Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

Sebastian Jarzina; Stefano Di Fiore; Bernhard Ellinger; Pia Reiser; Sabrina Frank; Markus Glaser; Jiaqing Wu; Femke J Taverne; Nynke I Kramer; Angela Mally

doi:10.3389/ftox.2022.864441

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

Front Toxicol. 2022 Apr 19:4:864441. doi: 10.3389/ftox.2022.864441. eCollection 2022.

Authors

Sebastian Jarzina¹, Stefano Di Fiore², Bernhard Ellinger³, Pia Reiser¹, Sabrina Frank¹, Markus Glaser¹, Jiaqing Wu^{4

5}, Femke J Taverne^{4

6}, Nynke I Kramer^{4

5}, Angela Mally¹

Affiliations

¹ Department of Toxicology, University of Würzburg, Würzburg, Germany.
² Fraunhofer Institute for Molecular Biology and Applied Ecology, Division Molecular Biotechnology Aachen, Aachen, Germany.
³ Fraunhofer Institute for Molecular Biology and Applied Ecology, Division Translational Medicine, ScreeningPort, Hamburg, Germany.
⁴ Institute for Risk Assessment Sciences, Utrecht University, Utrecht, Netherlands.
⁵ Toxicology Division, Wageningen University, Wageningen, Netherlands.
⁶ Host-microbe Interactions, Wageningen University, Wageningen, Netherlands.

Abstract

Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.

Keywords: In vitro toxicity testing; QIVIVE; adverse outcome pathway (AOP); key event relationship; nephrotoxicity; risk assessment.