1 McGill University-Montreal Children's Hospital Research Institute and Departments of 2 Pediatrics and 3 Human Genetics, McGill University, Montreal, Quebec, Canada H3Z 2Z3
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ABSTRACT |
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First published August 15, 2001;
10.1152/ajprenal.00092.2001.Na-phosphate (Pi)
cotransporters in the apical membrane of renal proximal tubular cells
play a major role in the maintenance of Pi homeostasis.
Although two such cotransporters, Npt1 and Npt2, have been
identified, little is known about the function and regulation of Npt1.
We cloned and characterized the murine (Npt1) and human
(NPT1) genes, isolated the 5'-flanking region of
Npt1, and analyzed its promoter activity. Npt1 is
~29 kb with 12 exons, whereas NPT1 is ~49 kb with one
additional exon. The Npt1 promoter has a TATA-like box but
no CAAT box, and the transcription start site was identified by primer
extension and 5'-rapid amplification of cDNA ends. Transfection of
opossum kidney cells with Npt1 promoter-reporter gene
constructs demonstrated significant activity in a 570-bp fragment that
was completely inhibited by cotransfection with the transcription
factor, hepatocyte nuclear factor (HNF)-3
. Deletion of 200 bp from
the 3'-end of the 570-bp fragment abrogated its promoter activity. In
addition, promoter activity of a 4.5-kb fragment, but not the 570-bp
fragment, was stimulated fourfold by cotransfection with HNF-1
.
Other well-characterized cis-acting elements were identified in the
Npt1 promoter. We suggest that Npt1 expression is
transcriptionally regulated and provide a basis for the investigation
of Npt1 function by targeted mutagenesis.
kidney; brush-border membrane; hepatocyte nuclear factor; transcription
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INTRODUCTION |
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THERE IS CONSIDERABLE EVIDENCE to suggest that the rate-limiting step in renal phosphate (Pi) reabsorption is mediated by Na-Pi cotransporters that reside in the brush-border membrane of proximal tubular cells where the bulk of filtered Pi is reabsorbed (19). Two classes of renal Na-Pi cotransporters, type I (Npt1) (31) and type II (Npt2) (16), have been identified by expression cloning and localized to the brush-border membrane of proximal tubular cells (2, 7). Studies in our laboratory demonstrated that Npt2 is by far the most abundant Na-Pi cotransporter in mouse kidney (29) and that disruption of the Npt2 gene in mice results in increased urinary Pi excretion, an 85% loss in brush-border membrane Na-Pi cotransport, and significant hypophosphatemia (1). Moreover, mice homozygous for the disrupted Npt2 gene fail to respond to Pi deprivation with an adaptive increase in brush-border membrane Na-Pi cotransport (12) and to parathyroid hormone (PTH) with a decrease in transport (34). These findings underscore the importance of Npt2 in the maintenance of Pi homeostasis and indicate that it is the target for regulation by dietary Pi and PTH, major regulators of renal Pi reabsorption.
In contrast to Npt2, the precise function of Npt1 is not clear. Npt1
accounts for ~13% of total Na-Pi cotransporter mRNAs in
mouse kidney (29) and is also expressed in liver
(15, 31) and brain (17). Stable transfection
of MDCK and LLC-PK1 cells with Npt1 cDNA results
in increased cellular uptake of Pi (25), and
electrophysiological studies in cRNA-injected oocytes demonstrated that
Npt1 mediates electrogenic Na-dependent transport of Pi
(4). However, Npt1 also induces a Cl
conductance that is inhibited by Cl
channel antagonists
and organic anions (4) and mediates the transport of
anionic drugs, such as benzylpenicillin (33). On the basis
of these findings, it was suggested that Npt1 not only functions as a
Na-Pi cotransporter but also serves as a channel for
Cl
transport and the excretion of anionic xenobiotics. In
addition, more recent studies have suggested that Npt1 may function as
a modulator of intrinsic Pi transport, rather than as a
Na-Pi cotransporter per se (3).
Consistent with the differences in Npt1- and Npt2-mediated transport function are the differences in their pattern of regulation. Renal Npt1 gene expression is not modulated by either dietary Pi intake (12, 29, 30) or PTH (34). In addition, renal abundance of Npt1 mRNA and protein is not upregulated by Npt2 gene disruption (12, 34). Of interest are the findings in rat hepatocyte cultures that Npt1 mRNA expression is increased by insulin in the presence of glucose and decreased by glucagon and cAMP (15). In addition, in intact rats, Npt1 mRNA expression in liver and kidney is decreased by fasting and increased by streptozotocin-induced diabetes (15). Moreover, studies in rat hepatoma cells demonstrated that insulin regulation of Npt1 mRNA abundance is mediated through the phosphatidyl 3-kinase/p70 ribosomal S6 kinase pathways (32). However, the mechanism whereby these signaling pathways increase Npt1 gene expression remains to be determined.
As a first step toward understanding the precise physiological role of
Npt1 and its regulation, we cloned and characterized the murine
(Npt1) and human (NPT1) genes, isolated the
5'-flanking region of Npt1, and analyzed its promoter
activity. We demonstrate the presence of well-characterized cis-acting
elements in the Npt1 promoter and provide evidence for
regulation of Npt1 promoter activity by hepatocyte nuclear
factor (HNF)-1 and HNF-3
, hepatocyte transcription factors that
regulate gene expression in a variety of tissues. Our data suggest that
Npt1 gene expression is regulated at the transcriptional
level and provide a basis for the investigation of Npt1 gene
function by targeted mutagenesis.
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MATERIALS AND METHODS |
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Southern blot analysis.
Genomic DNA, isolated from mouse (129/SvJ strain) liver and human
peripheral blood leukocytes (26), was digested with
restriction endonucleases (5 U/µg). The digests were resolved on
0.8% agarose gels and transferred to nitrocellulose membranes
[BA-(S)85, Schleicher & Schuell, Xymotech Biosystems, Montreal,
Quebec, Canada] (26). Full-length murine and human
Npt1/NPT1 cDNAs (gift of Dr. M. R. Hughes, Georgetown
University, Washington, DC) (5, 6) were radiolabeled with
[-32P]dCTP (3,000 Ci/mmol; ICN Biomedicals, Irvine,
CA), and hybridization was performed as previously described
(10). The blots were washed twice with 2× standard saline
citrate (SSC)/0.1% SDS at room temperature for 5 min each and exposed
to a Kodak Biomax MR film (Kodak) at
70°C.
PCR amplification and DNA sequencing.
The mouse and human Npt1/NPT1 genes were cloned and
characterized using Long Amplification-PCR (LA-PCR), and the primers
are listed in Table 1. Primer
sequences were derived from the Npt1/NPT1 (NaPi-1)
cDNA sequences (GenBank accession nos. X77241 and X71355). LA-PCR
was achieved with Elongase (Life Technologies) or Expand Long Template
enzyme mix (Roche Diagnostics, Laval, Quebec, Canada) as follows:
initial denaturation step at 94°C, 40 cycles of amplification, and a
final elongation step at 68°C for 20 min. The
template-primer annealing temperatures were generally 2°C
below Tm (melting temperature, Table 1).
PCR products <8 kb were purified and subcloned into pCR2.1 using the
TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Genomic Npt1
and NPT1 subclones were sequenced by an automated dye
termination method at the Sheldon Biotechnology Center (McGill
University) or at the DNA Synthesis and Sequencing Facility of the
Université Laval (Quebec, Quebec, Canada).
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Genomic NPT1 clones. To characterize the 3'-end of the NPT1 gene, we did a blast search in GenBank (nr database), using as probe a segment of DNA that encompasses the NPT1 promoter region (GenBank accession no. D83236). Two presequenced BAC clones containing a large fragment of the human gene (RPCI-11-317E16.TJ and HS3223A2E06) were identified, purchased from Research Genetics (Huntsville, AL), and subjected to PCR screening using primer pairs derived from the human NaPi-1 (NPT1) cDNA sequence. DNA from positive BAC clones was purified and sequenced to identify intron/exon boundaries by methods described above.
Isolation of Npt1 promoter region.
The 5'-flanking region of the Npt1 gene was isolated by
screening a 129/SvJ mouse genomic BAC library with a 205-bp Npt1
cDNA fragment derived from 5'-rapid amplification of cDNA ends
(5'-RACE; see 5'-RACE and primer extension). One positive
clone was identified by Southern blotting and confirmed by PCR, using
primers F-61/R25 (Table 1). To establish a restriction map of the
5'-region of Npt1 gene, the BAC clone was digested with
several restriction enzymes for Southern analysis and hybridized with
the 32P-labeled 205-bp 5'-RACE cDNA fragment. The
Npt1 BAC clone was digested with KpnI and
HindIII, and the DNA fragments were shotgun subcloned into
pBluescript KS() digested with the same enzymes. The resulting
recombinant clones were screened by PCR, and a clone containing a
4.8-kb fragment of the Npt1 promoter region (designated pKS-Npt1-4800) was purified and sequenced as
described above.
5'-RACE and primer extension.
5'-RACE was performed according to the method of Ranasinghe and Hobbs
(Elsevier Trends Journals Technical Tips online,
http://tto.biomednet. com) with minor modifications. Total RNA (15 µg), extracted from mouse kidney using Trizol reagent (Life
Technologies), was reverse transcribed with Superscript II RT (Life
Technologies) using a Npt1-specific antisense primer R255
(Table 1). The cDNA-RNA hybrid was treated with RNase A,
phenol-chloroform extracted, and ligated in pBluescript KS() cut with
EcoRV. PCR was performed using the pBluescript-cDNA-RNA
complex as template with T7 primer and Npt1-specific antisense primer R25 (Table 1). PCR products were subcloned into pCR2.1
and sequenced. Primer extension was accomplished with a Primer
Extension System (Promega, Madison, WI) as described by the supplier,
using two 32P-labeled Npt1-specific antisense
primers (R25 and R70; see Table 1) located in exons 2 and
3, respectively.
Preparation of Npt1 reporter plasmids.
Six different constructs were generated using the promoterless
luciferase reporter plasmid pGL3-basic (Promega) by PCR-based strategies using the 4.8-kb Npt1 promoter fragment as
template. PCR was performed with high-fidelity DNA polymerases
(ELONGASE or Pwo). A 570-bp fragment, which includes exon 1 and a short sequence of 5'-region, was PCR amplified with primers MP7
and MP8 (Table 2). The PCR product was
digested with MluI and BglII and introduced, in a
sense orientation, into the same sites of pGL3-basic, generating the
reporter plasmid Npt1-570. The primer pair MP5/MP11 (Table
2) was used to PCR amplify a 4,500-bp fragment, spanning exon
1 and the 5'-genomic region. The PCR product was digested with
MluI and HindIII, whose sites were engineered by the primers, subcloned in a sense orientation upstream of the luciferase gene in pGL3-basic, and designated Npt1-4500. The
same 4,500-bp fragment was inserted into pGL3-basic, in an antisense orientation, yielding Npt1-4500R. Three additional
Npt1 promoter constructs, Npt1-200,
Npt1-570200, and
Npt1-4500
200, were generated by PCR, using,
respectively, primer sets MP23/MP11, MP7/MP24, and MP5/MP24 (Table 2),
and inserted in the reporter plasmid pGL3-basic. The reporter
constructs Npt1-570
200 and
Npt1-4500
200 were lacking 200 bp at the 3'-end of
Npt1-570 and Npt1-4500,
respectively.
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Cell culture, transient transfection, and reporter assays.
Opossum kidney (OK) proximal tubule cell line OK/E13 (obtained from Dr.
J. Cole, University of Missouri, Columbia, MO), mouse distal convoluted
tubule (MDCT) cell line (28), hepatoma cell line HepG2
[obtained from Dr. C. Deal, Ste. Justine Hospital Research Centre,
Montreal, Quebec, Canada (20)], COS-1, and HEK-293 cells were maintained in DMEM supplemented with 10% serum (5% bovine serum + 5% fetal calf serum) at 37°C in a humidified atmosphere of 95% air-5% CO2. At 80% confluence, cells were
cotransfected with 0.4 µg of the Npt1 promoter-luciferase
reporter plasmid and pCMV-gal plasmid, an internal standard for
transfection efficiency, using LipofectAMINE 2000 (Life Technologies).
In experiments involving HNF-1
and HNF-3
[kindly provided by
Drs. C. Goodyer (McGill University, Montreal, Quebec, Canada), E. Holthuizen (University Medical Center, Utrecht, The Netherlands), J. Crabtree (Stanford University, Stanford, CA), and R. H. Costa
(University of Illinois, Chicago, IL)], cells were cotransfected with
Npt1 promoter-reporter gene constructs, HNF-1
or HNF-3
constructs, and pCMV-
gal. Positive and negative controls were
carried out using, respectively, pGL3-control (containing the
luciferase coding region under the control of the SV40 early promoter)
and pGL3-basic (promoterless reporter). For enzymatic assays,
transfected cells grown on 24-well plates for 24 h (48 h for MDCT)
were lysed with buffer provided with the luciferase reporter assay kit
(Roche Diagnostics).
-Galactosidase activity was measured
using the Galacto-Star kit (Tropix, Bedford, MA) according to the
manufacturer's protocol. An EG & G Berthold Luminometer (Fisher
Scientific, Montreal, Quebec, Canada) was used for all enzymatic
assays, and experiments in quadruplicate wells were repeated at least
three times.
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RESULTS |
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Organization of the Npt1 and NPT1 genes.
Southern blot analysis of murine (Fig. 1)
and human (data not shown) genomic DNA, using the corresponding
full-length cDNAs as probes (5, 6), was used to estimate
the size and complexity of the Npt1 and NPT1
genes. Based on digests with at least seven restriction enzymes, or
combinations thereof, an approximate size of 23 kb was estimated for
the Npt1 and NPT1 genes. This estimate is below
that determined by genomic cloning (see below), indicating that large
regions of the genes were not detected with the probes used. Blots
washed at both high and low stringency gave similar results, suggesting
that both the Npt1 and NPT1 genes are single-copy genes.
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Transcription initiation site and features of the 5'-flanking
region of the Npt1 gene.
Primer extension and 5'-RACE were performed to determine the Npt1
transcription start site. Products of 118 and 159 bp were detected
using antisense primers R25 and R70, respectively (Fig. 4A). This located the
transcription start site to position 95 relative to the first
nucleotide of the translation initiation site (designated
1). To confirm these findings, 5'-RACE was performed with
antisense primer R25. The sequence of the resulting product depicted in
Fig. 4B is consistent with the primer extension data.
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Transcriptional activity of the Npt1 promoter.
To characterize the transcriptional activity of the 5'-flanking region
of the Npt1 gene, Npt1 promoter-luciferase
reporter constructs were transfected into a variety of cell lines. OK
cells, derived from the renal proximal tubule of opossum kidney, were previously used to examine NPT1 (27) and
Npt2 promoter activity (11). MDCT cells were
derived from mouse distal tubule (21) and shown to express
Npt1 mRNA in addition to type III Na-Pi
cotransporter mRNAs (28). HepG2 cells, derived from a
human hepatoma, were also studied because Npt1 expression was
documented in rat hepatoma cells (32) and in rat
(15, 32) and mouse (33) hepatocytes. In OK
cells, a 570-bp fragment (Npt1-570) that includes
exon 1 was able to induce the highest luciferase activity
compared with constructs containing longer (Npt1-4500)
and shorter (Npt1-200) 5'-fragments (Fig.
5A). These data suggest that
negative regulatory elements are present upstream of
Npt1-570 and that the 200-bp 5'-fragment is not
sufficient to support transactivation. Npt1-570 was
also able to drive expression of the luciferase reporter gene in MDCT
and HepG2 cells, albeit at a lower level than in OK cells (Fig.
5A), but not in COS-1 cells (Fig. 5A) or HEK-293
cells (data not shown), suggesting tissue specificity of
Npt1 promoter activity. Npt1-200 was not
able to drive transcriptional activity in MDCT, HepG2, or COS-1 cells,
consistent with data in OK cells (Fig. 5A). Deletion of the
200 bp from Npt1-4500 and Npt1-570
significantly inhibited promoter activity in OK cells (Fig.
5B), indicating that this region is necessary for
transactivation.
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DISCUSSION |
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The type I Na-Pi cotransporter, Npt1/NPT1, is a
465/467-amino acid protein that resides in the brush-border membrane of
renal proximal tubular cells and is also expressed in liver and brain (19). Electrophysiological studies demonstrated that Npt1
mediates not only the Na-dependent transport of Pi but also
induces a Cl conductance that is inhibited by
Cl
channel blockers and organic anions (4).
As a first step to elucidate the physiological function of Npt1/NPT1,
we cloned and characterized the mouse and human genes and demonstrated
that, although they differ in size and exon number, both genes exhibit similar amino acids and codon usage at intron/exon boundaries. We also
characterized the 5'-flanking region of the mouse gene and show that
hepatic nuclear transcription factors HNF-1
and HNF-3
,
respectively, stimulate and inhibit Npt1 promoter activity. These findings and the identification of well-characterized cis-acting elements in the Npt1 promoter suggest that Npt1
gene expression is subject to transcriptional regulation.
In contrast to Npt1, there is little evidence to suggest that Npt2 gene expression is regulated at the transcriptional level (11, 18), despite the identification of several cis-acting elements in the promoter region (13). Rather, the regulation of Npt2-mediated Na-Pi cotransport occurs primarily at the posttranscriptional level (18). Functional studies (19), as well as targeted disruption of the Npt2 gene in mice (1), indicate that Npt2 is the target for regulation of renal Na-Pi cotransport by dietary phosphate and PTH, major determinants of renal phosphate reabsorption. In this regard, it is of interest in that neither dietary phosphate (12, 30), PTH (34), nor knockout of the Npt2 gene (12) has an effect on renal Npt1 gene expression.
It is well known that thyroid hormone and glucocorticoids are important regulators of renal phosphate handling (14) and that both hormones elicit their cellular effects via receptors that bind to specific DNA response elements in the 5'-region of target genes and thereby modulate gene transcription (9). Thus the demonstration of thyroid hormone and glucocorticoid response elements in the promoter region of the Npt1 gene suggests that Npt1 may be a target for this regulation. Consistent with this notion are the findings of a previous study showing that neither T3 nor dexamethasone had an effect on Npt2-promoter luciferase reporter gene expression, suggesting that other targets may be involved (11).
Several well-characterized cis-acting elements have also been
identified in a 1.4-kb promoter fragment of the NPT1 gene
(27), demonstrating species conservation of core promoter
elements. However, a comparison of Npt1 and NPT1
promoter sequences exhibited little homology (data not shown). The
physiological significance of these consensus sequences in the
Npt1/NPT1 genes requires further investigation. In the
present study, we examined the effect of two transcription factors,
HNF-1 and HNF-3
, for which recognition sequences were identified
in the Npt1 promoter, and demonstrate that both have the
ability to modulate Npt1 gene transcription in vitro.
Our findings that HNF-1 stimulates and HNF-3
inhibits
Npt1 gene transcription may be relevant to the regulation of
renal Pi handling in vivo. These transcription factors play
an important role in the regulation of a variety of genes and are
expressed in polarized epithelia of liver, digestive tract, pancreas,
and kidney (22). HNF-1
expression in the kidney is
confined to the proximal tubule (22), the segment of the
nephron where the bulk of filtered Pi is reabsorbed
(19) and where Npt1 mRNA (8) and protein
(2) have been localized.
Recent studies demonstrated that inactivation of the
HNF-1 gene in mice results in a renal Fanconi syndrome,
which is characterized by severe urinary wasting of Pi,
glucose, and amino acids (23). Moreover, patients with
maturity-onset diabetes type 3 (MODY3), which results from mutations in
the HNF-1
gene, also exhibit defects in the renal
reabsorption of Pi, glucose, and amino acids (24). Clearly, further studies are necessary to determine
whether the renal phosphate leak in HNF-1
-deficient mice and
patients with MODY3 can be attributed to decreased renal
Npt1/NPT1 gene expression. In this regard, it is of interest
that decreased renal expression of SGLT2, a proximal tubular Na-glucose
cotransporter that plays a key role in glucose reabsorption, can
account for the renal glucose leak in HNF-1
-deficient mice
(23). Moreover, HNF-1
has a direct effect on
SGLT2 gene transcription, as demonstrated in cells
cotransfected with SGLT2 promoter-reporter gene constructs and HNF-1
(23). These findings provide a molecular
basis for the increase in urinary excretion of glucose in the mutant
mouse strain and suggest that similar mechanisms may account for the renal Pi leak in the murine and human disorders. Finally,
HNF-1
may also play a role in the regulation of Npt1/NPT1
gene expression in the liver. Previous studies have shown that Npt1
mRNA abundance is modulated by changes in glucose metabolism in the
liver (15, 32) and that mutations in HNF-1
gene in mice and humans lead to severe disturbances in glucose
homeostasis (22).
In summary, we have cloned and characterized the mouse and human genes
encoding the type I Na-Pi cotransporter, Npt1/NPT1, and demonstrate that hepatic nuclear transcription factors
HNF-1 and HNF-3
, respectively, stimulate and inhibit
Npt1 promoter activity. In addition, we identified several
well-characterized cis-acting elements in the Npt1 promoter.
We suggest that Npt1 expression is transcriptionally
regulated and provide a basis for the investigation of Npt1
function by targeted mutagenesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. N. Zhao for initiating the promoter studies, D. Leclerc for advice on genome databases, and Y. Sabbagh for computer support.
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FOOTNOTES |
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This work was supported by Canadian Institutes for Health Research Grant GR-13297 to H. S. Tenenhouse.
Address for reprint requests and other correspondence: H. S. Tenenhouse, Montreal Children's Hospital Research Institute, Rm. 222, 4060 Ste. Catherine St. West, Montreal, Quebec, Canada H3Z 2Z3 (E-mail: mdht{at}www.debelle.mcgill.ca).
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 August 15, 2001;10.1152/ajprenal.00092.2001
Received 20 March 2001; accepted in final form 9 August 2001.
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