Renal principal cell-specific expression of green
fluorescent protein in transgenic mice
Ludmilla
Zharkikh1,
Xiaohong
Zhu1,
Peter K.
Stricklett2,
Donald E.
Kohan2,
Greg
Chipman1,
Sylvie
Breton3,4,
Dennis
Brown3, and
Raoul D.
Nelson1
Departments of 1 Pediatrics and 2 Medicine,
University of Utah, Salt Lake City, Utah 84132; and 3 Program in
Membrane Biology and Renal Unit, Massachusetts General Hospital,
and 4 Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02114
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ABSTRACT |
The purpose
of this study is to develop transgenic mice with principal
cell-specific expression of green fluorescent protein (GFP). After the
cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of
the promoter were used to drive expression of GFP in transgenic mice.
In transgenic mice, GFP was selectively expressed in principal cells of
the renal collecting duct and not in intercalated cells. Expression was
increased by dehydration of mice. AQP2 and GFP expression was
maintained in primary cultures of renal medulla that were stimulated
with cAMP or vasopressin analogs. GFP-expressing cells were then
isolated by fluorescence-activated cell sorting. RT-PCR analysis showed
expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and
cAMP response element binding protein but not H+-ATPase B1
subunit or anion exchanger 1. After expansion of these cells in
culture, RT-PCR analysis showed continued expression of the same genes.
This pattern of gene expression is that of principal cells rather than
intercalated cells. This transgenic mouse model can be used in future
studies of gene expression during the development, differentiation, and
maturation of renal principal cells.
renal collecting duct; aquaporin-2; gene expression
regulation
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INTRODUCTION |
THE RENAL
COLLECTING DUCT is responsible for regulation of extracellular
volume, osmolality, and pH and is involved in disorders of acid-base
and salt and water balance, including hypertension. The collecting duct
is composed of intercalated and principal cells. Intercalated cells
make up ~30-40% of the cortical and outer medullary collecting
duct cells and 10% of inner medullary collecting duct cells (10,
50). These cells are characterized by the expression of specific
genes, such as the vacuolar H+-ATPase B1 subunit
(37), anion exchanger 1 (AE1) (1), and carbonic anhydrase type II (45). These genes are essential
to the specialized function of intercalated cells, which includes regulated secretion and/or reabsorption of hydrogen and bicarbonate ions. Principal cells make up ~60-70% of the cortical and outer medullary collecting duct cells and 90% of inner medullary collecting duct cells (10, 50). They are characterized by the
expression of specific genes such as aquaporin-2 (AQP2) (19, 20,
39, 44), AQP3 (15, 16), AQP4 (18, 51,
53), and vasopressin type 2 receptors (V2R)
(41). AQP2 is expressed in the apical plasma membrane and
subapical vesicles of the entire collecting duct. AQP3 is expressed in
the basolateral plasma membrane of the entire collecting duct. AQP4 is
expressed throughout the entire collecting duct but is expressed at
higher levels in the inner medullary collecting duct. AQP4 is
also expressed in the S3 segment of the mouse proximal tubule
(34, 53). Ultimately, AQP2, AQP3, AQP4, and the
vasopressin receptor are involved in vasopressin-regulated water
reabsorption by the principal cells within the collecting duct
(35, 40).
The development and differentiation of the renal collecting duct have
been studied in some detail (47, 49), but the regulation of intercalated and principal cell development and
differentiation is less well understood. This process begins as the
ureteric bud, a derivative of the mesonephric duct, and primitive
mesenchyme, a derivative of the intermediate mesoderm, coinduce each
other to differentiate into the metanephric kidney. The ureteric
bud repeatedly branches and elongates. Each branch of the ureteric bud
induces the metanephric mesenchyme to form individual nephrons. Ultimately, the ureteric bud and mesenchyme collectively give rise to
the glomerulus and tubule. The terminal portion of the tubule is the
collecting duct. In the final stage, the collecting duct further
differentiates into intercalated and principal cells (3, 13,
33). The intercalated and principal cells likely mature further
after their initial formation (3, 6, 55). The factors that
control development, differentiation, and maturation of principal
cell-specific gene expression are incompletely understood.
One of the limiting factors in the study of principal cell development
and differentiation is the lack of model systems. The most powerful
approach to study regulation of gene expression in vivo is to use a
transgenic mouse model. Such a model would preserve the complex
cellular relationships found within kidney that are required for
principal cell-specific gene expression. However, the expense and time
required for analysis must be minimized by initial in vitro experiments
using a cell culture system, from the same species, that retains
differentiated features. The cell culture system that would most likely
retain differentiated features is a primary culture or cultured cells
that have only been passaged in vitro a limited number of times. The
use of such a system would then require one to be able to isolate
principal cells from the primary culture in a specific and efficient
manner. The development of model systems is essential to make further
progress in the study of collecting duct development, differentiation,
and maturation.
The purpose of this paper is to describe such a new model system for
the in vivo and in vitro study of the development, differentiation, and
maturation of principal cells within the renal collecting duct. Because
AQP2 is a specific marker of the principal cell phenotype, these
studies have focused on the principal cell-specific regulation of AQP2
gene expression. The mouse AQP2 gene was cloned and sequenced, and the
mouse AQP2 promoter was used to drive the expression of enhanced green
fluorescent protein (EGFP) in principal cells of transgenic mice.
Primary cultures of the medulla from these transgenic mice were then
optimized for the maintenance of AQP2 and EGFP transgene expression.
EGFP-expressing cells were isolated by fluorescence-activated cell
sorting (FACS). The sorted cells expressed principal cell-specific
marker genes, such as AQP2 and AQP3, but not intercalated cell-specific
marker genes, such as H+-ATPase and AE1. These sorted cells
were propagated and continued to express principal cell-specific genes
such as AQP2, AQP3, AQP4, and V2R. This transgenic mouse
model should prove useful in further studies of principal cell biology
both in vivo and in vitro.
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MATERIALS AND METHODS |
PCR.
Mouse DNA or cDNA were amplified by PCR using 0.05 units/µl
Taq DNA polymerase (Life Technologies, Rockville, MD), 200 µM each of dATP, dCTP, dGTP, and dTTP, 400 nM each of forward and reverse primers (Table 1), 1.5 mM
MgCl2, 50 mM KCl, and 10 mM Tris · HCl, pH 8.4, in
25 µl. The thermocycling program consisted of a 94°C hot start
followed by 20-35 cycles of 94°C for 20 s, 58-72°C
for 20 s, and 72°C for 60 s and a final extension at 72°C for 5 min (Table 2). The cycle number and
annealing temperature were varied depending on the primer set. The
cycle number was limited to maintain nonsaturating conditions of
amplification.
DNA sequencing.
Plasmids or PCR products were sequenced by the University of Utah DNA
Sequencing Facility. It utilizes the dye primer system for universal
primers and dye terminator system for other primers. The products were
analyzed by using the ABI PRISM 377 or 3700 DNA analyzers (Applied
Biosystems, Foster City, CA). The results were analyzed by using
Sequencher (Gene Codes, Ann Arbor, MI). The results have been submitted
to GenBank.
Cloning and sequencing of the mouse AQP2 gene.
A PCR method for library screening was developed. Mouse genomic DNA was
prepared by standard methods (26) and 100 ng were amplified by PCR using AQP2GF and AQP2GR2 (Tables 1 and 2). The PCR
product was isolated with the Wizard PCR Preps DNA purification system
(Promega, Madison, WI) and sequenced. These PCR primers were used to
screen an arrayed 129/OLA mouse genomic library that was packaged in a
P1 artificial chromosome (PAC) vector (Genome Systems, St. Louis, MO)
(46). P1 clone DNA was prepared by an alkaline lysis
method (4). Restriction digests of all clones were
initially analyzed by agarose gel electrophoresis. All clones were
further analyzed by PCR for the presence of the promoter and exon 1 using primers HAQP2F1 and AQP2G5PER and exon 4 by PCR using primers
AQP2G3PEF and AQP2G3PER (Tables 1 and 2). Clone 13430 was digested with
EcoRI and a 21-kb fragment subcloned into pKSII (Stratagene,
La Jolla, CA). Both strands of this plasmid were sequenced by primer
walking. The sequence was compared with a partial sequence for the
mouse promoter (accession no. D87129) and cDNA (accession no.
AF020519).
Construction of the mAQP2 promoter-EGFP transgene.
A 9.5-kb EcoRI-Eco47III fragment was ligated
into EcoRI and SmaI digested, calf intestinal
phosphatase-treated pEGFP-1 (Clontech, Palo Alto, CA). The product was
restriction mapped and the ligation junctions were sequenced. The
transgene was excised from the vector by digestion with
EcoRI and AflII and purified away from the vector by
electrophoresis through low-melting-point agarose gel. The linear
transgene was isolated by digestion of the gel slice with
-agarose
I, phenol/chloroform extraction, ethanol precipitation, and
purification with an Elutip-D column (Schleicher & Schuell, Keene, NH).
The DNA was quantitated by OD260 and agarose gel electrophoresis with a
quantitative standard.
Generation and breeding of transgenic mice.
Transgenic founder mice were created by the Transgenic and Gene
Targeting Mouse Facility at the University of Utah according to
standard methods (25). The transgene was injected into the male pronucleus of single-cell embryos derived from C57BL6 × CBA F1 mice, and the embryos were implanted into pseudopregnant females. Candidate transgenic mice were weaned, ears punched, and a tail clip
was obtained just before 21 days of age. After genotyping, the founder
mice were mated with C57BL6 × CBA F1 animals. F1-F4 transgenic mice were analyzed for transgene expression. Line 14 was
also mated with the Immortomouse, which contains the
H2Kb-SV40Tag transgene (32), to obtain double
transgenic mice. Transgenic mice were euthanized by exanguination after
methoxyfluorane or halothane anesthesia, and the kidneys or other
organs were dissected for the various studies described below.
Genotyping of transgenic mice.
Tail DNA was isolated by standard methods (26). Tail DNA
(100 ng) was amplified by PCR using the stated primers (Tables 1 and
2). The MAQP2F1 and EGFPN primers were used to detect the mAQP2
promoter-EGFP transgene, the SV40F2 and PCH110R3 primers were used to
detect the H2Kb-SV40Tag transgene, and the MAQP2GF and
MAQP2GR2 primers were used detect the endogenous AQP2 gene and,
therefore, control for equal loading and integrity of the genomic DNA
(Tables 1 and 2). The products were fractionated by agarose gel
electrophoresis and visualized by ethidium bromide staining. Normal
mouse DNA with 0, 1, 3, 10, 30, and 100 copies/cell equivalent of
transgene DNA were analyzed to estimate transgene copy number. The
results were captured by using the Eagle Eye Gel Documentation System (Stratagene).
RT-PCR analysis of transgene expression.
Organ RNA was isolated by using the acid-phenol-guanidine thiocyanate
method (12) that was modified to minimize DNA
contamination of RNA. Cultured cell RNA was isolated with the RNeasy
Mini system (Qiagen, Valencia, CA) with DNase I treatment of RNA on the
column. RNA was reverse transcribed by using Superscript II (Life
Sciences) with oligo(dT) (12-18) priming according to
manufacturer recommendations. cDNA was then amplified by PCR using the
stated primers (Tables 1 and 2). The AQP2F and AQPR2 primers were used
to amplify AQP2. The EGFPF2 and PCH110R3 primers were used to amplify
the EGFP. The GAPDHF and GAPDHR primers were used to amplify GAPDH from organ cDNA, and EF1
F and EF1
R were used to amplify elongation factor 1
(EF1
) from cellular cDNA. All PCR reactions were carried out in the presence and absence of RT to demonstrate that there was no
contaminating DNA.
Dehydration of mice.
Water was withheld from transgenic mice for 48 h. Age- and
sex-matched mice served as controls. Mice were euthanized and RNA was
prepared from organs as stated above.
Fluorescence microscopy of kidney.
Transgenic mice were anesthetized by inhalation of methoxyflurane or
halothane and fixed by cardiac perfusion with PBS (10 mM sodium
phosphate buffer containing 0.9% NaCl, pH 7.4) and 2% paraformaldehyde in PBS at room temperature. The kidneys were then
dissected and fixed by immersion for 4-6 h at room temperature.
Transgenic mice were initially screened for EGFP expression by
examining thick kidney sections. Kidneys were embedded in 3% agarose
in PBS and cut into 200- to 300-µm sections with an oscillating vibratome. The sections were viewed by epifluorescence microscopy using
a Nikon E800 microscope equipped with the PCM2000 confocal system
having filters specific for EGFP. The images were captured digitally by
using the SimplePCI imaging software package (Compix, Cramberry, PA). A
Z-series was collected at 10-µm intervals and a montage was created.
Thick kidney sections from transgenic mice expressing EGFP were then
immunostained for AQP2. The 200- to 300-µm sections were permeabilized by incubation with PBS-0.5 g/l saponin, 2 g/l BSA, 2 g/l
gelatin in PBS (SBG) for 30 min, and nonspecific binding was blocked by
incubation with 10% goat serum in PBS-SBG for 60 min. The sections
were incubated overnight with a 1:5,000 dilution of a purified rabbit
antibody to AQP2 (14) (provided by Dr. Mark Knepper,
National Institutes of Health) in PBS-SBG at 4°C and then washed four
times with PBS-SBG for 30 min. The sections were then incubated
overnight with a 1:200 dilution of CY5-conjugated goat anti-rabbit
antibody (Amersham Pharmacia Biotech, Piscataway, NJ) in PBS-SBG at
4°C and washed four times with PBS-SBG for 30 min. The sections were
mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed
with the same confocal microscope previously described with laser
excitation and filters specific for EGFP and CY5. The pseudocolor
images were generated by using SimplePCI (Compix, Cramberry, PA). EGFP
appears as green and Cy5 appears as red.
Thin kidney sections from transgenic mice expressing EGFP were
immunostained with antibodies to AQP2 and H+-ATPase.
Kidneys were fixed as previously described. The kidneys were then
cryoprotected by immersion in 30% sucrose in PBS for 4 h, mounted
in Tissue-Tek (Miles, Elkhart, IN), and frozen at
29°C in a
Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were
cut at 4 µm and picked up onto Superfrost/Plus microscope slides
(Fisher Scientific, Pittsburgh, PA).
Sections were hydrated 5 min in PBS and treated for 4 min with 1% SDS
in PBS, an antigen retrieval technique previously described (11). After three washes of 5 min each in PBS, nonspecific
staining was blocked by using a solution of 1% BSA in PBS for 15 min.
An affinity-purified chicken antibody raised against the COOH-terminal 14 amino acids of the 31-kDa subunit (E subunit) of the
H+-ATPase was applied at a concentration of 10 µg/ml for
1.5 h at room temperature. This antibody has been
characterized previously (9). Similarly, an
affinity-purified rabbit antibody raised against the COOH-terminal 14 amino acids of AQP2 was used in other experiments (8).
Sections were then washed two times for 5 min each time in PBS
containing 2.7% NaCl to reduce nonspecific binding, followed by one
wash in normal PBS. Secondary donkey anti-chicken IgG conjugated with
CY3 (Jackson Immunologicals) was applied at a dilution of 1:800 for
1 h at room temperature, and washes were performed as for the
primary antibody. Slides were mounted in Vectashield (Vector
Laboratories) diluted 1:2 in Tris · HCl buffer (1.5 M, pH 8.9).
The images were captured with a Hamamatsu Orca digital camera.
Pseudocolor images were merged by using IP Lab Spectrum software
(Scanalytics). Cy3 appears as red and EGFP appears as green.
For quantitative immunofluorescence studies, the pseudocolored images
were merged into a single file and a grid was overlaid by using Adobe
Photoshop. The EGFP- and AQP2-positive cells were counted in the
cortex, outer medulla, and inner medulla of kidneys from three control
mice and three dehydrated mice. The results were expressed as
means ± SE. Significance was determined by using two-sided
Student's t-test.
Cells cultured on coverslips were visualized by phase contrast and
fluorescent microscopy using a Nikon Eclipse E800 microscope equipped
with a CoolSNAP digital camera (Roper Scientific, Trenton, NJ) and by
fluorescence microscopy using a Nikon Elipse and PCM2000 confocal system.
Cell culture.
Primary cultures of medulla were obtained according to modifications of
published protocols (22, 30). The kidneys were dissected
from mice that were heterozygous for the mAQP2-EGFP and
H2Kb-SV40Tag transgenes. The medulla was dissected and
minced into 1-mm fragments with a razor blade. The fragments were
digested in a bicarbonate-free Krebs solution [(in mM) 145 NaCl, 10 HEPES, 5 KCl, 1 NaH2PO4, 2.5 CaCl2,
1.8 MgSO4, and 5 glucose, pH 7.3] with 1 mg/ml class IV
collagenase (Worthington, Lakewood, NJ) and 0.1 mg/ml DNase I (Sigma,
St. Louis, MO) at 37°C for 15-20 min until tubules were
dispersed into cylindrical fragments. The tubular fragments were
centrifuged at 400 g for 5 min in 10% albumin in PBS. The
pellet was resuspended in medium and cultured in six-well Primaria
plates or standard six-well plates with 23-mm-diameter polyethylene
terephthalate membrane inserts coated with mouse collagen IV (BD
Biosciences, San Jose, CA). The cells were cultured in DMEM/F-12 (1:1)
(Life Technologies) with 15 mM HEPES, 100 U/ml penicillin, 10 U/ml
streptomycin, 2 mM glutamine, 10 µg/ml insulin, 5.5 µg/ml
transferrin, 6.7 ng/ml selenium, 10
7 M dexamethasone,
10
7 M aldosterone, 5 nM T3, 10 ng/ml mouse EGF, 10 U/ml
mouse interferon-
, and 10% fetal bovine serum at 33°C until
confluent. The cells were then shifted to medium without serum or
interferon-
at 37°C for 24-48 h. The cells were then
stimulated with 500 µM 8-(4-chlorophenylthio) cAMP (CPT-cAMP) or 400 µM 3-isobutyl-1-methlyxanthine (IBMX) and 10
7 M DDAVP
for 72 h. FACS-derived cells were cultured with the same medium
and stimulated as stated. The cells were typically passaged by
treatment with trypsin/EDTA before confluence.
FACS.
After stimulation with CPT-cAMP or DDAVP and IBMX, primary cultured
cells were dispersed into a single-cell suspension by scraping and
treatment with 0.05% trypsin and 0.5 mM EDTA. The trypsin was quenched
with serum-containing medium. The cells were resuspended in ice-cold
medium and pushed through a 40-µM filter. The cells were sorted in
ice-cold medium with a FACSVantage Cell Sorter (BD Biosciences)
equipped to sort EGFP. Nontransgenic cells were used to determine the
level or autofluorescence. RNA was prepared from the cells directly or
after the cells were expanded by cell culture.
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RESULTS |
Cloning, sequencing, and structural analysis of the mouse AQP2
gene.
To perform further experiments to understand the basis for principal
cell-specific expression of AQP2, the mouse AQP2 gene was isolated from
a PAC library created from the SVJ/129 mouse strain. Oligonucleotide
primers were designed to amplify regions of the mouse AQP2 cDNA and
gene that were homologous to exons 2 and 3 from the human AQP2 gene
(accession no. Z29491). These primers were then used for PCR screening
of an arrayed mouse genomic library in the PAC vector pAD10SacBII
(42). The four clones contained inserts with common
patterns of restriction fragments (data not shown). Using the mouse
cDNA (accession no. AF020519), the human promoter (accession no.
U40369), and the human intron-exon boundaries (accession no. D31846,
Z29491), additional PCR primers were designed to amplify other regions
of the AQP2 gene. PCR analysis with these primers demonstrated that all
four clones contained sequences homologous to the human proximal
promoter exon 1 and exon 4 (Fig.
1). A 21-kb EcoRI fragment was
subcloned from the PAC clone into pBluescript KS II, and both strands
were sequenced by primer walking to establish the overall gene
structure (Fig. 2). The results were
submitted to GenBank (accession no. AY055468).

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Fig. 1.
PCR analysis showed that all 4 P1 artificial chromosome clones
contain all 4 coding exons and the promoter of the mouse aquaporin-2
(AQP2) gene. Four P1 artificial chromosome clones were analyzed by PCR
using primers that amplify the AQP2 genomic regions, including the
promoter and coding exon 1, coding exon 4 only, and coding exons 3 and
4. Mouse tail DNA and kidney cDNA are shown as positive controls.
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Fig. 2.
Structure of the mouse AQP2 gene and the mAQP2-enhanced
green fluorescent protein (EGFP) transgene. Top: 21-kb AQP2
gene. Sizes of the 5'- and 3'-flanking regions, 4 coding exons, and 3 introns are shown. Bottom: structure of the mAQP2-EGFP
transgene. AQP2 promoter (9.5 kb) was fused to the EGFP coding region
with the SV40 early region polyadenylation signal. Regions of the
transgene that were amplified by PCR for genotyping and by RT-PCR for
mRNA expression analysis are also shown.
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The identity of the mouse AQP2 gene was confirmed by comparison of the
DNA sequence to the human AQP2 gene, the proximal mouse AQP2 promoter,
and the mouse cDNA. The proximal 1.2 kb of promoter sequence is
identical to the published mouse AQP2 promoter (accession no. D87129),
and the coding exon sequences are identical to the published mouse AQP2
cDNA (accession no. AF020519), with the exception of several
single-nucleotide polymorphisms. The coding exon and intron boundaries
were conserved relative to the human gene (52) (accession
no. D31846). This 21-kb subclone of the mouse AQP2 gene was used for
further experiments designed to understand the basis for principal
cell-specific expression of the AQP2 gene.
Design of an EGFP transgene using the mouse AQP2 promoter.
Although we previously demonstrated that 14 kb of the human AQP2
promoter was sufficient to confer principal cell-specific expression of
a transgene, the expression was incomplete (38). We
therefore used the mouse AQP2 promoter in hopes of achieving more
complete expression of GFP. Mouse AQP2 promoter (9.5 kb) was fused to
an EGFP cassette that included the EGFP coding region with its own
translational initiation site and an SV40 early region polyadenylation
signal (Fig. 2). The mouse AQP2 promoter contained the TATA-box and the
predicted downstream transcription initiation site but did not contain
the translation initiation site for the mouse AQP2 gene. This transgene
was designed to achieve principal cell-specific transcription via the
AQP2 promoter and efficient translation utilizing the EGFP cassette. It
will be referred to as mAQP2 promoter-EGFP.
Creation, breeding, and genotyping of transgenic mice.
Linearized mAQP2 promoter-EGFP was used to create transgenic mice by
standard pronuclear microinjections (27). Founders were
identified by PCR analysis of tail DNA using oligonucleotide primers
that anneal within the promoter and EGFP cassette (Figs. 2 and
3). Primers specific for the endogenous
AQP2 gene were used to confirm integrity of the tail DNA (Fig. 3).
Single copy detection was demonstrated by analysis of transgene added
to normal mouse DNA to simulate 1-100 copies/cell equivalents
(Fig. 3). At least three F1 animals derived from each of the four
founders were analyzed for expression of the transgene. Only one
founder ultimately was found to express the transgene. This particular
founder transmitted the transgene to >50% of offspring, suggesting
more than one integration site. Several F1 animals were used to
generate F2 animals. An F1 animal that transmitted the transgene to
50% of offspring and whose offspring always expressed the transgene
was used to propagate the transgenic line of mice for further
experiments. The PCR genotyping of the tail from the original founder
and the F1 animal are shown in Fig. 3. The line of mice derived
from this F1 animal contains ~1-3 copies/cell equivalent of the
transgene. This copy number has remained stable for at least four
generations.

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Fig. 3.
PCR genotyping showed that the mAQP2-EGFP transgene was
present in founder tail DNA. Purified transgene DNA plus normal mouse
DNA or transgenic tail DNA were amplified by PCR using primers specific
for the mAQP2-EGFP transgene (top) and the endogenous mAQP2
gene (bottom). Left: results of purified
transgene DNA added to normal mouse DNA to simulate 1, 3, 10, 30, and
100 copies/cell equivalent. Right: results from PCR analysis
of tail DNA from the founder and the F1 animal that carried the active
transgene integration site.
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RT-PCR analysis for transgene expression.
Each transgenic line was screened by RT-PCR for expression of the
transgene in kidney. One of the four lines consistently expressed the
transgene in kidney. Organ panels were analyzed by RT-PCR for
expression of EGFP and AQP2 in two male and two female mice. A
representative organ panel for each sex is shown in Figs. 2 and 3. EGFP
was expressed in kidney but not other organs, including the male
reproductive system (Fig. 4). AQP2 not
only was expressed in kidney but also was expressed in the vas deferens of the male reproductive system. The integrity of the RNA and similar
loading was confirmed by RT-PCR analysis for GAPDH (accession no.
M32599). Parallel reactions carried out in the absence of RT verified
that the kidney-specific products did not result from amplification of
genomic DNA contaminating the RNA. The identity of the RT-PCR products
was verified by direct sequencing. These results show kidney-specific
expression of the mAQP2-EGFP transgene that parallels that of
endogenous AQP2 in the kidney but not in the male reproductive system.

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Fig. 4.
RT-PCR analysis showed that EGFP and AQP2 were expressed in the
kidney of male (A) and female (B) mAQP2-EGFP
transgenic mice and that GAPDH was expressed in all organs. Parallel
analyses performed in the absence of RT did not reveal any DNA
contamination of the RNA.
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Transgenic mice were then dehydrated for 48 h, and kidney RNA was
analyzed by RT-PCR for expression of AQP2. The results are shown for
three control and three dehydrated mice (Fig.
5). The results were replicated in an
additional experiment. Thirsting resulted in an increase in EGFP
expression in parallel with AQP2. There was no change in expression of
the housekeeping gene GAPDH. This is consistent with vasopressin
induction of transgene and AQP2 expression. This maneuver will be
potentially useful in future studies on gene regulation.

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Fig. 5.
RT-PCR analysis of kidney RNA showed that the expression
of EGFP and AQP2 was increased in dehydrated mAQP2-EGFP transgenic mice
compared with control mice. No change in GAPDH expression was observed.
Results are shown for 3 control and 3 dehydrated animals. Parallel
analyses performed in the absence of RT did not reveal any DNA
contamination of the RNA.
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Fluorescence microscopic analysis of kidney for EGFP expression.
All transgenic mouse lines were screened for expression of EGFP by
fluorescence microscopy of thick sections of mouse kidney. Mouse
kidneys were fixed and sectioned to 200-µm thickness with an
oscillating microtome. These thick sections were then examined by
fluorescence microscopy. This is a rapid and easy method to screen for
EGFP expression in mAQP2 promoter-EGFP transgenic mice. The transgenic
line that expressed EGFP in kidney by RT-PCR also expressed EGFP in
kidney by fluorescence microscopy. At least three animals from the
F1-F5 generations showed expression of EGFP in an identical
pattern. Representative images demonstrate that EGFP is expressed in a
radial pattern from the cortex through the outer medulla into the inner
medulla (Fig. 6). Close examination reveals variegated expression, which is what one would expect if the
transgene were expressed only in principal cells but not in
intercalated cells. This pattern of expression suggests that the mAQP2
promoter-EGFP transgene is expressed within the collecting duct and
perhaps only in AQP2-expressing principal cells.

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Fig. 6.
Confocal fluorescence microscopy of 200-µm transgenic kidney
sections showed that EGFP was expressed in the tubules in a pattern
characteristic of collecting ducts. Some cells within a collecting
tubule did not contain EGFP (G-I). A-C:
cortex, outer medulla, and inner medulla were viewed with ×4
objective. Bar = 615 µm. D-F: cortex, outer
medulla, and inner medulla were viewed with ×10 objective. Bar = 246 µm. G-H: cortex and outer medulla were viewed
with ×60 objective. Bar = 40 µm. I: outer medulla
was viewed with ×100 objective. Bar = 25 µm.
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Immunofluorescence studies were then performed on these thick sections
of transgenic kidney to determine whether EGFP was found only in
AQP2-expressing collecting ducts rather than other tubules that do not
express AQP2. Transgenic kidney slices were permeabilized with saponin
and immunostained for AQP2 by indirect immunofluorescence using a
CY5-conjugated secondary antibody (Fig. 7). The pattern of EGFP expression was
very similar to the pattern of endogenous AQP2 in the cortex, outer
medulla, and inner medulla. However, there were consistently fewer
EGFP-expressing tubules than AQP2-expressing tubules. These results
were seen in three different animals. Breeding the transgene to
homozygosity did not significantly change the pattern of expression
(data not shown). This pattern of expression suggests that mAQP2
promoter-EGFP transgene was expressed in AQP2-expressing collecting
ducts but that expression was incomplete.

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Fig. 7.
Confocal fluorescence
microscopy of 200-µm transgenic kidney sections showed that EGFP
(green) was expressed in the same tubules that express AQP2 (red). Some
AQP2-expressing cells do not express EGFP (K-N).
Sections of kidney (200 µm) were immunostained with a primary rabbit
antibody to AQP2 and a secondary CY5-conjugated anti-rabbit antibody.
A-C, G, K, and O:
cortex. D-F: medulla. H, L, and
P: outer medulla. I, M, and
Q: inner medulla. J, N, and
R: papilla. A, D, and
G-J: EGFP. C, F, and
O-R: AQP2. B, E, and
K-N: both EGFP and AQP2. Bars = 615 µm with ×4
objective (A-F); 62 µm with ×40 objective
(G-R).
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|
Immunofluorescence studies were performed on thin cryosections of
kidney to determine whether EGFP was expressed only in AQP2-expressing principal cells rather than in H+-ATPase-expressing
intercalated cells within the collecting duct (Fig.
8). EGFP retained fluorescence despite
formaldehyde fixation, cryosectioning, and immunostaining. EGFP
colocalized with AQP2 (Fig. 8, A-C) but did not
colocalize with H+-ATPase E subunit (Fig. 8,
C-E). However, EGFP was not expressed in all
AQP2-expressing cells (Fig. 8, A-C). These studies
demonstrate that EGFP is only expressed in AQP2-expressing principal
cells and not in the H+-ATPase-expressing intercalated
cells.

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Fig. 8.
Fluorescence microscopy of thin
cryosections of transgenic kidney showing that EGFP (green) was present
in AQP2-positive (red) cells (A-C) but was not present
in H+-ATPase-positive (red) intercalated cells
(D-E). Cryosections (4 µm) of transgenic kidney were
immunostained with a primary rabbit antibody to AQP2
(A-C) or H+-ATPase 31-kDa subunit
(D and E) and a secondary CY3-conjugated
antibody. A: AQP2 (red). C: EGFP (green). B: both AQP2 and
EGFP. D and E: both H+-ATPase E
subunit (green) and EGFP (red).
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|
Further quantitative immunofluorescence studies of AQP2 and EGFP
expression were performed in control and dehydrated mice to determine
the extent of expression of EGFP in principal cells and the effects of
dehydration on this expression. Mice were dehydrated for 48 h.
Then, the kidneys from three control and three dehydrated mice were
fixed, cryosectioned, and immunostained for AQP2. Digital images of
AQP2 and EGFP were merged and displayed with a grid overlay. There were
no EGFP-positive cells that were AQP2 negative. Therefore,
500-1,500 AQP2-positive cells were counted in each region of the
kidney. The percentage of AQP2-positive cells that were also
EGFP-positive was 10, 12, 18, and 34% in the cortex, outer stripe of
outer medulla, inner stripe of outer medulla, and inner medulla,
respectively, in control animals and 24, 36, 44, and 44% in the
cortex, outer stripe of outer medulla, inner stripe of outer medulla,
and inner medulla, respectively, of dehydrated animals (Fig.
9). Dehydration caused a statistically
significant increase in the percentage of EGFP-positive cells in the
cortex and outer medulla (P < 0.05, Student's
t-test) but not in the inner medulla (P = 0.06). These results indicate that EGFP expression in principal cells
was incomplete but could be increased significantly by dehydration.

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Fig. 9.
Quantitation of EGFP-positive principal cells (PC) in control and
dehydrated animals. Immunostaining and fluorescence microscopy were
performed as stated in Fig. 8. EGFP- and AQP2-positive cells were
counted. Values are means ± SE of the percentage of AQP2-positive
principal cells that were EGFP positive for the cortex, outer stripe of
outer medulla (OS), inner stripe of outer medulla (IS), and inner
medulla (IM). There is a significant difference between the control
(n = 3) and dehydrated (n = 3) animals
in the cortex, OS, and IS (P < 0.05, Student's
t-test) but not in the IM (P = 0.06).
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|
Expression of EGFP in primary cultures of renal medulla.
The mAQP2 promoter-EGFP transgenic mice were bred with
H2Kb-SV40Tag transgenic mice to generate mice with both
transgenes. Double transgenic kidneys were then used for the culture of
renal medulla. When induced in culture with interferon-
, the
H2Kb-SV40Tag transgene produces a temperature-sensitive
version of the SV40 virus T antigen in all cell types
(32). The SV40 T antigen should facilitate the passage of
principal cells expressing EGFP.
Therefore the renal medulla was cultured from mice that were transgenic
for both mAQP2 promoter-EGFP and H2Kb-SV40Tag by a modification of established methods (29). The tubule
fragments were cultured on standard tissue cultureware or type IV
collagen-coated polyethylene terephthalate filters. The cells were
cultured under conditions that result in high-level expression of
active SV40 T antigen (32) (33°C with interferon-
in
the medium). When the cells approached confluence, they were shifted to
conditions that would reduce the level of SV40 T antigen expression.
After 24-48 h, the cells were stimulated for 48-72 h with
10
7 M DDAVP plus 400 µM IBMX or with 500 µM
CPT-cAMP. RT-PCR analysis demonstrated that AQP2 and EGFP mRNA
were variably present in primary culture without stimulation but were
consistently present after stimulation with DDAVP and IBMX or
with CPT-cAMP (Fig. 10). The expression
of EF1
, a housekeeping gene, was detected with or without
stimulation (Fig. 10). In addition, islands of green fluorescence were
observed in these cells after stimulation with DDAVP and IBMX
or with CPT-cAMP (Fig. 11). These
results have been reproduced in at least three experiments. These
results demonstrate that EGFP- and AQP2-expressing principal cells
could be maintained in culture to carry out FACS.

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Fig. 10.
RT-PCR analysis showed increasing expression of AQP2 and
EGFP but no change in elongation factor 1 (EF1 ) in a primary
culture of medullary epithelial cells that were stimulated with
CPT-cAMP or with DDAVP and IBMX. Parallel reactions without RT did not
reveal genomic DNA of RNA.
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Fig. 11.
Fluorescence microscopy showing islands of EGFP (green) expression
in a primary culture of medulla from mAQP2-EGFP transgenic kidneys that
were stimulated with CPT-cAMP (A, D) or with
DDAVP and IBMX (B, E). Cells were confluent
according to phase contrast microscopy (C, F).
Bar = 100 or 50 µm with ×20 (D-F) or ×40
(A-C) objectives, respectively.
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|
FACS.
Cells were cultured on collagen IV-coated filters and stimulated with
DDAVP and IBMX or cultured in flasks and stimulated with CPT-cAMP, as
previously stated, dispersed into single cells, and subjected to FACS.
The data are shown for DDAVP- and IBMX-treated cells (Fig.
12). The results were identical for
stimulation with CPT-cAMP. The nontransgenic and transgenic cells are
similar in terms of the forward scattering and side scattering shown in
the scatter plots (Fig. 12, top left). However, there is a
difference between transgenic and nontransgenic cells on the basis of
forward scattering of light and fluorescence (GFP; Fig. 12, top
right). There is also a marked difference between transgenic and
nontransgenic cells on the basis of the EGFP fluorescence shown in the
histogram (Fig. 12, bottom). The cells with fluorescence
>102 units and forward scattering >20 units (regions M1
and R2) were collected under sterile conditions. Typically, this was
1-3% of the total cells. When a sample of these cells was
immediately reanalyzed, >95% of cells exhibited a high level of
fluorescence. Either RNA was prepared directly from these cells or the
cells were expanded by cell culture for further experiments.

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Fig. 12.
Fluorescence-activated cell sorting (FACS) showing that
fluorescent cells could be isolated from a primary culture of renal
medulla from transgenic, but not from nontransgenic, kidneys after
stimulation with CPT-cAMP. Left: data for cells derived from
nontransgenic renal medulla. Right: data for cells derived
from transgenic renal medulla; highly fluorescent cells are shown in
regions M1 and R2. Dot plots show side scatter vs. forward scatter and
forward scatter vs. GFP, and the histogram shows cell number vs GFP.
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Characterization of cells obtained by FACS.
EGFP-positive cells were initially analyzed directly. RNA was directly
prepared from 105-positive cells, and gene expression was
determined by RT-PCR. The cells expressed AQP2, AQP3, AQP4,
V2R, and cAMP response element binding protein (CREB) but
not the H+- ATPase B1 subunit or AE1, indicating they
retained the principal cell phenotype rather than the intercalated cell
phenotype (Fig. 13). Next, cells
prepared in the same way were passaged five times after reaching 80%
confluence. After the final passage, the cells were cultured on
collagen IV-coated polyethylene terephthalate filters until confluent
and then stimulated with DDAVP and IBMX for 72 h. Endogenous gene
expression was determined by RT-PCR analysis (Fig.
14). The cells clearly continued to
express AQP2, AQP3, AQP4, V2R, and CREB. AQP2 and AQP3
expression was increased by stimulation, whereas AQP4, V2R,
and CREB were not increased by stimulation. The housekeeping gene
EF1
was unchanged by stimulation. The cell population obtained by
FACS retains the ability to express AQP2, AQP3, AQP4, V2R,
and CREB for five passages.

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Fig. 13.
RT-PCR analysis of EGFP-positive (+) cells
isolated by FACS after CPT-cAMP stimulation, showing expression of
principal cell markers including AQP2, AQP3, AQP4, vasopressin type 2 receptor (V2R), and cAMP element binding protein (CREB) but
no expression of intercalated cell markers such as the
H+-ATPase 58-kDa subunit (B1) and anion exchanger 1 (AE1).
Parallel analysis EF1 without RT did not reveal DNA contamination of
RNA (data not shown). Kidney RNA was used as a positive control. EF1
was used as a housekeeping gene.
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Fig. 14.
RT-PCR analysis of EGFP-postive cells that were
passaged, showing continued expression of principal cell markers
including AQP2, AQP3, AQP4, V2R, and CREB. EGFP-positive
cells that were isolated by FACS were expanded in culture, plated on
collagen-coated filters, and stimulated with DDAVP and IBMX. The
expression of the stated genes was determined by RT-PCR analysis.
Parallel reactions without RT show that there was no DNA contamination
of the RNA. Kidney RNA was used as a positive control. EF1 was used
as a control housekeeping gene.
|
|
 |
DISCUSSION |
The goal of these studies was to develop a new transgenic mouse
model for parallel in vivo and in vitro studies of gene expression regulation in principal cells within the renal collecting duct. Transgenic mice were created with 9.5 kb of the mouse AQP2 promoter driving expression of an EGFP. The mouse AQP2 promoter was sufficient to drive EGFP expression in vivo in a kidney and principal
cell-specific manner. In addition, the mouse AQP2 promoter was also
sufficient to confer increased expression of EGFP in response to
dehydration, as has been demonstrated for AQP2 in rats (23,
39). This increase in expression is manifested as an increase in
the level of EGFP mRNA and abundance of principal cells expressing
EGFP. Finally, the mouse AQP2 promoter confers increased expression of
EGFP in response to stimulation with a vasopressin analog, DDAVP, in
the presence of a phosphodiesterase inhibitor, IBMX, or a cAMP analog, CPT-cAMP. These results agree with the previous demonstration that
vasopressin and cAMP stimulate expression in AQP2 reporter constructs
in vitro in transient transfection experiments using renal cell lines
(28, 56). These transgenic mouse experiments demonstrate
that the stimulation of AQP2 mRNA expression in vivo by dehydration and
by vasopressin or cAMP in vitro results from the stimulation of
transcription of the AQP2 gene. The cell-specific and temporal
regulation of EGFP expression in the kidney of this transgenic mouse
model suggest that the mouse model could be very useful in the study of
AQP2 gene expression and principal cell biology.
A potential limitation of this transgenic model is the incomplete
expression of EGFP in principal cells that is observed in intact
kidney. This incomplete expression likely explains the expression level
observed in primary cultures. Incomplete expression has been observed
with other transgenic models, including those that have achieved
kidney-specific gene expression (5, 31, 38). This type of
incomplete expression is often referred to as position effect
variegation. It may be due to the tandem arrays of the transgene at the
site of integration (21). In this case, Cre/lox technology
has been used to reduce the transgene copy number and enhance
expression of the transgene (21). Incomplete expression
may also be due to the lack of chromatin insulator sequences or
boundary elements in the transgene. Improved expression could be
accomplished by the flanking of the transgenes with insulator sequences
(43) or creating transgenic mice using BAC clones containing gene loci with intact insulator sequences (24,
54). These approaches are technically more difficult but will be
considered in the future design of transgenes in attempts to improve
the expression level of EGFP.
Incomplete expression of EGFP in this transgenic model can be partially
overcome. In vivo expression of EGFP can be increased by thirsting
animals. Expression in primary culture may be enhanced by stimulating
with DDAVP and IBMX or with CPT-cAMP for longer periods of time or more
selective dissection of inner medulla, where there is an increased
abundance of collecting ducts. Despite the incomplete
expression of EGFP, the expression of EGFP is very specific for
principal cells. This is the essential feature required for the use of
this transgenic model for future studies.
It is interesting to note that the EGFP transgene was not expressed in
the male reproductive system where AQP2 is expressed. In the male
reproductive system, AQP2 is expressed in the principal cells of the
distal vas deferens in a vasopressin-independent manner (38,
48). It is possible that the lack of expression of the transgene
was due to an unfavorable transgene integration site in this particular
line of mice. Alternatively, it is also possible that 9.5 kb of the
mouse AQP2 promoter did not contain the required vas deferens-specific
enhancer sequences. Further studies are in progress to determine the
mechanism of vas deferens-specific expression of AQP2. Such
investigations may contribute to a better understanding of the
mechanism of vasopressin-independent expression of AQP2 in the vas
deferens (48) and therefore will provide unique insight
into the development and differentiation of the principal cells of the
vas deferens.
This transgenic model was used to establish conditions that maintain
expression of EGFP in principal cells in culture. In culture, viable
EGFP-expressing principal cells can be identified by fluorescence
microscopy and isolated by FACS. Whether principal cells are analyzed
directly or after passage, the cells express marker genes typical of
principal cells after stimulation with CPT-cAMP or with DDAVP plus
IBMX. The marker genes include AQP2, AQP3, AQP4, and V2R.
Interestingly, the cells also expressed CREB, which is the
transcription factor that may mediate vasopressin and cAMP-induced
transcription via CRE sites within the AQP2 promoter (28, 36,
56). These studies illustrate that flow cytometry can be used to
isolate EGFP-expressing principal cells that can be used for a variety
of molecular studies.
This transgenic model could be used to investigate the development and
differentiation of renal collecting duct cells into principal and
intercalated cells. Although this area is incompletely understood,
there is evidence that the collecting duct phenotype exhibits
plasticity in terms of relative numbers of principal and intercalated
cells. For example, carbonic anhydrase II null mice have a marked
decrease in the prevalence of intercalated cells and increase in the
prevalence of principal cells in the inner medulla (7). In
addition, rats treated with acetazolamide, an inhibitor of carbonic
anhydrase, exhibit an increase in intercalated cells and a decrease in
principal cells in the outer medulla, whereas there was a decrease in
intercalated cells and increase in principal cells in the inner medulla
(2). These studies support the role of carbonic anhydrase
in the determination of the collecting duct phenotype. Finally, there
are studies in vitro that demonstrate that
-intercalated cells give
rise to
-intercalated and principal cells in culture
(17). The molecular mechanisms that determine principal
and intercalated cell phenotype in the collecting duct are incompletely
understood. Such molecular mechanisms will be investigated by using
this transgenic model.
This transgenic mouse model could also be used for in vivo studies of
gene expression in principal cells. It is possible that physiological,
pharmacological, or genetic manipulations may alter gene expression in
principal cells in collecting ducts in adult or neonatal mice. One
could create transgenic or gene-targeted animals that express
dominant-negative, altered function, or loss of function mutations of
transcription factors or members of signal transduction pathways that
might potentially be important in determining principal cell gene
expression. The examination of kidneys for EGFP expression in principal
cells of collecting ducts should provide an immediate impression of the
effects on AQP2 gene transcription and thus on the principal cell
phenotype. Furthermore, principal cells could be isolated by laser
capture microdissection and gene expression profiling performed by
using microarrays or quantitative RT-PCR.
The transgenic mouse model could also be used in in vitro studies of
gene expression regulation in principal cells. The cells could be
studied in primary culture or after a limited number of passages.
Compared with cell lines, cells studied in primary culture have the
advantage that the state of differentiation is more likely to resemble
that of cells in vivo. The EGFP-expressing cells could be
examined after alteration of culture conditions or after microinjection
or transfection with expression constructs. The effects on EGFP
expression, a surrogate marker for principal differentiation, could
then be examined directly. The results can be quantified by using FACS
analysis. Alternatively, FACS would allow the isolation of principal
cells from which RNA can be prepared. The RNA could be used for
subtraction cloning, differential display, or microarray studies to
investigate differential regulation of gene expression in principal cells.
In summary, we have shown that the mouse AQP2 promoter is sufficient to
drive renal principal cell-specific expression of EGFP and that the
expression is temporally modulated in vivo by dehydration and in vitro
by vasopressin and cAMP analogs. Thus we have created a transgenic
mouse model that should prove to be useful for a variety of in vivo and
in vitro studies of AQP2 gene expression and principal cell biology.
 |
ACKNOWLEDGEMENTS |
This work was funded in part by grants from the Primary Children's
Foundation (R. D. Nelson), The Kenneth E. & Becky H. Johnson Foundation (R. D. Nelson), American Society of Nephrology (R. D. Nelson), and National Institute of Diabetes and Digestive and Kidney
Diseases Grants 5-RO1-DK-53990 (R. D. Nelson) and DK-38452 (D. Brown and S. Breton).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. D. Nelson, Dept. of Pediatrics, Div. of Nephrology, Univ. of Utah, 50 North Medical Dr., Salt Lake City, UT 84132 (E-mail:
raoul.nelson{at}hsc.utah.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 6, 2002;10.1152/ajprenal.00224.2001
Received 18 July 2001; accepted in final form 30 July 2002.
 |
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