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1.
J Asian Nat Prod Res ; 21(8): 754-771, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30606060

ABSTRACT

Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.


Subject(s)
Diterpenes/pharmacology , Organic Anion Transport Protein 1/antagonists & inhibitors , Animals , CHO Cells , Cell Proliferation/drug effects , Cricetulus , Diterpenes/pharmacokinetics , Herb-Drug Interactions , Humans , Molecular Docking Simulation , Organic Anion Transport Protein 1/analysis , Organic Anion Transport Protein 1/chemistry , Organic Anion Transport Protein 1/genetics , Probenecid/chemistry , Probenecid/pharmacology , p-Aminohippuric Acid/pharmacokinetics
2.
Mol Pharm ; 13(9): 3130-40, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27467266

ABSTRACT

One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.


Subject(s)
Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Renal Insufficiency, Chronic/metabolism , Animals , Fluoresceins/metabolism , Humans , Models, Biological , Organic Anion Transport Protein 1/analysis , Organic Anion Transporters, Sodium-Independent/analysis
3.
J Pharm Sci ; 102(9): 3302-8, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23630107

ABSTRACT

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.


Subject(s)
Kidney/metabolism , Organic Anion Transport Protein 1/analysis , Organic Anion Transporters, Sodium-Independent/analysis , Organic Cation Transport Proteins/analysis , Aged , Gene Expression , Humans , Immunohistochemistry , Kidney/ultrastructure , Male , Middle Aged , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Organic Cation Transport Proteins/genetics , Organic Cation Transporter 2 , RNA, Messenger/analysis , RNA, Messenger/genetics
4.
Reprod Toxicol ; 26(3-4): 220-30, 2008.
Article in English | MEDLINE | ID: mdl-18926897

ABSTRACT

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1-3 and 6, Bsep and Bcrp, Oct1-2, Oat1-3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague-Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood-brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.


Subject(s)
ATP-Binding Cassette Transporters/analysis , Blood Proteins/analysis , Cytochrome P-450 Enzyme System/analysis , ATP-Binding Cassette Transporters/genetics , Age Factors , Animals , Animals, Newborn , Catecholamine Plasma Membrane Transport Proteins/analysis , Catecholamine Plasma Membrane Transport Proteins/genetics , Cytochrome P-450 Enzyme System/genetics , Female , Male , Microsomes, Liver/enzymology , Organic Anion Transport Protein 1/analysis , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Independent/analysis , Organic Anion Transporters, Sodium-Independent/genetics , Rats , Rats, Sprague-Dawley
5.
Am J Nephrol ; 28(5): 772-83, 2008.
Article in English | MEDLINE | ID: mdl-18441523

ABSTRACT

BACKGROUND/AIMS: Renal organic anion transporters (OAT1 and OAT3) localized in the basolateral membrane mediate the uptake of organic anions from the blood into proximal tubules. This study aimed to examine the effects of renal ischemia/reperfusion injury (IRI) on the expression of cortical renal OAT1 and OAT3 and the functional impact. METHODS: Male rats underwent a right nephrectomy and clamping of the left renal pedicle for 50 min or sham operation, followed by reperfusion for 1, 2, 4 and 6 days. The expression of OAT1 and OAT3 was detected by RT-PCR, immunohistochemistry and Western blot analysis. Na(+)-K(+)-ATPase activity was also estimated. RESULTS: The renal clearance of para-aminohippurate was significantly decreased on day 1 in IRI rats compared with sham-operated rats and returned to normal when the tubular injury recovered. There were significant increases in the mRNA and protein levels of OAT1 and OAT3 in renal cortex homogenates and basolateral membranes on day 1 after IRI, while on days 2 and 4 after IRI, the renal expression of OAT1 and OAT3 decreased gradually but was still significantly higher than that of the sham-operated rats. The Na(+)-K(+)-ATPase activity in renal cortex homogenates decreased significantly on day 1 after IRI but gradually increased on days 2, 4 and 6. CONCLUSIONS: Renal para-aminohippurate clearance was depressed in response to IRI; however, the expressions of renal cortex OAT1 and OAT3 were significantly elevated in the early stage of IRI which may have substantial impact on renal excretion of some drugs and toxic compounds.


Subject(s)
Kidney Cortex/chemistry , Organic Anion Transport Protein 1/analysis , Organic Anion Transporters, Sodium-Independent/analysis , Reperfusion Injury/metabolism , Up-Regulation , Animals , Blotting, Western , Immunohistochemistry , Male , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , p-Aminohippuric Acid/metabolism
6.
J Histochem Cytochem ; 55(6): 575-84, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17312013

ABSTRACT

We have previously shown that postischemic injury to renal allografts results in profound impairment of p-aminohippuric acid (PAH) extraction. To elucidate the cellular integrity of the human organic anion transporter 1 (hOAT1) in postischemic acute renal failure (ARF), immunohistochemical analysis of hOAT1 was performed in cadaveric renal allografts using confocal microscopy for three-dimensional reconstruction of serial optical images. Biopsy samples were obtained from 10 cadaveric renal allografts 1 hr after reperfusion during transplant operation. Control tissues were obtained from four living donors of healthy kidneys immediately before an arterial clamp was applied to the renal artery. Control tissues demonstrated hOAT1 distributed to basolateral membrane of proximal tubule cells. In contrast, maldistribution of hOAT1 to cytoplasm and/or diminution of the protein was noted in cadaveric allografts. Characteristics of maldistribution were variable: disappearance of lateral distribution, diffuse cytoplasmic aggregates, apical cytoplasmic aggregates, and disappearance of the staining. In addition, iothalamate and PAH clearances were performed on posttransplant days 3-7 in 18 recipients of a cadaveric renal allograft. PAH clearance was depressed <250 ml/min in all but three subjects. We conclude that reperfused, transplanted kidneys exhibit maldistribution of hOAT1 in proximal tubule cells, resulting in impairment of PAH clearance. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.


Subject(s)
Kidney Transplantation/methods , Kidney/metabolism , Organic Anion Transport Protein 1/analysis , Adult , Cadaver , Chromatography, High Pressure Liquid , Female , Humans , Immunohistochemistry , Iothalamic Acid/pharmacokinetics , Ischemia/physiopathology , Kidney/physiopathology , Kidney/surgery , Kidney Tubules, Proximal/metabolism , Living Donors , Male , Metabolic Clearance Rate , Microscopy, Fluorescence , Middle Aged , Reperfusion , Tissue Donors , Transplantation, Homologous , p-Aminohippuric Acid/pharmacokinetics
7.
J Ren Nutr ; 16(3): 199-203, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16825019

ABSTRACT

Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic renal failure (CRF). Indoxyl sulfate is taken up by renal proximal tubular cells through organic anion transporters 1 and 3 (OAT1/3), and is accumulated in the renal proximal tubular cells of uremic rats. To determine whether indoxyl sulfate is accumulated in human OAT1/3 (hOAT1/3)-positive renal proximal tubular cells, localization of indoxyl sulfate and hOAT1/3 in the kidneys of CRF patients was determined by immunohistochemistry. Kidney samples were obtained by autopsy from 9 CRF patients (mean serum creatinine 4.7 mg/dL, ranging from 2.0 to 14.5 mg/dL) and 9 patients with non-kidney disease (mean serum creatinine 0.6 mg/dL, ranging from 0.4 to 0.9 mg/dL). Immunohistochemistry was performed using antibodies against indoxyl sulfate, hOAT1, and hOAT3. Indoxyl sulfate was localized in the hOAT1- and hOAT3-positive renal tubular cells in the kidneys of CRF patients. The indoxyl sulfate-positive area in the kidneys was markedly increased in the kidneys of CRF patients compared with patients with non-kidney disease. The indoxyl sulfate-positive area was positively correlated with serum creatinine. In conclusion, in CRF patients, indoxyl sulfate is accumulated in the tubular cells with hOAT1 and/or hOAT3 localized at the basolateral membrane. The extent of indoxyl sulfate accumulation in the kidneys is more prominent in those patients with more severe CRF.


Subject(s)
Indican/metabolism , Kidney Failure, Chronic/metabolism , Kidney Tubules/metabolism , Organic Anion Transport Protein 1/analysis , Organic Anion Transporters, Sodium-Independent/analysis , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Indican/analysis , Kidney/chemistry , Kidney Tubules/chemistry , Kidney Tubules, Distal/chemistry , Kidney Tubules, Proximal/chemistry , Loop of Henle/chemistry , Male , Middle Aged
8.
Pflugers Arch ; 450(4): 269-79, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15895249

ABSTRACT

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.


Subject(s)
Kidney Cortex/cytology , Actins/analysis , Animals , Female , Immunohistochemistry , Kidney Cortex/physiology , Low Density Lipoprotein Receptor-Related Protein-2/analysis , Organic Anion Transport Protein 1/analysis , Organic Anion Transporters, Sodium-Independent/analysis , Rats , Rats, Wistar , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/analysis , Sodium-Potassium-Exchanging ATPase/analysis , Tubulin/analysis , p-Aminohippuric Acid/metabolism
9.
Exp Eye Res ; 80(1): 61-72, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15652527

ABSTRACT

PURPOSE: To identify and localize the expression of multispecific organic anion transporting polypeptides (Oatps/OATPs) in the ciliary body epithelium and to investigate their possible involvement in the transport of the antiglaucoma agent unoprostone. METHODS: Oatps/OATPs were detected by immunoblot analysis and by immunofluorescence microscopy in homogenized and fixed rat and human ciliary body samples using specific polyclonal antibodies. Transport of 3H-labelled unoprostone was measured in Oatp/OATP expressing Xenopus laevis oocytes. RESULTS: Immunoblots of ciliary body extracts were positive for rat Oatp1a4, Oatp1a5 and Oatp1b2 and for human OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. Confocal immunofluorescence microscopy localized Oatp1a4 and Oatp1b2 as well as all immunoblot positive human OATPs at the basolateral plasma membrane of the non-pigmented rat and human ciliary body epithelium, respectively. However, for human OATPs additional regional differences in expression were found with OATP1A2 and OATP1C1 being expressed only in the pars plana of human ciliary body epithelium. Furthermore, OATP1C1, OATP3A1 and OATP4A1 were also expressed at the basolateral plasma membrane of the pars plana pigmented epithelium. And finally, deesterified unoprostone carboxylate was found to be transported by OATP1A2, OATP2B1 and OATP4A1 with approximate K(m)-values of 93, 91 and 132 microm, respectively. CONCLUSIONS: Several multispecific organic anion transporting polypeptides are expressed at the basolateral plasma membrane of the non-pigmented, and to a lesser extent also of the pigmented, epithelium in rat and human ciliary body. These Oatps/OATPs can account for the previously suggested 'liver-like' transport functions of mammalian ciliary body epithelium.


Subject(s)
Ciliary Body/metabolism , Dinoprost/analogs & derivatives , Organic Anion Transporters/analysis , Adult , Aged , Aged, 80 and over , Animals , Antibody Specificity/immunology , Antihypertensive Agents/analysis , Biological Transport , Cell Line , Ciliary Body/immunology , Dinoprost/analysis , Epithelium/immunology , Epithelium/metabolism , Female , Fluorescent Antibody Technique/methods , Humans , Liver-Specific Organic Anion Transporter 1/analysis , Liver-Specific Organic Anion Transporter 1/metabolism , Male , Middle Aged , Organic Anion Transport Protein 1/analysis , Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters/metabolism , Peptides/metabolism , Rats , Rats, Inbred BN
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