Vectorial transport of the arginine derivatives asymmetric dimethylarginine (ADMA) and L-homoarginine by OATP4C1 and P-glycoprotein studied in double-transfected MDCK cells.

Elevated plasma concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with cardiovascular events and mortality. Key enzymes involved in the homeostasis of both arginine derivatives are expressed in proximal tubule cells of the kidney. To get access to these enzymes, transport proteins are important. One of the transporters mediating the transport of ADMA and L-homoarginine is the solute carrier superfamily (SLC) member OATP4C1, located in the basolateral membrane of proximal tubule cells. To gain insights into the role of export pumps in the transport of both substances, we established a double-transfected MDCK cell line expressing OATP4C1 and the export pump P-glycoprotein (P-gp). Using MDCK cell monolayers, we demonstrated in time-dependent and concentration-dependent vectorial transport experiments that ADMA and L-homoarginine are transported from the basolateral to the apical compartment of MDCK-OATP4C1-P-gp cells with significantly higher transport rates compared to single-transfected MDCK-OATP4C1, MDCK-P-gp and MDCK-VC (control) cells (e.g. transport ratio MDCK-OATP4C1-P-gp/MDCK-VC: for 50 µM ADMA = 2.0-fold, for 50 µM L-homoarginine = 3.4-fold). These results indicate that both OATP4C1 and P-gp transport the arginine derivatives ADMA and L-homoarginine and are, therefore, important for the homoeostasis of both substances.

The renal transport protein OATP4C1 mediates uptake of the uremic toxin asymmetric dimethylarginine (ADMA) and efflux of cardioprotective L-homoarginine.

Elevated plasma concentrations of the uremic toxin asymmetrical dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with cardiovascular events and total mortality. Enzymes degrading ADMA [dimethylaminohydrolase 1 (DDAH1)] and synthesizing L-homoarginine [L-arginine:glycine amidinotransferase (AGAT)] are expressed in human proximal tubule cells. So far, it is not known which transport protein in the basolateral membrane of proximal tubule cells is mediating the uptake of ADMA into the cells for subsequent degradation or the export of intracellularly synthesized L-homoarginine. One study suggested that the uptake transporter OATP4C1 (gene symbol SLCO4C1) may be involved in the transport of ADMA and other uremic toxins. OATP4C1 is a member of the SLCO/SLC21 family of solute carriers, localized in the basolateral membrane of human proximal tubule cells. By using stably-transfected HEK cells overexpressing human OATP4C1, we demonstrate that ADMA and L-homoarginine are substrates of OATP4C1 with Km values of 232.1 µM and 49.9 µM, respectively. ADMA and the structurally related uremic toxin SDMA (100 µM) inhibited OATP4C1-mediated L-homoarginine uptake (P < 0.01), whereas other tested uremic toxins such as urea and p-cresyl sulfate have no effect on OATP4C1-mediated transport. Preloading experiments (300 µM for 60 min) with subsequent efflux studies revealed that OATP4C1 also facilitates efflux e.g. of L-homoarginine. Both ADMA and L-homoarginine are substrates of human OATP4C1. Because proximal tubule cells are one site of ADMA metabolism and L-homoarginine synthesis, we postulate a protective role of OATP4C1 by mediating uptake of ADMA from and export of L-homoarginine into the systemic circulation.

Assays for Analyzing the Role of Transport Proteins in the Uptake and the Vectorial Transport of Substances Affecting Cell Viability.

Endogenous compounds, drugs, or other xenobiotics may affect cell viability. A prerequisite for intracellular cell damage is the uptake of such substances across the plasma membrane into cells. Furthermore, the subsequent transporter-mediated export out of cells may influence cell viability. Therefore, transport proteins mediating the uptake (uptake transporter) or export (export pumps) of substances in and out of cells are important determinants of cell viability. Uptake transporters mostly belong to the superfamily of solute carriers (SLC transporters), whereas export pumps are members of the ABC-transporter superfamily (ATP-binding cassette). Cell systems recombinantly overexpressing uptake transporters (single transfectants) or multiple-transfected cell models expressing simultaneously an uptake transporter together with an export pump (double transfectants) are important in vitro tools for analyzing protein-mediated transport of potentially cell toxic compounds.Here we describe different in vitro transport assays for the functional analysis of transport proteins. Using single-transfected HEK293 cells stably overexpressing an uptake transporter, substances can be tested as potential substrates (uptake assay) or potential transport inhibitors (inhibition assay) for the respective transport protein. Vectorial transport of substances with the uptake across the basolateral plasma membrane and the export across the apical membrane of polarized grown MDCKII cells can be analyzed using double-transfected cell models with the simultaneous overexpression of an uptake transport and an export pump in vectorial transport assays, thereby mimicking physiological transport processes, e.g., in liver or kidney.