386 research outputs found

    Fluorescent tagging of endogenous heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages

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    Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.Toxicolog

    Engineering functional kidney tissue using human iPS cells

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    Recent advances in the field of stem cell research have enabled the derivation of renal organoids from hiPSCs; these organoids might be a powerful tool with important implications for regenerative medicine, but meticulous assessment of the functional abilities of the induced nephrons is key to the use of these organoids in any application. Here, I show that hiPSC-derived renal organoids possess proximal tubular transporters and receptors; I present optimised techniques to assess the function of these receptors in-vitro and show that these organoids have anion and cation uptake capacities similar to what can be seen in foetal kidney tissue or isolated proximal tubules, implying tubular functional capacity, an aspect of renal physiology that has particular importance in the renal handling of drugs and toxins. Due to high blood flow and the primary role of the kidney in clearing toxins and metabolites, renal cells are highly vulnerable to drug toxicity. The lack of in-vitro high throughput models to screen pharmaceutical compounds for potential nephrotoxicity during drug development has always hindered the field of drug development and increased the cost of delivering drugs into the market; in this study, I demonstrate that hiPSCs-derived renal organoids are able to predict nephrotoxicity with reasonable accuracy. Combining this ability with the possibility of cryopreserving renal-differentiated cells and to the use of HMOX1 reporter cell line, to detect oxidative stress, could streamline the use of these organoids in nephrotoxicity screening and could potentially flourish the field of drug development. While the current model of renal organoids could be used for drug screening without further manipulation, the use of such tissue for therapeutic purposes necessitates a higher degree of organisation and complexity. In-vivo kidney function is based on the complex interplay of a range of highly specialised cells together with their three-dimensional structure and organisation. Scientists are adopting different strategies to build kidney tissue, from hiPSCs, that could be suitable for use in therapeutic applications. Common to any of these strategies is the need to generate the correct cell types in sufficient numbers and purity, and most important, in the right location. I aim in this study to isolate correctly differentiated ureteric bud (UB) structures from surrounding cells and to induce branching from single UB-like structure to recapitulate branching morphogenesis in-vitro. I conjugated GDNF protein to a fluorophore and used it to label the UB structures and isolate them. I show that the combination of GDNF, FGF1, CHIR99021 and RA was able to induce branching in the isolated UBlike structures. The ability to isolate pure differentiated UB structures from surrounding contaminant tissue and to induce them to branch forming contiguous collecting duct tree could be a step further towards engineering a more realistic kidney tissue with single continuous collecting duct system, yet optimising culture conditions and techniques to build such a tissue is still needed

    The Influence of Carbon Dioxide on Cellular Cyclic Adenosine Monophosphate

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    Inorganic carbon is fundamental to the physiology of all organisms, however elevated CO2 is generally detrimental. Numerous class III adenylyl cyclases (AC) from both prokaryotic and mammalian organisms have been shown to respond to inorganic carbon in vitro, however, at present there is limited evidence in vivo. This thesis demonstrates in cellulo evidence that hypercapnia CO2 blunts agonist induced cAMP signalling. The eect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signalling pathway, and is independent of the cell line employed. Through a combination of pharmacological and genetic tools this eect of elevated CO2 on cAMP signalling is demonstrated to require Ca2+ ion release from IP3 receptors in the endoplasmic reticulum. Consistent with these ndings, CO2 caused an increase in cytoplasmic Ca2+ concentrations which require the presence of active IP3 receptors and is absent under comparable acidotic conditions. Physiological relevance for this signalling mechanism is demonstrated through activity of the sodium dependant proton exchanger NHE3. This transporter exhibits well-characterised inhibition by cAMP dependant protein kinase PKA to increase bicarbonaturia in vivo. Overall these results provide conclusive evidence of potentially profound eects of inorganic carbon on intracellular cell signalling, which could lead to signicant insight into the pathophysiology and treatment of numerous disorders including metabolic acidosis, reperfusion injuries, acute lung injury and obesity

    Cellular and Molecular Mechanisms of Nephropathic Cystinosis

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    Nephropathic cystinosis (MIM # 219800) is a rare autosomal recessive disorder caused by mutations in the lysosomal cystine transporter cystinosin, encoded by the CTNS gene (17p13.2). This devastating condition initially affects kidneys and subsequently many other organs including eyes, thyroid, pancreas, muscles, and brain. While lysosomal cystine storage is a key feature of the disease and the main target of current therapy, recent groundbreaking research has revealed that cystinosin has diverse functions in cells, being involved in vesicle trafficking, energy homeostasis, and cell death mechanisms. These discoveries deepen our insights into the mechanisms of cystinosis and of lysosomal biology in general. In this Special Issue dedicated to the pioneer of cystinosis research Dr. Jerry Schneider, we highlight the state-of-the-art understanding of cellular and molecular mechanisms of various disease features, opening new horizons for innovative treatment strategies for cystinosis and potentially other lysosomal storage diseases

    RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells

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    The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells’ Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.publishedVersio

    Redox Imbalance and Mitochondrial Abnormalities in Kidney Disease

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    The kidney performs important functions in the human body and can inflict either acute kidney injury (AKI) or chronic kidney disease (CKD). AKI can be induced by kidney ischemia, drugs such as cisplatin, and heavy metals such as cadmium and arsenic. CKD can be induced by drugs, heavy metals, hypertension, and diabetes, as well as cancer. Importantly, nearly all kidney disorders have been shown to involve redox imbalance, reductive stress, oxidative stress, and mitochondrial abnormalities such as impaired mitochondrial homeostasis, including disrupted mitophagy and deranged mitochondrial unfolded protein responses. Understanding how these redox-related dysregulated pathways operate may give us new insights into how to design novel approaches to fighting kidney disease. This Special Issue of Biomolecules entitled “Redox imbalance and mitochondrial abnormalities in kidney disease” covers a variety of topics focusing on oxidative stress, mitochondrial dysfunction, and antioxidation enhancement implicated in kidney disease or kidney transplantation

    The Effects of Mycotoxins on Human and Animal Health—a Special Focus on the Cellular and Molecular Mechanisms Responsible for Mycotoxin Toxicity

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    This Special Issue gathers original studies (eight articles and one review) that aim to improve our knowledge concerning mycotoxin toxicity. The most recent research concerning the impact of zearalenone (ZEA) and the immune response is presented in a review that aims to explore the immunotoxicity produced by ZEA on different types of immune cells (phagocytes related to innate immunity and lymphocytes related to acquired immunity) as well as on immune organs, and also to identify the mechanism of action used by ZEA to modulate the immune response. The role of zearalenone in macrophage-mediated innate immunity after bacterial lipopolysaccharide (LPS) stimulation was the subject of another study, suggesting that the intake of ZEA-contaminated food might result in decreasing innate immunity. Another important study related to the co-exposure to mycotoxins and other food/feed contaminants showed that combining patulin with cadmium induces enhanced hepatotoxicity and nephrotoxicity both in vitro and in vivo, while the exposure to both deoxynivalenol and porcine circovirus has an additive effect on inflammatory cytokines by inducing the mRNA expression of interleukine-1 beta (IL-1β) and interleukine-6 (IL-6). The use of feed compounds rich in bioactive constituents for mitigating the mycotoxins effects in swine was the subject of other three studies herein, which have shown the efficacy of antioxidants from dietary grapeseed and sea buckthorn meal waste to decrease the toxicity of a diet contaminated with aflatoxin B1 or both aflatoxin B1 and ochratoxin A at the level of organs involved in the metabolism and excretion (liver and kidney), as well as at the level of secondary lymphoid organs (mesenteric lymph nodes). The use of different models (cell co-culture model, rat model) are recommended by another two studies in order to evaluate the ochratoxin A toxicity or for better prediction of the mycotoxins risk for human cancer

    3D Stem Cell Culture

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    Recently, stem cells have been drawing increasing interest in basic and translational research that aims to understand stem cell biology and generate new therapies for various disorders. Many stem cells can be cultured in 2D relatively easily using tissue culture plastic. However, many of these cultures do not represent the natural conditions of stem cells in the body. In the body, microenvironments include numerous supporting cells and molecules. Therefore, researchers and clinicians have sought ideal stem cell preparations for basic research and clinical applications, which may be attainable through 3D culture of stem cells. The 3D cultures mimic the conditions of the natural environment of stem cells better, as cells in 3D cultures exhibit many unique and desirable characteristics that could be beneficial for therapeutic interventions. 3D stem cell cultures may employ supporting structures, such as various matrices or scaffolds, in addition to stem cells, to support complex structures. This book brings together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cell culture techniques and also to highlight stem cell preparations for possible novel therapeutic applications
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