Abstract
The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 3.1 ± 0.2 µM). ASP+ uptake was inhibited by tetraethylammonium (TEA+), tetrapentylammonium (TPA+), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA+ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.
Highlights
Human organic cation transporter 2 is highly expressed on the basolateral membrane domain of renal proximal tubule cells [1,2,3]
Organic cations are excreted from the cells of proximal tubules into the urine by apically expressed transporters such as the Multidrug and Toxin Extrusion (MATE) proteins, which function as H+/organic cations (OCs) exchangers [3,6]
In Madin-Darby Canine Kidney (MDCK) cells transduced with Human organic cation transporter 2 (hOCT2)-GFP, bands corresponding to the hOCT2 transporter tagged with GFP were detected both using antibodies against GFP or hOCT2
Summary
Human organic cation transporter 2 (hOCT2) is highly expressed on the basolateral membrane domain (facing the interstitial space) of renal proximal tubule cells [1,2,3]. Organic cations are excreted from the cells of proximal tubules into the urine by apically expressed (facing the urine) transporters such as the Multidrug and Toxin Extrusion (MATE) proteins, which function as H+/OC exchangers [3,6]. Relevant information about hOCT2 functional properties, regulation and interaction with drugs has been obtained using expression systems such as human embryonic kidney cells (HEK293) (see, for example, [19,20]) and Xenopus oocytes (see, for example, [21,22,23]) These cellular systems do not have distinct apical and basolateral membrane domains, the formation of which is essential for proper function of the polarized renal tubule cells, where these transporters are abundantly expressed and functionally important. The functional properties of hOCT2 stably expressed in MDCK cysts and HEK293 cells were compared to study whether and how a polarized expression system could influence hOCT2 activity
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