Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line

Lara M. Mangravite1, Joshua H. Lipschutz2, Keith E. Mostov3, and Kathleen M. Giacomini1

Departments of 1 Biopharmaceutical Sciences, 2 Medicine and Anatomy, Cardiovascular Research Institute, and 3 Departments of Anatomy, Biochemistry, and Biophysics, University of California, San Francisco, California 94143-0446


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK1 cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.

renal tubule; renal reabsorption; nucleoside analogs; nucleosides; adenosine transport; green fluorescence protein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NUCLEOSIDES AND NUCLEOSIDE analogs are used in the treatment of neoplasms, viral infections, and cardiac arrhythmias. These compounds are hydrophilic and are transported across lipid membrane barriers by specific transporter proteins. There are two main classes of nucleoside transporters, equilibrative and concentrative. Concentrative nucleoside transporters are Na+ dependent and are present in renal and intestinal epithelium and in liver (39). On the basis of substrate selectivity, several types of concentrative nucleoside transporters have been characterized as purine selective (N1), pyrimidine selective (N2) (30, 38), and broadly selective (N3, N4, and N5) (7, 10, 11). Transporters exhibiting N1- and N2-type characteristics, termed sodium-dependent purine-selective nucleoside transporter (SPNT or CNT2) and concentrative nucleoside transporter 1 (CNT1), respectively, have been cloned from various mammalian species (human, rat, and pig) (8, 12, 28, 31, 40).

Nucleoside transporters appear to play major roles in the kidney. Both equilibrative and concentrative nucleoside transport activities have been observed in renal epithelium, where they are hypothesized to act sequentially to mediate the transepithelial flux of nucleosides. Equilibrative transport mechanisms have been primarily observed in basolateral membrane vesicle preparations whereas concentrative mechanisms have been observed in apical membrane vesicles, suggesting that nucleosides are transported in a reabsorptive direction (9, 10, 16-19). This model of reabsorptive flux is further substantiated by renal clearance studies in humans demonstrating active reabsorption of nucleosides (15, 27). In contrast, evidence of secretion of nucleoside analogs challenges such reabsorptive schemes but may be explained by interaction of these analogs with secretory transporters in the kidney (1, 25, 26).

In addition to their role in transepithelial flux, nucleoside transporters are also thought to modify the adenosine signaling in the kidney. Adenosine acts via adenosine receptors (A1, A2A, A2B, and A3) to modify kidney function and has been implicated in the metabolic regulation of glomerular filtration rate (GFR), renin release, erythropoietin production, adrenergic transmission, urine flow, and solute excretion. Receptor function has been localized to the renal tubule (42). Termination of receptor activity, by decreasing the level of adenosine in the vicinity of these receptors, is thought to occur via two mechanisms: 1) enzymatic deamination of adenosine and subsequent transporter-mediated internalization of inosine and 2) transporter-mediated internalization of adenosine (9). Recent studies describe A1-like receptor activity on the apical membrane and A2-like activity on the basolateral membrane (6, 33). Localization of nucleoside transporters to the apical or basolateral membrane is important in understanding their role in modulating adenosine action in the kidney.

To clearly understand the role of nucleoside transporters in the kidney in mediating the transepithelial flux of nucleosides and nucleoside analogs, it is critical to localize the transporters to the apical or basolateral membrane within renal epithelial cells. Such studies are also important in understanding the interrelationships between nucleoside transporters and adenosine receptor subtypes and the role of transporters in modulating the effects of adenosine in the kidney. Localization of transporters by using Madin-Darby canine kidney (MDCK) cells, a polarized renal cell line, has mimicked in vivo localization in many transporter protein studies (3, 23, 24, 37). In this study we tagged the cloned nucleoside transporters, SPNT and CNT1, with green fluorescent protein (GFP) and expressed the resultant fusion proteins in MDCK cells. Stable expression of SPNT-GFP and CNT1-GFP in MDCK cells provides a method of visualizing the transporters to conclusively determine localization. The transfected cells may also be used to study the functional characteristics of these transporters and for studies of intracellular sorting and regulation of expression of SPNT and CNT1 nucleoside transporters.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture media and supplements were purchased from the University of California, San Francisco (UCSF) Cell Culture Facility (San Francisco, CA). G418 and blasticidin were purchased from Calbiochem (La Jolla, CA). Clontech (Palo Alto, Ca) provided eGFP-C1, and pcDNA3 and pcDNA6/V5-His/lacZ were purchased from Invitrogen (Carlsbad, CA). The pADtet7 vector and tet-off MDCK were a generous gift from Dr. Yoram Altschuler (UCSF) but are also available from Clontech (2). The EMBL MDCK II strain was a kind gift from Dr. Karl Matlin. Texas-red-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Vectashield was supplied by Vector Laboratories (Burlingame, CA). Transwell polycarbonate cell culture filters and polycarbonate cell culture plates were purchased from Corning Costar (Corning, NY). Bradford reagent was supplied by Bio-Rad (Hercules, CA), and Pierce (Rockford, IL) provided albumin standard. Radiolabeled uridine, inosine, and thymidine were purchased from Moravek Biochemicals (Brea, CA). All other chemicals were purchased from Sigma (St. Louis, MO).

Plasmid construction. Rat SPNT and CNT1 were subcloned into the pcDNA3 vector by adding an EcoRV site to the 5'-end and a NotI site to the 3'-end using PCR. Both rat SPNT and CNT1 were subcloned into eGFP-C1 by adding a BglII site to the 5'-end and a SalI site to the 3'-end using the same method. SPNT-GFP was isolated from eGFP-C1 by adding an EcoRI site upstream to GFP and a XbaI site on the 3'-end of SPNT and was subcloned into the pADtet7 expression vector. All sequences were confirmed by automated sequence analysis at the Biomolecular Resource Center (UCSF).

Cell culture. All cells were maintained in MEM Eagle's with Earle's balanced salt solution (BSS) supplemented with 5% heat inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO2-95% air. Cells were transfected with pcDNA3-CNT1, pcDNA3-SPNT, eGFP-C1-CNT1, pADtet7-SPNT, or empty vector by the calcium phosphate method as previously described (4). pADtet7-SPNT was cotransfected with pcDNA6-V5-His-lacZ at a ratio of 100:1 to confer resistance to blasticidin. pcDNA3-CNT1 and pcDNA3-SPNT were cotransfected with eGFP-C1 at the same ratio so that positive clones could be selected by fluorescence. Three days after transfection, stable clones were selected in media containing both 10 µg/ml blasticidin and 20 ng/ml doxycycline (for the pADtet7-SPNT vector) or 0.7 mg/ml G418. After 10-14 days, individual stable clones were isolated and positive clones were further selected by immunocytochemistry and by [3H] uridine uptake.

Uptake measurements. Unpolarized cells were seeded at 5 × 104 cells/well in 24-well Costar polycarbonate plates and allowed to reach confluence over 3-4 days. Uptake measurements were made as previously described (35). Cells were washed at room temperature in either Na+ (128 mM NaCl, 4.73 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, 5 mM HEPES-Tris, pH 7.4) or Na+-free buffer in which choline chloride is substituted for NaCl. Buffer was aspirated, and cells were incubated in 10 µM nucleoside (0.1 µM radiolabeled nucleoside and 10 µM unlabeled nucleoside) for a specific time (1-2 min unless otherwise noted). All uptakes were carried out in the presence of 10 µM nitrobenzothioinosine (NBMPR) and in either the presence or the absence of Na+. Uptake was stopped by aspiration of reaction mix and three washes in ice-cold Na+-free buffer. Cells were solublized in 1 M NaOH for 2 h, neutralized with 1 M HCl, and counted on a Beckman scintillation counter. All studies were performed in triplicate. Data are reported as means ± SD. All assays are repeated with empty vector or untransfected controls. In all cases, empty vector controls mirrored untransfected controls and are not reported. For all studies, two to three wells per plate were solublized and assayed for protein content by using the Bradford method (35).

Inhibition studies. Inhibition assays were carried out for 1-4 min in triplicate. Briefly, [3H]uridine uptake was measured in the presence of unlabeled uridine at concentrations varying between 0 and 2 mM in the presence or absence of Na+. Data are presented as means ± SE. Data were fit to the equation V = Vo/[1 + (I/IC50)n], where V is the uptake of [3H]uridine in the presence of unlabeled uridine, Vo is [3H] uridine uptake in the absence of unlabeled uridine, I is the unlabeled uridine concentration, and n is the Hill coefficient.

Localization uptake studies. To determine the uptake of nucleosides across the apical or basolateral membrane, the following procedure was used. Individual stable clones were polarized by growth on Transwell filters at a confluent density for 7 days with regular media changes. Before the experiment, each filter was washed on both the apical and basolateral sides with either Na+ or Na+-free buffer. In some cases, transepithelial electrical resistance (TEER) values were taken before uptake by using a Millicell-ERS (Millipore, Bedford, MA) equlibrated in Na+ buffer. Radiolabeled nucleoside at the same concentrations as stated above was added to either the apical or basolateral side, and nucleoside-free buffer was added to the opposite side. NBMPR (10 µM) was added to both sides. Cells were incubated for 1-2 min. Radiolabeled nucleoside was aspirated, and filters were washed three times with ice-cold Na+-free buffer. Filters were air-dried, removed from plastic support, and counted on a Beckman scintillation counter. Two filters from each plate were solublized as described above and assayed for protein content.

Confocal microscopy. Samples grown on filters for 7 days as stated above were fixed with 4% paraformaldehyde, permeablized with 0.025% (wt/vol) saponin in phosphate buffered saline, stained with Texas-red conjugated phalloidin for visualization of actin, and mounted on slides in Vectashield mounting medium. Samples were analyzed using a BioRad MRC-1024 confocal microscope.

Transient transfection of LLC-PK1. LLC-PK1 cells were maintained in M-199 with Earle's BSS (UCSF Cell Culture Facility) supplemented with 3% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO2-95% air. Cells were grown on Transwell filters with a 0.4-µm pore diameter for 72 h and then transfected in Optimem Reduced Serum Medium (UCSF Cell Culture Facility) using LipofectAMINE 2000 (GIBCO-BRL, Rockville, MD). Cells were then grown 24 or 48 h before being fixed and stained for confocal microscopy as described above.

Data analysis. All experiments were performed in triplicate on at least three separate occasions. For determination of statistical significance, Student's unpaired t-test was used and P < 0.05 was considered significant. For determination of intracellular uridine concentrations after uptake on polarized MDCK, the following values were used for all calculations: an intracellular volume (total cell volume minus nucleus and vesicles) of 897 fl/cell as determined by Butor and Davoust (5) and 500,000 cells/Transwell filter as determined by counting via hemocytometer.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stable expression of SPNT-GFP and CNT1-GFP in epithelial cells. To study the intracellular distribution of SPNT and CNT1, we constructed GFP fusion proteins. Using a PCR-based strategy, we tagged the NH2 terminus of each transporter with a genetically stabilized form of the Aequorea victoria GFP to generate SPNT-GFP and CNT1-GFP. Each tagged transporter was stably expressed in MDCK cells (Fig. 1, A and B). MDCK cells have low background concentrative nucleoside transport activity and form uniform, tight monolayers. The SPNT-GFP clone was capable of being expressed in a doxycycline-repressible fashion but for the purpose of these studies was expressed continuously in the same manner as were the other clones. In addition, we constructed stable transfections of wild-type SPNT and CNT1 and all empty vectors. Functionality of SPNT-GFP and CNT1-GFP was investigated to determine (1) whether the tagged transporters were functionally active and (2) whether the GFP tag kinetically altered the activity of the transporters. Na+-dependent uptake of [3H]uridine in cells expressing SPNT-GFP or CNT1-GFP was significantly increased over uptake in untransfected or empty vector-containing cells (data not shown). TEER values did not differ significantly between transfected and untransfected cells, indicating no difference in monolayer tightness. Time-dependent Na+-stimulated uptake was linear at early times and plateaued at 10 min for both clones (data not shown). Subsequently, all activity assays were performed for 1-2 min unless otherwise noted. Na+-dependent [3H]uridine uptake by CNT1- and SPNT-transfected cells (both tagged and untagged) grown in Transwells was concentrative, with a final cellular uridine concentration ~10 times larger than the extracellular concentration.


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Fig. 1.   Localization of concentrative nucleoside transporters, concentrative nucleoside transporter 1-green fluorescence protein (CNT1-GFP) and SPNT-GFP, in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. CNT1-GFP (A), SPNT-GFP (B), isolated GFP (C), and untransfected (D) MDCK clones were polarized by growth on filters for 7 days. The cells were fixed, permeablized, stained for actin with Texas-red conjugated-X phalloidin, and visualized by confocal fluorescence microscopy. CNT1-GFP (E) and SPNT-GFP (F) LLC-PK1 cells were transiently transfected as described in MATERIALS AND METHODS and prepared for confocal microscopy in the same manner as the MDCK cells. Left column: GFP or GFP fusion protein. Middle column: actin. Right column: 2 images merged with GFP or GFP fusion protein (green) and actin (red). Vertical optical sections are shown with the apical membrane on top. Bar, 50 µm.

We examined the substrate selectivity of the tagged transporters by observing the uptake of several radiolabeled endogenous nucleosides ([3H]uridine, [3H] inosine, [3H]thymidine). The three were chosen because inosine is a model purine nucleoside, thymidine is a model pyrimidine nucleoside, and uridine is transported by all subtypes of nucleoside transporters. Significant uptake of [3H]uridine and [3H]inosine but not [3H]thymidine was observed in cells expressing SPNT-GFP whereas uptake of [3H]uridine and [3H]thymidine but not [3H]inosine was observed in cells expressing CNT1-GFP (Fig. 2). No significant uptake was observed in untransfected cells or empty vector clones. These results are consistent with the expected substrate selectivity of SPNT and CNT1 and indicate that the GFP tag does not alter selectivity of these transporters. The IC50 of uridine as an inhibitor of [3H]uridine uptake was 3.97 ± 0.86 µM for SPNT-GFP and 5.51 ± 1.45 µM for untagged SPNT (Fig. 3A). The IC50 of uridine was 11.0 ± 1.53 µM for CNT1-GFP and 3.13 ± 0.37 µM for untagged CNT1 (Fig. 3B). Although these values are statistically different, they represent only modest differences in transport activity.


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Fig. 2.   Uptake of [3H]uridine, [3H]inosine, and [3H]thymidine in transfected MDCK cells. Uptake of 0.1 µM 3H-nucleoside (Control, uridine; I, inosine; T, thymidine) was measured at 25°C in the presence of 10 µM nitrobenzothioinosine (NBMPR) and 10 µM of unlabeled nucleoside in SPNT1-GFP and untransfected cells (A; solid and hatched bars, respectively) and CNT1-GFP and untransfected cells (B; solid and hatched bars, respectively). All compounds were studied in the presence of Na+. Data are presented as percentage of [3H]uridine uptake (pmol · min-1 · mg protein-1) in the presence of Na+ and represent the average of 3 experiments (n = 3 for each).



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Fig. 3.   Inhibition of [3H]uridine uptake by unlabeled uridine in transfected MDCK cells. IC50 studies of [3H]uridine uptake were done in the presence of unlabeled uridine at increasing concentrations in SPNT1-GFP and SPNT1 cells (A; open circle  and , respectively) and CNT1-GFP and CNT1 cells (B; open circle  and , respectively). Each point represents the mean ± SE (n = 3) of 1 representative experiment. IC50 values were obtained by fitting the data to the equation V = Vo/[1 + (I/IC50)]n, where V is uptake of [3H]uridine in the presence of unlabeled uridine, Vo is [3H]uridine uptake in the absence of unlabeled uridine, I is unlabeled uridine concentration, and n is the Hill coefficient. All fits were carried out by using a nonlinear fitting routine in Kaleidagraph, and values were compared using Student's unpaired t-test (P < 0.05).

Localization of SPNT-GFP and CNT1-GFP in polarized MDCK cells. SPNT-GFP and CNT1-GFP transfected MDCK cells were grown as a polarized monolayer on a permeable filter support and examined by confocal fluorescence microscopy. Vertical optical sections of both clones visualized at 488 nm show that SPNT-GFP and CNT1-GFP predominantly stain on the apical membrane (Fig. 1, A and B). In the case of SPNT-GFP, those cells that are most highly transfected as determined by fluorescence levels show low levels of basolateral staining as well. In contrast, isolated GFP clones display a cytosolic pattern of expression, and untransfected cells do not display any GFP staining (Fig. 1, C and D). These data indicate the GFP-tagged transporters predominantly localize to the apical membrane domain in MDCK cells.

To determine whether CNT1-GFP and SPNT-GFP showed a similar pattern of distribution in a proximal tubule cell line, we transiently transfected LLC-PK1 cells and examined the proteins by confocal microscopy (Fig. 1, E and F). In general, we observed a transfection efficiency of ~20% for both SPNT-GFP and CNT1-GFP. The patterns of distribution for both fusion proteins as well as GFP were identical to those observed in stably transfected MDCK. That is, GFP showed a cytoplasmic distribution, whereas both CNT1-GFP and SPNT-GFP were predominately localized to the apical membrane, with SPNT-GFP displaying a small amount of basolateral signal.

To further confirm apical localization, [3H]uridine uptake from both apical and basolateral surfaces of polarized cells was examined. Na+-dependent transport was seen primarily at the apical membrane in SPNT-GFP and CNT1-GFP cells (Fig. 4, A and B) as well as in SPNT and CNT1 cells (data not shown). Low levels of Na+-dependent nucleoside transport at the basolateral membrane were observed for both tagged and untagged SPNT and CNT1. For CNT1-GFP and CNT1, Na+-dependent basolateral uptake was 10 times lower than Na+-dependent apical uptake. For SPNT-GFP and SPNT, Na+-dependent basolateral uptake was 25% of its respective apical uptake. Basolateral activity may indicate low levels of basolateral localization. None of the empty vector clones exhibited uridine uptake that was significantly different from that in untransfected cells.


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Fig. 4.   Functional localization of SPNT1-GFP and CNT1-GFP in MDCK cells. SPNT1-GFP and untransfected cells (A; solid bars and open bars, respectively) and CNT1-GFP and untransfected cells (B; solid bars and open bars, respectively) were polarized by growth on 0.4-µm filters for 7 days. [3H]uridine uptake was measured from either apical (AP) or basolateral (BL) side in the presence or absence of Na+. Data are presented as percentage of apical uptake in presence of Na+ and represent the average of 4 experiments (n = 3 for each). Control is [3H]uridine uptake from the apical side in the presence of Na+.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MDCK is the epithelial cell line most extensively used for studies of membrane trafficking pathways and has accurately predicted the in vivo localization of many transporters (3, 23, 24, 37). These cells are capable of direct and indirect protein targeting to both the basolateral and the apical membrane (32, 34). The present work describes the construction of two fusion proteins, SPNT-GFP and CNT1-GFP, and their stable transfection into MDCK cells. We demonstrated that the GFP tag does not alter the substrate selectivity or functional localization of these transporters and only modestly affects kinetic characteristics of the tagged transporters compared with untagged transporters. Confocal imaging and functional studies indicated that both tagged transporters are primarily localized to the apical membrane in MDCK and LLC-PK1 cells, although SPNT-GFP appears to have low levels of basolateral localization as well. This may be a result of saturation of pathway of membrane traffic to the apical membrane, as has been described previously for other proteins (22). It may also reflect a dual role for SPNT on both membranes. By stably expressing fluorescent-labeled SPNT and CNT1 in these cells, we have determined their subcellular localization (Fig. 1) and provided a model for addressing further questions regarding the cellular role of these transporters.

Renal clearance studies of nucleosides in humans indicate that adenosine is actively reabsorbed whereas deoxyadenosine and other nucleoside analog drugs (deoxyfluorouridine, zidovudine, and zalcitabine) are actively secreted (14, 15, 29). Secretory flux of deoxyadenosine as well as several other nucleoside analog drugs has been linked to xenobiotic transporters including organic cation and organic anion transporters (1, 25, 26). Our observation that SPNT and CNT1 predominantly localize to the apical membrane is consistent with studies in isolated renal apical membrane vesicles and suggests that these transporters play a role in the reabsorption of adenosine and other nucleosides rather than in the secretion of deoxyadenosine and nucleoside analog drugs.

These findings also implicate CNT1 and, in particular, SPNT in the adenosine pathway of renal autoregulation by placing them in proximity to the A1 receptor. Northern blot analysis indicates that A1 is the most abundant adenosine receptor type in the kidney (41). Functional studies in the presence of adenosine receptor agonists and antagonists indicate that A1-type activity stimulates a majority of the known renal adenosine effects, including GFR and renin release (20, 21). In addition, A1-receptor antagonists have been shown in animal studies to limit severity of acute renal failure (36). Nucleoside transporters may serve to terminate adenosine receptor signaling by decreasing local concentrations of extracellular adenosine (13). Interestingly, stable transfection of the A1 receptor in MDCK cells demonstrates that ~80% of this receptor localizes to the apical membrane with the remaining 20% appearing on the basolateral membrane, similar to the results found for SPNT (33). Although CNT1 is observed mainly in tissue where nucleoside salvage would be expected, SPNT has a much wider tissue distribution and is in high abundance within the heart, an organ with a large degree of adenosine receptor activity (7). Thus SPNT may serve the additional function of modulating A1 receptor-mediated effects of adenosine in the renal tubule and elsewhere by internalizing adenosine as well as inosine, its deaminated metabolite.

In summary, the localization of SPNT-GFP and CNT1-GFP to the apical membrane of MDCK and LLC-PK1 cells supports the model of renal nucleoside reabsorption and suggests a role for these transporters in the modification of adenosine signaling at the A1 receptor. In addition, the stably transfected cells provide a model for visualization of SPNT and CNT1 that may be used in the tracking of individual nucleoside transporters within mammalian cells. Localization under normal physiological conditions provides a starting point from which to begin exploring the trafficking pathways and regulatory responses of concentrative nucleoside transporters. For the first time, we are capable of observing the mobility of these transporters in response to physiological factors.


    ACKNOWLEDGEMENTS

This work was supported primarily by National Institutes of Health (NIH) Grant GM-42230. L. M. Mangravite was supported by Pharmaceutical Chemistry, Pharmacology, and Toxicology Training Grant GM-07175. Additional support for the work was provided by a National Kidney Foundation Young Investigator Grant and NIH Grant K08-DK-02509 (to J. H. Lipschutz) and other NIH grants (to K. E. Mostov).


    FOOTNOTES

Address for reprint requests and other correspondence: K. M. Giacomini, Box 0446, 513 Parnassus Ave., San Francisco CA 94143-0446 (E-mail: kmg{at}itsa.ucsf.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 February 2000; accepted in final form 20 December 2000.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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