Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

Abstract

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.

Introduction

The renal proximal tubule plays a pivotal role in the excretion of a wide range of xenobiotics and endogenous metabolites. Tubular secretion can be considered a two-step process consisting of xenobiotic uptake of across the basolateral membrane into the cell followed by exit across the apical membrane. For most molecules this process is achieved through the polar distribution of different sets of transporters to either the apical or basolateral membrane. In man, for example, the organic anion transporters; OAT1 and OAT3 and the organic cation transporter OCT2 are thought to be the predominant transporters of xenobiotics at the basolateral membrane of the proximal tubule cell (Ahsan and Burckhardt, 2007, Wright and Dantzler, 2004). In contrast, the apical exit step for many xenobiotics is more complicated, members of the ATP-binding cassette (ABC) family of transporters including MDR1 (P-glycoprotein, ABCB1), MRP2 (ABCC2), MRP4 (ABCC4) or BCRP (ABCG2) (Schinkel and Jonker, 2003, Smeets et al., 2004, Huls et al., 2008) along with organic cation transporters including OCTN1, OCTN2 and MATE-1 and organic anion transporters such as OAT4 or URAT1 have all been implicated in the apical exit step for a range of xenobiotics (Koepsell et al., 2003, Otsuka et al., 2005, Enomoto et al., 2002, Ekaratanawong et al., 2004).

From a molecular approach, we have gained a good understanding of the substrate profiles of the component transporters expressed in the proximal tubule, but we have little knowledge of either the contribution of individual apical and basolateral transporters, or how transporters integrate to produce an efficient secretory mechanism that can clear a wide range of xenobiotic molecules. To date, evidence for the role of transporters in the proximal tubule has been limited to studies of basolateral transporters in human cortical renal slices, or isolated human tubule fragments (De Kanter et al., 2002, Nozaki et al., 2007). Only recently have studies begun to investigate the properties and functions of either primary (Lash et al., 2006) or established human tubule cells (e.g. HK2 or Caki cells) grown as polarized monolayers on permeable filter supports (Ashman et al., 2006, Glube et al., 2007).

In the past, our laboratory developed a model of primary human tubular kidney cell cultures. The tubular cells can either be cultured as purified (cortical) proximal (PTC) and distal tubule/collecting duct cells (DTC) or as mixed cultures of these cells. These primary cell cultures in many aspects, perfectly mimic the in vivo human nephron, both at the physiological level; with expression of tubular segment specific cell surface markers, appropriate nephron-segment specific response to PTH and vasopressin and proximal tubule endocytotic capacity, and at the pathophysiological level; with cellular interleukin and osteopontin production and crystal retention capacity (Helbert et al., 1997, Helbert et al., 1999, Helbert et al., 2001, Van der Biest et al., 1994, Verhulst et al., 2002a, Verhulst et al., 2002b, Verhulst et al., 2004).

The aim of the present study was to validate human tubule cells grown on permeable filter supports as a robust, polarized primary cell culture model of the human proximal tubule with which to study the mechanisms and regulation of xenobiotic transporters in an integrated way. To achieve this, tubular cells were isolated and grown on permeable filter supports for up to 12 days. The expression of a range of key transport proteins was assessed at both the mRNA and protein expression level. The utility of the model to study transporters was confirmed by the demonstration of the net transepithelial fluxes of PAH and creatinine and identification of the transporters involved in the secretion of these prototypic proximal tubule substrates. Finally we demonstrated the functional expression of MDR1 (P-glycoprotein, ABCB1), MRP2 (ABCC2) and BCRP (ABCG2) at the apical membrane of polarized monolayers of tubule cells using either Hoechst 33342 or calcein high throughput dye efflux assays.