RESUMO
Kidneys are one of the main dose-limiting organs in radiotherapeutic procedures of lower abdomen. Likewise, the threat of exposure of radiosensitive organs such as kidneys in warfare or radiation accidents among military personal or due to terrorist activities in general public is of increasing concern. These events warrant the need for appropriate animal models to study the acute and chronic effects of low- and high-dose rate radiation exposures. In this study, for the first time, we validated Tsc2+/- mouse model to study whether radiation accelerates carcinogenesis in kidneys. Tsc2+/- mice at increasing age groups at 8 and 10 months were exposed to repeated doses of gamma radiation (0.4 Gy × 5) and assessed for aggravated kidney tumor formation at 2 months post-irradiation. Animals from irradiated group showed a significant increase in numbers of bilateral, multifocal tumors compared with mock-irradiated animals. Intra-glomerular reactive oxygen species (ROS) levels measured by dihydroethidium florescence showed significant increases in ROS production in irradiated Tsc2+/- mice compared with non-irradiated animals. Similarly, selective hematological parameters and glomerular filtration rate were further reduced significantly in irradiated Tsc2+/- mice. Tsc2 protein, tuberin in irradiated mice, however, remains at the same reduced levels as that of the mock-irradiated heterozygous Tsc2 mice. The results indicate that radiation alters kidney homeostatic function and influences high spontaneous incidence of renal cell carcinoma in this rodent model. Repurposing of Tsc2+/- mice model will, therefore, provide a unique opportunity to study acute and delayed effects of radiation in the development of kidney cancers.
Assuntos
Neoplasias Renais/radioterapia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Glomérulos Renais/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
Previous studies demonstrated that global deficiency of eNOS in diabetic mice exacerbated renal lesions and that overexpression of eNOS may protect against tissue injury. Our study revealed for the first time overexpression of eNOS leads to disease progression rather than protection. Transgenic mice selectively expressing eNOS in endothelial cells (eNOSTg) were cross bred with Ins2Akita type-1 (AK) diabetic mice to generate eNOS overexpressing eNOSTg/AK mice. Wild type, eNOSTg, AK and eNOSTg/AK mice were assessed for kidney function and blood glucose levels. Remarkably, overexpressing eNOSTg mice showed evidence of unpredicted glomerular injury with segmental mesangiolysis and occasional microaneurysms. Notably, in eNOSTg/AK mice overexpression of eNOS led to increased glomerular/endothelial injury that was associated with increased superoxide levels and renal dysfunction. Results indicate for the first time that overexpressing eNOS in endothelial cells cannot ameliorate diabetic lesions, but paradoxically leads to progression of nephropathy likely due to eNOS uncoupling and superoxide upsurge. This novel finding has a significant impact on current therapeutic strategies to improve endothelial function and prevent progression of diabetic renal disease. Further, the eNOSTg/AK model developed in this study has significant translational potentials for elucidating the underlying mechanism implicated in the deflected function of eNOS in diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Endotélio Vascular/metabolismo , Glomérulos Renais/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Animais , Nefropatias Diabéticas/diagnóstico por imagem , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/diagnóstico por imagem , Endotélio Vascular/patologia , Insulina/genética , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia EletrônicaRESUMO
Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.IMPORTANCEMycoplasma pneumoniae, a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate M. pneumoniae-associated airway and extrapulmonary pathologies.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Mycoplasma pneumoniae/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Transporte ProteicoRESUMO
Concerns on high-energy particle radiation-induced tumorigenic transformation of normal tissue in astronauts, and in cancer patients undergoing radiotherapy, emphasizes the significance of elucidating the mechanisms involved in radiogenic transformation processes. Mostly used genetically modified or tumor-prone models are less reliable in determining human health risk in space or protracted post-treatment normal tissue toxicity. Here, in wild type C57BL/6 mice, we related the deregulation of distinctive set of tissue-specific oncotargets in major organs upon 56Fe (600 MeV/amu; 0.5 Gy/min; 0.8 Gy) particle radiation and compared the response with low LET γ-radiation (137Cs; 0.5 Gy/min; 2 Gy). One of the novel findings is the 'tissue-independent' activation of TAL2 upon high-energy radiation, and thus qualifies TAL2 as a potential biomarker for particle and other qualities of radiation. Heightened expression of TAL2 gene transcript, which sustained over four weeks post-irradiation foster the concept of oncogene addiction signaling in radiogenic transformation. The positive/negative expression of other selected oncotargets that expresses tissue-dependent manner indicated their role as a secondary driving force that addresses the diversity of tissue-dependent characteristics of tumorigenesis. This study, while reporting novel findings on radiogenic transformation of normal tissue when exposed to particle radiation, it also provides a platform for further investigation into different radiation quality, LET and dose/dose rate effect in healthy organs.
Assuntos
Transformação Celular Neoplásica/genética , Vício Oncogênico , Radiação , Animais , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Íons , Queratina-5/metabolismo , Camundongos Endogâmicos C57BL , Micronúcleo Germinativo/metabolismo , Oncogenes , Especificidade de Órgãos/efeitos da radiação , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Transcriptoma/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
There are 160,000 cancer patients worldwide treated with particle radiotherapy (RT). With the advent of proton, and high (H) charge (Z) and energy (E) HZE ionizing particle RT, the cardiovascular diseases risk estimates are uncertain. In addition, future deep space exploratory-type missions will expose humans to unknown but low doses of particle irradiation (IR). We examined molecular responses using transcriptome profiling in left ventricular murine cardiomyocytes isolated from mice that were exposed to 90 cGy, 1 GeV proton ((1)H) and 15 cGy, 1 GeV/nucleon iron ((56)Fe) over 28 days after exposure. Unsupervised clustering analysis of gene expression segregated samples according to the IR response and time after exposure, with (56)Fe-IR showing the greatest level of gene modulation. (1)H-IR showed little differential transcript modulation. Network analysis categorized the major differentially expressed genes into cell cycle, oxidative responses, and transcriptional regulation functional groups. Transcriptional networks identified key nodes regulating expression. Validation of the signal transduction network by protein analysis and gel shift assay showed that particle IR clearly regulates a long-lived signaling mechanism for ERK1/2, p38 MAPK signaling and identified NFATc4, GATA4, STAT3, and NF-κB as regulators of the response at specific time points. These data suggest that the molecular responses and gene expression to (56)Fe-IR in cardiomyocytes are unique and long-lasting. Our study may have significant implications for the efforts of National Aeronautics and Space Administration to develop heart disease risk estimates for astronauts and for patients receiving conventional and particle RT via identification of specific HZE-IR molecular markers.
Assuntos
Redes Reguladoras de Genes/efeitos da radiação , Radioisótopos de Ferro/toxicidade , Miócitos Cardíacos/efeitos da radiação , Radioterapia de Alta Energia/efeitos adversos , Transdução de Sinais/efeitos da radiação , Animais , Células Cultivadas , Análise por Conglomerados , Ativação Enzimática , Fibrose , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos da radiação , Masculino , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos da radiação , Transcriptoma/efeitos da radiação , Irradiação Corporal TotalRESUMO
Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells' differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.