RESUMO
Pharmacokinetic changes induced by radiation following radiotherapy ("RT-PK" phenomenon) are of great significance to the effectiveness and safety of chemotherapeutic agents in clinical settings. The aims of this study were to clarify the organic anion transporters (Oats) involved in the "RT-PK" phenomenon of bestatin in rats following X-ray irradiation and to elucidate its potential mechanism via vitamin D signalling. Pharmacokinetic studies, uptake assays using rat kidney slices and primary proximal tubule cells, and molecular biological studies were performed. Significantly increased plasma concentrations and systemic exposure to bestatin were observed at 24 and 48 h following abdominal X-ray irradiation, regardless of oral or intravenous administration of the drugs in rats. Reduced renal clearance and cumulative urinary excretion of bestatin were observed at 24 and 48 h post-irradiation in rats following intravenous administration. The uptake of the probe substrates p-aminohippuric acid and oestrone 3-sulfate sodium in vitro and the expression of Oat1 and Oat3 in vivo were reduced in the corresponding models following irradiation. Moreover, the upregulation of the vitamin D receptor (Vdr) in mRNA and protein levels negatively correlated with the expressions and functions of Oat1 and Oat3 following irradiation. Additionally, elevated plasma urea nitrogen levels and histopathological changes were observed in rats after exposure to irradiation. The "RT-PK" phenomenon of bestatin occurs in rats after exposure to irradiation, possibly resulting in the regulation of the expressions and activities of renal Oats via activation of the Vdr signalling pathway.
Assuntos
Regulação para Baixo , Rim , Receptores de Calcitriol , Animais , Ratos , Receptores de Calcitriol/metabolismo , Masculino , Rim/metabolismo , Rim/efeitos da radiação , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Ratos Sprague-Dawley , Raios X , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/efeitos da radiação , Túbulos Renais Proximais/efeitos dos fármacos , Leucina/análogos & derivadosRESUMO
The organic anion transporters OAT1 (SLC22A6) and OAT3 (SLC22A8) are drug transporters that are expressed in the kidney, with well-established roles in the in vivo transport of drugs and endogenous metabolites. A comparatively unexplored potential function of these drug transporters is their contribution to the in vivo regulation of natural products (NPs) and their effects on endogenous metabolism. This is important for the evaluation of potential NP interactions with other compounds at the transporter site. Here, we have analyzed the NPs present in several well-established databases from Asian (Chinese, Indian Ayurvedic) and other traditions. Loss of OAT1 and OAT3 in murine knockouts caused serum alterations of many NPs, including flavonoids, vitamins, and indoles. OAT1- and OAT3-dependent NPs were largely separable based on a multivariate analysis of chemical properties. Direct binding to the transporter was confirmed using in vitro transport assays and protein binding assays. Our in vivo and in vitro results, considered in the context of previous data, demonstrate that OAT1 and OAT3 play a pivotal role in the handling of non-synthetic small molecule natural products, NP-derived antioxidants, phytochemicals, and nutrients (e.g., pantothenic acid, thiamine). As described by remote sensing and signaling theory, drug transporters help regulate redox states by meditating the movement of endogenous antioxidants and nutrients between organs and organisms. Our results demonstrate how dietary antioxidants and other NPs might feed into these inter-organ and inter-organismal pathways.
Assuntos
Antioxidantes , Produtos Biológicos , Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos Sódio-Independentes , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Animais , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Camundongos , Nutrientes/metabolismo , Camundongos Knockout , Humanos , Transporte Biológico , Rim/metabolismo , Flavonoides/farmacocinética , Flavonoides/metabolismoRESUMO
Hyperuricemia (HUA) is a metabolic disease characterized by the increase of serum uric acid (UA) level. Sargentodoxae Caulis (SC) is a commonly used herbal medicine for the treatment of gouty arthritis, traumatic swelling, and rheumatic arthritis in clinic. In this study, a total of fifteen compounds were identified in SC water extract using UHPLC-Q-TOF-MS/MS, including three phenolic acids, seven phenolic glycosides, four organic acids, and one lignan. Then, to study the hypouricemia effect of SC, a HUA mouse model was induced using a combination of PO, HX, and 20% yeast feed. After 14 days of treatment with the SC water extract, the levels of serum UA, creatinine (CRE), blood urea nitrogen (BUN) were reduced significantly, and the organ indexes were restored, the xanthine oxidase (XOD) activity were inhibited as well. Meanwhile, SC water extract could ameliorate the pathological status of kidneys and intestine of HUA mice. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting results showed that SC water extract could increase the expression of ATP binding cassette subfamily G member 2 (ABCG2), organic cation transporter 1 (OCT1), organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3), whereas decrease the expression of glucose transporter 9 (GLUT9). This study provided a data support for the clinical application of SC in the treatment of HUA.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Hiperuricemia , Ácido Úrico , Xantina Oxidase , Animais , Camundongos , Hiperuricemia/tratamento farmacológico , Masculino , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Úrico/sangue , Xantina Oxidase/metabolismo , Modelos Animais de Doenças , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Rim/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Transportadores de Ânions Orgânicos/metabolismo , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Hidroxibenzoatos/isolamento & purificação , Hidroxibenzoatos/farmacologiaRESUMO
PURPOSE: Adefovir (as dipivoxil) was selected as a probe drug in a previous transporter cocktail phenotyping study to assess renal organic anion transporter 1 (OAT1), with renal clearance (CLR) as the primary parameter describing renal elimination. An approximately 20% higher systemic exposure of adefovir was observed when combined with other cocktail components (metformin, sitagliptin, pitavastatin, and digoxin) compared to sole administration. The present evaluation applied a population pharmacokinetic (popPK) modeling approach to describe adefovir pharmacokinetics as a cocktail component in more detail. METHODS: Data from 24 healthy subjects were reanalyzed. After establishing a base model, covariate effects, including the impact of co-administered drugs, were assessed using forward inclusion then backward elimination. RESULTS: A one-compartment model with first-order absorption (including lag time) and a combination of nonlinear renal and linear nonrenal elimination best described the data. A significantly higher apparent bioavailability (73.6% vs. 59.0%) and a lower apparent absorption rate constant (2.29 h-1 vs. 5.18 h-1) were identified in the combined period compared to the sole administration period, while no difference was seen in renal elimination. The population estimate for the Michaelis-Menten constant (Km) of the nonlinear renal elimination was 170 nmol/L, exceeding the observed range of adefovir plasma maximum concentration, while the maximum rate (Vmax) of nonlinear renal elimination was 2.40 µmol/h at the median absolute estimated glomerular filtration rate of 105 mL/min. CONCLUSION: The popPK modeling approach indicated that the co-administration primarily affected the apparent absorption and/or prodrug conversion of adefovir dipivoxil, resulting in the minor drug-drug interaction observed for adefovir as a victim. However, renal elimination remained unaffected. The high Km value suggests that assessing renal OAT1 activity by CLR has no relevant misspecification error with the cocktail doses used.
Assuntos
Adenina , Modelos Biológicos , Organofosfonatos , Humanos , Organofosfonatos/farmacocinética , Organofosfonatos/sangue , Organofosfonatos/administração & dosagem , Adenina/análogos & derivados , Adenina/farmacocinética , Adenina/administração & dosagem , Masculino , Adulto , Feminino , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Interações Medicamentosas , Fenótipo , Pessoa de Meia-Idade , Adulto Jovem , Digoxina/farmacocinética , Digoxina/sangue , Digoxina/administração & dosagem , Metformina/farmacocinética , Metformina/administração & dosagem , Metformina/sangue , Fosfato de Sitagliptina/farmacocinética , Disponibilidade BiológicaRESUMO
Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.
Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes , Transportadores de Ânions Orgânicos , Animais , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Células Epiteliais/metabolismo , Transportadores de Ânions Orgânicos/metabolismoRESUMO
BACKGROUND: Disorders of purine metabolism are the main cause of hyperuricemia. Current drugs for the treatment of hyperuricemia usually cause a degree of cardiovascular damage. METHODS: This study aimed to investigate the therapeutic effects of Armillaria mellea fruiting body (AFB), Armillaria rhizomorph (AR) and Armillaria mellea fermentation product (after rhizomorphs removal) (AFP) on hyperuricemic mice. The hyperuricemia mouse model was established by oral administration of potassium oxonate 0.9 gâ kg-1 and hypoxanthine 0.5 gâ kg-1 for two weeks. Starting from the third week, the intragastric administration of the intervention drug group was as follows: Allopurinol 0.013 gâ kg-1, AFB (3.9 and 7.8 gâ kg-1), AR (3.9 and 7.8 gâ kg-1), AFP (1.95 and 3.9 gâ kg-1) once daily for 14 days. RESULTS: Results showed that AFB, AR, and AFP reduced the contents of serum uric acid, serum creatinine, and blood urea nitrogen in hyperuricemic mice and the mechanism of action might be through up-regulation of the expression levels of organic anion transporter 1/organic anion transporter 3 proteins in kidney tissue. AR and AFP both exhibited better uric acid-lowering effects than AFB, which may be due to the higher purine content of AFB. CONCLUSIONS: Armillaria mellea and its fermentation products can treat hyperuricemia by up-regulating OAT1 protein and OAT3 protein, reducing uric acid content in mice.
Assuntos
Armillaria , Mel , Hiperuricemia , Transportadores de Ânions Orgânicos , Camundongos , Animais , Hiperuricemia/induzido quimicamente , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Armillaria/metabolismo , Rim , Ácido Úrico/metabolismo , Ácido Úrico/farmacologia , Fermentação , alfa-Fetoproteínas , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/farmacologia , Transportadores de Ânions Orgânicos/uso terapêutico , Purinas/metabolismo , Purinas/farmacologia , Purinas/uso terapêuticoRESUMO
Organic anion transporters (OATs) of the SLC22 family have crucial roles in the transport of organic anions, including metabolites and therapeutic drugs, and in transporter-mediated drug-drug interactions. In the kidneys, OATs facilitate the elimination of metabolic waste products and xenobiotics. However, their transport activities can lead to the accumulation of certain toxic compounds within cells, causing kidney damage. Moreover, OATs are important drug targets, because their inhibition modulates the elimination or retention of substrates linked to diseases. Despite extensive research on OATs, the molecular basis of their substrate and inhibitor binding remains poorly understood. Here we report the cryo-EM structures of rat OAT1 (also known as SLC22A6) and its complexes with para-aminohippuric acid and probenecid at 2.1, 2.8 and 2.9 Å resolution, respectively. Our findings reveal a highly conserved substrate binding mechanism for SLC22 transporters, wherein four aromatic residues form a cage to accommodate the polyspecific binding of diverse compounds.
Assuntos
Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos , Ratos , Animais , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Microscopia Crioeletrônica , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Rim/metabolismoRESUMO
Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.
Assuntos
Transportadores de Ânions Orgânicos , Ratos , Animais , Transportadores de Ânions Orgânicos/metabolismo , Probenecid/farmacologia , Probenecid/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Eliminação Renal , Furosemida/farmacologia , Furosemida/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Ácido Piridóxico/metabolismo , Ácido Piridóxico/farmacologia , Interações Medicamentosas , Biomarcadores/metabolismo , Rim/metabolismoRESUMO
Cisplatin is a widely used chemotherapeutic agent to treat solid tumours in clinics. However, cisplatin-induced acute kidney injury (AKI) limits its clinical application. This study investigated the effect of hyperoside (a flavonol glycoside compound) on regulating AKI.The model of cisplatin-induced AKI was established, and hyperoside was preadministered to investigate its effect on improving kidney injury.Hyperoside ameliorated renal pathological damage, reduced the accumulation of SCr, BUN, Kim-1 and indoxyl sulphate in vivo, increased the excretion of indoxyl sulphate into the urine, and upregulated the expression of renal organic anion transporter 1 (Oat1). Moreover, evaluation of rat kidney slices demonstrated that hyperoside promoted the uptake of PAH (p-aminohippurate, the Oat1 substrate), which was confirmed by transient over-expression of OAT1 in HEK-293T cells. Additionally, hyperoside upregulated the mRNA expression of Oat1 upstream regulators hepatocyte nuclear factor-1α (HNF-1α) and pregnane X receptor (PXR).These findings indicated hyperoside could protect against cisplatin-induced AKI by promoting indoxyl sulphate excretion through regulating the expression and function of Oat1, suggesting hyperoside may offer a potential tactic for cisplatin-induced AKI treatment.
Assuntos
Injúria Renal Aguda , Cisplatino , Ratos , Animais , Cisplatino/efeitos adversos , Cisplatino/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Indicã/toxicidade , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Rim/metabolismoRESUMO
In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.
Assuntos
Cloretos , Proteína 1 Transportadora de Ânions Orgânicos , Animais , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Cloretos/metabolismo , Rim/metabolismo , Transporte Biológico , Ânions/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mamíferos/metabolismoRESUMO
Organic anion transporter 3 (OAT3) is predominantly expressed in the kidney and plays a vital role in drug clearance. Consequently, co-ingestion of two OAT3 substrates may alter the pharmacokinetics of the substrate. This review summarizes drug-drug interactions (DDIs) and herbal-drug interactions (HDIs) mediated by OAT3, and inhibitors of OAT3 in natural active compounds in the past decade. This provides a valuable reference for the combined use of substrate drugs/herbs for OAT3 in clinical practice in the future and for the screening of OAT3 inhibitors to avoid harmful interactions.
Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes , Medicamentos Sintéticos , Humanos , Rim , Interações Ervas-Drogas , Proteína 1 Transportadora de Ânions Orgânicos , Células HEK293RESUMO
The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.
Assuntos
Rim , Proteína 1 Transportadora de Ânions Orgânicos , Humanos , Rim/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Expressão Gênica , Células Epiteliais/metabolismoRESUMO
Organic anion transporters 1 and 3 (OAT1/3) occupy a key role in mediating renal elimination. Kynurenic acid (KYNA) was previously discovered as an effective endogenous biomarker to assess drug-drug interaction (DDI) for OAT inhibitors. Here, further in vitro and in vivo investigation was performed to characterize the elimination routes and feasibility of KYNA, along with other reported endogenous metabolites, as biomarkers of Oat1/3 inhibition in bile duct-cannulated (BDC) cynomolgus monkeys. Our results suggested that KYNA is a substrate of OAT1/3 and OAT2, but not OCT2, MATE1/2K, or NTCP, and that it shares comparable affinities between OAT1 and OAT3. Renal and biliary excretions and plasma concentration-time profiles of KYNA, pyridoxic acid (PDA), homovanillic acid (HVA), and coproporphyrin I (CP-I) were assessed in BDC monkeys dosed with either probenecid (PROB) at 100 mg/kg or the control vehicle. Renal excretion of KYNA, PDA, and HVA was determined to be the major elimination route. The maximum concentration and the area under the plasma concentration-time curve (Cmax and AUC0-24h) of KYNA were about 11.6- and 3.7-fold higher in the PROB group than in the vehicle group. Renal clearance of KYNA decreased by 3.2-fold, but biliary clearance (CLbile) was not altered after PROB administration. A similar trend was observed for PDA and HVA. Interestingly, an elevation of plasma concentration and reduction of CP-I CLbile were observed after PROB treatment, which suggested inhibition of the CP-I Oatp-Mrp2 transport axis by PROB. Overall, our results indicated that KYNA could potentially facilitate early and reliable assessment of DDI liabilities of Oat inhibition in monkeys. SIGNIFICANCE STATEMENT: This work reported renal excretion as the major elimination pathway for kynurenic acid, pyridoxic acid, and homovanillic acid. Administration of probenecid reduced renal clearance and increased plasma exposure of these biomarkers in monkeys, consistent with the observation in humans. These endogenous biomarkers discovered in monkeys could be potentially used to evaluate the clinical drug-drug interactions in the early phase of drug development.
Assuntos
Transportadores de Ânions Orgânicos , Probenecid , Humanos , Animais , Macaca fascicularis/metabolismo , Probenecid/farmacologia , Probenecid/metabolismo , Ácido Piridóxico , Ácido Homovanílico , Estudos de Viabilidade , Ácido Cinurênico , Transportadores de Ânions Orgânicos/metabolismo , Biomarcadores/metabolismo , Interações Medicamentosas , Rim/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismoRESUMO
Bentysrepinine (Y101) is a novel phenylalanine dipeptide for the treatment of hepatitis B virus. Renal excretion played an important role in the elimination of Y101 and its metabolites, M8 and M9, in healthy Chinese subjects, although the molecular mechanisms of renal excretion and potential drug-drug interactions (DDIs) remain unclear. The present study aimed to determine the organic anion transporters (OATs) involved in the renal disposition of Y101 and to predict the potential DDI between Y101 and entecavir, the first-line agent against HBV and a substrate of OAT1/3. Pharmacokinetic studies and uptake assays using rat kidney slices, as well as hOAT1/3-HEK293 cells, were performed to evaluate potential DDI. The co-administration of probenecid (an inhibitor of OATs) significantly increased the plasma concentrations and area under the plasma concentration-time curves of M8 and M9 but not Y101, while reduced renal clearance and the cumulative urinary excretion of M8 were observed in rats. The time course of Y101 and M8 uptake via rat kidney slices was temperature-dependent. Moreover, the uptake of M8 was inhibited significantly by probenecid and benzylpenicillin, but not by p-aminohippurate or tetraethyl ammonium. M8 was found to be a substrate of hOAT3, but Y101 is not a substrate of either hOAT1 or hOAT3. Additionally, the entecavir inhibited the uptake of M8 in the hOAT3-transfected cells and rat kidney slices in vitro. Interestingly, no significant changes were observed in the pharmacokinetic parameters of Y101, M8 or entecavir, regardless of intravenous or oral co-administration of Y101 and entecavir in rats. In conclusion, M8 is a substrate of OAT3 in rats and humans. Furthermore, M8 also mediates the DDI between Y101 and entecavir in vitro, mediated by OAT3. We speculate that it would be safe to use Y101 with entecavir in clinical practice. Our results provide useful information with which to predict the DDIs between Y101 and other drugs that act as substrates of OAT3.
Assuntos
Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos Sódio-Independentes , Humanos , Ratos , Animais , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Probenecid/metabolismo , Probenecid/farmacologia , Ratos Wistar , Células HEK293 , Dipeptídeos/metabolismo , Interações Medicamentosas , Rim/metabolismoRESUMO
The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.
Assuntos
Ácidos Cetoglutáricos , Proteína 1 Transportadora de Ânions Orgânicos , Humanos , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteínas de Membrana Transportadoras , LipídeosRESUMO
Organic anion transporter 1 (OAT1) expressed in the kidney plays an important role in the elimination of numerous anionic drugs used in the clinic. We report here that insulin, a pancreas-secreted hormone, regulated the expression and activity of kidney-specific OAT1 both in cultured cells and in rats. We showed that treatment of OAT1-expressing cells with insulin led to an increase in OAT1 expression, transport activity, and SUMOylation. Such insulin-induced increase was blocked by afuresertib, a specific inhibitor for protein kinase B (PKB), suggesting insulin regulates OAT1 through PKB signaling pathway. Furthermore, insulin stimulated transport activity and SUMOylation of endogenously expressed OAT1 in rat kidneys. In conclusion, our data support a remote sensing and signaling model, in which OAT1 plays an essential role in intercellular and inter-organ communication and in maintaining local and whole-body homeostasis. Such complex and dedicated communication is carried out by insulin, and PKB signaling and membrane sorting.
Assuntos
Insulina , Proteína 1 Transportadora de Ânions Orgânicos , Ratos , Animais , Insulina/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Tecnologia de Sensoriamento Remoto , Rim/metabolismo , Transdução de Sinais , Hormônios Pancreáticos/metabolismo , Insulina Regular Humana , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismoRESUMO
Organic anion transporter 3 (OAT3), an indispensable basolateral membrane transporter predominantly distributed in the kidney proximal tubules, mediated the systemic clearance of substrates including clinical drugs, nutrients, endogenous and exogenous metabolites, toxins, and critically sustains body homeostasis. Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3. Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms. The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. The upregulated OAT3 function by proteasome inhibition was attributed to the augment in maximum transport velocity and stability of membrane OAT3. Lastly, in vivo study using Sprague Dawley rats validated that proteasome inhibition using bortezomib induced enhancement of OAT3 ubiquitination and membrane expression in kidney. These data suggest that activity of proteasome but not lysosome could have an impact on the physiological function of OAT3, and proteasome displayed a promising target for OAT3 regulation in vitro and in vivo, and could be used in restoring OAT3 impairment under pathological conditions, avoiding OAT3-associated toxicity and diseases, ensuring drug efficacy and safety.
Assuntos
Antineoplásicos , Complexo de Endopeptidases do Proteassoma , Ratos , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Bortezomib/farmacologia , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ratos Sprague-Dawley , Proteína 1 Transportadora de Ânions OrgânicosRESUMO
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).
Assuntos
Microbioma Gastrointestinal , Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos Sódio-Independentes , Humanos , Redes e Vias Metabólicas , Metabolômica , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismoRESUMO
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 IC50 values close to or less than 10 µM. The valid analysis method and optimized systems developed in this study can be utilized to improve the predictability of drug interactions in humans and consequently aid in successful disease treatment by maintaining appropriate systemic exposures.
Assuntos
Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos Sódio-Independentes , Interações Medicamentosas , Fluoresceínas , HumanosRESUMO
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 (Km) was 94 µM for hOAT1 and 32 µM for mOat1. Inhibition experiments for hOAT1 and mOat1 provided IC50 values for glibenclamide of 5.1 and 6.4 µM and for probenecid of 31 and 11 µM. Than the interaction of hOAT1 and mOat1 with 23 drugs and 13 pesticides was examined. Three pesticides and thirteen drugs showed high inhibitory potency of 50% or more to both transporters. Furthermore, we identified rosiglitazone as a differential active inhibitor, with stronger inhibitory properties (IC50) to mOat1 (7.7 µM) than to hOAT1 (31 µM), and olmesartan with the most pronounced difference: The IC50 of hOAT1 (0.40 µM) was 48-fold lower than of mOat1 (19 µM). In conclusion, we found a strong correlation for the inhibitory effects of most drugs and pesticides on human and mouse OAT1. But the example of olmesartan shows that species differences have to be considered when extrapolating data from mouse to human.