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Parietal epithelial cells (PECs) are kidney progenitor cells with similarities to a bone marrow stem cell niche. In focal segmental glomerulosclerosis (FSGS) PECs become activated and contribute to extracellular matrix deposition. Colony stimulating factor-1 (CSF-1), a hematopoietic growth factor, acts via its specific receptor, CSF-1R, and has been implicated in several glomerular diseases, although its role on PEC activation is unknown. Here, we found that CSF-1R was upregulated in PECs and podocytes in biopsies from patients with FSGS. Through in vitro studies, PECs were found to constitutively express CSF-1R. Incubation with CSF-1 induced CSF-1R upregulation and significant transcriptional regulation of genes involved in pathways associated with PEC activation. Specifically, CSF-1/CSF-1R activated the ERK1/2 signaling pathway and upregulated CD44 in PECs, while both ERK and CSF-1R inhibitors reduced CD44 expression. Functional studies showed that CSF-1 induced PEC proliferation and migration, while reducing the differentiation of PECs into podocytes. These results were validated in the Adriamycin-induced FSGS experimental mouse model. Importantly, treatment with either the CSF-1R-specific inhibitor GW2580 or Ki20227 provided a robust therapeutic effect. Thus, we provide evidence of the role of the CSF-1/CSF-1R pathway in PEC activation in FSGS, paving the way for future clinical studies investigating the therapeutic effect of CSF-1R inhibitors on patients with FSGS.
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Glomerulosclerose Segmentar e Focal , Receptores de Hialuronatos , Fator Estimulador de Colônias de Macrófagos , Podócitos , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Animais , Humanos , Podócitos/metabolismo , Podócitos/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Camundongos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Glomérulos Renais/patologia , Glomérulos Renais/metabolismo , Masculino , Modelos Animais de Doenças , Células Cultivadas , Feminino , Regulação para Cima , Movimento Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transdução de Sinais , Camundongos Endogâmicos C57BL , Receptores de Fator Estimulador das Colônias de Granulócitos e MacrófagosRESUMO
Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.
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Senescência Celular , Fenótipo Secretor Associado à Senescência , Humanos , Ferro , Rim , FibroseRESUMO
The relevance of the human oral microbiome to our understanding of human health has grown in recent years as microbiome studies continue to develop. Given the links of the oral cavity with the digestive, respiratory and circulatory systems, the composition of the oral microbiome is relevant beyond just oral health, impacting systemic processes across the body. However, we still have a very limited understanding about intrinsic and extrinsic factors that shape the composition of the healthy oral microbiome. Here, we followed a citizen-science approach to assess the relative impact on the oral microbiome of selected biological, social, and lifestyle factors in 1648 Spanish individuals. We found that the oral microbiome changes across age, with middle ages showing a more homogeneous composition, and older ages showing more diverse microbiomes with increased representation of typically low abundance taxa. By measuring differences within and between groups of individuals sharing a given parameter, we were able to assess the relative impact of different factors in driving specific microbial compositions. Chronic health disorders present in the analyzed population were the most impactful factors, followed by smoking and the presence of yeasts in the oral cavity. Finally, we corroborate findings in the literature that relatives tend to have more similar oral microbiomes, and show for the first time a similar effect for classmates. Multiple intrinsic and extrinsic factors jointly shape the oral microbiome. Comparative analysis of metabarcoding data from a large sample set allows us to disentangle the individual effects.
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Bactérias , Microbiota , Bactérias/genética , Humanos , Estilo de Vida , Pessoa de Meia-Idade , Boca , EspanhaRESUMO
Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.
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Chronic kidney disease (CKD) will become the fifth global cause of death by 2040, thus emphasizing the need to better understand the molecular mechanisms of damage and regeneration in the kidney. CKD predisposes to acute kidney injury (AKI) which, in turn, promotes CKD progression. This implies that CKD or the AKI-to-CKD transition are associated with dysfunctional kidney repair mechanisms. Current therapeutic options slow CKD progression but fail to treat or accelerate recovery from AKI and are unable to promote kidney regeneration. Unraveling the cellular and molecular mechanisms involved in kidney injury and repair, including the failure of this process, may provide novel biomarkers and therapeutic tools. We now review the contribution of different molecular and cellular events to the AKI-to-CKD transition, focusing on the role of macrophages in kidney injury, the different forms of regulated cell death and necroinflammation, cellular senescence and the senescence-associated secretory phenotype (SAPS), polyploidization, and podocyte injury and activation of parietal epithelial cells. Next, we discuss key contributors to repair of kidney injury and opportunities for their therapeutic manipulation, with a focus on resident renal progenitor cells, stem cells and their reparative secretome, certain macrophage subphenotypes within the M2 phenotype and senescent cell clearance.
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Injúria Renal Aguda/metabolismo , Macrófagos/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Biomarcadores/metabolismo , Progressão da Doença , Humanos , Regeneração , Fenótipo Secretor Associado à SenescênciaRESUMO
Phagocytosis is an inherent function of tissue macrophages for the removal of apoptotic cells and cellular debris during acute and chronic injury; however, the dynamics of this event during fibrosis development is unknown. We aim to prove that during the development of kidney fibrosis in the unilateral ureteral obstruction (UUO) model, there are some populations of macrophage with a reduced ability to phagocytose, and whether the infusion of a population of phagocytic macrophages could reduce fibrosis in the murine model UUO. For this purpose, we have identified the macrophage populations during the development of fibrosis and have characterized their phagocytic ability and their expression of CPT1a. Furthermore, we have evaluated the therapeutic effect of macrophages overexpressing CPT1a with high phagocytic skills. We evidenced that the macrophage population which exhibits high phagocytic ability (F4/80low-CD11b) in fibrotic animals decreases during the progression of fibrosis while the macrophage population with lower phagocytic ability (F4/80high-CD11b) in fibrotic conditions, conversely, increases and CPT1a macrophage cell therapy with a strengthening phagocytic ability is associated with a therapeutic effect on kidney fibrosis. We have developed a therapeutic approach to reduce fibrosis in the UUO model by enrichment of the kidney resident macrophage population with a higher proportion of exogenous phagocytic macrophages overexpressing CPT1a.
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Carnitina O-Palmitoiltransferase/metabolismo , Rim/patologia , Macrófagos/transplante , Fagocitose , Obstrução Ureteral/complicações , Animais , Antígeno CD11b/metabolismo , Carnitina O-Palmitoiltransferase/genética , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Perfilação da Expressão Gênica , Masculino , Camundongos , Células RAW 264.7RESUMO
Introduction: Cystic fibrosis (CF) is an autosomal genetic disease, associated with the production of excessively thick mucosa and with life-threatening chronic lung infections. The microbiota of the oral cavity can act as a reservoir or as a barrier for infectious microorganisms that can colonize the lungs. However, the specific composition of the oral microbiome in CF is poorly understood.Methods: In collaboration with CF associations in Spain, we collected oral rinse samples from 31 CF persons (age range 7-47) and matched controls, and then performed 16S rRNA metabarcoding and high-throughput sequencing, combined with culture and proteomics-based identification of fungi to survey the bacterial and fungal oral microbiome.Results: We found that CF is associated with less diverse oral microbiomes, which were characterized by higher prevalence of Candida albicans and differential abundances of a number of bacterial taxa that have implications in both the connection to lung infections in CF, as well as potential oral health concerns, particularly periodontitis and dental caries.Conclusion: Overall, our study provides a first global snapshot of the oral microbiome in CF. Future studies are required to establish the relationships between the composition of the oral and lung microbiomes in CF.
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OBJECTIVE/HYPOTHESIS: The 10-item Eating-Assessment Tool (EAT-10) is a dysphagia screening test. In HNC patients, screening and diagnosis of dysphagia are not well-established. To determine the metrological properties of the EAT-10 compared with videofluoroscopy in non-surgical HNC-patients and to assess the relationship between EAT-10 scores and patients' self-reported symptoms. STUDY DESIGN: Prospective cohort study. METHODS: Forty-six HNC-patients recently diagnosed and referred to chemoradiotherapy (CRT). Main outcome was evidence of dysphagia according to EAT-10 score, self-perception on a Visual Analog Scale (VAS) of impaired swallowing, severity on the Penetration-Aspiration Scale (PAS), and the Functional Oral Intake Scale (FOIS). Patients were assessed at baseline, before-CRT, after-CRT, and at 3-month follow-up. RESULTS: A strong baseline correlation between EAT-10, VAS, and FOIS was observed. All 3 values decreased in weeks 6 to 9 after CRT initiation; a poor correlation of EAT-10 with VAS was observed at 3-month follow-up. A receiver operating characteristic curve determined new cut-off points (sensitivity/specificity) for safe swallowing: baseline 3 (86%, 77%); post-CRT, 15 (62.5%, 80%); and 3-month follow-up, 4 (83%, 75%). CONCLUSIONS: New safe-swallow EAT-10-points are suggested for this population during screening and the oncological follow-up. A poor correlation between EAT10-score and patient self-reported symptoms was observed at the end-RT and at 3-month follow-up, highlighting the need for an objective evaluation instrument.
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Quimiorradioterapia , Transtornos de Deglutição/diagnóstico , Deglutição , Neoplasias de Cabeça e Pescoço/fisiopatologia , Neoplasias de Cabeça e Pescoço/terapia , Adulto , Idoso , Correlação de Dados , Transtornos de Deglutição/etiologia , Técnicas de Diagnóstico do Sistema Digestório , Feminino , Fluoroscopia , Neoplasias de Cabeça e Pescoço/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos ProspectivosRESUMO
During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls' staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.
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Ferro/metabolismo , Lipocalina-2/metabolismo , Insuficiência Renal/metabolismo , Sepse/complicações , Animais , Biomarcadores/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lipocalina-2/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Insuficiência Renal/etiologia , Insuficiência Renal/patologiaRESUMO
Macrophages show remarkable phenotypic plasticity in response to environmental signals. Although it is generally less considered, cytoskeletal changes in macrophages influence their phenotype, including phagocytosis and secretion of soluble cytokines. Influenza virus NS1A-binding protein (Ivns1abp) belongs to the Kelch family of proteins that play a central role in actin cytoskeleton dynamics by directly associating with F-actin and by protecting against actin derangement. Due to its role in cytoskeleton preservation, the Ivns1abp gene might be a critical regulator of the macrophage phenotype and function under inflammatory conditions. In this study, we determine that the modulation of the Ivns1abp gene in macrophages could modify resistance to macrophages against inflammation and maintain functional phagocytosis. Our results indicate that inflammatory insults inhibit the Ivns1abp gene, whereby phagocytosis is inhibited and the ability of macrophages to induce proliferation and repair of damaged cells is compromised. Furthermore, our results show that inflammatory insults alter the activity of the transcription factor c-myc, a factor which directly modulates the expression of the Ivns1abp gene. In conclusion, this study demonstrates a central role of lvns1abp in promoting and preserving a reparative macrophage phenotype and resistance to this inflammatory environment.
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Citocinas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Orthomyxoviridae/metabolismo , Fagócitos/metabolismo , Fagocitose/fisiologia , Proteínas de Ligação a RNA/genética , Animais , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: To assess the performance of peak expiratory flow (PEF) for sarcopenia screening in patients with chronic obstructive pulmonary disease (COPD), using the revised European Working Group on Sarcopenia in Older People (EWGSOP-2) criteria as the reference standard in pulmonary rehabilitation patients; and second, to study the factors associated with low PEF in this population. METHODS: Diagnostic accuracy study conducted in consecutive community-dwelling COPD rehabilitation patients. Sensitivity, specificity, accuracy, likelihood ratios, predictive values, and area under the Receiver-Operating Characteristic curve were retrospectively calculated for PEF (index test) and compared with EWGSOP-2 criteria (reference standard). RESULTS: Of 151 potentially eligible patients, 79 (67.5 ± 7.1 years; 78.8% men) fulfilled inclusion criteria and 10 (12.7%) had a diagnosis of sarcopenia. The PEF cut-off with highest accuracy (65.8%) was PEF ≤ 200 L/min (sensitivity 90%, specificity 62.3%, and positive and negative likelihood ratios 2.39 and 0.16, respectively). Airway obstruction and muscle mass were significantly associated with PEF ≤ 200 L/min. CONCLUSIONS: Considering the EWGSOP-2 criteria as the reference standard, a cut-off of PEF ≤ 200 L/min showed only fair validity for detecting sarcopenia, so it cannot be recommended as a stand-alone screening tool in older rehabilitation patients with COPD.
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Sarcopenia , Idoso , Feminino , Humanos , Vida Independente , Masculino , Programas de Rastreamento , Curva ROC , Estudos Retrospectivos , Sarcopenia/diagnósticoRESUMO
BACKGROUND: Macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated. METHODS: Using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied. RESULTS: Our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles. CONCLUSIONS: Altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype.
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Carnitina O-Palmitoiltransferase/genética , Expressão Gênica/fisiologia , Inflamação , Cetocolesteróis/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Animais , Carnitina O-Palmitoiltransferase/fisiologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Cetocolesteróis/farmacologia , Ativação de Macrófagos/fisiologia , Camundongos , Células RAW 264.7 , TransfecçãoRESUMO
At present, Lupus Nephritis (LN) is still awaiting a biomarker to better monitor disease activity, guide clinical treatment, and predict a patient's long-term outcome. In the last decade, novel biomarkers have been identified to monitor the disease, but none have been incorporated into clinical practice. The transmembrane receptor neuropilin-1 (NRP-1) is highly expressed by mesangial cells and its genetic deletion results in proteinuric disease and glomerulosclerosis. NRP-1 is increased in kidney biopsies of LN. In this work we were interested in determining whether urinary NRP-1 levels could be a biomarker of clinical response in LN. Our results show that patients with active LN have increased levels of urinary NRP-1. When patients were divided according to clinical response, responders displayed higher urinary and tissue NRP-1 levels at the time of renal biopsy. Areas under the receiver operating characteristic curve, comparing baseline creatinine, proteinuria, urinary NRP-1, and VEGFA protein levels, showed NRP-1 to be an independent predictor for clinical response. In addition, in vitro studies suggest that NRP-1could promote renal recovery through endothelial proliferation and migration, mesangial migration and local T cell cytotoxicity. Based on these results, NRP-1 may be used as an early prognostic biomarker in LN.
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Biomarcadores , Nefrite Lúpica/metabolismo , Neuropilina-1/metabolismo , Adulto , Biópsia , Movimento Celular , Proliferação de Células/genética , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/genética , Nefrite Lúpica/urina , Masculino , Pessoa de Meia-Idade , Neuropilina-1/genética , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Proteinúria , RNA Mensageiro/genética , Curva ROC , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: Radiation-induced dysphagia is common in patients with head and neck cancer (HNC). Available evidence suggests that exercise therapy prior to oncological treatment could potentially improve deglutition and quality of life; however, a randomized clinical trial is needed to confirm this observation. METHODS/DESIGN: The Redyor study is a single-blind randomized clinical trial designed to compare the effect of prophylactic oropharyngeal exercises on quality of life and dysphagia of 52 patients with HNC referred to the Radiotherapy Department. The intervention will consist of respiratory muscle training (3 times/day, 5 days/week, 21 weeks) added to the standard swallow therapy. All patients will perform the same exercise intervention, but at different times: before chemoradiotherapy (CRT; early intervention group) or immediately after completing CRT (late intervention group). The main outcome will be change in dysphagia severity assessed with the Penetration-Aspiration Scale in videofluoroscopy study; quality of life will be assessed with the EORTC-QLQ-C30 and its Head and Neck Cancer Module (QLQ-H&N35) at 3, 6, and 12 months after completing CRT. DISCUSSION: This ongoing clinical trial, registered in 2016, is based on the hypothesis that undergoing a pre-radiotherapy rehabilitation (pre-habilitation) program will have greater benefits (less decrease in quality of life, less delay in swallowing parameters, and less severe dysphagia) compared to post-CRT rehabilitation. The main objective is to assess dysphagia severity in HNC patients; and secondly, to evaluate changes in dysphagia-related quality of life, and to determine the correlation between a clinical variable and instrumental parameters during this period. TRIAL REGISTRATION: NCT0209009911 .
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Quimiorradioterapia , Transtornos de Deglutição/prevenção & controle , Deglutição , Terapia por Exercício/métodos , Neoplasias de Cabeça e Pescoço/radioterapia , Qualidade de Vida , Quimiorradioterapia/efeitos adversos , Transtornos de Deglutição/diagnóstico , Transtornos de Deglutição/etiologia , Transtornos de Deglutição/fisiopatologia , Humanos , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Método Simples-Cego , Espanha , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: The costimulatory CD40-CD40L pathway plays a role in kidney inflammation. We have previously reported that renal CD40 upregulation precedes cellular interstitial infiltrate and fibrosis in the unilateral ureteral obstruction (UUO) model. Here we sought to evaluate whether the administration of siRNA-CD40 has a therapeutic effect in a reversible unilateral ureteral obstruction (D-UUO) mice model. METHODS: Eight week-old C57BL6J male mice were divided into four groups: Vehicle (Phosphate-buffered saline) (n = 8); siRNA SC (non-specific siRNA) (n = 6); siRNA-CD40 (n = 8) and WT (wild type) (n = 6) mice. UUO was performed with a microvascular clamp. At day 3 after surgery, the ureteral clamp was removed and nephrectomy of the contralateral kidney was performed. Immediately, PBS, siRNA SC (50µg) or siRNA-CD40 (50µg) was administrated via the tail vein. Mice were killed 48h hours after the siRNA or saline administration. Wild type (WT) mice were used as controls. Blood samples were collected for measuring creatinine and blood urea nitrogen (BUN). Histology and kidney mRNA expression were performed. RESULTS: The administration of siRNA-CD40 reduced significantly the severity of acute renal failure associated with UUO. Pathologic analysis showed reduction of tubular dilation, interstitial fibrosis, F4/80 macrophage and CD3 (T cell) infiltration in animals treated with siRNA-CD40. Furthermore, kidney mRNA gene expression analysis showed significantly lower levels of macrophage markers (F4/80 and Mannose receptor), fibrosis matrix proteins (Fibronectin, MMP-9, Collagen IV and α-SMA), pro-inflammatory cytokines (iNOS and MCP-1) and the pro-fibrotic molecule TGF-ß1 in siRNA-CD40. CONCLUSIONS: The administration of siRNA-CD40 therapy reduces the severity of the acute kidney injury induced by obstructive uropathy and promotes kidney repair. This strategy seems suitable to be tested in humans.
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Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/genética , Obstrução Ureteral/complicações , Obstrução Ureteral/terapia , Injúria Renal Aguda/patologia , Animais , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Inativação Gênica , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase-associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow-derived M2 (BM-ÑM2) and Ñ-NGAL macrophages in the db/db mice. Seventeen-week-old mice with established DKD were divided into five treatment groups with their controls: D+BM-ÑM2; D+Ñ-BM; D+Ñ-NGAL; D+Ñ-RAW; D+SHAM and non-diabetic (ND) (db/- and C57bl/6J) animals. We infused 1 × 106 macrophages twice, at baseline and 2 weeks thereafter. BM-ÑM2 did not show any therapeutic effect whereas Ñ-NGAL significantly reduced albuminuria and renal fibrosis. The Ñ-NGAL therapy increased the anti-inflammatory IL-10 and reduced some pro-inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF-ß1. Overall, our study provides evidence that Ñ-NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Diabetes Mellitus Experimental/terapia , Nefropatias Diabéticas/terapia , Lipocalina-2/genética , Macrófagos/transplante , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Apoptose , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Lipocalina-2/metabolismo , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Podócitos/metabolismo , Podócitos/patologia , Cultura Primária de Células , Células RAW 264.7 , Transdução de Sinais , Transdução Genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , TransgenesRESUMO
Long-term kidney transplant outcomes have reached mild improvements recently. Parietal epithelial cells (PECs) are progenitor cells located along the Bowman's capsule that can be isolated in urine, and display the capability to replace podocytes, but in certain situations cause glomerulosclerosis. In this study, a cohort of stable kidney transplant recipients with 6 months protocol biopsy was divided in two groups depending on the presence (uPEC+; n = 41) or absence (uPEC-; n = 25) of PECs in urine and followed for 2 years. No differences were found between groups at 6 months after transplantation considering clinical variables, alloimmune response, renal function, albuminuria and graft pathology. However, uPEC+ group showed increased podocyturia and a higher rate of proliferating PECs along the Bowman's capsule, without concomitant enhancement of the CD44 pro-sclerotic activation marker. Accordingly, 2 years follow up evidenced poorer outcomes in the uPEC+ group with worse renal function, increased albuminuria, wider mesangial expansion and more severe IFTA. In summary, chronic allograft damage can progress in certain stable-supposed grafts by podocyte detachment and reactive PECs proliferation, being the uPEC presence a biomarker of this process. This damage-response regenerative process, if sustained in time, might fail in preserve the allograft function and histology. Our study raises new prospects to overcome current limits on long-term allograft results.
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Macrophage-epithelial cross-talk regulates cell cycle progression and represents an important factor in rescuing epithelial cells from cell cycle arrest in order to maintain a healthy epithelial phenotype. However, the underlying mechanisms are still not well defined. We provide evidence that macrophage-secreted lipocalin-2 (Lcn-2) plays a key role during this process. In a co-culture setup using cell cycle arrested NRK52e renal epithelial cells and primary bone marrow-derived macrophages, Lcn-2 restores proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lcn-2 overexpression in macrophages overcomes epithelial cell cycle arrest and enhances epithelial markers via megalin and the downstream activation of PI3K/Akt signalling pathway, whereas a knockdown of Lcn-2 in macrophages prevented this effect. Our results show that macrophage-secreting Lcn-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation.
Assuntos
Proliferação de Células , Células Epiteliais/fisiologia , Lipocalina-2/fisiologia , PPAR gama/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Macrófagos/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-DawleyRESUMO
Epithelial to mesenchymal transition (EMT) has emerged as a key process in the development of renal fibrosis. In fact, EMT-derived fibroblasts contribute to the progression of chronic renal disease. In addition, anti-inflammatory M2 macrophages have exhibited a great influence on renal fibrosis. However, because of the high impact that the inputs of different environmental cytokines have on their phenotype, macrophages can easily lose this property. We aim to known if microencapsulated macrophages on M2-inducing alginate matrices could preserve macrophage phenotype and thus release factors able to act on epithelial cells to prevent the epithelial differentiation towards mesenchymal cells. We reproduced an in vitro model of EMT by treating adipose-derived stem cells with all-trans retinoic acid (ATRA) and induced their transformation toward epithelia. Dedifferentiation of epithelial cells into a mesenchymal phenotype occurred when ATRA was retired, thus simulating EMT. Results indicate that induction of M2 phenotype by IL-10 addition in the alginate matrix produces anti-inflammatory cytokines and increases the metabolic activity and the viability of the encapsulated macrophages. The released conditioned medium modulates EMT and maintains healthy epithelial phenotype. This could be used for in vivo cell transplantation, or alternatively as an external releaser able to prevent epithelial to mesenchymal transformation for future anti-fibrotic therapies.
Assuntos
Meios de Cultivo Condicionados/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Macrófagos/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Interleucina-10/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Fenótipo , Células RAW 264.7RESUMO
BACKGROUND/AIMS: Alternatively activated macrophages (AAM) have regenerative and anti-inflammatory characteristics. Here, we sought to evaluate whether AAM cell therapy reduces renal inflammation and fibrosis in the unilateral ureteral obstruction (UUO) mice model. METHODS: We stabilized macrophages by adenoviral vector NGAL (Neutrophil gelatinase-associated lipocalin-2) and infused them into UUO mice. To ascertain whether macrophages were capable of reaching the obstructed kidney, macrophages were stained and detected by in vivo cell tracking. RESULTS: We demonstrated that some infused macrophages reached the obstructed kidney and that infusion of macrophages overexpressing NGAL was associated with reduced kidney interstitial fibrosis and inflammation. This therapeutic effect was mainly associated with the phenotype and function preservation of the transferred macrophages isolated from the obstructed kidney Conclusions: Macrophage plasticity is a major hurdle for achieving macrophage therapy success in chronic nephropathies and could be overcome by transferring lipocalin-2.