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1.
ESC Heart Fail ; 11(4): 2272-2286, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38638083

RESUMO

AIM: Severe functional tricuspid regurgitation (FTR) is associated with high risk of cardiovascular events, particularly heart failure (HF) and mortality. MicroRNAs (miRNAs) have been recently identified as novel biomarkers in different cardiovascular conditions, but no studies have focused on FTR. We sought to (1) to identify and validate circulating miRNAs as regulators of FTR and (2) to test association of miRNA with heart failure and mortality in FTR. METHODS AND RESULTS: Consecutive patients with isolated severe FTR (n = 100) evaluated in the outpatient Heart Valve Clinic and age- and gender-matched subjects with no TR (controls, n = 50) were prospectively recruited. The experimental design included (1) a screening phase to identify candidate miRNA differentially expressed in FTR (n = 8) compared with controls (n = 8) through miRNA array profiling of 192 miRNAs using quantitative reverse transcription PCR arrays [qRT-PCR]) and (2) a validation phase in which candidate miRNAs identified in the initial screening were selected for further validation by qRT-PCR in a prospectively recruited cohort of FTR (n = 92) and controls (n = 42). Bioinformatics analysis was used to predict their potential target genes and functional pathways elicited. A combined endpoint of hospital admission due to heart failure (HF) and all-cause mortality was defined. Initial screening identified 16 differentially expressed miRNAs in FTR compared with controls, subsequently confirmed in the validation phase (n = 16 were excluded due to significant haemolysis). miR-186-5p, miR-30e-5p, and miR-152-3p identified FTR with high predictive value [AUC of 0.93 (0.88-0.97), 0.83 (0.75-0.91) and 0.84 (0.76-0.92), respectively]. During a median follow-up of 20.4 months (IQR 8-35 months), 32% of FTR patients reached the combined endpoint. Patients with low relative expression of miR-15a-5p, miR-92a-3p, miR101-3p, and miR-363-3p, miR-324-3p, and miR-22-3p showed significantly higher rates of events (log-rank test for all P < 0.01). Both miR-15a-5p [hazard ratio: 0.21 (0.06-0.649, P = 0.007) and miR-92a-3p (0.27 (0.09-0.76), P = 0.01] were associated with outcomes after adjusting for age, gender, and New York Heart Association functional class. CONCLUSIONS: Circulating miRNAs are novel diagnostic and prognostic biomarkers in severe FTR. The quantification of miR-186-5p, miR-30e-5p, and miR-152-3p held strong diagnostic value, and the quantification of miR-15a-5p and miR-92a-3p are independently associated with outcomes. The recognition of specific miRNAs offers a novel perspective for TR evaluation.


Assuntos
Biomarcadores , MicroRNA Circulante , Insuficiência Cardíaca , Insuficiência da Valva Tricúspide , Humanos , Masculino , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico , Projetos Piloto , Insuficiência da Valva Tricúspide/genética , Insuficiência da Valva Tricúspide/epidemiologia , Estudos Prospectivos , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Biomarcadores/sangue , Pessoa de Meia-Idade , Idoso , Prognóstico , MicroRNAs/sangue , MicroRNAs/genética , Seguimentos
2.
ESC Heart Fail ; 9(5): 3367-3379, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35837763

RESUMO

BACKGROUND: Patients with acute myocardial infarction (MI) are at high risk of upcoming events, in particular heart failure (HF), but reliable stratification methods are lacking. Our goal was to evaluate the potential role of circulating miRNAs as prognostic biomarkers in patients presenting with MI. METHODS AND RESULTS: We conducted a prospective study among 311 consecutive patients hospitalized with MI (65% ST-segment elevation MI & median age of 55 years) with long-term follow-up. An initial screening was conducted to select candidate miRNAs, with subsequent study of 14 candidate miRNAs. The primary outcome was the composite of hospital admission for HF or cardiovascular death. During a mean follow-up of 2.1 years miR-21-5p, miR-23a-3p, miR27b-3p, miR-122-5p, miR210-3p, and miR-221-3p reliably predicted the primary outcome. Multivariate Cox regression analyses highlighted that miR-210-3p [hazard ratio (HR) 2.65 per 1 SD increase, P < 0.001], miR-23a-3p (HR 2.11 per 1 SD increase, P < 0.001), and miR-221-3p (HR 2.03 per 1 SD increase, P < 0.001) were able to accurately predict the primary outcome, as well as cardiovascular death, HF hospitalizations, and long-term New York Heart Association (NYHA) functional class. These three miRNAs clearly improved the performance of multivariate clinical models: ΔC-statistic = 0.10 [95% confidence interval (CI), 0.03-0.17], continuous net reclassification index = 34.8% (95%CI, 5.8-57.4%), and integrated discrimination improvement (P < 0.001). CONCLUSIONS: This is the largest study evaluating the prognostic value of circulating miRNAs for HF-related events among patients with MI. We show that several miRNAs predict HF hospitalizations, cardiovascular mortality, and poor long-term NYHA status and improve current risk prediction methods.


Assuntos
MicroRNA Circulante , Insuficiência Cardíaca , MicroRNAs , Infarto do Miocárdio , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Biomarcadores
3.
Cancers (Basel) ; 13(7)2021 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-33916610

RESUMO

First-line treatment with regorafenib in frail metastatic colorectal cancer (mCRC) patients has shown some benefit. To accurately identify such patients before treatment, we studied blood biomarkers and primary tumor molecules. We unveiled serum microRNAs (miRNAs), single-nucleotide polymorphisms (SNPs) in angiogenic-related genes, and Notch 1 expression as biomarkers associated with response or toxicity. MicroRNA array profiling and genotyping of selected SNPs were performed in the blood of fragile mCRC patients treated with regorafenib. Notch 1 and CRC-associated miRNA expression was also analyzed in tumors. High levels of miR-185-5p in serum, rs7993418 in the vascular endothelial growth factor receptor 1 (VEGFR1) gene, and Notch 1 expression in biopsies were associated with a favorable response to treatment. Serum levels of miR-126-3p and miR-152-3p and tumor expression of miR-92a-1-5p were associated with treatment toxicity, particularly interesting in patients exhibiting comorbidities, and high levels of miR-362-3p were associated with asthenia. Additionally, several miRNAs were associated with the presence of metastasis, local recurrence, and peritoneal metastasis. Besides, miRNAs determined in primary tumors were associated with tumor-node-metastasis (TNM) staging. The rs2305948 and rs699947 SNPs in VEGFR2 and VEGFA, respectively, were markers of poor prognosis correlating with locoregional relapse, a higher N stage, and metastatic shedding. In conclusion, VEGF and VEGFR SNPs, miRNAs, and Notch 1 levels are potential useful biomarkers for the management of advanced CRC under regorafenib treatment.

4.
Cancers (Basel) ; 12(6)2020 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-32545884

RESUMO

Colon cancer is one of the most frequently diagnosed malignancies in adults, considering both its incidence and prevalence. Anatomically, the right colon is considered as being from the cecum to the splenic flexure, and the left colon is from the splenic flexure to the rectum. Sidedness is a surrogate of a wide spectrum of colorectal cancer (CRC) biology features (embryology, microbiome, methylation, microsatellite instability (MSI), BRAF, aging, KRAS, consensus molecular subtypes (CMS), etc.), which result in prognostic factors. Different molecular subtypes have been identified, according to genomic and transcriptomic criteria. A subgroup harboring a BRAF mutation has been described, and represents approximately 10% of the patients diagnosed with colon cancer. This subgroup has morphological, clinical, and therapeutic characteristics that differ substantially from patients who do not carry this genetic alteration. Unfortunately, there is no established standard of care for this particular cohort of patients. This manuscript aims to study the biology of this subgroup of colon cancer, to understand the current approach in clinical research.

5.
Hepatology ; 70(3): 925-938, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30414342

RESUMO

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.


Assuntos
Translocação Bacteriana/imunologia , Disbiose/imunologia , Interferon gama/metabolismo , Mucosa Intestinal/imunologia , Cirrose Hepática/patologia , Imunidade Adaptativa/fisiologia , Animais , Ascite/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Microbioma Gastrointestinal/imunologia , Humanos , Imunidade Inata/fisiologia , Mucosa Intestinal/microbiologia , Cirrose Hepática/microbiologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar
6.
J Cell Biol ; 216(3): 835-847, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28235946

RESUMO

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4ß1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.


Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/genética , Neoplasias Renais/imunologia , NF-kappa B/genética , Molécula 1 de Adesão de Célula Vascular/genética , Doença de von Hippel-Lindau/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Mutação/genética , Mutação/imunologia , NF-kappa B/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/imunologia , Doença de von Hippel-Lindau/genética
7.
Sci Rep ; 7: 41099, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28106131

RESUMO

Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFß, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1ß, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.


Assuntos
Injúria Renal Aguda/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/etiologia , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Modelos Animais de Doenças , Fibrose , Mediadores da Inflamação/metabolismo , Isquemia/complicações , Rim/irrigação sanguínea , Rim/patologia , Macrófagos/metabolismo , Masculino , Nefrite/complicações , Nefrite/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações
8.
Oncotarget ; 6(37): 39941-59, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26498686

RESUMO

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized.We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies.Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors.Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context.


Assuntos
Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Prostaglandinas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Oxirredutases Intramoleculares/genética , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Microscopia Confocal , Microssomos/enzimologia , Prostaglandina-E Sintases , Interferência de RNA , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Regulação para Cima
9.
PLoS One ; 10(6): e0127175, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26079930

RESUMO

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.


Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/genética , MicroRNAs/genética , Injúria Renal Aguda/sangue , Injúria Renal Aguda/complicações , Adulto , Procedimentos Cirúrgicos Cardíacos , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos/genética , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Projetos Piloto , Curva ROC
10.
Nefrologia ; 33(6): 826-34, 2013 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-24158125

RESUMO

microRNAs are small, endogenous RNA molecules which are critical for a new step in the regulation of the gene expression. They have become the most critical biological mediators characterized in the last ten years. microRNAs participate in almost every cellular process, therefore their deregulation is associated with the development of a wide range of pathologies, including kidney diseases. Increasing evidence demonstrates that microRNAs are key regulators of the normal kidney function and development, but they are also at the basis of several renal diseases. Recent works have established that these molecules can be secreted to extracellular environments, enabling their detection in peripheral body fluids such as urine and serum. Moreover, circulating miRNAs detected in body fluids turn into suitable biomarkers of kidney diseases, including acute kidney injury. This new generation of renal biomarkers could have a great impact in the clinical practice, significantly contributing to improve patient management. In this review, we discuss over the implication of microRNAs in normal kidney function and homeostasis as well as the role of circulating miRNAs as novel biomarkers of kidney diseases, focusing on their potential usefulness in acute kidney injury management.


Assuntos
Injúria Renal Aguda/sangue , Rim/química , MicroRNAs/análise , MicroRNAs/sangue , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/genética , Biomarcadores , Humanos , Rim/irrigação sanguínea , Nefropatias/genética , Nefropatias/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , Especificidade de Órgãos , Processamento Pós-Transcricional do RNA , Traumatismo por Reperfusão/metabolismo , Medição de Risco
11.
PLoS One ; 7(9): e44305, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22962609

RESUMO

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Cinesinas/metabolismo , MicroRNAs/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Adesão Celular , Biologia Computacional , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Túbulos Renais Proximais/patologia , Cinesinas/genética , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
12.
PLoS One ; 7(3): e33258, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22432008

RESUMO

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Adulto , Idoso , Animais , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Transplante de Rim , Necrose Tubular Aguda/complicações , Necrose Tubular Aguda/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Oxigênio/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transplante Homólogo , Adulto Jovem
13.
Kidney Int ; 77(9): 781-93, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20164827

RESUMO

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.


Assuntos
Isquemia/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Expressão Gênica , Hipóxia/genética , Hipóxia/metabolismo , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Isquemia/genética , Rim/metabolismo , Rim/fisiopatologia , Nefropatias/genética , Nefropatias/metabolismo , Testes de Função Renal , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
14.
Cell Physiol Biochem ; 23(4-6): 285-94, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19471096

RESUMO

ERK1/2 has been reported to be activated in the postischemic kidney but its precise role in ischemia/reperfusion (I/R) injury remains unclear. Therefore, we have studied the expression of ERK1/2 and its contribution to cytoskeleton organization and cell adhesion structures in proximal tubular cells, all affected during I/R. We observe ERK1/2 activation at 24 hours of reperfusion in an in vivo model of I/R, when acute tubular necrosis (ATN) is most prominent. In addition, by means of an in vitro model of hypoxia/reoxygenation (H/R) in rat proximal NRK-52E cells we show that p-ERK1/2 is strongly induced early during reoxygenation. Moreover, we also demonstrate that ROS generation contributed to this induction. ERK1/2 activation is contemporary with cell-cell adhesion disruption during reoxygenation but the use of U0126 did not have effect on adherens junctions (AJ) and tight junctions (TJ) disassembly, neither on epithelial monolayer permeability. On the contrary, ERK1/2 affects cytoskeleton organization and focal complexes assembly during H/R, since U0126 improved actin and tubulin cytoskeleton structure, reduced cell contraction and prevented paxillin redistribution. In summary, ERK1/2 signalling plays an essential role in I/R induced injury, mediating proximal cell adhesive alterations which lead to tubular damage and ultimately might compromise renal function.


Assuntos
Citoesqueleto/ultraestrutura , Adesões Focais/ultraestrutura , Isquemia/enzimologia , Túbulos Renais Proximais/enzimologia , Rim/irrigação sanguínea , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Células Cultivadas , Células Epiteliais/enzimologia , Células Epiteliais/ultraestrutura , Hipóxia/fisiopatologia , Isquemia/patologia , Túbulos Renais Proximais/ultraestrutura , Ratos , Espécies Reativas de Oxigênio/metabolismo , Reperfusão , Transdução de Sinais , Fatores de Tempo
15.
J Pharmacol Exp Ther ; 324(2): 454-62, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17977980

RESUMO

The inflammatory response is tightly regulated by several mediators that promote the adhesive and migratory capacities of different cell types, including peripheral blood mononuclear cells (PBMCs). Our laboratory has previously characterized the inflammatory response developed in the experimental model of mercuric chloride (HgCl(2))-induced nephritis in Brown Norway rats as an acute inflammatory response dependent on very late antigen (VLA)-4. This response can be modulated by all-trans-retinoic acid (at-RA), a vitamin A metabolite that regulates a broad range of biological processes and exhibits anti-inflammatory properties. Based on this in vivo experimental model, we have established a VLA-4-dependent ex vivo system to study the effect of at-RA on PBMC polarization, adhesion, and migration and to elicit new mechanisms triggered by at-RA for abrogating an inflammatory response. We found that at-RA significantly reduces the VLA-4-dependent migration of PBMCs activated in vivo. In addition, we demonstrated by spreading assays that in vivo at-RA treatment abrogates the acquisition of a polarized cell phenotype. In fact, at-RA inhibits the actin polymerization required for cell morphology changes, and it alters the distribution of F-actin and VLA-4 integrin in focal contacts, essential for cell adhesion. Moreover, we describe that at-RA also abrogates the redistribution of Rac1 and RhoA, important proteins implicated in the dynamic process of cell movement. In summary, we demonstrate the capacity of at-RA to block the acquisition of an appropriate migratory phenotype in PBMCs as a new mechanism underlying the anti-inflammatory effects of this compound.


Assuntos
Anti-Inflamatórios/farmacologia , Movimento Celular/fisiologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/fisiologia , Fenótipo , Tretinoína/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos BN , Tretinoína/uso terapêutico
16.
BMC Nephrol ; 8: 3, 2007 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-17250768

RESUMO

BACKGROUND: Mercuric chloride (HgCl2) induces an autoimmune nephritis in the Brown Norway (BN) rats characterized by anti-glomerular basement membrane antibodies (anti-GBM Ab) deposition, proteinuria and a severe interstitial nephritis, all evident at day 13 of the disease. We assessed the effects of all-trans retinoic acid (at-RA) in this experimental model. At-RA is a vitamin A metabolite which has shown beneficial effects on several nephropathies, even though no clear targets for at-RA were provided. METHODS: We separated animals in four different experimental groups (HgCl2, HgCl2+at-RA, at-RA and vehicle). From each animal we collected, at days 0 and 13, numerous biological samples: urine, to measure proteinuria by colorimetry; blood to determine VLA-4 expression by flow citometry; renal tissue to study the expression of VCAM-1 by Western blot, the presence of cellular infiltrates by immunohistochemistry, the IgG deposition by immunofluorescence, and the cytokines expression by RT-PCR. Additionally, adhesion assays to VCAM-1 were performed using K562 alpha4 transfectant cells. ANOVA tests were used for statistical significance estimation. RESULTS: We found that at-RA significantly decreased the serum levels of anti-GBM and consequently its deposition along the glomerular membrane. At-RA markedly reduced proteinuria as well as the number of cellular infiltrates in the renal interstitium, the levels of TNF-alpha and IL-1beta cytokines and VCAM-1 expression in renal tissue. Moreover, we reported here for the first time in an in vivo model that at-RA reduced, to basal levels, the expression of VLA-4 (alpha4beta1) integrin induced by mercury on peripheral blood leukocytes (PBLs). In addition, using K562 alpha4 stable transfectant cells, we found that at-RA inhibited VLA-4 dependent cell adhesion to VCAM-1. CONCLUSION: Here we demonstrate a therapeutic effect of at-RA on an autoimmune experimental nephritis model in rats. We report a significant reduction of the VLA-4 integrin expression on PBLs as well as the inhibition of the VLA4/VCAM1-dependent leukocyte adhesion by at-RA treatment. Thereby we point out the VLA-4 integrin as a target for at-RA in vivo.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Integrina alfa4beta1/imunologia , Nefrite/tratamento farmacológico , Nefrite/imunologia , Tretinoína/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Imunossupressores/administração & dosagem , Masculino , Cloreto de Mercúrio , Ratos , Resultado do Tratamento
17.
Exp Cell Res ; 312(19): 3711-27, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17026998

RESUMO

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.


Assuntos
Isquemia/metabolismo , Isquemia/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Rim/irrigação sanguínea , Actinas/metabolismo , Junções Aderentes/patologia , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Adesões Focais , Técnicas In Vitro , Rim/lesões , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Junções Íntimas/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
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