Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masereeuw, R.
Right arrow Articles by Russel, F. G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masereeuw, R.
Right arrow Articles by Russel, F. G. M.
J Am Soc Nephrol 14:2741-2749, 2003
© 2003 American Society of Nephrology

BASIC SCIENCE

Impaired Renal Secretion of Substrates for the Multidrug Resistance Protein 2 in Mutant Transport–Deficient (TR-) Rats

Rosalinde Masereeuw, Sylvia Notenboom, Pascal H. E. Smeets, Alfons C. Wouterse and Frans G. M. Russel

Department of Pharmacology and Toxicology, University Medical Center Nijmegen, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands.

Correspondence to Dr. Rosalinde Masereeuw, Department of Pharmacology and Toxicology 233, University Medical Center Nijmegen, Nijmegen Center for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: +31 24361 3730; FAX: +31 24361 4214;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Previous studies with mutant transport–deficient rats (TR-), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is also present in the luminal membrane of proximal tubule cells of the kidney, but little information is available on its role in the renal excretion of xenobiotics. The authors compared renal transport of the fluorescent Mrp2 substrates calcein, fluo-3, and lucifer yellow (LY) between perfused kidneys isolated from Wistar Hannover (WH) and TR- rats. Isolated rat kidneys were perfused with 100 nM of the nonfluorescent calcein-AM or 500 nM fluo3-AM, which enter the tubular cells by diffusion and are hydrolyzed intracellularly into the fluorescent anion. The urinary excretion rates of calcein and fluo-3 were 3 to 4 times lower in perfused kidneys from TR- rats compared with WH rats. In contrast, the renal excretion of LY (10 µM, free anion) was somewhat delayed but appeared unimpaired in TR- rats. Membrane vesicles from Sf9 cells expressing human MRP2 or human MRP4 indicated that MRP2 exhibits a preferential affinity for calcein and fluo-3, whereas LY is a better substrate for MRP4. We conclude that the renal clearance of the Mrp2 substrates calcein and fluo-3 is significantly reduced in TR- rat; for LY, the absence of the transporter may be compensated for by (an)other organic anion transporter(s). E-mail: R.Masereeuw@ncmls.kun.nl


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATP-binding cassette (ABC) proteins play an important role in the cellular defense against xenobiotics and their metabolites by active secretion of these compounds into bile, urine, and intestinal lumen. Overexpression of the ATP-driven transporters in tumor cells is associated with multidrug resistance, which is a major complication for successful cancer chemotherapy. The proteins couple ATP hydrolysis to the transport of specific substrates and include P-glycoprotein, which transports uncharged compounds and organic cations, and the multidrug resistance proteins (MRP), with a preference for organic anions (for reviews, see references 1–3). The canalicular membrane of hepatocytes contains an isoform of MRP called MRP2 or canalicular multispecific organic anion transporter (cMOAT) (4). In mutant transport-deficient (TR-) rats and Eisai hyperbilirubinemic rats (EHBR), the mRNA levels of Mrp2 are very low and the protein itself is absent (5,6). In TR- rats, this is due to a single nucleotide deletion in the gene resulting in a frame shift, which leads to an early stop codon. A human counterpart of the TR- rat is the Dubin-Johnson syndrome patient, who is lacking MRP2 (7). This defect is associated with a deficiency in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions.

Although the function of MRP2 in the liver has been studied extensively, the protein is also present in the luminal membrane of small intestine (8,9) and proximal tubule cells of the kidney (10), but little information is available on the mechanism governing renal and intestinal excretion of anionic xenobiotics by MRP2. All three organs have a main task in the excretion of xenobiotics, and multispecific organic anion transporters are supposed to play a key role in this process. Using killifish renal proximal tubules, we previously characterized an ATP-dependent efflux mechanism for the organic anions fluorescein methotrexate (FL-MTX) and Lucifer yellow (LY) (11,12). With a polyclonal antibody directed against rabbit Mrp2, an ortholog of the transporter could be identified in killifish at the proximal tubule brush border membrane (13). These findings are consistent with a teleost form of Mrp2, which mediates transport in killifish proximal tubules. Its importance in overall renal organic anion secretion is, however, still undefined. It has been demonstrated that the renal excretion of {alpha}-naphtyl-{beta}-D-glucuronide (14), leukotriene C4 (15), or glutathione-bimane (16) is not impaired in the TR- rat, whereas the biliary excretion of these compounds is markedly reduced. This indicates that (an)other MRP-like transport mechanism(s) exist for the renal excretion of organic anions, which compensate for the absence of Mrp2. A possible candidate may be Mrp4, which was recently identified by us as a novel organic anion transporter in the apical membrane of rat and human proximal tubule (17).

In the present study, we compared the renal excretion of three fluorescent anionic Mrp2 substrates in perfused kidneys isolated from normal Wistar Hannover (WH) rats and TR- rats. Our results indicate that the excretion of calcein and fluo-3 is significantly reduced in TR- rat kidneys, whereas LY excretion appeared unimpaired. In membrane vesicles from insect cells expressing human MRP2 and MRP4, calcein and fluo-3 were transported almost exclusively by MRP2. LY appeared to be a better substrate for MRP4 compared with MRP2. This suggests that for renal LY excretion, Mrp4 may compensate for the lack of Mrp2 in TR- rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Sodium pentobarbital was obtained from Apharmo (Arnhem, The Netherlands), Pluronic F108 was from BASF (Arnhem, The Netherlands), and heparin, aldosterone, and inulin were from Organon (Oss, The Netherlands). LY-CH dilithium salt, tetramethylrhodamine isothiocyanate-dextran (TRITC-dextran, Mw. 4,400), and dextran (Mw. 60,000 to 90,000) were purchased from Sigma (St. Louis, MO). Lysine-vasopressin was obtained from Sandoz Pharma Ltd. (Basel, Switzerland), angiotensin II from Beckman (Palo Alto, CA), and Synthamin 14 was from Travenol (Thetford, Norfolk, UK). Bovine serum albumin (BSA) was obtained from Boehringer Mannheim (Mannheim, Germany). Fluorescein methotrexate (FL-MTX), fluo-3, fluo-3-acetoxymethylester (fluo-3-AM), calcein-acetoxymethylester (calcein-AM), and calcein were from Molecular Probes (Eugene, OR). All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany) or Sigma (St. Louis, MO). The p-glycoprotein inhibitor, PSC-833, was a gift from Novartis (Arnhem, The Netherlands). The Groningen Yellow/transport mutant rat strain (GY/TR-; TR- rats) was kindly provided by the group of Jansen (14) and bred in Nijmegen.

Kidney Isolation and Perfusion
Rat kidneys were isolated and perfused as previously described in detail (18). Briefly, male Wistar-Hannover or TR- rats (225 to 275 g) were anesthetized intraperitoneally with pentobarbital (6 mg/100 g), and furosemide was injected intraperitoneally (1 mg/100 g) to prevent deterioration of the distal nephron. Heparin (125 IU/100 g) was injected in the femoral vein. The ureter of the right kidney was cannulated and the renal artery via the mesenteric artery without interruption of the blood flow. The kidney was then excised and placed in a fluid bath with a constant temperature of 37.5°C. The perfusate reservoir was placed also in a waterbath of 37.5°C, and fluid was gassed with 95% O2/5% CO2. The perfusion fluid had the following composition (mM, except indicated otherwise): NaCl 114.0, KCl 5.2, CaCl2 1.8, MgCl2 1.0, NaHCO3 22.5, Na2HPO4 0.84, KH2PO4 0.28, glucose 5.0, urea 4.0, Pluronic F108 25.0 g · l-1, glutathione 0.33, inositol 0.083, cysteine 0.50, glycine 2.3, Na-pyruvate 2.0, Na-acetate 1.22, Na-propionate 0.21, inosine 1.0, alanine 5.0, glutamine 0.11, L-glutamine acid 2.0, ascorbic acid 0.01, Na-lactate 1.0, choline chloride 1.0 mg/L, insulin 4 IU/L, aldosterone 2.0 µg · l-1, lysine-vasopressin 0.01 IU · l-1, and angiotensin II 15.0 ng · l-1. To this solution, 1.0% Synthamin 14, a mixture of 15 amino acids, was added. Pluronic F-108 was used as oncotic agent in the normal perfusion medium, whereas a combination of 3.3% dextran and 2% BSA was used in Pluronic-free perfusion fluid. For the determination of GFR, either inulin was added to the perfusion fluid in Pluronic experiments (100 µg · ml-1) or TRITC-dextran in dextran/BSA experiments (10 µg · ml-1). Because inulin would bind to BSA in Pluronic-free perfusion medium, TRITC-dextran was used. In our perfused kidney preparation, perfusion medium was recirculated at a constant flow rate (15 ml · min-1) with a perfusate pressure of approximately 90 mmHg. During the first 5 min of perfusion, the venous effluent was discarded; after this period, the perfusion fluid was recirculated, and the kidney was allowed to stabilize for 30 min. Then the experiment was started with a 30-min baseline period, after which the experiment was started by the addition of 0.23 mg of FL-MTX resulting in an initial perfusate concentration of 1 µM, calcein-AM with an initial perfusate concentration of 100 nM, fluo-3-AM (initial perfusate concentration of 500 nM), or LY (initial perfusate concentration of 10 µM). During the baseline period, the perfusate volume was 500 ml, from which a sample of 5 ml was drawn. After the baseline period, the experimental fluid was connected to the kidney, with a total volume of 250 ml in which FL-MTX, calcein-AM, fluo-3-AM, or LY were already dissolved. The experimental period was 120 min. Urine samples were collected during control and experimental periods over 10-min intervals. Perfusate samples (300 µl) were drawn at the midpoint of each urine collection interval. Two additional perfusate samples were taken: one at the beginning of the experimental period (t = 0), and one at the end of the experiment. Urine and perfusate samples were analyzed directly after the experiment. Perfusion fluid during experimental period and perfusion and urine samples were protected from light.

Uptake of Fluorescent Substrates in Membrane Fractions of Sf9-Cells Overexpressing MRP2 and MRP4
Cells from Spodoptera frugiperda (Sf9) expressing human MRP2 and MRP4 were generated by infection of cells using a recombinant baculovirus encoding MRP2 and MRP4 as described previously (17). For controls, Sf9 cells were infected with a baculovirus encoding the {beta}-subunit of rat H+/K+-ATPase. Crude membrane fractions from infected Sf9 cells were isolated. Protein concentration was determined using the BioRad protein assay (BioRad Laboratories, Veenendaal, The Netherlands). Uptake of fluorescent substrates was measured using a rapid filtration technique (19).

Analytical Methods
Urine and perfusate samples were analyzed for glucose, using the GLUCO-QUANT kit from Boehringer (Mannheim, Germany), and alkaline phosphatase (according to reference 20) to assess proximal tubule function. Inulin was determined according to a previously published method (21). The concentration of FL-MTX, calcein, fluo-3, LY, or TRITC-dextran in perfusate and urine samples were determined by means of fluorescence spectrophotometry. To this end, an aliquot of 50 µl of the perfusate sample was taken and adjusted to 600 µl with analysis buffer (Sörensen buffer, pH 7.36). Urine samples were diluted 10 times with buffer, from which an aliquot of 25 µl was taken and adjusted to 600 µl with 50 µl of blank perfusion fluid and 525 µl of buffer. Fluorescence in these prepared samples was measured using a Perkin Elmer LS50 luminescence spectrophotometer (Beaconsfield, Buckinghamshire, UK). Excitation and emission wavelengths were as follows: for FL-MTX, 491 and 516 nm, respectively; for calcein, 488 and 518 nm, respectively; for fluo-3, 506 and 524 nm, respectively; for LY, 425 and 525 nm, respectively; for the determination of TRITC-dextran, the wavelengths were set to 511 and 575 nm, respectively. In all cases, a bandwidth of 5 nm was used. Concentrations were calculated by comparing fluorescent intensity (in photomultiplier units) with a calibration curve of spiked samples of blank perfusion fluid with different concentrations of FL-MTX, calcein, fluo-3, LY, or TRITC-dextran.

Statistical Analyses
In membrane transport experiments, net ATP-dependent uptake was calculated by subtracting values in the absence of ATP from those in the presence of ATP. Furthermore, uptake values were corrected for control uptake measured in vesicles expressing the {beta}-subunit of H+,K+-ATPase. All data are expressed as mean ± SD unless stated otherwise. Statistical differences between means were determined with t test. Statistical differences between multiple means were determined with a one-way ANOVA followed by the least significant difference post test. Means were considered significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In recent studies using killifish proximal tubules, we showed that FL-MTX is a substrate for Mrp2 and that its luminal secretion was sensitive to inhibition by leukotriene C4, chlorodinitrobenzene (CDNB), and estradiol-17-{beta}-D-glucuronide (11,12). Here, we investigated the secretion of this fluorescent substrate by Mrp2 in the isolated perfused rat kidney. The viability of the perfused rat kidneys was assessed by monitoring fractional excretion of glucose, cumulative excretion of alkaline phosphatase, fractional reabsorption of water, urine flow and pH, GFR, and renal perfusate pressure. On the basis of these criteria, kidneys from WH rats as well as TR- rats showed good function over the 2-h time course of the clearance experiments (Table 1). Our attempts to measure active secretion of FL-MTX in perfused rat kidneys were unsuccessful. After perfusing WH rat kidneys for 2 h with 1 µM FL-MTX, a clearance over glomerular filtration ratio of 0.61 ± 0.11 (n = 3) was observed, indicating net reabsorption of the fluorescent dye. Most likely, the bulky organic anion is not able to enter tubule cells from the basolateral side of the cells.


View this table:
[in this window]
[in a new window]

  		Table 1. Functional parameters of isolated perfused kidneys from WH and TR- rats during control perfusionsa
 
To overcome cell entry problems as observed for FL-MTX, we used an esterified fluorescein derivative, calcein-AM. This hydrophobic nonfluorescent precursor enters the tubule cells by simple diffusion and is subsequently cleaved into calcein by cytoplasmic esterases. Evers et al. (22) showed that this organic anion is actively secreted by MRP2 in stably transfected cells. After addition of 100 nM calcein-AM to perfusion medium, the renal excretion rate of calcein was measured in urine and depicted in the upper panel of Figure 1 as a function of perfusion time. Evidently, the renal excretion of the organic anion was delayed and reduced in TR- rats, indicating the contribution of Mrp2 in renal clearance of calcein. Kidney functional parameters in calcein-AM perfusion experiments were not significantly different in TR- rats compared with WH rats (Table 2). The precursor, calcein-AM, is also a good substrate for P-glycoprotein (23). To avoid efflux of calcein-AM by P-glycoprotein, we performed perfusion experiments in the presence of 10 µM PSC-833; however, no differences in calcein excretion were found in comparison with the perfusions in absence of PSC-833 (data not shown).



View larger version (21K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Renal excretion rate of calcein as a function of time. A concentration of 100 nM of the hydrophobic, nonfluorescent compound calcein-AM was added to normal perfusion medium (upper panel) or Pluronic-free medium (lower panel), and secretion of the fluorescence calcein into urine was measured in Wistar Hannover (WH) rat kidneys (•, {circ}) and in mutant transport–deficient (TR-) rat kidneys ({blacksquare}, {square}). Data are means of four to five experiments ± SD. *significantly different from WH (P < 0.05). **significantly different from WH (P < 0.01).

 

View this table:
[in this window]
[in a new window]

  		Table 2. Functional parameters of isolated perfused rat kidneys with 100 nM calcein-AM in normal perfusion medium and/or after perfusion with Pluronic-free mediuma
 
It was demonstrated that Pluronic copolymers inhibit MRP1-mediated transport (24), although others found no significant effect on MRP1 or even an increase in MRP2-mediated transport (22). Since Pluronic F108 was added to perfusion medium as a colloid oncotic agent and to increase perfusion fluid viscosity, we investigated the effect of the copolymer on renal calcein excretion. For comparison, a combination of dextran (3.3% wt/vol) and BSA (2% wt/vol) was used as a colloid osmotic agent. The results shown in the lower panel of Figure 1 indicate that the renal excretion rate is somewhat faster during the first 60 min of the experiment. However, no differences in maximum calcein excretion rate were observed between the two perfusion media in WH rat kidneys. Although differences in GFR and urinary excretion rate were observed, kidney viability was not altered by the change in colloid osmotic agent (Table 2). GFR values were increased in Pluronic-free medium, either due to a decreased vascular resistance of the kidney when perfused with an albumin solution or due to a reduced oncotic pressure resulting in an increase in net filtration Starling forces (25). In TR- rat kidneys perfused with Pluronic-free medium, a steady-state calcein excretion rate of approximately 13 pmol · min-1 was observed after 80 min of perfusion. This was higher than the maximum excretion rate observed for calcein in normal medium perfusions, however, a steady-state was not yet achieved after 120 min of perfusion. Most likely, the faster onset of calcein excretion in Pluronic-free medium is responsible for this difference. Also renal perfusion flow was somewhat lower in normal medium compared with Pluronic-free medium (varying between 11 and 15 ml/min in normal medium versus 15 to 20 ml/min in Pluronic-free medium).

In addition to calcein, the calcium-dependent fluorescence indicator, fluo-3, was found to be an Mrp2 substrate (26). In agreement with calcein, fluo-3 excretion was significantly reduced in perfused kidneys from TR- rats compared with WH rats after perfusion with the nonfluorescent precursor, fluo-3-AM (Figure 2, upper panel). The use of the hydrophobic acetoxymethyl ester derivative was necessary for tubular cell loading of fluo-3, because the organic anion itself was not secreted in the perfused rat kidney. Most likely, the basolateral membrane functions as a diffusional barrier for fluo-3. The lower panel of Figure 2 shows that an overall clearance over GFR of approximately 0.6 was calculated in both the WH and TR- rat kidneys after perfusion with 500 nM fluo-3. This indicates that, similar to FL-MTX, a net reabsorption of fluo-3 was observed. No differences in kidney function during fluo-3 or fluo-3-AM perfusions were detected for both rat strains (Table 3).



View larger version (19K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Renal excretion rate of fluo-3 as a function of time. A concentration of 500 nM of the hydrophobic, nonfluorescent, ester derivative fluo-3-AM was added to perfusion medium (upper panel) or the free fluorescing organic anion fluo-3 (lower panel), and secretion of fluo-3 into urine was measured in WH rat kidneys (•, {circ}) and in TR- rat kidneys ({blacksquare}, {square}). In the upper panel, data are expressed as renal excretion rate; in the lower panel, clearance over GFR was calculated. Data are means ± SD of four experiments. **significantly different from WH (P < 0.01).

 

View this table:
[in this window]
[in a new window]

  		Table 3. Functional parameters of isolated perfused rat kidneys with 500 nM fluo-3-AM or 500 nM fluo-3 in normal perfusion mediuma
 
The reduced excretion of calcein and fluo-3 in TR- rat kidneys in comparison with WH rat kidneys appears to be due to the absence of Mrp2 and not a result of a reduced hydrolysis rate of the hydrophobic precursors into the fluorescent organic anions. Figure 3 shows the concentration of calcein (upper panel) and fluo-3 (lower panel) detected in perfusion medium in time after perfusion with the acetoxymethyl esters. No significant differences in concentrations of the hydrolyzed organic anions appearing in the recirculating medium between the two rat strains were detected.



View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Appearance of calcein (upper panel) and fluo-3 (lower panel) in kidney perfusate after perfusion with calcein-AM or fluo-3-AM. A concentration of 500 nM hydrophobic, nonfluorescent compound calcein-AM or fluo-3-AM was added to perfusion medium, and appearance of the fluorescence calcein or fluo-3 into perfusion medium was measured in WH rat kidneys (•) and in TR- rat kidneys ({circ}). Data are means ± SD of three experiments.

 
In a previous study, we showed that the renal secretion of the anionic fluorescent dye, LY, was sensitive to inhibition by leukotriene C4 and CDNB (12), suggesting the involvement of Mrp2 in luminal LY secretion. Figure 4 shows LY/inulin clearance ratios for kidneys perfused with buffer containing 10 µM LY. After exposing WH rat kidneys to a perfusate concentration of 10 µM LY, the LY/inulin clearance ratio exceeded unity, indicating net secretion of the fluorescent dye. In agreement with previous results (12), the clearance ratio increased during the first 40 min of the experiment and then gradually declined. In TR- rat kidneys, however, the clearance ratio increased slower and a maximum was observed 10 min later than in WH rats (at 70 min of perfusion) followed by a gradual decline. For both rat strains, the renal clearance ratio at maximum averaged about 2. No differences in kidney function during LY perfusions were detected for both rat strains (Table 4).



View larger version (22K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Renal clearance over GFR of LY as a function of time. A concentration of 10 µM LY was used in WH rat kidneys (•) and in TR- rat kidneys ({blacksquare}). Data are means ± SD of four experiments. *significantly different from WH (P < 0.05).

 

View this table:
[in this window]
[in a new window]

  		Table 4. Functional parameters of isolated perfused rat kidneys with 10 µM Lucifer yellow in normal perfusion mediuma
 
We recently demonstrated that renal proximal tubule expresses another apical organic anion ABC transporter, MRP4, the substrate specificity of which overlaps with that of MRP2 (17). To investigate whether Mrp4 may compensate for the lack of Mrp2 in TR- rat kidney, uptake studies were performed with membrane vesicles isolated from Sf9-MRP4 cells and compared with studies performed using Sf9-MRP2 membrane vesicles. The net ATP-dependent uptake of 10 µM calcein was clearly observed for Sf9-MRP2 vesicles (Figure 5) but was undetectable in Sf9-MRP4 vesicles. For Fluo-3, a fourfold higher uptake was found for Sf9-MRP2 vesicles compared with Sf9-MRP4 vesicles. On the other hand, Sf9-MRP4 vesicles exhibited a fourfold higher LY uptake in comparison with Sf9-MRP2 vesicles.



View larger version (14K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Net ATP-dependent uptake, corrected for control uptake, of fluorescent substrates in Sf9-MRP2 (gray bars) and Sf9-MRP4 (black bars) vesicles. Membrane vesicles were incubated at 37°C for 20 min with 10 µM calcein, 10 µM Fluo-3, and 100 µM LY and an ATP regenerating system. Data are given as mean ± SD, n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many of the transport systems for organic anions in the apical membrane of renal proximal tubule have initially been characterized in membrane vesicle studies (for recent reviews, see reference 27,28). Nevertheless, remarkably little information is available on the function of Mrp2 in the kidney, whereas the characteristics of Mrp2-mediated secretion in liver canalicular membranes have been studied extensively in normal and TR- rats. A major cause for this discrepancy is that isolated brush border membrane vesicles of proximal tubule cells are exclusively oriented right-side out (29) and are therefore unsuitable for measuring ATP-dependent uptake, as the ATP-binding site can only be reached by ATP when it is exposed to the extravesicular space.

In the present study, we investigated Mrp2-mediated renal excretion of fluorescent substrates using isolated perfused rat kidney, which allows an accurate determination of renal drug clearance under controlled conditions (30,31). Although we have previously shown that FL-MTX is a good substrate for Mrp2 using membrane vesicles from Sf9-cells overexpressing recombinant rabbit Mrp2 (32), our attempts to use the compound in perfused rat kidneys were unsuccessful. In killifish proximal tubules, we clearly observed a two-step process in FL-MTX secretion: (1) uptake into the tubular cells against the electrochemical gradient; (2) efflux into the tubular lumen by Mrp2 (11). In contrast, FL-MTX was not secreted by perfused rat kidney in the present study, most likely because this large anionic compound is not taken up from the peritubular side. Apparently, a basolateral uptake system for large lipophilic organic anions similar to that in killifish proximal tubules is not present in rat kidney. To overcome cell entry problems as observed for FL-MTX, the cell permeant derivatives calcein-AM and fluo-3-AM were applied. Renal secretion of the fluorescent organic anions, calcein and fluo-3, was clearly impaired in isolated TR- rat kidneys. These results are in agreement with previous findings that calcein and fluo-3 are actively transported by MRP2 (22,26). The steady increase in renal dye excretion in TR- rats may be a result of glomerular filtration of free calcein and fluo-3 in the recirculating system. It is unclear by what mechanism hydrolyzed organic anions appear in perfusion medium. Probably a carrier-mediated process is involved in efflux of the anionic compounds in perfusate, although passive back-diffusion cannot be excluded because the concentration gradient is in favor of this process. Using a polarized canine renal cell line (MDCKII) stably expressing MRP2, Evers et al. (22) found a stimulation of calcein transport by Pluronic L61, suggesting that the cells retained the precursor, calcein-AM, better. This was unexpected because others already reported a clear inhibition of MRP1-mediated transport by Pluronic block copolymers (24). Our results demonstrate that neither a stimulatory nor an inhibitory effect of the copolymer Pluronic F108 could be detected. In presence of the copolymer, present in normal perfusion medium, the renal excretion rate of calcein was somewhat lower compared with the perfusions with Pluronic-free medium. This can be explained by the low urinary flow and GFR in normal medium compared with Pluronic-free medium.

In TR- rat kidneys, the efflux of LY was only delayed in comparison with WH rats but not reduced. This contrasts with our previous findings for LY in killifish renal proximal tubules, where its secretion was found to be sensitive to the Mrp2 inhibitors, leukotriene C4, CDNB, and 17{beta}-estradiol-17{beta}-D-glucuronide (12,13). It has been shown that TR- rats have a dramatically reduced hepatic excretion of the conjugates {alpha}-naphtyl-{beta}-D-glucuronide and leukotriene C4, whereas their urinary excretion was almost unimpaired (14,15). Therefore, it may very well be possible that the absence of Mrp2 from the kidney can be compensated for by (an)other Mrp-like organic anion transporter(s). Mrp4 seems to be a good candidate because it is expressed in the apical membrane of renal proximal tubule, but not in hepatocyte canalicular membranes (17). However, the organic anion transport proteins (Oatp), Oatp1, Oatp3, Oat-k1, and Oat-k2, are also localized to the apical membrane of rat proximal tubules (17,33–35). These transporters accept Mrp2 substrates, like dinitrophenol-glutathione and methotrexate (17,36,37). Nevertheless, the Oatp probably have a preference for transport in the uptake direction (reabsorption), rather than that they are involved in excretion (38). In the present study, we demonstrate that Mrp4 indeed might compensate for the lack of Mrp2 in TR- rat kidneys. In close agreement with our findings in perfused rat kidney, MRP2 shows a preferential affinity for calcein and fluo-3, whereas LY is a better substrate for MRP4. Van Aubel et al. (17) showed previously that there is no difference in Mrp4 protein expression in isolated brush border proximal tubular membranes of TR- rat compared with control rats. Apparently, the basal expression level of Mrp4 in rat kidney is sufficient to compensate for the loss of Mrp2 in renal LY handling. In contrast, calcein and fluo-3 excretions are equally reduced in TR- rats compared with controls, but calcein seems not to be a substrate for MRP4, whereas fluo-3 is a poor substrate for the transporter. This might be a result of differences between rat Mrp4 and human MRP4 substrate specificity. Evidently, more research is needed to define the relative importance of all above-mentioned organic anion transporters in the facilitated efflux of anionic xenobiotics into the urine. Moreover, in this study we used marker compounds unimportant for Dubin-Johnson patients. In a forthcoming study, we will investigate the renal handling of clinically relevant drugs known to be substrates for Mrp2 (e.g., pravastatin, temocaprilate, cefpiramide, ceftriaxone, p-aminohippurate).

In conclusion, the results of the present study demonstrate that, in contrast with earlier findings (14–16), the renal secretion of some organic anions is clearly impaired in TR- rats, implying the contribution of Mrp2. For LY, absence of Mrp2 from the kidney may be compensated for by (an)other organic anion transporter(s), including Mrp4. However, the clearance of the fluorescent substrates, calcein and fluo-3, is significantly reduced in TR- rats.


   Acknowledgments
 
We gratefully acknowledge the aid of Miriam Huls in the membrane vesicle experiments. This study was supported by the Dutch Kidney Foundation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Leveille-Webster CR, Arias IM: The biology of P-glycoproteins. J Membr Biol 143: 89–102, 1995[Medline]
  2. Borst P, Evers R, Kool M, Wijnholds J: The multidrug resistance protein family. Biochim Biophys Acta 1461: 347–357, 1999[Medline]
  3. Dean M, Allikmets R: Complete characterization of the human ABC gene family. J Bioenerg Biomembr 33: 475–479, 2001[CrossRef][Medline]
  4. Paulusma CC, Bosma PJ, Zaman GJR, Bakker CTM, Otter M, Scheffer GL, Scheper RJ, Borst P, Oude Elferink RPJ: Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 271: 1126–1128, 1996[Abstract]
  5. Mayer R, Kartenbeck J, Buchler M, Jedlitschky G, Leier I, Keppler D: Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J Cell Biol 131: 137–150, 1995[Abstract/Free Full Text]
  6. Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, Sugiyama Y: Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am J Physiol 272: G16–G22, 1997
  7. Paulusma CC, Kool M, Bosma PJ, Scheffer GL, Borg Ft, Scheper RJ, Tytgat GNJ, Borst P, Baas F, Oude Elferink RPJ: A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 25: 1539–1542, 1998[CrossRef]
  8. Mottino AD, Hoffman T, Jennes L, Vore M: Expression and localization of multidrug resistant protein mrp2 in rat small intestine. J Pharmacol Exp Ther 293: 717–723, 2000[Abstract/Free Full Text]
  9. Van Aubel RAMH, Hartog A, Bindels RJM, van Os CH, Russel FGM: Expression and immunolocalization of multidrug resistance protein 2 in rabbit small intestine. Eur J Pharmacol 400: 195–198, 2000[CrossRef][Medline]
  10. Schaub TP, Kartenbeck J, Konig J, Vogel O, Witzgall R, Kriz W, Keppler D: Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J Am Soc Nephrol 8: 1213–1221, 1997[Abstract]
  11. Masereeuw R, Russel FGM, Miller DS: Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy. Am J Physiol 271: F1173–F1182, 1996
  12. Masereeuw R, Moons MM, Toomey BH, Russel FGM, Miller DS: Active Lucifer yellow secretion in renal proximal tubule: Evidence for organic anion transport system crossover. J Pharmacol Exp Ther 289: 1104–1111, 1999[Abstract/Free Full Text]
  13. Masereeuw R, Terlouw SA, van Aubel RAMH, Russel FGM, Miller DS: Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol Pharmacol 57: 59–67, 2000[Abstract/Free Full Text]
  14. de Vries MH, Redegeld FA, Koster AS, Noordhoek J, de Haan JG, Oude Elferink RPJ, Jansen PLM: Hepatic, intestinal and renal transport of 1-naphthol-beta-D-glucuronide in mutant rats with hereditary-conjugated hyperbilirubinemia. Naunyn Schmiedebergs Arch Pharmacol 340: 588–592, 1989[Medline]
  15. Keppler D, Muller M, Klunemann C, Guhlmann A, Krauss K, Muller J, Berger U, Leier I, Mayatepek E: Transport and in vivo elimination of cysteinyl leukotrienes. Adv Enzyme Regul 32: 107–116, 1992[CrossRef][Medline]
  16. Terlouw SA, Masereeuw R, van Den Broek PHH, Notenboom S, Russel FGM: Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells. Br J Pharmacol 134: 931–938, 2001[CrossRef][Medline]
  17. van Aubel RAMH, Smeets PHE, Peters JGP, Bindels RJM, Russel FGM: The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 13: 595–603, 2002[Abstract/Free Full Text]
  18. Cox PGF, Moons MM, Slegers JFG, Russel FGM, van Ginneken CAM: Isolated perfused rat kidney as a tool in the investigation of renal handling and effects of nonsteroidal antiinflammatory drugs. J Pharmacol Meth 24: 89–103, 1990[CrossRef][Medline]
  19. van Aubel RAMH, van Kuijck MA, Koenderink JB, Deen PMT, van Os CH, Russel FGM: Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells. Mol Pharmacol 53: 1062–1067, 1998[Abstract/Free Full Text]
  20. Mircheff AK, Wright EM: Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities. J Membr Biol 28: 309–333, 1976[CrossRef][Medline]
  21. Heyrovski A: A new method for the determination of inulin in plasma and urine. Clin Chem Acta 1: 470–474, 1956[CrossRef][Medline]
  22. Evers R, Kool M, Smith AJ, van Deemter L, de Haas M, Borst P: Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer 83: 366–374, 2000[CrossRef][Medline]
  23. Homolya L, Hollo Z, Germann UA, Pastan I, Gottesman MM, Sarkadi B: Fluorescent cellular indicators are extruded by the multidrug resistance protein. J Biol Chem 268: 21493–21496, 1993[Abstract/Free Full Text]
  24. Miller DW, Batrakova EV, Kabanov AV: Inhibition of multidrug resistance-associated protein (MRP) functional activity with pluronic block copolymers. Pharm Res 16: 396–401, 1999[CrossRef][Medline]
  25. Maack T: Renal clearance and isolated kidney perfusion techniques. Kidney Int 30: 142–151, 1986[Medline]
  26. Nies AT, Cantz T, Brom M, Leier I, Keppler D: Expression of the apical conjugate export pump. Mrp 2: In the polarized hepatoma cell line, WIF-B. Hepatology 28: 1332–1340, 1998[CrossRef][Medline]
  27. Masereeuw R, Russel FGM: Mechanisms and clinical implications of renal drug excretion. Drug Metab Rev 33: 299–351, 2001[CrossRef][Medline]
  28. Russel FGM, Masereeuw R, van Aubel RAMH: Molecular aspects of renal anionic drug transport. Annu Rev Physiol 64: 563–594, 2002[CrossRef][Medline]
  29. Haase W, Schafer A, Murer H, Kinne R: Studies on the orientation of brush-border membrane vesicles. Biochem J 172: 57–62, 1978[Medline]
  30. Masereeuw R, Moons MM, Russel FGM: Renal excretion and accumulation kinetics of 2-methylbenzoylgycine in the isolated perfused rat kidney. J Pharm Pharmacol 48: 560–565, 1996[Medline]
  31. Masereeuw R, Moons MM, Russel FGM: Rhodamine 123 accumulates extensively in the isolated perfused rat kidney and is secreted by the organic cation system. Eur J Pharmacol 321: 315–323, 1997[CrossRef][Medline]
  32. Terlouw SA, Graeff C, Smeets PHE, Fricker G, Russel FGM, Masereeuw R, Miller DS: Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule. J Pharmacol Exp Ther 301: 578–585, 2002[Abstract/Free Full Text]
  33. Bergwerk AJ, Shi X, Ford AC, Kanai N, Jacquemin E, Burk RD, Bai S, Novikoff PM, Stieger B, Meier PJ, Schuster VL, Wolkoff AW: Immunologic distribution of an organic anion transport protein in rat liver and kidney. Am J Physiol 271: G231–G238, 1996
  34. Masuda S, Saito H, Nonoguchi H, Tomita K, Inui K: mRNA distribution and membrane localization of the OAT-K1 organic anion transporter in rat renal tubules. FEBS Lett 407: 127–131, 1997[CrossRef][Medline]
  35. Masuda S, Ibaramoto K, Takeuchi A, Saito H, Hashimoto Y, Inui K: Cloning and functional characterization of a new multispecific organic anion transporter. Oat-k 2: in rat kidney. Mol Pharmacol 55: 743–753, 1999[Abstract/Free Full Text]
  36. Li L, Lee TK, Meier PJ, Ballatori N: Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273: 16184–16191, 1998[Abstract/Free Full Text]
  37. Saito H, Masuda S, Inui K: Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J Biol Chem 271: 20719–20725, 1996[Abstract/Free Full Text]
  38. Hagenbuch B, Meier PJ: The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 1–18, 2003[Medline]
Received for publication March 10, 2003. Accepted for publication August 16, 2003.




This article has been cited by other articles:


Home page
 Toxicol SciHome page
C. C. Bridges, L. Joshee, and R. K. Zalups
MRP2 and the DMPS- and DMSA-Mediated Elimination of Mercury in TR- and Control Rats Exposed to Thiol S-Conjugates of Inorganic Mercury
Toxicol. Sci., September 1, 2008; 105(1): 211 - 220.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
C. C. Bridges, L. Joshee, and R. K. Zalups
Multidrug Resistance Proteins and the Renal Elimination of Inorganic Mercury Mediated by 2,3-Dimercaptopropane-1-Sulfonic Acid and Meso-2,3-dimercaptosuccinic Acid
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 383 - 390.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
M. Hasegawa, H. Kusuhara, M. Adachi, J. D. Schuetz, K. Takeuchi, and Y. Sugiyama
Multidrug Resistance-Associated Protein 4 Is Involved in the Urinary Excretion of Hydrochlorothiazide and Furosemide
J. Am. Soc. Nephrol., January 1, 2007; 18(1): 37 - 45.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S. Notenboom, A. C. Wouterse, B. Peters, L. H. Kuik, S. Heemskerk, F. G. M. Russel, and R. Masereeuw
Increased Apical Insertion of the Multidrug Resistance Protein 2 (MRP2/ABCC2) in Renal Proximal Tubules following Gentamicin Exposure
J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 1194 - 1202.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
F. M. van de Water, O. C. Boerman, A. C. Wouterse, J. G. P. Peters, F. G. M. Russel, and R. Masereeuw
INTRAVENOUSLY ADMINISTERED SHORT INTERFERING RNA ACCUMULATES IN THE KIDNEY AND SELECTIVELY SUPPRESSES GENE FUNCTION IN RENAL PROXIMAL TUBULES
Drug Metab. Dispos., August 1, 2006; 34(8): 1393 - 1397.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
P. H.E. Smeets, R. A.M.H. van Aubel, A. C. Wouterse, J. J.M.W. van den Heuvel, and F. G.M. Russel
Contribution of Multidrug Resistance Protein 2 (MRP2/ABCC2) to the Renal Excretion of p-aminohippurate (PAH) and Identification of MRP4 (ABCC4) as a Novel PAH Transporter
J. Am. Soc. Nephrol., November 1, 2004; 15(11): 2828 - 2835.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
N. Mizuno, M. Suzuki, H. Kusuhara, H. Suzuki, K. Takeuchi, T. Niwa, J. W. Jonker, and Y. Sugiyama
IMPAIRED RENAL EXCRETION OF 6-HYDROXY-5,7-DIMETHYL-2-METHYLAMINO-4-(3-PYRIDYLMETHYL) BENZOTHIAZOLE (E3040) SULFATE IN BREAST CANCER RESISTANCE PROTEIN (BCRP1/ABCG2) KNOCKOUT MICE
Drug Metab. Dispos., September 1, 2004; 32(9): 898 - 901.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
T. Imaoka, H. Kusuhara, S. Adachi-Akahane, M. Hasegawa, N. Morita, H. Endou, and Y. Sugiyama
The Renal-Specific Transporter Mediates Facilitative Transport of Organic Anions at the Brush Border Membrane of Mouse Renal Tubules
J. Am. Soc. Nephrol., August 1, 2004; 15(8): 2012 - 2022.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masereeuw, R.
Right arrow Articles by Russel, F. G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masereeuw, R.
Right arrow Articles by Russel, F. G. M.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP