Date Published: March 13, 2019
Publisher: Public Library of Science
Author(s): Emir Taghikhani, Renke Maas, Martin F. Fromm, Jörg König, Rudolf E. Stauber.
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.
L-homoarginine is an endogenous, non-proteinogenic amino acid, which may increase nitric oxide availability and enhance endothelial function . In recent years, low urinary and circulation L-homoarginine concentrations emerged as a risk marker for renal and cardiovascular diseases and mortality [2,3]. L-homoarginine is endogenously formed from lysine and occurs in various body fluids and organs . As demonstrated by Genome Wide Association studies (GWAS studies), one single-nucleotid polymorphism (SNP) in the gene encoding for the L-arginine:glycine amidinotransferase (AGAT) is associated with circulating L-homoarginine concentrations [5,6], indicating that AGAT is one major enzyme for endogenous L-homoarginine synthesis. The kidney is one important site of AGAT expression  and L-homoarginine plasma concentration is closely linked to glomerular filtration rate. Lower L-homoarginine concentrations were found in patients with impaired kidney function , suggesting that less L-homoarginine is synthesized in proximal tubule cells and exported into plasma in patients with reduced kidney function thereby indicating an important role of the kidney in L-homoarginine metabolism and homeostasis .
Toyohara and coworkers  showed in a landmark study that the transport protein OATP4C1 could be involved in the renal secretion of selected uremic toxins. It was therefore the aim of our study to investigate the role of OATP4C1 for the transport of the uremic toxin ADMA and the structurally related substances L-homoarginine and L-arginine. We established stably-transfected HEK cells recombinantly overexpressing the human OATP4C1 protein 100% identical to the protein encoded by the reference sequence (NM_180991.4). Since overexpression of human OATP4C1 in transgenic rats decreased plasma concentrations of the uremic toxin ADMA , we first tested OATP4C1-mediated uptake of the uremic toxin ADMA, of L-arginine and of the cardioprotective biomarker L-homoarginine. All three substances could be identified as substrates of human OATP4C1 (Figs 2 and 4). Furthermore, ADMA and the closely related uremic toxin SDMA inhibited OATP4C1-mediated L-homoarginine uptake in a dose-dependent manner (Fig 5) with IC50 values of 116 μM and 54 μM, respectively, whereas other known uremic toxins had no effect on OATP4C1-mediated L-homoarginine uptake (Fig 6). L-homoarginine can also be exported by OATP4C1 out of cells as demonstrated by preloading experiments (Figs 7 and 8).