Mononuclear phagocyte sub-types in vitro display diverse transcriptional responses to dust mite exposure.

Mononuclear phagocytes (MNP), including macrophages and dendritic cells form an essential component of primary responses to environmental hazards and toxic exposures. This is particularly important in disease conditions such as asthma and allergic airway disease, where many different cell types are present. In this study, we differentiated CD34+ haematopoietic stem cells towards different populations of MNP in an effort to understand how different cell subtypes present in inflammatory disease microenvironments respond to the common allergen house dust mite (HDM). Using single cell mRNA sequencing, we demonstrate that macrophage subtypes MCSPP1+ and MLCMARCO+ display different patterns of gene expression after HDM challenge, noted especially for the chemokines CXCL5, CXCL8, CCL5 and CCL15. MLCCD206Hi alternatively activated macrophages displayed the greatest changes in expression, while neutrophil and monocyte populations did not respond. Further work investigated how pollutant diesel exhaust particles could modify these transcriptional responses and revealed that CXC but not CC type chemokines were further upregulated. Through the use of diesel particles with adsorbed material removed, we suggest that soluble pollutants on these particles are the active constituents responsible for the modifying effects on HDM. This study highlights that environmental exposures may influence tissue responses dependent on which MNP cell type is present, and that these should be considerations when modelling such events in vitro. Understanding the nuanced responsiveness of different immune cell types to allergen and pollutant exposure also contributes to a better understanding of how these exposures influence the development and exacerbation of human disease.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Induced pluripotent and CD34+ stem cell derived myeloid cells display differential responses to particle and dust mite exposure.

Myeloid cells form an essential component of initial responses to environmental hazards and toxic exposures. The ability to model these responses in vitro is central to efforts tasked with identifying hazardous materials and understanding mechanisms of injury and disease. Induced pluripotent stem cell (iPSC) derived cells have been suggested as alternatives to more established primary cell testing systems for these purposes. iPSC derived macrophage and dendritic like cells were compared to CD34+ haematopoietic stem cell derived populations using transcriptomic analysis. Using single cell sequencing-based characterisation of iPSC derived myeloid cells, we identified transitional, mature and M2 like macrophages as well as dendritic like antigen presenting cells and fibrocytes. Direct transcriptomic comparisons between iPSC and CD34+ cell derived populations revealed higher expression of myeloid differentiation genes such as MNDA, CSF1R and CSF2RB in CD34+ cells, while iPSC populations had higher fibroblastic and proliferative markers. Exposure of differentiated macrophage populations to nanoparticle alone or in combination with dust mite, resulted in differential gene expression on combination only, with responses markedly absent in iPSC compared to CD34+ derived cells. The lack of responsiveness in iPSC derived cells may be attributable to lower levels of dust mite component receptors CD14, TLR4, CLEC7A and CD36. In summary, iPSC derived myeloid cells display typical characteristics of immune cells but may lack a fully mature phenotype to adequately respond to environmental exposures.

Direct transcriptomic comparison of xenobiotic metabolism and toxicity pathway induction of airway epithelium models at an air-liquid interface generated from induced pluripotent stem cells and primary bronchial epithelial cells.

The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development.

Biokinetics and Internal Dosimetry of Tritiated Steel Particles.

Decommissioning fission and fusion facilities can result in the production of airborne particles containing tritium that could inadvertently be inhaled by workers directly involved in the operations, and potentially others, resulting in internal exposures to tritium. Of particular interest in this context, given the potentially large masses of material involved, is tritiated steel. The International Commission on Radiological Protection (ICRP) has recommended committed effective dose coefficients for inhalation of some tritiated materials, but not specifically for tritiated steel. The lack of a dose coefficient for tritiated steel is a concern given the potential importance of the material. To address this knowledge gap, a "dissolution" study, in vivo biokinetic study in a rodent model (1 MBq intratracheal instillation, 3-month follow-up) and associated state-of-the-art modelling were undertaken to derive dose coefficients for model tritiated steel particles. A committed effective dose coefficient for the inhalation of 3.3 × 10-12 Sv Bq-1 was evaluated for the particles, reflecting an activity median aerodynamic diameter (AMAD) of 13.3 µm, with the value for a reference AMAD for workers (5 µm) of 5.6 × 10-12 Sv Bq-1 that may be applied to occupational inhalation exposure to tritiated steel particles.

CD34+ derived macrophage and dendritic cells display differential responses to paraquat.

Paraquat (PQ) is a redox cycling herbicide known for its acute toxicity in humans. Airway parenchymal cells have been identified as primary sites for PQ accumulation, tissue inflammation and cellular injury. However, the role of immune cells in PQ induced tissue injury is largely unknown. To explore this further, primary cultures of human CD34+ stem cell derived macrophages (MCcd34) and dendritic cells (DCcd34) were established and characterised using RNA-Seq profiling. The impact of PQ on DCcd34 and MCcd34 cytotoxicity revealed increased effect within DCcd34 cultures. PQ toxicity mechanisms were examined using sub-cytotoxic concentrations and TempO-seq transcriptomic assays. Comparable increases for several stress response pathway (NFE2L2, NF-kB and HSF) dependent genes were observed across both cell types. Interestingly, PQ induced unfolded protein response (UPR), p53, Irf and DC maturation genes in DCcd34 but not in MCcd34. Further exploration of the immune modifying potential of PQ was performed using the common allergen house dust mite (HD). Co-treatment of PQ and HD resulted in enhanced inflammatory responses within MCcd34 but not DCcd34. These results demonstrate immune cell type differential responses to PQ, that may underlie aspects of acute toxicity and susceptibility to inflammatory disease.

Diesel exhaust particle and dust mite induced airway inflammation is modified by cerium dioxide nanoparticles.

Cerium dioxide nanoparticles (CeO2NPs) have been used as diesel fuel-borne catalysts for improved efficiency and pollutant emissions. Concerns that such material may influence diesel exhaust particle (DEP) effects within the lung upon inhalation, prompted us to examine particle responses in mice in the presence and absence of the common allergen house dust mite (HDM). Repeated intranasal instillation of combined HDM and DEP increased airway mucin, eosinophils, lymphocytes, IL-5, IL-13, IL-17A and plasma IgE, which were further increased with CeO2NPs co-exposure. A single co-exposure of CeO2NPs and DEP after repeated HDM exposure increased macrophage and IL-17A levels above DEP induced levels. CeO2NPs exposure in the absence of HDM also resulted in increased levels of plasma IgE and airway mucin staining, changes not observed with repeated DEP exposure alone. These observations indicate that CeO2NPs can modify exhaust particulate and allergen induced inflammatory events in the lung with the potential to influence conditions such as allergic airway disease.

Pulmonary toxicity of inhaled nano-sized cerium oxide aerosols in Sprague-Dawley rats.

Cerium oxide nanoparticles (CeO2NPs), used in some diesel fuel additives to improve fuel combustion efficiency and exhaust filter operation, have been detected in ambient air and concerns have been raised about their potential human health impact. The majority of CeO2NP inhalation studies undertaken to date have used aerosol particles of larger sizes than the evidence suggests are emitted from vehicles using such fuel additives. Hence, the objective of this study was to investigate the effects of inhaled CeO2NP aerosols of a more environmentally relevant size, utilizing a combination of methods, including untargeted multi-omics to enable the broadest possible survey of molecular responses and synchrotron X-ray spectroscopy to investigate cerium speciation. Male Sprague-Dawley rats were exposed by nose-only inhalation to aerosolized CeO2NPs (mass concentration 1.8 mg/m3, aerosol count median diameter 40 nm) for 3 h/d for 4 d/week, for 1 or 2 weeks and sacrificed at 3 and 7 d post-exposure. Markers of inflammation changed significantly in a dose- and time-dependent manner, which, combined with results from lung histopathology and gene expression analyses suggest an inflammatory response greater than that seen in studies using micron-sized ceria aerosols. Lipidomics of lung tissue revealed changes to minor lipid species, implying specific rather than general cellular effects. Cerium speciation analysis indicated a change in Ce3+/Ce4+ ratio within lung tissue. Collectively, these results in conjunction with earlier studies emphasize the importance of aerosol particle size on toxicity determination. Furthermore, the limited effect resolution within 7 d suggested the possibility of longer-term effects.

Cerium dioxide nanoparticles exacerbate house dust mite induced type II airway inflammation.

BACKGROUND: Nanomaterial inhalation represents a potential hazard for respiratory conditions such as asthma. Cerium dioxide nanoparticles (CeO2NPs) have the ability to modify disease outcome but have not been investigated for their effect on models of asthma and inflammatory lung disease. The aim of this study was to examine the impact of CeO2NPs in a house dust mite (HDM) induced murine model of asthma. RESULTS: Repeated intranasal instillation of CeO2NPs in the presence of HDM caused the induction of a type II inflammatory response, characterised by increased bronchoalveolar lavage eosinophils, mast cells, total plasma IgE and goblet cell metaplasia. This was accompanied by increases in IL-4, CCL11 and MCPT1 gene expression together with increases in the mucin and inflammatory regulators CLCA1 and SLC26A4. CLCA1 and SLC26A4 were also induced by CeO2NPs + HDM co-exposure in air liquid interface cultures of human primary bronchial epithelial cells. HDM induced airway hyperresponsiveness and airway remodelling in mice were not altered with CeO2NPs co-exposure. Repeated HMD instillations followed by a single exposure to CeO2NPs failed to produce changes in type II inflammatory endpoints but did result in alterations in the neutrophil marker CD177. Treatment of mice with CeO2NPs in the absence of HDM did not have any significant effects. RNA-SEQ was used to explore early effects 24 h after single treatment exposures. Changes in SAA3 expression paralleled increased neutrophil BAL levels, while no changes in eosinophil or lymphocyte levels were observed. HDM resulted in a strong induction of type I interferon and IRF3 dependent gene expression, which was inhibited with CeO2NPs co-exposure. Changes in the expression of genes including CCL20, CXCL10, NLRC5, IRF7 and CLEC10A suggest regulation of dendritic cells, macrophage functionality and IRF3 modulation as key early events in how CeO2NPs may guide pulmonary responses to HDM towards type II inflammation. CONCLUSIONS: CeO2NPs were observed to modulate the murine pulmonary response to house dust mite allergen exposure towards a type II inflammatory environment. As this type of response is present within asthmatic endotypes this finding may have implications for how occupational or incidental exposure to CeO2NPs should be considered for those susceptible to disease.

The small airway epithelium as a target for the adverse pulmonary effects of silver nanoparticle inhalation.

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine 'inhalation-like' nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag+) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects.

Multi-Method Characterization of the Human Circulating Microbiome.

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonization of various body niches (e.g., the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other "classically sterile" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a "blood microbiome" by providing a comprehensive description of bacterially derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilized a set of plasma samples from healthy subjects (n = 5) and asthmatic subjects (n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs. RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA/RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

Mechanistic insight into the impact of nanomaterials on asthma and allergic airway disease.

Asthma is a chronic respiratory disease known for its high susceptibility to environmental exposure. Inadvertent inhalation of engineered or incidental nanomaterials is a concern for human health, particularly for those with underlying disease susceptibility. In this review we provide a comprehensive analysis of those studies focussed on safety assessment of different nanomaterials and their unique characteristics on asthma and allergic airway disease. These include in vivo and in vitro approaches as well as human and population studies. The weight of evidence presented supports a modifying role for nanomaterial exposure on established asthma as well as the development of the condition. Due to the variability in modelling approaches, nanomaterial characterisation and endpoints used for assessment in these studies, there is insufficient information for how one may assign relative hazard potential to individual nanoscale properties. New developments including the adoption of standardised models and focussed in vitro and in silico approaches have the potential to more reliably identify properties of concern through comparative analysis across robust and select testing systems. Importantly, key to refinement and choice of the most appropriate testing systems is a more complete understanding of how these materials may influence disease at the cellular and molecular level. Detailed mechanistic insight also brings with it opportunities to build important population and exposure susceptibilities into models. Ultimately, such approaches have the potential to more clearly extrapolate relevant toxicological information, which can be used to improve nanomaterial safety assessment for human disease susceptibility.

Diesel exhaust particulate associated chemicals attenuate expression of CXCL10 in human primary bronchial epithelial cells.

Air pollution affects a large proportion of the population particularly in urban areas, with diesel particulates recognised as particular causes for concern in respiratory conditions such as asthma. In this study we examined the response of human primary airway epithelial cells to diesel particulate chemical extracts (DE) and characterised gene expression alterations using RNA-SEQ. Using the antagonist CH223191, DE induced CYP1A1 and attenuation of CXCL10 among other genes were observed to be aryl hydrocarbon receptor dependent. Basal and toll like receptor dependent protein levels for CXCL10 were markedly reduced. Investigation of similar regulation in plasmacytoid dendritic GEN2.2 cells did not show DE dependent regulation of CXCL10. Instillation of DE into mice to recapitulate airway epithelial exposure to chemical extracts in an in vivo setting failed to demonstrate a reduction in CXCL10. There was however an increase in the Th2 type epithelial cell derived inflammatory mediators TSLP and SERPINB2. We also observed an increased macrophages and a decrease in the proportion of lymphocytes in bronchoalveolar lavage fluid. CXCL10 can play a role in allergic airway disease through recruitment of Th1 type CD4+ T-cells, which can act to counterbalance Th2 type allergic responses. Modulation of such chemokines within the airway epithelium may represent a mechanism through which pollutant material can modify respiratory conditions such as allergic asthma.

Bronchial epithelial innate and adaptive immunity signals are induced by polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons including Benzo[a]pyrene have been recognised as important pollutant chemicals with the potential to influence the respiratory system in disease. Airway epithelial cells are an integral component of how immune responses are directed as a consequence of exposure to inhaled material. It was aim of this study to examine how such cells respond to PAH exposure and to characterise the immune response. Human primary bronchial epithelial cells (HPBECs) were exposed to Benzo[a]pyrene, Benzo[e]pyrene, Fluoranthene and Benzo[b]fluoranthene for 24 h and a repeat exposure up to 7 days, and examined for global gene expression using RNA-Seq. In addition to increased expression of CYP1A1 and other AHR dependent changes, we identified significant increases in innate and adaptive immune signals including, IL-1A, IL-19, SERPINB2, STAT6, HLA-DMB and HLA-DRA. We also observed increased expression of HMOX1 and NQO1, genes involved in the response to oxidative stress. Immune system related gene expression was differentially induced by each compound with Benzo[a]pyrene and Benzo[b]fluoranthene demonstrating the most potent responses. Differential induction paralleled the level to which AHR dependent gene expression and oxidative stress markers were induced. We also observed similar levels of gene expression when cells were exposed to organic extracts from diesel exhaust particles. In conclusion, hazard characterisation of responses to PAH exposure in HPBECs highlights specific responses of both innate and adaptive immunity.

Ligation of TLR7 on CD19(+) CD1d(hi) B cells suppresses allergic lung inflammation via regulatory T cells.

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B-cell population is defined as IL-10-producing CD19(+) CD1d(hi) cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19(+) CD1d(hi) B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19(+) CD1d(hi) B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL-10 reporter, Il10(-/-) and Tlr7(-/-) mice, we formally demonstrate that TLR7 ligation of CD19(+) CD1d(hi) B cells increases their capacity to produce IL-10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7-elicited CD19(+) CD1d(hi) B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3(+) T regulatory cells in allergen-sensitized mice, we show that that TLR7-elicited CD19(+) CD1d(hi) B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL-10-producing CD19(+) CD1d(hi) B cells, which can suppress allergic lung inflammation via T regulatory cells.

Transcriptomics hit the target: Monitoring of ligand-activated and stress response pathways for chemical testing.

High content omic methods provide a deep insight into cellular events occurring upon chemical exposure of a cell population or tissue. However, this improvement in analytic precision is not yet matched by a thorough understanding of molecular mechanisms that would allow an optimal interpretation of these biological changes. For transcriptomics (TCX), one type of molecular effects that can be assessed already is the modulation of the transcriptional activity of a transcription factor (TF). As more ChIP-seq datasets reporting genes specifically bound by a TF become publicly available for mining, the generation of target gene lists of TFs of toxicological relevance becomes possible, based on actual protein-DNA interaction and modulation of gene expression. In this study, we generated target gene signatures for Nrf2, ATF4, XBP1, p53, HIF1a, AhR and PPAR gamma and tracked TF modulation in a large collection of in vitro TCX datasets from renal and hepatic cell models exposed to clinical nephro- and hepato-toxins. The result is a global monitoring of TF modulation with great promise as a mechanistically based tool for chemical hazard identification.

Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 µM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity.

Application of RPTEC/TERT1 cells for investigation of repeat dose nephrotoxicity: A transcriptomic study.

The kidney is a major target organ for toxicity. Incidence of chronic kidney disease (CKD) is increasing at an alarming rate due to factors such as increasing population age and increased prevalence of heart disease and diabetes. There is a major effort ongoing to develop superior predictive models of renal injury and early renal biomarkers that can predict onset of CKD. In the EU FP7 funded project, Predict-IV, we investigated the human renal proximal tubule cells line, RPTEC/TERT1 for their applicability to long term nephrotoxic mechanistic studies. To this end, we used a tiered strategy to optimise dosing regimes for 9 nephrotoxins. Our final testing protocol utilised differentiated RPTEC/TERT1 cells cultured on filter inserts treated with compounds at both the apical and basolateral side, at concentrations not exceeding IC10, for 14 days in a 24 h repeat application. Transepithelial electrical resistance and supernatant lactate were measured over the duration of the experiments and genome wide transcriptomic profiles were assayed at day 1, 3 and 14. The effect of hypoxia was investigated for a subset of compounds. The transcriptomic data were analysed to investigate compound-specific effects, global responses and mechanistically informative signatures. In addition, several potential clinically useful renal injury biomarkers were identified.

Development of an in vitro renal epithelial disease state model for xenobiotic toxicity testing.

There is a growing impetus to develop more accurate, predictive and relevant in vitro models of renal xenobiotic exposure. As part of the EU-FP7, Predict-IV project, a major aim was to develop models that recapitulate not only normal tissue physiology but also aspects of disease conditions that exist as predisposing risk factors for xenobiotic toxicity. Hypoxia, as a common micro-environmental alteration associated with pathophysiology in renal disease, was investigated for its effect on the toxicity profile of a panel of 14 nephrotoxins, using the human proximal tubular epithelial RPTECT/TERT1 cell line. Changes in ATP, glutathione and resazurin reduction, after 14 days of daily repeat exposure, revealed a number of compounds, including adefovir dipivoxil with enhanced toxicity in hypoxia. We observed intracellular accumulation of adefovir in hypoxia and suggest decreases in the efflux transport proteins MRP4, MRP5, NHERF1 and NHERF3 as a possible explanation. MRP5 and NHERF3 were also down-regulated upon treatment with the HIF-1 activator, dimethyloxalylglycine. Interestingly, adefovir dependent gene expression shifted from alterations in cell cycle gene expression to an inflammatory response in hypoxia. The ability to investigate aspects of disease states and their influence on renal toxin handling is a key advantage of in vitro systems developed here. They also allow for detailed investigations into mechanisms of compound toxicity of potential importance for compromised tissue exposure.

Interleukin-19 as a translational indicator of renal injury.

Accurate detection and prediction of renal injury are central not only to improving renal disease management but also for the development of new strategies to assess drug safety in pre-clinical and clinical testing. In this study, we utilised the well-characterised and differentiated human renal proximal tubule cell line, RPTEC/TERT1 in an attempt to identify markers of renal injury, independent of the mechanism of toxicity. We chose zoledronate as a representative nephrotoxic agent to examine global transcriptomic alterations using a daily repeat bolus protocol over 14 days, reflective of sub-acute or chronic injury. We identified alterations in targets of the cholesterol and mevalonate biosynthetic pathways reflective of zoledronate specific effects. We also identified interleukin-19 (IL-19) among other inflammatory signals such as SERPINA3 and DEFB4 utilising microarray analysis. Release of IL-19 protein was highly induced by an additional four nephrotoxic agents, at magnitudes greater than the characterised marker of renal injury, lipocalin-2. We also demonstrate a large increase in levels of IL-19 in urine of patients with chronic kidney disease, which significantly correlated with estimated glomerular filtration rate levels. We suggest IL-19 as a potential new translational marker of renal injury.