Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland
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ABSTRACT |
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The luminal uptake of L-cystine and cationic amino acids by (re)absorptive epithelia, as found in the small intestine and the proximal kidney tubule, is mediated by the transport system b0,+, which is defective in cystinuria. Expression studies in Xenopus laevis oocytes and other nonepithelial cells as well as genetic studies on cystinuria patients have demonstrated that two gene products, the glycoprotein rBAT and the multitransmembrane-domain protein b0,+AT, are required for system b0,+ function. To study the biosynthesis, surface expression, polarity, and function of this heterodimer in an epithelial context, we established stable Madin-Darby canine kidney (MDCK) cell lines expressing rBAT and/or b0,+AT. Confocal immunofluorescence microscopy shows that both subunits depend on each other for apical surface expression. Immunoprecipitation of biosynthetically labeled proteins indicates that b0,+AT is stable in the absence of rBAT, whereas rBAT is rapidly degraded in the absence of b0,+AT. When both are coexpressed, they associate covalently and rBAT becomes fully glycosylated and more stable. Functional experiments show that the expressed transport is of the high-affinity b0,+-type and is restricted to the apical side of the epithelia. In conclusion, coexpression experiments in MDCK cell epithelia strongly suggest that the intracellular association of rBAT and b0,+AT is required for the surface expression of either subunit, which together form a functional heterocomplex at the apical cell membrane.
glycoprotein-associated amino acid transporter; cystinuria; exchanger; epithelial cell polarity; kidney transport; intestinal absorption; Madin-Darby canine kidney cells
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INTRODUCTION |
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STUDIES OF CYSTINURIA PATIENTS have revealed that the underlying defect is a lack of cystine and cationic amino acid (re)absorption across small intestine and proximal kidney tubule epithelia due to missing transport across their luminal membranes (see Ref. 21 for a review). The molecular identification of the defective transporter has been initiated by the cloning of the glycoprotein rBAT (D2) that induces a b0,+-type transport (Na+-independent uptake of dicationic and large neutral amino acids) when expressed in Xenopus laevis oocytes (3, 28, 32). Genetic studies have shown that defects in the gene encoding this glycoprotein (SLC3A1) were indeed responsible for type I cystinuria (6). However, it became clear that rBAT itself was not the entire transporter and that it had to associate with an endogenous oocyte protein to transport amino acids. This conclusion was based on the fact that rBAT had the predicted structure of a type II single transmembrane-domain glycoprotein that was homologous to the heavy chain of 4F2 (CD98), known to form a heterodimer with an unknown hydrophobic protein called "light chain." Furthermore, immunoprecipitation experiments had shown that rBAT similarly formed a covalently bound heterodimer with an unknown protein (see Ref. 21 for a review).
Many functional amino acid uptake and two-electrode voltage-clamp studies were performed in oocytes expressing rBAT, presumably associated with an endogenous X. laevis light chain, that defined the substrate uptake specificity of the expressed amphibian b0,+-type transport and revealed that many of the rBAT mutations found in cystinuria lead to a lack of transporter surface expression (9, 20). In view of the physiological role played by the epithelial b0,+ transport system for vectorial amino acid (re)absorption, it was particularly interesting to learn that it functions as an obligatory exchanger that preferentially exchanges extracellular dicationic amino acids for intracellular neutral amino acids (5, 10, 22). This exchange mode indicates that, for net vectorial amino acid transport, the b0,+ system has to function in parallel with another transporter that unidirectionally recycles the exchanged neutral amino acids back into the cell, such as the B0 system, which has not been clearly identified as yet at a molecular level (see Refs. 30 and 31 for a discussion).
After the identification of light chains of 4F2hc, which define the new family of glycoprotein-associated amino acid transporters (gpaATs) (30), another mammalian member of that family, b0,+AT (BAT1), was shown to associate specifically with rBAT in transient expression systems. The b0,+-type uptake property of the resulting heterodimer was characterized in COS cells (that do not express endogenous b0,+AT) and in X. laevis oocytes (7, 19, 22). In this latter expression system, the problem of the endogenous light chains was circumvented by expressing a functional fusion protein [human rBAT (hrBAT)-mouse b0,+AT (mb0,+AT)] (22). Genetic studies have demonstrated that most cases of non-type I cystinuria could be explained by a mutation at the level of the b0,+AT gene SLC7A9 (11, 12).
In the present study, we have addressed in an epithelial expression system the questions of the requirement of rBAT and b0,+AT interaction for their biosynthesis, surface expression, apical polarity, and function.
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EXPERIMENTAL PROCEDURES |
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Cell culture. MDCK cells (strain II) were cultured at 37°C and 5% CO2 in DMEM (52100-013, Life Technologies, Basel, Switzerland) with 22 mM NaHCO3, 5 × 104 U/l penicillin, 50 mg/l streptomycin, 2 mM L-glutamine, 1% nonessential amino acids (11140-035, Life Technologies), and 10% FCS.
cDNA constructs and transfection.
The mb0,+AT cDNA (22) coding sequence
was subcloned in the vector pcDNA3.1()Hygro (Invitrogen,
Carlsbad, CA). The hrBAT cDNA (2) coding sequence was
subcloned in the vector pLKneo, in which its transcription is
controlled by a glucocorticoid-inducible promoter (16).
MDCK cells were transfected with these constructs using the
Lipofectamine Plus reagent (Life Technologies), according to the
manufacturer's protocol. Selection of transfected cells was carried
out with 200 µg/ml of G418 and/or 150 µg/ml of Hygromycine, beginning 48 h after transfection. Resistant clones were isolated after ~3 wk of selection using cloning rings.
Northern blot and RT-PCR analyses. Total cellular RNA was isolated according to the manufacturer's protocol using TRIzol Reagent (Life Technologies). Transcription of hrBAT was tested by Northern blot analysis, as previously described (27). Alternatively, transcription of mb0,+AT was tested by RT-PCR as follows: first-strand cDNA was synthesized from 100 ng of total RNA with Moloney murine leukemia virus RT (Promega, Madison, WI) and 50 pmol of random hexamer primers (Life Technologies). One-tenth of the first-strand cDNA was used as a template for PCR amplification using 50 pmol of mb0,+AT primers [forward primer 5'-TTCACAGTGATGACCCCAACGGAGCT-3', reverse primer 5'-TTTGTGACCGGCCTGGAGATTCTCTG-3' (Microsynth, Balgach, Switzerland)] and 2 U of recombinant Taq polymerase (Biofinix, Praroman, Switzerland) in a total volume of 30 µl of reaction buffer. Transcription of hrBAT was confirmed by performing PCR on the same first-strand cDNA using hrBAT primers [forward primer 5'-GGCACTTTGACGAAGTGCGAAACCA-3', reverse primer 5'-AACGCGAAGTCAGCCGTGAACTGTCT-3' (Microsynth)].
Pulse-labeling of MDCK cells. MDCK cells cultivated to confluency on plastic dishes (Corning-Costar, Acton, MA) were washed three times with PBS containing 2 mM EDTA. Cells were incubated for 30 min in methionine-free DMEM (D3916, Sigma, St. Louis, MO) supplemented with 22 mM NaHCO3, 0.5 × 106 U/l penicillin, 0.5 g/l streptomycin, 62.6 mg/l L-cystine · 2HCl, and 10% FCS. The medium was then exchanged for the above-mentioned medium containing 1.0-2.5 mCi/ml of 35S-methionine (NEN, Boston, MA) for 30 min or 2 h at 37°C, 5% CO2. For chase experiments, cells were subsequently incubated in completed culture medium for the times indicated. The fact that the precipitated bands are stronger after a 2-h chase period (see Fig. 3, A and B, lanes 5 and 8) than just after the 2-h pulse-labeling (see Fig. 3, A and B, lanes 4 and 7) is due to the continuing incorporation, during the chase period, of labeled L-methionine that accumulated within the cell during the pulse period. Cells were then washed three times with PBS containing 0.1 CaCl2 and 1 mM MgCl2 and lysed on ice with (in mM) 50 Tris · HCl, pH 8.0, 120 NaCl, 0.2 polymethylsulfonyl fluoride, and 10 diamide, as well as 0.5% Nonidet P-40 (NP-40) and a 1% protease inhibitor cocktail (Sigma). Incorporated radioactivity was determined by TCA precipitation and liquid scintillation.
Antisera. Polyclonal rabbit antibodies were raised against synthetic peptides corresponding to the NH2 terminus of hrBAT, MAEDKSKRDSIEMSMKGC, and the COOH terminus of mb0,+AT, CHLQMLEVVPEKDPE, coupled to keyhole limpet hemocyanin (Eurogentech, Seraing, Belgium).
Immunoprecipitation of pulse-labeled MDCK cells with polyclonal
antibodies (serum).
Lysates (each sample containing the same amount of incorporated
methionine) were precleared by two incubations with uncoated beads.
Polyclonal antibodies were added to the lysates and incubated overnight
at 4°C. Beads (protein G plus protein A-agarose, Calbiochem, La
Jolla, CA) were added and incubated for 4 h at 4°C. The beads were washed three times each in 20 mM Tris · HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40 with and without 500 mM LiCl. SDS-PAGE sample buffer was added, and samples were heated to 65°C for
15 min, with -mercaptoethanol added where indicated, and SDS-PAGE
was performed on 10% gels. Gels were stained in Coomassie blue, fixed,
incubated in Amplify (Amersham, Arlington Heights, IL), and exposed to film.
Double immunofluorescence staining of cotransfected MDCK cells. Cells were seeded on filters (35 mm, Corning Costar Transwell filters) at 100% confluency and cultivated for 7 days preceding the experiments. rBAT expression was induced 3 days before the experiments with 1 µM dexamethasone. Filters were washed three times using PBS containing 2 mM EDTA. Cells were fixed using 3% paraformaldehyde and 0.2% Triton X-100 for 15 min at room temperature. Filters were washed three times and cut into squares. The cells were first incubated with rabbit anti-b0,+AT serum at 1:500 dilution in PBS containing 0.5% BSA overnight at 4°C and, after being washed, were then incubated for 6 h at room temperature with 1 µg/ml of Texas red-labeled anti-rabbit-IgG Fab fragments (Rockland, Gilbertsville, PA). After being washed, free binding sites on anti-b0,+AT antibodies were blocked by incubating the filter pieces overnight at 4°C with 150 µg/ml of unlabeled anti-rabbit-IgG Fab fragments (Rockland). After another round of washes, filter pieces were incubated for 6 h with anti-rBAT serum diluted 1:50 in PBS containing 0.5% BSA and washed again. Filter pieces were subsequently incubated overnight at 4°C with FITC-labeled anti-rabbit-IgG antibody (Sigma), washed, and mounted in DAKO-glycergel (DAKO, Glostrup, Denmark) containing 2.5% 1,4-diazabicyclo[2,2,2]octane (DABCO) as a fading retardant. Confocal images were taken using a Leica laser scanning microscope (TCSSP, Wetzlar, Germany) equipped with a ×63 oil-immersion objective. The appropriate controls were performed without the first and/or second primary (serum) antibodies: omission of the rBAT antibody did not yield any detectable FITC signal, demonstrating the absence of cross-reaction with the b0,+AT antibody.
Filter uptake experiments.
MDCK cells were passaged to 35-mm Corning Costar Transwell filters at
100% confluency and cultivated for 7 days. rBAT expression was induced
24 h before the experiments with 1 µM dexamethasone. The
integrity of the monolayer was checked by resistance measurement using
the Millicell device (Millipore, Bedford, MA). Filters were washed
three times with uptake buffer [in mM: 150 NaCl (+Na condition) or
cholin-Cl (Na condition), 10 HEPES, pH 7.4, 1 CaCl2, 5 KCl, 1 MgCl2, and 10 glucose] at 37°C and incubated in
uptake buffer for 10 min. The buffer was replaced unilaterally with
buffer supplemented with amino acid at the indicated concentrations and
the corresponding 3H-labeled L-amino acid as
the tracer (except for L-[14C]cystine and
L-[14C]isoleucine); the contralateral
compartment received the same solution without the labeled
L-amino acid tracer. Uptake experiments were performed for
1 min (the linearity of uptake was verified in preliminary time course
experiments). Diamide (10 mM) was added to the solution for experiments
with L-cystine. The uptake was stopped by replacing the
amino acid uptake solution with ice-cold uptake buffer and washed four
times. The filters were excised and placed into scintillation vials
containing scintillation fluid (Packard, Meriden, CT). After vials were
shaked overnight at room temperature, radioactivity was determined by
scintillation counting.
Statistics. Data are expressed as means ± SE. The difference between control and test values was evaluated using Student's t-test (2 tailed, unpaired, or 1 sample).
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RESULTS |
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Stable cell lines expressing rBAT and/or b0,+AT. MDCK cell lines stably expressing human rBAT under the control of a glucocorticoid-inducible promoter were generated and tested for dexamethasone-inducible rBAT mRNA expression by Northern blotting. In a second round of transfections, mouse b0,+AT cDNA, placed under the control of a constitutively active promoter, was introduced into rBAT-expressing cell lines and wild-type MDCK cells, together with a second selection marker. Double-transfected cell lines with the highest b0,+AT mRNA expression levels were identified by RT-PCR. Another series of MDCK cell lines was generated that constitutively express an rBAT-b0,+AT fusion protein. The function of this fused heterodimer has been characterized previously in X. laevis oocytes (22).
Coexpression is required for the apical surface expression of both
subunits.
The subcellular localization of rBAT and b0,+AT was
analyzed by confocal immunofluorescence microscopy in selected cell
lines cultivated on filters (Fig. 1). In
cell lines expressing only b0,+AT, a diffuse
cytosolic signal was observed, the intensity of which was strong on
average but highly variable among cells (Fig. 1A). Cell
lines expressing rBAT alone also showed, after induction with
dexamethasone, cytosolic staining that was variable but, on average, of
a much lower intensity (Fig. 1B).
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Stabilization and maturation of rBAT depends on association with
b0,+AT.
To analyze the interaction between the subunits, rBAT and
b0,+AT were immunoprecipitated separately from
biosynthetically pulse-labeled cells expressing both proteins and
analyzed by SDS-PAGE fluorography (Fig.
2). b0,+AT appears in
lanes 2 (sample reduced) and 6 (nonreduced) as a band with a relative mass (Mr) of 40. This value
is lower than expected from its calculated molecular weight (53.7 kDa),
similarly to what has been observed previously for other gpaATs (for
glycoprotein-associated amino acid transporters). The bands
observed at ~80 and 90 kDa in lane 2 correspond to the
coprecipitated core-glycosylated and terminally glycosylated forms of
rBAT, respectively (9, 22). Disulfide linkage of rBAT with
b0,+AT can be inferred from the fact that in nonreducing
conditions (lane 6), the rBAT bands are replaced by a band
at ~135 kDa, which corresponds to the heterodimer (doublet due to
differential rBAT glycosylation not resolved because of compression by
immunoglobulins). The labeled material close to the top of the
separating gel likely represents higher order aggregates. The fact that
the b0,+AT band (Mr 40) precipitated
by the anti-b0,+AT antibody is nearly as strong in
lane 6 (sample not reduced) as in lane 2 (reduced
conditions) indicates that much of the precipitated b0,+AT
is not associated with rBAT, thus suggesting that b0,+AT is
present in great excess over rBAT. On the other hand, when the
precipitation is made with an anti-rBAT antibody, all terminally glycosylated and nearly all core-glycosylated rBAT migrate, in nonreducing conditions (lane 8), at a higher
Mr corresponding to the heterodimer or higher
order aggregates. Thus nearly all of the expressed rBAT is associated
with b0,+AT.
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Apically restricted amino acid transport by the
b0,+AT-rBAT heterodimer.
To demonstrate that the apically localized rBAT-b0,+AT
heterodimer is functional, we performed amino acid uptake experiments on epithelia cultivated on permeable supports (Fig.
4). All assays were carried out for 1 min, a time period that is within the linear phase of uptake, as
determined in preliminary time course experiments (data not shown). The
polarity of the expressed transport was tested by measuring the
L-arginine-inhibitable L-cystine uptake from
both sides of the epithelia in the absence of Na+. This
typical b0,+ transport was observed only at the apical side
of epithelia expressing both rBAT and b0,+AT. As expected,
cells transfected with either rBAT or b0,+AT alone did not
exhibit any notable b0,+-type transport. Expression of the
fusion protein rBAT-b0,+AT induced apical
L-cystine transport similarly to the coexpression of both
subunits. However, this construct also induced some transport at the
basolateral side, indicating that the link constructed between
b0,+AT and rBAT somehow interferes with mechanisms leading
to polar distribution.
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DISCUSSION |
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To address questions of rBAT and b0,+AT heterooligomerization, maturation, surface expression, polarity, and transport properties in the context of an epithelium, we expressed these proteins in MDCK cells. We chose this cell line of distal nephron origin as the recipient because it has no endogenous proximal tubule-like amino acid transport properties and represents a well-established model for epithelial polarity studies. In terms of amino acid transport, untransfected MDCK cell epithelia cultured on filters display only very low apical Na+-independent uptake rates of the dicationic amino acids L-cystine, L-arginine, and L-lysine, when tested at the concentration of 100 µM (Fig. 5). In addition, they express some partially Na+-dependent apical transport of neutral amino acids (Fig. 5) that probably corresponds to that attributed earlier to system ASC plus an operationally defined "general transport system G" (4). On the basolateral side, wild-type MDCK cells are known to express systems L and ASC, as well as the inducible transporters A, TAUT (taurine), and BGT (betain and GABA) which do not interfere with short apical uptake experiments (4, 29, 33).
In contrast to the present study with transfected MDCK epithelia, previous studies of the heterogeneously expressed rBAT-b0,+AT complex were made in nonepithelial systems, i.e., X. laevis oocytes (22) (with an rBAT-b0,+AT fusion protein to prevent heterodimerization with an endogenous b0,+AT), COS cells (7, 11, 19), and HELA cells (12), such that it was not clear whether heterodimerization of rBAT and b0,+AT in epithelial cells would also be a condition for surface expression and the resulting transport properties would be identical. We have to mention, however, that one other group has described transport that surprisingly depends on coexpression of b0,+AT with 4F2hc (23, 24). The physiological relevance of this observation is questionable in view of the strict basolateral localization of 4F2hc and the clear apical restriction of b0,+AT in the same cells (proximal tubule, small intestine) (13, 22, 25) as well as the lack of biochemical interaction of the two transporter chains (22).
In the present study, we show by immunofluorescence that both rBAT and b0,+AT remain intracellular unless they are coexpressed, at which point they reach the apical cell surface (Fig. 1). From the immunoprecipitation experiments, we can infer that it is the heterooligomerization that permits exit from the endoplasmatic reticulum (ER) and surface expression. Indeed, in the absence of b0,+AT, rBAT is rapidly degraded, presumably in the ER, as none of it is terminally glycosylated (Fig. 2). In contrast, in the presence of b0,+AT, it is rapidly shifted to a terminally glycosylated form that remains stable over an 8-h chase period (Fig. 3). This mature rBAT form is shown to be entirely heterooligomerized on nonreducing gels (Fig. 2).
The need for association of b0,+AT to a glycoprotein for surface expression is analogous to that of other glycoprotein-associated amino acid transporters that require heterooligomerization with 4F2hc to reach the basolateral surface (18). However, unlike rBAT, 4F2hc can reach the cell surface of X. laevis oocytes also without an associated light chain.
The requirement for heterodimeric association for surface expression of
both rBAT and b0,+AT subunits is reminiscent of the
situation of the Na,K-ATPase that is also composed of a type II
glycoprotein (-subunit) and a catalytic multimembrane-spanning
protein (
-subunit) (14). In the case of the
Na,K-ATPase, the site of intracellular subunit retention (ER), the
involved chaperones and the intersubunit interaction sites have been
studied extensively, such that the Na,K-ATPase represents a useful
paradigm for heterooligomeric amino acid transporters (1,
15).
Besides the data presented in this study, transport specificity and
kinetic data from the cloned rBAT-b0,+AT complex have been
previously generated in our laboratory using a fusion protein made of
mouse b0,+AT linked to human rBAT. In terms of the amino
acid uptake specificity range (measured at fixed amino acid
concentrations), there was no difference between the results obtained
previously with the fusion protein expressed in oocytes and the same
chains coexpressed in MDCK cells. Two other publications (7,
19) show other sets of kinetic values obtained in COS cells
expressing rat and human rBAT-b0,+AT, respectively. In
terms of amino acid uptake, all four studies agree on the fact that,
relative to L-cystine, cationic amino acids
(L-lysine, L-arginine) as well as some large
neutral amino acids (L-leucine, L-tyrosine) are
transported with a similar efficiency (±3-fold difference) and that
glycine is (nearly) not transported. There is, however, a clear
difference in the transport of the -branched amino acids
L-isoleucine and L-valine, which are not significantly transported in our two studies (mouse
b0,+AT-human rBAT in oocytes and MDCK cells), whereas they
are well transported by the rat and human complexes expressed in COS
cells. The significance of this difference is not yet clear.
Our results obtained in MDCK epithelia show a requirement of rBAT- b0,+AT association for the surface expression of each subunit. However, the general validity of this result is questioned by the differential pattern of rBAT and b0,+AT localization in the proximal tubule. Indeed, we and others have observed opposed (and not parallel) expression gradients of b0,+AT and rBAT in the brush-border membrane of the proximal tubule, with a maximum of b0,+AT in the first portions (S1, S2) (7, 19, 22) and a maximum of rBAT in the last part (S3) of the proximal tubule (13, 17, 22). This suggests the possibility that yet another protein that allows the surface expression of b0,+AT in the absence of rBAT, or vice versa, is present in the proximal tubule and not in MDCK cells.
Older in vivo and ex vivo studies suggest the possibility that in the early portions of the proximal tubule (S1, S2), L-cystine is transported by a low-affinity transport system and that a higher affinity system is situated in the later portion (S3) (see Ref. 26). In view of the relatively high apparent affinities measured for the L-cystine transport by the rBAT-b0,+AT complex, it has been suggested that this heterooligomer would be the transporter expressed in S3 (see discussion in Ref. 31). This raises the possibility that another b0,+AT-associated protein is expressed in the S1 and S2 segments, also revealing the necessity of reevaluating amino acid transport in in vivo and/or ex vivo systems.
The functional role of rBAT-b0,+AT in vectorial amino acid transport can only be understood when one considers its complementarity with other transporters. The fact that it functions as an exchanger of extracellular cationic against intracellular neutral amino acids (8, 31) already indicates that there must be, in the same membrane, a unidirectional transporter that recycles the neutral amino acids which the exchanger transports out of the cells (for instance, system B0, the molecular identity of which is not yet clear) (31). Furthermore, amino acids brought into the cell via these apical transporters have then to leave across the basolateral membrane into the extracellular space. Two rBAT-b0,+AT-related exchangers, 4F2-y+LAT1 and 4F2-LAT2, have been localized to the basolateral membrane of the proximal tubule (25, 30), and a parallel unidirectional transport system has not yet been described. Expressing these transporters in an epithelium already expressing rBAT-b0,+AT, such as the cell line presented in this study, will allow us to investigate the functional interactions of these transporters.
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ACKNOWLEDGEMENTS |
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The authors thank Christian Gasser for the artwork.
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FOOTNOTES |
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This work was supported by Swiss National Science Foundation Grant 31-59141.99 (to F. Verrey).
Address for reprint requests and other correspondence: F. Verrey, Institute of Physiology, Univ. of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (E-mail: verrey{at}access.unizh.ch).
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.
First published February 12, 2002;10.1152/ajprenal.00212.2001
Received 6 July 2001; accepted in final form 6 February 2002.
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