RESUMO
First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic "cycling" of KRAS between its guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145). Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Camundongos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Guanosina Difosfato/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Guanosina Trifosfato/metabolismo , Cinética , Piperazinas , Piridinas , PirimidinasRESUMO
INTRODUCTION: We previously reported the significant upregulation of eight circulating exosomal microRNAs (miRNAs) in patients with diabetic kidney disease (DKD). However, their specific roles and molecular mechanisms in the kidney remain unknown. Among the eight miRNAs, we evaluated the effects of miR-5010-5p on renal tubular epithelial cells under diabetic conditions in this study. RESEARCH DESIGN AND METHODS: We transfected the renal tubular epithelial cell line, HK-2, with an miR-5010-5p mimic using recombinant plasmids. The target gene of hsa-miR-5010-5p was identified using a dual-luciferase assay. Cell viability was assessed via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Moreover, mRNA and protein expression levels were determined via real-time PCR and western blotting, respectively. RESULTS: High glucose levels did not significantly affect the intracellular expression of miR-5010-5p in HK-2 cells. Transfection of the miR-5010-5p mimic caused no change in cell viability. However, miR-5010-5p-transfected HK-2 cells exhibited significantly decreased expression levels of inflammatory cytokines, such as the monocyte chemoattractant protein-1, interleukin-1ß, and tumor necrosis factor-É, under high-glucose conditions. These changes were accompanied by the restored expression of phosphorylated AMP-activated protein kinase (AMPK) and decreased phosphorylation of nuclear factor-kappa B. Dual-luciferase assay revealed that miR-5010-5p targeted the gene, protein phosphatase 2 regulatory subunit B delta (PPP2R2D), a subunit of protein phosphatase 2A, which modulates AMPK phosphorylation. CONCLUSIONS: Our findings suggest that increased miR-5010-5p expression reduces high glucose-induced inflammatory responses in renal tubular epithelial cells via the regulation of the target gene, PPP2R2D, which modulates AMPK phosphorylation. Therefore, miR-5010-5p may be a promising therapeutic target for DKD.
Assuntos
Proteínas Quinases Ativadas por AMP , MicroRNAs , Proteína Fosfatase 2 , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais , Glucose/metabolismo , Inflamação/metabolismo , Luciferases , MicroRNAs/metabolismo , Proteína Fosfatase 2/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologiaRESUMO
PURPOSE: MET amplification is a frequent mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI) in patients with EGFR-mutated non-small cell lung cancer (NSCLC), and combined treatment with EGFR TKIs and MET TKIs has been explored as a strategy to overcome resistance. However, durable response is invariably limited by the emergence of acquired resistance. Here, we investigated the preclinical activity of REGN5093-M114, a novel antibody-drug conjugate targeting MET in MET-driven patient-derived models. EXPERIMENTAL DESIGN: Patient-derived organoids, patient-derived cells, or ATCC cell lines were used to investigate the in vitro/in vivo activity of REGN5093-M114. RESULTS: REGN5093-M114 exhibited significant antitumor efficacy compared with MET TKI or unconjugated METxMET biparatopic antibody (REGN5093). Regardless of MET gene copy number, MET-overexpressed TKI-naïve EGFR-mutant NSCLC cells responded to REGN5093-M114 treatment. Cell surface MET expression had the most predictive power in determining the efficacy of REGN5093-M114. REGN5093-M114 potently reduced tumor growth of EGFR-mutant NSCLC with PTEN loss or MET Y1230C mutation after progression on prior osimertinib and savolitinib treatment. CONCLUSIONS: Altogether, REGN5093-M114 is a promising candidate to overcome the challenges facing functional MET pathway blockade.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Imunoconjugados , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Imunoconjugados/uso terapêutico , Receptores ErbB , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas c-met , Mutação , Linhagem Celular TumoralRESUMO
Mesenchymal stem cells (MSCs) are promising source of cell-based regenerative therapy. In consideration of the risk of allosensitization, autologous MSC-based therapy is preferred over allogenic transplantation in patients with chronic kidney disease (CKD). However, it remains uncertain whether adequate cell functionality is maintained under uremic conditions. As chronic inflammation and oxidative stress in CKD may lead to the accumulation of senescent cells, we investigated cellular senescence of CKD MSCs and determined the effects of metformin on CKD-associated cellular senescence in bone marrow MSCs from sham-operated and subtotal nephrectomized mice and further explored in adipose tissue-derived MSCs from healthy kidney donors and patients with CKD. CKD MSCs showed reduced proliferation, accelerated senescence, and increased DNA damage as compared to control MSCs. These changes were significantly attenuated following metformin treatment. Lipopolysaccharide and transforming growth factor ß1-treated HK2 cells showed lower tubular expression of proinflammatory and fibrogenesis markers upon co-culture with metformin-treated CKD MSCs than with untreated CKD MSCs, suggestive of enhanced paracrine action of CKD MSCs mediated by metformin. In unilateral ureteral obstruction kidneys, metformin-treated CKD MSCs more effectively attenuated inflammation and fibrosis as compared to untreated CKD MSCs. Thus, metformin preconditioning may exhibit a therapeutic benefit by targeting accelerated senescence of CKD MSCs.
Assuntos
Hipoglicemiantes/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Metformina/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Técnicas de Cocultura , Dano ao DNA , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Insuficiência Renal Crônica/metabolismoRESUMO
Recently, it has been reported that Fusobacterium nucleatum, a major pathogen involved in chronic periodontitis, may play an important role in colorectal cancer (CRC) progression. In addition, inflammatory bowel diseases such as ulcerative colitis and Crohn's disease represent major predisposing conditions for the development of CRC, and this subtype of cancer is called colitis-associated cancer (CAC). Although the importance of F. nucleatum in CRC has attracted attention, its exact role and related mechanism in CAC progression remain unclear. In this study, we investigated the effects of F. nucleatum in experimental colitis induced with dextran sodium sulfate (DSS), which is a well-known colitis-inducing chemical, on the aggressiveness of CAC and its related mechanism in both in vitro and in vivo models. F. nucleatum synergistically increased the aggressiveness and epithelial-mesenchymal transition (EMT) characteristics of CRC cells that were treated with DSS compared to those in non-treated CRC cells. The role of F. nucleatum in CAC progression was further confirmed in mouse models, as F. nucleatum was found to significantly increase the malignancy of azoxymethane (AOM)/DSS-induced colon cancer. This promoting effect of F. nucleatum was based on activation of the EGFR signaling pathways, including protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), and epidermal growth factor receptor (EGFR) inhibition significantly reduced the F. nucleatum-induced EMT alteration. In conclusion, F. nucleatum accelerates the progression of CAC by promoting EMT through the EGFR signaling pathway.
RESUMO
Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia-inducible factor (HIF)-1α is largely unknown. In this study, we investigated whether HIF-1α could be a deacetylation target of Sirt1 and the effect of their interaction on age-associated renal injury. Five-week-old (young) and 24-month-old (old) C57Bl/6J mice were assessed for their age-associated changes. Kidneys from aged mice showed increased infiltration of CD68-positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF-1α. The level of Bcl-2/adenovirus E1B-interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor-ß1, which are regulated by HIF-1α, was significantly higher in aged mice suggesting that HIF-1α activity was increased. In HK-2 cells, Sirt1 inhibitor sirtinol and siRNA-mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down-regulated, which allowed the acetylation and activation of HIF-1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia-induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF-1α activity by Sirt1-induced deacetylation of HIF-1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF-1α-transfected HK-2 cells. Finally, we confirmed that chronic activation of HIF-1α promoted apoptosis and fibrosis, using tubular cell-specific HIF-1α transgenic mice. Taken together, our data suggest that Sirt1-induced deacetylation of HIF-1α may have protective effects against tubulointerstitial damage in aged kidney.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Nefrite Intersticial/metabolismo , Sirtuína 1/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nefrite Intersticial/patologia , Sirtuína 1/químicaRESUMO
Macrophage activation is increased in diabetes and correlated with the onset and progression of vascular complications. To identify drugs that could inhibit macrophage activation, we developed a cell-based assay and screened a 1,040 compound library for anti-inflammatory effects. Beta2-adrenergic receptor (ß2AR) agonists were identified as the most potent inhibitors of phorbol myristate acetate-induced tumor necrosis factor-α production in rat bone marrow macrophages. In peripheral blood mononuclear cells isolated from streptozotocin-induced diabetic rats, ß2AR agonists inhibited diabetes-induced tumor necrosis factor-α production, which was prevented by co-treatment with a selective ß2AR blocker. To clarify the underlying mechanisms, THP-1 cells and bone marrow macrophages were exposed to high glucose. High glucose reduced ß-arrestin2, a negative regulator of NF-κB activation, and its interaction with IκBα. This subsequently enhanced phosphorylation of IκBα and activation of NF-κB. The ß2AR agonists enhanced ß-arrestin2 and its interaction with IκBα, leading to downregulation of NF-κB. A siRNA specific for ß-arrestin2 reversed ß2AR agonist-mediated inhibition of NF-κB activation and inflammatory cytokine production. Treatment of Zucker diabetic fatty rats with a ß2AR agonist for 12 weeks attenuated monocyte activation as well as pro-inflammatory and pro-fibrotic responses in the kidneys and heart. Thus, ß2AR agonists might have protective effects against diabetic renal and cardiovascular complications.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Anti-Inflamatórios/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Nefropatias Diabéticas/prevenção & controle , Rim/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Miocárdio/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Fibrose , Humanos , Rim/metabolismo , Rim/patologia , Macrófagos/metabolismo , Masculino , Miocárdio/patologia , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Ratos Zucker , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Estreptozocina , Células THP-1 , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismoRESUMO
We reported a functional incompetence in mesenchymal stem cells (MSCs) under uremia, but the mechanisms have not been explored. To study the mechanisms of dysfunctional MSCs induced by uremia, we characterized insulin signaling in MSCs and investigated the effect of uremic toxin, p-cresol, on the proangiogenic actions of insulin. In MSCs, insulin induced hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor, and stromal cell-derived factor 1α expressions via PI3K/Akt-dependent pathway. MSCs treated with p-cresol exhibited altered insulin signaling in a selective manner for insulin receptor substrate-1/PI3K/Akt pathway, whereas ERK pathway remained active. The insulin-induced increase of HIF-1α was blunted by p-cresol treatment. This Akt-selective insulin resistance was also observed in MSCs isolated from chronic kidney disease (CKD) mice. In mice model of hindlimb ischemia, blood flow recovery, capillary density, and local production of angiogenic factors in the ischemic limb treated with CKD MSCs were significantly inferior to those promoted by control MSCs. However, modifying CKD MSCs by overexpression of HIF-1α restored all of these changes. Taken together, these data suggest that p-cresol contributes to insulin resistance in a selective manner for Akt pathway. This might be a biological explanation for the functional incompetence of MSCs under uremia through defects in the insulin-induced elevation of HIF-1α protein expression.
Assuntos
Medula Óssea/metabolismo , Cresóis/farmacologia , Resistência à Insulina , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Uremia/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Transfecção , Uremia/metabolismoRESUMO
BACKGROUND: Chronic kidney disease (CKD) is associated with increased risk for cardiovascular diseases (CVD). We hypothesized that inadequate angiogenic response in uremic patients could result from dysfunction of bone marrow-derived stromal cells [mesenchymal stem cells (MSCs)]. METHODS: We investigated whether MSCs are functionally competent in uremia induced by partial kidney ablation in C57Bl/6J mice. RESULTS: Uremic MSCs showed decreased expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1 and stromal cell-derived factor (SDF)-1α, increased cellular senescence, decreased proliferation, defects in migration in response to VEGF and SDF-1α and in vitro tube formation. Interestingly, the expression of fibroblast-specific protein-1 was higher in uremic MSCs. Uremia decreased hypoxia-inducible factor-1α, VEGF and VEGFR1 expression under hypoxia and Akt phosphorylation in both basal and VEGF-stimulated states. A diminished mitogenic effect on endothelial proliferation was observed in conditioned media from uremic MSCs. In addition, intravital microscopic analysis showed decreased angiogenesis in uremic MSCs. CONCLUSION: These results clearly demonstrate the functional incompetence in MSCs under uremic conditions and may significantly contribute to the disproportionately high risk for CVD in patients with CKD.
Assuntos
Medula Óssea/patologia , Falência Renal Crônica/complicações , Células-Tronco Mesenquimais/patologia , Neovascularização Patológica , Uremia/etiologia , Uremia/patologia , Animais , Biomarcadores/metabolismo , Western Blotting , Medula Óssea/metabolismo , Adesão Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Falência Renal Crônica/etiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Uremia/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Excessive accumulation of extracellular matrix (ECM) in the kidneys and epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells contributes to the renal fibrosis that is associated with diabetic nephropathy. Histone deacetylase (HDAC) determines the acetylation status of histones and thereby controls the regulation of gene expression. This study examined the effect of HDAC inhibition on renal fibrosis induced by diabetes or transforming growth factor (TGF)-beta1 and determined the role of reactive oxygen species (ROS) as mediators of HDAC activation. In streptozotocin (STZ)-induced diabetic kidneys and TGF-beta1-treated normal rat kidney tubular epithelial cells (NRK52-E), we found that trichostatin A, a nonselective HDAC inhibitor, decreased mRNA and protein expressions of ECM components and prevented EMT. Valproic acid and class I-selective HDAC inhibitor SK-7041 also showed similar effects in NRK52-E cells. Among the six HDACs tested (HDAC-1 through -5 and HDAC-8), HDAC-2 activity significantly increased in the kidneys of STZ-induced diabetic rats and db/db mice and TGF-beta1-treated NRK52-E cells. Levels of mRNA expression of fibronectin and alpha-smooth muscle actin were decreased, whereas E-cadherin mRNA was increased when HDAC-2 was knocked down using RNA interference in NRK52-E cells. Interestingly, hydrogen peroxide increased HDAC-2 activity, and the treatment with an antioxidant, N-acetylcysteine, almost completely reduced TGF-beta1-induced activation of HDAC-2. These findings suggest that HDAC-2 plays an important role in the development of ECM accumulation and EMT in diabetic kidney and that ROS mediate TGF-beta1-induced activation of HDAC-2.
Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/enzimologia , Histona Desacetilases/metabolismo , Rim/enzimologia , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Acetilcisteína/farmacologia , Amidas/farmacologia , Animais , Antioxidantes/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular , Transdiferenciação Celular , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/genética , Fibrose , Regulação da Expressão Gênica , Histona Desacetilase 2 , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Ácido Valproico/farmacologiaRESUMO
BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in remodeling of extracellular matrix (ECM) in the glomeruli. PAI-1 is up-regulated by high glucose and is overexpressed in diabetic kidney. Since reactive oxygen species (ROS) mediate ECM accumulation in diabetic glomeruli and was recently found to mediate transforming growth factor-beta1 (TGF-beta1)-induced PAI-1 up-regulation in glomerular mesangial cells, we examined the role of ROS in high glucose-induced PAI-1 expression in cultured glomerular mesangial cells and in streptozotocin-induced diabetic rat glomeruli. METHODS: Growth arrested and synchronized primary rat mesangial cells were treated with different concentrations of glucose in the presence or absence of N-acetylcysteine (NAC) or trolox, or after cellular reduced form of glutathione (GSH) depleted with DL-buthionine-(S,R)-sulfoximine (BSO). Taurine was administered to diabetic rats from 2 days to 4 weeks after streptozotocin injection. Urinary protein excretion, glomerular volume, and fractional mesangial area were measured as markers of renal injury and lipid peroxide (LPO) as an oxidative stress marker. PAI-1 mRNA expression was measured by Northern blot analysis in mesangial cells and reverse transcription-polymerase chain reaction (RT-PCR) in glomeruli, PAI-1 protein by Western blot analysis and enzyme-linked immunosorbent assay (ELISA), and plasmin activity by fluorometry. RESULTS: High glucose significantly increased PAI-1 mRNA and protein expression and decreased plasmin activity in mesangial cells. Equimolar concentrations of l-glucose or mannitol did not affect PAI-1 expression. BSO pretreatment significantly increased basal PAI-1 expression and amplified the response to high glucose. NAC effectively inhibited high glucose-induced, but not basal, PAI-1 expression. Reduced plasmin activity in mesangial cells by high glucose was rescued by antioxidants. Anti-TGF-beta antibody inhibited both high glucose- and H(2)O(2)-induced PAI-1 up-regulation. Taurine significantly reduced plasma LPO, glomerular PAI-1 expression, glomerular volume, fractional mesangial area, and proteinuria in streptozotocin-induced diabetic rats. CONCLUSION: These results demonstrate that ROS mediate high glucose-induced up-regulation of PAI-1 expression in cultured mesangial cells and in diabetic glomeruli. Since both high glucose and TGF-beta1 induce cellular ROS and ROS mediate both high glucose- and TGF-beta1-induced PAI-1, ROS appear to amplify TGF-beta1 signaling in high glucose-induced PAI-1 up-regulation. Antioxidants can prevent accumulation of ECM protein in diabetic glomeruli partly by abrogating up-regulation of PAI-1 and suppression of plasmin activity.
Assuntos
Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Mesângio Glomerular/metabolismo , Glucose/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Sequência de Bases , Butionina Sulfoximina/farmacologia , Células Cultivadas , DNA Complementar/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Fibrinolisina/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Glucose/farmacologia , Glutationa/metabolismo , Masculino , Estresse Oxidativo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Taurina/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To demonstrate the presence of an independent renin-angiotensin system (RAS) in the peritoneum and to determine the role of locally produced angiotensin (Ang) II in high glucose-induced upregulation of transforming growth factor (TGF)-beta1 and fibronectin by human peritoneal mesothelial cells (HPMC). METHODS: In cultured HPMC, the expression of mRNAs for angiotensinogen, angiotensin-converting enzyme (ACE), Ang II type 1 receptor (AT1), and TGF-beta1 was evaluated by real-time polymerase chain reaction; ACE, AT1, and fibronectin proteins by Western blot analysis; and Ang I, Ang II, and TGF-beta1 proteins by ELISA. Dichlorofluorescein (DCF)-sensitive cellular reactive oxygen species (ROS) were measured by fluorometry. RESULTS: HPMC constitutively expressed all the components of RAS, and 50 mmol/L D-glucose (high glucose) significantly increased angiotensinogen, ACE, and AT1 mRNAs and ACE, AT1, and Ang II proteins. Ang II increased TGF-beta1 and fibronectin protein expression and DCF-sensitive cellular ROS. Losartan prevented Ang II-induced increase in cellular ROS. Both losartan and captopril inhibited high glucose-induced upregulation of TGF-beta1 and fibronectin expression in HPMC in a dose-dependent manner. Antioxidant catalase and NADPH oxidase inhibitor diphenyleneiodinium effectively inhibited Ang II-induced TGF-beta1 and fibronectin protein expression. CONCLUSIONS: The present data demonstrate that HPMC constitutively express RAS, that Ang II produced by HPMC mediates high glucose-induced upregulation of TGF-beta1 and fibronectin expression, and that Ang II-induced TGF-beta1 and fibronectin expression in HPMC is mediated by NADPH oxidase-dependent ROS. These data suggest that locally produced Ang II and ROS in the peritoneum may be potential therapeutic targets in peritoneal fibrosis during long-term peritoneal dialysis.
Assuntos
Angiotensina II/fisiologia , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Peritônio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Técnicas de Cultura de Células , Soluções para Diálise/administração & dosagem , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Fibronectinas/genética , Glucose/administração & dosagem , Humanos , Peritônio/efeitos dos fármacos , Peritônio/patologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1 , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC. METHODS: The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H(2)O(2)) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism. RESULTS: D-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H(2)O(2) or H(2)O(2) continuously generated by glucose oxidase up-regulated fibronectin expression [corrected]. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose-and H(2)O(2)-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion. CONCLUSION: The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions.
Assuntos
Fibronectinas/metabolismo , Glucose/administração & dosagem , Peritônio/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Antioxidantes/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , NADPH Oxidases/fisiologia , Peritônio/citologiaRESUMO
Although high glucose (HG) has been shown to induce nuclear factor-kappaB (NF-kappaB) activation in vascular cells, the upstream regulation and the biologic significance of NF-kappaB activation in diabetic renal injury are not clear. It was, therefore, examined if HG-induced generation of reactive oxygen species (ROS) and protein kinase C (PKC) activation are involved in NF-kappaB activation in mesangial cells (MC), and the role of NF-kappaB activation in HG-induced monocyte chemoattractant protein-1 (MCP-1) expression by MC was further investigated. Recent observations suggest that MCP-1 may play a role in the development and progression of diabetic nephropathy. HG rapidly induced NF-kappaB activation in MC as estimated by electrophoretic mobility shift assay. Supershift assay suggests that most of the binding activity arose from p50/p50 and p50/p65 dimers. Antioxidants, pyrrolidine dithiocarbamate, N-acetyl-L-cystein, and trolox effectively inhibited HG-induced NF-kappaB activation in MC. HG rapidly generated dichlorofluorescin-sensitive intracellular ROS in MC as measured by laser-scanning confocal microscopy. HG also activated PKC rapidly in MC. Inhibition of PKC effectively blocked HG-induced intracellular ROS generation and NF-kappaB activation in MC. HG increased MCP-1 mRNA expression by 1.9-fold and protein secretion by 1.6-fold that of control glucose in MC transfected with control vector but not in MC transfected with dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). Inhibition of either PKC or ROS effectively blocked HG-induced, but not basal, MCP-1 protein secretion by MC transfected with control vector. Thus this study demonstrates that HG rapidly activates NF-kappaB in MC through PKC and ROS and suggests that HG-induced NF-kappaB activation in MC may play a role in diabetic renal injury through upregulation of MCP-1 mRNA and protein expression.