| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 281, Issue 8, 5072-5083, February 24, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
¶¶||**1
From the
Departments of Medicine, Pharmacology, ||Pediatrics, and **Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093 and the ¶Department of Medicine, San Diego Veterans Affairs Healthcare System, San Diego, California 92161
Received for publication, July 22, 2005 , and in revised form, October 20, 2005.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The proximal tubule of the mammalian kidney contains a highly potent system for the secretion of organic anions; many of its substrates are cleared upon the "first pass" of the circulation through the kidney. Basolateral uptake, the driving force for this secretion, operates by coupling the entry of substrate to the exit of dicarboxylates (particularly 
-ketoglutarate) along their concentration gradient. Dicarboxylates are maintained at high intracellular concentrations through the action of a Na+-dicarboxylate cotransporter, in turn driven by the Na+ gradient. The overall process is accordingly referred to as tertiary active transport. This "classical" organic anion transport pathway is responsible for the tubular secretion (and therefore renal excretion), and sometimes nephrotoxicity, of a strikingly diverse array of important pharmaceuticals and other xenobiotics, as well as, potentially, endogenous organic anions (23, 24). Notable among its substrates are 
-lactam antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs),4 antivirals, and loop and thiazide diuretics; the latter largely utilize this pathway to gain access to their site of action in the lumen of the distal nephron (25–27).
OAT1 has been demonstrated to mediate the transport of many of these same compounds when heterologously expressed (e.g. following microinjection of OAT1 cRNA into Xenopus oocytes or transfection of OAT1 cDNA into epithelial cell lines), including of the prototypical substrate of the classical pathway, para-aminohippurate (PAH) (reviewed in Refs. 28–30). Furthermore, OAT1 couples organic anion entry to dicarboxylate exit (7) and has been immunolocalized to the basolateral surface of the proximal tubule (31, 32). Thus, OAT1 manifests physiological properties consistent with a role in the basolateral uptake step of organic anion secretion by the classical pathway. However, functional data on OAT1 have derived mainly from in vitro studies, and the actual activity of OAT1 in vivo, in the context of the whole kidney and/or the entire organism, might be quite different. Moreover, several other transporters have also been proposed to mediate PAH/organic anion secretion in the proximal tubule on the basis of in vitro studies similar to those used in the functional characterization of OAT1. These include the basolateral proximal tubular transporters OAT2 (33, 34), OAT3 (35–37), and MRP1 (38, 39), as well as apical transporters, such as NPT1 (40) and MRP2 and -4 (41–43). Thus, the actual relative contribution of OAT1 to the net secretion of organic anions in the proximal tubule has remained unclear.
We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in vivo. We find that loss of OAT1 leads to a striking decrease in the tubular secretion of organic anions, including reduced secretion of the loop diuretic, furosemide, resulting in a rightward shift of the dose-natriuresis response curve for this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway and thus in the excretion of the numerous clinically important compounds counted among its substrates. We previously reported the generation and characterization of an OAT3 knock-out mouse with defects in organic anion transport. In that study, renal transport of substrate was only determined "ex vivo" (i.e. via experiments conducted on isolated tissue (specifically, renal cortical slices) removed from wild-type and knock-out mice). Accordingly, the possibility that mechanisms operating at the level of the whole kidney (or the entire organism) might supersede or otherwise compensate for the loss of OAT function could not be excluded. The current data indicate that, regardless of the potential contribution of OAT3 (or of OAT2 and MRP1), the bulk of organic anion transport during the basolateral uptake step of the classical pathway is mediated by OAT1. We have also analyzed the plasma and urine of wild-type and OAT1 knock-out mice for content of endogenous organic anions. Several compounds were markedly increased in the plasma and/or decreased in the urine of knock-out mice, consistent with loss of their renal secretion. Moreover, we have demonstrated that some of these compounds interact with OAT1 in vitro. These latter compounds might therefore represent physiological substrates of OAT1.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-galactosidase and a neomycin-selectable marker (LacZ-Neo), resulting effectively in the loss of the ability to either transcribe or translate OAT1 (for details, see "Results"). Mice heterozygous for the OAT1 null allele (F1 generation) were generated at Deltagen, Inc. (Redwood City, CA) and back-crossed to C57BL/6J for a total of four generations. Heterozygous animals from the final back-cross were bred to each other, yielding knock-out and wild-type littermates of the same mixed genetic background, from which all animals used in the experiments described were descended. Mice were group-housed at up to five mice per cage under a 12-h light-dark cycle and were provided access to food and water ad libitum. All experiments were performed on male age-matched (within 2 weeks) mice that were between 12 and 20 weeks of age. Experimental protocols were in accordance with Ref. 79 and were approved by the Institutional Animal Care and Use Committee.
Materials—[3H]PAH (4 Ci/mmol), [3H]estrone sulfate (ES; 46 Ci/mmol), and [3H]inulin (50 Ci/mmol) were obtained from PerkinElmer Life Sciences. Fluorescein was purchased from Molecular Probes, Inc. (Eugene, OR). Other unlabeled organic anions (probenecid, PAH, N-acetyl aspartate, benzoate, 3-hydroxybutyrate, 4-hydroxyphenyllactate, and 4-hydroxyphenylpyruvate) were obtained from Sigma. All chemicals were >98% pure except for 3-hydroxybutyrate, which was >95% pure.
Genotyping—Mice were genotyped by PCR analysis of genomic DNA isolated from tail snips as previously described (44). Twenty ng of genomic DNA were used as template in multiplex PCRs using a single reverse primer recognizing a sequence downstream of the site of recombination and two forward primers recognizing sequences within the recombined region, one targeting the endogenous sequence and the other the inserted transgenic sequence; the respective sequences of these are 5'-TTGCTGAGGTTGGCATTAGCAGGTG-3', 5'-TAGGACTGGAGGTCCTCAGGCATTG-3', 5'-GGGTGGGATTAGATAAATGCCTGCTCT-3'. Cycle parameters were as follows: denaturing at 95 °C for 15 min followed by 35 cycles of 94 °C denaturing for 20 s, 60 °C annealing for 20 s, and 72 °C extension for 20 s. Amplification from the endogenous sequence (present in wild-type and heterozygous mice) yields a 223-bp PCR product, whereas amplification from the recombined sequence (present in knock-out and heterozygous mice) yields a 493-bp product. PCRs were visualized by electrophoresis through 1.3% agarose gels stained with ethidium bromide.
Northern Analysis, Histology, and Immunohistochemistry—Northern analysis was performed as previously described (16, 44). Briefly, 5 µg of total kidney RNA from three each of wild-type and OAT1 knock-out male mice were electrophoresed in a denaturing gel and then transferred to a charged nylon membrane. The OAT1 probe template (a 2.1-kb fragment corresponding to the entire cDNA sequence) was derived from the Image clone BC021647 [GenBank] by restriction digestion followed by gel isolation. 32P-labeled probes generated by random priming were hybridized to the Northern membrane overnight at 42 °C in Ultra-Hyb buffer (Ambion Inc., Austin, TX), and the membrane was then washed at high stringency prior to autoradiography.
Tissues for histological analysis were collected from three each of wild-type and OAT1 knock-out male mice and either processed for hematoxylin and eosin staining or quick frozen. -Galactosidase staining on frozen sections was performed by the University of California San Diego Cancer Center Histology Shared Resource facility (La Jolla, CA). Frozen sections for immunohistochemistry were treated with boiling water for 15 min to expose cryptic antigenic sites and then incubated with the OAT1 antibody (diluted 1:500; Alpha Diagnostic Inc., San Antonio, TX) at 4 °C overnight, followed by binding of secondary antibody (goat anti-rabbit IgG conjugated to Alexa-fluor 568 (diluted 1:1000; Molecular Probes) at room temperature for 2 h.
General Phenotypic Analysis—Urine for chemical analysis was obtained by picking up the mice to elicit reflex urination and then holding them over a Petri dish for sample collection. Generally, it was necessary to combine collections performed on multiple occasions (on the same animal) to yield quantities sufficient for chemical analysis (200–300 µl). Plasma for chemical analysis was obtained by cardiac puncture with a heparinized syringe, while mice were under terminal pentobarbital anesthesia, typically yielding 600–700 µl of blood, which was then centrifuged to yield 300 µl of plasma. All analyses were performed by the Clinical Laboratory of the University of California San Diego Medical Center (San Diego, CA), except where otherwise noted. Determination of general metabolic parameters was performed in indirect, open circuit calorimeter chambers (Columbus Instruments, Columbus OH). These chambers provided measures of oxygen consumption, carbon dioxide production, and locomotor activity as well as food and water intake. Data were collected every 30 min over three 12-h dark cycles and two 12-h light cycles and collapsed across these 12-h intervals for subsequent analysis.
Uptake in Renal Slices—Determination of uptake in renal slices was performed essentially as described previously (44). Briefly, mice received terminal anesthesia with pentobarbital, and kidneys were immediately dissected and placed into freshly oxygenated ice-cold saline. Tissue slices (0.5 mm in thickness; 10 mg, wet weight) were cut with a Stadie-Riggs microtome and maintained in ice-cold modified Cross and Taggart saline until uptake assays were performed (generally <15 min). Slices were then incubated with substrate (0.25 µM [3H]PAH, 0.25 µM [3H]estrone sulfate, or 1 µM fluorescein, in the presence and absence of 1 mM probenecid) for 1 h at room temperature. After incubation, the slices were removed from the uptake medium and blotted, weighed, and dissolved in 1 ml of 1 M NaOH, followed by neutralization with 1 ml of 1 M HCl. [3H]PAH and [3H]ES were quantified by liquid scintillation counting. Fluorescein was quantified by determination of fluorescence intensity (emission 520 nm, excitation 485 nm), with correction for autofluorescence as measured in aliquots of dissolved tissue from slices that were not incubated in fluorescein. Duplicate medium samples were also assayed, and data are presented as the ratio of concentration in tissue to concentration in medium (T/M ratios). For each of the 12 uptake conditions (uptake of each of three substrates, by knock-out or wild-type slices, in the presence or absence of probenecid), measurements were made in two independent experiments and in a total of six slices, derived from 4–6 different wild-type matched to 4–6 different knock-out animals (a total of 16 wild-type and 16 knock-out mice were used).
In Vivo Analysis of Kidney Function, Organic Anion Clearance, and Diuretic Response—Measurement of basal kidney function and in vivo clearance was performed as previously described (45, 46). Briefly, a total of 11 wild-type and 12 knock-out mice were anesthetized for terminal clearance experiments with 100 mg/kg inactin intraperitoneally and 100 mg/kg ketamine intramuscularly; 20% of this dose was readministered intraperitoneally every hour to maintain anesthesia until euthanasia. The femoral artery was cannulated for blood pressure measurement and blood sample withdrawal, the jugular vein was cannulated for continuous maintenance infusion of 2.25 g/dl bovine serum albumin in 0.85% NaCl at a rate of 0.5 ml/h/30 g, body weight, and the bladder was catheterized for collection of urine. In one set of mice, unlabeled PAH was infused into six matched pairs of wild-type and OAT1 knock-out mice at doses ranging from 35 to 4200 ng/min/g, body weight, for determination of PAH clearance (each pair of mice receiving a different dose), whereas [3H]inulin was infused at the uniform rate of 20 µCi/h/30 g, body weight, for assessment of the glomerular filtration rate. In a separate set of mice (five wild-type and six knock-out), 3H-labeled ES was infused at a rate of 15 ng/min/g for determination of ES clearance. After surgery, the mice were allowed to stabilize for 60 min. Then a timed urine collection was performed for 30 min using a bladder catheter, with blood withdrawn at the beginning and end of the collection period. Concentrations of inulin and ES were determined by liquid scintillation counting, and concentration of PAH was determined with a colorimetric assay following precipitation of plasma proteins, as previously described (47, 48). In the case of plasma ES, which, unlike PAH (49, 50), is highly protein-bound (51, 52), both protein-free and total concentrations were measured for each individual mouse in the experiment (protein-free concentrations were determined using centrifugal filter devices (Microton; Millipore Corp., Bedford, MA)).
Approximately 30 min after conclusion of the PAH clearance experiments, the natriuretic response to intravenous application of furosemide was assessed. Since PAH clearance differed between genotypes, it is important to note that in both knock-out and wild-type mice, the ED50 values of the natriuretic response were independent of the PAH dose applied in the initial clearance experiment. This is consistent with the assumption that any PAH remaining in the circulation (after letting the kidneys clear PAH for 30 min before application of diuretic) did not exert a significant effect on the response to furosemide. Slow bolus application of vehicle (0.85% NaCl, 30 µl/25 g, body weight, over 1 min) was followed by bolus application of increasing doses of furosemide (0.1, 0.3, 1, 3, and 10 mg/kg). After each bolus and allowing 2 min for drug distribution, urine was collected via a bladder catheter for 5–10 min, depending on urinary flow rate. Arterial blood pressure was recorded at the midpoint of every collection period. Finally, before euthanizing the mice by intravenous application of saturated KCl at the end of the experiment, in some cases, kidneys were excised and decapsulated, and the wet weight was determined.
Concentrations of Na+ and K+ in plasma and urine were determined using a flame photometer (Cole-Parmer Instrument Co., Vernon Hills, IL). Furosemide concentrations in urine were determined by modification of the PAH assay (which, as reported before (53), can detect sulfonamide therapeutic agents); standards and samples were diluted in 0.9% sodium chloride to a 100-µl final volume, acid-hydrolyzed (by the addition of 5 µl of 37% HCl, followed by incubation at 70 °C for 10 min), and centrifuged (16,000 x g for 1 min). To 100 µl of supernatant, 20 µl of distilled water and 20 µl of 0.1 g/dl sodium nitrite (NaNO2) were added, and reactions were incubated for 3 min at room temperature; then 20 µl of 0.5 g/dl ammonium sulfamate (NH4SO3NH2) were added, followed by incubation for an additional 3 min at room temperature; finally, 20 µl of coupling reagent (N-1-naphthyl-ethylendiamine-dehydrochloride (0.1 g/dl)) were added, and reactions were incubated for 10 min at room temperature before absorbance was read at 544 nm. The furosemide calibration curve was linear between 0.001 and 0.1 mg/ml. Spiking of standards with furosemide-free urine from knock-out and wild-type mice confirmed that there was no background interference.
Sufficient urine for colorimetric detection of furosemide in both genotypes was obtained after the 3- and 10-mg/kg doses of the diuretic. At these time points, plasma PAH (and therefore probably urinary PAH as well) was estimated to be reduced to less than 1% of initial levels in the knock-out mice (and to 10-10 of initial levels in wild-type mice). Nevertheless, to avoid any potential interference from PAH, only samples from the mice receiving the three lowest doses of PAH were analyzed. Consistent with primary detection of furosemide, absorbance readings manifested an essentially linear increase with increasing diuretic doses in both wild-type and knock-out samples (Fig. 6C).
Quantification of Endogenous Organic Anions—The concentrations of endogenous organic anions in plasma and urine of wild-type and knock-out mice were determined by gas chromatography-mass spectrometry (GC-MS), following solid phase batch extraction (54), with alterations to permit miniaturization. Plasma samples of 100 µl or urine volumes equivalent to 0.25–1 µmol of creatinine were reacted with pentafluorobenzylhydroxylamine to form oximes of oxo-acids, aldehydes, and ketones. The lyophilized reaction products were extracted in 42% t-amyl-alcohol/chloroform over a column of silicic acid, and the dried eluate was reacted with BSTFA/TRISIL to form trimethylsilyl derivatives. These were injected (0.5–1 µl) on a bonded phase (DB5) capillary column (30 m x 0.25 mm) in an Agilent 6890/5973 GC-MS. Electron impact mass spectra were obtained in scan mode (50–650 atomic mass units at 2.4 cycles/s), and species were quantified using calibrated response curves of selected ions. 4-Nitrophenol and 2-oxocaproic acid were used as internal standards.
|
0.25 µM of [3H]PAH, in the absence or presence of various concentrations of unlabeled organic anions. Subsequently, oocytes were washed, and radioactivity was measured by scintillation counting. Ki was determined by nonlinear regression using Prism 3.0 (GraphPad Inc., San Diego, CA).
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-galactosidase and a neomycin-selectable marker (LacZ-Neo) (Fig. 1A). This sequence contains two transcriptional termination sites as well as multiple stop codons downstream of the LacZ-Neo cassette. Therefore, transcription initiated at the OAT1 promoter would be likely to terminate prior to entry into the OAT1 coding sequences downstream of the recombined region. In the event that there were a failure of appropriate transcriptional termination, the presence of the stop codons would serve to ensure that downstream OAT1 sequences, if transcribed, would not be translated. The minuscule portion of OAT1 upstream of the LacZ-Neo cassette would only encode 10 amino acids, insufficient for function. Finally, a splice acceptor site is present upstream of the cassette, guarding against the possibility that aberrantly initiated transcripts could "splice around" the recombined sequence.
PCR amplification from genomic DNA confirmed the presence of the expected recombination event (Fig. 1B). When paired with a "reverse" primer recognizing a sequence downstream of the targeted locus, a "forward" primer specific to the deleted sequence did not form a product in amplifications from genomic DNA from knock-out mice but did form a product in wild-type or heterozygous mice, whereas a primer specific to the inserted LacZ-Neo sequence formed a product in knock-out and heterozygous mice but not in wild-type mice. Northern blotting of RNA from kidney tissue confirmed the loss of OAT1 transcript in the knock-out, whereas the expected signal of 2 kb was detected in the wild-type (Fig. 2). Furthermore, immunohistochemical analysis confirmed the absence of OAT1 protein in the knock-out. Incubation of kidney sections with an OAT1-specific antibody revealed strong cortical staining (apparently confined to the basolateral membrane of proximal tubular cells) in wild-type animals, whereas no specific staining was noted in knock-out mice (Fig. 3, A and B). Finally, -galactosidase staining of kidney sections revealed significant staining in the renal cortex of knock-out mice, greater than the low level of staining due to endogenous galactosidase-like activity in the cortex of wild-type mice (Fig. 3C). This expression of -galactosidase in the same location as usual OAT1 expression is consistent with the specific recombination of the LacZ transgene into the OAT1 locus, resulting in regulation of expression by native OAT1 promoter and enhancer elements.
Phenotypic Analysis of OAT1 Knock-out Mice—There is clear sexual dimorphism in organic anion transport and OAT1 expression; renal transport of PAH, both ex vivo (as determined using cortical slices) and in vivo, is greater in male rats than in females (56), and expression of OAT1 at the level of both mRNA (57–59) and protein (60) is greater in male mice or rats than in females. Therefore, the experiments described here utilized male animals, unless otherwise specified.
OAT1 knock-out mice, both male and female, are viable and fertile and appear healthy without any obvious anatomical or developmental abnormalities. Histological examination of the kidney as well as of multiple other tissues (liver, heart, spleen, muscle, and brain) indicated that loss of OAT1 does not result in gross morphological alterations (not shown). Analysis in metabolism chambers did not reveal significant differences between wild-type and knock-out mice in weight, ingestion of food or water, oxygen consumption, CO2 production, or locomotor activity (Fig. 4). The measurements in metabolism chambers were repeated with a second set of mice, yielding similar results (not shown). Chemical analysis of plasma and urine did not reveal any significant alterations in electrolytes, creatinine, or glucose. The anion gap (the difference between milliequivalents of the routinely measured serum cations and anions, 
) was also unaffected, indicating no significant increase in total plasma organic anions in the knock-out mice (Table 1).
|
|
We found that uptake of a 0.25 µM concentration of the prototypical OAT substrate, PAH, was markedly diminished in renal slices from knock-out in comparison with slices from wild-type mice, with the T/M ratio being 1.51 ± 0.07 in wild-type and 0.90 ± 0.04 in knock-out (n = 6; p < 0.001) (Fig. 5A). Uptake in the presence of probenecid (representing probenecid-insensitive uptake) corresponded to a T/M ratio of 0.74 ± 0.01 in wild-type and 0.71 ± 0.04 in knock-out. Therefore, the difference in uptake is even more striking when considering only the OAT-mediated (i.e. probenecid-sensitive) component of uptake, which is given by the difference between uptake in the absence versus in the presence of probenecid. This difference was estimated (see "Statistics") to correspond to a T/M ratio of 0.77 ± 0.07 in wild-type and 0.20 ± 0.05 in knock-out (p < 0.001), suggesting that approximately three-quarters of OAT-specific PAH uptake is lost in the knock-out. The residual OAT-specific uptake is likely to be due to OAT3 (see "Discussion").
Uptake of 1 µM fluorescein (FL; detected fluorometrically), a substrate for both OAT1 (61–63) and OAT3 (44, 64), was also significantly reduced in knock-out slices, although not to the same degree as uptake of PAH: T/M ratio of 2.77 ± 0.13 in wild-type, 2.01 ± 0.17 in knock-out (n = 6; p < 0.01) (Fig. 5B). T/M ratios in the presence of probenecid (probenecid-insensitive uptake) were 1.77 ± 0.12 in wild-type and 1.62 ± 0.11 in knock-out. Thus, probenecid-sensitive uptake of FL was reduced from an estimated 1.01 ± 0.18 in wild-type to 0.40 ± 0.20 in knock-out (p < 0.05) (i.e. by around three-fifths). In contrast, uptake of 0.25 µM ES, which is a well characterized substrate for OAT3 but is apparently not transported by OAT1 (on the basis of prior in vitro studies (reviewed in Refs. 28–30)), was not significantly different in slices from knock-out and wild-type mice (T/M ratio of 5.20 ± 0.51 in wild type; 4.79 ± 0.27 in knock-out (n = 6)) (Fig. 5C). This finding of preserved ES transport corroborates the specificity of the transport defect in the knock-out, indicating that diminished PAH and FL uptake is not the result of a more general loss of the capacity for transport.
Renal Excretion of Organic Anions in Vivo—The above data demonstrate the presence of specific deficits in the capacity for basolateral uptake of organic anions into the renal cortex of the OAT1 knock-out mouse. In order to determine whether these transport deficits translated to diminished renal secretion in vivo, we compared the urinary excretion of organic anions in knock-out and wild-type mice, performing clearance experiments under inactin/ketamine anesthesia (these anesthetics are not OAT substrates). As noted earlier, there were no significant differences between genotypes in body weight or in plasma or urinary electrolytes. In addition, there were no significant differences in kidney weight, arterial hematocrit, hemodynamic parameters (mean arterial blood pressure and heart rate), or general indices of renal function, including glomerular filtration rate and absolute or fractional urinary excretion of fluid, Na+, or K+ (Table 2). In contrast, there was a specific reduction in renal secretion of PAH.
|
25 µM and greater (lower plasma concentrations could not be reliably detected by the colorimetric PAH assay), renal clearance of PAH was markedly lower in OAT1 knock-out mice: 62.5 ± 6.4 µl/min/g body weight in wild-type (n = 4) and 15.2 ± 2.1 in knock-out mice (n = 5) (p < 0.001; Fig. 6). Renal excretion is the sum of glomerular filtration and net tubular secretion. The clearance of inulin, a substance that does not undergo net secretion, was similar in the two genotypes, 10.0 ± 0.4 µl/min/g body weight in wild-type and 12.1 ± 0.7 in OAT1 knock-out mice (n = 6; Fig. 6), indicating comparable glomerular filtration. Thus, all of the reduction in PAH clearance is attributable to loss of net renal secretion. The knock-out mice do manifest some residual PAH secretion (PAH clearance greater than inulin clearance), potentially reflecting the contribution of other organic anionic transporters (see "Discussion").
|
0.5 µM (corresponding to 2 µM total concentration in either genotype, both of which manifested similar protein binding of ES (not shown)) and determined urinary excretion rates. As expected, given the results of the slice experiments presented above, there was no significant difference in renal clearance of ES between wild-type and knock-out mice: 19.1 ± 5.6 (n = 5) and 16.0 ± 4.0 (n = 6) µl/min/g body weight, respectively (Fig. 6). Diuretic Responsiveness in Wild-type and OAT1 Knock-out Mice— Loop diuretics act on ion transporters located on the luminal surface of the thick ascending limb of the loop of Henle (distal to the proximal tubule) to block Na+ reabsorption, leading to increased excretion of Na+ and water in the urine (termed natriuresis). As noted earlier, these diuretics are substrates for the classical pathway of organic anion secretion and, in fact, predominantly gain entry to their site of action in the lumen through this secretory pathway rather than through glomerular filtration (25–27). Accordingly, we sought to determine whether OAT1 knock-out mice manifested reduced renal secretion of the loop diuretic, furosemide, resulting in diminished acute natriuresis.
For each individual mouse in these experiments, we assayed acute natriuretic responsiveness by infusing successively higher intravenous doses of furosemide, followed shortly by successive urine collections for measurement of urinary excretion of Na+ and of the diuretic (see "Experimental Procedures" for details). This permitted calculation of the ED50 of furosemide (the dose at which the half-maximal Na+-excretory response was attained) for each mouse. Whereas there were no significant differences in arterial blood pressure between the genotypes, both at base line and following infusion of diuretic (Fig. 7B), the OAT1 knock-out mice did indeed manifest a rightward shift of the dose-natriuresis curve; the ED50 averaged 0.70 ± 0.06 mg/kg in wild-type mice as compared with 3.1 ± 0.5 mg/kg in knock-out mice (n = 6, p < 0.001; Fig. 7A).
|
Quantification of Endogenous Organic Anions in Wild-type and OAT1 Knock-out Mice—In order to identify potential endogenous (i.e. physiological) substrates of OAT1, we used GC-MS to determine the levels of 60 of the most abundant organic anions in samples of plasma and urine from wild-type and knock-out mice. (No significant peaks other than those corresponding to these compounds were observed in any of the samples.) We identified 18 compounds that were present at significantly higher plasma concentrations and/or lower urinary concentrations in the knock-out, suggesting the involvement of OAT1 in their renal secretion (Table 3). Since we were performing multiple comparisons (specifically, 60 comparisons each in plasma and urine for a total of 120 comparisons), we expect that some of these compounds represent "false positives." (For example, under the most conservative assumption, that there are no differences between wild-type and knock-out mice in the concentrations of any of the measured organic anions, the expected number of false positives would be 6 if the significance threshold were set at the customary p < 0.05 (0.05 x 120 = 6)). However, as discussed below, three subsets of these compounds are more likely than others to represent authentic OAT1 substrates (indicated by boldface type in Table 3).
|
-ketoglutarate (discussed below), are depicted in Fig. 8. Altered plasma clearance for benzoate, 4-hydroxyphenyllactate, 4-hydroxyphenylacetate, and 4-hydroxyphenylpyruvate may be explained by their structural similarity to PAH, and there is also steric similarity to N-acetylaspartate. The structural relationship to the group of short-chain hydroxyacids (3-hydroxybutyrate, 3-hydroxyisobutyrate, and 3-hydroxypropionate) is less evident, but the structures of those compounds are all very similar among themselves. Thus, all of these compounds might be natural substrates for OAT1.
|
-ketoglutarate, which was significantly increased in the urine of knock-out mice (Fig. 8 and boldface type in Table 3). As discussed earlier (see also Refs. 21 and 66), this dicarboxylate is a well characterized counterion for basolateral uptake of organic anions by the classical pathway. Since it is therefore transported in the "opposite" direction as OAT substrates, the knock-out would in fact be expected to manifest increased renal excretion (resulting in increased urinary and/or decreased plasma concentration), provided there were a pathway for apical exit of this compound. The existence of such an apical exit pathway is indicated by the fact that -ketoglutarate has been demonstrated to undergo net secretion in renal tubules (67, 68).
Interaction of Putative Endogenous Substrates with OAT1 in Vitro— Five of the seven organic anions that we identified as potential novel endogenous OAT1 substrates are routinely available from chemical suppliers: 4-hydroxyphenylpyruvate, benzoate, 4-hydroxyphenyllactate, N-acetylaspartate, and 3-hydroxybutyrate. We tested the ability of (mouse) OAT1 to interact with these compounds by assessing inhibition by the latter of uptake of labeled PAH into OAT1-expressing Xenopus oocytes (Fig. 9). (Expression of mouse OAT1 in oocytes was induced by microinjection of the corresponding in vitro transcribed cRNA.) As expected, co-incubation of oocytes with unlabeled PAH resulted in dose-dependent inhibition of uptake of labeled PAH, with an estimated Ki of 13 µM (this is similar to previously reported values for the affinity of PAH for OAT1 (28, 69)).
We found that each of the test compounds also inhibited OAT1-mediated PAH uptake in a dose-dependent manner, consistent with competitive inhibition of transport. The Ki of 4-hydroxyphenylpyruvate was estimated to be 56 µM, the Ki of benzoate was 253 µM, the Ki of 4-hydroxyphenyllactate was 390 µM, the Ki of N-acetylaspartate was 841 µM, and the Ki of 3-hydroxybutyrate was 3.3 mM. Whereas some of these Ki values are substantially higher than that of PAH, indicating lower affinity for OAT1 (at least in vitro), they are comparable with the OAT1 Ki values of other well characterized classical pathway substrates, such as the -lactam antibiotics benzylpenicillin and cephaloridine (Ki 1.7 and 2.3 mM, respectively (70)), the diuretic hydrochlorothiazide (Ki 150 µM (26)), and the NSAID salicylate (Ki 340 µM (71)). Thus, these results, in conjunction with the findings from the GC-MS analysis, indicate that 4-hydroxyphenylpyruvate, benzoate, 4-hydroxyphenyllactate, N-acetylaspartate, and 3-hydroxybutyrate might be physiological substrates of OAT1.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
OAT1, originally identified by us as NKT (1, 3),2 is but one member of a large family of proteins, the OATs (21, 22), many of which handle similar substrates in vitro. Family members include OAT2 (8, 34), OAT3 (11, 35, 37), OAT4 (12), mOAT5 (20), RST/URAT1 (9, 15), and UST1 (10). Each of these is expressed in the kidney, and, with the exception of UST1, each has been demonstrated to transport organic anions. (OAT homologs that are apparently not expressed in kidney have also been identified: hOAT5 (not the ortholog of mOAT5), UST3 and UST6 (which are liver-specific), and OAT6, which is restricted to olfactory mucosa (13, 14, 16, 18, 19).) Moreover, other renal transporters, MRP1, -2, and -4 (38, 41–43), and NPT1 (40) have also been demonstrated to mediate transport of organic anions, including of the prototypic classical pathway substrate, PAH. As has OAT1, some of the above proteins have been localized immunohistochemically to the basolateral membrane of the proximal tubule (OAT3 (31, 32, 60), human OAT2 (73), and MRP1 (38)). Thus, there exists a multiplicity of renal transporters that could potentially participate in the classical pathway of organic anion secretion, so that the relative contribution of OAT1 to net secretion has remained unclear. Now our findings in the knock-out mouse clarify the role of OAT1; regardless of the contribution of the other transporters noted above, the bulk of organic anion transport during the basolateral uptake step of secretion by the classical pathway is mediated by OAT1.
We previously characterized an OAT3 knock-out mouse, examining renal transport ex vivo (in kidney slices (44)). We found nearly complete loss of OAT-mediated (probenecid-inhibitible) uptake of ES (which was expected for this substrate, since in vitro studies had suggested that it was relatively specific for OAT3 (reviewed in Ref. 28)) and partial loss of uptake of PAH (the residual uptake was presumed to have been due to OAT1). The current ex vivo findings, of no significant loss of ES uptake but marked, although not complete, loss of PAH uptake, are essentially compatible with the previous results. However, whereas renal slices would be expected to recapitulate "native" basolateral OAT activity to a closer degree than heterologous expression systems, such as Xenopus oocytes, ex vivo findings might not necessarily reflect function in vivo,in the context of the entire organism or even of the whole kidney. Transport in the latter situation might be expected to be affected by rates of blood and urine flow, binding of substrates by plasma proteins, and the contributions of apical transporters in the renal tubule (which are not exposed to medium in renal slices), and these factors might impact different transporters to differing degrees. In the present instance, the profound loss of urinary excretion of PAH in the OAT1 knock-out suggests, by inference, that the contribution of OAT3 to PAH transport in vivo is relatively minor.
A long body of physiological work supports the notion that PAH is the prototype of a highly diverse set of organic anions that are handled in common by the classical pathway of renal organic anion secretion. Thus, our finding that OAT1 is responsible for the bulk of renal secretion of PAH in vivo suggests an integral role in the excretion of the many other substrates of the classical pathway. These substrates comprise commonly prescribed drugs (-lactam antibiotics, nucleoside-analog antivirals, NSAIDs, and diuretics), other xenobiotics, and, potentially, endogenous organic anions (23, 24, 28). Moreover, it appears that the basolateral uptake step is frequently directly responsible for the nephrotoxicity due to some of these compounds (e.g. ochratoxin A (74) and cephaloridine (75)) by bringing about their tubular accumulation. Conversely, inhibition of basolateral uptake of a potentially toxic compound, although protecting against nephrotoxicity, might lead to delayed clearance and, therefore, to increased extrarenal toxicity (reviewed in Refs. 76 and 77). OAT1 has previously been shown to interact with many of these compounds in vitro (nearly 200 apparent substrates were listed in a recent review (28)), including several potential nephrotoxins. Thus, study of OAT1 knock-out mice will be useful for investigations of the mechanisms of xenobiotic toxicity as well as for pharmacokinetic analyses of many clinically important compounds. One might predict that the knock-outs would be relatively protected from nephrotoxicity due to classical pathway substrates but potentially more susceptible to extrarenal toxicity.
Transport by OAT1 might also subserve physiological functions quite apart from excretion of the xenobiotics and potential toxins listed above. Identification of endogenous substrates will help elucidate such functions. Our study provides direct evidence by inhibition of PAH transport that benzoate, 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate, N-acetylaspartate, and 3-hydroxybutyrate (and/or similar molecules) are "natural" substrates of OAT1, and similar variations in renal excretion in vivo suggest that 4-hydroxyphenylacetate, 3-hydroxyisobutyrate, and 3-hydroxypropionate are also substrates. Benzoate arises from metabolism by bacteria in the gastrointestinal tract. 4-Hydroxyphenyllactate, 4-hydroxyphenylacetate, and 4-hydroxyphenylpyruvate are intermediates of tyrosine catabolism. N-Acetylaspartate is an abundant component of neural white matter which may be important in osmoregulation. 3-Hydroxybutyrate is an important metabolic fuel in ketone metabolism; 3-hydroxyisobutyrate is structurally similar but very distinct metabolically, arising as an intermediate in the catabolic pathway of valine. Finally, 3-hydroxypropionate is a compound that arises from propionate catabolism (78). Thus, transport by OAT1 might appreciably influence metabolism of amino and fatty acids. However, the metabolism chamber data (which revealed no significant differences between wild-type and knock-out mice in food consumption or energy utilization) suggest that, at least under basal conditions, loss of OAT1 function does not grossly perturb intermediary metabolism. Conceivably, differences might be unmasked under conditions of physiological stress, where buildup might lead to toxic effects, for example.
Finally, it is worth noting that whereas expression of OAT1 in adults is virtually restricted to the proximal tubule of the kidney (1), there is detectable, albeit much lower, expression in other barrier epithelia, including choroid plexus, retina, and olfactory mucosa (14, 19, 44). Such expression is potentially relevant to accumulation of organic anions in, or elimination from, various body fluid compartments, such as cerebro-spinal fluid and the vitreous humor of the eye, with implications for drug delivery and/or localized toxicity. Furthermore, OAT1 in olfactory mucosa might participate in the trans-epithelial absorption of nasally administered substances, which could provide a direct route into the central nervous system circumventing the blood-brain barrier (reviewed in Ref. 2). The OAT1 knock-out mice could be of considerable utility in the investigation of OAT function in these various other tissues as well.
|
FOOTNOTES |
|---|
1 To whom correspondence should be addressed: Dept. of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 858-822-3482; Fax: 858-822-3483; E-mail: snigam{at}ucsd.edu.
2 C. E. Lopez-Nieto, GenBankTM accession number MMU52842.
3 H. Takanaga, S. Ohtsuki, and T. Terasaki, GenBankTM accession number AB062418
[GenBank]
.
4 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; PAH, para-aminohippurate; ES, estrone sulfate; FL, fluorescein; GC-MS, gas chromatography-mass spectrometry; T/M, ratio of concentration in tissue to concentration in medium.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
