Isolation of mouse THP gene promoter and
demonstration of its kidney-specific activity in transgenic
mice
Xinhua
Zhu1,
Jin
Cheng1,
Jing
Gao1,
Herbert
Lepor1,
Zhong-Ting
Zhang1,
Joanne
Pak3, and
Xue-Ru
Wu1,2,3
Departments of 1 Urology and 2 Microbiology, Kaplan
Comprehensive Cancer Center, New York University School of
Medicine, New York 10016; and 3 Department of Veterans
Affairs Medical Center, New York, New York 10010
 |
ABSTRACT |
Tamm-Horsfall protein (THP), the most
abundant urinary protein synthesized by the kidney epithelial cells, is
believed to play important and diverse roles in the urinary system,
including renal water balance, immunosuppression, urinary stone
formation, and inhibition of bacterial adhesion. In the present study,
we describe the isolation of a 9.3-kb, 5'-region of the mouse THP gene
and show the highly conserved nature of its proximal 589-bp, 5'-flanking sequence with that in rats, cattle, and humans. We also
demonstrate using the transgenic mouse approach that a 3.0-kb, proximal
5'-flanking sequence is sufficient to drive the kidney-specific expression of a heterologous reporter gene. Within the kidney, transgene expression was confined to the renal tubules that
endogenously expressed the THP protein, which suggests specific
transgene activity in the thick ascending limb of the loop of Henle and
early distal convoluted tubules. Our results establish the kidney- and
nephron-segment-specific expression of the mouse THP gene. The
availability of the mouse THP gene promoter that functions in vivo
should facilitate additional studies of the molecular mechanisms of
kidney-specific gene regulation and should provide new molecular tools
for better understanding renal physiology and disease through
nephron-specific gene targeting.
Tamm-Horsfall protein; expression; thick ascending limb of Henle's
loop; reporter gene
 |
INTRODUCTION |
TAMM-HORSFALL PROTEIN
(THP), also named uromodulin, is an 85- to 95-kDa glycoprotein
that is synthesized by the kidney epithelial cells of all placental
mammals (19, 20). Within the kidney, THP has been
localized by immunohistochemistry and in situ hybridization to the
thick ascending limb of Henle's loop (TALH) and the early distal
convoluted tubules (2, 3, 17, 27, 40). At these locations,
THP is believed to be membrane-anchored via its COOH-terminal glycophosphatidylinositol linkage, but the protein can be released into
the urine by the action of phospholipases or proteases (7, 9, 15,
28, 34). The released THP constitutes the most abundant protein
in normal human urine with a daily excretion rate of 50-200 mg
(19, 20).
Because of its abundance, species conservation, and unique nephron
assocation, THP is believed to play critical roles in urinary physiology. First, there has been tremendous interest surrounding the
role of THP in immunoregulation, because THP binds avidly to
recombinant interleukin (IL)-1, IL-2, tumor necrosis factor (TNF),
complement 1q, and immunoglobulins and inhibits lectin- and IL-induced
T-cell activation (15, 33, 51, 55). Such inhibitory
activity is larely attributed to the oligosaccharid moieties of THP
(8, 37, 43). Because the kidney is the main site of IL
catabolism, it has been suggested (15, 20) that THP might
act as a potent immunosuppressant. Second, THP has been found to be
involved in regulating urinary stone formation, although it is
controversial whether it promotes or inhibits the stone formation
(13). Nevertheless, some in vitro studies indicate that
purified THP is capable of inhibiting the growth of calcium oxalate
crystals (14, 52). Consistent with this, patients who are
prone to forming renal stones have appreciably lower urinary THP levels
than healthy controls (5, 10, 11, 35). Third, via its high
mannose residues, THP can bind to type-1 fimbriated Escherichia
coli, which is the most common pathogen to cause urinary tract
infection (26, 32). We have recently shown that THP at a
physiological concentration can effectively block type-1 fimbriated
E. coli from binding to uroplakins, the latter of which are
putative urothelial receptors (25, 41, 53). These data indicate that THP can serve as a potentially defensive factor in the
urinary tract against uropathogenic E. coli. Fourth, it has
been suggested that the gel-forming capability of THP within the TALH
may contribute to the water impermeability of this nephron segment
(20). Finally, molecular cloning and sequencing of THP has
revealed several domains that are shared by many important molecules.
Thus the NH2-terminal region of THP contains four epidermal growth factor-like domains that are present in epidermal growth factor
precursor, LDL receptor, thrombomodulin, and tissue plasminogen activator (28). THP also contains at the COOH-terminal
region a ZP domain that is found in zona pellucida proteins ZP-2 and ZP-3, betaglycan, and pancreatic protein GP-2 (16, 30).
Although currently unclear, it is likely that the epidermal growth
factor and ZP domains may play a functional role in renal physiology.
Under normal conditions, the kidney synthesizes large amounts of THP.
This implies that the reduced synthesis of this protein may cause or
reflect renal dysfunction. Indeed, urinary THP reduction has been
associated with certain pathological conditions, including acute
tubular necrosis, diabetic nephropathy, hyperprostaglandin E syndrome,
and active lupus nephritis (4, 23, 39, 45, 47). Although
THP has been frequently used as an indicator for renal tubular function
(46, 57), the mechanisms by which THP gene expression are
regulated have not been studied.
In this paper, we describe the isolation and sequencing of the
5'-region of the mouse THP gene and characterization of the gene
structure. We show that, like its human counterpart, mouse THP
expression is highly kidney specific. In addition, we have generated
transgenic mice that harbor a 3.0-kb, 5'-flanking region of the mouse
THP gene and an enhanced green fluorescence reporter gene and have
demonstrated that the 3.0-kb sequence contains all the necessary
elements to direct kidney-specific expression of the reporter gene.
Within the kidney, transgene expression is colocalized with the
endogenous THP, thus establishing that transgene expression is
restricted to the TALH. The availability of a kidney- and
segment-specific gene promoter opens new avenues for studying the
molecular mechanisms of kidney-specific gene regulation, renal physiology, and disease.
 |
METHODS |
Extraction of total RNA and PCR.
For the determination of THP gene expression in mice, total RNA was
extracted from various mouse tissues using a total RNA extraction kit
(Promega). Total RNA (2 µg) was reverse-transcribed and PCR amplified
using one pair of primers corresponding to mouse THP cDNA (sense:
5'-AGGGCTTTACAGGGGATGGTTG-3'; anti-sense: 5'-GATTGCACTCAGGGGGCTCTGT-3') (30). PCR was performed as follows: 94°C for 5 min,
55°C for 30 s, 72°C for 1 min for 35 cycles; and 94°C for 5 min, 55°C for 30 s, 72°C for 8 min for the last cycle.
Amplification with glyceraldehyde-6-phosphate dehydrogenase gene
primers was used as a normalization control.
Isolation of genomic clones containing the mouse
THP gene.
A mouse genomic library constructed with bacterial artificial
chromosome (BAC) as a vector and 129/SVJ mouse genomic DNA as inserts
(average insert size, 50-240 kb; Incyte Genomics) was mass
screened with PCR using oligonucleotide primers designed according to
mouse THP cDNA (sense: 5'-AGGGCTTTACAGGGGATGGTTG-3'; anti-sense:
5'-GATTGCACTCAGGGGGCTCTGT-3'). Positive clones
identified in the initial screen were verified with a second round of
PCR using nested primers (sense: 5'-GCCTCAGGGCCCGGATGGAAAG-3';
antisense: 5'-GCAGCAGTGGTCGCTCCAGTGT-3'). For the identification of the
5'-portion of the THP gene encompassing the 5'-flanking region, the
large genomic clones from PCR screening were subjected to restriction digestion followed by Southern blotting using three different cDNA
probes located at the 5'-end, middle, and 3'-end of the mouse THP cDNA.
Restriction fragments that reacted with the 5'-end probe but not the
middle or 3'-end probes were chosen, as these most likely contained the
5'-coding region as well as the upstream region. These fragments were
subcloned into pBluescript (Stratagene) and fully sequenced, and the
genomic structure was delineated by restriction digestion and
comparison with the existing mouse THP cDNA sequence.
Construction of the THP-enhanced green fluorescent
protein chimeric gene and expression in transgenic mice.
One of the two isolated genomic clones (C2, Fig. 2A) was
used as a template for PCR amplification using a sense primer located at
3.0 kb (5'-GGGCCCCCAAGAGATCCAAGTCTCCT-3') in relation to the first
base of exon 1 and an anti-sense primer ending at the ninth base of
exon 1 (5'-GGGCCCCTGGTCCAGTCACAAGTAAG-3'). The A of the first ATG,
although noncoding in endogenous THP, was mutated to C to avoid
potential translation interference with the initiation codon of the
reporter gene. The ends of each primer were supplemented with an
ApaI restriction sequence to facilitate cloning. After the
3.0-kb PCR product was subcloned into the TA cloning vector (Invitrogen) and its authenticity was verified by DNA sequencing, it
was retrieved by ApaI digestion and cloned into the same
site of the pEGFP vector (Clontech). Restriction digestion and DNA sequencing of the fusion-gene junction were carried out to verify the
correct orientation. The 4.0-kb THP-enhanced green fluorescent protein
(EGFP) chimeric gene was then excised en bloc by
KpnI/AflII digestion, gel-purified, and
microinjected into fertilized eggs of FVB/N inbred mice for transgenic
mouse production according to established protocols (6).
Southern blot analysis of mouse-tail DNA.
Transgene-bearing founder animals and their germ-line transmission to
offspring were determined by Southern blotting analysis. Briefly,
mouse-tail genomic DNA was extracted using proteinase K and a
salt-precipitation method. The purified DNA was then digested with
HindIII, electrophoresed, transferred onto a nylon membrane, and hybridized with a 520-bp, 32P-labeled
BamHI/NcoI fragment of the 5'-upstream sequence
of the mouse THP gene, which would allow the detection of both
endogenous and transgene fragments.
Northern blot analysis.
For the assessment of transgene expression on a messenger RNA level,
Northern blotting was performed using various tissues from a transgenic
line that harbored the THP-EGFP transgene. Total RNA was extracted,
resolved by agarose gel electrophoresis, transferred onto a nylon
membrane, and hybridized with a 32P-labeled, 720-bp
BamHI/NotI restriction fragment of the EGFP gene.
After autoradiography, the probe was stripped by boiling the membrane
in a high-stringency buffer and rehybridized with a mouse
-actin probe.
Fluorescence microscopy.
Freshly dissected mouse tissues were fixed in Zamboni's fixative (2%
paraformaldehyde and 15% picric acid prepared in phosphate-buffered saline) for 2 h, embedded in optimum cutting temperature (OCT) embedding medium, frozen in liquid nitrogen, and sectioned using a
cryostat into 5-µm-thick sections. The sectioned tissues were either
directly examined by fluorescence microscopy for the expression of
green fluorescence protein or were stained with a polyclonal antibody
against THP after a 1:100 dilution (Biodesign International) followed
by a rhodamine-conjugated secondary antibody.
 |
RESULTS |
THP is kidney specific in mice.
Although previous immunohistochemical staining and Northern blotting
could detect THP only in the kidney (3, 28), more sensitive assays had not been performed to verify these observations. We extracted total RNA from multiple mouse tissues and carried out
RT-PCR using oligonucleotide primers specific for mouse THP cDNA
(30). A 440-bp product that matched the predicted length was amplified from the mouse-kidney RNA preparation but not from that
of the urinary bladder, liver, esophagus, spleen, skin, stomach, small
intestine, large intestine, lungs, heart, testes, brain, skeletal
muscle, thymus, forestomach, and seminal vesicles (Fig. 1A). This result firmly
established the kidney specificity of THP gene expression in mice.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Tissue-specific expression of Tamm-Horsfall protein (THP)
gene in the mouse. Total RNA was isolated from strain 129/SVJ
mouse tissues including kidneys (lane 1), bladder
(lane 2), liver (lane 3), esophagus (lane
4), spleen (lane 5), skin (lane 6), stomach
(lane 7), small intestine (lane 8), large
intestine (lane 9), lungs (lane 10), heart
(lane 11), testes (lane 12), brain (lane
13), skeletal muscle (lane 14), thymus (lane
15), forestomach (lane 16), and seminal vesicles
(lane 17). A: RT-PCR was carried out using a pair
of oligonucleotide primers designed according to mouse THP cDNA. Note
that the THP gene is highly expressed in the mouse kidneys (lane
1) but not in any other epithelial or mesenchymal tissues.
B: RT-PCR controls using primers specific for
glyceraldehyde-6-phosphate dehydrogenase gene.
|
|
Isolation and characterization of the 5'-upstream region of the
mouse THP gene.
The remarkable tissue specificity of THP prompted us to isolate its
regulatory sequence for further study of the molecular mechanisms of
its gene expression. A pair of oligonucleotide primers was synthesized
according to the previously published mouse THP cDNA and was used to
mass screen a 129/SVJ-mouse genomic DNA library constructed with BAC
vectors (see METHODS). The primary screening yielded two
positive clones, each of which harbored a 60- to 70-kb insert. The
identity of both clones was verified by a secondary screening using
nested primers. One of two positive clones was chosen for further
characterization. Because of the large insert size, this clone was
digested with multiple restriction enzymes and probed with three
different cDNA fragments located in the 5'-end, middle, and 3'-end of
the mouse THP cDNA (data not shown). A 9.3-kb KpnI fragment
and an 8.0-kb ApaI fragment hybridized strongly with the
5'-end probe but not the middle and 3'-end probes, which suggests that
these fragments contained the 5'-portion of the coding region as well
as a 5'-flanking sequence. These two fragments (C1 and C2; Fig.
2A) were subcloned and
completely sequenced (GenBank accession no. AF420599).

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 2.
Organization and nucleotide sequence of the 5'-region of
the mouse THP gene. A: C1 (9.3 kb) and C2 (8.0 kb) represent
the two overlapping genomic clones isolated from a bacterial artificial
chromosome mouse genomic library. Comparison of genomic DNA sequence
with that of mouse THP cDNA reveals the existence in the 3'-portion of
the genomic clones of exons 1 and 2 (solid bars), introns 1 and 2 (thick lines connecting the exons), and partial exon 3. Upstream of the
coding region is a 7.0-kb, 5'-flanking sequence (distal portion is
truncated with double slashes for presentation). Restriction sites: K,
KpnI; A, ApaI; P, PstI; X,
XbaI; and S, SpeI. B: nucleotide
sequence (GenBank accession no. AF420599) of the proximal 5'-flanking
region ( 2.0 kb) is shown with exon (bold letters) and abbreviated
intron sequences (between the slashes in lower-case letters). +1, Start
of exon 1; arrow, translation start site. Consensus recognition
sequences that are potentially important for transcription-factor
binding are boxed and include a proximal TATA box, a CCAAT box, binding
sites for activator protein-2 (AP-2; 2 sites), heat shock transcription
factor (5 sites), AP-1, GATA-1 (2 sites), hepatocyte nuclear factor-5
(2 sites), keratinocyte enhancer (1 site), and AML-1a (1 site). Mouse
CA(46) (double underline) and Alu-Sx (single
underline) repetitive sequences are also indicated.
|
|
Comparison of the sequence of the genomic clones with that of the
published mouse THP cDNA revealed that the longer genomic clone (C1)
contained the first two exons and the partial exon 3 intervened by the
first two introns, and that the shorter clone (C2) had the less-distal
5'-flanking sequence and terminated within the second intron (Fig.
2A). A BLAST nucleotide search of GenBank using the coding
regions of the isolated mouse THP gene fragment as a query
yielded two highly homologous sequences, both of which represented the
mouse THP cDNA clones. The first (GenBank accession no. BC012973)
showed 100% identity (641/641 nucleotides), and the second (GenBank
accession no. L33406), which showed 97% identity (713/732
nucleotides), was the previously published mouse THP cDNA
(30). Because the majority of sequence discrepancies between our genomic sequence and the second THP cDNA clone resided in
the third position of a codon, they were most likely due to the
polymorphisms between the mouse strains that were previously used for
cDNA library screening and those presently used for genomic DNA screening.
The 5'-flanking region contained a proximal TATA box that was 30 bp
before the first exon (designated as +1) and a canonical CCAAT enhancer
binding site at
209 bp (Fig. 2B). In the more distal
regions resided three stretches of CA(46)
repeat and an Alu-Sx repetitive sequence. A computer-assisted sequence
search of the 5'-flanking region of the mouse THP gene against the
Web-based TRANSFEC database and FindPatterns of SeqWeb 1.2 revealed a
number of consensus-recognition sequences for known transcription
factors. These included two activator protein-2 (AP-2) sites arranged
in tandem neighboring the TATA box; one AP-1 site at
779 bp; two GATA-1 sites at
527 and
870 bp, respectively; two hepatocyte nuclear factor-5 (HNF-5) sites at
419 and
635 bp, respectively; one
keratinocyte-enhancer binding site at
421 bp; one AML-1a site at
952 bp; and five heat shock transcription factor (HSF) binding sites
scattered throughout the proximal region (Fig. 2B). It is
noteworthy that most of these sites were located downstream of the
repetitive sequences and are thus potentially important for regulating
THP gene transcription (see DISCUSSION). Finally, sequence
alignment of the 5'-flanking regions of THP genes from mice, rats,
cattle, and humans (56) revealed a high degree of cross-species conservation (Fig. 3).
Mouse and rat sequences were 90% identical, those from humans
and cattle were 75% identical, and those from mice and humans were
66% identical. In addition, there existed several highly conserved
regions that could be important for kidney-specific and
nephron-segment-specific gene expression (Fig. 3).

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 3.
Alignment of the proximal 5'-flanking sequences of THP
genes from different species. Sequences (589 bp) from mouse, rat
(GenBank accession no. S75965; Ref. 56), cow (GenBank
accession no. S75961; Ref. 56), and human (GenBank
accession no. S75968; Ref. 28) were aligned using PileUp
software (SegWeb 1.2). Consensus sequences for known transcription
factors are boxed. Nucleotides identical in all four species are marked
by asterisks. Note the highly conserved nature of the 5'-upstream
sequences of THP in different species.
|
|
In vivo activity of the 5'-flanking region of the mouse
THP gene.
To test whether the 5'-flanking region of the mouse THP gene can confer
kidney specificity, we linked a 3.0-kb THP 5'-flanking sequence to a
downstream EGFP reporter gene (Fig.
4A). This chimeric construct
was microinjected into one-cell mouse embryos of the inbred FVB/N
strain for transgenic mouse production. Southern blotting of the
live-born animals identified three founder animals, all of which
transmitted the transgenes to their offspring (Fig. 4B). The
sizes of the HindIII-digested fragments of the THP-EGFP transgene in lines 1 and 6 precisely matched the
predicted 4.0-kb (corresponding to head-to-tail orientation of two
transgene copies inserted in tandem) and 5.5-kb (tail-to-tail
orientation) sizes, respectively. The size of the transgene fragment in
line 11 (12.0 kb) did not match predicted sizes, which
suggests that the transgene was inserted as a single copy into the
mouse genome.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Generation of transgenic mice
harboring the THP-enhanced green fluorescent protein (EGFP) chimeric
gene. A: transgene construct. A 3.0-kb PCR product
containing a sequence from 3.0 kb of the 5'-flanking region to +9 bp
of the first exon was inserted 5'-upstream of an EGFP reporter gene,
the latter of which was supplemented with an SV40 poly A tail.
Restriction enzymes include HindIII (H); ApaI
(A); KpnI (K); NotI (N); and AflII
(Af). B: Southern blotting identification of transgenic
mice. Genomic DNA purified from mouse-tail biopsies was digested with
HindIII, resolved by agarose gel, transferred onto nylon
membrane, and hybridized with a 32P-labeled probe located
at the 5'-flanking region of the THP gene ("probe" in
A). Note the detection of a predicted 1.8-kb endogenous DNA
fragment (arrow) in all mice including the nontransgenic control
(lane 1) and transgene fragments of 4.0 kb in line
1 (lane 2; head-to-tail orientation), 5.5 kb in
line 6 (lane 3; tail-to-tail orientation), and
12.0 kb in line 11 (lane 4; most likely a single
transgene copy with unpredictable size). *Band (10 kb) present in all
mice (most likely an incompletely digested endogenous THP gene
fragment).
|
|
To assess the pattern of transgene expression, we used Northern
blotting to examine a variety of tissues from transgenic mice for the
expression of the EGFP gene. EGFP mRNA was detected exclusively in the
kidney and not in any other tissues of the transgenic mice (Fig.
5). In addition, kidneys of the
nontransgenic controls were completely negative for EGFP. The
expression of EGFP was also assessed on a protein level by fluorescence
microscopy. Cross sections of kidneys from all three transgenic lines
but not from nontransgenic control mice exhibited strong fluorescence
in the tubular cells (Fig. 6,
A and B). The fluorescence was concentrated along
the deep cortex and cortex-medulla junction, which is consistent with
the localization of TALH and early distal tubules (2, 3,
40). Within the tubular cells, the fluorescence was uniformly cytoplasmic with the nuclei being entirely negative (Fig. 6). This was
consistent with the fact that EGFP existed as a cytoplasmic protein
when expressed in animal cells (29). Importantly, green fluorescence was not detected in any other tissues examined, including urinary bladder, liver, brain, small intestine, colon, stomach, prostate, testes, spleen, lungs, thyroid gland, heart, and thymus. To
further establish the nephron-segment-specific expression of the
5'-flanking sequence, we stained the kidney sections of transgenic mice
with an anti-THP antibody followed by a secondary antibody conjugated
with rhodamine. The anti-THP staining colocalized precisely with the
green fluorescence and thus established transgene expression only in
those cells that endogenously express the THP protein; specifically,
the TALH and early distal tubules (Fig.
7). The expression of EGFP in the
functionally important TALH did not appear to have any untoward effect
on renal function, because all transgenic animals thrived and bred
normally.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of EGFP reporter gene
in transgenic mouse tissues. Total RNA was isolated from the kidneys of
two nontransgenic littermates (lanes 1 and 3) and
the kidney of a transgenic mouse of line 1 (lane
2) as well as from other tissues of a male and a female transgenic
mouse including forestomach (lane 4), stomach (lane
5), spleen (lane 6), small intestine (lane
7), large intestine (lane 8), forebrain (lane
9), hindbrain (lane 10), lung (lane 11),
thymus (lane 12), liver (lane 13), urinary
bladder (lane 14), heart (lane 15), uterus
(lane 16), seminal vesicle (lane 17), testis
(lane 18), thyroid gland (lane 19), and skin
(lane 20). A sample of total RNA (15 µg) from each tissue
was subjected to Northern blotting using an EGFP probe. Note the
specific detection of EGFP mRNA in the kidney but not in any other
tissues of the transgenic mice. EGFP was also absent in the kidneys of
the nontransgenic controls.
|
|

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 6.
Detection of EGFP in transgenic
mice. Frozen sections of paraformaldehyde-fixed mouse tissues were
directly examined with fluorescence microscopy after a brief nuclear
staining with propidium iodine (red). Renal tubules in deep cortex and
cortex-medulla junction of the two transgenic F1 mice [line
1 (A); and line 6 (B)] but not
those of a nontransgenic control mouse (C), were strongly
positive for green fluorescence. Fluorescence was uniformly cytoplasmic
with the nuclei being completely negative. Other tissues from the
transgenic mice including urinary bladder (D), liver
(E), brain (F), and stomach (G) were
all negative for green fluorescence. All panels were of the same
magnification (×400).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Segment-specific expression of
THP-EGFP transgene. Kidney sections of the transgenic mice were stained
with an anti-THP antibody followed by a rhodamine-conjugated secondary
antibody. Digital images from the same field were taken using a green
(A) or red (B) filter, and the two images were
overlaid (C). Transgene expression (represented by green
fluorescence) colocalized precisely with the endogenous THP expression
(shown by red fluorescence). EGFP, the transgene product, showed
uniform cytoplasmic staining, whereas the endogenous THP showed
preferentially apical membrane staining (arrowheads). All panels were
of the same magnification (×600).
|
|
 |
DISCUSSION |
Identification of a kidney- and nephron-segment-specific promoter.
Via gene cloning and transgenic mouse analysis, the present study
demonstrates that a 3.0-kb, 5'-flanking sequence of the mouse THP gene
is capable of driving a heterologous reporter gene to express in a
kidney-specific manner. Because such kidney specificity occurred
consistently in all three transgenic lineages, it is highly unlikely
that the tissue-specific gene expression was due to chromosomal
insert-site-dependent events of the THP-EGFP transgene; rather, it
reflects the genuine promoter activity of the 5'-flanking sequence.
These in vivo expression data therefore strongly indicate that the
3.0-kb upstream sequence of the mouse THP gene contains all required
cis elements that govern the kidney-specific gene transcription. Moreover, the transgene was expressed exclusively in the
renal tubular cells that endogenously express the THP protein, namely,
the TALH and early distal tubules. This latter finding indicates that
cis elements that control the nephron segment-specific expression must also reside within the 3.0-kb, 5'-flanking sequence. The existence of several stretches of repetitive sequences, which can
potentially serve as "insulators" for position-independent gene
transcription (44, 50), further supports the idea that the
most critical cis elements of the THP gene are located
within the 3-kb upstream region.
Although the specific cis elements responsible for the
above-mentioned kidney- and nephron-segment-restricted expression are yet to be identified, the proximal promoter of the mouse THP gene does
contain several canonical binding sites for known transcription factors. These include the TATA and CAATT boxes, both of which are
frequently present in promoters of tissue-specific genes and are
usually absent in ubiquitously expressed genes. Also of interest is the
presence of the AP-2 binding sites, which are in close proximity to the
TATA box. It is worth noting that the TATA and CAATT boxes and the
first (distal) AP-2 site are also species conserved (see Fig. 3), which
suggests a possible role in conferring tissue specificity. There is
mounting experimental evidence which indicates that these
cis elements are indeed indispensable for keratinocyte-specific gene expression (22). Given that
cells of the TALH are keratinocytes in nature and thus most likely
express the keratinocyte-specific transcription factors, these proximal cis elements may well be involved in renal tubule-specific
transcription. Finally, the THP gene promoter contains, in the more
distal portion (
952 bp), an AML-1a binding element that could
potentially interact with Runt-domain-containing transcription factors.
Initially identified in the hemopoietic cells, the Runt-domain proteins
are capable of regulating a variety of tissue-specific genes by acting
as an organizer to bring together other transcription factors
(49). In addition to these putative tissue-specific
elements, the mouse THP gene promoter harbors several interspersed heat
shock factor binding motifs. It is possible that some of these motifs
may be involved in stress-related modulation of THP gene expression, given the fact that THP can undergo quantitative changes during oxidative stress such as acute renal failure (23, 24, 42). The functional significance of any of these known cis
elements along with those unknown but highly species-conserved
sequences (see Fig. 3) will require further experimental verification.
By further dissection of the promoter region of the THP gene using deletion and mutation approaches, it should be possible to narrow down
the minimal promoter elements that are necessary for THP gene
expression. Such experiments may also unveil novel cis
elements and transcription factors for kidney-specific and
segment-restricted expression. The identification of the THP gene
promoter that functions in vivo has set a stage for studying
kidney-specific gene regulation.
Although it is well known that different segments of the nephronal
tubules perform distinct functions in absorption and secretion, it was
only relatively recently that segment-specific markers began to be
identified and characterized. Representative examples include the
renal-specific oxidoreductase specific for the proximal convoluted
tubules (54), the ClC-K1 chloride channel for the thin
ascending limb of Henle's loop (48), and the aquaporin-2 water channel for the collecting duct (1, 31). Besides the THP gene, the only other known gene that is specifically expressed in
the TALH is the NKCC2 gene (18). It encodes a (diuretic) bumbetanide-sensitive Na+-K+-Cl
cotransporter that is located at the apical membrane of the TALH (18). This multiple membrane-spanning protein mediates
coupled transport of Na+, K+, and
Cl
. Igarashi and co-workers (18) recently
isolated the mouse NKCC2 gene promoter and found that a 280-bp proximal
DNA fragment was sufficient to confer specific reporter gene expression
in a TALH-derived cell line. It is of interest that the NKCC2 gene
promoter also contains the TATA and CAATT boxes and the AP-1 and AP-2
binding sites. It is presently unknown (but will certainly be
interesting to examine) whether these shared binding sites will turn
out to be important for TALH-specific transcription.
Potential applications of the mouse THP gene
promoter.
The availability of the mouse THP gene promoter should enhance our
understanding of the molecular mechanisms underlying kidney-specific gene transcription and facilitate kidney- and nephron-segment-specific gene targeting. Biologically active molecules can be specifically introduced into the TALH using the transgenic mouse approach, and the
effects on renal physiology and pathology can be investigated systematically in an in vivo setting. For example, genes that are not
normally expressed in the TALH can be ectopically targeted to evaluate
their ability to alter the TALH-associated functions. In addition,
genes that are naturally expressed at the TALH such as the
Na+-K+-Cl
cotransporter can be
overexpressed to determine the impact on ion reabsorption. Conversely,
mutated or truncated molecules that exert dominant negative effects can
be expressed in the TALH to examine the pathophysiology as a result of
their deficiency. The THP gene promoter can also be used to drive
oncogenes to study the contribution of TALH cells in the tumorigenesis
of renal cell carcinoma, for which the cellular origin remains elusive
(12). Finally, genes of particular importance but having
wide tissue distribution can be ablated in a TALH-specific fashion.
This can be accomplished by generating a transgenic mouse expressing
the Cre recombinase under the control of the THP gene promoter and then
by breeding such a mouse with another transgenic mouse having a
loxP-flanked target gene (21). Many of these transgenic
approaches can be carried out in conjunction with an inducible gene
expression strategy, so that the analysis can be done at postnatal
stages (36, 38). Together, these studies will undoubtedly
shed new light on renal-specific gene expression and renal functions.
 |
ACKNOWLEDGEMENTS |
This work is supported in part by a Merit Review Award from the
Department of Veterans Affairs Medical Research Service and Grant
5-R01-DK-56903 from the National Institute of Diabetes and Digestive
and Kidney Diseases.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: X.-R.
Wu, Dept. of Urology, New York Univ. School of Medicine, 550 First
Ave., Rm. Skirball 10U, New York, NY 10016 (E-mail:
xue-ru.wu{at}med.nyu.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.
10.1152/ajprenal.00297.2001
Received 19 September 2001; accepted in final form 5 November 2001.
 |
REFERENCES |
1.
Agre, P.
Homer W. Smith award lecture: aquaporin water channels in kidney.
J Am Soc Nephrol
11:
764-777,
2000[Free Full Text].
2.
Bachmann, S,
Koeppen-Hagemann I,
and
Kriz W.
Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry.
Histochemistry
83:
531-538,
1985[ISI][Medline].
3.
Bachmann, S,
Metzger R,
and
Bunnemann B.
Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney.
Histochemistry
94:
517-523,
1990[ISI][Medline].
4.
Bernard, AM,
Ouled AA,
Lauwerys RR,
Lambert A,
and
Vandeleene B.
Pronounced decrease of Tamm-Horsfall proteinuria in diabetics.
Clin Chem
33:
1264,
1987[ISI][Medline].
5.
Bichler, K,
Mittermuller B,
Strohmaier WL,
Feil G,
and
Eipper E.
Excretion of Tamm-Horsfall protein in patients with uric acid stones.
Urol Int
62:
87-92,
1999[ISI][Medline].
6.
Brinster, RL,
Chen HY,
Trumbauer M,
Senear AW,
Warren R,
and
Palmiter RD.
Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs.
Cell
27:
223-231,
1981[ISI][Medline].
7.
Cavallone, D,
Malagolini N,
and
Serafini-Cessi F.
Mechanism of release of urinary Tamm-Horsfall glycoprotein from the kidney GPI-anchored counterpart.
Biochem Biophys Res Commun
280:
110-114,
2001[ISI][Medline].
8.
Easton, RL,
Patankar MS,
Clark GF,
Morris HR,
and
Dell A.
Pregnancy-associated changes in the glycosylation of Tamm-Horsfall glycoprotein. Expression of sialyl Lewis(x) sequences on core 2 type O-glycans derived from uromodulin.
J Biol Chem
275:
21928-21938,
2000[Abstract/Free Full Text].
9.
Fukuoka, S,
Freedman SD,
Yu H,
Sukhatme VP,
and
Scheele GA.
GP-2/THP gene family encodes self-binding glycosylphosphatidylinositol-anchored proteins in apical secretory compartments of pancreas and kidney.
Proc Natl Acad Sci USA
89:
1189-1193,
1992[Abstract].
10.
Ganter, K,
Bongartz D,
and
Hesse A.
Tamm-Horsfall protein excretion and its relation to citrate in urine of stone-forming patients.
Urology
53:
492-495,
1999[ISI][Medline].
11.
Glauser, A,
Hochreiter W,
Jaeger P,
and
Hess B.
Determinants of urinary excretion of Tamm-Horsfall protein in nonselected kidney stone formers and healthy subjects.
Nephrol Dial Transplant
15:
1580-1587,
2000[Abstract/Free Full Text].
12.
Gu, FL,
Cai SL,
Cai BJ,
and
Wu CP.
Cellular origin of renal cell carcinoma: an immunohistological study on monoclonal antibodies.
Scand J Urol Nephrol Suppl
138:
203-206,
1991[Medline].
13.
Hess, B.
Tamm-Horsfall glycoprotein: inhibitor or promoter of calcium oxalate monohydrate crystallization processes?
Urol Res
20:
83-86,
1992[ISI][Medline].
14.
Hess, B,
Nakagawa Y,
and
Coe FL.
Inhibition of calcium oxalate monohydrate crystal aggregation by urine proteins.
Am J Physiol Renal Fluid Electrolyte Physiol
257:
F99-F106,
1989[Abstract/Free Full Text].
15.
Hession, C,
Decker JM,
Sherblom AP,
Kumar S,
Yue CC,
Mattaliano RJ,
Tizard R,
Kawashima E,
Schmeissner U,
and
Heletky S.
Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines.
Science
237:
1479-1484,
1987[ISI][Medline].
16.
Hoops, TC,
and
Rindler MJ.
Isolation of the cDNA encoding glycoprotein-2 (GP-2), the major zymogen granule membrane protein. Homology to uromodulin/Tamm-Horsfall protein.
J Biol Chem
266:
4257-4263,
1991[Abstract/Free Full Text].
17.
Hoyer, JR,
Sisson SP,
and
Vernier RL.
Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney.
Lab Invest
41:
168-173,
1979[ISI][Medline].
18.
Igarashi, P,
Whyte DA,
Li K,
and
Nagami GT.
Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene.
J Biol Chem
271:
9666-9674,
1996[Abstract/Free Full Text].
19.
Kokot, F,
and
Dulawa J.
Tamm-Horsfall protein updated.
Nephron
85:
97-102,
2000[ISI][Medline].
20.
Kumar, S,
and
Muchmore A.
Tamm-Horsfall protein: uromodulin (1950-1990).
Kidney Int
37:
1395-1401,
1990[ISI][Medline].
21.
Le, Y,
and
Sauer B.
Conditional gene knockout using cre recombinase.
Methods Mol Biol
136:
477-485,
2000[Medline].
22.
Magnaldo, T,
Vidal RG,
Ohtsuki M,
Freedberg IM,
and
Blumenberg M.
On the role of AP2 in epithelial-specific gene expression.
Gene Expr
3:
307-315,
1993[Medline].
23.
McLaughlin, PJ,
Aikawa A,
Davies HM,
Ward RG,
Bakran A,
Sells RA,
and
Johnson PM.
Uromodulin levels are decreased in urine during acute tubular necrosis but not during immune rejection after renal transplantation.
Clin Sci (Colch)
84:
243-246,
1993[ISI][Medline].
24.
Morimoto, RI,
Kroeger PE,
and
Cotto JJ.
The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions.
EXS
77:
139-163,
1996[Medline].
25.
Pak, J,
Pu Y,
Zhang ZT,
Hasty DL,
and
Wu XR.
Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors.
J Biol Chem
276:
9924-9930,
2001[Abstract/Free Full Text].
26.
Parkkinen, J,
Virkola R,
and
Korhonen TK.
Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.
Infect Immun
56:
2623-2630,
1988[ISI][Medline].
27.
Peach, RJ,
Day WA,
Ellingsen PJ,
and
McGiven AR.
Ultrastructural localization of Tamm-Horsfall protein in human kidney using immunogold electron microscopy.
Histochem J
20:
156-164,
1988[ISI][Medline].
28.
Pennica, D,
Kohr WJ,
Kuang WJ,
Glaister D,
Aggarwal BB,
Chen EY,
and
Goeddel DV.
Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.
Science
236:
83-88,
1987[ISI][Medline].
29.
Pines, J.
GFP in mammalian cells.
Trends Genet
11:
326-327,
1995[ISI][Medline].
30.
Prasadan, K,
Bates J,
Badgett A,
Dell M,
Sukhatme V,
Yu H,
and
Kumar S.
Nucleotide sequence and peptide motifs of mouse uromodulin (Tamm-Horsfall protein): the most abundant protein in mammalian urine.
Biochim Biophys Acta
1260:
328-332,
1995[ISI][Medline].
31.
Rai, T,
Uchida S,
Marumo F,
and
Sasaki S.
Cloning of rat and mouse aquaporin-2 gene promoters and identification of a negative cis-regulatory element.
Am J Physiol Renal Physiol
273:
F264-F273,
1997[Abstract/Free Full Text].
32.
Reinhart, HH,
Obedeanu N,
and
Sobel JD.
Quantitation of Tamm-Horsfall protein binding to uropathogenic Escherichia coli and lectins.
J Infect Dis
162:
1335-1340,
1990[ISI][Medline].
33.
Rhodes, DC.
Binding of Tamm-Horsfall protein to complement 1q measured by ELISA and resonant mirror biosensor techniques under various ionic-strength conditions.
Immunol Cell Biol
78:
474-482,
2000[ISI][Medline].
34.
Rindler, MJ,
Naik SS,
Li N,
Hoops TC,
and
Peraldi MN.
Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein.
J Biol Chem
265:
20784-20789,
1990[Abstract/Free Full Text].
35.
Romero, MC,
Nocera S,
and
Nesse AB.
Decreased Tamm-Horsfall protein in lithiasic patients.
Clin Biochem
30:
63-67,
1997[ISI][Medline].
36.
Rossant, J,
and
Nagy A.
Genome engineering: the new mouse genetics.
Nat Med
1:
592-594,
1995[ISI][Medline].
37.
Sathyamoorthy, N,
Decker JM,
Sherblom AP,
and
Muchmore A.
Evidence that specific high mannose structures directly regulate multiple cellular activities.
Mol Cell Biochem
102:
139-147,
1991[ISI][Medline].
38.
Sauer, B.
Inducible gene targeting in mice using the Cre/lox system.
Methods
14:
381-392,
1998[ISI][Medline].
39.
Schroter, J,
Timmermans G,
Seyberth HW,
Greven J,
and
Bachmann S.
Marked reduction of Tamm-Horsfall protein synthesis in hyperprostaglandin E-syndrome.
Kidney Int
44:
401-410,
1993[ISI][Medline].
40.
Sikri, KL,
Foster CL,
MacHugh N,
and
Marshall RD.
Localization of Tamm-Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques.
J Anat
132:
597-605,
1981[ISI][Medline].
41.
Sokurenko, EV,
Chesnokova V,
Dykhuizen DE,
Ofek I,
Wu XR,
Krogfelt KA,
Struve C,
Schembri MA,
and
Hasty DL.
Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin.
Proc Natl Acad Sci USA
95:
8922-8926,
1998[Abstract/Free Full Text].
42.
Tacchini, L,
Pogliaghi G,
Radice L,
Anzon E,
and
Bernelli-Zazzera A.
Differential activation of heat-shock and oxidation-specific stress genes in chemically induced oxidative stress.
Biochem J
309:
453-459,
1995[ISI][Medline].
43.
Tandai-Hiruma, M,
Endo T,
and
Kobata A.
Detection of novel carbohydrate binding activity of interleukin-1.
J Biol Chem
274:
4459-4466,
1999[Abstract/Free Full Text].
44.
Thorey, IS,
Cecena G,
Reynolds W,
and
Oshima RG.
Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.
Mol Cell Biol
13:
6742-6751,
1993[Abstract].
45.
Torffvit, O,
and
Agardh CD.
Tubular secretion of Tamm-Horsfall protein is decreased in type 1 (insulin-dependent) diabetic patients with diabetic nephropathy.
Nephron
65:
227-231,
1993[ISI][Medline].
46.
Torffvit, O,
Jorgensen PE,
Kamper AL,
Holstein-Rathlou NH,
Leyssac PP,
Poulsen SS,
and
Strandgaard S.
Urinary excretion of Tamm-Horsfall protein and epidermal growth factor in chronic nephropathy.
Nephron
79:
167-172,
1998[ISI][Medline].
47.
Tsai, CY,
Wu TH,
Yu CL,
Lu JY,
and
Tsai YY.
Increased excretions of
2-microglobulin, IL-6, and IL-8 and decreased excretion of Tamm-Horsfall glycoprotein in urine of patients with active lupus nephritis.
Nephron
85:
207-214,
2000[ISI][Medline].
48.
Uchida, S,
Rai T,
Yatsushige H,
Matsumura Y,
Kawasaki M,
Sasaki S,
and
Marumo F.
Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter.
Am J Physiol Renal Physiol
274:
F602-F610,
1998[Abstract/Free Full Text].
49.
Westendorf, JJ,
and
Hiebert SW.
Mammalian runt-domain proteins and their roles in hematopoiesis, osteogenesis, and leukemia.
J Cell Biochem
32, Suppl 33:
51-58,
1999.
50.
Willoughby, DA,
Vilalta A,
and
Oshima RG.
An Alu element from the K18 gene confers position-independent expression in transgenic mice.
J Biol Chem
275:
759-768,
2000[Abstract/Free Full Text].
51.
Winkelstein, A,
Muchmore AV,
Decker JM,
and
Blaese RM.
Uromodulin: a specific inhibitor of IL-1-initiated human T-cell colony formation.
Immunopharmacology
20:
201-205,
1990[ISI][Medline].
52.
Worcester, EM.
Inhibitors of stone formation.
Semin Nephrol
16:
474-486,
1996[ISI][Medline].
53.
Wu, XR,
Sun TT,
and
Medina JJ.
In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections.
Proc Natl Acad Sci USA
93:
9630-9635,
1996[Abstract/Free Full Text].
54.
Yang, Q,
Dixit B,
Wada J,
Tian Y,
Wallner EI,
Srivastva SK,
and
Kanwar YS.
Identification of a renal-specific oxido-reductase in newborn diabetic mice.
Proc Natl Acad Sci USA
97:
9896-9901,
2000[Abstract/Free Full Text].
55.
Ying, WZ,
and
Sanders PW.
Mapping the binding domain of immunoglobulin light chains for Tamm-Horsfall protein.
Am J Pathol
158:
1859-1866,
2001[Abstract/Free Full Text].
56.
Yu, H,
Papa F,
and
Sukhatme VP.
Bovine and rodent Tamm-Horsfall protein (THP) genes: cloning, structural analysis, and promoter identification.
Gene Expr
4:
63-75,
1994[Medline].
57.
Zimmerhackl, LB.
Evaluation of nephrotoxicity with renal antigens in children: role of Tamm-Horsfall protein.
Eur J Clin Pharmacol
44, Suppl1:
S39-S42,
1993[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 282(4):F608-F617