Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

Abstract

Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 μM amiodarone, 10 μM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFβ-type I receptor kinase inhibitor GW788388 (1 μM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1, ATF3, DDIT3, and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFβ inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1. This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.

Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells

“…ATF4 is a basic leucine-zipper (bZIP) transcription factor, which binds to C/EBP-ATF response elements (CARE) elements in DNA leading to the expression of several downstream genes, including DNA Damage Inducible Transcript 3 ( DDIT3 , aka GADD153 and CHOP), TRIB3 , ATF3 , ASNS , HERPUD1 and PPP1R15A (GADD34), all of which were induced here upon exposure to ETC inhibitors. Links between metabolic state and ATF4 induction have been previously observed in other in vitro studies, including the observation that ATF4 is induced upon ETC inhibition in neuronal, liver cancer cells and iPSC induced proximal tubular-like cells (Krug et al 2014 ; Jennings et al 2022 ; van der Stel et al 2022 ). There is an established link between mitochondrial disturbances and Parkinsonian motor disease, where UPR has been implicated as a protective mechanism (Costa et al 2020 ).…”

Characterization of Organic Anion and Cation Transport in Three Human Renal Proximal Tubular Epithelial Models

“…In the present study, we have utilised a previously characterised two-week strategy to differentiate iPSC cells into proximal tubular-like cells (PTL) [ 23 , 33 ]. These cells exhibit certain characteristics of proximal tubule cells, such as the megalin-mediated uptake of albumin and functional P-gp activity [ 33 ], and have been shown to be a promising model for toxicity prediction and assessment in several transcriptomic studies [ 37 , 38 , 39 , 40 ].…”

Differentiation and Subculturing of Renal Proximal Tubular‐like Cells Derived from Human iPSC

“…The human iPSC‐derived PTL model was exposed to several model compounds, including cadmium (Singh et al., 2021), paraquat (Nunes et al., 2022), and bardoxolone‐methyl (Snijders et al., 2021), to investigate toxicity mechanisms. Furthermore, the PTL cells were employed to study temporal effects of stress response pathways by exposure to a range of compounds known to activate specific known stress response pathways, including sodium arsenite, tunicamycin, amiodarone, and rotenone (Jennings et al., 2022). In addition to transcriptomics, activation of the HMOX1 stress response gene was monitored by exposing PTL differentiated from the fluorescent HMOX1 reporter human iPSC line (Snijders et al., 2021) to compounds reported to induce oxidative stress (Jennings et al., 2022).…”

“…Furthermore, the PTL cells were employed to study temporal effects of stress response pathways by exposure to a range of compounds known to activate specific known stress response pathways, including sodium arsenite, tunicamycin, amiodarone, and rotenone (Jennings et al., 2022). In addition to transcriptomics, activation of the HMOX1 stress response gene was monitored by exposing PTL differentiated from the fluorescent HMOX1 reporter human iPSC line (Snijders et al., 2021) to compounds reported to induce oxidative stress (Jennings et al., 2022). Overall, the human iPSC‐derived PTL model shows to be promising in studying and predicting drug‐induced toxicity.…”

“…These iPSC‐derived PTL cells display a polarized phenotype, express proximal tubular‐specific proteins, such as megalin, and show functional characteristics of proximal tubule cells, including response to parathyroid hormone, uptake of albumin and ABCB1 transport activity (Chandrasekaran et al., 2021). PTL have been used in several transcriptomic studies which supported the utilization of these cells in toxicity assessment and prediction (Jennings et al., 2022; Nunes et al., 2022; Singh et al., 2021; Snijders et al., 2021). The human iPSC‐derived PTL model was exposed to several model compounds, including cadmium (Singh et al., 2021), paraquat (Nunes et al., 2022), and bardoxolone‐methyl (Snijders et al., 2021), to investigate toxicity mechanisms.…”