Development of an in vitro rat proximal tubule cell model as a platform for drug transporter and drug-drug interaction studies

Abstract

The kidney plays a vital role in the elimination of many endogenous metabolites and xenobiotics. Drug transporters expressed in the proximal tubule cells are key factors in the ability of the organ to successfully carry out its function. Previously, primary human proximal tubule cells have been shown to retain a remarkable degree of differentiation in culture and provide a realistic model of the proximal tubule. To address the challenge of extrapolation of drug transporter data from animal and human, this project was set out to develop a parallel rat proximal tubule cell model. This would allow direct comparison of the handling of candidate drugs in both species, and provide better understanding of the mechanisms of drug transport. A technique to isolate primary rat proximal tubule cells (PTCs) was successfully developed using a collagenase digest/Percoll gradient approach. Rat PTCs cultured for 6 days were shown to exhibit cobberstone morphology, typical of many epithelial cells. A range of transport proteins including Mdr1a/b, Bcrp, Mrp2, Oat1, Oct2, Oatp4c1, Slc2a9, Urat1, Mate1, and Mct1 were detected at the mRNA level in these cells. Functional expression of Mdr1a/b, Bcrp, Mrp2, Oct2 and Mct1 was also detected using fluorescence substrate retention assays. In addition, Mdr1a/b, Bcrp and Mrp2 transporters were found localised on the apical membrane of polarised rat PTC monolayer, and Oct2 was found on the basolateral membrane. The handling of urate by rat PTC monolayers was investigated. The monolayers showed absorptive and secretory pathways for urate, although the absorptive pathway was 3.2-fold higher in magnitude. Similarly, 3.4-times more urate was predominant across the apical than across the basolateral membrane. Oat1 and Bcrp were deduced as the transporters responsible for the secretory pathway, and Urat1 and Slc2a9 in the absorptive pathway. This was in accordance with the human PTC monolayers, and both models were representative of urate transport in vivo. Digoxin transport exhibited a net absorptive flux in rat PTC monolayers; absorptive flux was 1.7-fold higher in magnitude than the secretory flux. In contrast, in human PTC monolayers, digoxin secretory flux was 4.2-fold higher than the absorptive flux. In human PTC monolayers, digoxin secretion consisted of OATP4C1-mediated digoxin uptake by the basolateral membrane and MDR1-mediated efflux across the apical membrane. In rat PTC monolayers in addition to these pathways, a significant Oatp-mediated absorptive flux of digoxin located on the apical membrane of rat PTC monolayer was identified as the difference between rat and human digoxin handling, resulting in a dominant absorptive flux of digoxin in rat compared to net secretion in human PTC monolayers. These data alone highlight the importance of developing realistic in vitro human and rat PTC models to understand species difference in renal drug handling.