1Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Germany2Deptartment of Vegetative Physiology and Pathophysiology, Georg August University Göttingen, Germany

RT-PCR-based evidence for the in vivo stimulationof renal tubular p-aminohippurate (PAH) transportby triiodothyronine (T3) or dexamethasone (DEXA)in kidney tissue of immature and adult rats*

ANDREW BAHN2, ACHIM HAUSS1, DOROTHEAAPPENROTH1, DIANA EBBINGHAUS2, YOHANNESHAGOS2,PETERSTEINMETZER1, GERHARD BURCKHARDT2,and CHRISTIAN FLECK1

With 3 figures

Received: January 31, 2003; Accepted: February 24, 2003

Address for correspondence: Prof. Dr. CH. FLECK, Klinikum der Friedrich-Schiller-Universität Jena, Institut für Pharma-kologie und Toxikologie, D-07740 Jena, Germany; Fax: ++49+3641+938702, E-mail: Christian.Fleck@mti.uni-jena.de

Key words: Renal tubular transport; renal cortical slices; stimulation; OAT1; RT-PCR; dexamethasone; triiodothyronine;p-aminohippurate, age; rat.

Summary

Our previous studies have shown that a pre-treatment ofrats with triiodothyronine (T3) or dexamethasone (DEXA)increases renal PAH excretion significantly. This stimula-tion was accompanied by an enhanced protein synthesiswithin the renal cortex. To explore the molecular basis forthis sub-chronic induction process, we investigated thestimulation of PAH accumulation in renal cortical slices aswell as the expression level of organic anion transporter 1(OAT1), the recently cloned renal basolateral PAH-trans-porter, using RT-PCR techniques under the applied condi-tions. 10- and 55-day-old Han:WIST rats were treated invivo with T3 (20 µg/100 g b.wt.) or DEXA (60 µg/100 gb.wt.), both for 3 days, once daily. Renal cortical sliceswere incubated for 2 hours in Cross-Taggart medium andPAH uptake into kidney tissue was measured time depen-dently (slice to medium ratio, QS/M). The accumulation ca-pacity is comparable between immature and mature rats(control-QS/M: 6.7 ± 0.1 vs. 6.9 ± 0.2, respectively). Bothage groups showed a significant increase of PAH accumu-lation capacity after T3 treatment (10-day-old rats: 15.0 ±0.2; 55-day-old rats: 11.7 ± 1.3). After DEXA pre-treat-ment, PAH accumulation was only slightly changed (10-

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day-old rats: 5.9 ± 0.2; 55-day-old rats: 8.2 ± 1.3). Semi-quantitative measurements of OAT1 mRNA expressionlevel showed a significant increase of OAT1 mRNA afterpre-treatment with both T3 and DEXA in the two agegroups. Thus, this is the first evidence that T3 and DEXApre-treatment induces the expression of OAT1.

Introduction

Many endogenous metabolites, xenobiotics and drugs,which are permanently negatively charged under physio-logical conditions, are called organic anions (OA). Thekidneys are the main target for the elimination of these insome cases harmful substances. Since about two decadesit was demonstrated in our laboratory that the renal ex-cretion capacity for organic anions like p-aminohippu-rate (PAH) can be stimulated sub-chronically(after atleast 24 h) by hormones, e. g. by triiodothyronine (T3) inboth young and adult rats (BRÄUNLICH 1987), and bydrugs such as dexamethasone (DEXA) especially in im-mature rats (BRÄUNLICH et al. 1986). PAH excretion intothe urine increased after T3 as well as after DEXA treat-

*Dedicated to Prof. Dr. WOLFGANG KLINGER on occasion of his 70th birthday on July 03, 2003.

ment by up to 60% (BRÄUNLICH 1988). Further in vitroinvestigations on renal cortical slices demonstrated thatthe PAH accumulation capacity was significantly in-creased after in vivoadministration of T3, whereas the invivo application of DEXA did not result in a stimulationof the PAH accumulation capacity (BRÄUNLICH et al.1986; BRÄUNLICH 1987). A different effect was visible, ifthe stimulation was induced in vitro. In this case DEXAshowed a significant stimulatory effect on PAH accumu-lation capacity in young and adult rats (FLECK et al.1998). The simultaneous treatment of rats with both hor-mones resulted in a synergistic effect, leading to the as-sumption of two separate ways of action for T3 or dex-amethasone on the renal organic anion secretion capacity(BRÄUNLICH and PILS 1992). In a recent approach look-ing for strategies to overcome multi-drug resistance inrenal cell carcinoma we provided evidence that thesestimulation effects are also relevant in man indicatingspecies independent sub-chronicregulatory mechanismsof OA excretion (FLECK et al. 2002).

A new family of transport proteins mediating the secre-tion of organic anions in the kidneys, the organic aniontransporter (OAT), has recently been discovered (BURCK-HARDT and PRITCHARD 2000). At present, five membershave been functionally described, OAT1-4 and URAT1(SEKINE et al. 2000; ENOMOTO et al. 2002). They all pos-sess broad and in part overlapping substrate specificities,entailing that drug-drug interaction can influence renaldrug excretion (for review see DRESSERet al. 2001). Twoof the transporters, OAT1 and OAT3, were localized to thebasolateral side of the proximal tubule cells (TOJO et al.1999; HOSOYAMADA et al. 1999; KOJIMA et al. 2002; CHA

et al. 2001) and were shown to transport PAH (for a re-view on OAT1 see BURCKHARDT et al. 2001; KUSUHARA

et al. 1999; CHA et al. 2001) with apparent Km’s of3.5–16 µM for OAT1 and 65–87 µM for OAT3, indicatingthat OAT1 represents the high affinity, so-called ‘classic’PAH transporter. The release of PAH at the luminal cellside is still not well characterized at the molecular level.

Acute regulation of organic anion transport activitywas demonstrated for human and rat OAT1 (LU et al.1999; UWAI et al. 1998) and rat OAT3 (TAKEDA et al.2000). An activation of protein kinase C (PKC) resultedin a reduction of their transport activity. The same effectswere documented for the PAH-secreting opossum kidney(OK) cell line (TAKANO et al. 1996). SAUVANT andcoworkers (2001) showed that the basolateral as well asthe apical PAH transport activity in OK cells were down-regulated by PKC, whereas the mitogen-activated proteinkinase (MAPK) kinase MEK activated both processes, ofwhich the basolateral step was further stimulated by theepidermal growth factor (EGF). A hormonal impact onthe regulation of organic anion transport activity wasnoted for parathyroid hormone (PTH) and ACTH. PTHhas been shown to act on OK cells in an acute manner viaan activation of PKA and PKC resulting in the mentionedreduction of transepithelial organic anion transport activi-ty (NAGAI et al. 1997). Recently it was demonstrated that

ACTH-stimulated cortisol release from primary bovineadrenocortical cells was inhibited by probenecid andtrans-stimulated by PAH (STEFFGEN et al. 1999;ROHRBACH et al. 1997). Moreover, a marked reduction ofthe expression level of OAT1 was observed in the adrenalcortex in hypophysectomized rats (STEFFGENet al. 2001)suggesting a “non-acute” sub-chronicalregulation of or-ganic anion transport activity. Further proofs for a “non-acute” hormonal control of organic anion transporterswere illustrated for OAT1- and OAT3-mRNA expressionin the rat kidney. The expression levels for both mRNA’sincreased after birth and reached a maximum at day 35(BUIST et al. 2002). In older rats, OAT1 declined in fe-male animals, but not in males. Earlier investigations toexplore the molecular basis for the stimulation effects onrenal PAH accumulation capacity in vivo and in vitro re-vealed a de novoprotein synthesis as one possible expla-nation (BRÄUNLICH 1981; ORTWEILER et al. 1987).

Little is known about these induction/sub-chronicreg-ulation processes of the OAT family of transporters. Thequestion arose whether the stimulation of PAH excre-tion/accumulation capacity processes is related tochanges at the transcriptional level. To address this ques-tion we performed animal studies on the basis of theknown induction phenomena and examined additionallysemi-quantitative RT-PCR studies to look for the tran-scriptional activity of the ‘classic’ renal PAH transporter,the OAT1 gene (BAHN et al. 2000).

Material and methods

Animals Experiments were carried out on female Wistar rats

(Han: WIST) of our institute’s own out-bred stock; as 10-day-old animals both sexes were used, as 55-day-old ani-mals only females were used. The litters were reduced to 6animals. Young animals were nursed by their dams. Adultrats were fed a standard diet (Altromin 1316, Lage, Ger-many) and tap water ad libitum. Animals were housedunder standardized conditions in plastic cages, light-darkcycle 12/12 hours, temperature 22 ± 2 °C, humidity 50 ±10%. Animal care and treatment were conducted in confor-mity with institutional guidelines that are in compliancewith international laws.

Stimulation of PAH excretion capacity60 µg/100 g b.wt. dexamethasone (DEXA; Fortecortin

Mono, E. Merck, Darmstadt, Germany) or 20 µg/100 gb.wt. T3 (Triiodothyronine, Berlin-Chemie, Berlin, Ger-many) were given i.p. for 3 days, once daily. Hormoneswere dissolved in normal saline (1 ml/100 g b.wt.). Con-trols received the solvent only.

Clearance experiments were performed 24 h after thelast treatment with either DEXA or T3. Renal excretion ofPAH (E. Merck, Darmstadt) was measured after the admin-istration of doses which can reliably saturate the transportcapacity in rats of different ages: 150 (10-day-old) or 300

368 Exp Toxic Pathol 54 (2003) 5-6

(55-day-old) mg PAH/100 g b.wt. i.p., dissolved in 2ml/100 g b.wt. distilled water (BRÄUNLICH et al. 1983). Ani-mals were placed in metabolic cages for one hour and urinewas collected. The urine of two 10-day-old rats was pooledto obtain a sufficient amount for PAH determination.

PAH-accumulation experiments

DEXA or T3 treated and control rats were anesthetizedwith ether, killed by decapitation, bled and the kidneyswere removed and stored in normal saline on ice. Renalcortical slices with pool sizes of about 100 mg (< 0.5 mmthick) were incubated in PAH containing 50 ml-Erlenmeyerflasks with bidirectional shaking (about 100 rpm) in 3 mlCross-Taggart buffer (pH 7.4; 30 °C; oxygen gassing (2.5l/hour/sample); incubation time 120 minutes; concentrationof PAH: 8.5 × 10–5 M). Following incubation, PAH was de-termined in the supernatant fraction of the tissue ho-mogenate and in the incubation medium. The active uptakeof PAH was expressed by the ratio between PAH concen-tration in the tissue and in the medium after the end of incu-bation (slice to medium ratio = QS/M) according to STOPP

and BRÄUNLICH (1975). Concentrations of PAH were deter-mined using the colorimetric method introduced by BRAT-TON et al. (1939).

Cloning of the rat OAT1-competitor

To determine the amount of rat organic anion transporter1 (rOAT1)-mRNA expression we cloned a cDNA with adeletion of 92 bp specific for the same rOAT1-primers asthose used for amplification of rOAT1 out of the rat tissues.Briefly, we amplified rOAT1-pcDNA3.1 cloned previouslyin our laboratory with rOAT1-U: 5′-GGTGCTGATCT-TGAACTACC-3′ and rComp-R: 5′-AGTAGGCAAAGC-TAGTGGCAGTTCCTTCTGCAGGC-3′ including therOAT1-R sequence (5′-TAGTAGGCAAAGCTAGTGG-3′)within a standard PCR (94 °C for 2 min, 94 °C for 30 s,55 °C for 30 s, 72 °C for 1 min, for 35 cycles). This resultedin a PCR-product of 481 bp, which was cut out of an 2%agarose gel, extracted with the Nucleotrap-kit (Machereyand Nagel, Düren, Germany) and ligated into p57 plasmid(MBI Fermentas, St. Leon-Rot, Germany) using T4 ligase(Roche Diagnostics, Mannheim, Germany). This final con-struct was used in different amounts in the following RT-PCR reactions.

Total RNA extraction and competitive RT-PCR

Approximately 100 mg of rat tissue was homogenizedwith a mortar and pestle in liquid nitrogen and transferredto the extraction buffer of the RNeasy total RNA extractionkit (QIAGEN, Hilden, Germany). Total RNA was extractedaccording to the manufacturer’s protocol. One microgramof this RNA was used for reverse transcription with omni-script reverse transcriptase (QIAGEN) and an oligo(dT)-anchor primer (5′-GACCACGCGTATCGATGTCGAC(T)18(AGC)-3′) at 37 °C for 1 h. 2–4 microliters of the RTreaction together with different amounts of the rat OAT1-competitor were taken for a standard PCR (see above) ap-plying rOAT1-U: 5′-GGTGCTGATCTTGAACTACC-3′ asa forward primer and rOAT1-R: 5′-TAGTAGGCAAAGC-

TAGTGG-3′ as a reverse primer. This gave a rOAT1-spe-cific PCR-product of 573 bp confirmed by sequence analy-sis using the dye-terminator cycle sequencing kit and anautomatic ABI377 sequencer (Applied Biosystems, Weiter-stadt, Germany). Densitometric calculations of OAT1-cDNA were performed with ONE-Dscan software (MWG,München, Germany).

Data analysisResults are given as arithmetic means ± SEM of four to

six independent slice preparations (4–6 kidneys). In thePAH excretion experiments n = 6 were used. IndividualPCR assays were carried out in triplicate and duplicate, re-spectively.

Statistical differences were assessed by analysis of vari-ance (ANOVA) and post hocmultiple t-test with Bonfer-roni correction; differences were taken to be significantwhen P ≤ 0.05. Calculations were performed using com-mercial software (SPSS Science, Chicago, U.S.A.).

Results

In figure 1, the influence of in vivo administration ofDEXA (60 µg/100 g b. wt.) or T3 (20 µg/100 g b. wt.) onrenal PAH excretion and PAH accumulation capacity of

Exp Toxic Pathol 54 (2003) 5-6 369

Fig. 1. Influence of in vivoT3 or dexamethasone (DEXA)treatment (cp. method) on urinary PAH excretion and PAHaccumulation in renal cortical slices of 10- and 55-day-oldrats. Arithmetic means ± S.E.M.; n = 4; * – significant hor-mone effects; o – significant age differences (p <0.05).

In adult as well as 10-day-old rats treated with T3, uri-nary PAH excretion and PAH accumulation in renalcortical slices correlate substancially with the content ofOAT1 encoding mRNA (1.9-fold increase). But despitethe increase in OAT1 encoding mRNA after DEXAtreatment, the PAH accumulation capacity is evidentlyunchanged in both age groups, whereas urinary PAHexcretion is – compared to OAT1-mRNA content – sig-nificantly more enhanced, especially in 10-day-old ratsby up to 4-fold.

Discussion

In earlier studies we demonstrated a significant in-crease of PAH accumulation capacity as a consequenceof a pre-treatment of rats with hormones like T3 or drugslike DEXA (BRÄUNLICH et al. 1986; BRÄUNLICH 1987;BRÄUNLICH 1988; FLECK et al. 2002). We also providedevidence that an increased protein synthesis might be re-

renal cortical slices is shown, both given for three daysonce daily. In 10-day-old rats the renal PAH excretionwas significantly lower than in adult animals, but in-creased significantly after in vivo pre-treatment with T3and, more pronounced, with DEXA. In 55-day-old ratsthis stimulatory effect could, in principle, be reproduced.In accordance with previous findings (BRÄUNLICH et al.1986) the PAH accumulation capacity of slices was notinfluenced by the in vivopre-treatment with DEXA, nei-ther in immature nor in adult rats. On the other hand, thestimulatory effect of T3 on renal PAH handling is reflect-ed in both age groups by enhanced QS/M values in the ac-cumulation experiments.

To further clarify the reason for the different hor-mone effects, semi-quantitative RT-PCR investigationswere performed shown in representative gel elec-trophoresis pictures (fig. 2). The question arose whetherthese OAT1-mRNA contents reflect changes in OAT1mediated transport capacity. For this reason relativechanges were calculated with controls = 100% (fig. 3).

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Fig. 2. Gel electrophoresis after competitive RT-PCR for semi-quantitative determination of OAT1-mRNA in relation to com-petitor-OAT1-cDNA content in renal cortical slices of 10- and 55-day-old rats after pre-treatment with dexamethasone or T3(see method). One representative example for each group; Total n = 6 in triplicate. At the center margin of all figures: DNA-length standard (100 bp-ladder); upper band (573 bp) represents OAT1-cDNA in comparative concentration per sample; lowerband (481 bp) corresponds to the concentration of competitor-cDNA in decreasing order with highest concentration left.

sponsible for these stimulation processes (ORTWEILER etal. 1987). Besides tubular filtration, efficient renal secre-tion of organic anions including several drugs and xeno-biotics is mediated by membrane transport proteins. Inthis regard, the basolateral uptake of these substancesinto the proximal tubule cells is the rate limiting step inthe excretion process. In the meantime, several proteinsinvolved in the basolateral uptake of organic anions likePAH were identified on the molecular level (for reviewsee BURCKHARDT and WOLFF 2000; SEKINE et al. 2000).Of these proteins, organic anion transporter 1 (OAT1) isknown as the high affinity PAH-transporter (BURCK-HARDT et al. 2001). To address the question whether theobserved stimulated increase of PAH excretion capacityis due to a de-novosynthesis of transport proteins andtherefore a “ non-acute” effect, we treated immature (10-day-old) and adult (55-day-old) rats with T3 and DEXA,respectively, and measured renal PAH secretion, in vitroPAH accumulation and expression of OAT1 mRNAusing RT-PCR techniques.

The results for PAH excretion capacity and accumula-tion are in good accordance with our previous findings:in all experimental groups PAH excretion was faster afterhormone administration. Surprisingly, this effect wasstronger after DEXA than after T3 treatment (cf. fig. 1).On the other hand, in renal cortical slices the PAH accu-mulation capacity was distinctly more enhanced after T3treatment, both in immature and in adult rats. DEXA hadno effect on PAH uptake into renal tissue in young ratsand its effect was only moderate in adult animals. In aprevious study, we have administered DEXA directly incontact with the renal cortical slices in vitro, so that phar-macokinetic interferences can be excluded (FLECK et al.1998). Thein vitro stimulation of kidney tissue withDEXA was followed by a significant increase in PAH ac-cumulation capacity both in immature and in adult ani-mals. It remains open whether this effect is mediated viainteractions at the glucocorticoid receptors, or by a sub-strate stimulation because glucocorticoids may be sub-strates for organic anion transporters (OATs, ULLRICH etal. 1991). One possible reason for the lack of effect of anin vivopre-treatment with DEXA on the in vitro PAH ac-cumulation capacity has been explained by BRÄUNLICH

et al. (1993) as a result of an increase in kidney weightfollowed by an increase in total transported PAHamounts in vivo, whereas per 1 g kidney weight therewas no increase in PAH uptake capacity. Altogether itcan be concluded, that both in vivo and in vitro stimula-tion with DEXA increases the renal PAH transport ca-pacity.

RT-PCR analysis revealed a significantly enhancedexpression of OAT1 mRNA in both age groups and afterpre-treatment with T3 and DEXA corresponding well tothe data of PAH excretion in vivo (cf. fig. 3). Conse-quently, this supports the notion of a “ non-acute” effectof hormone and/or drug treatment in rats in vivo as pos-tulated from the observations on the protein level(BRÄUNLICH 1981). These evidences are nice prerequi-

sites and a basis for a therapeutic approach in the treat-ment of malfunction of renal organic anion transport.

Acute down-regulation of renal organic anion trans-port in situ was shown in isolated killifish proximaltubules, perfused S2 segments of rabbit proximal tubulesand opossum kidney epithelial cells (OK, MILLER 1998;SHUPRISHAet al. 2000; TAKANO et al. 1996) to be mediat-ed by protein kinase C (PKC). Investigations on themouse and human ortholog of OAT1 revealed a direct ef-fect on the phosphorylation status and transport activityof the protein (LU et al. 1999; YOU et al. 2000). More re-cent work of SAUVANT and coworkers on OK cells (2001)and of MILLER (2002) on isolated killifish proximaltubules let assume that a more complex mechanism regu-lates organic anion transport in the kidneys. These datadocument a remarkable impact of acute and sub-chronicregulation of transport proteins on the secretion of en-dogenous as well as exogenous substances includingseveral drugs.

In summary, our own work in the past decades and inthis report showed that an in vitro as well as an in vivostimulation of organic anion transport, measured with the

Exp Toxic Pathol 54 (2003) 5-6 371

Fig. 3. Relative changes of urinary PAH excretion, PAHaccumulation in renal cortical slices (QS/M), and in OAT1encoding mRNA content after pre-treatment with dexam-ethasone (DEXA) or T3 in 10- and 55-day-old rats withcontrols = 100%. Arithmetic means ± S.E.M.; n = 5–6 mea-surements in triplicate; * – significant hormone effects(p <0.05).

‘classic’ substrate p-aminohippurate (PAH), could bemediated by T3 and dexamethasone. We were able tocorrelate these induction processes in part with anenhanced mRNA synthesis indicating that sub-chronicregulation includes regulation on the transcriptionallevel. Moreover, we can speculate from our data, thatdifferent induction pathways are responsible for theobserved effects.

Acknowledgement: The authors want to express theirthanks to Anne Berthold and A. Hillemann for their excel-lent technical assistance, and A. Nolte (Dept. of Biochem-istry, Universität Göttingen, Germany) for nucleotide se-quencing.

References

BAHN A, PRAWITT D, BUTTLER D, et al.: Genomic structureand in vivo expression of the human organic anion trans-porter 1 (hOAT1) gene. Biochem Biophys Res Commun.2000; 275: 623–630.

BRATTON AC, MARSHALL EK Jr., BABBITT D, et al.: A newcoupling component for sulfonamide determination. JBiol Chem 1939; 128: 537–550.

BRÄUNLICH H: Influence of protein biosynthesis inhibitorson stimulated renal p-aminohippurate excretion in rats.Acta Physiol Acad Sci Hung 1981;58: 321–326.

BRÄUNLICH H, FLECK C, BAJANOWSKI T, et al.: Dosisab-hängigkeit und Altersabhängigkeit des renalen tubulärenTransportes von p-Aminohippursäure (PAH) bei Rattennach Injektion von Einzeldosen. Pharmazie 1983;38:483–485.

BRÄUNLICH H, KÖHLER A, SCHMIDT I: Acceleration of p-aminohippurate excretion in immature rats by dexam-ethasone treatment. Med Biol 1986; 64: 267–270.

BRÄUNLICH H: Transport of p-aminohippurate in renal cor-tical slices of rats of different ages following treatmentwith thyroid hormones. Biomed Biochim Acta 1987; 46:251–257.

BRÄUNLICH H: Hormonal control of postnatal developmentof renal tubular transport of weak organic acids. PediatrNephrol 1988; 2: 151–155.

BRÄUNLICH H, PILS W: Synergistic effect of triiodothyro-nine and dexamethasone on renal tubular transport of p-aminohippurate in rats of different ages. Dev PharmacolTher 1992; 19: 50–56.

BRÄUNLICH H, RÖSSLERS, GERHARDT C: Influence of sym-pathetic nervous system on dexamethasone-stimulatedrenal tubular transport of p-aminohippurate in youngrats. Dev Pharmacol Ther 1993; 20: 86–92.

BUIST SC, CHERRINGTONNJ, CHOUDHURI S, et al.: Gender-specific and developmental influences on the expressionof rat organic anion transporters. J Pharmacol Exp Ther2002; 301: 145–151.

BURCKHARDT G, PRITCHARD JB: Organic anion and cationantiporters. In: The Kidney, Physiology & Pathophysiol-ogy. Ed.: SELDIN DW, GIEBISCH G. – 3rd ed. – Phila-delphia et al.: Lippincott Williams & Wilkins, 2000;193–222.

BURCKHARDT G, WOLFF NA: Structure of renal organicanion and cation transporters. Am J Physiol Renal Phy-siol 2000; 278: F853–F866.

BURCKHARDT G, BAHN A, WOLFF NA: Molecular physiolo-gy of renal p-aminohippurate secretion. News PhysiolSci 2001; 16: 114–118.

CHA SH, SEKINE T, FUKUSHIMA JI, et al.: Identification andcharacterization of human organic anion transporter 3expressing predominantly in the kidney. Mol Pharmacol2001; 59: 1277–1286.

DRESSERMJ, LEABMAN MK, GIACOMINI KM: Transportersinvolved in the elimination of drugs in the kidney:organic anion transporters and organic cation trans-porters. J Pharm Sci 2001; 90: 397–421.

ENOMOTO A, KIMURA H, CHAIROUNGDUA A, et al.: Molecu-lar identification of a renal urate anion exchanger thatregulates blood urate levels. Nature 2002; 417: 447–452.

FLECK C, KRATOCHWIL E, WINTERSTEIN K, et al.: In vitrostimulation of renal tubular p-aminohippurate transportby dexamethasone in rat kidneys and in intact kidney tis-sue of patients suffering from renal cell carcinoma. UrolRes 1998; 26: 143–148.

FLECK C, HILGER R, JURKUTAT S, et al.: Ex vivostimulationof renal transport of the cytostatic drugs methotrexate,cisplatin, topotecan (Hycamtin) and raltitrexed (To-mudex) by dexamethasone, T3 and EGF in intact humanand rat kidney tissue and in human renal cell carcinoma.Urol Res 2002; 30: 256–262.

HOSOYAMADA M, SEKINE T, KANAI Y, et al.: Molecularcloning and functional expression of a multispecific or-ganic anion transporter from human kidney. Am J Physi-ol 1999; 276: F122–F128.

KOJIMA R, SEKINE T, KAWACHI M, et al.: Immunolocaliza-tion of multispecific organic anion transporters, OAT1,OAT2, and OAT3, in rat kidney. J Am Soc Nephrol 2002;13: 848–857.

KUSUHARA H, SEKINE T, UTSUNOMIYA-TATE N, et al.:Molecular cloning and characterization of a new multi-specific organic anion transporter from rat brain. J BiolChem 1999; 274: 13675–13680.

LU R, CHAN BS, SCHUSTERVL: Cloning of the human kid-ney PAH transporter: narrow substrate specificity andregulation by protein kinase C. Am J Physiol 1999; 276:F295–F303.

MILLER DS: Protein kinase C regulation of organic aniontransport in renal proximal tubule. Am J Physiol RenalPhysiol 1998; 274: F156–F164.

MILLER DS: Xenobiotic export pumps, endothelin signal-ing, and tubular nephrotoxicants – a case of molecularhijacking. J Biochem Mol Toxicol 2002; 16: 121–127.

NAGAI J, YANO I, HASHIMOTO Y, et al.: Inhibition of PAHtransport by parathyroid hormone in OK cells: involve-ment of protein kinase C pathway. Am J Physiol 1997;273: F674–F679.

ORTWEILER W, JAHN F, BRÄUNLICH H: Increase of 14C-leucineuptake following stimulation of renal tubular transportprocesses. Biomed Biochim Acta 1987; 46: 271–276.

ROHRBACH S, JARRY H, METTEN M, et al.: Demonstration ofa probenecid inhibitable anion exchanger involved incortisol release and PAH uptake in adrenocortical cells.Pflügers Arch-Eur J Physiol 1997; 433: R26.

SAUVANT C, HOLZINGER H, GEKLE M: Modulation of thebasolateral and apical step of transepithelial organicanion secretion in proximal tubular opossum kidneycells. Acute effects of epidermal growth factor and mito-gen-activated protein kinase. J Biol Chem 2001; 276:14695–14703.

372 Exp Toxic Pathol 54 (2003) 5-6

kidney epithelial cells. Am J Physiol 1996; 271:F469–F475.

TAKEDA M, SEKINE T, ENDOU H: Regulation by proteinkinase C of organic anion transport driven by rat organicanion transporter 3 (rOAT3). Life Sci 2000; 67:1087–1093.

TOJOA, SEKINE T, NAKAJIMA N, et al.: Immunohistochemi-cal localization of multispecific renal organic aniontransporter 1 in rat kidney. J Am Soc Nephrol 1999; 10:464–71.

ULLRICH KJ, RUMRICH G, PAPAVASSILIOU F, et al.: Contralu-minal p-aminohippurate transport in the proximale tubuleof the rat kidney. VIII. Transport of corticosteroids.Pflügers Arch-Eur J Physiol 1991; 418: 371–382.

UWAI Y, OKUDA M, TAKAMI K, et al: Functional characteri-zation of the rat multispecific organic anion transporterOAT1 mediating basolateral uptake of anionic drugs inthe kidney. FEBS Lett 1998; 438: 321–324.

YOU G, KUZE K, KOHANSKI RA, et al.: Regulation ofmOAT-mediated organic anion transport by okadaic acidand protein kinase C in LLC-PK(1) cells. J Biol Chem2000; 275: 10278–10284.

SEKINE T, CHA SH, ENDOU H: The multispecific organicanion transporter (OAT) family. Pflügers Arch 2000;440: 337–350.

STEFFGEN J, ROHRBACH S, BEERY E, et al.: Demonstrationof a probenecid-inhibitable anion exchanger involved inthe release of cortisol and cAMP and in the uptake of p-aminohippurate in bovine adrenocortical cells. CellPhysiol Biochem 1999; 9: 72–80.

STEFFGEN J, HAGOS Y, KOEPSELLH, et al. : Regulated ex-pression and zonation of transport proteins in the ratadrenal gland. Kidney Blood Pres Res 2001; 24: 349.

STOPPM, BRÄUNLICH H: Die Akkumulation von p-Amino-hippursäure und Zyklopenthiazid in Nierenrindenschnit-ten verschieden alter Ratten und ihre Abhängigkeit vonder Energiebereitstellung. Acta biol med germ 1975; 34:89–98.

SHUPRISHAA, LYNCH RM, WRIGHT SH, et al.: PKC regula-tion of organic anion secretion in perfused S2 segmentsof rabbit proximal tubules. Am J Physiol Renal Physiol2000; 278: F104–F109.

TAKANO M, NAGAI J, YASUHARA M, et al.: Regulation ofp-aminohippurate transport by protein kinase C in OK

Exp Toxic Pathol 54 (2003) 5-6 373