Functional approaches to study renal and mitochondrial toxicity in vitro

Abstract

Nephrotoxicity is a common adverse effect of drugs that accounts for a substantial part of drug development discontinuations and market withdrawals. Within the kidney, the renal proximal tubule epithelial cells (RPTEC) are commonly affected by drug-induced nephrotoxicity, which is mainly due to their prominent role in secretion of xenobiotics. These transport processes not only result in high toxin exposures but also imply high energy metabolic rates, portrayed by a high mitochondrial density in these cells. Indeed, nephrotoxins are often substrates for renal secretory transporters and frequently interfere with mitochondrial functions. Unfortunately, nephrotoxicity of drug candidates is rarely detected during the preclinical development, which is attributed to the poor predictivity of animal experiments on the one hand and, on the other hand, the lack of predictive human in vitro test systems. The latter is mainly because most established RPTEC cell lines lost the characteristic expression of xenobiotic transporters rendering these cells unsuitable for sensitive nephrotoxicity detection. However, more recently presented cell lines including the RPTEC/TERT1 cells promise maintained functionality and polarization. Therefore, the work presented in this thesis describes the usability of the RPTEC/TERT1 cell line for in vitro nephrotoxicity testing. The key functionalities of the proximal tubule epithelium, reabsorption of water and secretion of cationic xenobiotics were characterized and quantified in these cells and subsequently translated into sensitive toxicity readouts. Highlighting the usefulness of the latter, impaired cellular water reabsorption was detected for several known nephrotoxins and even revealed nephrotoxins not affecting cell viability. Furthermore, the described vectorial transport of a well-established substrate of the OCT/MATE transporters for cations by RPTEC/TERT1 cells provides evidence that the cells represent a valuable tool to identify substrates of this important secretion axis. Next, a 3D cultivation protocol for RPTEC/TERT1 cells was established by embedding the cells in an extracellular matrix and subsequently characterized. Interestingly, solely culturing RPTEC/TERT1 cells between two layers of Matrigel induced the formation of stable tubular structures. These tubuli were shown to consist of a monolayer of cells that are connected by tight junctions and exhibit an in vivo-like polarization including apical microvilli and basolateral expression of Na+/K+-ATPase. Strikingly, RPTEC/TERT1 cells cultured in 3D showed a pronounced increase in expression of many xenobiotic transporters when compared to their counterparts cultured on plastics or Transwells. Finally, the RPTEC/TERT1 cell line was utilized in a mechanistic toxicology study to investigate the recent problem of gliflozin-induced renal tubular tumors (RTTs) in rodents and cases of acute kidney injury (AKI) and diabetic ketoacidosis (DKA) in human patients. It was demonstrated that canagliflozin but not dapagliflozin or empagliflozin is a dual inhibitor of glutamate dehydrogenase (GDH) and electron transport chain complex I, in combination restricting glutaminolysis, thereby damaging RPTECs. This effect might well contribute to the increased incidence of RTTs in rats exposed to canagliflozin likely related to degenerative/regenerative proliferation and, since ammonia release via GDH is an important renal countermeasure against acidosis, the results present an explanation for the predominance of canagliflozin medication among DKA and AKI cases. To conclude, the present thesis advances the field of in vitro nephrotoxicity testing by characterization of novel, functional and sensitive cellular readouts, by establishment and characterization of an innovative 3D cultivation protocol that improved cellular differentiation status and by delineation of RPTEC/TERT1 energy and mitochondrial metabolism and the adverse effects of impairments thereof.