title: Fluorescence-Based Transport Assays Revisited in a Human Renal Proximal Tubule Cell Line.
abstract: Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of tools we describe are also compatible with innovative kidney culture models and allows studying transport mechanisms that are central to drug absorption, disposition, and detoxification.
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.|Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences , 3508 TB Utrecht, The Netherlands.
authors.forename: Pedro
authors.initials: P
authors.lastname: Caetano-Pinto
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.|Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences , 3508 TB Utrecht, The Netherlands.
authors.forename: Manoe J
authors.initials: MJ
authors.lastname: Janssen
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: Gijzen
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.
authors.forename: Laurens
authors.initials: L
authors.lastname: Verscheijden
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.
authors.forename: Martijn J G
authors.initials: MJ
authors.lastname: Wilmer
authors.affiliation: Department of Pharmacology and Toxicology, Radboud university medical center, Radboud Institute for Molecular Life Sciences , 6500 HB Nijmegen, The Netherlands.|Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences , 3508 TB Utrecht, The Netherlands.
authors.forename: Rosalinde
authors.initials: R
authors.lastname: Masereeuw
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D005453:Fluorescence; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; C073492:ABCC4 protein, human; C116614:ABCG2 protein, human
keywords: ABC membrane transporters; advanced in vitro models; fluorescence functional assays; human proximal tubule cells; receptor-mediated endocytosis