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. 2024 Jun 9;13(12):1008.
doi: 10.3390/cells13121008.

Characterization of Organic Anion and Cation Transport in Three Human Renal Proximal Tubular Epithelial Models

Tamara Meijer  1   2 , Daniel da Costa Pereira  1   2 , Olivia C Klatt  1   2 , Joanne Buitenhuis  1   2 , Paul Jennings  1   2 , Anja Wilmes  1   2
Affiliations

Characterization of Organic Anion and Cation Transport in Three Human Renal Proximal Tubular Epithelial Models

Tamara Meijer et al. Cells. .

Abstract

The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.

Keywords: human induced pluripotent stem cells; organic anion transport; organic cation transport; renal proximal tubule.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The morphology and tight barrier assessment of primary PTEC, RPTEC/TERT1, and PTL cells cultured on transwells. (a) The phase contrast images of matured primary PTEC #1, RPTEC/TERT1, and PTL SBAD3 cultured on transwells. The scale bar represents 50 µm. (b) Transepithelial electrical resistance (TEER) measurements of primary PTEC #1, primary PTEC #2, RPTEC/TERT1, PTL SBAD3, and PTL SBAD2 cultured on transwells. (c) The barrier integrity assessment by the lucifer yellow rejection of primary PTEC #1, primary PTEC #2, RPTEC/TERT1, PTL SBAD3, and PTL SBAD2 cultured on transwells. Cells were incubated for 1 h with 60 µM lucifer yellow added apically. Both the TEER values and the percentage rejection of lucifer yellow are shown as the mean ± standard deviation (SD) of n independent experiments (n = 7 for RPTEC/TERT1; n = 3 for PTL SBAD3 and SBAD2; n = 2 for primary PTEC #1 and #2).
Figure 2
Figure 2
The expression of proteins involved in organic cation transport. (a) The immunofluorescent images of primary PTEC #1, RPTEC/TERT1, PTL SBAD3, PTL SBAD2, and iPSC SBAD2 stained with the nuclear stain Hoechst 33342 (blue) and antibodies against ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) (green), organic cation transporter 2 (OCT2) (red), and organic cation/carnitine transporter 2 (OCTN2) (red). Images were obtained using 63× water confocal imaging. Auto contrast was set per antibody staining to allow for a comparison between the different cell models. The scale bar represents 50 µm. (b) The Western blot of OCT2 in primary PTEC #1, RPTEC/TERT1, PTL SBAD3, and iPSC SBAD2. Actin was used as an internal control.
Figure 3
Figure 3
The uptake and transfer of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) in primary PTEC, RPTEC/TERT1, and PTL cells cultured on transwells. Cells were pre-incubated for 1 h in the absence or presence of the inhibitor quinidine applied basolaterally (BL), followed by a 1 h incubation with the substrate ASP applied BL or apically (AP) in the absence or presence of quinidine applied BL. (a) The time-dependent accumulation of ASP in cells in the absence or presence of quinidine shown as ASP fluorescence intensity per cell. Data are shown as the mean ± SD of 2 independent experiments. A two-tailed unpaired Student’s t-test was performed between the area under the curves (AUCs) of the three different conditions, which were all significantly different from one another in all cell models (p-value < 0.05). (b-i) The accumulation of ASP in cells in the absence or presence of quinidine, all applied BL, after 1 h incubation. Data are shown as the mean ± SD of n independent experiments (n = 4 for RPTEC/TERT1; n = 3 for PTL SBAD3 and SBAD2; n = 2 for primary PTEC #1 and #2), depicted as % control (no inhibitor added). Statistical significance was calculated using a two-tailed unpaired Student’s t-test. * represents a p-value < 0.05 (no inhibitor versus inhibitor added). (b-ii) The representative images of ASP accumulation in cells after 1 h incubation with or without quinidine. To visualise the best effect of the inhibitor, auto contrast was set per cell type to compare ASP accumulation with and without an inhibitor. The scale bar represents 100 µm. (c) The transport of ASP from basolateral to apical supernatant in the absence or presence of quinidine applied BL after 1 h incubation. Data are shown as the mean ± SD of n independent experiments (n = 4 for RPTEC/TERT1; n = 3 for PTL SBAD3 and SBAD2; n = 2 for primary PTEC #1 and #2), depicted as % control (no inhibitor added). Statistical significance was determined as described under (b-i).
Figure 4
Figure 4
The expression and function of organic anion transporter 1 (OAT1) and 3 (OAT3). (a) The mRNA expression of OAT1 and OAT3 in ciPTEC-OAT1, ciPTEC-OAT3, primary PTEC #2, RPTEC/TERT1, PTL SBAD2, and iPSC SBAD2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. Representative images from the gel electrophoresis of polymerase chain reaction (PCR) products are shown. (b) The cellular uptake of the fluorescent substrate 6-carboxyfluorescein (6-CF). ciPTEC-OAT1, ciPTEC, primary PTEC #2, RPTEC/TERT1, and PTL SBAD2 were cultured on microplates. Cells were pre-incubated for 1 h in the absence or presence of the inhibitor probenecid, followed by a 1 h incubation with the substrate 6-CF alongside cyclosporine A (CsA) to block efflux and in the absence or presence of probenecid. The uptake of 6-CF was determined using the BioTek Cytation 1 Cell Imaging System with a 4× imaging objective. Representative images are shown. The scale bar represents 100 µm.
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