RESUMO
The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.
Assuntos
Alternativas ao Uso de Animais , Bem-Estar do Animal , Animais de Laboratório , Animais , Europa (Continente)RESUMO
Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.
Assuntos
Experimentação Animal , Alternativas aos Testes com Animais , Animais , Europa (Continente)RESUMO
Elevated mechanical stress in glomerular hypertension is thought to damage podocytes, the loss of which leads to development of glomerulosclerosis. Applying cDNA array analysis to mechanically stressed podocytes, we have recently identified TSG101 as a stretch-induced candidate gene among others. TSG101, which is part of the ESCRT-I complex, is involved in multivesicular body (MVB) formation. Here we demonstrate that TSG101 mRNA is strongly upregulated in conditionally immortalized mouse podocytes by cyclic mechanical stress. Differentiation of podocytes does not affect TSG101 mRNA levels. TSG101 immunofluorescence is distributed in a vesicular pattern in podocytes, the staining intensity being enhanced by mechanical stress. In DOCA/salt treated rats, a model of glomerular hypertension, glomerular TSG101 mRNA levels are elevated, and an increased number of MVBs is observed by electron microscopy in podocyte processes. Our data demonstrate that mechanical stress upregulates TSG101 in podocytes, suggesting that glomerular hypertension enhances sorting of cell surface proteins and their ligands into the degradative pathway in podocytes.
Assuntos
Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Podócitos/metabolismo , Podócitos/patologia , Estresse Mecânico , Fatores de Transcrição/genética , Regulação para Cima/genética , Animais , Diferenciação Celular/genética , Acetato de Desoxicorticosterona , Masculino , Camundongos , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Podócitos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos WistarRESUMO
Evaluation of renal function is crucial for a number of clinical situations. Here, we reported a novel exogenous fluorescent marker (FITC-HPßCD) to real-time assess renal function by using a transcutaneous fluorescent detection technique. FITC-HPßCD was designed based on the principle of renal clearance of designed drugs. It displays favorable fluorescent properties, high hydrophilicity, low plasma protein binding, and high stability in porcine liver esterase as well as in plasma and nontoxicity. More importantly, FITC-HPßCD can be efficiently and rapidly filtered by glomerulus and completely excreted into urine without proximal tubular reabsorption or secretion in rat models. Additionally, the marker was well-tolerated, with nearly 100% urinary recovery of the given doses, and no metabolism were found. Relying on this novel kidney function marker and transcutaneous devices, we demonstrate a rapid, robust, and convenient approach for real-time assessing renal function without the need of time-consuming blood and urine sample preparation. Our work provides a promising tool for noninvasive real-time monitoring of renal function in vivo.
Assuntos
Biomarcadores/metabolismo , Biomarcadores/urina , Ciclodextrinas/química , Testes de Função Renal/métodos , Animais , Biomarcadores/química , Proteínas Sanguíneas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Ciclodextrinas/farmacocinética , Ciclodextrinas/urina , Estabilidade de Medicamentos , Esterases/metabolismo , Fluoresceína/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Humanos , Testes de Função Renal/instrumentação , Óptica e Fotônica/métodos , Ratos Sprague-Dawley , SuínosRESUMO
Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.
Assuntos
Moléculas de Adesão Celular/metabolismo , Túbulos Renais Proximais/metabolismo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Animais , Biomarcadores/metabolismo , Desdiferenciação Celular , Modelos Animais de Doenças , Túbulos Renais Proximais/patologia , Masculino , Especificidade de Órgãos , Ratos , Regulação para CimaRESUMO
Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,ß) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.
Assuntos
Hiperglicemia/patologia , Rim/patologia , Metabolismo dos Lipídeos , Receptores Nucleares Órfãos/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Fibrose , Humanos , Hiperglicemia/complicações , Hiperglicemia/genética , Hiperglicemia/fisiopatologia , Hiperlipidemias/complicações , Hiperlipidemias/genética , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Rim/ultraestrutura , Testes de Função Renal , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Células Mesangiais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Podócitos/ultraestruturaRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. METHODS: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. RESULTS: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. CONCLUSIONS: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.
Assuntos
Benzofenonas/farmacologia , Inibidores de Catecol O-Metiltransferase , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Nitrofenóis/farmacologia , Doenças Renais Policísticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Doenças Renais Policísticas/patologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TolcaponaRESUMO
Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking-derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking-derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor-free urothelium from patients with nonmuscle-invasive urothelial bladder cancer (NMIBC) or muscle-invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor-free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor-free urothelium from patients with NMIBC. Growth factors and oncogenes were up-regulated in tumor-free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up-regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor-free tissue.
Assuntos
Regulação da Expressão Gênica , Fumar/efeitos adversos , Fumar/genética , Neoplasias da Bexiga Urinária/patologia , Bexiga Urinária/patologia , Urotélio/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Progressão da Doença , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais , Fumar/metabolismo , Fumar/patologia , Abandono do Hábito de Fumar , Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/metabolismoRESUMO
Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.
Assuntos
Anormalidades Múltiplas/genética , Morte Fetal/genética , Doenças Renais Císticas/genética , Cinesinas/genética , Situs Inversus/genética , Adolescente , Animais , Criança , Feminino , Humanos , Recém-Nascido , Rim/anormalidades , Cinesinas/metabolismo , Fígado/anormalidades , Masculino , Camundongos , Camundongos Mutantes , Mutação , Pâncreas/anormalidades , Linhagem , Síndrome , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Xenopus laevisRESUMO
The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.
Assuntos
Proteínas Nucleares/genética , Doenças Renais Policísticas/genética , Substituição de Aminoácidos/genética , Animais , Arginina/genética , Expressão Gênica/fisiologia , Predisposição Genética para Doença , Masculino , Proteínas Mutantes/genética , Doenças Renais Policísticas/patologia , Polimorfismo de Nucleotídeo Único/fisiologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Triptofano/genética , Regulação para Cima/fisiologiaRESUMO
Telomeres of most somatic cells progressively shorten, compromising the regenerative capacity of human tissues during aging and chronic diseases and after acute injury. Whether telomere shortening reduces renal regeneration after acute injury is unknown. Here, renal ischemia-reperfusion injury led to greater impairment of renal function and increased acute and chronic histopathologic damage in fourth-generation telomerase-deficient mice compared with both wild-type and first-generation telomerase-deficient mice. Critically short telomeres, increased expression of the cell-cycle inhibitor p21, and more apoptotic renal cells accompanied the pronounced damage in fourth-generation telomerase-deficient mice. These mice also demonstrated significantly reduced proliferative capacity in tubular, glomerular, and interstitial cells. These data suggest that critical telomere shortening in the kidney leads to increased senescence and apoptosis, thereby limiting regenerative capacity in response to injury.
Assuntos
Injúria Renal Aguda/fisiopatologia , Rim/fisiologia , Regeneração/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Telômero/ultraestrutura , Injúria Renal Aguda/metabolismo , Animais , Apoptose , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Rim/patologia , Rim/cirurgia , Masculino , Camundongos , Camundongos Knockout , Nefrectomia , RNA/genética , RNA/metabolismo , Traumatismo por Reperfusão/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development. METHODS: In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters. RESULTS: Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Specifically, gene expression analysis demonstrated that at day 0 the RAS system is involved. This is altered at day 6, when Wnt signaling and focal adhesion pathways are affected. However, at and after 24 days, proliferation, apoptosis, altered ECM signaling and many other factors become involved. CONCLUSIONS: Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation.
Assuntos
Rim/patologia , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/genética , Animais , Divisão Celular , Progressão da Doença , Células Epiteliais/patologia , Adesões Focais/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fenótipo , Rim Policístico Autossômico Dominante/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Sistema Renina-Angiotensina/fisiologia , Canais de Cátion TRPP/fisiologia , Proteínas Wnt/fisiologiaRESUMO
Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.
RESUMO
A long-standing hypothesis in radiotherapy is that intensity-modulated radiotherapy (IMRT) increases the risk of second cancer due to low-dose exposure of large volumes of normal tissue. Therefore, young patients are still treated with conventional techniques rather than with modern IMRT. We challenged this hypothesis in first-of-its-kind experiments using an animal model. Cancer-prone Tp53+/C273X knockout rats received mediastinal irradiation with 3 × 5 or 3 × 8 Gy using volumetric-modulated arc therapy (VMAT, an advanced IMRT) or conventional anterior-posterior/posterior-anterior (AP/PA) beams using non-irradiated rats as controls (n = 15/group, ntotal = 90). Tumors were assigned to volumes receiving 90-107%, 50-90%, 5-50%, and <5% of the target dose and characterized by histology and loss-of-heterozygosity (LOH). Irradiated rats predominantly developed lymphomas and sarcomas in areas receiving 50-107% (n = 26) rather than 5-50% (n = 7) of the target dose. Latency was significantly shortened only after 3 × 8 Gy vs. controls (p < 0.0001). The frequency (14/28 vs. 19/29; p = 0.29) and latency (218 vs. 189 days; p = 0.17) of radiation-associated tumors were similar after VMAT vs. AP/PA. LOH was strongly associated with sarcoma but not with treatment. The results do not support the hypothesis that IMRT increases the risk of second cancer. Thus the current practice of withholding dose-sparing IMRT from young patients may need to be re-evaluated.
Assuntos
Modelos Animais de Doenças , Mediastino/efeitos da radiação , Neoplasias Induzidas por Radiação/epidemiologia , Radioterapia de Intensidade Modulada/métodos , Animais , Feminino , Masculino , Órgãos em Risco , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/métodos , Ratos , Ratos Transgênicos , Fatores de Risco , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Looking for a candidate substance inducing hepatobiliary dysfunction under parenteral nutrition (PN) in newborns, we recently discovered that newborn infusions extract large amounts of the plasticizer diethylhexylphthalate (DEHP) from commonly used polyvinylchloride (PVC) infusion lines. This plasticizer is well known to be genotoxic and teratogenic in animals and to cause changes in various organs and enzyme systems even in humans. The aim of this study was to examine the effect of DEHP, extracted in the same way and in the same amount as in newborns, on livers of young rabbits. METHODS: Prepubertal rabbits received lipid emulsion through central IV lines continuously for 3 weeks either via PVC or polyethylene (PE) infusion systems. Livers were examined after 1 and 3 weeks by light and electron microscopy. RESULTS: By light microscopy, hydropic degeneration, single-cell necrosis, fibrosis, and bile duct proliferation were observed more in the PVC group. Electron microscopy revealed multiple nuclear changes, clusters and atypical forms of peroxisomes, proliferation of smooth endoplasmic reticulum, increased deposition of lipofuscin, and a mild perisinusoidal fibrosis only in the PVC group. These changes, which are generally regarded as reaction upon a toxic stimulus, could be exclusively attributed to DEHP. CONCLUSIONS: This investigation proved that DEHP produces toxin-like changes in livers of young rabbits in the same dose, duration, and method of administration as in newborn infants. For this reason, it is likely that DEHP is the substance that causes hepatobiliary dysfunction in newborns under PN. Possible modes of action of DEHP are proposed.
Assuntos
Dietilexilftalato/toxicidade , Fígado/efeitos dos fármacos , Nutrição Parenteral Total/instrumentação , Nutrição Parenteral Total/métodos , Plastificantes/toxicidade , Animais , Animais Recém-Nascidos , Emulsões Gordurosas Intravenosas , Bombas de Infusão , Fígado/patologia , Fígado/ultraestrutura , Microscopia Eletrônica , Polivinil , Coelhos , Distribuição AleatóriaRESUMO
Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Animais , Modelos Animais de Doenças , Fibrose , Rim/metabolismo , Rim/patologia , Masculino , Rim Policístico Autossômico Dominante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
There is an evident and growing medical need for an accurate determination of kidney function for a broad spectrum of indications. The glomerular filtration rate (GFR) is the most accepted indicator of renal function. Due to difficulties in performing the test, GFR is currently determined rarely in clinical practice. A procedure for such GFR determination has to be safe, accurate and easy to handle. By using the new compound fluorescein isothiocyanate-sinistrin (FS) these requirements are met. The pharmacological profile and tolerability of FS, selected from among various newly synthesized, labelled compounds intended for use as GFR markers, was characterized in male Sprague-Dawley rats following i.v. application. Using the newly described fluorometric method, FS can be determined much more easily in serum and urine than with the established enzymatic method. After i.v. dosing, FS concentrations in serum declined rapidly in various experimental groups to a comparable extent (t (1/2), mean+/-SD: 22.4+/-8.3 to 26.2+/-5.4 min). Its increase after unilateral nephrectomy reflects the loss of filtration capacity. Comparable concentration-time curves of FS in serum measured fluorometrically and enzymatically suggest no relevant alteration of pharmacokinetic behaviour by the labelling. This notion is supported by the high urinary excretion rate and absence of biliary excretion. The higher sensitivity of the fluorometric method suggests a dose of FS of 100 mg in humans compared with 5 g of sinistrin or inulin. FS was well tolerated after single and multiple applications. On the basis of these results, the kinetics of FS are comparable with the gold standard inulin or sinistrin, but FS is superior in handling. Providing the data can be transferred from rat to human, determination of GFR using the new method should result in an improvement of acceptance by both physicians and patients.
Assuntos
Fluoresceína/química , Taxa de Filtração Glomerular/efeitos dos fármacos , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Animais , Biomarcadores , Masculino , Estrutura Molecular , Oligossacarídeos/metabolismo , Oligossacarídeos/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Glomerular hypertension is a major determinant advancing progression to end-stage renal failure. Podocytes, which are thought to counteract pressure-mediated capillary expansion, are increasingly challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowman's capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell-matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up-regulated by stretch (approximately threefold). By reverse transcriptase-polymer chain reaction, up-regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate-salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV-coated membranes accelerated stretch-induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch-adapting molecule in podocytes.