Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model

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.

Exacerbation of Cisplatin Cellular Toxicity by Regulation of the Human Organic Cation Transporter 2 through Angiotensin II

“…A similar activating effect of AII on transport mediated by OCT was observed in freshly isolated renal mouse proximal tubules [ 51 ]. Using other experimental setups, for example, human embryonic kidney cells stably overexpressing hOCT2, such regulation by AII was not observed ( Supplementary Figure S1 ), probably due to the fact that these cells do not express AT1R endogenously or because regulation pathways can be different in strongly polarized cells, such as the MDCK cells [ 26 ]. In this study, AII stimulation of hOCT2 activity increased CDDP cellular toxicity and, therefore, may be also involved in the development of CDDP-induced nephrotoxicity.…”

“…The inhibition of OCT2 by substances such as cimetidine and pantoprazole may represent a useful approach to protect cells against undesired toxicity caused by Platinum agents [ 14 , 18 , 19 , 20 , 21 , 22 , 23 ] since cancer cells seem to express no or only low levels of this transporter [ 14 ]. Interestingly, the activity of OCT2 can be rapidly regulated by several signaling pathways [ 24 , 25 , 26 ] and by pathological states [ 27 , 28 , 29 ], suggesting a possible role of transporter regulation for modulating side effects of chemotherapy with Platinum agents. The hOCT2 regulation can be dependent on cell polarization [ 26 ], that is the formation of distinct apical and basolateral membrane domains, which is typical for renal epithelial cells.…”

“…Interestingly, the activity of OCT2 can be rapidly regulated by several signaling pathways [ 24 , 25 , 26 ] and by pathological states [ 27 , 28 , 29 ], suggesting a possible role of transporter regulation for modulating side effects of chemotherapy with Platinum agents. The hOCT2 regulation can be dependent on cell polarization [ 26 ], that is the formation of distinct apical and basolateral membrane domains, which is typical for renal epithelial cells.…”

Substrate-Dependent Trans-Stimulation of Organic Cation Transporter 2 Activity

“…The chemical structures of the OCT2 substrates are indicated in Figure S2 . The fluorescent dye DiASP, used here as a reference tracer substrate for OCT2 [ 59 , 60 ], was purchased from Invitrogen (Carlsbad, CA, USA).…”

Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations 2.0

“…In epithelial tissues, organic cation transporters show a polar distribution, being mainly localized in the basolateral plasma membrane domain. Therefore, Koepp et al [ 5 ] in their work “Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model” 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). In this system, hOCT2 showed a specific localization in the basolateral membrane domain.…”