1 School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom; and 2 Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany
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ABSTRACT |
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Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent Pi cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number AW423104). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.
inorganic phosphate; electrophysiology
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INTRODUCTION |
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THE HOMEOSTASIS OF INORGANIC PHOSPHATE (Pi) is maintained by intestinal absorption and tightly controlled renal excretion (1). A family of Na-dependent Pi transport systems, denoted NaPi-II, is involved in the apical translocation steps in both renal and intestinal epithelia (20). NaPi-II homologs from several species have been characterized with respect to their structure, function, and tissue distribution (3, 19).
The type II transporter family can be subdivided into the functionally divergent isoforms NaPi-IIa and NaPi-IIb (27). In mammals, NaPi-IIa is expressed mainly in the apical membrane of the renal proximal tubule cells and is vital for controlled Pi reabsorption (19, 28). NaPi-IIb is expressed in the apical membrane of the small intestine and mediates Pi absorption from the lumen (15). The IIb transporter is also expressed in lung and secretory tissues (9).
Pi transport in fish is of interest because carp and winter flounder (Pleuronectes americanus) show renal Pi secretion and reabsorption with NaPi-II transporters likely to be involved in both translocation steps (13, 23, 27). The cloning and functional characterization of a NaPi-IIb (NaPi-IIb1) isoform from the intestine of the zebrafish (Danio rerio), a stenohaline freshwater teleost, has been reported (21). After expression in Xenopus laevis oocytes, the protein transported Pi in a sodium-, voltage-, and pH-dependent manner with functional properties comparable to the mammalian NaPi-IIa and flounder NaPi-IIb isoforms. A second NaPi-IIb-related mRNA, denoted NaPi-IIb2, was detected in zebrafish kidney, but the entire sequence has not yet been determined.
The tissue distribution of the two NaPi-IIb isoforms has previously been investigated by RT-PCR (21). However, the intracellular localization of the transporters is of prime importance in defining the physiological role of the proteins. Basolateral expression of NaPi-IIb indicates Pi secretion whereas apical expression would represent sites of Pi reabsorption. Accordingly, a single NaPi-IIb isoform is expressed in the apical and the basolateral membrane of different renal tubular segments in winter flounder (8, 16).
The aim of this study was to obtain a functional NaPi-IIb2 clone and to determine the intracellular localization and functional characteristics.
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MATERIALS AND METHODS |
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Animals
Zebrafish (Danio rerio) were bred in house. For the extraction of RNA, adult animals were anesthetized on ice and decapitated, and organs of interest were isolated.Female clawed frogs (X. laevis) were purchased from H. Kähler (Hamburg, Germany). The removal and preparation of oocytes has previously been described in detail (26).
The care and use of experimental animals was carried out in accordance with the guidelines of the Department of Animal Care, Nordrhein-Westfalen, Germany.
Rapid Amplification of cDNA Ends
To complete the cDNA sequence of NaPi-IIb2 first 3'- and then 5'-rapid amplification of cDNA ends (RACE) was performed. For 3' RACE, the reverse transcription was primed with an adaptor primer containing 18 T residues and a NotI adaptor (Pharmacia, Freiburg, Germany). The following PCR included the adaptor primer and the forward primer 5'-CCGTTTTCACCTCCGCC-3'. Because no distinct fragment was amplified, a nested reaction was done with the primer 5'-GCTGGTATCCTGCTGTGGT-3'. The fragment of ~900 bp was cloned and sequenced. A kit from Gibco BRL (Neu-Isenburg, Germany) was used to amplify the 5'-end of NaPi-IIb2. Total RNA (1 µg) from zebrafish kidney was used. Reverse transcription was primed with a gene-specific oligonucleotide (5'-GTCTGTAGAGCATCC-3'), and subsequently the supplier's protocol was followed. The first PCR using the G-rich adaptor primer and the primer 5'-ACAGAAGTGCCGATATTTGCAC-3' resulted in a specific fragment of 550 bp. The fragment was cloned (TA cloning, Invitrogen, Groningen, Netherlands) and sequenced.Library screening. A zebrafish genomic DNA library (Clontech, Heidelberg, Germany) was screened to identify the 5' end of the NaPi-IIb2 gene. A probe of 560 bp was generated by using PCR including digoxigenin-labeled nucleotides (Roche, Mannheim, Germany) using the primers 5'-GGATGCTCTACAGACTCACC-3' and 5'-TCCCGGCTCCCACGAGGATG-3'. In total, 1.8 × 105 clones on six plates were screened. Hybridization was performed overnight in DIG Easy Hyb (Roche) with 10 ng/ml probe added. The nylon membranes (Roche) were washed at low stringency (0.2× SSC at 42°C). Positive clones were visualized by using antidigoxygenin antibodies coupled to alkaline phosphatase according to the manufacturer's manual (Roche). Candidate areas were excised from the mother plate, and the phages were rescreened. Positive clones were eluted (Qiagen DNA-extraction kit), and the inserts were sequenced.
Immunocytochemistry
Custom antisera were purchased from Eurogentec (Cologne, Germany), including peptide synthesis, coupling to keyhole limpet hemocyanin, and the immunization of two rabbits per antigen. Standard protocols were followed. The peptide sequences were NH2-YDNPALGIEDEAKVT-COOH (NaPi-IIb2) and NH2-IIEPKKTVDSCEILK-COOH (NaPi-IIb1). Epitopes are highlighted in Fig. 2 (green shading).Cryosections from X. laevis oocytes were prepared according to Terada et al. (25). Briefly, cRNA-injected oocytes were fixed in 3% paraformaldehyde in PBS for 1 h, rinsed with PBS, and incubated in 30% sucrose for at least 16 h. Oocytes were embedded in TissueTec (Miles Scientific, Naperville, IL) and frozen, and 5-µm sections were cut. Sections were rinsed twice with PBS and blocked with 10% normal serum for 10 min. Sections were washed three times with PBS, incubated with the first antibody for 1 h (17.5 µg in PBS, 3% dry nonfat milk, 1% saponin), washed with PBS, and incubated with the secondary Cy3-labeled antibody (Dianova, Hamburg, Germany) for 1 h.
Adult female zebrafish tissue was fixed for 10-15 min in ice-cold fixative (2% paraformaldehyde and 0.5% picric acid in 80% ethanol), rinsed with 0.1 mol/l cacodylate buffer, and embedded in paraffin for sectioning. Sections of 4-10 µm were rinsed in PBS and preincubated with NH4Cl for 10 min. Nonspecific binding was blocked with fish-gel mix (2% fetal calf serum; 2% BSA; and 0.2% fish-gelatin 45%, Sigma, Steinheim, Germany; in PBS) for 45 min. Sections were labeled by incubating with primary antibody (diluted 1:2,000), washing with PBS, and then incubating with Cy-3-conjugated secondary antibody. Nuclei were counterstained by addition of 4',6-diamidino-2-phenylindole dihydrochloride (Sigma) to the last incubation step.
Two-Electrode Voltage Clamp
cRNA was synthesized by using the mMESSAGEmMACHINE T7 kit (Ambion, Austin, TX) according to the supplier's protocol. Typically, X. laevis oocytes were injected with 10 ng cRNA by standard protocols (26). Transport activity was evaluated by the two-electrode voltage-clamp technique 2-3 days after cRNA injection.Microelectrodes were pulled from borosilicate glass capillaries (Clark
Electromedical Instruments) on a horizontal puller (model P-97,
Sutter Instruments) and filled with 4 M potassium acetate. Electrodes
showed resistance in the range of 0.5-1.5 M when immersed in
control ND96 solution containing (in mmol/l) 96 NaCl, 2 KCl,
1.8 CaCl2, 1 MgCl2, and 5 HEPES; pH adjusted to 7.4 by titration with KOH. Steady-state membrane potentials were typically between
40 and
60 mV in control solution. Current was
induced by addition of Pi to the bath medium. Current
recordings were made either with membrane potential clamped (at
50
mV) or with voltage step protocols [from Vtest =
120 to 20 mV, holding potential
(Vh) =
50 mV,
Vtest = 10 mV]. Experiments were
repeated with at least three oocytes per batch, and at least three
batches were used.
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RESULTS |
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We have previously identified two NaPi-IIb-related cDNA fragments originally isolated from zebrafish intestinal and renal tissues, named NaPi-IIb1 and NaPi-IIb2, respectively (21). The NaPi-IIb1 isoform was readily cloned and characterized; however, NaPi-IIb2 resisted several attempts of full-length cloning. Here, we report the cloning of NaPi-IIb2 and compare the two transcripts in terms of their localization and functional properties.
Cloning of NaPi-IIb2
A PCR-related strategy was envisaged to clone NaPi-IIb2. The COOH terminus deduced from the 3'-RACE product showed the cysteine cluster characteristic of NaPi-IIb isoforms as well as the PDZ (PSD95-Dlg-zona occludens-1-domain) binding motif (27). Several different approaches were performed to clone the 5'-end of the cDNA with unsatisfactory results. Either truncated fragments were amplified or a stop codon interrupted the open reading frame. The DNA sequence, corresponding to a 3'-fragment of NaPi-IIb2, was deposited as an expressed sequence tag (accession number AF297180). We isolated and sequenced two
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Zebrafish NaPi-IIb1 and NaPi-IIb2 are 66% identical at the amino acid
level (Fig. 2). The membrane-spanning
domains and intracellular loop 1 (ICL1) and extracellular loop 3 (ECL3)
are well conserved between different members of the NaPi-IIb family,
with major differences localized to the large extracellular loop 2 and
the NH2 and COOH termini.
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NaPi-IIb1 and -IIb2 have an overlapping expression pattern: RT-PCR experiments revealed a rather broad expression pattern of the two isoforms. NaPi-IIb1 is expressed in intestine, eye, and kidney, and NaPi-IIb2 is expressed in intestine, eye, kidney, brain, liver, heart, and testis (21). Here we have used immunocytochemistry to further elucidate the cellular location of NaPi-IIb1 and NaPi-IIb2.
Immunocytochemistry
The COOH terminus of various NaPi-II proteins has proven suitable for antiserum production (4); consequently, we chose comparable epitopes in zebrafish NaPi-IIb1 and NaPi-IIb2. The specificity of the two antisera were tested by using thin sections of cRNA-injected X. laevis oocytes expressing the zebrafish NaPi-IIb proteins. Both antisera recognized the cognate epitope without cross-reacting with the other isoform (Fig. 3).
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Figure 4, A and B,
shows serial sections of zebrafish intestine labeled with NaPi-IIb1-
and NaPi-IIb2-specific antisera, respectively. The sections show a
villus (longitudinal axis denoted by dashed line) with the luminal
space marked L. Both NaPi-IIb1 and NaPi-IIb2 are strongly
localized to the apical membrane of the enterocytes with no signal
detected intracellularly or in basolateral membranes. Incubation of
serial sections with the cognate preimmune sera did not yield a
specific signal (Fig. 4, C and D).
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Figure 4, E and F, shows serial sections of zebrafish kidney labeled with NaPi-IIb1- and NaPi-IIb2-specific antisera, respectively. The longitudinal axis of a specific nephron fragment is marked by dashed lines. Expression of NaPi-IIb1 and NaPi-IIb2 was largely apical (arrows), and a high degree of overlap between the two proteins was evident. However, NaPi-IIb2 was detected in a larger number of nephron segments, suggesting that a more widespread expression than NaPi-IIb1 although differences in antisera affinity cannot be ruled out.
In addition to the expression of NaPi-IIb transcripts in renal and intestinal epithelium, a distinct signal was also exclusively detected in the apical membrane of bile ducts (Fig. 4, G and H).
Functional Characterization of NaPi-IIb2
Previous experiments have demonstrated that the NaPi-IIb1 isoform operates in a sodium-, phosphate-, pH-, and voltage-dependent manner (21). We used NaPi-IIb1-injected oocytes as positive controls. These data are not shown in this paper but were found to be in accordance with previously reported findings.Phosphate dependence.
Addition of phosphate to the external medium triggered an inward
current in a dose-dependent manner (Fig. 5, A and
B). Water-injected control
oocytes did not exhibit phosphate-induced currents (data not shown).
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Sodium dependence.
Like NaPi-IIb1, it was found that the NaPi-IIb2 isoform functions in a
sodium-dependent manner (Fig. 6, A and
B). Mean current was plotted
as a function of sodium concentration and fitted with the Hill equation
(Fig. 6C). The relative affinity for sodium, K
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pH dependence. In mammals, the NaPi-IIa cotransporters display maximal transport rates in basic conditions whereas the intestinal isoform NaPi-IIb shows maximal transport rates in acidic conditions, although the effect of pH on function is less pronounced (15). The fish intestine is thought to be less acidic than the mammalian intestine; therefore, the effect of pH on fish NaPi cotransporter function was of interest. Flounder NaPi-IIb is expressed in both renal and intestinal tissues and displays pH-dependent function similar to mammalian NaPi-IIa (transport rate at pH 8 is greater than at pH 6.5) (12). Zebrafish NaPi-IIb1 function exhibits a similar pH profile (21); therefore, we investigated the role of pH on NaPi-IIb2 transporter function.
Figure 7A displays typical current-voltage relationships for a NaPi-IIb2-injected oocyte bathed in 1 mmol/l Pi at pH 6.0-8.0. Note that at Vtest =
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Voltage dependence.
Figure 8 shows that there is a marked
functional difference of NaPi-IIb1 and NaPi-IIb2 with respect to
membrane potential. NaPi-IIb1 function is relatively insensitive to
membrane potential with current ~75% of maximum at a membrane
potential of 20 mV. However, as the membrane potential increases,
NaPi-IIb2 function decreases rapidly to ~20% of maximal current at
Vtest = 20 mV. Therefore, NaPi-IIb2 transporter
function is more dependent on membrane potential, a characteristic
similar to that of winter flounder (12, 21) and mammalian
sodium-phosphate cotransporters (10).
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DISCUSSION |
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Mammalian phosphate homeostasis is maintained by uptake from the small intestine followed by tightly regulated renal reabsorption (1). The sodium-phosphate cotransporters involved in these processes are structurally related but functionally distinct (20). NaPi-IIa is expressed in the kidney and exhibits maximal transport rates under alkaline conditions, whereas the intestinal NaPi-IIb function is less sensitive to external pH (15). This ensures that intestinal reabsorption of Pi occurs over a wide pH range, for example, when challenged by acidic output from the stomach and by neutralization in the duodenum.
The direction of renal transepithelial Pi transport in fish
is closely related to the glomerular filtration rate (GFR) and thus to the habitat of the species. Freshwater fish are hypertonic to
their surroundings and so water and ion balance is achieved by a high
GFR followed by reabsorption of sodium, phosphate, and other ions.
Marine fish are hypotonic to their surroundings; consequently, GFR is
relatively low to maintain water levels and controlled renal secretion
contributes to maintaining ionic balance. Therefore, in freshwater
teleosts net absorption of Pi seems to prevail whereas net
secretion occurs in marine teleosts (5). Recently, it
emerged that members of the NaPi-II protein family play a pivotal role in both Pi secretion and reabsorption (8, 28).
Micropuncture studies identified the proximal tubular segment PII of
winter flounder (P. americanus) and skate (Raja
erinacea) as the major site of tubular secretion (5).
Experiments carried out in flounder suggested that a basolaterally
sorted NaPi-II homolog drives Pi secretion in this segment.
In adjacent collecting tubules, the same transporter was found in the
apical membrane, possibly involved in the tuned reabsorption of
Pi (8). In zebrafish, two distinct NaPi-II
homologs have been reported; however, their physiological role was
unclear (21). Figure 9
summarizes the localization of NaPi-IIb cotransporters in fish and
NaPi-IIa in the mammalian kidney. Here, we describe the functional
characterization of the second isoform NaPi-IIb2 as well as the
immunohistochemical localization of NaPi-IIb1 and NaPi-IIb2.
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NaPi-IIb1 and NaPi-IIb2 Have Overlapping Expression Patterns
The presence of two NaPi-IIb isoforms represents a common feature in both freshwater and marine species, although the physiological significance of the closely related transporters remains to be established (28). Our specific antisera localized both transporters in the apical membrane of renal tubular cells, intestinal enterocytes, and bile duct epithelia. The data suggest that both isoforms play a role in accumulating body Pi by mediating (renal) reabsorption and intestinal uptake. Expression of NaPi-II in bile ducts has not been reported previously.Elevated concentrations of Pi and/or Ca2+ in urine or bile increase the risk of stone formation. We hypothesize that the physiological role of NaPi-IIb in bile ducts is to scavenge Pi to prevent formation of precipitates. Pi may be not only secreted or filtered into bile but also generated by the breakdown of hormones or other extracellular compounds.
The two NaPi-IIb isoforms are coexpressed in the same organs. The markedly different affinities for Pi indicate that the two transporters complement each other to function efficiently over a large range of extracellular Pi concentrations. Thus NaPi-IIb1 would represent a low-affinity, high-capacity system absorbing the bulk of Pi, whereas NaPi-IIb2 would be responsible for efficient transport at low external Pi concentrations (high affinity, low capacity). The renal reabsorption of glucose in mammals by the sodium-glucose cotransporters (SGLT) follows a similar strategy. That is, uptake of glucose is mediated predominantly in the proximal convoluted tubule by the low-affinity, high-capacity transporter SGLT2 with fine control of reabsorption in the straight proximal tubule by the high-affinity, low-capacity SGLT1 (29).
The colocalization of the two transporters represents a puzzling fact. However, our experiments do not exclude a partial overlap with flanking regions expressing only one of the isoforms. Coexpression of the two isoforms could prove beneficial if a relatively short tubular fragment had to cope with highly variable loads of Pi.
A second renal NaPi-II-related isoform (NaPi-IIc) has recently been reported in rats and humans (24). NaPi-IIc is predominantly expressed in weaning rats and to a lower level in adult animals. In contrast to all other NaPi-II transporters, NaPi-IIc mediates electroneutral cotransport of sodium and Pi. Comparison of NaPi-II protein sequences did not reveal a significant phylogenetic link between zebrafish NaPi-IIb2 and rat or human NaPi-IIc (not shown).
Zebrafish NaPi-IIb2 Is Functionally Distinct From NaPi-IIb1
Many cloned NaPi-II cotransporters have been functionally characterized by expressing the protein in X. laevis oocytes and analyzing currents with the two-electrode voltage-clamp technique. All data quoted in this paper were acquired under standard conditions, i.e., 1 mmol/l Pi, 100 mmol/l sodium, and Vtest =Phosphate dependence of the NaPi-II cotransporters.
Zebrafish NaPi-IIb1 and NaPi-IIb2 exhibit significantly different
apparent affinities for external phosphate ions;
K
Sodium dependence of the NaPi-II cotransporters.
The reported values for the sodium affinity of NaPi-II cotransporters
display a wide range. Reported values of
K
pH dependence of the NaPi-II cotransporters. The differences in pH sensitivity of mammalian NaPi-IIa and NaPi-IIb have been investigated at the molecular level. Site-directed mutagenesis experiments have nominated a candidate motif for the pH-dependent nature of NaPi-IIa function. It was found that switching the charged amino acids REK (in ECL3) of NaPi-IIa with the uncharged residues GNT of NaPi-IIb led to the loss of pH-dependent transport of mammalian NaPi-IIa and gain of pH sensitivity of NaPi-IIb (refer to Fig. 2, red overscore) (7). Flounder NaPi-IIb has only two charged amino acids in the same region (AEK) and is shown to exhibit pH dependence similar to mammalian NaPi-IIa (12). A similar phenomenon was reported for zebrafish NaPi-IIb1, which contains only one charged amino acid (GET). NaPi-IIb2 has this same motif (GET) and so was also predicted to exhibit pH dependence in a NaPi-IIa like manner. However, NaPi-IIb2 exhibits pH-dependent characteristics, unlike other members of the NaPi family. In the pH range 6.5-8.0, NaPi-IIb2 function is largely pH independent (a type IIb characteristic), but function was severely curtailed between pH 6.0 and 6.5 (a type IIa characteristic). Therefore, we suggest that another, as yet unidentified, motif must play a role in determining the pH sensitivity of transport in zebrafish NaPi-II cotransporters.
Voltage dependence.
NaPi-IIb2 function was found to be strongly dependent on membrane
potential (cotransport was reduced by 70% on depolarizing the oocyte
membrane from 120 to 0 mV). This finding is conserved between all
cloned NaPi cotransporters with the exception of zebrafish NaPi-IIb1,
which is relatively voltage insensitive.
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ACKNOWLEDGEMENTS |
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The authors thank Ursula Strunck, Brian Burtle, and Heike Rimpel for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the Wellcome Trust, The Max-Planck Society, and the University of Newcastle upon Tyne.
Address for reprint requests and other correspondence: A. Werner, School of Cellular and Molecular Biosciences, Univ. of Newcastle, Newcastle upon Tyne, NE2 4HH, UK (E-mail: andreas.werner{at}ncl.ac.uk).
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 December 17, 2002;10.1152/ajprenal.00356.2002
Received 4 October 2002; accepted in final form 10 December 2002.
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