Qualitative and quantitative concentration-response modelling of gene co-expression networks to unlock hepatotoxic mechanisms for next generation chemical safety assessment

Next generation risk assessment of chemicals revolves around the use of mechanistic information without animal experimentation. In this regard, toxicogenomics has proven to be a useful tool to elucidate the underlying mechanisms of adverse effects of xenobiotics. In the present study, two widely used human in vitro hepatocyte culture systems, namely primary human hepatocytes (PHH) and human hepatoma HepaRG cells, were exposed to liver toxicants known to induce liver cholestasis, steatosis or necrosis. Benchmark concentration-response modelling was applied to transcriptomics gene co-expression networks (modules) to derive benchmark concentrations (BMCs) and to gain mechanistic insight into the hepatotoxic effects. BMCs derived by concentration-response modelling of gene co-expression modules recapitulated concentration-response modelling of individual genes. Although PHH and HepaRG cells showed overlap in deregulated genes and modules by the liver toxicants, PHH demonstrated a higher responsiveness, based on the lower BMCs of co-regulated gene modules. Such BMCs can be used as transcriptomics point of departure (tPOD) for assessing module-associated cellular (stress) pathways/processes. This approach identified clear tPODs of around maximum systemic concentration (Cmax) levels for the tested drugs, while for cosmetics ingredients the BMCs were 10-100-fold higher than the estimated plasma concentrations. This approach could serve next generation risk assessment practice to identify early responsive modules at low BMCs, that could be linked to key events in liver adverse outcome pathways. In turn, this can assist in delineating potential hazards of new test chemicals using in vitro systems and used in a risk assessment when BMCs are paired with chemical exposure assessment.Toxicolog

Application of high-throughput transcriptomics for mechanism-based biological read-across of short-chain carboxylic acid analogues of valproic acid

Chemical read-across is commonly evaluated without particular knowledge of the biological mechanisms leading to observed adverse outcomes in vivo. Integrating data that indicate shared modes of action in humans will strengthen read-across cases. Here we studied transcriptomic responses of primary human hepatocytes (PHH) to a large panel of carboxylic acids to include detailed mode-of-action data as a proof-of-concept for read-across in risk assessment. In rodents, some carboxylic acids, including valproic acid (VPA), are known to cause hepatic steatosis, whereas others do not. We investigated transcriptomics responses of PHHs stimulated for 24 h by 18 structurally different VPA analogues in a concentration range to determine biological similarity in relation to in vivo steatotic potential. Using a targeted high-throughput screening assay we assessed the differential expression of ~3,000 genes covering relevant biological pathways. Differentially expressed gene analysis revealed differences in potency of carboxylic acids and expression patterns were highly similar for structurally similar compounds. Strong clustering occurred for steatosis-positive versus steatosis-negative carboxylic acids. To quantitatively define biological read-across, we combined pathway analysis and weighted gene co-expression network analysis. Active carboxylic acids displayed high similarity in gene network modulation. Importantly, free fatty acid synthesis modulation and stress pathway responses are affected by active carboxylic acids, providing coherent mechanistic underpinning for our findings. Our work shows that transcriptomic analysis of cultured human hepatocytes can reinforce the prediction of liver injury outcome based on quantitative and mechanistic biological data and support the application in read-across.Toxicolog

Shear stress regulated signaling in renal epithelial cells and polycystic kidney disease

Several cellular processes and pathways were altered both by fluid shear stress and Pkd1 gene disruption in renal epithelial cells. Many of these signaling pathways are implicated in ADPKD as well. However, more than 20 years after the discovery of PKD1 and PKD2 as genetic cause of ADPKD, the exact cellular function of the polycystins still remains unclear. Our data indicate that polycystin-1 is not a direct mechano-sensor, but it restrains shear stress induced gene expression via an unknown mechanism. Additional research is required to identify the cellular function of polycystins and the mechanism of mechanotransduction. This is needed to refine the mechanism of cyst formation in ADPKD and other ciliopathies, which could identify potential targets for therapy. Nevertheless, we showed that inhibition of activin signaling is a promising therapy to slow cyst progression in Pkd1del mice. Although other treatment strategies have been tested successfully to reduce PKD progression in pre-clinical studies, the efficacy in human patients is sometimes minimal or absent. Therefore, it has been suggested to target multiple signaling pathways affected in ADPKD. These combined therapies should reestablish the balance in cellular signaling of renal epithelial cells and maintain cellular homeostasis within physiological boundaries. Nederlandse organisatie voor wetenschappelijk onderzoekLUMC / Geneeskund

Shear stress regulated signaling in renal epithelial cells and polycystic kidney disease

Several cellular processes and pathways were altered both by fluid shear stress and Pkd1 gene disruption in renal epithelial cells. Many of these signaling pathways are implicated in ADPKD as well. However, more than 20 years after the discovery of PKD1 and PKD2 as genetic cause of ADPKD, the exact cellular function of the polycystins still remains unclear. Our data indicate that polycystin-1 is not a direct mechano-sensor, but it restrains shear stress induced gene expression via an unknown mechanism. Additional research is required to identify the cellular function of polycystins and the mechanism of mechanotransduction. This is needed to refine the mechanism of cyst formation in ADPKD and other ciliopathies, which could identify potential targets for therapy. Nevertheless, we showed that inhibition of activin signaling is a promising therapy to slow cyst progression in Pkd1del mice. Although other treatment strategies have been tested successfully to reduce PKD progression in pre-clinical studies, the efficacy in human patients is sometimes minimal or absent. Therefore, it has been suggested to target multiple signaling pathways affected in ADPKD. These combined therapies should reestablish the balance in cellular signaling of renal epithelial cells and maintain cellular homeostasis within physiological boundaries. </p

Shear stress regulated signaling in renal epithelial cells and polycystic kidney disease

Several cellular processes and pathways were altered both by fluid shear stress and Pkd1 gene disruption in renal epithelial cells. Many of these signaling pathways are implicated in ADPKD as well. However, more than 20 years after the discovery of PKD1 and PKD2 as genetic cause of ADPKD, the exact cellular function of the polycystins still remains unclear. Our data indicate that polycystin-1 is not a direct mechano-sensor, but it restrains shear stress induced gene expression via an unknown mechanism. Additional research is required to identify the cellular function of polycystins and the mechanism of mechanotransduction. This is needed to refine the mechanism of cyst formation in ADPKD and other ciliopathies, which could identify potential targets for therapy. Nevertheless, we showed that inhibition of activin signaling is a promising therapy to slow cyst progression in Pkd1del mice. Although other treatment strategies have been tested successfully to reduce PKD progression in pre-clinical studies, the efficacy in human patients is sometimes minimal or absent. Therefore, it has been suggested to target multiple signaling pathways affected in ADPKD. These combined therapies should reestablish the balance in cellular signaling of renal epithelial cells and maintain cellular homeostasis within physiological boundaries. </p

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells

Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. Shear stress is altered in renal diseases caused by tubular dilation, obstruction, and hyperfiltration, which occur to compensate for lost nephrons. Fundamental in regulation of shear stress are primary cilia and other mechano-sensors, and defects in cilia formation and function have profound effects on development and physiology of kidneys and other organs. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in renal epithelial cells. Functional enrichment-analysis revealed TGF-β, MAPK, and Wnt signaling as core signaling pathways up-regulated by shear. Inhibitors of TGF-β and MAPK/ERK signaling modulate a wide range of mechanosensitive genes, identifying these pathways as master regulators of shear-induced gene expression. However, the main down-regulated pathway, that is, JAK/STAT, is independent of TGF-β and MAPK/ERK. Other up-regulated cytokine pathways include FGF, HB-EGF, PDGF, and CXC. Cellular responses to shear are modified at several levels, indicated by altered expression of genes involved in cell-matrix, cytoskeleton, and glycocalyx remodeling, as well as glycolysis and cholesterol metabolism. Cilia ablation abolished shear induced expression of a subset of genes, but genes involved in TGF-β, MAPK, and Wnt signaling were hardly affected, suggesting that other mechano-sensors play a prominent role in the shear stress response of renal epithelial cells. Modulations in signaling due to variations in fluid shear stress are relevant for renal physiology and pathology, as suggested by elevated gene expression at pathological levels of shear stress compared to physiological shear

Comparative transcriptomics of shear stress treated Pkd1(-/-) cells and pre-cystic kidneys reveals pathways involved in early polycystic kidney disease

Molecular Technology and Informatics for Personalised Medicine and HealthFunctional Genomics of Systemic Disorder

High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

Functional Genomics of Systemic Disorder

Inhibition of Activin Signaling Slows Progression of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by the formation of numerous kidney cysts, is caused byPKD1orPKD2mutations and affects 0.1% of the population. Although recent clinical studies indicate that reduction of cAMP levels slows progression of PKD, this finding has not led to an established safe and effective therapy for patients, indicating the need to find new therapeutic targets. The role of TGF-βin PKD is not clearly understood, but nuclear accumulation of phosphorylated SMAD2/3 in cyst-lining cells suggests the involvement of TGF-βsignaling in this disease. In this study, we ablated the TGF-βtype 1 receptor (also termed activin receptor-like kinase 5) in renal epithelial cells of PKD mice, which had little to no effect on the expression ofSMAD2/3target genes or the progression of PKD. Therefore, we investigated whether alternative TGF-βsuperfamily ligands account for SMAD2/3 activation in cystic epithelial cells. Activins are members of the TGF-βsuperfamily and drive SMAD2/3 phosphorylationviaactivin receptors, but activins have not been studied in the context of PKD. Mice with PKD had increased expression of activin ligands, even at early stages of disease. In addition, treatment with a soluble activin receptor IIB fusion (sActRIIB-Fc) protein, which acts as a soluble trap to sequester activin ligands, effectively inhibited cyst formation in three distinct mouse models of PKD. These data point to activin signaling as a key pathway in PKD and a promising target for therapy