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.

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

“…Human-derived immortalized renal cell models such as human embryonic kidney 293 (HEK 293) and human kidney 2 (HK 2) cells have shown to be a valuable tool in screenings in drug development, but have low or no endogenous expression of relevant drug transporters (48,49). To overcome these limitations, we have developed ciPTEC-OAT1 with proven suitability for nephrotoxicity and drug-transporter interactions studies (10)(11)(12)(13)(14). The broad range of drug transporter expression in ciPTEC-OAT1 provides a strong basis for highly predictive renal drug interaction studies.…”

“…Functionality of P-gp and MRP2/4 was assessed based upon accumulation of substrates, as previously described for ciPTEC in 2D ( Fig. 1c) (12). Prior to measuring efflux activity of P-gp and MRP2/4 in the OrganoPlate, medium-channel inlet and outlets were washed with 50 μl KHH at 37°C.…”

“…CMFDA is then metabolized to the fluorescent 5-chloromethylfluorescein (CMF) via esterase activity and then further metabolized into carboxylfluorescein-glutathione (GS-MF) through direct interaction with glutathione (GSH) or via transferase activity. CMF is completely transferred into GS-MF during incubation time used here (12). GS-MF is predominantly a substrate for Fig.…”

“…Gene expression levels were normalized to GAPDH, used as reference gene, and here calculated a fold difference compared to gene expression levels in 2D static cell culture (6-well plate). **p < 0.01 compared to mRNA expression in ciPTEC-OAT1 cultured in a 6-well plate (12). We therefore used a combination of inhibitors, as described in the BMaterials and Methodsŝ ection.…”

“…Further transduction resulted in stable expression and functionality of OAT1 or OAT3 (10). Multiple studies have shown the potential of ciPTEC for studying drug transport and drug-induced toxicity cultured under conventional static conditions, often referred to as two-dimensional (2D) cell culture (10)(11)(12)(13). In addition, transepithelial barrier function, polarization, and transport of cations and anions have been demonstrated in ciPTEC cultured in a threedimensional (3D) model using a tailor-made hollow fiber membrane system exposed to flow (13,14).…”