Prostaglandin E2 Inhibits Its Own Renal Transport by Downregulation of Organic Anion Transporters rOAT1 and rOAT3
Christoph Sauvant,
Hildegard Holzinger and
Michael Gekle
Physiologisches Institut, Universität Würzburg, Würzburg, Germany
Address correspondence to: Dr. Christoph Sauvant, Physiologisches Institut der Universität Würzburg, Röntgenring 9, 97070 Würzburg, Germany. Phone: +49-931-31-2724; Fax: +49-931-31-2741; E-mail: christoph.sauvant{at}mail.uni-wuerzburg.de
Received for publication July 14, 2005.
Accepted for publication October 11, 2005.
Prostaglandin E2 (PGE2) is the principal mediator of fever andinflammation. Recently, evidence emerged that during febrileresponse, PGE2 that is generated in the periphery enters thehypothalamus and contributes to the maintenance of fever. Ina rat model of fever generation, peripheral PGE2 is increased,whereas clearance by metabolism of peripheral PGE2 is downregulated.The major route of PGE2 excretion is via the renal proximaltubular organic anion secretory system, where basolateral uptakethat is mediated by renal organic anion transporter 1 (rOAT1)and rOAT3 is rate limiting. Therefore, it was hypothesized thatPGE2 itself will abolish its excretion by rOAT1 or rOAT3. Fluoresceinwas used as a prototypic organic anion, and NRK-52E cells fromrat served as a proximal tubular model system. PGE2 time-dependentlydownregulates basolateral organic anion uptake, without affectingcell volume or cell protein, recirculation of counter ions,or proximal tubular transport systems in general. In addition,PGE2 diminishes expression of both rOAT1 and rOAT3. Both organicanion uptake and expression of rOAT1 and rOAT3 are dose-dependentlydownregulated by PGE2. These findings suggest that during feveror inflammation, renal secretory transport of PGE2 is reduced,contributing to elevated PGE2 levels in blood. These data fitinto the hypothetical concept of peripheral PGE2’s playinga significant role in fever. The described regulatory mechanismmay also be of relevance in chronic inflammatory events. Moreover,the data presented could explain why increased plasma uratelevels occur in diseases that go along with increased levelsof PGE2.
The organic anion transport system of the renal proximal tubuleplays a crucial role in the excretion of a variety of potentiallytoxic compounds. This system consists of organic anion exchangersthat are located at the basolateral membrane and a less well-characterizedtransport step at the apical membrane. The classical basolateralorganic anion exchanger is the terminal step in a tertiary activetransport system, dependent on an inward-directed Na+ gradientto drive the uptake of -ketoglutarate, which then is exchangedfor organic anions. It has been shown that OAT1 represents characteristicsof the basolateral, polyspecific transporter for organic anions,which had been functionally described for some time (1). Recently,new evidence has indicated that organic anion transporter 3(OAT3) (2), which also is located in the basolateral renal proximaltubular membrane, also works as an appropriate exchanger fororganic anions and dicarboxylates (3,4). Moreover, additionalhomologues have been cloned and were called OAT2 (5), OAT4 (6),OAT5 (7), and OAT6 (8). These clones show 40 to 60% homologyin amino acid sequence compared with OAT1 or OAT3, and theydiffer in substrate specificity and expression pattern. Furthermore,these latter proteins are not anion exchangers like OAT1 orOAT3 but seem to work as facilitators of anion diffusion. Insummary, the classical renal basolateral polyspecific uptaketransporter for organic anions is represented by OAT1 and OAT3(1).
Meanwhile, there is increasing evidence that renal basolateralorganic anion uptake is regulated. A growing number of studiesconcentrate on acute regulatory phenomena and deal with regulationby protein kinase C and protein kinase A (for review, see refs.[1,9]). With respect to long-term regulation of renal organicanion transport, fewer data are available. There is evidencethat renal organic anion transport is regulated by sex steroids(10), which now is shown to be most probably due to regulationof OAT1 and OAT3 (11,12) and maybe OAT2 (12). Moreover, hyperuricemiawas shown to reversibly downregulate OAT1 and OAT3 (13) in rats.
Prostaglandin E2 (PGE2) is a substrate for both OAT1 and OAT3(14) and is considered to be the principal downstream mediatorof fever (15). In general, fever-generating PGE2 is thoughtto be produced in the brain (16). Nevertheless, it is shownthat substantial amounts of peripheral PGE2 enter hypothalamicstructures (17), and recent data support the idea of blood-derivedPGE2’s playing an important role in fever (18,19). Theauthors showed that in a rat fever model, the main enzymes thatlead to generation of PGE2 are dramatically upregulated in liverand lung (and hypothalamus) (20). Conversely, the transportersthat mediate uptake into catabolizing tissues (liver and lung)and the enzymes that mediate catabolism therein are tremendouslydiminished (20), whereas there is no indication for a diminisheduptake of PGE2 into the brain.
No data concerning kidney function or excretory clearance werepresented in the latter studies. However, it is known that prostaglandins(and their respective metabolites) are excreted to a major extentby the kidneys, and blood levels are highly dependent on renalprostaglandin excretion (21). Because of protein binding, proximaltubular secretion of PGE2 plays a major role in renal excretion(22). Because PGE2 is a substrate for both OAT1 and OAT3 (14),these transporters represent the rate-limiting basolateral uptakestep of proximal tubule PGE2 secretion. Therefore, it seemsreasonable to speculate that renal secretion of PGE2 also couldbe impaired in the above-mentioned fever model. Thus, we hypothesizedthat PGE2 inhibits the rate-limiting step of proximal tubularorganic anion secretory transport during long-term exposure,the opposite of what was observed during acute exposure (23–25).To gain evidence to support this hypothesis, we determined thelong-term effect of PGE2 on the basolateral uptake step in ratproximal tubule cells (NRK-52E cells).
If not stated otherwise, chemicals were from Sigma (St. Louis,MO).
Cell Culture
NRK-52E cells were obtained from the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (Braunschweig, Germany). NRK-52E cellswere cultured in DMEM that was enriched with 26 mmol/L NaHCO3and 5% (vol/vol) FCS. NRK-52E cells were cultured on Petri dishesor permeable supports (Falcon; Becton Dickinson Labware, FranklinLakes, NJ). The cells were maintained at pH 7.4 and 37°Cand gassed with 95% O2/5% CO2. All studies were performed betweenpassages 20 and 40. The seeding density was 0.4 x 106/cm2. Themedium was changed every third day, and the cells were usedfor experiments at day 10 after seeding. All experiments wereperformed with cells that were serum starved for 24 h beforethe experiments.
Transport Measurements General Setup.
For transport experiments, NRK-52E cells were seeded on 24-wellfilters (Falcon; Becton Dickinson Labware) and grown until confluence.The volumes of the apical and basolateral compartment were 0.5and 1.0 ml, respectively, to avoid hydrostatic pressure differences.Before each experiment, the cells were washed three times withPBS (138 mmol/L NaCl, 1 mmol/L NaH2PO4, 4 mmol/L Na2HPO4, 4mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 5 mmol/L glucose[pH 7.4]). Transport measurements were performed in PBS at pH7.4 and 37°C.
Fluorescein Uptake Method.
OA transport was determined by measuring the uptake of 10 x10–6 M fluorescein after 2 min, as similarly describedpreviously (25). After that, cells where washed four times withice-cold PBS until no fluorescein was detectable in the washingsolution. After that, the cells were lysed in 1 ml of 0.1% Triton-X100in 20 mM MOPS [3-(N-morpholino)propanedulfonic acid], and fluorescencewas counted in a multiwell plate reader (Victor2; Wallac Instruments,Turku, Finland). Counts were corrected for extracellular bindingand unspecific adhesion to the cells by subtraction of fluoresceincounts on cells at 4°C. Fluorescein counts were normalizedto protein content in the lysate measured by BCA protein assay(Pierce, Rockford, IL).
Evaluation of Fluorescein Uptake into NRK-52E Cells.
Proximal tubular NRK-52E cells up to now were not used as amodel for organic anion transport. Therefore, we first investigatedwhether this cell line is suitable for organic anion transportstudies. As we used 24-well filters herein (growth area 0.3cm2), we increased fluorescein concentration 10-fold as comparedwith what was described before (25) for a setup using six-wellfilters (4.2 cm2). We initially investigated basolateral fluoresceinuptake into proximal tubular NRK-52E cells compared with basolateraluptake into renal fibroblast NRK-52F cells. NRK-52E cells showedsaturable uptake of fluorescein, which is not the case in NRK-52Ffibroblasts. In NRK-52E cells, fluorescein uptake was linearfor up to 5 min; therefore, we chose a 2-min uptake time, whichis well within the linear range. As indicated in a previousstudy (26), OAT1 and OAT3 have different substrate specificitiesfor para-aminohippuric acid and -estrone sulfate, whereas theiraffinity to fluorescein is almost equal (27). Para-aminohippuricacid (10 mM) and -estrone sulfate (1000 µM) competitivelyinhibited fluorescein uptake into proximal tubular NRK-52E cellsto a similar extent, which was first evidence that NRK-52E cellsexpress both renal OAT1 (rOAT1) and rOAT3. Therefore, we decidedto investigate further organic anion uptake in NRK-52E cells.
Fluorescein Efflux Method
For investigating apical fluorescein efflux, the cells wereseeded on 24-well plates and incubated with 10 x 10–6mol/L fluorescein for 60 min. After washing, the efflux intothe apical compartment was determined for up to 15 min at 37or 4°C. Apical solutions and the cellular compartment werecollected separately. Fluorescein was detected as describedabove in the fluorescein uptake method. The total amount ofcounted fluorescein in both compartments was set as amount offluorescein in the cells at time 0 of the efflux experiments.Uptake rates were calculated over the linear efflux phase.
Tracer Flux Studies
Uptake of cycloleucine and glutarate into NRK-52E cells wasdone using radiolabeled substances, as described previously(28). Apical uptake of cycloleucine was determined using cellsthat were seeded on Petri dishes, whereas glutarate uptake wasby using cells that were grown on filter membranes. The concentrationswere as follows: [14C]glutarate, 1.5 x 10–6 mol/L; [14C]cycloleucine,1.5 x 10–6 mol/L. Unlabeled phenylalanine (58 x 10–3mol/L) was used as competing substrate for [14C]cycloleucine,and unlabeled glutarate (15 x 10–3 mol/L) was used ascompeting substrate for [14C]glutarate. [3H]mannitol (55 x 10–9mol/L) was used for correction of paracellular fluxes and measurementof extracellular water space. At the end of the experiment,the cells were washed twice with ice-cold PBS. Radioactivityof the solutions and the cells was measured using a liquid scintillationcounter (Packard Instruments, Frankfurt, Germany).
Reverse Transcription–PCR
RNA from cells that were seeded on Petri dishes was extractedusing AquaPure RNA Isolation Kit (BioRad, Hercules, CA). Inbrief, reverse transcription–PCR (RT-PCR) was performedaccording to Superscript One-Step RT-PCR system protocol (Invitrogen,Carlsbad, CA). cDNA was generated at 50°C for 30 min, andthen the samples were denatured at 94°C for 2 min. PCR amplificationwas performed in 40 cycles of 94°C for 15 s, then 55°Cfor 30 s and 72°C for 60 s. The final elongation step was72°C for 10 min. For rOAT1, the primers were 5'-tgg cataat acc gaa gag cc-3' (sense) and 5'-tgc tgc tgt tga ttc tgctt-3' (antisense), resulting in a 340-bp RT-PCR product. ForrOAT3, the primers were 5'-tgg agg acc tgt gat tgg aga a-3'(sense) and 5'-ata gaa cca gcc agc gta tgg a-3' (antisense),resulting in a 391-bp RT-PCR product.
Western Blot Analysis
Western blot analysis was done as described previously (29).Cells on Petri dishes were rinsed three times with PBS. Subsequently,cells were washed with ice-cold PBS three times and lysed inice-cold Triton X-100 lysis buffer (50 mM Tris-HCl [pH 7.5],100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM -glycerophosphate,200 µM sodium orthovanadate, 0.1 mM PMSF, 1 µg/mlleupeptin, 1 µM pepstatin A, and 1% Triton X-100) for25 min at 4°C. Insoluble material was removed by centrifugationat 12,000 x g for 15 min at 4°C. The protein content wasdetermined using a microbicinchoninic acid assay (Pierce) withBSA as a standard. Cell lysates were matched for protein, separatedby 12% SDS-PAGE and transferred to a polyvinylidene difluoridemicroporous membrane. Subsequently, membranes were blotted withrabbit anti-rOAT1 antibody or rabbit anti-rOAT3 antibody (bothfrom Alpha Diagnostics International, San Antonio, TX). Theprimary antibody was detected using horseradish peroxidase–conjugatedgoat anti-rabbit IgG and visualized by ECL Western blottingreagents and Hyperfilm ECL (Amersham Life Sciences International,Buckinghamshire, UK). According to the manufacturer’shandbook, Hyperfilm ECL exhibits a linear response to the lightproduced from enhanced chemiluminescence. In addition, linearitywas verified for our experimental conditions by a dilution serieswith increasing amounts of total cell protein (30). Blots wereanalyzed using SigmaGel 2000 Software (Jandel Scientific, CorteMadera, CA).
Analysis of Kinetic Data
Kinetic data (affinity, transport maximum) were obtained byfitting the values to the Michaelis-Menten equation accordingto the least-square method, using Sigma Plot Software (JandelScientific). IC50 values were determined according to DeLeanet al. (31) by the following equation: V = {V0/[1 + ([I]/IC50)]n}+ K, where V is the transport rate at any given [I], [I] isthe concentration of the inhibiting substance (PGE2), V0 isthe transport rate at [I] = 0, and K is the transport rate atmaximum inhibition. The data were fitted using the least-squaremethod (Sigma Plot, Jandel Scientific).
Other Methods
Cell number and cell volume were determined using a Coultercounter. Protein content was measured by BCA protein assay (Pierce;see Results or the figure legends for additional details onexperimental protocols).
Statistical Analyses
Data are presented as mean ± SEM, except for IC50, whichare presented as mean ± SD. The n value is given in thetext or in the figures and stands for the number of supportsused for the respective experiments (e.g., wells, filters, petridishes). For all experiments, n equals the number of cultureplates or filters used to perform the measurements. Statisticalsignificance was determined by unpaired t test or ANOVA as appropriate.Differences were considered statistically significant when P< 0.05.
Departing from a study of Ziemann et al. (32), which investigatedthe effect of PGE2 on MDR1 expression in rat primary hepatocytes,we decided to incubate proximal tubular NRK-52E cells with 10µM PGE2 for up to 72 h first. As indicated in Figure 1A,incubation with 10 µM PGE2 led to almost complete inhibitionof basolateral fluorescein uptake into NRK-52E cells after 72h, with the half-maximal effect taking place after approximately30 h. This effect was absent in cells that were incubated withserum-free growth medium only (Figure 1A).
Figure 1. Time course (up to 72 h) of the effect of 10 µM prostaglandin E2 (PGE2) on NRK-52E cells. (A) Time course of PGE2 effect on basolateral fluorescein uptake (10 µM; 37°C to 4°C) into proximal tubular NRK-52E cells. Basolateral fluorescein uptake rate is given as pmol/mg cell protein and 2 min. Cells were kept in serum-free growth medium without PGE2 for up to 72 h (untreated control cells []) or in serum-free growth medium with 10 µM PGE2 for up to 72 h (PGE2-treated cells [•]). (B) Time course of PGE2 on apical efflux rate of fluorescein into the apical (luminal) compartment out of preloaded NRK-52E cells. Cells were preloaded with fluorescein as indicated in detail in Materials and Methods (60 min; 10 µM). Efflux was measured for up to 15 min and at 37 and 4°C. Efflux is expressed as 37°C to 4°C and was linear up to 6 min. Efflux rate was calculated for the linear phase as pmol/min and pmol/mg cell protein. Efflux is given in fluorescein amount per minute expressed at % of the fluorescein amount in the cells after preloading. n is given beside the respective symbols.
As a lower cellular fluorescein content in a polarized cellcan be due either to reduced basolateral uptake or to increasedapical efflux, we investigated the effect of PGE2 on apicalefflux from cells that were preloaded with fluorescein. As indicatedin Figure 1B, the apical efflux rate of fluorescein was notaffected by PGE2 (10 µM) even after 72 h. Thus, PGE2 doesnot influence the luminal exit step for organic anions in oursetup. As PGE2 did not affect apical efflux of fluorescein,we exclude that the decrease in organic anion content is dueto an increase in luminal efflux. Thus, PGE2 does not affectthe apical part (described in [1,33]) of the organic anion secretorysystem in NRK-52E cells.
To exclude the possibility that the reduced fluorescein contentis due to a decrease in volume of the cellular compartment orto a loss of cell protein in general, we investigated totalcellular volume (Figure 2A) and protein content per cell (Figure 2B)in NRK-52E cells after incubation with 10 µM PGE2for 72 h. Because both parameters were not affected after PGE2exposure, the decrease in fluorescein content is due neitherto a PGE2 effect on NRK-52E cell volume nor to an effect ontotal NRK-52E cellular protein content.
Figure 2. Time course (up to 72 h) of the effect of 10 µM PGE2 on cellular volume and protein of NRK-52E cells. (B) Time course of PGE2 on cell volume per area of proximal tubular NRK-52E cells. Total cell volume is given as µl/cm2 effective growth area. (C) Time course of PGE2 on protein content per single NRK-52E cell. Protein content per cell is given as ng per single cell. n is given beside the respective symbols.
As both rOAT1 and rOAT3 mediate uptake of organic anions inexchange to dicarboxylates, organic anion uptake into proximaltubular cells indirectly depends on the activity of the basolateralsodium dicarboxylate co-transporter. Therefore, we investigatedwhether basolateral uptake of glutarate into NRK-52E cells isaffected by 10 µM PGE2 for up to 72 h. Glutarate is anonmetabolizable analogue of -ketoglutarate, which is takenup into the cells by co-transport with Na+via the sodium dicarboxylateco-transporter and acts as a counter ion for the organic aniontransporter (34). Inhibitory action of PGE2 on basolateral organicanion uptake could in principle be mediated indirectly via inhibitionof the latter transporter. As shown in Figure 3A, PGE2 incubationwas without effect on basolateral uptake of glutarate into NRK-52Ecells, indicating that PGE2 does not act by impairing countersubstrate recycling. In addition, PGE2 (10 µM for up to72 h; Figure 3B) also failed to inhibit apical uptake of cycloleucineinto NRK-52E cells. Because neither the apical uptake of theneutral amino acid cycloleucine nor the basolateral uptake ofglutarate was impaired, PGE2 does not affect transport systemsin general. Furthermore, the apical membrane potential–dependentuptake of cycloleucine (35) still occurs to the same extentas compared with controls, indicating that PGE2 does not disturbmembrane potential. Taken together, these data indicate thatPGE2 incubation affects rOAT1 and rOAT3 specifically and hasno general, nonspecific effect on rat proximal tubular NRK-52Ecells.
Figure 3. Time course (up to 72 h) of the effect of 10 µM PGE2 on apical uptake of the amino acid cycloleucine and basolateral uptake of the nonmetabolizable dicarboxylate glutarate. (A) Time course of PGE2 on basolateral uptake of glutarate into proximal tubular NRK-52E cells. Cells were grown on filter membranes, and labeled glutarate was added exclusively to the basolateral bath. Glutarate uptake was determined as uptake of 1.5 x 10–6 mol/L [14C]glutarate inhibitable by 1.5 x 10–3 mol/L nonlabeled glutarate at 37°C. Data are presented as pmol/cm2 per 5 min. n = 6 for every bar. (B) Time course of PGE2 on apical uptake of cycloleucine into proximal tubular NRK-52E cells. Cells were grown on Petri dishes, and labeled cycloleucine was added to the apical bath. Cycloleucine uptake was determined as uptake 1.5 x 10–6 mol/L [14C]cycloleucine inhibitable by 58 mM phenylalanine at 37°C. Data are presented as pmol/cm2 per 5 min. n = 6 for every bar.
Because all evidence indicated a specific inhibitory effectof PGE2 incubation on basolateral organic anion transport activity,we investigated whether this may be due to a reduced expressionof rOAT1 and/or rOAT3 in NRK-52E cells. As already indicatedby competitive inhibition experiments (see Materials and Methods),RT-PCR experiments (Figure 4) showed that NRK-52E cells expressboth rOAT1 and rOAT3 under control conditions. However, no RT-PCRsignal from either rOAT1 or rOAT3 was obtained after incubationof NRK-25E cells with 10 µM PGE2 for 48 h. Thus, incubationof proximal tubular NRK-52E cells with PGE2 leads to decreasedtranscription of the mRNA for both rOAT1 and rOAT3 and thereforemost probably will lead to reduced expression of rOAT1 and rOAT3.
Figure 4. Effect of incubation with 10 µM PGE2 on mRNA levels of renal organic anion transporter 1 (rOAT1) and rOAT3 in NRK-52E cells after 48 h. Reverse transcription–PCR was performed as indicated in Materials and Methods. M, marker; con, untreated control; 48 h, cells incubated with 10 µM PGE2 for 48 h.
To characterize further the effect of PGE2 on proximal tubularbasolateral organic anion uptake, we performed dose-responsestudies to investigate the effect of PGE2 on fluorescein uptakeinto NRK-52E cells. As half-maximal effect of 10 µM PGE2on basolateral fluorescein uptake takes place after 30 h, wedecided to incubate the cells for 48 h for dose-response experiments.PGE2 exerted a sigmoidal dose-response effect after 48 h (Figure 5)showing the maximum effect of PGE2 already at 1 µM.Half maximum action of PGE2 on basolateral fluorescein uptakeafter 48 h took place in the nanomolar range (IC50 = 31 nM ±9.8 nM).
Figure 5. Dose-response effect of PGE2 on basolateral uptake of fluorescein into proximal tubular NRK-52E cells after 48 h. NRK-52E cells were incubated with up to 1 µM PGE2 for 48 h. Basolateral fluorescein uptake (10 µM; 37°C to 4°C) into proximal tubular NRK-52E cells was determined afterward, mentioned in Materials and Methods. Basolateral fluorescein uptake rate is given as pmol/mg cell protein and 2 min. IC50 value is presented as mean ± SD.
To investigate whether the latter inhibition is due to reducedexpression of rOAT1 or rOAT3 or both, we performed Western blotexperiments using NRK-52E cells that were incubated with upto 1 µM PGE2 for 48 h (Figures 6 and 7). As indicatedin Figure 6A, expression of rOAT1 protein dose-dependently decreasedin NRK-52E cells showing half-maximal inhibition at 23 nM (±3.6 nM) PGE2 (Figure 6B), which is well within the range ofthe IC50 calculated from fluorescein uptake experiments (seeabove). PGE2 incubation also decreased the expression of rOAT3protein (Figure 7A) dose dependently, with half-maximal inhibitionoccurring at 27 nM (± 15 nM) PGE2 (Figure 7B). Thus,PGE2 shows equivalent inhibitory kinetics for rOAT1 and rOAT3expression. Furthermore, the half-maximal inhibition constantsare well within the range of inhibitory kinetics of PGE2 onbasolateral fluorescein uptake. Anti-rOAT1 antibody recognizeda band in the range just below 60 kD, which is well within therange of data published before for OAT1 (36,37). Anti-rOAT3antibody recognized a band of approximately 130 kD, which iswell within the range of OAT3 Western blot data (with a different,noncommercial anti-OAT3 antibody) published before (38). However,there are also Western blot data from rat kidney cortex publishedfrom another group using the same noncommercial antibody aspublished previously (38) showing two bands (approximately 60and 116 kD) (11). We do not have any explanation for the discrepancybetween these two publications. However, that two antibodiesof different origin directed against the same protein show aband of identical molecular weight (work of Kojima et al. [38]and our data) is strong evidence that it is the correct proteinthat is detected. That the half-maximal inhibition constantsare well within the range of inhibitory kinetics of PGE2 onbasolateral fluorescein uptake in proximal tubular NRK-52E cellssupports this conclusion.
Figure 6. Dose-response effect of PGE2 on the expression of rOAT1 in proximal tubular NRK-52E cells after 48 h. Expression was determined by Western blotting as described in Materials and Methods. (A) Single Western blot showing dose-response effect of PGE2 on the expression of rOAT1 in proximal tubular NRK-52E cells after 48 h. (B) Dose-response curve of PGE2 on the expression of rOAT1 in proximal tubular NRK-52E cells after 48 h including four distinct Western blotting experiments from four different cell passages. n is given beside the respective symbol. IC50 values are presented as mean ± SD.
Figure 7. Dose-response effect of PGE2 on the expression of rOAT3 in proximal tubular NRK-52E cells after 48 h. Expression was determined by Western blotting as described in Materials and Methods. (A) Single Western blot showing dose-response effect of PGE2 on the expression of rOAT3 in proximal tubular NRK-52E cells after 48 h. (B) Dose-response curve of PGE2 on the expression of rOAT3 in proximal tubular NRK-52E cells after 48 h including four distinct Western blotting experiments from five different cell passages. n is given beside the respective symbol. IC50 values are presented as mean ± SD.
PGE2 is a well-known inductor of fever (16). In a rat modelof LPS-induced fever (19), upregulation of the enzymes thatmediate its generation was shown. At the same time, a downregulationof the PGE2 transporters in liver and lung occurs, thereby disablingthese tissues to metabolize PGE2 and thus leading to elevatedplasma levels. However, PGE2 itself was not determined. In general,information on plasma concentrations of PGE2 in fever is meagre.The mean plasma concentration of PGE2 is approximately 1 to10 nM in resting and healthy humans (39). Plasma levels in rabbitLPS fever models rise approximately four-fold after a singleinjection of LPS (17). If a four-fold increase also would bethe case in fever in humans, PGE2 concentrations possibly couldreach some tens of nanomolar that is in the range of PGE2 actionon OAT in this study. Protein binding of PGE2 in plasma is approximately60% (22). Therefore, protein binding does not change the freeconcentration of PGE2 by orders of magnitude but only by half.Thus, the expected concentration of free PGE2 is likely to bein a range that suffices to generate an effect on OAT1 and OAT3.PGE2 is a substrate for both OAT mentioned (14). With respectto a role of peripheral PGE2 in fever (as suggested by Ivanovet al. [19]), it would make sense that renal clearance is impairedtogether with metabolic clearance to achieve high plasma levelsof PGE2. Nevertheless, as no data on PGE2 plasma levels in humansduring fever are available (Medline search August 2005), thisis still a speculation. Future studies will have to investigatethis important question in detail. With respect to the above-mentioneddata showing downregulation of PGE2 uptake transporters in liverand lungs (19,20), it is of interest whether this downregulationis also mediated by PGE2 itself. This has to be investigatedin future studies.
Nonetheless, there is some evidence indicating that PGE2 inhibitsits own renal excretion. In rat arthritis models, PGE2 is shownto be elevated in plasma and to be involved in both acute andchronic inflammatory events (40). In collagen-induced arthritisin mice, plasma levels of PGE2 are elevated up to three-fold,whereas urinary excretion is not different from controls after12 wk of arthritis (41), indicating a decrease in renal PGE2clearance. As PGE2 increases glomerular filtration, decreasedrenal clearance can be due only to reduced tubular secretion.As OAT1 and OAT3 mediate the rate-limiting step of organic aniontubular secretion, this is strong evidence that PGE2 downregulates(or at least inhibits) these proteins also in vivo. This isevidence that the PGE2-induced regulation of renal OAT alsomay be of importance in vivo and also during local inflammatoryevents.
There is indirect evidence that downregulation of OAT1 or OAT3also occurs in humans in vivo. It is well known that PGE2 levelsin the kidney are elevated in individuals who have Bartter syndrome,nephrogenic diabetes insipidus, and hyperprostaglandin E syndrome(42). Moreover, plasma urate levels are elevated in all of thesecircumstances. In humans, urate is eliminated via the kidney,where it is taken up by proximal tubular cells via OAT1 andOAT3 (43). Thus, increased urate plasma levels in the mentioneddiseases may be due, at least in part, to a downregulation ofOAT1 or OAT3 by PGE2. Thus, it seems reasonable that under thesecircumstances, PGE2 may lead to downregulation of rOAT1 andrOAT3 and thereby increases plasma urate levels. The investigationof this hypothesized mechanism will be the scope of future studies.Moreover, as in rat hyperuricemic kidney rOAT1 and rOAT3 arealso downregulated (13), the question arises whether urate itselfleads to downregulation of rOAT1 and rOAT3 or whether PGE2 isinvolved under these circumstances, too.
In addition, our data give rise to a very attractive hypothesison the persistence of chronic inflammation as PGE2 is knownto be elevated in chronic inflammatory events (40), as mentionedbefore. Thus, PGE2-induced downregulation of its own renal excretionwill contribute to high plasma levels of PGE2 driving chronicinflammation, which leads to further generation of PGE2 andto a further reduction of its renal clearance. Future studieswill have to test this hypothesis.
With respect to the kidney, receptors for PGE2 have been detectedin the renal cortex (44). In addition, PGE2 is considered tobe the major renal cyclo-oxygenase metabolite of arachidonicacid (44), which is produced in the renal cortex (45), particularlyin proximal tubule cells (46). In inflammatory disease of thekidney, PGE2 and its receptors are upregulated (47). In proximaltubule cells from pigs and humans, PGE2 is generated by reactiveoxygen species induced by stress (e.g., ischemia [48]) and maycontribute directly to renal lesions and loss of kidney function.Increased PGE2 levels in renal tissue may result in part fromreduced elimination via OAT1 and OAT3, which are downregulatedby PGE2 itself. Because PGE2 is an important mediator of inflammation,this may represent a vicious circle mechanism to drive PGE2-inducedinflammatory events in the kidney cortex interstitium. However,data published only recently indicate that prostanoids maintainrenal circulation in (Thy-1)-nephritis (49). If so, then inhibitionof prostanoid secretion by prostanoids (e.g., PGE2) would preventa prostanoid washout and thus would be involved in maintainingrenal circulation during nephritis, at least in rats.
In summary, we could show that PGE2 downregulates the rate-limitingstep of proximal tubular organic anion secretion mediated byOAT1 and OAT3. This may contribute to elevated levels of urate,PGE2, and other harmful metabolites or xenobiotics in renaldisease. This mechanism also may play an important role, onthe one hand, in keeping up PGE2 levels in acute and chronicinflammation or, on the other hand, in maintaining renal circulationduring nephritis. Moreover, this is the first report to indicatea transcriptional (down) regulation of rOAT1 and rOAT3, whichare rate-limiting for renal organic anion secretion by a distinctstimulus that is important in physiology and pathophysiology.
Acknowledgments
This work was supported in part by the Wilhelm-Sander-Stiftungand the Deutsche Forschungsgemeinschaft DFG Ge 905/8-1.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-ketoglutarate, which then is exchanged for organic anions. It has been shown that OAT1 represents characteristics of the basolateral, polyspecific transporter for organic anions, which had been functionally described for some time (1). Recently, new evidence has indicated that organic anion transporter 3 (OAT3) (2), which also is located in the basolateral renal proximal tubular membrane, also works as an appropriate exchanger for organic anions and dicarboxylates (3,4). Moreover, additional homologues have been cloned and were called OAT2 (5), OAT4 (6), OAT5 (7), and OAT6 (8). These clones show 40 to 60% homology in amino acid sequence compared with OAT1 or OAT3, and they differ in substrate specificity and expression pattern. Furthermore, these latter proteins are not anion exchangers like OAT1 or OAT3 but seem to work as facilitators of anion diffusion. In summary, the classical renal basolateral polyspecific uptake transporter for organic anions is represented by OAT1 and OAT3 (1). 
-estrone sulfate, whereas their affinity to fluorescein is almost equal (27). Para-aminohippuric acid (10 mM) and 
37°C to 4°C) into proximal tubular NRK-52E cells. Basolateral fluorescein uptake rate is given as pmol/mg cell protein and 2 min. Cells were kept in serum-free growth medium without PGE2 for up to 72 h (untreated control cells [
]) or in serum-free growth medium with 10 µM PGE2 for up to 72 h (PGE2-treated cells [•]). (B) Time course of PGE2 on apical efflux rate of fluorescein into the apical (luminal) compartment out of preloaded NRK-52E cells. Cells were preloaded with fluorescein as indicated in detail in Materials and Methods (60 min; 10 µM). Efflux was measured for up to 15 min and at 37 and 4°C. Efflux is expressed as 
