RPTEC/TERT1 cells form highly differentiated tubules when cultured in a 3D matrix.

Philipp Secker

doi:10.14573/altex.1710181

Abstract

The proximal tubule is the primary site for renal solute reabsorption and secretion and thus a main target for drug-induced toxicity. Current nonclinical methods using 2D cell cultures are unable to fully recapitulate clinical drug responses mainly due to limited in vitro functional lifespan. Since extracellular matrices are known to be key regulators of cell development, culturing cells on classic 2D plastic surfaces inevitably results in loss of differentiation. Hence, 3D models of the human proximal tubule that recapitulate the in vivo morphology would allow for improved drug screening and disease modeling. Here, the development and characterization of a 3D proximal tubule model using RPTEC/TERT1 cells is presented. RPTEC/TERT1 cells self-assembled in matrigel to form highly differentiated and stable 3D tubular structures characterized by a branched network of monolayered cells encircling a cell-free lumen thus mimicking the proximal tubule. In vitro tubuli resembled the polarity of a proximal tubule epithelium as indicated by polar expression of Na+/K+- ATPase and ZO-3. Furthermore, 3D cultured RPTEC/TERT1 cells showed overall increased mRNA expression of xenobiotic transporters e.g. OCTs and MATEs and de novo expression of OAT3 when compared to cultures on plastics or membrane inserts. Finally, this model was used to assess delayed cisplatin-induced nephrotoxicity and demonstrated increased sensitivity when compared to 2D culture. Thus, the easy-to-use model described here may prove to be useful for mechanistic investigations, e.g. in discovery of compounds interfering with tubule formation, differentiation and polarization, as well for the detection and understanding of pharmaceutical induced nephrotoxicity.

Highlights

Toxicology still heavily relies on animal experiments to predict adverse effects of drugs and chemicals in a human population
3D cultured renal proximal tubule epithelial cells (RPTECs)/TERT1 cells showed overall increased mRNA expression of xenobiotic transporters, e.g., organic cation transporter (OCT) and multidrug and toxin extrusion proteins (MATE) and de novo expression of OAT3 when compared to cultures on plastics or membrane inserts
This model was used to assess delayed cisplatin-induced nephrotoxicity and demonstrated increased sensitivity when compared to 2D culture

Summary

Introduction

Toxicology still heavily relies on animal experiments to predict adverse effects of drugs and chemicals in a human population. Translatability of rodent findings towards the human situation is known to be often very poor (Hackam and Redelmeier, 2006; Leist and Hartung, 2013; Matthews, 2008; Olson et al, 2000; van der Worp et al, 2010), potentially resulting in undetected adverse effects (false negatives) or loss of valuable compounds due to species-specific side effects (false positives) (Hartung, 2009). E.g., the 3R (reduce, replace, refine) principle of animal testing (Russell and Burch, 1959), these disenchanting numbers have boosted the development of alternative, human cell-based test methods. The use of human cells overcomes the burden of translating results obtained from animal experiments to humans. Results obtained from in vitro toxicology studies were often more than disappointing. One comprehensive study by Lin and Will with hundreds of either hepatotoxic, cardiotoxic or nephrotoxic compounds and three frequently used cell lines originating from the three organs found that only 5% of compounds exhibited differential toxicity and – even worse – in case of a differential response, the most sensitive cell line did not necessarily match with the most susceptible organ (Lin and Will, 2012)

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