Search

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A cisplatina é um quimioterápico largamente utilizado no tratamento de tumores sólidos e tem na nefrotoxicidade sua maior limitação de uso. Apesar dos esforços, os mecanismos de toxicidade ainda não estão completamente elucidados. Neste trabalho, a modulação da expressão gênica foi avaliada em rins de camundongos tratados com cisplatina a partir de dados publicamente disponíveis no Gene Expression Omnibus (GEO) sob os números de acesso GSE69652 e GSE106993. Resumidamente, os camundongos foram tratados com cisplatina ou com salina (controle). Após o tratamento os animais foram eutanasiados e tiveram os rins removidos, o RNA foi então extraído e sequenciado. A análise de expressão diferencial de transcritos identificou aumento na resposta celular ao dano de DNA, especialmente relacionada à apoptose por vias intrínsecas e diminuição da expressão de genes relacionados ao metabolismo basal celular, como metabolismo de aminoácidos e de ácidos graxos. A análise ao nível de transcritos revelou mudança nos padrões de splicing alternativo, onde genes codificadores expressaram transcritos em suas isoformas codificadoras e não codificadoras de proteínas. A expressão diferencial revelou, ainda, alteração em dezenas de RNAs longos não codificadores (lncRNA). A identificação destes lncRNAs adicionou novas informações aos mecanismos de regulação da morte celular por apoptose e da inflamação decorrentes da nefrotoxicidade induzida por cisplatina. Em conjunto, a expressão dos lncRNAs lincRNA-p21 e Gm26917 e de isoformas não codificadoras do gene codificador Mdm2 revelaram forte regulação negativa de Mdm2, apesar da superexpressão desse gene, culminando na manutenção da apoptose. Pela primeira vez a transcrição readthrough, que acontece quando a transcrição continua após o sítio de terminação, está sendo relatada na nefrotoxicidade induzida por cisplatina. Os genes que sofreram transcrição readthrough estão envolvidos no processamento do mRNA. Evidências de que os RNAs de genes que sofrem transcrição readthrough não são traduzidos mostram que esse tipo de transcrição pode estar relacionado à modulação do splicing devido ao tratamento com cisplatina. Cisplatin is a chemotherapy drug widely used in the treatment of solid tumors and has in nephrotoxicity its greatest limitation. Despite efforts, the mechanisms of toxicity are not yet completely elucidated. In this work, the modulation of gene expression was evaluated in the kidneys of mice treated with cisplatin using publicly available data in the Gene Expression Omnibus (GEO) under access numbers GSE69652 and GSE106993. Briefly, the mice were treated with cisplatin or saline (control). At the end of the treatment, the animals were euthanized and the kidneys were removed, then the RNA was extracted and sequenced. Differential transcript expression analysis identified an increase in the cellular response to DNA damage, especially related to apoptosis by intrinsic pathways and a decrease in the expression of genes related to cellular basal metabolisms, such as amino acid and fatty acid metabolism. The analysis at the level of transcripts revealed a change in alternative splicing patterns, where coding genes expressed transcripts in their coding and non-coding protein isoforms. The differential expression also revealed changes in dozens of long non-coding RNAs (lncRNA). The identification of these lncRNAs added new information to the mechanisms of regulation of cell death by apoptosis and inflammation resulting from cisplatin-induced nephrotoxicity. Together, the expression of the lincRNA-p21 and Gm26917 lncRNAs and non-coding isoforms of the Mdm2 protein-coding gene, revealed strong negative regulation of Mdm2, despite the overexpression of this gene, culminating in the maintenance of apoptosis. For the first time, readthrough transcription, which occurs when transcription continues after the termination site, is being reported in cisplatin-induced nephrotoxicity. The genes that have undergone readthrough transcription are involved in the processing of mRNA. Evidence that the RNAs of genes undergoing readthrough transcription are not translated shows that this type of transcription may be related to the modulation of splicing due to treatment with cisplatin. more...

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs. more...

Leptospirosis is caused by pathogenic bacteria of the genus Leptospira spp. This neglected re-emergent disease has global distribution and relevance in veterinary production. Here, we report the whole-genome sequence and annotation of Leptospira interrogans serovar Hardjo subtype Hardjoprajitno strain Norma, isolated from cattle in a livestock leptospirosis outbreak in Brazil. more...

Os ensaios toxicológicos são obrigatórios durante o desenvolvimento de novos fármacos. No entanto, a falta de preditividade dos modelos in vivo tem aumentado a taxa de falha dos candidatos a fármacos, elevando o tempo e os custos para o desenvolvimento de um novo produto. Os métodos alternativos in vitro podem levar ao desenvolvimento de medicamentos mais seguros e com redução de custos, podendo ser utilizados nos ensaios pré-clínicos de novas moléculas. A falta de sensibilidade dos marcadores atuais para nefrotoxicidade faz com que estes efeitos sejam descobertos tardiamente. Assim, é fundamental a busca por melhores biomarcadores precoces. O sistema renal recebe grande fluxo sanguíneo devido a suas funções e, por isso, é importante alvo de toxicidade de muitos medicamentos. Considerando que os danos moleculares e celulares precedem as alterações histopatológicas, o estudo da expressão gênica diferencial induzida por medicamentos torna-se importante. Uma vez que a expressão de um conjunto de genes é consistentemente alterada após a exposição a um xenobiótico, a expressão destes genes pode ser utilizada como biomarcadores. O uso de medicamentos reconhecidamente nefrotóxicos em estudos de biomarcadores é uma maneira de identificar tais genes. A cisplatina é um quimioterápico amplamente utilizado na prática médica que tem na nefrotoxicidade sua maior limitação. As principais células renais atingidas pela nefrotoxicidade induzida por cisplatina são as do túbulo proximal. No presente estudo, células LLC-PK1 foram expostas a cisplatina por 48 horas e as concentrações de 1 µM, 6 µM e 15 µM foram escolhidas após o ensaio de MTT. A expressão diferencial de genes relacionados a nefrotoxicidade foi analisada através de PCR em tempo real nas células expostas às concentrações citadas. A diminuição da viabilidade celular observada nas concentrações de 1 µM e 6 µM não foi acompanhada de alteração na expressão dos genes estudados e necessitam de mais estudos para a compreensão de como a cisplatina afetou o crescimento celular. Os genes HAVCR1 (KIM-1), BAX, CASP9, CASP3 e ICAM-1 tiveram a expressão aumentada após exposição a 15 µM de cisplatina quando comparada a células não expostas. Nossos resultados sugerem que esses genes podem ser utilizados como biomarcadores in vitro de nefrotoxicidade, necessitando de mais estudos para validação dos mesmos. Toxicological tests are required to develop new drugs. However, the lack of predictability of in vivo models has increased the failure rate of drug candidates, increasing time and cost for developing a new medicine. In vitro alternative methods may lead to the development of safer drugs, reduce cost, and can be used in preclinical trials of new molecules. The lack of sensitivity of current markers for nephrotoxicity makes its effects are discovered later. Therefore, it is crucial to search for better early biomarkers. Renal system receives large blood flow due to its functions, and therefore it is important target to many drugs. Whereas the molecular and cellular damage precede the histopathological changes, the study of gene expression induced by drugs has becoming important. Once the expression of a set of genes is consistently changed after the exposure to xenobiotics, these genes can be used as biomarkers. Hence, nephrotoxic drugs have been used such as biomarker studies to identify these genes. Cisplatin is a chemotherapeutic agent widely used in medical practice that has in nephrotoxicity its biggest limitation. The major cells affected by cisplatin-induced nephrotoxicity are the proximal tubule. In this study, LLC-PK1 cells were exposed to cisplatin for 48 hours and the concentrations at 1 µM, 6 µM and 15 µM were selected after the MTT assay. The expression of nephrotoxicity related genes was analyzed by real-time PCR in cells exposed to the aforementioned concentrations. Decrease in cell viability observed at concentrations of 1 µM and 6 µM and was not accompanied by changes in gene expression studies and require further research for understanding such as cisplatin affect cell growth. The HAVCR1 (KIM-1), BAX, CASP9, CASP3 and ICAM-1 genes have increased expression after exposure to 15 µM of cisplatin when compared to unexposed cells. The present studys results suggest that these genes may be used as biomarkers in vitro nephrotoxicity, requiring further studies for validation. more...

Acute kidney injury (AKI) is a global public health concern, impacting nearly 13.3 million patients and resulting in three million deaths per year. Chronic kidney disease has increased by 135% since 1990, representing the pathology with the fastest growth rate worldwide. The annual costs of dialysis and kidney transplants range between US$35,000 and US$100,000 per patient. Despite its great impact, kidney disease has remained mostly asymptomatic for many years. AKI continues to be a major, unmet medical condition for which there are no pharmacological treatments available, while animal models are limited to provide direction for therapeutic translation into humans. Currently, serum creatinine is the standard biomarker to identify nephrotoxicity; however, it is a late stage biomarker. Hence, there is a pressing need to study in vitro biomarkers for the assessment of nephrotoxicity in order to develop new and safer drugs. Understanding of the mechanisms by which molecules produce nephrotoxicity is vital in order to both prevent adversity and treat kidney injury. In this review, we address new technologies and models that may be used to identify earlier biomarkers and pathways involved in nephrotoxicity, such as cell culture, omics, bioinformatics platform, CRISPR/Cas9 genome-editing, in silico, organoids and 3D bioprinting, considering AOP. more...