción Sp1/genética , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/genéticaRESUMEN
(-)-Epigallocatechin 3-gallate (EGCG) is a potent antioxidant polyphenol in green tea that acts as an anticancer agent via both direct and indirect pathways. Although the relationship between EGCG's anticancer effects and its antioxidant activity is not fully understood, it is known that EGCG stimulates production of reactive oxygen species (ROS), which induce oxidative stress leading to cell death. In IM9 multiple myeloma cells, EGCG acted in a dose- and time-dependent manner to induce apoptotic cell death. Among the antioxidant enzymes expressed in IM9 cells, levels of peroxiredoxin V (PrdxV) were selectively and significantly reduced by EGCG. Moreover, the ROS scavenger NAC completely inhibited EGCG-induced apoptosis and PrdxV reduction, while overexpression of PrdxV, but not a Prdx(VC48S) mutant, protected IM9 cells from EGCG-induced apoptosis. EGCG-induced reductions in cell viability and PrdxV levels were also observed in primary CD138+ multiple myeloma cells from patients. These results suggest that PrdxV is a key target via which EGCG mediates its anticancer effects.
Asunto(s)
Apoptosis/efectos de los fármacos , Catequina/análogos & derivados , Mieloma Múltiple/enzimología , Peroxirredoxinas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Acetilcisteína/farmacología , Catequina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Peroxirredoxinas/metabolismo , Fosforilación/efectos de los fármacos , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Especies Reactivas de Oxígeno , Sindecano-1 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Autophagy is important for cells to break down and recycle cellular proteins, remove damaged organelles, and especially, for recovery from acute kidney injury (AKI). Despite research on the role and cellular mechanism of autophagy in AKI, the role of autophagy in the progression to chronic kidney disease (CKD) remains poorly understood. Here, using farnesoid X receptor (FXR) knockout (KO) mice, we determined whether FXR prevents the progression of AKI to CKD after renal ischemic-reperfusion (such as I/R) injury through the regulation of renal autophagy and apoptosis. FXR regulated genes that participate in renal autophagy under feeding and fasting conditions, such as hepatic autophagy, and the activation of FXR by agonists, such as GW4064 and INT-747, attenuated the increased autophagy and apoptosis of hypoxia-induced human renal proximal tubule epithelial (HK2) cells. The expression levels of autophagy-related and apoptosis-related proteins in FXR KO mice were increased compared with those in wild-type (WT) mice. We also showed that the increase in reactive oxidative species (ROS) in hypoxia-treated HK2 cells was attenuated by treatment with FXR agonist or by FXR overexpression, and that the level of ROS was elevated in FXR-deficient cells and mice. At 28 days after I/R injury, the autophagy levels were still elevated in FXR KO mice, and the expression levels of fibrosis-related proteins and ROS deposits were higher than those in WT mice. In conclusion, the regulation of renal autophagy and apoptosis by FXR may be a therapeutic target for the early stages of kidney damage, and the progression of AKI to CKD.
Asunto(s)
Lesión Renal Aguda/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Insuficiencia Renal Crónica/metabolismo , Lesión Renal Aguda/patología , Animales , Apoptosis/fisiología , Autofagia/fisiología , Línea Celular , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Humanos , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Insuficiencia Renal Crónica/patologíaRESUMEN
Maslinic acid (MA), also named crategolic acid, is a pentacyclic triterpene extracted from fruits and vegetables. Although various beneficial pharmacological effects of MA have been revealed, its effect on renal fibrosis remains unclear. This study was designed to clarify whether MA could attenuate renal fibrosis and determine the putative underlying molecular mechanisms. We demonstrated that MA-treated mice with unilateral ureteral obstruction (UUO) developed a histological injury of low severity and exhibited downregulated expression of fibrotic markers, including α-smooth muscle actin (α-SMA), vimentin, and fibronectin by 38, 44 and 40%, and upregulated expression of E-cadherin by 70% as compared with untreated UUO mice. Moreover, MA treatment restored the expression levels of α-SMA, connective tissue growth factor, and vimentin to 10, 7.8 and 38% of those induced by transforming growth factor (TGF)-ß in NRK49F cells. MA decreased expression of Smad2/3 phosphorylation and Smad4 in UUO kidneys and TGF-ß treated NRK49F cells (p < 0.05, respectively). Notably, MA specifically interferes with MyD88, an adaptor protein, thereby mitigating Smad4 nuclear expression (p < 0.01 compared to TGF-ß treated group) and ameliorating renal fibrotic changes (p < 0.01 for each fibrotic markers compared to TGF-ß induced cells). In addition, in the UUO model and lipopolysaccharide-induced NRK49F cells, MA treatment decreased the expression of IL-1ß, TGF-α and MCP-1, ICAM-1, associated with the suppression of NF-κB signaling. These findings suggest that MA is a potential agent that can reduce renal interstitial fibrosis, to some extent, via targeting TGF-ß/Smad and MyD88 signaling.
RESUMEN
Peroxiredoxin V, an atypical thioredoxin peroxidase, is widely expressed in mammalian tissues. In addition, Prdx V is localized in mitochondria, peroxisome, cytosol, and the nucleus. Prdx V has been reported to protect a wide range of cellular environments as an antioxidant enzyme, and its dysfunctions may be implicated in several diseases, such as cancer, inflammation, and neurodegenerative disease. Identification and relative quantification of proteins affected by Prdx V may help identify novel signaling mechanisms that are important for oxidative stress response. However, the role of Prdx V in the modulation of hypoxia-related cellular response is not studied yet. To examine the function of endogenous Prdx V in hypoxic condition in vivo, we generated a transgenic mouse model with Prdx V siRNA expression controlled by U6 promoter. Of many tissues, the knockdown of Prdx V expression was displayed in the kidney, lung, and liver but not the spleen and skin. We conducted on the basis of nano-UPLC-MS(E) proteomic study to identify the Prdx V-affected protein networks in hypoxic kidneys. In this study, we identified protein networks associated with oxidative stress, fatty acid metabolism, and mitochondrial dysfunction. Our results indicated that Prdx V affected to regulation of kidney homeostasis under hypoxia stress.
Asunto(s)
Hipoxia de la Célula/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Riñón/metabolismo , Estrés Oxidativo/fisiología , Peroxirredoxinas/metabolismo , Proteómica/métodos , Transducción de Señal/fisiología , Animales , Western Blotting , Cromatografía Liquida , Biología Computacional , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Ratones , Ratones Transgénicos , Peroxirredoxinas/deficiencia , Espectrometría de Masas en TándemRESUMEN
BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. METHODS: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF- κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. RESULTS: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. CONCLUSION: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Ergocalciferoles/farmacología , Indicán/antagonistas & inhibidores , Indicán/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación , Sustancias Protectoras/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidoresRESUMEN
Activation of the epidermal growth factor receptor (EGFR)/signal transducer and activator of transcription 3 (Stat3) signaling pathway has been reported to be associated with renal fibrosis. We have recently demonstrated that peroxiredoxin V (PrdxV) acted as an antifibrotic effector by inhibiting the activity of Stat3 in TGF-ß-treated NRK49F cells. However, the underlying mechanism of PrdxV remains poorly understood. To investigate molecular mechanism of PrdxV, we used a transgenic mouse model expressing PrdxV siRNA (PrdxVsi mice) and performed unilateral ureteral obstruction (UUO) for 7 days. 209/MDCT cells were transiently transfected with HA-tagged WT PrdxV and C48S PrdxV. Transgenic PrdxVsi mice displayed an exacerbated epithelial-to-mesenchymal transition (EMT) as well as an increase in oxidative stress induced by UUO. In the UUO kidney of the PrdxVsi mouse, knockdown of PrdxV increased Tyr1068-specific EGFR and Stat3 phosphorylation, whereas overexpression of WT PrdxV in 209/MDCT cells showed the opposite results. Immunoprecipitation revealed the specific interaction between WT PrdxV and Stat3 in the absence or presence of TGF-ß stimulation, whereas no PrdxV-EGFR or C48S PrdxV-Stat3 interactions were detected under any conditions. In conclusion, PrdxV is an antifibrotic effector that sustains renal physiology. Direct interaction between PrdxV and Stat3 through Cys48 is a major molecular mechanism.
Asunto(s)
Receptores ErbB/metabolismo , Enfermedades Renales/metabolismo , Peroxirredoxinas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Línea Celular , Cisteína/genética , Cisteína/metabolismo , Receptores ErbB/genética , Fibrosis , Enfermedades Renales/genética , Enfermedades Renales/patología , Ratones , Ratones Noqueados , Peroxirredoxinas/genética , Factor de Transcripción STAT3/genética , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
The p-cresyl sulfate accumulates in kidney disease and may be involved in renal injury. α-Lipoic acid (α-LA) acts as an antioxidant in cell injury. We investigated the effects of α-LA treatment on p-cresyl sulfate-induced renal tubular injury. p-Cresyl sulfate induced cell death, and increased Bax/Bcl-2, cleaved caspase-3, Beclin-1, and LC3BII/LC3BI in human renal proximal tubular epithelial (HK-2) cells, which was counteracted by α-LA treatment. p-Cresyl sulfate-induced apoptosis was reduced by autophagy inhibitor 3-methyladenine, and p-cresyl sulfate induced autophagy was reduced by pan-caspase inhibitor Z-VAD-FMK. Moreover, p-cresyl sulfate treatment increased the expression of ER stress proteins and decreased the expression of baculoviral IAP repeat-containing proteins 6; these effects were prevented by α-LA treatment. Apoptosis and autophagy were associated with the phosphorylation of mitogen-activated protein kinase and nuclear translocation of the nuclear factor-κB p65 subunit. Pretreatment inhibitors of p38 and JNK, and knockdown of ATF4 gene reduced apoptosis- and autophagy-related protein expressions in p-cresyl sulfate treated HK-2 cells. These results demonstrate that α-lipoic acid attenuated p-cresyl sulfate-induced cell death by suppression of apoptosis and autophagy via regulation of ER stress in HK-2 cells.