Proteasome Inhibitors Bortezomib and Carfilzomib Stimulate the Transport Activity of Human Organic Anion Transporter 1. Yunzhou Fan;Guofeng You. 2020. Mol Pharmacol. 97. PMID: 32234809

Organic anion transporter 1 (OAT1), expressed at the basolateral membrane of renal proximal tubule epithelial cells, mediates the renal excretion of many clinically important drugs. Previous study in our laboratory demonstrated that ubiquitin conjugation to OAT1 leads to OAT1 internalization from the cell surface and subsequent degradation. The current study showed that the ubiquitinated OAT1 accumulated in the presence of the proteasomal inhibitors MG132 and ALLN rather than the lysosomal inhibitors leupeptin and pepstatin A, suggesting that ubiquitinated OAT1 degrades through proteasomes. Anticancer drugs bortezomib and carfilzomib target the ubiquitin-proteasome pathway. We therefore investigate the roles of bortezomib and carfilzomib in reversing the ubiquitination-induced downregulation of OAT1 expression and transport activity. We showed that bortezomib and carfilzomib extremely increased the ubiquitinated OAT1, which correlated well with an enhanced OAT1-mediated transport of p-aminohippuric acid and an enhanced OAT1 surface expression. The augmented OAT1 expression and transport activity after the treatment with bortezomib and carfilzomib resulted from a reduced rate of OAT1 degradation. Consistent with this, we found decreased 20S proteasomal activity in cells that were exposed to bortezomib and carfilzomib. In conclusion, this study identified the pathway in which ubiquitinated OAT1 degrades and unveiled a novel role of anticancer drugs bortezomib and carfilzomib in their regulation of OAT1 expression and transport activity. SIGNIFICANCE STATEMENT: Bortezomib and carfilzomib are two Food and Drug Administration-approved anticancer drugs, and proteasome is the drug target. In this study, we unveiled a new role of bortezomib and carfilzomib in enhancing OAT1 expression and transport activity by preventing the degradation of ubiquitinated OAT1 in proteasomes. This finding provides a new strategy in regulating OAT1 function that can be used to accelerate the clearance of drugs, metabolites, or toxins and reverse the decreased expression under disease conditions.
Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin. Takahito Furuya;Issey Takehara;Asuka Shimura;Hisanao Kishimoto;Tomoya Yasujima;Kinya Ohta;Yoshiyuki Shirasaka;Hiroaki Yuasa;Katsuhisa Inoue. 2017. Biochem Biophys Res Commun. 495. PMID: 29273507

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.
Organic anion transporter OAT1 undergoes constitutive and protein kinase C-regulated trafficking through a dynamin- and clathrin-dependent pathway. Qiang Zhang;Mei Hong;Peng Duan;Zui Pan;Jianjie Ma;Guofeng You. 2008. J Biol Chem. 283. PMID: 18818201

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity V(max) without significant change in the substrate affinity K(m) of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K(+) from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrin-dependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway.
Distribution of the organic anion transporters Oat1 and Oat3 between renal membrane microdomains in obstructive jaundice. Evangelina Cecilia Nosetto;Romina Valeria Campagno;Adriana Mónica Torres;Anabel Brandoni. 2020. Pflugers Arch. 472. PMID: 32451678

Relation between the renal function and the membrane environment where the organic anion transporters Oat1 and Oat3 are localized is scarce. The aim of this study was to examine the Oat1 and Oat3 distribution in different cellular fractions under physiological conditions as well as the effects of extrahepatic cholestasis on membrane distribution of both proteins. Besides, the potential role of jaundice serum on the Oat1 and Oat3 expression in suspensions of renal tubular cells was evaluated. Cellular and membrane fractions of renal cortex were obtained from control rats to evaluate Oat1 and Oat3 protein expressions. Other rats were subjected to bile duct ligation (BDL) or Sham operation to determine the membrane distribution of Oat1 and Oat3 between lipid raft domains (LRD) and non-LRD. Incubation of renal cortical cells with serum from Sham and BDL were also performed to study Oat1 and Oat3 protein expressions. In physiological conditions, Oat1 and Oat3 were concentrated in LRD. The pathology induced a shift of Oat1 from LRD to non-LRD, while Oat3 showed no changes in its distribution. In cells exposed to BDL serum, Oat1 protein expression in membranes significantly increased. For Oat3, no difference between groups was observed. The Oat1 redistribution to non-LRD in BDL could be favoring the increase in renal transport of organic anions previously observed. This change was specific to Oat1. The in vitro experiment allows to conclude that some component present in BDL serum is responsible for the alterations observed in Oat1 expression in cortical membranes.
Developmental changes in multispecific organic anion transporter 1 expression in the rat kidney. N Nakajima;T Sekine;S H Cha;A Tojo;M Hosoyamada;Y Kanai;K Yan;S Awa;H Endou. 2000. Kidney Int. 57. PMID: 10760096

BACKGROUND: The cDNA of the multispecific organic anion transporter 1 (OAT1) responsible for the tubular secretion of organic anions was recently isolated. In the current study, we investigated the developmental changes in OAT1 expression in the rat kidney. METHODS: Ontogenic expression of rat OAT1 was investigated by Northern blot, in situ hybridization, Western blot, and immunohistochemical analysis. In addition, para-aminohippurate (PAH) accumulation was measured using fetal, neonatal, and adult rat kidney slices. RESULTS: In Northern blot analysis, OAT1 was detected as early as on embryonic day 18 in the fetal kidney. The expression level of OAT1 mRNA increased remarkably just after birth (postnatal day 0). In situ hybridization revealed OAT1 expression on embryonic day 19. In both the fetal and neonatal kidneys, OAT1 mRNA was localized in a relatively deep region in the cortex. Western blot analysis detected OAT1 protein on embryonic day 20, and the expression level increased after birth. Immunohistochemical analysis did not reveal OAT1 staining in the fetal kidneys. A faint signal of OAT1 protein was detected on postnatal day 0; thereafter, the expression level increased. In the functional study using kidney slices, low but definite probenecid-sensitive PAH accumulation was noted in fetal rat kidney on embryonic day 20. After birth, probenecid-sensitive PAH uptake was increased. CONCLUSIONS: The present study consistently demonstrates the remarkable increase of OAT1 expression after birth, and the immature excretory capacity of the proximal tubules of the neonatal kidney can be attributed, at least in part, to the low expression level of OAT1.
Renal organic anion transporter 1 is maldistributed and diminishes in proximal tubule cells but increases in vasculature after ischemia and reperfusion. Osun Kwon;Wei-Wei Wang;Shane Miller. 2008. Am J Physiol Renal Physiol. 295. PMID: 18922885

Renal solute clearances are reduced in ischemic acute kidney injury. However, the mechanisms explaining how solute clearance is impaired have not been clarified. Recently, we reported that cadaveric renal allografts exhibit maldistribution of organic anion transporter 1 (OAT1) in proximal tubule cells after ischemia and reperfusion, resulting in impairment of PAH clearance. In the present study, we characterized renal OAT1 in detail after ischemia-reperfusion using a rat model. We analyzed renal OAT1 using confocal microscopy with a three-dimensional reconstruction of serial optical images, Western blot, and quantitative real-time RT-PCR. OAT1 was distributed to basolateral membranes of proximal tubule cells in controls. With ischemia, OAT1 decreased in basolateral membrane, especially in the lateral membrane domain, and appeared diffusely in cytoplasm. After reperfusion following 60-min ischemia, OAT1 often formed cytoplasmic aggregates. The staining for OAT1 started reappearing in lateral membrane domain 1 h after reperfusion. The basolateral membrane staining was relatively well discernable at 240 h of reperfusion. Of note, a distinct increase in OAT1 expression was noted in vasculature early after ischemia and after reperfusion. The total amount of OAT1 protein expression in the kidney diminished after ischemia-reperfusion in a duration-dependent manner until 72 h, when they began to recover. However, even at 240 h, the amount of OAT1 did not reach control levels. The kidney tissues tended to show a remarkable but transient increase in mRNA expression for OAT1 at 5 min of ischemia. Our findings may provide insights of renal OAT1 in its cellular localization and response during ischemic acute kidney injury and recovery from it.
Lysine 48-linked polyubiquitination of organic anion transporter-1 is essential for its protein kinase C-regulated endocytosis. Qiang Zhang;Shanshan Li;Cam Patterson;Guofeng You. 2012. Mol Pharmacol. 83. PMID: 23087261

Organic anion transporter-1 (OAT1) mediates the body's disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We had previously established that OAT1 undergoes constitutive internalization from and recycling back to the cell surface and that acute activation of protein kinase C (PKC) inhibits OAT1 activity by reducing OAT1 cell-surface expression through accelerating its internalization from cell surface to intracellular compartments. However, the underlying mechanisms are poorly understood. In the current study, we provide novel evidence that acute activation of PKC significantly enhances OAT1 ubiquitination both in vitro and ex vivo. We further show that ubiquitination of cell-surface OAT1 increases in cells transfected with dominant negative mutant of dynamin-2, a maneuver blocking OAT1 internalization, which suggests that OAT1 ubiquitination proceeds before OAT1 internalization. Mass spectroscopy has revealed that ubiquitination of OAT1 consists of polyubiquitin chains, primarily through lysine 48 linkage. Transfection of cells with the dominant negative mutant of ubiquitin Ub-K48R, which prevents the formation of Lys48-linked polyubiquitin chains, abolishes PKC-stimulated OAT1 ubiquitination and internalization. Together, our findings demonstrate for the first time that Lys48-linked polyubiquitination is essential for PKC-regulated OAT1 trafficking.
Transcriptional regulation of human organic anion transporter 1 by B-cell CLL/lymphoma 6. Waja Wegner;Gerhard Burckhardt;Maja Henjakovic. 2014. Am J Physiol Renal Physiol. 307. PMID: 25234312

The human organic anion transporter 1 (OAT1) is crucial for the excretion of organic anions in renal proximal tubular cells and has been classified as a clinically relevant transporter in the kidneys. Our previous study indicated that renal male-predominant expression of rat Oat1 and Oat3 appears to be regulated by transcription factor B-cell CLL/lymphoma 6 (BCL6). The aim of this study was to characterize the effect of BCL6 on human OAT1 promoter and on the transcription of OAT1 mediated by hepatocyte nuclear factor-1α (HNF-1α). Luciferase assays were carried out in opossum kidney (OK) cells transiently transfected with promoter constructs of OAT1, expression vectors for BCL6 and HNF-1α, and the empty control vectors. BCL6 and HNF-1α binding on OAT1 promoter was analyzed using electrophoretic mobility shift assay (EMSA). Protein expression of HNF-1α was investigated by Western blot analysis. Site-directed mutagenesis was used to introduce mutations into BCL6 and HNF-1α binding sites within the OAT1 promoter. BCL6 enhanced the promoter activity of OAT1 independently of predicted BCL6 binding sites but was dependent on HNF-1α response element and HNF-1α protein. Coexpression of BCL6 and HNF-1α induced an additive effect on OAT1 promoter activation compared with BCL6 or HNF-1α alone. BCL6 does not bind directly or indirectly to OAT1 promoter but increases the protein expression of HNF-1α and thereby indirectly enhances OAT1 gene transcription. BCL6 constitutes a promising candidate gene for the regulation of human OAT1 transcription and other renal and/or hepatic drug transporters that have been already shown to be activated by HNF-1α.
Estrogen receptor α (ERα) indirectly induces transcription of human renal organic anion transporter 1 (OAT1). Anna M Euteneuer;Tamina Seeger-Nukpezah;Hendrik Nolte;Maja Henjakovic. 2019. Physiol Rep. 7. PMID: 31724834

Organic anion transporter 1 (OAT1) is a polyspecific transport protein located in the basolateral membrane of renal proximal tubule cells. OAT1 plays a pivotal role in drug clearance. Adverse drug reactions (ADR) are observed more frequently in women than in men, especially ADR are higher in women for drugs which are known interactors of OAT1. Sex-dependent expression of Oat1 has been observed in rodents with a tendency to male-dominant expression. This study aims at elucidating the transcriptional regulation of human OAT1 and tests the effect of estrogen receptor α (ERα). Promoter activation of OAT1 was assessed by luciferase assays carried out by Opossum kidney (OK) cells, transiently transfected with promoter constructs of human OAT1 and expression vectors for ERα and exposed to 100 nmol/L 17β-estradiol. Furthermore, a transcription factor array and proteomic analysis was performed to identify estrogen-induced transcription factors. Human OAT1 was significantly activated by ligand activated ERα. However, activation occurred without a direct interaction of ERα with the OAT1 promoter. Our data rather show an activation of the transcription factors CCAAT-box-binding transcription factor (CBF) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ERα, which in turn bind and initiate OAT1 promoter activity. Herewith, we provide novel evidence of estrogen-dependent, transcriptional regulation of polyspecific drug transporters including the estrogen-induced transcription factors CBF and HNRNPK.
Hepatocyte nuclear factor-4{alpha} regulates the human organic anion transporter 1 gene in the kidney. Ken Ogasawara;Tomohiro Terada;Jun-ichi Asaka;Toshiya Katsura;Ken-ichi Inui. 2007. Am J Physiol Renal Physiol. 292. PMID: 17344191

Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4alpha markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning -1191 to -700 base pairs (bp) and -140 to -79 bp were essential for the transactivation by HNF-4alpha. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4alpha, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4alpha, respectively. An electrophoretic mobility shift assay showed that HNF-4alpha bound to DR-2 and IR-8 under the conditions of HNF-4alpha overexpression. Furthermore, under normal conditions, HNF-4alpha bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4alpha regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4alpha.
Transcriptional regulation of human and mouse organic anion transporter 1 by hepatocyte nuclear factor 1 alpha/beta. Takami Saji;Ryota Kikuchi;Hiroyuki Kusuhara;Insook Kim;Frank J Gonzalez;Yuichi Sugiyama. 2007. J Pharmacol Exp Ther. 324. PMID: 18029548

Organic anion transporter 1 (OAT1/SLC22A6) is predominantly expressed in the proximal tubules of the kidney. Cumulative studies have shown its critical role in the tubular secretion of a variety of organic anions, including several clinically important drugs. In addition, OAT1 is also involved in the pharmacological effect of diuretics and the nephrotoxicity of antiviral drugs. In contrast to these functional characterizations, the regulatory mechanism of OAT1 expression is poorly understood. It was recently demonstrated that the expression of Oat1 was markedly reduced in the kidneys of hepatocyte nuclear factor 1alpha (Hnf1alpha)-null mice. However, in vitro evidence for the involvement of HNF1alpha and further analyses are required to illustrate the transcriptional regulation of OAT1 genes in more detail. Computational analysis of the potential transcription factor binding sites revealed that the HNF1-motif was conserved in the proximal-promoter region of human and mouse OAT1 genes. The mRNA expression of mouse organic anion transporter 1 was drastically reduced in Hnf1alpha-null mice compared with that in wild-type mice, which was consistent with a previous report (Maher et al., 2006). Forced expression of HNF1alpha alone or both HNF1alpha and HNF1beta enhanced the activity of human and mouse OAT1 promoters in the transactivation assays, whereas HNF1beta alone was not active. Mutations in the HNF1-motif significantly reduced this transactivation. Direct binding of HNF1alpha/HNF1alpha homodimer and HNF1alpha/HNF1beta heterodimer to the HNF1-motif found in the human OAT1 promoter was demonstrated by electrophoretic mobility shift assays. These results provide convincing evidence for the involvement of HNF1alpha/beta in the constitutive expression of human and mouse OAT1 in the kidney.
Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics. Volker Vallon;Timo Rieg;Sun Young Ahn;Wei Wu;Satish A Eraly;Sanjay K Nigam. 2008. Am J Physiol Renal Physiol. 294. PMID: 18216144

Organic anion transporter (OAT) genes have been implicated in renal secretion of organic anions, but the individual in vivo contributions of OAT1 (first identified as NKT) and OAT3 remain unclear. Potential substrates include loop diuretics (e.g., furosemide) and thiazide diuretics (e.g., bendroflumethiazide), which reach their tubular sites of action mainly by proximal tubular secretion. Previous experiments in Oat1 knockout (-/-) mice revealed an almost complete loss of renal secretion of the prototypic organic anion p-aminohippurate (PAH) and a role of OAT1 in tubular secretion of furosemide (Eraly SA, Vallon V, Vaughn D, Gangoiti JA, Richter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK. J Biol Chem 281: 5072-5083, 2006). In this study we found that both furosemide and bendroflumethiazide inhibited mOat1- and mOat3-mediated uptake of a labeled tracer in Xenopus oocytes injected with cRNA, consistent with their being substrates for mouse OAT1 and OAT3. Experiments in Oat3(-/-) mice revealed intact renal secretion of PAH, but the dose-natriuresis curves for furosemide and bendroflumethiazide were shifted to the right and urinary furosemide excretion was impaired similar to the defect in Oat1(-/-) mice. Thus, whereas OAT1 (in contrast to OAT3) is the classic basolateral PAH transporter of the proximal tubule, both OAT1 and OAT3 contribute similarly to normal renal secretion of furosemide and bendroflumethiazide, and a lack of either one is not fully compensated by the other. Although microarray expression analysis in the kidneys of Oat1(-/-) and Oat3(-/-) mice revealed somewhat altered expression of a small number of transport-related genes, none were common to both knockout models. When searching for polymorphisms involved in human diuretic responsiveness, it may be necessary to consider both OAT1 and OAT3, among other genes.
Organic anion transport pathways in antiviral handling in choroid plexus in Oat1 (Slc22a6) and Oat3 (Slc22a8) deficient tissue. Megha A Nagle;Wei Wu;Satish A Eraly;Sanjay K Nigam. 2012. Neurosci Lett. 534. PMID: 23196129

Transporters in the choroid plexus (CP) regulate transport of numerous compounds of physiological and therapeutic interest between blood and CSF and thus likely play a key role in determining CNS levels of drugs, toxins and metabolites. Here, high CP expression was noted for the organic anion transporters, Oat1 (SLC22A6 or NKT) and Oat3 (SLC22A8) which are also the principal Oats in the renal proximal tubule, as well as SLC22A17, hypothesized to be involved in iron transport. Because Oat1 and Oat3 have overlapping substrate specificity, ex vivo preparations of CP from Oat1((-/-)) and Oat3((-/-)) mice were used to isolate the individual transport function of each, respectively. Tissue from either knockout mouse mediated the probenecid-inhibitable transport of the Oat substrate, 6-carboxyfluorescein (6CF), confirming the presence of Oat1 and Oat3 function. Because many antiviral medications are Oat substrates, including those crucial in the treatment of HIV infections, the interaction of the antivirals zidovudine, acyclovir, tenofovir, lamivudine, and stavudine, with Oat1 and Oat3 in CP, was investigated by determining the inhibition of 6CF uptake. All the antivirals tested manifested significant interaction with both Oat1 and Oat3, with the exception of stavudine which did not significantly affect Oat1 function. These results could have important implications for antiretroviral (and other drugs) penetration into or retention within the CNS, a major reservoir for virus during HIV infection. Apart from any effect at the blood brain barrier (BBB), designing specific inhibitors of Oat1 and Oat3 may be helpful in altering CNS drug levels by blocking organic anion transporters in the CP. The role of SLC22A17 in the CP deserves further exploration. The ability of Oats to regulate the movement of small molecules across the BBB, CP, proximal tubule and other tissues may also be important for their role in remote sensing and signaling [1,21]).
Human organic anion transporter OAT1 is not responsible for glutathione transport but mediates transport of glutamate derivatives. Yohannes Hagos;Gerhard Burckhardt;Birgitta C Burckhardt. 2012. Am J Physiol Renal Physiol. 304. PMID: 23255614

Due to their clearance function, the kidneys are exposed to high concentrations of oxidants and potentially toxic substances. To maintain cellular integrity, renal cells have to be protected by sufficient concentrations of the antioxidant glutathione (GSH). We tested whether GSH or its precursors are taken up by human organic anion transporters 1 (OAT1) and 3 (OAT3) stably expressed in HEK293 cells. GSH did not inhibit uptake of p-aminohippurate (PAH) or of estrone sulfate (ES) in OAT3-transfected HEK293 cells. In OAT1-transfected cells, GSH reduced the uptake of PAH marginally. Among the GSH constituent amino acids, glutamate, cysteine, and glycine, only glutamate inhibited OAT1, but labeled glutamate was not taken up by a probenecid-inhibitable transport system. Thus OAT1 binds glutamate but is unable to translocate it. The GSH precursor dipeptide, cysteinyl glycine (cysgly), and the glutamate derivative N-acetyl glutamate (NAG), inhibited uptake of PAH when present in the medium and trans-stimulated uptake of PAH from the intracellular side, indicating that they are hitherto unrecognized transported substrates of OAT1. N-acetyl aspartate weakly interacted with OAT1, but aspartate did not. NAG inhibited also OAT3, albeit with much lower affinity compared with OAT1, and glutamate did not interact with OAT3 at all. Taken together, human OAT3 and OAT1 cannot be involved in renal GSH extraction from the blood. However, OAT1 could support intracellular GSH synthesis by taking up cysteinyl glycine.
Organic Anion Transporter 1 Is Inhibited by Multiple Mechanisms and Shows a Transport Mode Independent of Exchange. Adam G Hotchkiss;Tiandai Gao;Usman Khan;Liam Berrigan;Mansong Li;Leslie Ingraham;Ryan M Pelis. 2015. Drug Metab Dispos. 43. PMID: 26370539

The mechanism by which drugs inhibit organic anion transporter 1 (OAT1) was examined. OAT1 was stably expressed in Chinese hamster ovary (CHO) cells, and para-aminohippurate (PAH) and 6-carboxyfluorescein were the substrates. Most compounds (10 of 14) inhibited competitively, increasing the Michaelis constant (Km) without affecting the maximal transport rate (Jmax). Others were mixed-type (lowering Jmax and increasing Km) or noncompetitive (lowering Jmax only) inhibitors. The interaction of a noncompetitive inhibitor (telmisartan) with OAT1 was examined further. Binding of telmisartan to OAT1 was observed, but translocation was not. Telmisartan did not alter the plasma membrane expression of OAT1, indicating that it lowers Jmax by reducing the turnover number. PAH transport after telmisartan treatment and its washout recovered faster in the presence of 10% fetal bovine serum in the washout buffer, indicating that binding of telmisartan to OAT1 and its inhibitory effect are reversible. Together, these data suggest that telmisartan binds reversibly to a site distinct from substrate and stabilizes the transporter in a conformation unfavorable for translocation. In the absence of an exchangeable extracellular substrate, PAH efflux from CHO-OAT1 cells was relatively rapid. Telmisartan slowed PAH efflux, suggesting that some transporter-mediated efflux occurs independent of exchange. Although drug-drug interaction predictions at OAT1 assume competitive inhibition, these data show that OAT1 can be inhibited by other mechanisms, which could influence the accuracy of drug-drug interaction predictions at the transporter. Telmisartan was useful for examining how a noncompetitive inhibitor can alter OAT1 transport activity and for uncovering a transport mode independent of exchange.
From the Cover: Identification of Natural Products as Inhibitors of Human Organic Anion Transporters (OAT1 and OAT3) and Their Protective Effect on Mercury-Induced Toxicity. Xue Wang;Lifeng Han;Gentao Li;Wei Peng;Xiumei Gao;Curtis D Klaassen;Guanwei Fan;Youcai Zhang. 2017. Toxicol Sci. 161. PMID: 29045746

Mercury accumulates in kidneys and produces acute kidney injury. Semen cassiae (SC), a widely consumed tea and herbal medicine in Eastern Asia, has been reported to have protective effects on kidneys. In this study, SC extract was shown to almost abolish the histological alterations induced by mercuric chloride in rat kidneys. A total of 22 compounds were isolated from SC, and 1,7,8-methoxyl-2-hydroxyl-3-methyl-anthraquinone was detected in SC for the first time. Among the eight compounds identified in the blood of rats after SC treatment, six were strong inhibitors of human organic anion transporter 1 and 3 (OAT1 and OAT3). Inhibitory studies revealed that OAT1 and OAT3 were inhibited by SC constituents, in both a competitive and noncompetitive manner. Both OAT1- and OAT3-overexpressing cells were susceptible to the cytotoxicity of the cysteine-mercury conjugate, but only OAT1-overexpressing cells could be protected by 200 μM probenecid or 10 μM of the eight inhibitors in SC, suggesting that OAT1 is the major determinant in the cellular uptake of mercury. To facilitate the identification of inhibitors of OAT1 and OAT3, models of OAT1 and OAT3 were constructed using recently determined protein templates. By combining in silico and in vitro methods, inhibitors of OAT1 and OAT3 were predicted and validated from SC constituents. Collectively, the present study suggests that additional inhibitors of OAT1 and OAT3 can be predicted and validated from natural products by combining docking and in vitro screening, and could be a source of pharmaceutical compounds for developing treatments for mercury-induced kidney injury.
Apigenin, a novel candidate involving herb-drug interaction (HDI), interacts with organic anion transporter 1 (OAT1). Ting Wu;Haixin Li;Jiasheng Chen;Ying Cao;Weimin Fu;Pingzheng Zhou;Jianxin Pang. 2017. Pharmacol Rep. 69. PMID: 29128807

BACKGROUND: Apigenin is a flavonoid compound, widely distributed in natural plants. Various studies have suggested that apigenin has inhibitory effects towards several drug transporters, such as the organic anion transporting (OAT) polypeptides, 1B1 and 1B3 (OATP1B1 and OATP1B3). However, the mechanism by which apigenin interacts with OAT1 has not been well studied. METHODS: MDCK cells stably-expressing OAT1 were used to examine the inhibitory effects of apigenin on OAT1. UPLC-MS/MS was used to evaluate the in vitro and in vivo effects of apigenin on the uptake of acyclovir by OAT1. Cytotoxicity was determined by the cell viability, MTT assays. RESULTS: Apigenin effectively inhibited the activity of OAT1 in a dose-dependent manner with an IC50 value of 0.737μM. Pre-incubation of cells with apigenin caused a time-dependent inhibition (TDI) of OAT1. Additionally, we examined the interactions between apigenin and acyclovir or adefovir. Data showed that apigenin (1μM) significantly blocked the uptake of acyclovir by OAT1 in vitro with an inhibition rate of 55%. In vivo, apigenin could increase the concentration of acyclovir in plasma when co-administered with acyclovir. Importantly, the MTT assays showed that, at a dose of 50μM, apigenin significantly reduced the cytotoxicity of adefovir and substantially increased cell viability from 50.6% to 112.62%. CONCLUSION: Our results demonstrate that apigenin regulates OAT1, and can cause TDI or herb-drug interaction (HDI) when used in combination with acyclovir or adefovir. Therefore, apigenin could be used as a nephroprotective agent when used in combination with the substrates of OAT1.
The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts. Lan-Ying Pan;Kui Zeng;Li Li;Yan Lou;Su Zeng. 2019. Chin J Nat Med. 17. PMID: 31526503

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 μmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.
Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney. Gheun-Ho Kim;Ki Young Na;So-Young Kim;Kwon Wook Joo;Yoon Kyu Oh;Seoung-Wan Chae;Hitoshi Endou;Jin Suk Han. 2003. Nephrol Dial Transplant. 18. PMID: 12897087

BACKGROUND: Thiazide and loop diuretics are secreted from the proximal tubule via the organic anion transport system to reach their principal sites of action. Recently, a multispecific organic anion transporter 1 (OAT1) was identified in rat kidney and was localized to the basolateral membrane of the S2 segment in the proximal tubule. We postulated that interactions between thiazide or loop diuretics and OAT1 may play a role in the adaptation to long-term diuretic use, and investigated whether OAT1 is regulated in vivo by chronic administration of diuretics at the protein level. METHODS: Semi-quantitative immunoblotting and immunohistochemistry were carried out in kidneys from male Sprague-Dawley rats using a polyclonal peptide-derived antibody to OAT1. Furosemide (12 mg/day/rat, n = 6), hydrochlorothiazide (3.75 mg/day/rat, n = 6) or vehicle (1.7% ethanolamine, n = 6) were infused subcutaneously for 7 days using osmotic minipumps. Experimental and vehicle-control rats were pair-fed, and two bottles of drinking water were provided, one containing tap water and the other containing a solution of 0.8% NaCl with 0.1% KCl. RESULTS: Overt diuretic responses were observed to both furosemide and hydrochlorothiazide infusions. There were no differences in body weight or creatinine clearance between the experimental and control rats. Although OAT1 protein abundance in cortical homogenates was increased by furosemide infusion (271 +/- 35 vs 100 +/- 15%, P < 0.05), Na-K-ATPase alpha1 subunit protein abundance was not affected (113 +/- 14 vs 100 +/- 8%, P = 0.42). Immunohistochemical localization in tissue sections confirmed a strong increase in OAT1 expression in the basolateral membrane of the S2 segment of proximal tubule. OAT1 protein abundance in cortical homogenates was also increased by hydrochlorothiazide infusion (181 +/- 25 vs 100 +/- 7%, P < 0.01), whereas Na-K-ATPase alpha1 subunit protein abundance was not affected (105 +/- 4 vs 100 +/- 4%, P = 0.34). CONCLUSION: Chronic furosemide or hydrochlorothiazide infusion caused increases in OAT1 protein abundance in rat kidney. These results suggest that OAT1 may be up-regulated in vivo by substrate stimulation at the protein level.
Transport of 99mTc-MAG3 via rat renal organic anion transporter 1. Naoto Shikano;Yoshikatsu Kanai;Keiichi Kawai;Nobuyoshi Ishikawa;Hitoshi Endou. 2004. J Nucl Med. 45. PMID: 14734677

UNLABELLED: Recently, complementary DNA (cDNA) encoding a p-aminohippurate (PAH) transporter designated rat organic anion transporter 1 (OAT1) was isolated. OAT1, a multispecific organic anion transporter at the basolateral membrane, is exclusively expressed in the middle segment of the proximal tubule in the rat kidney. It has been proposed that OAT1 is indirectly involved in PAH uptake via the Na(+) dicarboxylate cotransporter. In this study, in molecular biologic experiments using OAT1-expressing Xenopus laevis oocytes, we obtained evidence that (99m)Tc-mercaptoacetylglycylglycylglycine (MAG3) is transported via OAT1. METHODS: Capped OAT1 complementary RNA (cRNA) was synthesized from library plasmid cDNA linearized with BamHI using in vitro transcription. Defolliculated oocytes were injected with 10 ng of OAT1 cRNA. Two to 3 d after injection, uptake of (99m)Tc-MAG3 was measured using ND96 solution containing 18.5 kBq of (99m)Tc-MAG3. Before the uptake experiments, OAT1-expressing oocytes were preincubated for 2 h with 1 mmol/L glutarate (a dicarboxylate), to generate an outwardly directed glutarate gradient. Then, after incubation for 60 min at room temperature, radioactivity of oocytes was determined. For the inhibition experiments, uptake was assessed in the absence or presence of inhibitor: 2 mmol/L of PAH, o-iodohippurate (OIH), probenecid, 3,5-diiodo-4-pyridone-N-acetate (iodopyracet), furosemide, ethacrynic acid, glucoheptonate, maleic acid, L-Tyr, or tetraethylammonium (TEA) or 0.1 mmol/L of 2,4-dinitrophenol (DNP). RESULTS: Na(+) had a significant effect on (99m)Tc-MAG3 uptake (P < 0.05). Accumulated glutarate stimulated simultaneous (99m)Tc-MAG3 uptake and glutarate excretion (P < 0.001). The following compounds significantly inhibited (99m)Tc-MAG3 uptake: PAH, 8.5% +/- 16.2% of (99m)Tc-MAG3 uptake in the absence of an inhibitor; OIH, 26.4% +/- 21.7%; probenecid, 29.1% +/- 12.4%; iodopyracet, 15.8% +/- 7.9%; furosemide, 30.5% +/- 15.7%; ethacrynic acid, 21.6% +/- 10.6%; glucoheptonate, 35.6% +/- 22.6%; and maleic acid, 60.1% +/- 18.7%. (99m)Tc-MAG3 accumulation in Xenopus laevis oocytes was not significantly inhibited by TEA, L-Tyr, or DNP. CONCLUSION: The following substances had a cis-inhibitory effect on (99m)Tc-MAG3 transport: PAH, OIH, probenecid, iodopyracet, furosemide, ethacrynic acid, and glucoheptonate. Glutarate had a trans-stimulative effect on (99m)Tc-MAG3 transport. (99m)Tc-MAG3 acts as a substrate of OAT1, an organic anion/dicarboxylate exchanger.
Ontogeny of expression of organic anion transporters 1 and 3 in ovine fetal and neonatal kidney. Charles E Wood;Roderick Cousins;Daying Zhang;Maureen Keller-Wood. 2005. Exp Biol Med (Maywood). 230. PMID: 16179735

Organic ions are excreted into the urine via the action of organic anion transporters (OATs). In adult kidney, both OAT1 and OAT3, both multispecific transporters, are abundant; OAT1 is a known transporter of para-aminohippurate (PAH) and OAT3 is a known transporter of sulfoconjugated estrogens. The present study was designed to test the hypotheses that the expression of both OAT1 and OAT3 are developmentally regulated and that the expression increases in late gestation. Fetal kidneys were collected at sacrifice of fetal sheep at 80, 100, 120, 130, and 145 days of gestation, as well as 1 day and 1 week after birth (n=4-5 per group). Renal tissue was separated into cortex and medulla and snap-frozen in liquid nitrogen for later extraction of mRNA. The expression levels of OAT1 and OAT3 were measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR), with specific probes and primers designed in our laboratory. Cellular distribution of protein expression was identified using immunohistochemistry with commercially available antisera. The OAT1 and OAT3 mRNA in renal cortex was increased in the more mature animals. At 145 days of gestation, OAT1 mRNA abundance was increased and remained elevated postnatally. Compared with prenatal ages, OAT3 mRNA was increased postnatally. The expression of both transporters was not significantly changed as a function of development in the renal medulla. The protein expression of OAT1 and OAT3 was identified in tubular epithelium in renal cortex, although the immunoreactivity for OAT1 was greater than for OAT3. We conclude that there is a developmental pattern of expression of both OAT1 and OAT3 in ovine renal cortex, and that the pattern of expression suggests that the function of both transporters is likely to be greater starting in late gestation.
Deletion of multispecific organic anion transporter Oat1/Slc22a6 protects against mercury-induced kidney injury. Adriana M Torres;Ankur V Dnyanmote;Kevin T Bush;Wei Wu;Sanjay K Nigam. 2011. J Biol Chem. 286. PMID: 21652719

The primary site of mercury-induced injury is the kidney due to uptake of the reactive Hg(2+)-conjugated organic anions in the proximal tubule. Here, we investigated the in vivo role of Oat1 (organic anion transporter 1; originally NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478)) in handling of known nephrotoxic doses of HgCl(2). Oat1 (Slc22a6) is a multispecific organic anion drug transporter that is expressed on the basolateral aspects of renal proximal tubule cells and that mediates the initial steps of elimination of a broad range of endogenous metabolites and commonly prescribed pharmaceuticals. Mercury-induced nephrotoxicity was observed in a wild-type model. We then used the Oat1 knock-out to determine in vivo whether the renal injury effects of mercury are mediated by Oat1. Most of the renal injury (both histologically and biochemically as measured by blood urea nitrogen and creatinine) was abolished following HgCl(2) treatment of Oat1 knock-outs. Thus, acute kidney injury by HgCl(2) was found to be mediated mainly by Oat1. Our findings raise the possibility that pharmacological modulation of the expression and/or function of Oat1 might be an effective therapeutic strategy for reducing renal injury by mercury. This is one of the most striking phenotypes so far identified in the Oat1 knock-out. (Eraly, S. A., Vallon, V., Vaughn, D. A., Gangoiti, J. A., Richter, K., Nagle, M., Monte, J. C., Rieg, T., Truong, D. M., Long, J. M., Barshop, B. A., Kaler, G., and Nigam, S. K. (2006) J. Biol. Chem. 281, 5072-5083).
Differential Interaction of Dantrolene, Glafenine, Nalidixic Acid, and Prazosin with Human Organic Anion Transporters 1 and 3. Birgitta C Burckhardt;Maja Henjakovic;Yohannes Hagos;Gerhard Burckhardt. 2017. J Pharmacol Exp Ther. 362. PMID: 28630284

In renal proximal tubule cells, the organic anion transporters 1 and 3 (OAT1 and OAT3) in the basolateral membrane and the multidrug resistance-associated protein 4 (MRP4) in the apical membrane share substrates and co-operate in renal drug secretion. We hypothesized that recently identified MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin also interact with human OAT1 and/or OAT3 stably transfected in human embryonic kidney 293 cells. These four drugs were tested as possible inhibitors of p-[3H]aminohippurate (PAH) and [14C]glutarate uptake by OAT1, and of [3H]estrone-3-sulfate (ES) uptake by OAT3. In addition, we explored whether these drugs decrease the equilibrium distribution of radiolabeled PAH, glutarate, or ES, an approach intended to indirectly suggest drug/substrate exchange through OAT1 and OAT3. With OAT3, a dose-dependent inhibition of [3H]ES uptake and a downward shift in [3H]ES equilibrium were observed, indicating that all four drugs bind to OAT3 and may possibly be translocated. In contrast, the interaction with OAT1 was more complex. With [14C]glutarate as substrate, all four drugs inhibited uptake but only glafenine and nalidixic acid shifted glutarate equilibrium. Using [3H]PAH as a substrate of OAT1, nalidixic acid inhibited but dantrolene, glafenine, and prazosin stimulated uptake. Nalidixic acid decreased equilibrium content of [3H]PAH, suggesting that it may possibly be exchanged by OAT1. Taken together, OAT1 and OAT3 interact with the MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin, indicating overlapping specificities. At OAT1, more than one binding site must be assumed to explain substrate and drug-dependent stimulation and inhibition of transport activity.
Analyses of 5' regulatory region polymorphisms in human SLC22A6 (OAT1) and SLC22A8 (OAT3). Vibha Bhatnagar;Gang Xu;Bruce A Hamilton;David M Truong;Satish A Eraly;Wei Wu;Sanjay K Nigam. 2006. J Hum Genet. 51. PMID: 16648942

Kidney excretion of numerous organic anionic drugs and endogenous metabolites is carried out by a family of multispecific organic anion transporters (OATs). Two closely related transporters, SLC22A6, initially identified by us as NKT and also known as OAT1, and SLC22A8, also known as OAT3 and ROCT, are thought to mediate the initial steps in the transport of organic anionic drugs between the blood and proximal tubule cells of the kidney. Coding region polymorphisms in these genes are infrequent and pairing of these genes in the genome suggests they may be coordinately regulated. Hence, 5' regulatory regions of these genes may be important factors in human variation in organic anionic drug handling. We have analyzed novel single nucleotide polymorphisms in the evolutionarily conserved 5' regulatory regions of the SLC22A6 and SLC22A8 genes (phylogenetic footprints) in an ethnically diverse sample of 96 individuals (192 haploid genomes). Only one polymorphism was found in the SLC22A6 5' regulatory region. In contrast, seven polymorphisms were found in the SLC22A8 5' regulatory region, two of which were common to all ethnic groups studied. Computational analysis permitted phase and haplotype reconstruction. Proximity of these non-coding polymorphisms to transcriptional regulatory elements (including potential sex steroid response elements) suggests a potential influence on the level of transcription of the SLC22A6 and/or SLC22A8 genes and will help define their role in variation in human drug, metabolite and toxin excretion. The clustering of OAT genes in the genome raises the possibility that nucleotide polymorphisms in SLC22A6 could also effect SLC22A8 expression, and vice versa.
Linkage of organic anion transporter-1 to metabolic pathways through integrated "omics"-driven network and functional analysis. Sun-Young Ahn;Neema Jamshidi;Monica L Mo;Wei Wu;Satish A Eraly;Ankur Dnyanmote;Kevin T Bush;Tom F Gallegos;Douglas H Sweet;Bernhard Ø Palsson;Sanjay K Nigam. 2011. J Biol Chem. 286. PMID: 21757732

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481-490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795-805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.
Male-dominant activation of rat renal organic anion transporter 1 (Oat1) and 3 (Oat3) expression by transcription factor BCL6. Waja Wegner;Birgitta Christina Burckhardt;Gerhard Burckhardt;Maja Henjakovic. 2012. PLoS One. 7. PMID: 22530049

BACKGROUND: Organic anion transporters 1 (Oat1) and 3 (Oat3) mediate the transport of organic anions, including frequently prescribed drugs, across cell membranes in kidney proximal tubule cells. In rats, these transporters are known to be male-dominant and testosterone-dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in male-dominant regulation of Oat1 and Oat3. METHODOLOGY/PRINCIPAL FINDINGS: Promoter activities of Oat1 and Oat3 were analyzed using luciferase assays. Expression profiling was done using a SurePrint G3 rat GE 8 × 60K microarray. RNA was isolated from renal cortical slices of four adult rats per sex. To filter the achieved microarray data for genes expressed in proximal tubule cells, transcription database alignment was carried out. We demonstrate that predicted androgen response elements in the promoters of Oat1 and Oat3 are not functional when the promoters were expressed in OK cells. Using microarray analyses we analyzed 17,406 different genes. Out of these genes, 56 exhibit a sex-dependent expression in rat proximal tubule cells. As genes potentially involved in the regulation of Oat1 and Oat3 expression, we identified, amongst others, the male-dominant hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1), B-cell CLL/lymphoma 6 (BCL6), and polymerase (RNA) III (DNA directed) polypeptide G (Polr3g). Moreover, our results revealed that the transcription factor BCL6 activates promoter constructs of Oat1 and Oat3. CONCLUSION: The results indicate that the male-dominant expression of both transporters, Oat1 and Oat3, is possibly not directly regulated by the classical androgen receptor mediated transcriptional pathway but appears to be regulated by the transcription factor BCL6.
Folic acid reverses uric acid crystal-induced surface OAT1 internalization by inhibiting RhoA activity in uric acid nephropathy. Xinlin Wu;Jianxiang Liu;Jianqing Zhang;Heng Liu;Miansheng Yan;Birong Liang;Hongbo Xie;Shijun Zhang;Baoguo Sun;Houming Zhou. 2016. Mol Med Rep. 13. PMID: 26846716

To investigate how organic anion transporter (OAT)-1 is involved in uric acid nephropathy (UAN), a rat model for UAN was established and the serum uric acid, blood urea nitrogen and serum creatinine levels were all measured, and observed to be increased. It was additionally identified that in UAN rats the surface OAT1 expression levels were reduced. By treating HEK cells with monosodium urate (MSU) crystals, it was observed that the cells exhibited a reduction in OAT1 levels. Furthermore, MSU crystals were observed to recruit Ras homolog family member A (RhoA), a small guanosine triphosphatase, to the membrane and activate it. Following RhoA activation, the OAT1 internalization rate was identified to be increased. The dominant‑negative RhoA N19 mutation was able to block MSU‑induced OAT1 internalization, indicating that the process was RhoA‑dependent. Finally, the results indicated that folic acid, a daily nutritional supplement, was capable of rescuing MSU‑induced nephropathy and OAT1 internalization. These observations indicated that uric acid crystals were able to reduce the OAT1 membrane distribution through activating RhoA, and that folic acid was capable of preventing MSU-induced OAT1 relocation by inhibiting the RhoA signaling pathway.
An Organic Anion Transporter 1 (OAT1)-centered Metabolic Network. Henry C Liu;Neema Jamshidi;Yuchen Chen;Satish A Eraly;Sai Yee Cho;Vibha Bhatnagar;Wei Wu;Kevin T Bush;Ruben Abagyan;Bernhard O Palsson;Sanjay K Nigam. 2016. J Biol Chem. 291. PMID: 27440044

There has been a recent interest in the broader physiological importance of multispecific "drug" transporters of the SLC and ABC transporter families. Here, a novel multi-tiered systems biology approach was used to predict metabolites and signaling molecules potentially affected by the in vivo deletion of organic anion transporter 1 (Oat1, Slc22a6, originally NKT), a major kidney-expressed drug transporter. Validation of some predictions in wet-lab assays, together with re-evaluation of existing transport and knock-out metabolomics data, generated an experimentally validated, confidence ranked set of OAT1-interacting endogenous compounds enabling construction of an "OAT1-centered metabolic interaction network." Pathway and enrichment analysis indicated an important role for OAT1 in metabolism involving: the TCA cycle, tryptophan and other amino acids, fatty acids, prostaglandins, cyclic nucleotides, odorants, polyamines, and vitamins. The partly validated reconstructed network is also consistent with a major role for OAT1 in modulating metabolic and signaling pathways involving uric acid, gut microbiome products, and so-called uremic toxins accumulating in chronic kidney disease. Together, the findings are compatible with the hypothesized role of drug transporters in remote inter-organ and inter-organismal communication: The Remote Sensing and Signaling Hypothesis (Nigam, S. K. (2015) Nat. Rev. Drug Disc. 14, 29). The fact that OAT1 can affect many systemic biological pathways suggests that drug-metabolite interactions need to be considered beyond simple competition for the drug transporter itself and may explain aspects of drug-induced metabolic syndrome. Our approach should provide novel mechanistic insights into the role of OAT1 and other drug transporters implicated in metabolic diseases like gout, diabetes, and chronic kidney disease.
Impaired renal organic anion transport 1 (SLC22A6) and its regulation following acute myocardial infarction and reperfusion injury in rats. Kungsadal Sirijariyawat;Atcharaporn Ontawong;Siripong Palee;Savitree Thummasorn;Chayodom Maneechote;Oranit Boonphang;Varanuj Chatsudthipong;Nipon Chattipakorn;Chutima Srimaroeng. 2019. Biochim Biophys Acta Mol Basis Dis. 1865. PMID: 31125610

Acute kidney injury (AKI) is a high frequent and common complication following acute myocardial infarction (AMI). This study examined and identified the effect of AMI-induced AKI on organic anion transporter 1 (Oat1) and Oat3 transport using clinical setting of pre-renal AKI in vivo. Cardiac ischaemia (CI) and cardiac ischaemia and reperfusion (CIR) were induced in rats by 30-min left anterior descending coronary artery occlusion and 30-min occlusion followed by 120-min reperfusion, respectively. Renal hemodynamic parameters, mitochondrial function and Oat1/Oat3 expression and function were determined along with biochemical markers. Results showed that CI markedly reduced renal blood flow and pressure by approximately 40%, while these parameters were recovered during reperfusion. CI and CIR progressively attenuated renal function and induced oxidative stress by increasing plasma BUN, creatinine and malondialdehyde levels. Correspondingly, SOD, GPx, CAT mRNAs were decreased, while TNFα, IL1β, COX2, iNOS, NOX2, NOX4, and xanthine oxidase were increased. Mitochondrial dysfunction as indicated by increasing ROS, membrane depolarisation, swelling and caspase3 activation were shown. Early significant detection of AKI; KIM1, IL18, was found. All of which deteriorated para-aminohippurate transport by down-regulating Oat1 during sudden ischaemia. This consequent blunted the trafficking rate of Oat1/Oat3 transport via down-regulating PKCζ/Akt and up-regulating PKCα/NFκB during CI and CIR. Thus, this promising study indicates that CI and CIR abruptly impaired renal Oat1 and regulatory proteins of Oat1/Oat3, which supports dysregulation of remote sensing and signalling and inter-organ/organismal communication. Oat1, therefore, could potentially worsen AKI and might be a potential therapeutic target for early reversal of such injury.
[Effect of acupuncture stimulation of "Shenshu"(BL23)-"Taixi"(KI3)on levels of serum uric acid and renal URAT1 and OAT1 protein expression in hyperuricemia rats]. Xu-Feng Liu;Hong-Yi Chen;Jing Wang;Su Yun;Xiao-Qian He;Xue-Qing Tang;Ming-He Sui. 2019. Zhen Ci Yan Jiu. 44. PMID: 31155862

OBJECTIVE: To observe the effect of acupuncture at "Shenshu"(BL23)-"Taixi"(KI3)on the levels of serum uric acid (SUA) and expression of renal urate-anion transporter 1 (URAT1) and organic anion transporter 1 (OAT1) proteins in hyperuricemia (HUA) rats, so as to explore its underlying mechanisms in improving HUA. METHODS: A total of 25 male Wistar rats were divided into 4 groups: control (n=6), HUA model (n=7), BL23-KI3 (n=6) and Ganshu (BL18)-Taichong (LR3, BL18-LR3 in short, n=6). The HUA model was established by gavage of Oteracil Potassium (2 g/kg), once daily for 10 days, then once every other day. For rats of the BL23-KI3 group, BL23 and KI3 were stimulated with filiform needles which were rotated for 10 s at a frequency about 100 r/min, and for rats of the BL18-LR3 group, BL18 and LR3 were stimulated with the same methods to those of the BL23-KI3 group. The treatment of both acupuncture groups was conducted once daily, 6 times a week (except Sundays) for 3 weeks. The contents of SUA and serum creatinine (SCr) were assayed by using an automatic biochemical analyzer. The pathological changes of the right kidney tissue were observed under light microscope after hematoxylin eosin (H.E.) staining, the immunoactivity of URAT1 and OAT1 of the right kidney tissue was determined by immunohistochemistry, and the expression of URAT1 and OAT1 proteins of the left kidney tissue detected by Western blot (WB). RESULTS: After modeling, the content of SUA and the expression of renal URAT1 protein (shown by both immunoactivity and WB) were significantly increased (P<0.01), but that of renal OAT1 protein was obviously decreased in the model group compared with the control group (P<0.01). There was no notably change in the level of SCr in the model group relevant to the control group (P>0.05). Following acupuncture intervention, the SUA content and URAT1 expression in both BL18-LR3 and BL23-KI3 groups were considerably down-regulated (P<0.05, P<0.01), and the expression of OAT1 protein in the BL23-KI3 group (not the BL18-LR3 group) were obviously up-regulated relevant to the model group (P<0.01). The effects of BL23-KI3 were significant superior to those of BL18-LR3 in down-regulating the expression of URAT1 and up-regulating OAT1 protein (P<0.01, P<0.05). CONCLUSION: Acupuncture of "BL23" and "KI3" can effectively down-regulate SUA level in HUA rats, which may be related to its effects in down-regulating the expression of URAT1 and up-regulating the expression of OAT1 in the kidney tissue.
Molecular characterization of the renal organic anion transporter 1. Gerhard Burckhardt;Natascha A Wolff;Andrew Bahn. 2002. Cell Biochem Biophys. 36. PMID: 12139402

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular alpha-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.
Downregulation of organic anion transporters OAT1 and OAT3 correlates with impaired secretion of para-aminohippurate after ischemic acute renal failure in rats. R Schneider;C Sauvant;B Betz;M Otremba;D Fischer;H Holzinger;C Wanner;J Galle;M Gekle. 2007. Am J Physiol Renal Physiol. 292. PMID: 17244891

Ischemic acute renal failure (iARF) was described to reduce renal extraction of the organic anion para-aminohippurate (PAH) in humans. The rate-limiting step of renal organic anion secretion is its basolateral uptake into proximal tubular cells. This process is mediated by the organic anion transporters OAT1 and OAT3, which both have a broad spectrum of substrates including a variety of pharmaceutics and toxins. Using a rat model of iARF, we investigated whether impairing the secretion of the organic anion PAH might be associated with downregulation of OAT1 or OAT3. Inulin and PAH clearance was determined starting from 6 up to 336 h after ischemia-reperfusion (I/R) injury. Net secretion of PAH was calculated and OAT1 as well as OAT3 expression was analyzed by RT-PCR and Western blotting. Inulin and PAH clearance along with PAH net secretion were initially diminished after I/R injury with a gradual recovery during follow-up. This initial impairment after iARF was accompanied by decreased mRNA and protein levels of OAT1 and OAT3 in clamped animals compared with sham-operated controls. In correlation to the improvement of kidney function, both mRNA and protein levels of OAT1 and OAT3 were upregulated during the follow-up. Thus decreased expression of OAT1 and OAT3 is sufficient to explain the decline of PAH secretion after iARF. As a result, this may have substantial impact on excretion kinetics and half-life of organic anions. As a consequence, the biological effects of a variety of organic anions may be affected after iARF.
Upregulation of rat renal cortical organic anion transporter (OAT1 and OAT3) expression in response to ischemia/reperfusion injury. Rui Zhang;Xiao Yang;Jun Li;Jun Wu;Wen-Xing Peng;Xiu-Qing Dong;Shu-Feng Zhou;Xue-Qing Yu. 2008. Am J Nephrol. 28. PMID: 18441523

BACKGROUND/AIMS: Renal organic anion transporters (OAT1 and OAT3) localized in the basolateral membrane mediate the uptake of organic anions from the blood into proximal tubules. This study aimed to examine the effects of renal ischemia/reperfusion injury (IRI) on the expression of cortical renal OAT1 and OAT3 and the functional impact. METHODS: Male rats underwent a right nephrectomy and clamping of the left renal pedicle for 50 min or sham operation, followed by reperfusion for 1, 2, 4 and 6 days. The expression of OAT1 and OAT3 was detected by RT-PCR, immunohistochemistry and Western blot analysis. Na(+)-K(+)-ATPase activity was also estimated. RESULTS: The renal clearance of para-aminohippurate was significantly decreased on day 1 in IRI rats compared with sham-operated rats and returned to normal when the tubular injury recovered. There were significant increases in the mRNA and protein levels of OAT1 and OAT3 in renal cortex homogenates and basolateral membranes on day 1 after IRI, while on days 2 and 4 after IRI, the renal expression of OAT1 and OAT3 decreased gradually but was still significantly higher than that of the sham-operated rats. The Na(+)-K(+)-ATPase activity in renal cortex homogenates decreased significantly on day 1 after IRI but gradually increased on days 2, 4 and 6. CONCLUSIONS: Renal para-aminohippurate clearance was depressed in response to IRI; however, the expressions of renal cortex OAT1 and OAT3 were significantly elevated in the early stage of IRI which may have substantial impact on renal excretion of some drugs and toxic compounds.
Raltegravir is a substrate for SLC22A6: a putative mechanism for the interaction between raltegravir and tenofovir. Darren M Moss;Wai San Kwan;Neill J Liptrott;Darren L Smith;Marco Siccardi;Saye H Khoo;David J Back;Andrew Owen. 2010. Antimicrob Agents Chemother. 55. PMID: 21078936

The identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEM(VBL100), and Caco-2 cells. Transport by uptake transporters was assessed by using a Xenopus laevis oocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEM(VBL100), and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. The K(m) and V(max) for SLC22A6 transport were 150 μM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 μM tenofovir with a 50% inhibitory concentration (IC(50)) of 14.0 μM, and tenofovir inhibited 1 μM raltegravir with an IC(50) of 27.3 μM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.
Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats. Tao Liu;Xinjin Guo;Qiang Meng;Changyuan Wang;Qi Liu;Huijun Sun;Xiaochi Ma;Taiichi Kaku;Kexin Liu. 2012. Peptides. 36. PMID: 22521734

The objective was to determine whether protective effects of JBP485 on biliary obstruction induced by alpha-naphthylisothiocyanate (ANIT) are mediated by the organic anion transporters Oat1, Oat3 and the multidrug resistance-associated protein Mrp2. The ANIT-induced increases in bilirubin (BIL), alanine aminotransferase (ALT) and aspartate transaminase (AST) in rat serum were inhibited significantly by oral administration of JBP485. The plasma concentration of JBP485 which is the substrate of Oat1 and Oat3 determined by LC-MS/MS was markedly increased after intravenous administration in ANIT-treated rats, whereas cumulative urinary excretion of JBP485 in vivo and the uptake of JBP485 in kidney slices were decreased remarkably. RT-PCR and Western blot showed the decreased expression of Oat1 and Oat3, increased expression of Mrp2 in ANIT-induced rats, meanwhile, the expression levels of Mrp2 and Oat1 were up-regulated after administration of JBP485. The up-regulation of Mrp2 and Oat1 was associated with a concomitant increase in urinary BIL after treatment with JBP485 in ANIT-treated rats. The mechanism for JBP485 to restore liver function might be related to improvement of the expression and function for Oat1 and Mrp2 as well as facilitation of urinary excretion for hepatoxic substance.
Unique metabolite preferences of the drug transporters OAT1 and OAT3 analyzed by machine learning. Anisha K Nigam;Julia G Li;Kaustubh Lall;Da Shi;Kevin T Bush;Vibha Bhatnagar;Ruben Abagyan;Sanjay K Nigam. 2020. J Biol Chem. 295. PMID: 31896576

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.
Potent Inhibitors of Organic Anion Transporters 1 and 3 From Natural Compounds and Their Protective Effect on Aristolochic Acid Nephropathy. Caiyu Li;Xue Wang;Yajuan Bi;Heshui Yu;Jing Wei;Yi Zhang;Lifeng Han;Youcai Zhang. 2020. Toxicol Sci. 175. PMID: 32159797

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a critical role in renal drug-drug interactions and are involved in the nephrotoxicity of many anionic xenobiotics. To date, relatively little is known about the interaction of natural compounds with OAT1 and OAT3. Of the 270 natural compounds screened in the present study, 21 compounds inhibited OAT1 and 45 compounds inhibited OAT3. Further concentration-dependent studies identified 7 OAT1 inhibitors and 10 OAT3 inhibitors with IC50 values of <10 μM, and most of them were flavonoids, the most commonly ingested polyphenolic compounds in the diet and herbal products. Computational modeling of OAT1 and OAT3 revealed the important residues for the recognition of inhibitors. The two strong OAT inhibitors, namely wedelolactone and wogonin, were evaluated for their in vivo interactions with the OAT substrate aristolochic acid I (AAI), a natural compound causing aristolochic acid-induced nephropathy (AAN) in many species. The cytotoxicity of AAI increased in two OAT-overexpressing cell lines, with more cytotoxicity in OAT1-overexpressing cells, suggesting a more important role of OAT1 than OAT3 in AAN. Both wedelolactone and wogonin markedly increased serum AAI concentrations in AAI-treated rats and ameliorated kidney injuries in AAI-treated mice. To conclude, the present findings are of significant value in understanding natural compound-drug interactions and provide a natural source for developing treatments for AAN.
The SLC Family Are Candidate Diagnostic and Prognostic Biomarkers in Clear Cell Renal Cell Carcinoma. Weiting Kang;Meng Zhang;Qiang Wang;Da Gu;Zhilong Huang;Hanbo Wang;Yuzhu Xiang;Qinghua Xia;Zilian Cui;Xunbo Jin. 2020. Biomed Res Int. 2020. PMID: 32461965

Clear cell renal cell carcinoma (ccRCC) is the most common lethal subtype of renal cancer, and changes in tumor metabolism play a key role in its development. Solute carriers (SLCs) are important in the transport of small molecules in humans, and defects in SLC transporters can lead to serious diseases. The expression patterns and prognostic values of SLC family transporters in the development of ccRCC are still unclear. The current study analyzed the expression levels of SLC family members and their correlation with prognosis in ccRCC patients with data from Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), The Cancer Genome Atlas (TCGA), cBioPortal, the Human Protein Atlas (HPA), the International Cancer Genome Consortium (ICGC), and the Gene Expression Omnibus (GEO). We found that the mRNA expression levels of SLC22A6, SLC22A7, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 were significantly lower in ccRCC tissues than in normal tissues and the protein expression levels of SLC22A6, SLC22A7, SLC22A13, and SLC34A1 were also significantly lower. Except for SLC22A7, the expression levels of SLC22A6, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 were correlated with the clinical stage of ccRCC patients. The lower the expression levels of SLC22A6, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 were, the later the clinical stage of ccRCC patients was. Further experiments revealed that the expression levels of SLC22A6, SLC22A7, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 were significantly associated with overall survival (OS) and disease-free survival (DFS) in ccRCC patients. High SLC22A6, SLC22A7, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 expression predicted improved OS and DFS. Finally, GSE53757 and ICGC were used to revalidate the differential expression and clinical prognostic value. This study suggests that SLC22A6, SLC22A7, SLC22A13, SLC25A4, SLC34A1, and SLC44A4 may be potential targets for the clinical diagnosis, prognosis, and treatment of ccRCC patients.
Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis oocytes. N Apiwattanakul;T Sekine;A Chairoungdua;Y Kanai;N Nakajima;S Sophasan;H Endou. 1999. Mol Pharmacol. 55. PMID: 10220563

Organic anion transporter 1 (OAT1) is the para-aminohippurate (PAH) transporter in the basolateral membrane of the proximal tubule. The present study investigated whether or not nonsteroidal anti-inflammatory drugs (NSAIDs) are transported by OAT1. All of the NSAIDs tested inhibited [14C]PAH uptake via OAT1 expressed in Xenopus laevis oocytes. Ibuprofen, indomethacin, salicylurate, and naproxen showed the strongest potency to inhibit [14C]PAH uptake (Ki approximately 2-10 microM); acetylsalicylate, salicylate, and phenacetin exhibited moderate potency (Ki approximately 300-400 microM), and acetaminophen (paracetamol) exhibited the weakest inhibitory potency (Ki approximately 2 mM). Radiolabeled acetylsalicylate, salicylate, and indomethacin were taken up by OAT1 and the uptake rate of these three NSAIDs was enhanced by the outwardly directed dicarboxylate gradient. The efflux of the preloaded [14C]PAH from the oocytes via OAT1 was trans-stimulated by PAH and glutarate added to the media. The addition of salicylate, acetylsalicylate, or salicylurate into the media also trans-stimulated the efflux of PAH, whereas indomethacin did not. The present study indicates that OAT1 is responsible for the renal uptake and secretion of NSAIDs.
The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules. William H Dantzler;Stephen H Wright. 2004. Biochim Biophys Acta. 1618. PMID: 14729155

Basolateral transport of organic anions (OAs) into mammalian renal proximal tubule cells is a tertiary active transport process. The final step in this process involves movement of OA into the cells against its electrochemical gradient in exchange for alpha-ketoglutarate (alphaKG) moving down its electrochemical gradient. Two homologous transport proteins (OAT1 and OAT3) that function as basolateral OA/alphaKG exchangers have been cloned and sequenced. We are in the process of determining the functional distribution and regulation of OAT1 and OAT3 in renal tubules. We are using rabbit OAT1 (rbOAT1) and OAT3 (rbOAT3) expressed in heterologous cell systems to determine substrate specificity and putative regulatory steps and isolated rabbit proximal renal tubule segments to determine functional distribution and physiological regulation of these transporters within their native epithelium. Rabbit OAT1 and OAT3 differ distinctly in substrate specificity. For example, rbOAT1 has a high affinity for the classical renal OA transport substrate, p-aminohippurate (PAH), whereas rbOAT3 has no affinity for PAH. In contrast, rbOAT3 has a high affinity for estrone sulfate (ES), whereas rbOAT1 has only a very slight affinity for ES. Both rbOAT1 and rbOAT3 appear to have about the same affinity for fluorescein (FL). These differences and similarities in substrate affinities make it possible to functionally map transporters along the renal tubules. Initial data indicate that OAT1 predominates in S2 segments of the rabbit proximal tubules, but studies of other segments are just beginning. Transport of a given substrate in any tubule segment depends on both the affinity of each transporter which can accept that substrate as well as the level of expression of each of those processes in that particular tubule segment. Basolateral PAH transport (presumably OAT1 activity) appears to be down-regulated by activation of protein kinase C (PKC) and up-regulated via mitogen-activated protein kinase (MAPK) through phospholipase A(2) (PLA(2)), prostaglandin E(2) (PGE(2)), cyclic AMP, and protein kinase A (PKA) activation.
Altered expression of rat renal cortical OAT1 and OAT3 in response to bilateral ureteral obstruction. Silvina R Villar;Anabel Brandoni;Naohiko Anzai;Hitoshi Endou;Adriana M Torres. 2005. Kidney Int. 68. PMID: 16316345

BACKGROUND: Bilateral ureteral obstruction (BUO) is characterized by the development of hemodynamic and tubular lesions. However, little is known about the expression of organic anion renal transporters. The objective of this work was to study the renal excretion of p-aminohippurate (PAH) and the cortical renal expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) in BUO rats. METHODS: Male Wistar rats underwent bilateral obstruction of the proximal ureters for 24 hours (BUO) or sham operation. After 24 hours of ureteral releasing, the following studies were performed: PAH renal excretion employing conventional clearance techniques and OAT1 and OAT3 abundance (homogenates, intracellular and basolateral plasma membrane fractions from renal cortex) using immunoblotting and immunocytochemical techniques (light microscopic and confocal immunofluorescence microscopic analysis). RESULTS: BUO rats showed a lower renal excretion of PAH. In obstructed kidneys, immunoblotting revealed a significant decrease in the abundance of both OAT1 and OAT3 in basolateral plasma membranes from renal cortex. An increase of OAT1 expression was observed in homogenates and in intracellular membrane fractions in kidneys from BUO rats compared with sham-operated ones, indicating an internalization of this carrier. Immunocytochemical techniques confirmed these results. On the contrary, OAT3 expression was reduced both in homogenates and in intracellular membrane fractions in obstructed kidneys. CONCLUSION: BUO was associated with down-regulation of OAT1 and OAT3 in basolateral plasma membranes from proximal tubule cells, thus these carriers may play important roles in the impaired organic anion excretion displayed in the obstructed kidney.
Expression of OAT1 and OAT3 in differentiating proximal tubules of the mouse kidney. Jin-Sun Hwang;Eun-Young Park;Wan-Young Kim;Chul-Woo Yang;Jin Kim. 2009. Histol Histopathol. 25. PMID: 19924639

Organic anion transporter 1 (OAT1) and OAT3 in the proximal tubules (PT) of the kidney play important roles in the elimination of harmful endogenous compounds and xenobiotics from the body. We investigated the temporal and spatial expression of OAT1 and OAT3 in the differentiating PT in mouse kidney. Ontogenic expression of OAT1 and OAT3 was investigated by immunohistochemical analysis. The S1, S2, and S3 segments of the PT were identified using antibodies to aquaporin 1 (AQP1), Na+-HCO3- cotransporter 1 (kNBC1), and AQP4. OAT1 immunoreactivity was first detected at PT in the inner cortex of 15-day-old fetuses (F15) and in the outer cortex of 7-day old pups. OAT3 was first observed in the distal tubule of F14 and in S2 segment of the PT of F16 and in S1 and S3 segments around the time of birth; expression increased through postpartum day 21. The ontogenic pattern of expression of OAT1 and OAT3 in the differentiating PT suggests that both transporters may function in the S2 segment in the fetus, but not until after birth in S1 and S3 segments.
Inhibitory effects of ketoconazole and rifampin on OAT1 and OATP1B1 transport activities: considerations on drug-drug interactions. Min-Koo Choi;Qing-Ri Jin;Yeong-Lim Choi;Sung-Hoon Ahn;Myung-Ae Bae;Im-Sook Song. 2011. Biopharm Drug Dispos. 32. PMID: 21456052

Ketoconazole and rifampin are the most widely used compounds examined in recent drug-drug interaction (DDI) studies, and they have multiple roles in modulating drug metabolizing enzymes and transporters. To determine the underlying mechanisms of DDI, this study was performed to investigate the inhibitory effects of ketoconazole and rifampin on the functions of OAT1 and OATP1B1, and to evaluate the potential of ketoconazole and rifampin for DDI with substrate drugs for these transporters in a clinical setting. Ketoconazole inhibited OATP1B1-mediated transport activity, while rifampin inhibited OAT1 and OATP1B1. Inhibition by rifampin and ketoconazole of the uptake of olmesartan, a substrate for OAT1 and OATP1B1, was evaluated in oocytes overexpressing these transporters. The K(i) values for rifampin on OAT1 and OATP1B1-mediated olmesartan uptake were 62.2 and 4.42 µM, respectively, and the K(i) value for ketoconazole on OATP1B1-mediated olmesartan uptake was 66.1 µM. As measured plasma concentrations of rifampin and ketoconazole were 7.29 and 6.4-13.3 µM, respectively, the likelihood of an OATP1B1-mediated drug-drug interaction between rifampin and olmesartan is thought to be possible, whereas OAT1 or OATP1B1-mediated DDI between rifampin or ketoconazole and olmesartan appears unlikely in the clinical setting.
α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4). Yohannes Hagos;Gunnar Schley;Johannes Schödel;Wolfgang Krick;Gerhard Burckhardt;Carsten Willam;Birgitta C Burckhardt. 2012. Pflugers Arch. 464. PMID: 22875277

2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.
Altered Renal Expression of Relevant Clinical Drug Transporters in Different Models of Acute Uremia in Rats. Role of Urea Levels. Anabel Brandoni;Adriana M Torres. 2015. Cell Physiol Biochem. 36. PMID: 26065488

BACKGROUND/AIMS: Organic anion transporter 1 (Oat1) and 3 (Oat3) are organic anion transporters that play critical roles in the body disposition of numerous clinically important drugs. We investigated the effects of acute uremia on the renal expression of Oat1 and Oat3 in three in vivo experimental models of acute kidney injury (AKI): induced by ischemia, by ureteral obstruction and by the administration of HgCl2. We also evaluated the influence of urea in the expression of these transporters in proximal tubular cells suspensions. METHODS: Membranes were isolated from kidneys of each experimental group and from cell suspensions incubated with different urea concentrations. Oat1 and Oat3 expressions were performed by immunoblotting. RESULTS: A good correlation between uremia and the renal protein expression of Oat1 and Oat3 was observed in vivo. Moreover, the incubation of isolated proximal tubular cells with different concentrations of urea decreases protein expression of Oat1 and Oat3 in plasma membranes in a dose-dependent manner. CONCLUSION: The more severe the renal failure, the more important is the decrease in protein expression of the transporters in renal membranes where they are functional. The in vitro study demonstrates that urea accounts, at least in part, for the decreased expression of Oat1 and Oat3 in proximal tubule plasma membranes.
Inhibition of Methotrexate Uptake via Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs. Masahiro Iwaki;Hiroaki Shimada;Yuri Irino;Manami Take;Sachiko Egashira. 2017. Biol Pharm Bull. 40. PMID: 28566636

Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDs-Glu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [3H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5- to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.
Time course of organic anion excretion in rats with bilateral ureteral obstruction: role of organic anion transporters (Oat1 and Oat3). Silvina R Villar;Anabel Brandoni;Adriana M Torres. 2008. Nephron Physiol. 110. PMID: 18953184

BACKGROUND: Urinary tract obstruction is a common cause of renal failure. In this study, we evaluated the time course of P-aminohippurate (PAH) renal excretion and the cortical expression of organic anion transporters (Oat1 and Oat3) at 1 (BUO-1), 2 (BUO-2) and 7 (BUO-7) days after release of 24-hour bilateral ureteral obstruction (BUO) in the rat. METHODS: Conventional clearance technique, differential centrifugation, semiquantitative immunoblotting and immunohistochemical techniques have been employed. RESULTS: These studies showed that Oat1 and Oat3 in basolateral membranes were downregulated both at BUO-1 and BUO-2. Concomitantly, the rats developed a reduction in PAH renal elimination. In contrast, total recovery in PAH renal excretion and in the expression of Oat1 and Oat3 were observed at BUO-7, as compared with the sham group. A direct correlation was observed between the secretory clearance of PAH and Oat1 (r(2) = 0.88) and Oat3 (r(2) = 0.83) expression in basolateral membranes. CONCLUSION: These results indicate that the differential expression of organic anion transporters is one of the main molecular mechanisms contributing to the organic anion excretion modifications observed during the time course of obstructive nephropathy. This study provides evidence regarding the importance of adjusting the dose regimens of negatively charged drugs during the different time phases of this pathology.
Renal expression and function of oat1 and oat3 in rats with vascular calcification. Romina Bulacio;María Herminia Hazelhoff;Adriana Mónica Torres. 2012. Pharmacology. 90. PMID: 22759781

BACKGROUND/AIMS: Calcium overload in vascular smooth muscle is a highly pathogenic event, which progresses with advancing age. Old patients are polymedicated, and several pharmacotherapeutic agents circulate in the plasma as organic anions. The organic anion transporters 1 and 3 (Oat1 and Oat3) are present in renal basolateral membranes, which transport organic anions of pharmacological and physiological interest. This study was designed to evaluate the renal expression and function of Oat1 and Oat3 in rats with vascular calcification. METHODS: Vascular calcification was induced by administration of a single dose of vitamin D(3) (300,000 UI/ kg b.w., i.m.) to male Wistar rats 10 days before the experiments. Oat1 and Oat3 expression was assessed by immunoblotting, immunohistochemistry and reverse-transcriptase polymerase chain reaction. The renal clearance of p-aminohippurate (PAH, a prototypical organic anion, substrate of Oat1 and Oat3) was measured by conventional clearance techniques. RESULTS: Oat1 and Oat3 protein levels showed an increase in plasma membranes of renal proximal tubules of treated animals, where both transporters are functional. This could explain the increase observed in the renal clearance of PAH in treated rats. CONCLUSIONS: These results suggest the relevance of considering the existence of vascular calcification, which is common in ageing, when organic anion drugs are prescribed.
Investigation of the differential transport mechanism of cinnabar and mercury containing compounds. Yang Wang;Shaoyu Zhou;Honghong Ma;Jing-Shan Shi;Yuan-Fu Lu. 2019. Environ Toxicol Pharmacol. 66. PMID: 30639899

BACKGROUND: Cinnabar has a long history of uses in Chinese traditional medicines as an ingredient in various remedies. However, the detailed mechanism of cinnabar in medication remains unclear, and the toxicity of cinnabar has been a debate due to its containing mercury sulfide. This study was designed to investigate the differential transport mechanism of cinnabar and other Hg-containing compounds HgCl2, MeHg and HgS, and to determine if organic anion transporters OAT1 and OAT3 were involved in the differential transport mechanism. MATERIALS AND METHODS: The 293T cells were employed to investigate and compare the differential transport mechanism of cinnabar and HgCl2, MeHg and HgS. Cells were incubated with a low dose (5 μM HgCl2 and MeHg, 200 μM HgS and cinnabar), medium dose (10 μM HgCl2 and MeHg, 400 μM HgS and cinnabar), and high dose (20 μM HgCl2 and MeHg, 800 μM HgS and cinnabar) of HgCl2, MeHg, HgS and cinnabar for 24 h. Following treatment, the cells were collected and the cell viability was determined by MTT assay. The intracellular mercury content was measured at 1, 4, and 24 h after treatment with 10 μM of the tested agents by an atomic fluorescence spectrophotometer. The effect of these tested agents on mitochondrial respiration was determined in a high-resolution oxygraphyat 24 h following treatment. Furthermore, the effect of modulation of expression of transporters OAT1 and OAT3 on the transport and cytotoxicity of the tested agents was evaluated. The up and down regulation of OAT1 and OAT3 were achieved by overexpression and siRNA transfection, respectively. RESULTS: Compared with HgCl2 and MeHg, the cytotoxicity of cinnabar and HgS was lower, with cell viability at the high dose cinnabar and HgS being about 65%, while MeHg and HgCl2 were 40% and 20%, respectively. The intracellular mercury accumulation was time-dependent. At 24 h the intracellular concentrations of HgCl2 and MeHg were about 7 and 5 times higher, respectively, than that of cinnabar. No significant difference was found in the intracellular mercury content in cells treated with cinnabar compared to HgS. The knockdown and overexpression of the transporter OAT1 resulted in significant reduction and increase, respectively, in mercury accumulation in HgCl2 -treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar, MeHg, and HgS. In addition, the knockdown and overexpression of the transporter OAT3 caused significant reduction and increase, respectively, in mercury accumulation in both HgCl2 and MeHg-treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar and HgS. Furthermore, it was found that cells transfected with siOAT1 caused significant resistance to the cytotoxicity induced by HgCl2, while no noticeable changes in cell viability were observed in cells treated with other tested agents. Additionally, cells transfected with OAT3 did not change cell sensitivity to cytotoxicity induced by all of the four tested agents. CONCLUSION: This study demonstrates that differential transport and accumulation of mercury in 293T cells exists among cinnabar and the three mercury-containing compounds HgCl2, MeHg and HgS, leading to distinct sensitivity to mercury induced cytotoxicity. The kidney organic anion transporters OAT1 and OAT3 are partially involved in the regulation of the transport of HgCl2 and MeHg, but not in the regulation of the transport of cinnabar.
Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition. Marija Ljubojevic;Carol M Herak-Kramberger;Yohannes Hagos;Andrew Bahn;Hitoshi Endou;Gerhard Burckhardt;Ivan Sabolic. 2004. Am J Physiol Renal Physiol. 287. PMID: 15010355

In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens.