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High-throughput microfluidic platform for culture of 3D-kidney tissue models
S138
Abstracts / Toxicology Letters 280S (2017) S137–S140
P-04-04-03 Direct activated factor X inhibitor attenuates renal fibrosis on unilateral ureteral obstruction-induced nephrotoxicity Yuya Horinouchi 1 , Yasumasa Ikeda 1 , Masaki Imanishi 2 , Yoshito Zamami 1 , Yuki Izawa-Ishizawa 1 , Keisuke Ishizawa 1 , Tsuchiya Koichiro 1 , Tamaki Toshiaki 1 1
Institute of Biomedical Sciences, The University of Tokushima Graduate School, Tokushima, Japan 2 Tokushima University Hospital, Tokushima, Japan Introduction and aims: Recent studies have suggested that activated factor X (FXa) or its receptor, protease-activated receptor (PAR), plays an important role in the pathophysiology of inflammatory diseases. Renal fibrosis plays an important role in the progression of nephrotoxicity. However, the involvement of FXa in renal fibrosis has remained unclear. In this study, we investigated whether the expression levels of FX and PAR increase in the kidney of unilateral ureteral obstruction (UUO) mice and whether a direct FXa inhibitor, edoxaban (EDO), attenuates renal fibrosis on UUO-induced nephrotoxicity in mice. Methods: The C57BL/6J mice were divided into 3 groups: UUO with vehicle, UUO with EDO, and sham operation with vehicle. The mice were sacrificed and examined at one week after surgery. Results: The expression levels of FX and receptors for FXa, PAR-1 and PAR-2, increased in the kidney of UUO mice compared with sham-operated mice. EDO treatment inhibited UUO-induced upregulation of the expression of the TGF-, collagen I, III and fibronectin. Moreover, UUO-induced upregulation of inflammatory cytokines were also abrogated by EDO treatment. In histological analysis, UUO-induced tubulointerstitial fibrosis and macrophage infiltration were suppressed in EDO-treated mice. Conclusions: Direct FXa inhibitor, EDO, attenuates renal fibrosis by inhibition of inflammatory responses on UUO-induced nephrotoxicity. These results suggest that EDO may be particularly beneficial for the inhibition of nephrotoxicity, in addition to its antithrombotic activity and, FXa is a potential pharmacological target in nephrotoxicity. http://dx.doi.org/10.1016/j.toxlet.2017.07.385 P-04-04-04 Development of a high content assay in ciPTEC-OAT1 as an in vitro model to predict drug-induced nephrotoxicity Anna-Karin Sjogren 1 , Katarina Breitholtz 1 , Malin Forsgard 1 , Lucas Milton 1 , Simone Stahl 2 , Katherine Fenner 2 , Martijn Wilmer 3 , Mikael Persson 1 , Jorrit Hornberg 1 1
Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca R&D, Gothenburg, Sweden 2 Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca R&D, Cambridge, United Kingdom 3 Department of Pharmacology and Toxicology, Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands There are currently no standard in vitro screens available to predict drug-induced kidney injury. We hypothesized one reason is the lack of integration of assays detecting early and/or subtle cellular changes upon a toxic insult in a cell system with adequate physiological relevance, including functional transporters. The aim of this
study was to develop an in vitro screening assay that will identify the risk of nephrotoxicity during drug discovery. As cell model, we used conditionally immortalized proximal tubular epithelial cells that were transfected with the drug transporter OAT1 (ciPTEC-OAT1). We developed a high content imaging assay using 6 fluorescent dyes to score parameters associated with nuclei, mitochondrial function, actin cytoskeleton, cell membrane permeability and lysosome function. Perturbations to these parameters were assessed upon treatment with 38 nephrotoxic and 24 non-nephrotoxic drugs (no environmental contaminants, industrial solvents etc were included). First, we confirmed that ciPTEC-OAT1 was more sensitive to the nephrotoxic compound cidofovir, a known substrate for OAT1, as compared to non-transfected ciPTEC. Next, we found that parameters related to nuclear structure and mitochondrial health were more sensitive than nuclei counts. By applying a therapeutic index-based cutoff of 250-fold, nuclei intensity variance alone classified 60% of the nephrotoxic compounds as positive, and 80% of non-nephrotoxic compounds as negative. Next, computational approaches will be applied to identify the most predictive combinations of high content imaging parameters (out of several hundreds), together with HO1, NGAL, IL6 and IL8 gene expression. http://dx.doi.org/10.1016/j.toxlet.2017.07.386 P-04-04-05 High-throughput microfluidic platform for culture of 3D-kidney tissue models Marianne Vormann, Henriëtte Lanz, Remko van Vught, Linda Gijzen, Sebastiaan Trietsch, Jos Joore, Paul Vulto Mimetas, Leiden, Netherlands Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) ® model in Mimetas’ OrganoPlates to predict kidney toxicity. The ® OrganoPlate is a microfluidic platform, which enables highthroughput culture of boundary tissues in miniaturized organ models. The goal of developing a perfused RPTC model is to reconstruct viable and leak-tight boundaries for performing cytotoxicity, as well as transport and efficacy studies. Human RPTC (SA7K clone, Sigma) were grown against an ECM ® in a 3channel OrganoPlate , yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. Staining showed tight junction formations (ZO-1), cilia pointing into the lumen (acetylated tubulin) and correct polarization with microvilli on the apical side of the tubule (ezrin). Tightness of the boundary over several days was shown by diffusion of a dextran dye added to the lumen of the tubule. Addition of toxic compounds resulted in disruption of the barrier which could be monitored in time. The time point of loss of integrity corresponds with the concentration and the toxic effect of the compound. Furthermore, fluorescent transport assays showed functional transport activity of in- and efflux transporters. ® The 3D proximal tubules cultured in the OrganoPlate are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment. http://dx.doi.org/10.1016/j.toxlet.2017.07.387
Abstracts / Toxicology Letters 280S (2017) S137–S140
P-04-04-03 Direct activated factor X inhibitor attenuates renal fibrosis on unilateral ureteral obstruction-induced nephrotoxicity Yuya Horinouchi 1 , Yasumasa Ikeda 1 , Masaki Imanishi 2 , Yoshito Zamami 1 , Yuki Izawa-Ishizawa 1 , Keisuke Ishizawa 1 , Tsuchiya Koichiro 1 , Tamaki Toshiaki 1 1
Institute of Biomedical Sciences, The University of Tokushima Graduate School, Tokushima, Japan 2 Tokushima University Hospital, Tokushima, Japan Introduction and aims: Recent studies have suggested that activated factor X (FXa) or its receptor, protease-activated receptor (PAR), plays an important role in the pathophysiology of inflammatory diseases. Renal fibrosis plays an important role in the progression of nephrotoxicity. However, the involvement of FXa in renal fibrosis has remained unclear. In this study, we investigated whether the expression levels of FX and PAR increase in the kidney of unilateral ureteral obstruction (UUO) mice and whether a direct FXa inhibitor, edoxaban (EDO), attenuates renal fibrosis on UUO-induced nephrotoxicity in mice. Methods: The C57BL/6J mice were divided into 3 groups: UUO with vehicle, UUO with EDO, and sham operation with vehicle. The mice were sacrificed and examined at one week after surgery. Results: The expression levels of FX and receptors for FXa, PAR-1 and PAR-2, increased in the kidney of UUO mice compared with sham-operated mice. EDO treatment inhibited UUO-induced upregulation of the expression of the TGF-, collagen I, III and fibronectin. Moreover, UUO-induced upregulation of inflammatory cytokines were also abrogated by EDO treatment. In histological analysis, UUO-induced tubulointerstitial fibrosis and macrophage infiltration were suppressed in EDO-treated mice. Conclusions: Direct FXa inhibitor, EDO, attenuates renal fibrosis by inhibition of inflammatory responses on UUO-induced nephrotoxicity. These results suggest that EDO may be particularly beneficial for the inhibition of nephrotoxicity, in addition to its antithrombotic activity and, FXa is a potential pharmacological target in nephrotoxicity. http://dx.doi.org/10.1016/j.toxlet.2017.07.385 P-04-04-04 Development of a high content assay in ciPTEC-OAT1 as an in vitro model to predict drug-induced nephrotoxicity Anna-Karin Sjogren 1 , Katarina Breitholtz 1 , Malin Forsgard 1 , Lucas Milton 1 , Simone Stahl 2 , Katherine Fenner 2 , Martijn Wilmer 3 , Mikael Persson 1 , Jorrit Hornberg 1 1
Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca R&D, Gothenburg, Sweden 2 Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca R&D, Cambridge, United Kingdom 3 Department of Pharmacology and Toxicology, Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands There are currently no standard in vitro screens available to predict drug-induced kidney injury. We hypothesized one reason is the lack of integration of assays detecting early and/or subtle cellular changes upon a toxic insult in a cell system with adequate physiological relevance, including functional transporters. The aim of this
study was to develop an in vitro screening assay that will identify the risk of nephrotoxicity during drug discovery. As cell model, we used conditionally immortalized proximal tubular epithelial cells that were transfected with the drug transporter OAT1 (ciPTEC-OAT1). We developed a high content imaging assay using 6 fluorescent dyes to score parameters associated with nuclei, mitochondrial function, actin cytoskeleton, cell membrane permeability and lysosome function. Perturbations to these parameters were assessed upon treatment with 38 nephrotoxic and 24 non-nephrotoxic drugs (no environmental contaminants, industrial solvents etc were included). First, we confirmed that ciPTEC-OAT1 was more sensitive to the nephrotoxic compound cidofovir, a known substrate for OAT1, as compared to non-transfected ciPTEC. Next, we found that parameters related to nuclear structure and mitochondrial health were more sensitive than nuclei counts. By applying a therapeutic index-based cutoff of 250-fold, nuclei intensity variance alone classified 60% of the nephrotoxic compounds as positive, and 80% of non-nephrotoxic compounds as negative. Next, computational approaches will be applied to identify the most predictive combinations of high content imaging parameters (out of several hundreds), together with HO1, NGAL, IL6 and IL8 gene expression. http://dx.doi.org/10.1016/j.toxlet.2017.07.386 P-04-04-05 High-throughput microfluidic platform for culture of 3D-kidney tissue models Marianne Vormann, Henriëtte Lanz, Remko van Vught, Linda Gijzen, Sebastiaan Trietsch, Jos Joore, Paul Vulto Mimetas, Leiden, Netherlands Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) ® model in Mimetas’ OrganoPlates to predict kidney toxicity. The ® OrganoPlate is a microfluidic platform, which enables highthroughput culture of boundary tissues in miniaturized organ models. The goal of developing a perfused RPTC model is to reconstruct viable and leak-tight boundaries for performing cytotoxicity, as well as transport and efficacy studies. Human RPTC (SA7K clone, Sigma) were grown against an ECM ® in a 3channel OrganoPlate , yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. Staining showed tight junction formations (ZO-1), cilia pointing into the lumen (acetylated tubulin) and correct polarization with microvilli on the apical side of the tubule (ezrin). Tightness of the boundary over several days was shown by diffusion of a dextran dye added to the lumen of the tubule. Addition of toxic compounds resulted in disruption of the barrier which could be monitored in time. The time point of loss of integrity corresponds with the concentration and the toxic effect of the compound. Furthermore, fluorescent transport assays showed functional transport activity of in- and efflux transporters. ® The 3D proximal tubules cultured in the OrganoPlate are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment. http://dx.doi.org/10.1016/j.toxlet.2017.07.387