Anion Transport System Research Articles

Comparative renal excretion of VX-702, a novel p38 MAPK inhibitor, and methotrexate in the perfused rat kidney model

Context: VX-702 is a novel p38 mitogen-activated protein kinase inhibitor being developed to treat rheumatoid arthritis. Objective: To characterize the renal excretion profile of VX-702 using the isolated perfused rat kidney (IPRK) model. Methods: Studies were performed to assess the dose linearity of VX-702 excretion and to evaluate the effect of inhibitors of organic anion (probenecid) and organic cation (cimetidine) transport systems on VX-702 disposition. VX-702 excretion was studied over a range of doses targeting concentrations between 100 and 600 ng/mL. VX-702 (600 ng/mL) was also co-perfused with probenecid (1 mM) and cimetidine (2 mM). The results were compared to parallel experiments performed with methotrexate (MTX). Results: VX-702 excretion was linear over the range of doses studied, and clearance data were consistent with net reabsorption by the kidney. Transport inhibition studies indicate that VX-702 is not a substrate for renal organic anion and organic cation transport systems. MTX (500 ng/mL) also displayed net reabsorption in the IPRK, but secretory transport was inhibited upon co-administration with probenecid. This finding is consistent with previous IPRK studies that demonstrated inhibitory effects of NSAIDS on MTX excretion. Conclusion: Overall, this study suggests that a renal drug-drug interaction between VX-702 and MTX would be unlikely if these medications were co-administered.

Active intestinal secretion of new quinolone antimicrobials and the partial contribution of P-glycoprotein.

Transport of quinolone antimicrobials and the contribution of the secretory transporter P-glycoprotein were studied in-vivo and in-vitro. In rat intestinal tissue (Ussing chambers method) and human Caco-2 cells (Transwell method), grepafloxacin showed secretory-directed transport. In both experimental systems, the secretory-directed transport was decreased by ciclosporin A, an inhibitor of P-glycoprotein, and probenecid, an inhibitor of anion transport systems. This suggested the contribution of P-glycoprotein and anion-sensitive transporter(s). The involvement of P-glycoprotein was investigated by using a P-glycoprotein over-expressing cell line, LLC-GA5-COL150, and P-glycoprotein-gene-deficient mice (mdr1a(-/-)/1b(-/-) mice). LLC-GA5-COL150 cells showed secretory-directed transport of grepafloxacin, while the parent cell line, LLC-PK1, did not. The secretory-directed transport of sparfloxacin and levofloxacin was also detected in LLC-GA5-COL150 cells. In the mdr1a(-/-)/1b(-/-) mice, the intestinal secretory clearance was smaller than that in wild-type mice after intravenous administration of grepafloxacin. Moreover, the absorption from an intestinal loop in mdr1a(-/-)/1b(-/-) mice was larger than that in wild-type mice. Accordingly, it appears that some quinolones are transported by secretory transporters, including P-glycoprotein. The involved transporters function in-vivo not only to transport grepafloxacin from blood to intestine but also to limit its intestinal absorption.

Molecular Mechanisms of Zinc-mediated Induction and Chromium(VI)-mediated Inhibition of Mouse Metallothionein-I Gene Transcription

The expression of metallothionein (MT), a heavy-metal-binding protein, is induced by heavy metals such as zinc, copper, cadmium, and mercury. This induction of MT maintains zinc homeostasis and defends against toxic heavy metals by sequestering these metals and lowering their concentrations at critical intracellular sites. However, MT cannot bind chromium(VI), a heavy metal that has been known for over 100 years to be a human carcinogen. Chromium(VI) enters cells via the sulfate anion transporter system and is reduced to intermediate oxidation states, such as chromium(V) and chromium(IV), in the process of forming stable chromium(III) forms. Chromium(VI) is known to inhibit MT gene transcription, and recently my colleagues and I reported that chromium(VI) inhibits zinc-induced MT gene transcription by modifying the transactivation potential of metal response element-binding transcription factor-1 (MTF-1), a zinc-finger transcription factor. Inhibition of MT gene transcription may therefore be involved in the carcinogenicity of chromium(VI). In this review, I briefly summarize the molecular mechanisms of heavy metal-induced MT gene transcription and discuss the current status of research on chromium(VI) toxicity and chromium(VI)-mediated inhibition of MT gene transcription.

Efflux Transporter‐Mediated Interactions With Atorvastatin—Interesting Findings With Multiple Substrates: Istradefylline, Verapamil, and Rifampicin

Efflux Transporter‐Mediated Interactions With Atorvastatin—Interesting Findings With Multiple Substrates: Istradefylline, Verapamil, and Rifampicin

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of "prototypical" Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.

Liquid–liquid ion transport junctions based on paired gold electrodes in generator–collector mode

Simultaneous electrochemically driven double anion transfer across liquid-liquid interfaces is demonstrated at a gold-gold junction electrode. In the presence of two closely spaced electrodes (generator and collector), anion uptake into the organic phase (oxidation) and anion expulsion into the aqueous phase (reduction) can be combined to result in a generator-collector anion transport system across the liquid-liquid interface. In this report we are employing a paired gold junction grown by electro-deposition to ca. 5 microm gap size with the N,N-diethyl-N',N'-didodecyl-phenylene-diamine water immiscible redox liquid immobilized into the gap to demonstrate simultaneous perchlorate anion uptake and expulsion. The effects of redox liquid volume and scan rate on the magnitude of currents and two mechanistic pathways for ion transport are discussed in the context of micro-electrophoretic processes.

Renal excretion of emtricitabine I: Effects of organic anion, organic cation, and nucleoside transport inhibitors on emtricitabine excretion

The excretion of emtricitabine (FTC) was characterized using isolated perfused rat kidney (IPK) model. Studies were performed to assess the dose‐linearity of FTC excretion, to evaluate the effect of inhibitors of organic anion (probenecid, PBC), organic cation (tetraethylammonium, TEA; cimetidine, CMD) and nucleoside (uridine, URD) transport systems on FTC excretion, and to determine the potential interaction between FTC and trimethoprim (TMP). FTC excretion was studied over a range of doses (80–1600 µg), targeting concentrations encompassing the therapeutic range of FTC (1–20 µg/mL). FTC (2 µg/mL) was also coperfused with PBC (500 µM), TEA (500 µM), CMD (2 mM), URD (500 µM), and TMP (13.7 µM). FTC dose‐linearity studies revealed that excretion parameters were not significantly different among dosing groups. Of the transport inhibitors tested, FTC XR decreased more than twofold in the presence of CMD (0.32 ± 0.099). PBC, TEA, and URD had no observed effect on FTC excretion. TMP coadministration significantly inhibited FTC excretion (XR = 0.43 ± 0.052). The results suggest that FTC renal transport is likely mediated by a CMD‐sensitive organic cation transporter (OCT) in the kidney. TMP may inhibit the renal excretion of FTC when the two compounds are coadministered in vivo. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5401–5410, 2008

Segment‐specific proximal tubule injury in tripterygium glycosides intoxicated rats

Tripterygium glycosides (TG), extracted from the Chinese herb Tripterygium wilfordii Hook. f, is a widely used anti-inflammatory and immunosuppressive agent with definite nephrotoxicity. However, its toxic mechanism remained undiscovered. The aim of this study was to characterize the potential toxicity of TG on segments of proximal tubule in rats. Six hundred and 1200 mg/kg of TG was administered by daily intragastric instillation for 16 days. A significant reduction of p-aminohippurate accumulation by renal cortical slices indicated that TG damaged organic anion transporter (OAT) system that localized at the proximal tubule, especially S(2) segment. A dramatic loss of kidney glutamine synthetase (GS) activity induced by TG reflected S(3) segment damage. Because mRNA expression of OAT1, OAT3, and GS was decreased substantially, we ascribe the fall of p-aminohippurate accumulation and GS activity to alterations at the transcriptional level. A dose-related diminution of kidney glutathione S-transferase activity was noted simultaneously, suggesting oxidative stress involvement. Histopathological lesions were observed in the TG intoxicated rat kidney even though there were no obvious changes of serum urea and creatinine at this dose level. In summary, we provided evidences supporting that TG caused segment-specific dysfunction in the kidney proximal tubule.

JNK activation mediates the apoptosis of xCT-deficient cells

System X c − is an anionic amino acid transport system highly specific for cystine and glutamate. The underlying mechanism of cell death of cultured cells from the subtle gray ( sut) mouse which contains an xCT null mutation remains elucidated. Our results show that the death of sut cells is likely caused by apoptosis mediated by c-Jun N-terminal kinase (JNK). The JNK activation triggers both a caspase-dependent (caspases-9 and -3) and an ER stress-mediated (eIF2 and CHOP) pathway to induce apoptosis. These findings suggest the possible pathways involved in the cell death of xCT-deficient cells.

Involvement of organic anion transport system in transdermal absorption of flurbiprofen

We previously demonstrated that transdermal permeation of flurbiprofen is mediated by a nonlinear transport mechanism(s). Here, we aimed to characterize this transport mechanism by employing an Ussing-type chamber method with tape-stripped hairless mouse skin. Transdermal permeation of [ 3H]flurbipofen was vectorial, saturable and energy-dependent, suggesting the involvement of a carrier-mediated transport system. Transdermal permeation and uptake from the epidermal side of [ 3H]flurbiprofen were inhibited by various nonsteroidal anti-inflammatory drugs (NSAIDs). The inhibitory potency did not correlate well with lipophilicity; anionic NSAIDs tended to be more potent inhibitors than non-anionic NSAIDs. The inhibition profile of both [ 3H]flurbiprofen permeation and uptake, and the Michaelis constants, were similar for a given anionic compound. These results suggest that an organic anion transport system is involved in flurbiprofen uptake from the epidermal side during the process of transdermal absorption. Efflux of [ 3H]flurbiprofen from the skin to the epidermal side, but not to the hypodermal side, increased in the presence of flurbiprofen or several anionic compounds. Such trans-stimulation may suggest the involvement of an organic anion exchanger system. Organic anion transporter 2 (OAT2) is a candidate for the exchanger involved in uptake and/or efflux of flurbiprofen in the skin.

亜鉛応答性転写因子MTF-1とCr（VI）毒性</br>—6価クロムの発がん性と遺伝子発現抑制作用との関係—

Chromium exists in many different oxidation states in the environment, Cr(VI) and Cr(III) being the most stable forms. Chromium has been known for over 100 years to be a human carcinogen. The greatest risk of cancer from chromium exposure is associated with Cr(VI). Cr(VI) enters cells via the sulfate anion transporter system and is reduced to intermediate oxidation states, such as Cr(V) and Cr(IV), in the process of forming stable Cr(III) forms. It is known that Cr(VI) affects expression of various genes. Metal responsive element-binding transcription factor-1 (MTF-1) is involved in sensing heavy metal load and the induced transcription of several protective genes, including metallothionein (MT)-I, MT-II, zinc transporter-1, and gamma-glutamylcysteine synthetase. Cr(VI) inhibits zinc-induced MT transcription via modifying transactivation potential of MTF-1. However, the molecular mechanism for the Cr(VI)-mediated inhibition of MTF-1 has not been fully elucidated. In this review, I briefly summarize the previous studies and discuss the current status of research on Cr(VI) toxicity and Cr(VI)-mediated inhibition against transcription.

Uptake of 3H-cAMP by retinal pigment epithelium isolated from bluegill sunfish (Lepomis macrochirus)

BackgroundIn bluegill sunfish, the melanin-containing pigment granules of the retinal pigment epithelium undergo cyclic movements in response both to ambient lighting and circadian cues. Pigment granules aggregate into the cell body at night (in the dark), and disperse into apical processes during the day (in the light). Regulation of pigment granule aggregation in a number of fishes depends on modulating the intracellular levels of cyclic adenosine monophosphate.ResultsHere we show isolated RPE takes up cyclic adenosine monophosphate (cAMP) in a saturable manner, exogenously applied cAMP induces pigment granule aggregation in retinal pigment epithelium isolated from bluegill, and aggregation induced in this manner is inhibited by treatment with probenecid, an organic anion transport inhibitor.ConclusionOur results raise the possibility that cAMP functions as a messenger secreted from the neural retina to signal darkness to the RPE, which takes it up. It further suggests that organic anion transport systems are the route by which cAMP crosses RPE cell membranes since probenecid inhibits extracellular cAMP from causing pigment granule aggregation.

Indomethacin decreases furosemide-induced natriuresis and diuresis on the neonatal kidney

Indomethacin is used to pharmacologically occlude patent ductus arteriosus in preterm infants. It induces renal untoward effects and furosemide is administered simultaneously to counteract them. The effect of furosemide is blunted by indomethacin. We analyzed comparatively the interactions of furosemide and indomethacin at the organic anion transport system in adult and newborn individuals. Adult and 5-day-old Wistar rats were allocated into three groups: (1) indomethacin (10 mg/kg, ip); (2) furosemide (2 mg/kg, ip); and (3) indomethacin/furosemide, at the same doses. Urinary flow, glomerular filtration rate (GFR), sodium and potassium fractional excretions, and free-water and osmolal clearances were estimated. Para-aminohippuric acid (PAH) uptake was measured in renal cortical slices to study the organic anion's secretory pathway. In adult and newborn rats, furosemide-induced increments in urinary fluxes and excretions of sodium and potassium were blunted by indomethacin administered simultaneously. PAH uptake was decreased to a further extent by indomethacin than by furosemide, suggesting that inhibition of the diuretic effect might be related to competition in the secretion of furosemide. Inhibitory interaction between indomethacin and furosemide was achieved at approximately 10-fold lower concentrations in the newborn than in the adult rats, suggesting that tubular secretion in the neonate is more sensitive to the action of these drugs than in the adult individual.

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

Hormonal and cytokine effects of uric acid

Current evidence supports the role of soluble uric acid as a true mediator of injury, exerting its effects through the induction of growth factors, cytokines, hormones and autacoids. In the present review, we summarize recent studies on the mechanisms involved in the uric acid deleterious effects. Although uric acid is considered an antioxidant in plasma, recent clinical and epidemiological studies have found that hyperuricemia is associated with mortality and development of hypertension, cardiovascular and chronic renal diseases. Experimental studies suggest that uric acid induce its detrimental effects at the cellular level entering to vascular smooth muscle cells (VSMC) via an organic anion transport system, and followed by the activation of specific MAP kinases, nuclear transcription factors, with stimulation of COX-2, PDGF A and C chain, PDGF alpha receptor, and various inflammatory mediators, including C-reactive protein and monocyte chemoattractant protein-1. Physiologically, these effects translate into a rise of arterial pressure, VSMC hypertrophy, tubulointerstitial infiltration and glomerular hypertension in the setting of renal vasoconstriction. Uric acid also promotes endothelial dysfunction through inactivation of NO and arresting the proliferation of endothelial cells. Thus, arteriosclerosis induced by hyperuricemia may be a novel mechanism for the development of essential hypertension. Soluble uric acid has important biologic roles. While it acts as an antioxidant, there is also evidence that uric acid has pro-inflammatory and proliferative effects on VSMC, and causes dysfunction of endothelial cells. These cellular mechanisms may translate into why uric acid is associated with renal and cardiovascular disease.

Interactions of Stevioside and Steviol with Renal Organic Anion Transporters in S2 Cells and Mouse Renal Cortical Slices

Our previous studies have shown that both stevioside and steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with organic anion transport system. The current study examined the direct interactions of stevioside and steviol with specific organic anion transporters. S2 cells expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion transport. Stevioside at 0.5-1 mM showed no interaction with any OAT. In contrast, steviol markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC50 for hOAT1 (11.4 microM) and hOAT3 (36.5 microM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 microM) and hOAT4 (285 microM) was much higher. Results obtained in mouse renal cortical slices were very similar; that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone sulfate (ES) transport. Stevioside has no interaction with human or mouse OATs. In contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter renal drug clearance.

Role of cGMP in natriuresis and pressure-natriuresis

Cyclic GMP (cGMP) is synthesized by renal proximal tubule (RPT) cells and is exported from these cells into the renal interstitial (RI) compartment by an organic anion transporter system. We have demonstrated that RI gGMP inhibits renal Na+ reabsorption at the renal tubule. We have further demonstrated that cGMP-induced natriuresis can be blocked by intrarenal pharmacological inhibition of protein kinase G (PKG). In order to demonstrate that RI cGMP is a potent physiological regulator of Na+ excretion, we selectively decreased RI cGMP using an RI infusion of cGMP-specific phosphodiesterase (PDE), which does not cross the cell membrane and is confined to the extracellular space because of its molecular size. cGMP PDE selectively decreased extracellular RI cGMP levels, without influencing cAMP levels, and caused significant antinatriuresis. On the other hand, blockade of cGMP degradation with selective cGMP PDE inhibitor, zaprinast, increased both RI cGMP and sodium excretion, independently of systemic or renal hemodynamic changes. Therefore, extracellular RI cGMP plays an important role in the regulation of renal Na+ excretion.

Characterization of Secretory Intestinal Transport of Phenolsulfonphthalein

It is known that secretory transport limits the oral bioavailability of certain drugs. However, there is little information on the secretion of anionic compounds in the intestine. Phenolsulfonphthalein (PSP) and p-aminohippuric acid (PAH) have been used widely as substrates for organic anion transport systems. PAH is transported in the secretory direction in the intestine. It is possible that PSP and PAH share the same transport system at the mucosal membrane. The purpose of this study was to characterize the transport system for PSP in the intestine. In the jejunum, the serosal-to-mucosal permeation rate of PSP was significantly reduced in an ATP-depleted condition, whereas a significant difference was not observed in the ileum. Some multidrug resistance-associated protein 2 (Mrp2) inhibitors inhibited PSP permeation in the jejunum. However, pravastatin, a substrate of Mrp2, did not inhibit the PSP permeation. The jejunal secretory transport of pravastatin was significantly reduced in an ATP-depleted condition and by addition of probenecid, but PSP did not affect the jejunal permeation of pravastatin. These results suggest that PSP is secreted into the intestinal lumen by Mrp2-like transporter and that two Mrp2 substrates, PSP and pravastatin, are likely to be transported by different transport systems at the mucosal membrane.

Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation

Recent studies have demonstrated that depletion of amino acids results in the induction of several genes and that a genomic cis-element termed amino acid response element (AARE) is required for the induction. System xc- is an anionic amino acid transport system highly specific for cystine and glutamate, and its activity is known to be induced by cystine deprivation. This transporter is composed of two protein components, xCT and 4F2 heavy chain, and xCT is thought to mediate the transport activity. In the present study, the molecular mechanism for the induction of xCT by amino acid deprivation has been investigated. In mouse NIH3T3 cells, the activity of system xc- and xCT mRNA is induced not only by deprivation of cystine but also by deprivation of other amino acids. Two AAREs, each located in the opposite direction with an intervening sequence, were found in the 5′-flanking region of the mouse xCT gene. Promoter analysis revealed that both AAREs were necessary for the maximal induction of xCT mRNA in response to the amino acid deprivation. Glucose deprivation had no effect on the induction of the activity of system xc-. Electrophoretic mobility shift assay showed that ATF4, but not ATF2, is involved in the amino acid control of xCT expression. These results demonstrate that xCT is a new member of the proteins whose transcriptional control by the amino acid deprivation is mediated by AARE.

Characterization of renal excretion mechanism for a novel diuretic, M17055, in rats

M17055 was developed as a novel diuretic that inhibits both Na+, K+, and 2Cl− cotransport at the thick ascending Henle's loop and Na+ reuptake at the distal tubule. It is secreted at the renal proximal tubules. The purpose of the present study was to characterize the renal excretion mechanism of M17055. We used the renal cortical slices and brush border membrane vesicles (BBMVs) to investigate the transport mechanisms across the basolateral and brush border membranes, respectively. M17055 uptake by rat renal slices increased with time and was saturable. Several organic anions including probenecid, para-aminohippurate (PAH), and estrone-3-sulfate, decreased M17055 uptake. The uptake of M17055 was also observed into HEK293 cells expressing rat OAT1, and was inhibited by PAH. M17055 uptake by BBMVs was time-dependent, saturable, osmolarity-sensitive, and inhibited by several organic anions, but not by PAH. These results suggest that plural organic anion transport systems are involved in M17055 transport via both basolateral and brush border membranes of proximal tubule epithelial cells, a part of the renal uptake being mediated by OAT1.