Expression of an AQP2 Cre recombinase transgene in kidney and
male reproductive system of transgenic mice
Raoul D.
Nelson1,
Peter
Stricklett1,2,
Corrine
Gustafson3,
Anna
Stevens3,
Dennis
Ausiello3,
Dennis
Brown3, and
Donald E.
Kohan1,2
1 University of Utah Medical
School, Salt Lake City 84132;
2 Veterans Affairs Medical
Center, Salt Lake City, Utah 84148; and
3 Massachusetts General Hospital
and Harvard Medical School, Charlestown, Massachusetts
02129
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ABSTRACT |
A transgenic mouse
approach was used to examine the mechanism of principal cell-specific
expression of aquaporin-2 (AQP2) within the renal collecting
duct. RT-PCR and immunocytochemistry revealed that murine AQP2
was expressed in principal cells in the renal collecting duct,
epithelial cells of the vas deferens, and seminiferous tubules within
testis. The vas deferens expression was confirmed in rats. RT-PCR and
immunocytochemistry showed that 14 kb of the human 5'-flanking
region confers specific expression of a nucleus-targeted and
epitope-tagged Cre recombinase in the principal cells within the renal
collecting duct, in the epithelial cells of the vas deferens, and
within the testis of transgenic mice. These results suggest that
cell-specific expression of AQP2 is mediated at the transcriptional
level and that 14 kb of the human AQP2 5'-flanking region contain
cis elements that are sufficient for
cell-specific expression of AQP2. Finally, renal principal cell
expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of
gene function in this cell type.
water-electrolyte balance; kidney collecting tubule; ion channel
physiology; gene expression regulation; promoter region; aquaporin
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INTRODUCTION |
AQUAPORIN-2 (AQP2)
belongs to a family of water channel proteins and is fundamentally
important in regulation of renal water excretion (1, 28). The renal
site of AQP2 expression is crucial to its function. AQP2 is expressed
exclusively in collecting duct principal cells (12, 13, 35). These
cells are located in the distal and terminal nephron, the region of the
kidney where final adjustments to urine concentration are made. In
addition, principal cells are concentrated in the renal medulla, where
water reabsorption is facilitated by high interstitial tonicity.
It is evident, therefore, that principal cell-specific expression of
AQP2 is of primary importance to the regulation of renal water excretion.
AQP2 is regulated in two ways. Long-term regulation occurs by arginine
vasopressin (AVP) augmentation of AQP2 mRNA and protein levels
(6, 20, 21, 29, 35), resulting in increased numbers of water channels
in the kidney. Short-term regulation occurs by vasopressin-induced
movement of AQP2 protein from subapical-cytosolic vesicles to the
apical plasma membrane by exocytic insertion of AQP2-containing
vesicles into the plasma membrane (35, 39). These processes result in
enhanced water permeability of the collecting duct, thereby allowing
water to be osmotically reabsorbed by the kidney across collecting duct
epithelium as a result of the interstitial hypertonicity that is
generated in the medulla. To date, AQP2 is the only known mechanism by
which AVP directly controls renal water excretion.
Although factors modulating AQP2 expression are beginning to be
identified, very little is known about the mechanism of principal cell-specific expression of this AVP-sensitive water channel. One
potential mechanism of cell-specific expression involves cell-specific gene transcription mediated by
cis-acting sequences in the human AQP2
gene and transcription factors expressed in principal cells. In this
context, regulatory regions of the AQP2 gene have been examined. The
human AQP2 5'-flanking region contains a TATA box with
transcription initiation sites 92 and 93 bp upstream from the
translational initiation site (44). Further sequence analysis reveals
putative transcriptional regulatory sequences, including several cAMP
response elements (CREs), several GATA consensus sites, E-boxes, and
AP-1, AP-2, and SP1 sites (24, 44). Studies have demonstrated a role
for the CREs and GATA sites in altered levels of AQP2 gene
transcription by cAMP-responsive element binding protein and GATA-3
(24, 33, 43), respectively. It is unclear whether these sequences play
a role in kidney- and principal cell-specific expression of AQP2.
To begin to examine how principal cell-specific expression of AQP2
occurs, a transgenic mouse approach was utilized. A 14-kb section of
the human AQP2 gene 5'-flanking region was used to determine
whether transcriptional elements contained within this region were
responsible for cell-specific expression. We report that this reporter
region does indeed confer principal cell-specific expression within the
kidney. An unanticipated result was transgene expression in the vas
deferens and testis, where AQP2 is also expressed. Finally, an added
feature of the transgene was the inclusion of Cre recombinase and an
epitope tag as the reporter (CreTag). As described, this system will
ultimately be used to attempt principal cell-specific gene targeting
within the kidney.
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METHODS |
Construction of the AQP2-CreTag transgene.
All cloning and DNA manipulations were done using conventional methods
(32). pMC-Cre (gift from Kirk Thomas, Howard Hughes Medical Institute,
University of Utah, Salt Lake City, UT) includes a Cre recombinase gene
that was modified at the amino-terminal end to include the nuclear
localization signal from the simian virus 40 (SV40) large T antigen
(18). pMC-Cre was further modified at the carboxy-terminal end to
include an 11-amino acid herpes simplex virus (HSV) glycoprotein D
epitope tag (QPELAPEDPED) (27). To do this, the Cre gene was cut from
pMC-Cre with EcoR
I/Xho I and subcloned into
EcoR
I/Xho I-digested pBluescript KS II
(Stratagene, La Jolla, CA) without the
BspH I sites in the vector, resulting in pBS-Cre. Overlapping and complementary 80-mer oligonucleotides were
synthesized (Bob Schackman, DNA/Peptide Facility, University of Utah),
annealed, and extended with Klenow enzyme. The resulting double-stranded DNA contains sequence encoding the carboxy-terminal Cre
extending from a BspH I site, the
carboxy-terminal 11-amino acid epitope tag, an added stop codon, and
Xba I and
Xho I sites. This DNA was digested
with BspH
I/Xho I and ligated into
BspH I/Xho I-digested pBS-Cre, resulting in
pBS-CreTag. An Xba I linker was
ligated into pBS-CreTag that was digested with
Mlu I and blunted with Klenow enzyme.
This modified pBS-CreTag was digested from the vector with
Xba I and ligated into the
Xba I site of pUHD 10-3 (16), becoming
pUHD-CreTag. pUHD provided a SV40 late region polyadenylation signal
downstream from CreTag. CreTag with the polyadenylation signal was
digested from pUHD-CreTag with HinD III, blunted with Klenow enzyme, and cut with
Sma I. This fragment was ligated into
the Sma I site of pBluescript KS II
that resides downstream from the 14-kb human AQP2 5'-flanking
region, resulting in pAQP2-CreTag. Cloning of the human AQP2
5'-flanking region was previously described (24). pAQP2-CreTag
was digested with Xho
I/Not I, and the AQP2-CreTag transgene
was separated from the vector sequences by electrophoresis through
low-melting-point agarose. The transgene was purified with an Elutip-D
column (Schleicher & Schuell, Keene, NH) after digestion of the agarose
with
-agarase I (New England Biolabs) and suspended in injection
buffer. All ligation junctions, the entire CreTag sequence and the
5' and 3' 300 bp of the AQP2 5'-flanking region were
sequenced to verify the structural integrity of the transgene. Finally,
to express CreTag in cultured cells, CreTag was subcloned downstream
from a cytomegalovirus (CMV) promoter.
Generation and breeding of transgenic mice.
Transgenic mice were created by the Transgenic Mouse Core Facility at
the University of Utah. The linearized transgene was injected into
pronuclei of C57BL/CBA single cell embryos, and the injected embryos
were transferred into pseudopregnant mice according to standard
techniques (22). Pups were analyzed for the presence of the transgene
by PCR amplification of tail DNA. Three founders were each bred to
nontransgenic C57BL/CBA mice. F1
and F2 animals from each founder
line were identified by PCR of tail DNA and used for analysis.
Identification of transgenic animals.
Tail DNA was prepared by standard methods (23). The transgene was
detected by PCR amplification of tail DNA using oligonucleotide primers
AQP2+310 (5'-GGA CGT CAG TCC TTA TCT GGA G-3') and CreDown (5'-GCG AAC ATC TTC AGG TTC TGC GG-3'), which span 625 bp
of the junction between the AQP2 promoter and CreTag reporter. Normal mouse DNA with 1-100 copies/cell equivalent of the transgene DNA was always run as a control to estimate copy number. Equal loading and
amplification efficiency of genomic DNA was controlled for by
amplification with RAPSYN(+) (5'-AGG ACT GGG TGG CTT CCA ACT CCC
AGA CAC-3') and RAPSYN(
) (5'-AGC TTC TCA TTG CTG CGC
GCC AGG TTC AGG-3'), which amplify 590 bp of the endogenous gene
RAPSYN (19). The products were electrophoresed through 2% agarose and were visualized by ethidium bromide staining and ultraviolet (UV) transillumination.
Expression of CreTag by transient transfection into cultured cells.
Two micrograms of the plasmid containing CreTag and a CMV promoter
(CMV-CreTag) were transiently transfected into a 35-mm dish of 3T3
cells using Lipofectamine (GIBCO BRL, Gaithersburg, CA) according to
the manufacturer's protocol. RNA was prepared with the acid phenol
method (5) 48 h posttransfection.
RT-PCR determination of AQP2, CreTag, and
glyceraldehyde-3-phosphate dehydrogenase mRNA expression.
RNA was prepared from transgenic mouse organs with the acid phenol
method (5); 2.5 µg total RNA from organs or cells was reverse
transcribed using
oligo(dT)12-18 and
Superscript II according to the manufacturer's procedure (GIBCO BRL).
The following oligonucleotide primers were used to PCR amplify AQP2, CreTag, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from one-tenth of the reverse transcription reaction: AQP2F (5'-GTG GCT GCC CAG CTG CTG GG-3') and AQP2R2 (5'-AGC TCC ACC GAC
TGC CGC CG-3') were used to amplify 500 bp of the mouse AQP2
cDNA; CreTagUP (5'-GGC TCT AGC GTT CGA ACG CAC TGA TTT
CGA-3') and SV40LateR (5'-T24 GTT GTT AA-3')
were used to amplify 844 bp of CreTag cDNA; CreTagUP and CreTagDown2
(5'-GGC TAT CGC CAT CTT CCA GCA-3') amplify the 635 bp of
Cre recombinase cDNA or gene, and GAPDHF (5'-CCT TCA TTG ACC TCA
ACT ACA TGG-3') and GAPDHR (5'-GCA GTG ATG GCA TGG ACT GTG
GT-3') were used to amplify 442 bp of the mouse GAPDH cDNA. All
RT-PCR reactions were carried out with and without RT to establish
whether cDNA or genomic DNA was being amplified. A negative control
without template was also run with each set of reactions to demonstrate
that there was no contamination of the PCR reaction. In selected
experiments, 10 ng of the CMV-CreTag plasmid DNA was used as a template
for PCR amplification of CreTag. The products were electrophoresed
through 2% agarose and were visualized by ethidium bromide staining
and UV transillumination. PCR products were directly sequenced using a
dye terminator cycle sequencing system with Amplitaq DNA polymerase FS
(Perkin Elmer, Norwalk, CT) in conjunction with an ABI fluorescent
sequencing system (DNA Sequencing Core Facility, University of Utah).
Single- and double-label immunohistochemistry.
Kidneys and male reproductive organs were fixed by cardiac perfusion
and immersion in 4% paraformaldehyde in Dulbecco's PBS at 4°C,
dehydrated with graded ethanol, paraffin embedded, and sectioned to 5 µm thickness. The sections were deparaffinized and microwaved in an
antigen retrieval solution (Biogenex, San Ramon, CA) (41).
Single-label immunohistochemical visualization of AQP2 and CreTag was
accomplished by the following protocol. Sections were blocked with 10%
goat serum in PBS (blocking solution) for 2 h and incubated overnight
at 4°C with 1:2,000 dilution of an affinity-purified polyclonal
rabbit antiserum to AQP2 (35) (gift from Mark Knepper, National
Institutes of Health, Bethesda, MD) or a 1:1,000 dilution of a mouse
monoclonal antibody to the HSV epitope tag (Novagen, Madison, WI) in
blocking solution. Sections were washed with 0.1% Triton X-100 in PBS
and incubated with a 1:100 dilution of a biotinylated goat anti-mouse
or goat anti-rabbit antibody in blocking solution for 2 h at 4°C.
Sections were washed, incubated with preformed complexes of
streptavidin and biotinylated-peroxidase conjugate (Vector
Laboratories, Burlingame, CA) for 30 min at room temperature, washed
again, and incubated with 3',3'-diaminobenzidine and
H2O2 with metal enhancement (Vector Laboratories). Selected sections were
counterstained with hematoxylin.
Double-label immunofluorescent visualization was accomplished using the
following protocol. Sections were blocked with 5% goat serum and 5%
horse serum in PBS (blocking solution) and incubated with a 1:2,000
dilution of an affinity-purified rabbit polyclonal antibody to AQP2 and
a 1:1,000 dilution of a mouse monoclonal antibody to the HSV epitope
tag in blocking solution overnight at 4°C. Sections were washed and
incubated with a 1:200 dilution of fluorescein-conjugated goat
anti-rabbit IgG antibody (Vector Laboratories) and a 1:200 dilution of
a Texas red-conjugated horse anti-mouse IgG antibody (Vector
Laboratories) in blocking solution for 2 h at 4°C. Sections were
washed, mounted in Vectashield (Vector Laboratories) and viewed with an
Olympus BX50 fluorescent microscope equipped with a CH250 Photometrics
charge-coupled device (CCD) camera. Images were digitally acquired with
Visis Smartcapture version 2.4.
Immunofluorescent visualization in the rat vas deferens was
accomplished as previously described (3). The tissue was fixed in
paraformaldehyde lysine periodate. After cryoprotection in 30%
sucrose, 5-µm sections were cut and incubated with a polyclonal anti-AQP2 carboxy-terminal peptide antibody that had been previously characterized (39). After they were washed, the sections were incubated with goat anti-rabbit IgG-FITC and counterstained with Evan's blue. Sections were examined on a Nikon FXA photomicroscope equipped with an Optronics three-bit color CCD camera. Images were
acquired digitally with IP Lab Spectrum software.
Immunoblotting analysis for CreTag in kidney and testis.
Kidney and testis from AQP2-CreTag transgenic and nontransgenic male
mice were minced and nuclei isolated by a modification of a previously
described method (8). The tissues were homogenized using a Dounce
homogenizer and pushed through a nylon mesh (Small Parts, Miami, FL).
Nuclei were pelleted and nuclear proteins isolated using previously
described methods (7, 36). In brief, the nuclear pellet was resuspended
in 20 mM HEPES (pH 7.9), 420 mM NaCl, 0.2 mM EDTA (pH 8), 1.5 mM
MgCl2, 25% (vol/vol) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT), incubated on ice for 15 min, and centrifuged at 15,000 g for 15 min at 4°C. The
supernatant was diluted 1:5 in 20 mM HEPES (pH 7.9), 0.2 mM EDTA (pH
8), 50 mM KCl, 20% (vol/vol) glycerol, 0.5 mM PMSF, and 0.5 mM DTT,
and aliquots were removed for protein determination using the Bradford
protein assay (Bio-Rad Laboratories, Hercules, CA). Nuclear proteins
were electrophoresed through a 12% SDS-PAGE gel and transferred to a
polyvinylidene difluoride-plus membrane (Micron Separations, Westboro,
MA) using standard protocols (14). Immunoblotting was performed using
the enhanced chemiluminescence protocol (Amersham, Arlington Heights,
IL). A mouse monoclonal antibody to the HSV epitope tag (Novagen,
Madison, WI) was used as primary antibody at a dilution of 1:20,000,
and a horseradish peroxidase-conjugated sheep anti-mouse antibody was
used as a secondary antibody at a dilution of 1:5,000 (Amersham).
Detection was accomplished by enhanced chemiluminescence, using
Hyperfilm-MP (Amersham). The relative molecular mass was calculated for
CreTag by comparison to the standards.
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RESULTS |
Transgene design.
To achieve principal cell-specific gene expression in the kidney and to
begin to achieve principal cell-specific gene targeting, we designed a
transgene with the AQP2 5'-flanking region driving expression of
a modified Cre cassette that was designated CreTag (Fig.
1). The original Cre cassette included an
SV40 nuclear localization signal on the amino terminus
(18). An 11-amino acid epitope tag, derived from HSV glycoprotein D
(27), was added to the carboxy-terminal end to facilitate detection of
Cre by immunoblotting and immunohistochemistry using a mouse monoclonal
antibody (Novagen). The 362-amino acid CreTag has a predicted molecular
mass of 41 kDa. The polyadenylation signal from the SV40 late region,
without an intron, was also added for efficient expression (accession no. V01380). The CreTag cassette was inserted into a CMV expression vector, resulting in CMV-CreTag, and was transiently transfected into
3T3 cells. CreTag mRNA was detectable by RT-PCR, and CreTag protein was
detectable in the nucleus of cells by immunocytochemistry and
immunoblotting analysis (Stricklett, Nelson, and Kohan, unpublished results). In addition, nuclear extracts from the cells expressing CreTag exhibit loxP site-specific recombinase activity in vitro (Stricklett and Kohan, unpublished results). A transgene was then constructed with 14 kb of the human AQP2 5'-flanking region (24) driving expression of the CreTag cassette (Fig. 1). The AQP2
5'-flanking region included the transcription initiation sites
from the human AQP2 gene but excluded the translational initiation
site. This transgene is referred to as AQP2-CreTag.

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Fig. 1.
Aquaporin-2 (AQP2)-CreTag transgene: 14 kb of human AQP2
5'-flanking region includes TATA box and transcription initiation
sites from human AQP2 gene. Cre recombinase cassette (CreTag cassette)
includes an amino-terminal simian virus 40 (SV40) nuclear localization
signal, Cre recombinase coding region, carboxy-terminal herpes simplex
virus (HSV) epitope tag, and SV40 virus late region polyadenylation
signal. ATG is translational initiation site, and TAG is translational
stop site. Transgenic animals were genotyped by PCR amplification of
junction between AQP2 5'-flanking region and CreTag cassette (625 bp). AQP2-CreTag mRNA expression was determined by RT-PCR analysis for
3' end of mRNA produced by CreTag cassette (850 bp).
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Generation of AQP2-CreTag transgenic mice.
Three independent founders were obtained following pronuclear
injections of the AQP2-CreTag transgene. PCR analysis of founder tail
DNA demonstrated that the CreTag transgene varied between 2 and 100 copies/cell equivalent among the different founder lines compared with
standards of transgene mixed with normal mouse genomic DNA (Fig.
2). The transgene was transmitted according
to Mendelian genetics. Transgenic
F1 and
F2 animals from each founder line and nontransgenic animals were analyzed for expression of CreTag and
AQP2.

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Fig. 2.
PCR determination of AQP2-CreTag transgene copy number in 3 founder
animals. A: results of PCR
amplification of tail DNA from transgenic founder
lines
1-3
and 0-200 copies/cell equivalent of transgene DNA added to normal
mouse DNA. Product size is 625 bp. B:
results of PCR amplification of endogenous RAPSYN gene from transgenic
founder line and control mouse DNA. Product size is 590 bp. Equal
intensity of RAPSYN amplification products indicates equal
amplification efficiency of tail DNA preparations. Relative
intensity of AQP2-CreTag transgene indicates that transgene is present
at 2, 100, and 2 copies/cell in lines
1-3,
respectively.
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AQP2 expression in mice.
Mouse AQP2 cDNA and amino acid sequences had not been reported when
these studies were initiated, but rat and human AQP2 cDNA and amino
acid sequences were known to be highly conserved (13, 40). Comparison
of rat and human AQP2 cDNA sequences (accession nos. L28112 and
Z29491) identified regions that were 100% conserved but differed
from rat AQP1 (accession nos. X67948, X70257), rat AQP3 (accession no.
D17695), rat AQP4 (accession no. U14077), rat AQP5 (accession no.
U16245), human AQP6 (accession no. AB006190), rat AQP7 (accession no.
AB000507), and rat AQP8 (accession no. AB005547). PCR primers were
designed to anneal to these regions and selectively amplify 510 bp of
mouse AQP2 cDNA from kidney. The internal 450 bp of the PCR products were directly sequenced, and the sequence was translated. The products
predict an amino acid sequence that is 98% identical to rat AQP2, but
only 42, 20, 44, 60, 33, 40, and 40% identical to rat AQP1, AQP3,
AQP4, AQP5, AQP7, and AQP8, and human AQP6, respectively, for 150 amino
acids that were compared. Furthermore, the sequence was 100% identical
to a sequence that was recently entered into GenBank (accession no.
AF020159). The PCR products generated by these primers, therefore, were
derived from mouse AQP2 cDNA.
The pattern of mouse AQP2 mRNA expression was next determined by RT-PCR
analysis of RNA derived from a panel of mouse organs (Fig.
3). The correct sized products were
observed in kidney, vas deferens, and testis. No products were present
in other organs, such as the brain, liver, heart, intestine, stomach,
spleen, ovary, fallopian tube, or uterus. The cDNA sequence of the PCR
products was identical in kidney, vas deferens, and testis. These
results indicated that AQP2 was selectively expressed in kidney, vas
deferens, and testis.

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Fig. 3.
RT-PCR determination of AQP2, CreTag, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA expression in transgenic mouse
line
2. AQP2 and CreTag are expressed in
kidney, testes, and vas deferens and epididymis. GAPDH is expressed in
all organs. No product is observed in absence of RT ( ).
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Immunohistochemistry was used to confirm the cellular pattern of
expression of AQP2 in kidney and to determine the cellular pattern of
expression in the male reproductive system. An affinity-purified antibody to rat AQP2 was used (35). Immunoperoxidase (Fig.
4) and immunofluorescent staining (Fig.
5) showed mouse AQP2 in the cytoplasm and
apical pole of 75-100% of collecting duct cells in the cortex,
outer medulla, and inner medulla of mouse kidney. The appearance of
less predominant apical staining in the immunofluorescence studies,
compared with prior published studies of rat, may be due to the image
capture techniques or redistribution during fixation. Otherwise, the
pattern of staining is identical to that observed in rat (35) and mouse
(2), suggesting that the observed staining is in principal cells. The
collecting duct cells that do not stain for AQP2 are intercalated
cells, as previously reported in the mouse kidney (2). In the male
reproductive system, immunoperoxidase staining showed mouse AQP2 in
apical and subapical regions of the principal cells in the vas deferens
and the central regions of seminiferous tubules but not in the mature
sperm in the epididymis or in the epididymis itself (Fig.
6). The pattern of staining suggests that
AQP2 is expressed in postmeiotic sperm. However, further investigations
will be required to define the exact stage of spermatogenesis at which
AQP2 is expressed. This is the first reported immunostaining for AQP2
in the male reproductive system.

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Fig. 4.
Immunohistochemical localization of AQP2 and CreTag in transgenic and
nontransgenic mouse kidney. A,
D, G,
J, and
M: immunostaining for AQP2 in
transgenic renal collecting duct within cortex, outer stripe of outer
medulla, inner stripe of outer medulla, outer half of inner medulla,
and papilla, respectively. Arrows, AQP2-negative intercalated cells
within collecting ducts. B,
E, H,
K, and
N: immunostaining for CreTag in renal
collecting duct within cortex, outer stripe of outer medulla, inner
stripe of outer medulla, outer half of inner medulla, and papilla,
respectively, of a kidney from transgenic mouse
line
2. Arrows with triangular heads denote
nuclear CreTag immunostaining in collecting duct of transgenic but not
nontransgenic animals. C,
F, I,
L, and
O: immunostaining for CreTag in renal
collecting duct within cortex, outer stripe of outer medulla, inner
stripe of outer medulla, outer half of inner medulla, and papilla,
respectively, of a kidney from a nontransgenic mouse. Asterisks,
nonspecific immunostaining for CreTag in nonnuclear regions in and
around tubules and glomeruli; CD, collecting ducts; G, glomeruli; PT,
proximal tubule.
A-O:
×360 magnification.
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Fig. 5.
Immunofluorescent colocalization of AQP2 and CreTag in kidney.
A-D:
collecting duct in cortex, outer stripe of outer medulla, inner stripe
of medulla, and inner medulla, respectively. AQP2 was immunostained
with a rabbit polyclonal antibody to AQP2 and a Texas red-conjugated
goat anti-rabbit antibody. CreTag was immunostained with a mouse
monoclonal antibody to HSV epitope tag and a fluorescein-conjugated
horse anti-mouse antibody. Note that nuclear CreTag immunostaining
(green) was always associated with AQP2 immunostaining (red). Arrows
with triangular heads, CreTag-positive cells. Regular arrows,
AQP2-negative intercalated cells. No nuclear CreTag staining was
associated with AQP2-negative intercalated cells. Asterisks,
nonspecific immunostaining for CreTag in nonnuclear regions around
tubules and glomeruli; r, nonspecific staining of red blood cells.
A-D:
×540 magnification.
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Fig. 6.
Immunocytochemical localization of AQP2 and CreTag in male reproductive
system. A and
C: immunostaining with a rabbit
polyclonal antibody for AQP2 in principal cells of vas deferens and in
seminiferous tubules of testis, respectively.
E and
G: minimal background immunostaining
with an irrelevant rabbit polyclonal antibody in vas deferens and
seminiferous tubules of testis, respectively.
H: there is no immunostaining for AQP2
in epididymis. B: CreTag
immunostaining with a mouse monoclonal antibody to HSV epitope tag is
localized to some of principal cells of vas deferens. Arrows, nuclear
Cre immunostaining. F: minimal
background immunostaining with an irrelevant mouse monoclonal antibody
in vas deferens. D: strong background
immunostaining in nontransgenic testis with HSV epitope tag antibody.
CreTag could not be localized in testis for this reason. PC, principal
cells in vas deferens; Sp, mature sperm; GS, germ cells and Sertoli's
cells; L, lumen; CT, connective tissue.
A-H:
×360 magnification.
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AQP2 expression in the rat vas deferens.
To determine whether the unexpected presence of AQP2 in the vas
deferens was restricted to the mouse, we also examined sections of the
rat vas deferens. As shown in Fig. 7, the
apical membrane domain and microvilli of principal cells
of this species were also strongly labeled with anti-AQP2 antibodies.
Such staining was not observed with preimmune or normal rabbit serum.
The presence of AQP2 in rat vas deferens was confirmed by PCR analysis.
A total of 750 bp were obtained, and the sequence was identical to that reported for rat AQP2 (results not shown) (13)

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Fig. 7.
Immunocytochemical localization of AQP2 in rat vas deferens. A cross
section of rat vas deferens was immunostained with anti-AQP2 antibodies
and counterstained with Evan's blue (red staining). Specific
yellow/green AQP2 staining is restricted to apical pole of principal
cells of vas deferens epithelium. Apical stereocilia that project from
these cells into lumen appear to be heavily stained. No basolateral or
extensive intracellular vesicular staining was detectable in these
cells. Arrows, apical AGP2 immunostaining of vas deferens epithelium.
Bar, 30 µm.
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RT-PCR method for detection of CreTag mRNA.
Initial RT-PCR studies of transgene expression were complicated by
contaminating genomic DNA amplification products derived from the
intronless AQP2-CreTag transgene, which were indistinguishable in size
from cDNA amplification products. An alternative RT-PCR method was used
to amplify cDNA without amplifying contaminating genomic DNA. This was
accomplished with a novel antisense PCR primer. This primer included an
oligo(dT) sequence at the 5' end that anneals to the poly(A) tail
of mRNA and a 6-bp anchor sequence at the 3' end that anneals to
the site upstream of the SV40 late region polyadenylation site (11) in
the CreTag reporter cassette. This antisense primer was used
in combination with a sense primer that annealed upstream in the CreTag
coding region. At the optimized annealing temperature, these primers
amplify CreTag cDNA without amplifying the CreTag transgene or genomic
DNA.
To demonstrate that this method works, a CMV expression vector
containing CreTag was transiently transfected into 3T3 cells. RT-PCR
analysis was performed using a standard and a novel antisense primer in
combination with a common sense primer (Fig.
8). The standard antisense primer amplified
product from a cDNA reaction carried out in the absence of RT as well
as from a plasmid template (Fig. 8,
middle). In contrast, the novel
antisense primer only amplified product in the presence of RT and did
not amplify plasmid template (Fig. 8,
top). This validates this RT-PCR
method for detection of authentic CreTag cDNA.

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Fig. 8.
Testing of method for RT-PCR analysis to determine CreTag mRNA. Murine
3T3 cells were transiently transfected with pCMV-CreTag, which has a
cytomegalovirus (CMV) promoter and enhancer driving expression of
CreTag cassette, or pKS, which contains no promoter or expression
cassette. RNA was reverse transcribed. PCR analysis was performed on
cDNA or plasmid DNA using oligonucleotide primer pairs.
Top: PCR analysis using specialized
primers CreTagUP and SV40LateR. Products are only observed in presence
of RT. Middle: PCR analysis using
conventional primers CreTagUP and CreTagDown. Products are observed in
presence and absence of RT and in presence of pCMVCreTag plasmid DNA.
Bottom: PCR analysis using primers
that amplify GAPDH. Molecular mass markers are shown at
left.
|
|
CreTag expression in transgenic mice.
CreTag transgene mRNA expression was compared with endogenous AQP2 mRNA
expression in multiple mouse organs. RT-PCR analysis was used to detect
mRNA expression by the CreTag transgene and AQP2 in
F1 transgenic animals derived from
each of the three founders.
CreTag and AQP2 mRNA expression was determined by RT-PCR of total RNA
from F1 transgenic mouse organs
derived from each of the three transgenic founder lines. CreTag was
expressed in kidney of all three lines of transgenic mice but was not
expressed in nontransgenic mice. Lines
1 and
2 exhibited expression of CreTag mRNA
in kidney, vas deferens, and testis, which paralleled the expression of
the AQP2. The RT-PCR results for line
2 are shown (Fig. 3).
Line
2 showed much stronger CreTag
expression than line 1 in kidney as well as vas deferens
and testis, perhaps reflecting the relative copy number of the
transgene. However, despite a relatively lower transgene copy number,
line
3 showed high-level CreTag expression
in kidney and many other organs. This is likely due to transgene
integration near strong enhancer elements.
The cellular patterns of CreTag and AQP2 expression within kidney were
determined with immunohistochemistry. Single-label peroxidase
immunohistochemistry for CreTag and AQP2 was performed on nontransgenic
and transgenic mouse kidneys from line
2 (Fig. 4). Nuclear CreTag
immunostaining was observed in collecting duct nuclei in the cortex,
outer medulla, and inner medulla of transgenic kidney but not in
nontransgenic kidney. Extranuclear staining was observed in both
transgenic and nontransgenic kidneys when the monoclonal antibody to
the HSV epitope tag or an irrelevant mouse monoclonal primary antibody
was used, suggesting that nonnuclear staining is due to staining
of endogenous mouse immunoglobulin. Nuclear immunostaining for CreTag
was observed in fewer cells than would be expected if CreTag were
expressed in all AQP2-expressing principal cells. This indicated that
expression of the AQP2-CreTag transgene was variegated.
Double-label immunofluorescent staining was performed on transgenic
kidney from line
2 to demonstrate that CreTag was
expressed in the nucleus of AQP2-expressing principal cells within the
collecting duct rather than in intercalated cells (Fig. 5). Nonnuclear
CreTag immunofluorescent staining was present even in nontransgenic
animals, again suggesting that it represented nonspecific background
immunostaining due to endogenous mouse immunoglobulin. However, it was
easy to distinguish this background staining from specific nuclear
staining. Nuclear CreTag immunofluorescent staining always colocalized
with nonnuclear AQP2 immunofluorescent staining. No cells lacking AQP2 immunofluorescent staining, including intercalated cells, exhibited nuclear CreTag immunofluorescent staining. In addition, not all renal
principal cells displaying AQP2 immunofluorescent staining also stained
for CreTag. This again indicates that the transgene expression is
variegated.
Next, single-label peroxidase immunohistochemistry was performed for
CreTag and AQP2 in nontransgenic and transgenic mouse vas deferens,
epididymis, and testis from line
2 (Fig. 6). Nuclear CreTag
immunostaining was observed in principal cells of the vas deferens but
not the epididymis. Immunostaining for CreTag could not be demonstrated
in the testis because the monoclonal antibody to the HSV epitope tag
exhibited nonspecific immunostaining of all nontransgenic germ cell
nuclei (Fig. 6).
Finally, immunoblotting analysis was performed to determine whether
CreTag was expressed in the testis of AQP2-CreTag transgenic mice.
Nuclear proteins from the kidney and testis of male transgenic and
nontransgenic mice were fractionated by SDS-PAGE, transferred to
membranes, and immunoblotted for CreTag using the murine monoclonal antibody to the HSV epitope tag. The relative molecular mass was estimated on the basis of comparison to molecular mass standards. In
kidney and testis of transgenic AQP2-CreTag mice, but not of nontransgenic mice, a polypeptide with relative molecular mass of 43 kDa was detected (Fig. 9). The size of this
polypeptide was in agreement with the mass predicted on the basis of
the primary structure. Other polypeptides of different relative
molecular mass were observed in transgenic and nontransgenic testis,
which were likely the nonspecific polypeptides found in transgenic and nontransgenic seminiferous tubules by immunocytochemistry.

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|
Fig. 9.
Immunoblotting analysis of nuclear proteins from transgenic and
nontransgenic mouse kidney medulla and testis. Nuclear proteins from
testis and kidney medulla were fractionated by SDS-PAGE,
transferred to a membrane, and probed for CreTag by immunoblotting
using a murine monoclonal antibody to HSV epitope tag using enhanced
chemiluminescence. Relative molecular mass of markers is shown at
right. Arrow, CreTag protein. CreTag
is only found in transgenic kidney and testis.
|
|
 |
DISCUSSION |
Before the current study, AQP2 expression had been demonstrated in
principal cells of mouse kidney by immunocytochemistry, but neither the
cDNA sequence nor the pattern of mRNA expression had been determined.
Hence, before commencing mouse transgenic studies, it was first
necessary to confirm that mice express AQP2 and to determine the tissue
pattern of such expression. Partial cDNA sequence analysis confirmed
that AQP2 mRNA was expressed in mice. RT-PCR analysis of
whole-organ RNA demonstrated that AQP2 mRNA was selectively expressed
in kidney and male reproductive organs. Immunocytochemical analysis
revealed that AQP2 protein was only expressed in principal cells of the
collecting ducts within the kidney, principal cells in the vas
deferens, and seminiferous tubules in the testis. The kidney- and renal
principal cell-specific expression of AQP2 was identical to that
reported in the past for the mouse (2) and paralleled rat (13,
35) and human (40). These findings suggest that AQP2 is involved in
water reabsorption in the collecting duct of the mouse, as in other species.
AQP2 expression in testis and vas deferens has not previously been
reported. The functional significance of AQP2 in seminiferous tubules
of the mouse testis remains unclear, as does the reason that studies of
rat testis did not reveal AQP2 (13). Interestingly, AQP7 and AQP8 are
expressed in postmeiotic sperm (25, 26). These aquaporins could play a
role in sperm maturation. The presence of AQP2 in the vas deferens may
reflect a function of this tubule segment in modifying the luminal
fluid content in a hormone-sensitive manner. The potential
physiological regulation of AQP2 in the vas deferens is the subject of
ongoing studies in our laboratory. In previous studies, AQP1 was most
abundant in the efferent ducts of the testis, in which much of the
fluid secreted by the seminiferous tubules is reabsorbed. It was not
present in epithelial cells of the epididymis or most of the vas
deferens, but it was present in cells of the terminal ampulla of the
vas deferens, seminal vesicle, and prostate (4). The proposed role of
AQP1 was to modulate the luminal fluid content in the male reproductive
system. Studies are clearly needed to determine the physiological role of aquaporins in the male reproductive system.
The mechanism of kidney- and principal cell-specific expression of the
AQP2 gene was investigated in transgenic mice. Fourteen kilobases of
the human AQP2 gene 5'-flanking region conferred kidney-, vas
deferens-, and testis-specific expression of a CreTag reporter cassette
in transgenic mice. Furthermore, CreTag was localized to
AQP2-expressing principal cells in the kidney and vas deferens.
Finally, CreTag was targeted to the nucleus. These results indicate
that organ- and cell-specific expression of AQP2 results from regulated
AQP2 gene transcription. In addition, the AQP2 gene 5'-flanking
region contains the cis-acting
sequences that are sufficient to confer organ- and cell-specific
expression.
Not all AQP2-expressing cells contained detectable CreTag, indicating a
variegated pattern of transgene activity. Such variegated cellular
expression patterns have been observed with many transgenes (10, 37,
38). Several explanations have been proposed. First, the promoter may
not be expressed efficiently in mice because of species differences
within the AQP2 promoter. Second, the 14-kb AQP2 5'-flanking
region may be missing upstream or downstream regulatory elements. Such
elements may include enhancers (45), chromatin boundary elements,
insulator DNA sequences (9, 15), or locus-controlling regions (10).
Third, bacterial reporter sequences may be inactivated by some as yet
unknown mechanism. Inactivation by methylation of hemizygous loci may
be one such mechanism (34). Further studies are needed to examine these possibilities.
Sequence analysis of the human AQP2 gene proximal promoter reveals
several consensus sites for transcription factors that may be involved
in cell-specific transcription (24, 44). These elements include CREs
and several GATA sites. cAMP and AVP, which acts through cAMP, have
been implicated in the regulation of AQP2 gene expression. For example,
cAMP and AVP increase AQP2 promoter-reporter activity and AQP2 mRNA
levels in cell lines (24, 33). Similarly, AVP treatment increases APQ2
levels in kidneys of Brattleboro rats with congenital diabetes
insipidus (6). Furthermore, mutation or deletion of the CRE sites in
the AQP2 promoter ablates cAMP and AVP-induced promoter activity (24,
33). There is also evidence to suggest a role for GATA sites in
transcriptional regulation of AQP2. GATA-3 is the only GATA
transcription factor that is known to be expressed in the renal
collecting duct (43). The finding that overexpression of GATA-3
increases AQP2 promoter-reporter activity in kidney cell lines suggests
a role for GATA-3 in principal cell-specific expression of AQP2 (43).
Studies in transgenic mice with reporter genes containing 14 kb of the
AQP2 5'-flanking region with GATA or CRE site mutations will be
required to demonstrate their role in mediating principal cell-specific
expression of AQP2.
A potential application for transgenic mice expressing AQP2-CreTag is
in principal cell-specific gene targeting. The AQP2-CreTag-expressing mouse could be mated with a mouse containing loxP sites flanking the
gene of interest. The progeny having both Cre recombinase expressed in
principal cells and a loxP-flanked gene would theoretically contain
deletion of the gene of interest in principal cells (17, 42). These
animals could then be used to establish the principal cell function of
a given gene. In addition, this system avoids lethal mutations that
result from loss of function of the gene of interest in other cell
types. For example, deletion of the endothelin-1 gene by conventional
gene targeting is lethal in the newborn period due to pharyngeal arch
malformation (31). Principal cell-specific gene targeting would allow
one to examine the role of endothelin-1 production in the collecting
duct, a region where this peptide is known to be produced in large
amounts (30). Clearly, before such physiological studies are
successful, it will first be necessary to optimize expression of Cre
recombinase. As alluded to earlier, this requires efforts to reduce the
variegation of Cre expression in principal cells.
Finally, it should be emphasized that variegated Cre expression within
the collecting duct may prove to be useful. In this scenario, a subset
of principals would contain deletions of the gene of interest. One
could compare principal cells with and without expression of the gene
of interest using histological, biochemical, or physiological
techniques that can discriminate between cells.
In summary, we confirm that AQP2 is expressed in principal cells in the
collecting duct of mouse kidney. Unexpectedly, AQP2 is also expressed
in principal cells of the vas deferens and seminiferous tubules of the
testis. The function of AQP2 in the male reproductive system is
unknown. Fourteen kilobases of the human AQP2 5'-flanking region
confers principal cell-specific expression in the collecting duct of
the kidney, principal cell-specific expression in the vas deferens, and
seminiferous tubule-specific expression in the testis of a CreTag
reporter gene in transgenic mice. These results suggest that
cell-specific expression of AQP2 is mediated at the transcriptional
level and that 14 kb of the human AQP2 gene contain cis elements that are sufficient for
expression of AQP2 in principal cells of the kidney and vas deferens
and in seminiferous tubules of the testis.
 |
ACKNOWLEDGEMENTS |
We acknowledge Steve Johnson for excellent technical assistance. We
thank Mark Knepper for kindly providing the rabbit antibody to AQP2. We
thank Peggy Moody and Brad Preston in the University of Utah Transgenic
Mouse Core Facility for helping in the creation of the transgenic mice.
 |
FOOTNOTES |
Portions of this work were funded by the Primary Children's Research
Foundation (R. D. Nelson), National Institutes of Health Grants
K08-DK-02132-06 (R. D. Nelson), RO1-DK-52043 (D. E. Kohan), RO1-HL-256857 (D. E. Kohan), DK-19406 (D. Ausiello), and
DK-38452 (D. Brown), and National Cancer Institute Grant 5-P30-CA42014 (R. D. Nelson and D. E. Kohan).
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. §1734 solely to indicate this fact.
Address for reprint requests: D. E. Kohan, Medicine Care Center (111),
Veteran Affairs Medical Center, 500 Foothill Dr., Salt Lake City, UT
84148.
Received 7 January 1998; accepted in final form 10 April 1998.
 |
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