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The renal excretion of drugs via organic anion transporters 1 and 3 (OAT1/3) is significantly decreased in patients with renal impairment. This study uses physiologically based pharmacokinetic models to quantify the reduction in OAT1/3-mediated secretion of drugs throughout varying stages of chronic kidney disease.Physiologically based pharmacokinetic models were constructed for four OAT1/3 substrates in healthy individuals: acyclovir, meropenem, furosemide, and ciprofloxacin. Observed data from drug-drug interaction studies with probenecid, a potent OAT1/3 inhibitor, were used to parameterize the contribution of OAT1/3 to the renal elimination of each drug. The models were then translated to patients with chronic kidney disease by accounting for changes in glomerular filtration rate, kidney volume, renal blood flow, plasma protein binding, and hematocrit. Additionally, a relationship was derived between the estimated glomerular filtration rate and the reduction in OAT1/3-mediated secretion of drugs based on the renal extraction ratios of ƿ-aminohippuric acid in patients with varying degrees of renal impairment. The relationship was evaluated in silico by evaluating the predictive performance of each final model in describing the pharmacokinetics (PK) of drugs across stages of chronic kidney disease.OAT1/3-mediated renal excretion of drugs was found to be decreased by 27-49%, 50-68%, and 70-96% in stage 3, stage 4, and stage 5 of chronic kidney disease, respectively. In support of the parameterization, physiologically based pharmacokinetic models of four OAT1/3 substrates were able to adequately characterize the PK in patients with different degrees of renal impairment. Total exposure after intravenous administration was predicted within a 1.5-fold error and 85% of the observed data points fell within a 1.5-fold prediction error. The models modestly under-predicted plasma concentrations in patients with end-stage renal disease undergoing intermittent hemodialysis. However, results should be interpreted with caution because of the limited number of molecules analyzed and the sparse sampling in observed chronic kidney disease pharmacokinetic studies.A quantitative understanding of the reduction in OAT1/3-mediated excretion of drugs in differing stages of renal impairment will contribute to better predictive accuracy for physiologically based pharmacokinetic models in drug development, assisting with clinical trial planning and potentially sparing this population from unnecessary toxic exposures.

Organic anion transporters (OATs) belong to a subgroup of the solute carrier 22 transporter family. OATs have a central role in xenobiotic disposition affecting the toxicokinetics of its substrates and inter-individual differences in their expression, activity and function impact both toxicokinetics and toxicodynamics. Amongst OATs, OAT1 (solute carrier family 22 member 6) is involved in the urinary excretion of many xenobiotics bringing substrates into renal proximal tubular cells which can then be secreted across the apical membrane into the tubule lumen. The mycotoxin ochratoxin A has been shown to have a high affinity for OAT1, which is an important renal transporter involved in its urinary excretion. Nowadays, molecular modeling techniques are widely applied to assess protein-ligand interactions and may provide a tool to depict the mechanistic of xenobiotic action be it toxicokinetics or toxicodynamics. This work provides a structured pipeline consisting of docking and molecular dynamic simulations to study OAT1-ligand interactions and the impact of OAT1 polymorphisms on such interactions. Such a computational structure-based analytical framework allowed to: i) model OAT1-substrate complex formation and depict the features correlating its sequence, structure and its capability to recruit substrates; and ii) investigate the impact of OAT1 missense mutations on substrate recruitment. Perspectives on applying such a structured pipeline to xenobiotic-metabolising enzymes are discussed.

As an expansion investigation of drug-drug interaction (DDI) from previous clinical trials, additional plasma endogenous metabolites were quantitated in the same subjects to further identify the potential biomarkers of organic anion transporter (OAT) 1/3 inhibition. In the single dose, open label, three-phase with fixed order of treatments study, 14 healthy human volunteers orally received 1000 mg probenecid alone, or 40 mg furosemide alone, or 40 mg furosemide at 1 hour after receiving 1000 mg probenecid on days 1, 8, and 15, respectively. Endogenous metabolites including kynurenic acid, xanthurenic acid, indo-3-acetic acid, pantothenic acid, p-cresol sulfate, and bile acids in the plasma were measured by liquid chromatography–tandem mass spectrometry. The Cmax of kynurenic acids was significantly increased about 3.3- and 3.7-fold over the baseline values at predose followed by the treatment of probenecid alone or in combination with furosemide respectively. In comparison with the furosemide-alone group, the Cmax and area under the plasma concentration–time curve (AUC) up to 12 hours of kynurenic acid were significantly increased about 2.4- and 2.5-fold by probenecid alone, and 2.7- and 2.9-fold by probenecid plus furosemide, respectively. The increases in Cmax and AUC of plasma kynurenic acid by probenecid are comparable to the increases of furosemide Cmax and AUC reported previously. Additionally, the plasma concentrations of xanthurenic acid, indo-3-acetic acid, pantothenic acid, and p-cresol sulfate, but not bile acids, were also significantly elevated by probenecid treatments. The magnitude of effect size analysis for known potential endogenous biomarkers demonstrated that kynurenic acid in the plasma offers promise as a superior addition for early DDI assessment involving OAT1/3 inhibition. SIGNIFICANCE STATEMENT This article reports that probenecid, an organic anion transporter (OAT) 1 and OAT3 inhibitor, significantly increased the plasma concentrations of kynurenic acid and several uremic acids in human subjects. Of those, the increases of plasma kynurenic acid exposure are comparable to the increases of furosemide by OAT1/3 inhibition. Effect size analysis for known potential endogenous biomarkers revealed that plasma kynurenic acid is a superior addition for early drug-drug interaction assessment involving OAT1/3 inhibition.

In vitro and in vivo studies have established the organic anion transporters OAT1 (SLC22A6, NKT) and OAT3 (SLC22A8) among the main multi-specific “drug” transporters. They also transport numerous endogenous metabolites, raising the possibility of drug-metabolite interactions (DMI). To help understand the role of these drug transporters on metabolism across scales ranging from organ systems to organelles, a formal multi-scale analysis was performed. Metabolic network reconstructions of the omics-alterations resulting from Oat1 and Oat3 gene knockouts revealed links between the microbiome and human metabolism including reactions involving small organic molecules such as dihydroxyacetone, alanine, xanthine, and p-cresol—key metabolites in independent pathways. Interestingly, pairwise organ-organ interactions were also disrupted in the two Oat knockouts, with altered liver, intestine, microbiome, and skin-related metabolism. Compared to older models focused on the “one transporter-one organ” concept, these more sophisticated reconstructions, combined with integration of a multi-microbial model and more comprehensive metabolomics data for the two transporters, provide a considerably more complex picture of how renal “drug” transporters regulate metabolism across the organelle (e.g. endoplasmic reticulum, Golgi, peroxisome), cellular, organ, inter-organ, and inter-organismal scales. The results suggest that drugs interacting with OAT1 and OAT3 can have far reaching consequences on metabolism in organs (e.g. skin) beyond the kidney. Consistent with the Remote Sensing and Signaling Theory (RSST), the analysis demonstrates how transporter-dependent metabolic signals mediate organ crosstalk (e.g., gut-liver-kidney) and inter-organismal communication (e.g., gut microbiome-host).

Anthracyclines, chemotherapeutic agents that have contributed to significant improvements in cancer survival, also carry risk of both acute and chronic cardiotoxicity. This has led to significantly elevated risks of cardiac morbidity and mortality among cancer survivors treated with these agents. Certain treatment related, demographic, and medical factors increase an individual's risk of anthracycline induced cardiotoxicity; however, significant variability among those affected suggests that there is an underlying genetic predisposition to anthracycline induced cardiotoxicity. The current narrative review seeks to summarize the literature to date that has identified genetic variants associated with anthracycline induced cardiotoxicity. These include variants found in genes that encode proteins associated with anthracycline transportation and metabolism, those that encode proteins associated with the generation of reactive oxygen species, and those known to be associated with cardiac disease. While there is strong evidence that susceptibility to anthracycline induced cardiotoxicity has genetic underpinnings, the majority of work to date has been candidate gene analyses. Future work should focus on genome-wide analyses including genome-wide association and sequencing-based studies to confirm and expand these findings.

BackgroundEndogenous biomarkers are promising tools to assess transporter-mediated drug–drug interactions early in humans.MethodsWe evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition.ResultsPDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter.ConclusionsThe current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction.

Context Relinqing granules (RLQ) are being used alone or in combination with antibacterial drugs to treat urological disorders. Objective This study investigates the pharmacokinetics of RLQ in humans and the potential for RLQ-perpetrated interactions on transporters. Materials and methods Twelve healthy subjects (six women and six men) participated to compare single- and multiple-dose pharmacokinetics of RLQ. In the single-dose study, all 12 subjects received 8 g of RLQ orally. After a 7-d washout period, the subjects received 8 g of RLQ for seven consecutive days (t.i.d.) and then a single dose. Gallic acid (GA) and protocatechuic acid (PCA) in plasma and urine samples were analysed using LC-MS/MS. The transfected cells were used to study the inhibitory effect of GA (50–5000 μg/L) and PCA (10–1000 μg/L) on transporters OAT1, OAT3, OCT2, OATP1B1, P-gp and BCRP. Results GA and PCA were absorbed into the blood within 1 h after administration and rapidly eliminated with a half-life of less than 2 h. The mean peak concentrations of GA (102 and 176 μg/L) and PCA (4.54 and 7.58 μg/L) were lower in males than females, respectively. The 24 h urine recovery rates of GA and PCA were about 10% and 5%, respectively. The steady-state was reached in 7 d without accumulation. GA was a potent inhibitor of OAT1 (IC50 = 3.73 μM) and OAT3 (IC50 = 29.41 μM), but not OCT2, OATP1B1, P-gp or BCRP. Discussion and conclusions GA and PCA are recommended as PK-markers in RLQ-related pharmacokinetic and drug interaction studies. We should pay more attention to the potential for RLQ-perpetrated interactions on transporters.

Drug interactions can induce significant clinical impacts, either by increasing adverse effects or by decreasing the therapeutic effect of drugs, and thus, need to be explored thoroughly. Clinically significant drug interactions can be induced by organic anion transporter 1 (OAT1) and OAT3 when concomitant medications competitively interact with the transporters. The purposes of this study were to develop and validate a sensitive and selective analytical method for 5-carboxyfluorescein (5-CF) and optimize the experimental conditions for interaction studies. An analytical method using high-performance liquid chromatography (HPLC) equipped with a fluorescence detector was validated for accuracy, precision, matrix effect, recovery, stability, dilutional integrity, and carry-over effect. In addition, the 5-CF concentration, incubation period, and washing conditions for interaction study were optimized. Using a valid analytical method and optimized conditions, we performed an interaction study for OAT1 and OAT3 using 26 test articles. Some of the test articles showed strong inhibitory potency for the transporters, with IC

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by μs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called “charge-relay system” that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

Hydrochlorothiazide (HCTZ) is a thiazide diuretic used in adults and children for the treatment of hypertension and edema. The pharmacokinetic (PK) properties of HCTZ in children are not well characterized, particularly among children with obesity who frequently suffer from hypertension and may, therefore, benefit from HCTZ therapy. HCTZ is excreted in the kidney via organic anion transporters 1 and 3 (OAT1 and OAT3). The ontogeny of OAT1 and OAT3 remain unknown, but HCTZ clearance may serve as a surrogate marker of OAT1 and OAT3 maturation. Population PK modeling was performed in NONMEM, and the model was leveraged to conduct dose-exposure simulations. This study examined 83 plasma samples from 49 participants (69% male) taking enteral HCTZ. The median (range) postnatal age was 6.7 years (0.03-19.5 years), and 17 (34%) participants were obese or morbidly obese. The median (range) dose of HCTZ was 0.654 mg/kg (0.11-1.8 kg) and the median number of doses recorded per participant was 5 (1-8). HCTZ PK was well characterized by a 1-compartment PK model. Body weight and a maturation model based on postmenstrual age were significant covariates for apparent clearance, but the presence of obesity was not. Dosing simulations were performed with a standardized 1mg/kg. Simulated exposure (area under the curve and maximum HCTZ concentrations) decreased with age and was likely due to older children receiving the maximum absolute doses of HCTZ. Further studies with more patients in each age group are required to confirm these PK findings of HCTZ in the children.

Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a K m of 171.9 μM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.

Mercury is a widespread pollutant. Mercuric ions uptake into tubular cells is supported by the Organic anion transporter 1 (Oat1) and 3 (Oat3) and its elimination into urine is through the Multidrug resistance-associated protein 2 (Mrp2). We investigated the effect of recombinant human erythropoietin (Epo) on renal function and on renal expression of Oat1, Oat3, and Mrp2 in a model of mercuric chloride (HgCl2)-induced renal damage. Four experimental groups of adult male Wistar rats were used: Control, Epo, HgCl2, and Epo + HgCl2. Epo (3000 IU/kg, b.w., i.p.) was administered 24 h before HgCl2 (4 mg/kg, b.w., i.p.). Experiments were performed 18 h after the HgCl2 dose. Parameters of renal function and structure were evaluated. The protein expression of Oat1, Oat3 and Mrp2 in renal tissue was assessed by immunoblotting techniques. Mercury levels were determined by cold vapor atomic absorption spectrometry. Pretreatment with Epo ameliorated the HgCl2-induced tubular injury as assessed by histopathology and urinary biomarkers. Immunoblotting showed that pretreatment with Epo regulated the renal expression of mercury transporters in a way to decrease mercury content in the kidney. Epo pretreatment ameliorates HgCl2-induced renal tubular injury by modulation of mercury transporters expression in the kidneys.

Background and Objectives Citrus flavonoids are not only components of daily nutrition, they are also promoted as dietary supplements and are important ingredients in traditional medicines. Interactions of flavonoids with synthetic drugs represent an often neglected issue. We therefore investigated in vitro whether the polymethoxyflavones nobiletin, sinensetin, and tangeretin and the flavonoid rutinosides didymin, hesperidin, and narirutin can inhibit human organic anion transporting polypeptides (OATP) 1B1, 1B3, and 2B1, which are important transporters mediating drug-drug and food-drug interactions. Methods Inhibition was investigated by quantifying the decreased uptake of the fluorescent OATP1B1 and OATP1B3 substrate 8-fluorescein-cAMP in HEK293 cells overexpressing OATP1B1 or OATP1B3 and of the fluorescent OATP2B1 substrate 4′,5′-dibromofluorescein in HEK293 cells overexpressing OATP2B1. Results We demonstrate that all flavonoids investigated inhibit OATP2B1 in the lower micromolar range (IC50 between 1.6 and 14.2 µM), but only the polymethoxyflavones also inhibit OATP1B1 and 1B3 (IC50 between 2.1 and 21 µM). Conclusions All flavonoids investigated might contribute to the intestinal OATP2B1-based interactions with drugs observed with citrus juices or fruits. In contrast, the concentration of the polymethoxyflavones after consumption of citrus juices or fruits is most likely too low to reach relevant systemic concentrations and thus to inhibit hepatic OATP1B1 and OATP1B3, but there might be a risk when they are consumed as medicines or as dietary supplements.

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.

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.

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.

Probenecid is used to treat gout and hyperuricemia as well as increase plasma levels of antiviral drugs and antibiotics. In vivo, probenecid mainly inhibits the renal SLC22 organic anion transporters OAT1 (SLC22A6), OAT3 (SLC22A8), and URAT1 (SLC22A12). To understand the endogenous role of these transporters in humans, we administered probenecid to 20 healthy participants and metabolically profiled the plasma and urine before and after dosage. Hundreds of metabolites were significantly altered, indicating numerous drug-metabolite interactions. We focused on potential OAT1 substrates by identifying 97 metabolites that were significantly elevated in the plasma and decreased in the urine, indicating OAT-mediated clearance. These included signaling molecules, antioxidants, and gut microbiome products. In contrast, urate was the only metabolite significantly decreased in the plasma and elevated in the urine, consistent with an effect on renal reuptake by URAT1. Additional support comes from metabolomics analyses of our Oat1 and Oat3 knockout mice, where over 50% of the metabolites that were likely OAT substrates in humans were elevated in the serum of the mice. Fifteen of these compounds were elevated in both knockout mice, whereas six were exclusive to the Oat1 knockout and 4 to the Oat3 knockout. These may be endogenous biomarkers of OAT function. We also propose a probenecid stress test to evaluate kidney proximal tubule organic anion transport function in kidney disease. Consistent with the Remote Sensing and Signaling Theory, the profound changes in metabolite levels following probenecid treatment support the view that SLC22 transporters are hubs in the regulation of systemic human metabolism.

Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.

The OAT1 (

The aim of our study was to define the genotype-phenotype correlations of mutations in theDemographic characteristics of 108 patients with hyperphenylalaninemia (HPA) and 94 patients whose diagnosis was confirmed byMild HPA-not-requiring-treatment (NT) was found in 50.9% of the patients, and a classical phenylketonuria (PKU) phenotype was found in 25.9%. Forty-seven types of variants were identified. The predominant variants were p.Ala403Val (9.9%), p.Ala300Ser (9.4%), and c.1066-11GA (splicing) (9.4%). Missense mutations accounted for 68% of mutations and attenuated the clinical impact; splice variations were found in 14.8% of cases with severe features. The p.Thr380Met allele was specific to the mild HPA-NT group. The c.1066-11GA (splicing) allele was associated with classical PKU, whereas the p.Arg408Trp allele was linked to severe symptoms. Three variations of unknown clinical significance were discovered: c.706+4AT (splicing), c.843-5TC (splicing), and p.Thr323=. Of these variants, the patient who was homozygous for the c.843-5TC (splicing) allele related to the classical PKU phenotype. 70% of the patients who underwent tetrahydrobiopterin (BH4) test were responsive. Phenotypes that responded to BH4 treatment were mostly mild phenotypes.The

Tranilast, N-(3',4'-dimethoxycinnamoyl)-anthranilic acid, is an anti-allergic drug and is considered for use in the treatment of rheumatoid arthritis. Methotrexate, an antimetabolite and folate antagonist to treat some cancers, is also a first-line drug for RA. The aim of this study was to understand whether tranilast could inhibit renal uptake transporters (Oat1, Oat3, and Oct2) and whether MTX combined with TL would have drug-drug interactions. The results of kidney slices and HEK293T-OAT3 cell uptake experiments showed that TL (10 μM) could inhibit the uptake of penicillin G and MTX, which are substrates of OAT3. When TL (10 mg/kg) was combined with MTX (5 mg/kg), the area under the curve and peak concentration of MTX increased by 46.46% and 113.51%, respectively, while the pharmacokinetic process of tranilast (10 mg/kg) was not changed by methotrexate (5 mg/kg). TL could increase plasma exposure of MTX by inhibiting Oat3 in vitro and in vivo.

Febuxostat is a second-line xanthine oxidase inhibitor that undergoes extensive hepatic metabolism to yield its major acyl-β-D-glucuronide metabolite (febuxostat AG). It was recently reported that febuxostat inhibited organic anion transporter 3 (OAT3)-mediated uptake of enalaprilat. Here, we investigated the inhibition of febuxostat and febuxostat AG on OAT3 in transfected human embryonic kidney 293 cells. Our transporter inhibition assays confirmed the potent noncompetitive and competitive inhibition of OAT3-mediated estrone-3-sulfate transport by febuxostat and febuxostat AG with corresponding apparent K

The renal elimination of uremic toxins (UTs) can be potentially altered by drugs that inhibit organic anion transporters 1/3 (OAT1/OAT3). The objective of the present study was to determine whether the prescription of at least one OAT1/OAT3 inhibitor was associated with the plasma accumulation of certain UTs in kidney transplant recipients. We included 403 kidney transplant recipients. For each patient, we recorded all prescription drugs known to inhibit OAT1/OAT3. Plasma levels of four UTs (trimethylamine N-oxide (TMAO), indole acetic acid (IAA), para-cresylsulfate (pCS), and indoxylsulfate (IxS) were assayed using liquid chromatography-tandem mass spectrometry. Plasma UT levels were significantly higher among patients prescribed at least one OAT inhibitor (n = 311) than among patients not prescribed any OAT inhibitors (n = 92). Multivariate analysis revealed that after adjustment for age, estimated glomerular filtration rate (eGFR), plasma level of albumin and time since transplantation, prescription of an OAT1/OAT3 inhibitor was independently associated with the plasma accumulation of pCS (adjusted odds ratio (95% confidence interval): 2.11 (1.26; 3.61]). Our results emphasize the importance of understanding the interactions between drugs and UTs and those involving UT transporters in particular.

Small changes to molecules can have profound effects on their pharmacological activity as exemplified by the addition of the two-carbon acetyl group to make drugs more effective by enhancing their pharmacokinetic or pharmacodynamic properties. N-acetyl-d,l-leucine is approved in France for vertigo and its l-enantiomer is being developed as a drug for rare and common neurological disorders. However, the precise mechanistic details of how acetylation converts leucine into a drug are unknown. Here we show that acetylation of leucine switches its uptake into cells from the l-type amino acid transporter (LAT1) used by leucine to organic anion transporters (OAT1 and OAT3) and the monocarboxylate transporter type 1 (MCT1). Both the kinetics of MCT1 (lower affinity compared to LAT1) and the ubiquitous tissue expression of MCT1 make it well suited for uptake and distribution of N-acetyl-l-leucine. MCT1-mediated uptake of a N-acetyl-l-leucine as a prodrug of leucine bypasses LAT1, the rate-limiting step in activation of leucine-mediated signalling and metabolic process inside cells such as mTOR. Converting an amino acid into an anion through acetylation reveals a way for the rational design of drugs to target anion transporters.

Background This study aimed to evaluate the urate‐lowering effects of Yi‐Suan‐Cha and explore its underlying mechanisms in experimental hyperuricemia induced in rats. Methods Forty‐eight male SD rats were randomly allocated into normal control, model, allopurinol, benzbromarone, low‐dose Yi‐Suan‐Cha (0.2 g/ml), and high‐dose Yi‐Suan‐Cha (0.4 g/ml) groups (n = 8 rats per group). Rat models of hyperuricemia were established through intragastric administration of adenine 25 mg/kg + potassium oxalate 300 mg/kg for 3 weeks. After the last administration, serum uric acid, creatinine, and urea nitrogen levels were measured. Renal histopathology was observed by hematoxylin‐eosin staining. Xanthine oxidase level in serum and liver homogenates was measured by ELISA. The protein and mRNA expression of URAT1, ABCG2, OAT1, and GLUT9 in the kidney was detected by Western blotting and RT‐PCR, respectively. Results The serum uric acid levels were significantly lowered in all medication groups than in the model group. The benzbromarone and both Yi‐Suan‐Cha groups showed clear kidney structures with no obvious abnormalities. Compared with the normal control group, the model group showed increased URAT1/GLUT9 protein expression and decreased ABCG2/OAT1 protein expression. Compared with the model group, both Yi‐Suan‐Cha groups showed decreased URAT1/GLUT9 protein expression and increased ABCG2/OAT1 protein expression. Compared with that in the normal control group, URAT1/GLUT9 mRNA expression increased in the model group. Compared with the model group, the low‐dose and high‐dose Yi‐Suan‐Cha groups showed decreased URAT1/GLUT9 mRNA expression and increased ABCG2/OAT1 mRNA expression. Conclusion Yi‐Suan‐Cha may lower uric acid level by downregulating URAT1/GLUT9 expression and upregulating ABCG2/OAT1 expression., Effects of Yi‐Suan‐Cha on the protein levels of renal transporters. Proteins were extracted from the kidney for Western blot analysis of URAT1 (A, B), ABCG2 (A, C), OAT1 (A, D), and GLUT9 (A, E).

Camellia japonica bee pollen is one of the major types of bee pollen in China and exhibits antioxidant and anti-inflammatory activities. The aims of our study were to evaluate the effects and the possible mechanism of Camellia japonica bee pollen polyphenols on the treatment of hyperuricemia induced by potassium oxonate (PO). The results showed that Camellia japonica bee pollen ethyl acetate extract (CPE-E) owned abundant phenolic compounds and strong antioxidant capabilities. Administration with CPE-E for two weeks greatly reduced serum uric acid and improved renal function. It inhibited liver xanthine oxidase (XOD) activity and regulated the expression of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporter 1 (OAT1), organic cation transporter 1 (OCT1) and ATP-binding cassette superfamily gmember 2 (ABCG2) in kidneys. Moreover, CPE-E suppressed the activation of the toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB (TLR4/MyD88/NF-κB) signaling pathway and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in PO-treated mice, and related inflammatory cytokines were reduced. CPE-E also modulated gut microbiota structure, showing that the abundance of Lactobacillus and Clostridiaceae increased in hyperuicemic mice. This study was conducted to explore the protective effect of CPE-E on hyperuricemia and provide new thoughts for the exploitation of Camellia japonica bee pollen.

Multiple doses of tenofovir disoproxil fumarate (TDF) together with emtricitabine is effective for HIV preexposure prophylaxis (PrEP). TDF is converted to tenofovir (TFV) in circulation, which is subsequently cleared via tubular secretion by organic ion transporters (OATs; OAT1 and OAT3). Using in vitro kinetic parameters for TFV and the OAT1 and OAT3 inhibitor probenecid, a bottom‐up physiologically‐based pharmacokinetic model was successfully developed for the first time that accurately describes the probenecid–TFV interaction. This model predicted an increase in TFV plasma exposure by 60%, which was within 15% of the observed clinical pharmacokinetic data, and a threefold decrease in renal cells exposure following coadministration of a 600 mg TDF dose with 2 g probenecid. When compared with multiple‐dose regimens, a single‐dose probenecid‐boosted TDF regimen may be effective for HIV PrEP and improve adherence and safety by minimizing TFV‐induced nephrotoxicity by reducing TFV accumulation in renal cells.

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.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF’s active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5−100 μmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC 50 value of 4.91 μmol/L. TER (5 μmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells’ uptake of acyclovir and increased the plasma concentration.

Hyperuricaemia, which results from the overproduction and underexcretion of uric acid, has been linked with chronic renal dysfunction, cardiovascular diseases, diabetes and metabolic syndrome. However, available clinical drugs cannot simultaneously target the production and excretion of uric acid. Norathyriol, a natural xanthone, was expected to have the dual actions. We previously reported that norathyriol possessed potent anti-hyperuricaemic activity related to the inhibition of uric acid production. Here, we investigated the uricosuric actions in hyperuricaemic animals and explored the possible molecular mechanisms associated with renal urate transporters and xanthine oxidase (XO). The results showed that norathyriol (0.5–4.0 mg/kg) dose- and time-dependently decreased serum urate levels in uric acid-induced hyperuricaemic mice and markedly increased the fractional excretion of urate in oxonate-induced hyperuricaemic rats, demonstrating a promotion of urate excretion in the kidney. Further evidence showed that norathyriol markedly increased renal mRNA and protein expression of the secretory organic anion transporter 1 (OAT1) in hyperuricaemic mice and strengthened its transport function in vitro. Moreover, norathyriol also upregulated the mRNA expression of the secretory transporters OAT3, ATP-binding cassette transporter G2 and multidrug resistance protein 4, but not their protein expression. Changes in the expression of the reabsorptive transporters were not observed. This is the first report that norathyriol has uricosuric effects by targeting OAT1. Moreover, norathyriol significantly inhibited XO activity in an uncompetitive manner. Taken together, these findings suggest that norathyriol has the potential to be developed as a new anti-hyperuricaemic agent with dual actions that activate OAT1 and inhibit XO activity.

A bioactivity guided program exploring the interaction of phytochemicals in the entire plant Primula macrocalyx with the organic anion transporters (OAT1 and OAT3) and microorganisms led to the elucidation of ten known flavones (1–4, 6–10, 12) and two previously undescribed flavones (5, 11). The structures of the compounds were determined by extensive analysis of spectroscopic data, as well as by comparison with data from previous reports. Two known flavones (9, 12) are reported for the first time from the family Primulaceae. All compounds were evaluated for inhibition of OAT1 and OAT3. Six flavones (2, 3, 6–8, 12) showed potent inhibitory activity on OAT1, while seven flavones (2, 3, 6–9, 12) showed marked inhibitory activity on OAT3, with IC50 ≤ 10.0 µM. Antimicrobial activities of crude fractions against sixteen microorganisms were tested to give a target yeast strain Candida rugosa for further evaluation of MICs on the isolates. Three flavones (7, 8, 12) showed marked antifungal activity with MIC µM. To our knowledge, this study is the first to evaluate these flavones as inhibitors of the OAT1 and OAT3, and as antifungal agents.

The organic anion transporter 1 (OAT1) is mainly expressed in proximal tubule cells, where it mediates the renal uptake of endogenous and exogenous compounds. Thereby, it has enormous clinical relevance particularly in drug-drug interactions. The aim of the present in vitro study was to elucidate potential species dependent disparity of human and mouse OAT1 in handling of structural diverse drugs and pesticides. A basic functional comparison of the two transporters showed a similar time-dependent uptake of the substrate para-aminohippuric acid (PAH), the affinity (K

Phellodendri Chinensis Cortex (PC) is a traditional medicinal material used to treat gout and hyperuricemia (HUA) in China. Berberine (BBR), the main component of PC, possesses anti-hyperuricemic and anti-gout effects. However, BBR exhibits low bioavailability due to its extensive metabolism and limited absorption. Thus, the metabolites of BBR are believed to be the potential active forms responsible for its in vivo biological activities. Berberrubine (BRB), one of the major metabolites of BBR, exhibits appreciable biological activities even superior to BBR. In this work, the anti-hyperuricemic efficacy of BRB was investigated in HUA model mice induced by co-administration with intraperitoneal potassium oxonate (PO) and oral hypoxanthine (HX) for 7 days. Results showed that administration with BRB (6.25, 12.5, and 25.0 mg/kg) significantly decreased the serum levels of uric acid (UA) by 49.70%, 75.35%, and 75.96% respectively, when compared to the HUA group. In addition, BRB sharply decreased the levels of serum creatinine (CRE) (by 19.62%, 28.98%, and 38.72%, respectively) and blood urea nitrogen (BUN) (by 16.19%, 25.07%, and 52.08%, respectively) and reversed the PO/HX-induced renal histopathological damage dose-dependently. Additionally, BRB lowered the hepatic XOD activity, downregulated the expressions of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), upregulated expressions of organic anion transporter 1/3 (OAT1/3) and ATP-binding cassette transporter subfamily G member 2 (ABCG2) at both protein and mRNA levels, and suppressed the activation of the JAK2/STAT3 signaling pathway. In addition, BRB significantly decreased the levels of inflammatory mediators (IL-1β, IL-6, and TNF-α). In conclusion, our study indicated that BRB exerted anti-hyperuricemic effect, at least in part, via regulating the urate transporter expressions and suppressing the JAK2/STAT3 signaling pathway. BRB was believed to be promising for further development into a potential therapeutic agent for HUA treatment.