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1.
Toxins (Basel) ; 13(10)2021 09 22.
Article in English | MEDLINE | ID: mdl-34678967

ABSTRACT

Research has shown that traditional dialysis is an insufficient long-term therapy for patients suffering from end-stage kidney disease due to the high retention of uremic toxins in the blood as a result of the absence of the active transport functionality of the proximal tubule (PT). The PT's function is defined by the epithelial membrane transporters, which have an integral role in toxin clearance. However, the intricate PT transporter-toxin interactions are not fully explored, and it is challenging to decouple their effects in toxin removal in vitro. Computational models are necessary to unravel and quantify the toxin-transporter interactions and develop an alternative therapy to dialysis. This includes the bioartificial kidney, where the hollow dialysis fibers are covered with kidney epithelial cells. In this integrated experimental-computational study, we developed a PT computational model that focuses on indoxyl sulfate (IS) transport by organic anionic transporter 1 (OAT1), capturing the transporter density in detail along the basolateral cell membrane as well as the activity of the transporter and the inward boundary flux. The unknown parameter values of the OAT1 density (1.15×107 transporters µm-2), IS uptake (1.75×10-5 µM-1 s-1), and dissociation (4.18×10-4 s-1) were fitted and validated with experimental LC-MS/MS time-series data of the IS concentration. The computational model was expanded to incorporate albumin conformational changes present in uremic patients. The results suggest that IS removal in the physiological model was influenced mainly by transporter density and IS dissociation rate from OAT1 and not by the initial albumin concentration. While in uremic conditions considering albumin conformational changes, the rate-limiting factors were the transporter density and IS uptake rate, which were followed closely by the albumin-binding rate and IS dissociation rate. In summary, the results of this study provide an exciting avenue to help understand the toxin-transporter complexities in the PT and make better-informed decisions on bioartificial kidney designs and the underlining transporter-related issues in uremic patients.


Subject(s)
Indican/metabolism , Kidney Tubules, Proximal/physiology , Organic Anion Transport Protein 1/physiology , Albumins/metabolism , Biological Transport , Computer Simulation , Humans , Membrane Transport Proteins/physiology , Toxins, Biological/metabolism , Uremia/metabolism
2.
J Pharmacol Exp Ther ; 368(1): 136-145, 2019 01.
Article in English | MEDLINE | ID: mdl-30361237

ABSTRACT

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of organic anion transporter (OAT) 1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single-dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 hour after receiving PROB (40 and 1000 mg orally) on days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased the area under the plasma concentration-time curve (AUC) of PDA by 3.1-fold (dosed alone; P < 0.05), and 3.2-fold (coadministered with FSM; P < 0.01), compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (P > 0.05) and 2.1-fold (P < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC, whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 vs. 3.3, respectively). PDA and HVA renal clearance (CL R) values were decreased by PROB to smaller extents compared with FSM (0.35-0.37 and 0.67-0.73 vs. 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and that its plasma exposure responds in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CL R values between subjects is more favorable relative to HVA.


Subject(s)
Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Pyridoxic Acid/blood , Adolescent , Adult , Biomarkers/blood , Cross-Over Studies , Healthy Volunteers , Humans , Male , Middle Aged , Reproducibility of Results , Young Adult
3.
Drug Metabol Drug Interact ; 29(3): 203-6, 2014.
Article in English | MEDLINE | ID: mdl-24870608

ABSTRACT

Selective cyclooxygenase-2 inhibitor lumiracoxib was shown to have the strong inhibitory potencies on the renal organic anion transporter (OAT)1 and also on OAT3 from drug transport experiments. The purpose of this study was to examine the effect of lumiracoxib on disposition of phenolsulfonphthalein (PSP) - which is mainly excreted into urine via OATs - from in vivo experiments. After the intravenous injection of PSP and lumiracoxib into rats, pharmacokinetic analysis was performed. After the intravenous injection of PSP as a bolus, its plasma concentration decreased time-dependently. Until 60 min after the injection, 51.1% of the dose was recovered into urine. The simultaneous administration of lumiracoxib increased the plasma levels of PSP and reduced its urinary recovery to 23.6% of the dose. The pharmacokinetic analysis using a two-compartment model exhibited that lumiracoxib affected the parameters implying the elimination of PSP. The present study demonstrates that lumiracoxib interfered with urinary excretion of PSP in rats.


Subject(s)
Coloring Agents/pharmacokinetics , Cyclooxygenase 2 Inhibitors/pharmacology , Diclofenac/analogs & derivatives , Phenolsulfonphthalein/pharmacokinetics , Animals , Diclofenac/pharmacology , Kidney/metabolism , Male , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Random Allocation , Rats, Wistar
4.
Pharm Res ; 31(5): 1120-32, 2014 May.
Article in English | MEDLINE | ID: mdl-24242937

ABSTRACT

PURPOSE: To conduct in vivo and in vitro experiments to investigate puerarin (PUR), an isoflavone C-glyoside, and elucidate its ability to alter methotrexate (MTX) transport and pharmacokinetics. METHODS: In vivo absorption studies, in vitro everted intestinal sac preparation, kidney slices in rats and bi-directional transport assay with mock-/MDCK-MDR1 cells, uptake studies in HEK293-OAT1/3 cells were employed to evaluate the interaction. RESULTS: In vivo and in vitro MTX absorption in rats were enhanced in combination with PUR. PUR inhibited digoxin efflux transport in MDCK-MDR1 monolayers with an IC50 value of 1.6 ± 0.3 µM, suggesting that the first target of drug interaction was MDR1 in the intestine during the absorption process. MTX renal clearance decreased significantly after simultaneous intravenous administration. MTX uptake in rat kidney slices and HEK293-OAT1/3 cells were markedly inhibited by PUR, suggesting that the second target of drug interaction was OATs located in the kidney. Moreover, concomitant administration of PUR reduced renal MTX accumulation and plasma levels of creatinine and BUN. CONCLUSIONS: Co-administration of PUR enhanced MTX exposure by inhibition of intestinal MDR1 and renal OAT1/3. Although the renal damage of MTX was improved by PUR, the high level exposure of MTX should be cautious in the clinical usage.


Subject(s)
Isoflavones/pharmacology , Methotrexate/pharmacology , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , ATP Binding Cassette Transporter, Subfamily B/physiology , Animals , Cell Line , Chromatography, Liquid , Dogs , Drug Interactions , HEK293 Cells , Humans , Male , Rats , Rats, Wistar , Tandem Mass Spectrometry
5.
Pharmacology ; 90(1-2): 66-77, 2012.
Article in English | MEDLINE | ID: mdl-22759781

ABSTRACT

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.


Subject(s)
Cell Membrane/metabolism , Kidney/physiology , Muscle, Smooth, Vascular/physiopathology , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Vascular Calcification/physiopathology , Animals , Aorta, Abdominal/metabolism , Arterial Pressure , Calcium/metabolism , Cholecalciferol , Disease Models, Animal , Kidney/pathology , Male , RNA, Messenger/metabolism , Rats , Rats, Wistar , Vascular Calcification/chemically induced , Vascular Calcification/metabolism , p-Aminohippuric Acid/metabolism
7.
J Vet Pharmacol Ther ; 35(3): 209-15, 2012 Jun.
Article in English | MEDLINE | ID: mdl-21623837

ABSTRACT

Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.


Subject(s)
Mammary Glands, Animal/cytology , Organic Anion Transporters/physiology , Animals , Cattle , Cells, Cultured , Epithelium/metabolism , Female , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/physiology , Organic Anion Transport Protein 1/genetics , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters/genetics , Organic Anion Transporters, Sodium-Dependent/genetics , Organic Anion Transporters, Sodium-Dependent/physiology , Protein Isoforms/genetics , Protein Isoforms/physiology , Real-Time Polymerase Chain Reaction
8.
Toxicol Appl Pharmacol ; 255(1): 48-56, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21640743

ABSTRACT

An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea. An LC-MS assay for quantitative determination of 3IS in mouse matrices was also developed. Following treatment with SCH 900424, 3IS levels were markedly increased in murine plasma and brain, thereby potentially contributing to renal- and central nervous system (CNS)-related rapid onset of toxicities. Furthermore, significant decrease in urinary excretion of 3IS in those animals due to compromised renal function may be associated with the elevation of 3IS in plasma and brain. These data suggest that 3IS has a potential to be a marker of renal and CNS toxicities during chemically-induced AKI in mice. In addition, based on the metabolomic analysis other statistically significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurenate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. This comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development.


Subject(s)
Acute Kidney Injury/chemically induced , Brain/metabolism , Indican/analysis , Metabolomics , Nicotinic Agonists/toxicity , Acute Kidney Injury/metabolism , Animals , Biomarkers , Indican/blood , Kidney/drug effects , Male , Mice , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology
9.
J Clin Invest ; 120(8): 2942-52, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20644253

ABSTRACT

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.


Subject(s)
Bile Acids and Salts/metabolism , Bilirubin/metabolism , Liver/metabolism , Methotrexate/pharmacokinetics , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Terfenadine/analogs & derivatives , Animals , Female , Liver-Specific Organic Anion Transporter 1 , Male , Mice , Mice, Knockout , Organic Anion Transport Protein 1/genetics , Rifampin/pharmacology , Terfenadine/pharmacokinetics
10.
J Pharmacol Exp Ther ; 332(2): 650-8, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19892921

ABSTRACT

Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. In transmembrane segment (TM) 12, residues Tyr-490 and dileucine Leu-503/Leu-504 were identified to be critical for hOAT1 function. Substitution of Tyr-490 with alanine led to a dramatic reduction in protein expression of hOAT1 and its transport activity. The contribution of the side chain of Tyr-490 to transport activity was then evaluated by replacing this residue with Trp or Phe. Substitution of Tyr-490 with Trp or Phe partially or fully recovered the protein expression of hOAT1 and its transport activity, respectively, that were lost by substitution of Tyr-490 with alanine, suggesting that the aromatic ring and the size of the side chain of Tyr-490 are critical for hOAT1 expression and function. Studies with protease inhibitors and pulse-chase labeling further showed that the loss of expression of hOAT1 and its transport activity by replacing Tyr-490 with alanine resulted from accelerated degradation of the transporter, whereas its maturation efficiency was not affected. In contrast to Tyr-490, substitution of Leu-503/Leu-504 with alanine also resulted in complete loss of protein expression of hOAT1 and its transport activity. However, such loss of protein expression could not be prevented by treating mutant-expressing cells with protease inhibitors. Pulse-chase experiments showed that the mutant transporter (L503/L504A) was trapped in the endoplasmic reticulum without conversion into mature form of the transporter. Our results are the first to highlight the central role of TM 12 in maintaining the stability and in promoting the maturation efficiency of hOAT1.


Subject(s)
Organic Anion Transport Protein 1/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , COS Cells , Chlorocebus aethiops , Humans , LLC-PK1 Cells , Mutagenesis, Site-Directed , Mutant Proteins , Organic Anion Transport Protein 1/biosynthesis , Organic Anion Transport Protein 1/genetics , Organic Anion Transport Protein 1/physiology , Swine , Transfection
11.
J Toxicol Sci ; 34 Suppl 2: SP259-65, 2009.
Article in English | MEDLINE | ID: mdl-19571478

ABSTRACT

The kidney is susceptible to drugs and environmental substances because of its anatomical and functional reasons, one of which is the existence of drug transport systems in proximal tubular cells. Among those, Organic anion transporter family (OAT family) plays the central role in elimination of drugs from the kidney and development of nephrotoxicity. Regarding drug nephrotoxicity in children, development of the child and the kidney should also be taken into account. This review focuses on the mechanisms of toxic nephropathy in children with special attention to the OAT family.


Subject(s)
Drug-Related Side Effects and Adverse Reactions , Kidney/drug effects , Organic Anion Transporters/physiology , Pharmaceutical Preparations/metabolism , Adolescent , Child , Child, Preschool , Humans , Infant , Infant, Newborn , Kidney/metabolism , Organic Anion Transport Protein 1/metabolism , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters/metabolism
12.
Toxicology ; 258(1): 56-63, 2009 Apr 05.
Article in English | MEDLINE | ID: mdl-19378467

ABSTRACT

PF1, an anti-inflammatory drug candidate, was nephrotoxic in cynomolgus monkeys in a manner that was qualitatively comparable to that observed with the two previous exploratory drug candidates (PF2and PF3). Based on the severity of nephrotoxicity, PF1 ranked between the other two compounds, withPF2 inducing mortality at all doses and PF3 eliciting only mild nephrotoxicity. To further characterize nephrotoxicity in monkeys and enable direct comparisons with humans, primary cultures of proximal tubular (PT) cells from monkey and human kidneys were used as in vitro tools, using lactate dehydrogenase release as the biomarker of cytotoxicity. In both human and monkey PT cells, PF2was by far the most cytotoxic compound of the three drugs. PF1 exhibited modest cytotoxicity at the highest concentration tested in human PT cells but none in monkey kidney cells whereas PF3 exhibited the reverse pattern.Because these drugs are organic anions, mechanistic studies using human organic anion transporters 1 and 3 (hOAT1 andhOAT3) transfected cell lines were pursued to evaluate the potential of these compounds to interact with these transporters. All three drugs exhibited high affinity for hOAT3 (PF1 exhibited the lowest IC50 of 6M) but only weakly interacted with hOAT1 (with no interaction found for PF2). PF2 was a strong hOAT3 (not hOAT1) substrate, whereas PF1 and PF3 were substrates for both hOAT1 and hOAT3.Upon pretreatment of monkeys with the OAT substrate probenecid, PF3 systemic exposure (AUC) and half-life (t1/2) increased approximately 2-fold whereas clearance (CL) and volume of distribution (Vdss) decreased, as compared to naïve monkeys. This indicated that PF3 competed with probenecid for hOAT1 and/or hOAT3mediated elimination of PF3. Thus, hOAT1 and/or hOAT3 may be responsible for the uptake of this series of drugs in renal PT cells, which may directly or indirectly lead to the observed nephrotoxicity in vivo.


Subject(s)
Anti-Inflammatory Agents/toxicity , Drugs, Investigational/toxicity , Kidney Tubules, Proximal/drug effects , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Cells, Cultured , Drug Evaluation, Preclinical/methods , Drugs, Investigational/chemistry , Drugs, Investigational/pharmacokinetics , Humans , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Macaca fascicularis , Organic Anion Transport Protein 1/genetics , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/genetics , Organic Anion Transporters, Sodium-Independent/physiology , Predictive Value of Tests , Transfection
13.
J Biol Chem ; 284(5): 2672-2679, 2009 Jan 30.
Article in English | MEDLINE | ID: mdl-19028678

ABSTRACT

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.


Subject(s)
Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Protein Kinase C/metabolism , Animals , Biological Transport , Enzyme Activation , In Vitro Techniques , Mice , Mice, Knockout , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Rats , Rats, Sprague-Dawley
14.
Am J Physiol Endocrinol Metab ; 296(2): E378-83, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19088254

ABSTRACT

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney. In the current study, we examined the regulation of hOAT1 by ANG II in kidney COS-7 cells. ANG II induced a concentration- and time-dependent inhibition of hOAT1 transport activity. Such inhibition mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased maximal velocity without significant change in Michaelis constant. ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine, a general protein kinase C (PKC) inhibitor. To obtain further information on which PKC isoform mediates ANG II regulation of hOAT1 activity, cellular distribution of various PKC isoforms was examined in cells treated with or without ANG II. We showed that ANG II treatment resulted in a significant translocation of PKCalpha from cytosol to membrane, and such translocation was blocked by treating hOAT1-expressing cells with Gö-6976, a PKCalpha-specific inhibitor. We further showed that ANG II-induced inhibition of hOAT1 activity and retrieval of hOAT1 from the cell surface could also be prevented by treating hOAT1-expressing cells with Gö-6976. We concluded that ANG II inhibited hOAT1 activity through activation of PKCalpha, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.


Subject(s)
Angiotensin II/pharmacology , Organic Anion Transport Protein 1/metabolism , Protein Kinase C-alpha/physiology , Animals , Antigens, Surface/metabolism , COS Cells , Carbazoles/pharmacology , Chlorocebus aethiops , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/physiology , Organic Anion Transport Protein 1/physiology , Protein Kinase C-alpha/antagonists & inhibitors , Protein Transport/drug effects , p-Aminohippuric Acid/pharmacokinetics
15.
Am J Physiol Renal Physiol ; 295(1): F247-52, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18480179

ABSTRACT

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of beta-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.


Subject(s)
Cholestasis/physiopathology , Hyperbilirubinemia/physiopathology , Kidney/physiopathology , Organic Anion Transporters, Sodium-Independent/physiology , Adaptation, Biological , Animals , Bile Acids and Salts/metabolism , Cefotiam/pharmacokinetics , Humans , Male , Organic Anion Transport Protein 1/physiology , Rats
16.
Basic Clin Pharmacol Toxicol ; 103(1): 1-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18373647

ABSTRACT

Perfluorooctanoic acid, an environmental contaminant, is found in both wild animals and human beings. There are large species and sex differences in the renal excretion of perfluorooctanoic acid. In the present study, we aimed to characterize organic anion transporters 1-3 (OAT1-3) in human beings and rats to investigate whether the species differences in the elimination kinetics of perfluorooctanoic acid from the kidneys can be attributed to differences in the affinities of these transporters for perfluorooctanoic acid. We used human (h) and rat (r) OAT transient expression cell systems and measured the [(14)C] perfluorooctanoic acid transport activities. Both human and rat OAT1 and OAT3 mediated perfluorooctanoic acid transport to similar degrees. Specifically, the kinetic parameters, K(m), were 48.0 +/- 6.4 microM for h OAT1; 51.0 +/- 12.0 microM for rOAT1; 49.1 +/- 21.4 microM for hOAT3 and 80.2 +/- 17.8 microM for rOAT3, respectively. These data indicate that both human and rat OAT1 and OAT3 have high affinities for perfluorooctanoic acid and that the species differences in its renal elimination are not attributable to affinity differences in these OATs between human beings and rats. In contrast, neither hOAT2 nor rOAT2 transported perfluorooctanoic acid. In conclusion, OAT1 and OAT3 mediated perfluorooctanoic acid transport in vitro, suggesting that these transporters also transport perfluorooctanoic acid through the basolateral membrane of proximal tubular cells in vivo in both human beings and rats. Neither human nor rat OAT2 mediated perfluorooctanoic acid transport. Collectively, the difference between the perfluorooctanoic acid half-lives in human beings and rats is not likely to be attributable to differences in the affinities of these transporters for perfluorooctanoic acid.


Subject(s)
Caprylates/pharmacokinetics , Caprylates/urine , Fluorocarbons/pharmacokinetics , Fluorocarbons/urine , Kidney/metabolism , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Animals , Biological Transport , Cell Line , Humans , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Rats , Species Specificity , Transfection
17.
Pflugers Arch ; 457(1): 223-31, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18365245

ABSTRACT

Glutaric acidurias are rare inherited neurodegenerative disorders accompanied by accumulation of the metabolites glutarate (GA) and 3-hydroxyglutarate (3OHGA), glutaconate, L: -, or D: -2-hydroxyglutarate (L: -2OHGA, D: -2OHGA) in all body fluids. Oocytes expressing the human (h) sodium-dicarboxylate cotransporter (NaDC3) showed sodium-dependent inward currents mediated by GA, 3OHGA, L: -, and D: -2OHGA. The organic anion transporters (OATs) were examined as additional transporters for GA derivatives. The uptake of [(3)H]p-aminohippurate in hOAT1-transfected human embryonic kidney (HEK293) cells was inhibited by GA, 3OHGA, D: -, or L: -2OHGA in a concentration-dependent manner. None of these compounds affected the hOAT3-mediated uptake of [(3)H]estrone sulfate (ES). In hOAT4-expressing cells and oocytes, ES uptake was strongly increased by intracellular GA derivatives. The data provide a model for the concerted action of OAT1 and NaDC3 mediating the basolateral uptake, and OAT4 mediating apical secretion of GA derivatives from proximal tubule cells and therefore contribute to the renal clearance of these compounds.


Subject(s)
Glutarates/metabolism , Glutarates/urine , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Animals , Cell Line , Dicarboxylic Acid Transporters/metabolism , Estrone/physiology , Female , Glutarates/pharmacology , Humans , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Kinetics , Oocytes/drug effects , Oocytes/metabolism , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Dependent/metabolism , Organic Anion Transporters, Sodium-Independent/genetics , RNA, Complementary/biosynthesis , RNA, Complementary/genetics , Sodium Channels/drug effects , Sodium Channels/metabolism , Sodium Channels/physiology , Substrate Specificity/physiology , Symporters/metabolism , Transfection , Xenopus laevis , p-Aminohippuric Acid/metabolism
18.
Am J Physiol Renal Physiol ; 294(4): F867-73, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18216144

ABSTRACT

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.


Subject(s)
Oocytes/physiology , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Sodium Chloride Symporter Inhibitors/pharmacology , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Animals , Bendroflumethiazide/pharmacology , Diuretics/pharmacology , Female , Furosemide/pharmacology , Insulin/pharmacology , Oocytes/drug effects , Organic Anion Transport Protein 1/drug effects , Organic Anion Transporters, Sodium-Independent/drug effects , Xenopus , p-Aminohippuric Acid/pharmacology
19.
J Pharmacol Exp Ther ; 324(2): 784-90, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18029548

ABSTRACT

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.


Subject(s)
Hepatocyte Nuclear Factor 1-alpha/physiology , Hepatocyte Nuclear Factor 1-beta/physiology , Organic Anion Transport Protein 1/physiology , Transcription, Genetic/physiology , Animals , Base Sequence/physiology , Cell Line , Female , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-beta/genetics , Humans , Male , Mice , Mice, Knockout , Molecular Sequence Data , Organic Anion Transport Protein 1/genetics , Protein Binding/physiology
20.
J Pharm Pharm Sci ; 10(1): 71-85, 2007.
Article in English | MEDLINE | ID: mdl-17498396

ABSTRACT

PURPOSE: Mycophenolic acid (MPA), an immunosuppressant, is excreted as its glucuronized form, MPAG. In humans, MPAG is mostly excreted into urine, whereas more than 80% of the dose is excreted into bile in rats. The aim of this study was to clarify the cause of the species difference. We investigated whether MPAG is a substrate of human organic anion transporters (hOATs), and we compared the affinities of multi-drug resistance-associated protein 2 (MRP2) for MPAG in rats and humans. METHODS: The inhibitory effects of MPAG on the uptake of typical substrates via hOAT1 and hOAT3 were determined using HeLa cells heterologously expressing hOAT1 and Xenopus laevis oocytes heterologously expressing hOAT3. MPAG transport activity via hOAT1 and hOAT3 was determined by the two-microelectrode voltage-clamp technique using Xenopus laevis oocytes expressing hOAT1 and hOAT3. The affinities of MPAG for hMRP2 and rMrp2 were determined by the inhibitory effects of MPAG on p-aminohippuric acid (a typical substrate) uptake using membrane vesicles expressing hMRP2 or rMrp2. RESULTS: MPAG inhibited the uptake of PAH via hOAT1 and hOAT3, and calculated IC50 values were 222.6+/-26.6 microM and 41.5+/-11.5 microM, respectively. However, MPAG was not transported by hOAT1 and hOAT3. MPAG strongly inhibited the uptake of PAH via both rMrp2 and hMRP2. However, the magnitudes of inhibitory effects were different. The calculated IC50 values were 286.2+/-157.3 microM and 1036.8+/-330.5 microM, respectively. CONCLUSION: MPAG is not a substrate but is an inhibitor of hOAT1 and hOAT3. The affinity of rMRP2 to MPAG was about 3.6 times as high as that of hMRP2. Therefore, the difference of affinity between hMRP2 and rMrp2 is a possible mechanism of the difference of excretion ratio of MPAG between rats and human.


Subject(s)
Glucuronides/metabolism , Immunosuppressive Agents/pharmacokinetics , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Mycophenolic Acid/pharmacokinetics , Animals , Bile/metabolism , HeLa Cells , Humans , Male , Multidrug Resistance-Associated Protein 2 , Organic Anion Transport Protein 1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Rats , Rats, Wistar , Species Specificity , Xenopus laevis , p-Aminohippuric Acid/pharmacokinetics
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