Mapping Nephrotoxicity: Advancing Human Proximal Tubule Models for Mechanistic Kidney Safety Assessment

The renal proximal tubule is a primary target of drug-induced toxicity due to its high metabolic activity, dense mitochondrial content, and central role in xenobiotic uptake and handling. Injury to this nephron segment is a major contributor to acute kidney injury development, which is associated with substantial morbidity and an increased risk of progression into chronic kidney disease. Despite its clinical relevance, accurately predicting nephrotoxicity remains challenging, as traditional animal-based approaches often fail to capture human-specific mechanisms of injury. In recent years, New Approach Methodologies (NAMs) have emerged as promising alternatives, offering the potential for more mechanistically informed and human-relevant toxicity assessment. The overarching aim of this thesis was to enhance the mechanistic understanding and predictive capacity of in vitro approaches for drug-induced nephrotoxicity, with a specific focus on the human renal proximal tubule. Through the integration of model development, functional assessment, and transcriptomic profiling, this work contributes to the advancement of human-relevant NAM-based strategies for kidney safety assessment. Human in vitro proximal tubular models encompass primary cells, immortalised cell lines, and iPSC-derived systems, each with distinct strengths and limitations. Model suitability is contingent on the scientific question being addressed and the biological features required to answer it. Accordingly, this work employed multiple human renal in vitro models to enable comparative assessment of functional and mechanistic responses relevant to nephrotoxicity. In Chapter 2, a reproducible, step-by-step protocol for differentiating human iPSC into proximal tubular-like cells (PTL) was established. The resulting PTL cells displayed key phenotypic features of the human proximal tubule, including markers associated with endocytic capacity (megalin) and epithelial polarity (ZO-3), and were suitable for downstream functional and toxicological applications. This work provided a robust experimental foundation for the subsequent functional characterisation and mechanistic toxicity studies presented in this thesis and offers a framework that can be built upon in future nephrotoxicity in vitro research. Chapter 3 focused on the functional characterisation of renal transport processes relevant to proximal tubule drug handling and nephrotoxicity. While organic cation transport could be consistently demonstrated across primary, immortalised, and iPSC-derived proximal tubular models, organic anion transport was lacking. These results highlight a persistent gap in recapitulating this critical aspect of proximal tubule biology in vitro and emphasise the importance of future model development efforts aimed at restoring organic anion transport functionality. In Chapter 4, transcriptomic profiling was applied to investigate molecular responses to aminoglycoside antibiotics across different human proximal tubule models. Comparative analysis revealed pronounced model-dependent differences in transcriptional responses. In iPSC-derived PTL and primary cells, aminoglycoside exposure induced a pronounced and coherent interferon-stimulated gene response, whereas immortalised cell lines exhibited weaker and less coherent responses. These findings demonstrate that transcriptomic responses and their mechanistic interpretation are strongly influenced by the underlying model, underscoring the value of biologically relevant renal systems for mechanistic toxicity assessment. Chapter 5 examined the influence of treatment regimen on mechanistic insight using brequinar in a repeated-dose transcriptomics case study. At concentrations where single exposure produced only weak transcriptional responses, repeated dosing elicited a pronounced activation of cellular stress response pathways. These findings indicate that single dose exposure strategies may fail to capture cumulative or time-dependent toxicity mechanisms that become apparent under repeated dose conditions. Taken together, this thesis demonstrates that advancing human-relevant, mechanism-driven nephrotoxicity assessment within a NAM framework requires careful alignment of model biology, functional competence, and treatment regimen with the defined context of use. An important next step will be to further improve differentiation and functional maturation of the proximal tubule phenotype in vitro, particularly with respect to restoring organic anion transporter activity, for which microphysiological systems and endothelial co-culture represent promising avenues.