Vol. 275, Issue 6, F928-F937, December 1998
Kid-1 expression is high in differentiated renal proximal
tubule cells and suppressed in cyst epithelia
Ralph
Witzgall1,
Nicholas
Obermüller2,
Ulrike
Bölitz1,
James P.
Calvet3,
Benjamin D.
Cowley Jr.3,
Cheryl
Walker4,
Wilhelm
Kriz1,
Norbert
Gretz2, and
Joseph V.
Bonventre5
1 Institute of Anatomy and Cell
Biology I, University of Heidelberg, 69120 Heidelberg;
2 Medical Research Center,
Klinikum Mannheim, University of Heidelberg, 68167 Mannheim, Germany;
3 Kansas University Medical
Center, Kansas City, Kansas 66160;
4 University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957; and
5 Renal Unit and Department of
Medicine, Massachusetts General Hospital and Harvard Medical School,
Charlestown, Massachusetts 02129
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ABSTRACT |
The cDNA coding for the transcriptional repressor protein Kid-1
was cloned in a screen for zinc finger proteins, which are regulated
during renal development and after renal ischemia. Kid-1 mRNA
levels increase in the course of postnatal renal development and
decrease after acute renal injury caused by ischemia or
administration of folic acid. We have raised a monoclonal anti-Kid-1
antibody and demonstrate that the Kid-1 protein is strongly expressed
in the proximal tubule of the adult rat kidney. During nephron
development, the Kid-1 protein appears after the S-shaped body stage
concomitantly with the brush-border enzyme alkaline phosphatase. In two
animal models of polycystic kidney disease, the expression of Kid-1 is downregulated. The loss of expression of Kid-1 in cyst wall cells correlates with the loss of alkaline phosphatase histochemical staining. Kid-1 mRNA levels are also reduced in rodent renal cell carcinomas, another condition characterized by epithelial cell dedifferentiation and increased proliferation. We propose that Kid-1
plays an important role during the differentiation of the proximal tubule.
renal development; renal cell carcinoma; polycystic kidney disease; gene regulation; transcriptional repression
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INTRODUCTION |
THE MORPHOLOGICAL PROCESSES leading to the formation of
the fully differentiated kidney have been well described (29), whereas the cellular and molecular events responsible for the ordered pattern
of renal development are poorly understood. Gene targeting experiments
have revealed that transcription factors play critical roles at
different stages of nephrogenesis, and it can therefore be assumed that
a hierarchy of genetic regulation exists. Inactivation of the
WT-1 (21),
Pax-2 (34), and
Emx-2 (24) genes leads to renal
agenesis, indicating that the proteins encoded by these genes act at a
very early stage of renal development. When the BF-2 gene is inactivated, renal tissue
is present but the number of nephrons that develop totals only ~10%
of the number present in normal mouse kidneys (16), suggesting that
BF-2 controls genes necessary at a stage of metanephrogenesis later
than the stages in which WT-1, Pax-2, and Emx-2 are critical. The
protein encoded by the hepatocyte nuclear factor
(HNF)-1 gene appears to play a role
at a very late stage of nephron development, since the inactivation of
the HNF-1 gene leads to defects
specifically in the proximal tubule (27).
We have cloned a rat cDNA encoding a zinc finger protein that we named
Kid-1 [predominantly found in the kidney ("k"), suppressed after
renal ischemia ("i"), and appearing late in renal
development ("d")] (38). The mRNA for Kid-1 is barely detectable
at the time of birth in the rat kidney, but levels increase after birth and reach the highest levels in the adult kidney (38). After ischemic
or toxic injury to the adult kidney, at a time when many kidney cells
dedifferentiate and undergo mitosis, Kid-1 mRNA levels decline to a
degree comparable to that in the newborn kidney (38). Northern blot and
RT-PCR analysis of a variety of rat organs have shown that among all
the organs tested, the Kid-1 mRNA is predominantly expressed in the
kidney (38). This result was subsequently confirmed in the mouse (3),
where in addition comparable Kid-1 mRNA levels could also be detected
in the eye (expression of Kid-1 mRNA in the eye was not analyzed in the
rat). A molecular analysis of the Kid-1 protein led to the finding that
the non-zinc finger region of Kid-1 was able to confer transcriptional
repressor activity (38). More specifically, the transcriptional
repressor activity of Kid-1 is conferred by the
Krüppel-associated box (KRAB)-domain at the
NH2-terminal of the protein, a
widely distributed motif among
Cys2His2-zinc
finger proteins (23, 39).
To learn more about the potential role of Kid-1 in the kidney, we
evaluated the expression of Kid-1 during nephron development, in two
models of polycystic kidney disease and in rat renal cell carcinomas.
Polycystic kidney disease and renal cell carcinomas are two conditions
in the adult kidney that are characterized by an increased rate of
epithelial cell proliferation and a loss of differentiation (4). One of
the polycystic kidney disease models investigated was the C57BL/6J
(cpk/cpk)
mouse, a model with an autosomal recessive pattern of inheritance (12,
13, 28); the other model was the Han:SPRD
(cy/+) rat, a model with an autosomal
dominant pattern of inheritance (9, 30). In addition, to further test
our hypothesis that Kid-1 expression is suppressed in dedifferentiated
epithelia, we examined the expression pattern of Kid-1 in the Eker rat
model of hereditary renal cell carcinoma (11) and in transformed rat
kidney epithelial cell lines (35). Our results indicate that the
expression of the Kid-1 protein is induced at a late stage of
differentiation of the proximal tubule and that it is downregulated in
cyst wall cells of polycystic kidneys and in renal cells tumors. This
downregulation is likely related to the dedifferentiated state of these
cells. Kid-1 may control genes important for the abnormal phenotype in polycystic disease.
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MATERIALS AND METHODS |
Preparation of a monoclonal anti-Kid-1
antibody. A fragment coding for amino acids 72-173
of the rat Kid-1 protein was subcloned into the plasmid pET-21b
(Novagen, Madison, WI). The resulting plasmid, pET-21b/Kid-1, was used
to express a fragment of the rat Kid-1 protein without the highly
conserved KRAB and zinc finger domains in Escherichia
coli. Because the recombinant protein contained a tail
of six histidine residues at its COOH terminal, it could easily be
purified over a Ni2+ column
according to the manufacturer's instructions (Novagen). Approximately
75 µg of the purified protein was injected subcutaneously into BALB/c
mice in 3-wk intervals. After four injections, splenic lymphocytes were
harvested and hybridomas were prepared according to standard protocols
(1). Culture supernatants were assayed on a dot blot with purified
glutathione S-transferase (GST)/Kid-1 fusion protein. Supernatants yielding a positive signal in the dot blot
assay were further tested in a Western blot. To obtain a clonal
population of cells, hybridomas were subcloned by limiting dilution in
a 96-well plate.
Hybridoma cell culture supernatants were combined with saturated
(NH4)2SO4
in a ratio of 55:45 (vol:vol). After an incubation of 2-4 h at
4°C, the antibody suspension was centrifuged 20 min at 4°C and
12,000 g. The antibody pellet was
dissolved in PBS (
of the original cell culture volume) and then dialyzed against PBS (1).
Epitope mapping. Rat Kid-1 cDNA
fragments coding for amino acids 81-195 (region between the KRAB
domain and the zinc finger domain), amino acids 1-195 (Kid-1
without the zinc finger domain), amino acids 53-576 (Kid-1 without
the KRAB-A domain), and amino acids 174-576 (zinc finger domain
only) were subcloned into the plasmid pGEX-KG (15), which resulted in
the expression of fusion proteins with GST in E. coli. Equal amounts of bacterial extracts were run on
SDS-PAGE gels and either stained with Coomassie brilliant blue or
subjected to Western blot analysis.
Transient transfection of COS-7 cells.
The cDNA coding for the full-length Kid-1 protein was subcloned into
the mammalian expression vector pMT3 [pMT2 (18) modified to
encode the nine-amino acid hemagglutinin (HA) epitope tag of the
influenza virus at the NH2
terminal of the insert]; transcription is driven by the adenovirus major late promoter. COS-7 cells were grown in Dulbecco's minimal essential medium supplemented with 10% calf serum. One day
before transfection, cells were plated at a density of 2.5-4 × 105 cells per 100-mm dish.
For transfection, cells were exposed to 20 µg of DNA in 5 ml of
Dulbecco's minimal essential medium-400 µg/ml DEAE-dextran (Sigma,
Deisenhofen, Germany)-0.1 mM chloroquine (Sigma). Three hours after the
addition of DNA, the medium was removed and the cells were shocked for
2 min at room temperature with 10% dimethyl sulfoxide (Sigma) in
1× PBS. After the shock treatment, the cells were washed once
with PBS and new medium was added. Cells were harvested at the
indicated times after transfection.
Preparation of nuclear extracts.
Nuclear extracts were prepared as described by Hoppe-Seyler et al.
(17). Two to three days after transfection, COS-7 cells were washed
twice with PBS and scraped into a microcentrifuge tube. The cells were
centrifuged for 5 min at 1,250 g, and
the pellet was resuspended in lysis buffer (150 mM NaCl; 10 mM Tris, pH
7.9; 1 mM EDTA, pH 8.0; 0.6% Nonidet P-40). After an incubation of 5 min on ice, the cells were centrifuged 5 min at 1,250 g. The supernatant (corresponding to
the cytoplasmic fraction) was saved, and the nuclear pellet was
resuspended in nuclear extract buffer [1.5 mM
MgCl2; 10 mM HEPES, pH 7.9; 0.1 mM
EGTA; 0.1 mM EDTA; 0.5 mM dithiothreitol (DTT); 0.5 mM
phenylmethylsulfonyl fluoride; 25% glycerol; 420 mM NaCl]. The
nuclear suspension was incubated 20 min on ice and then centrifuged 5 min at 14,000 g. The supernatant
(corresponding to the soluble nuclear fraction) was saved, and the
remaining pellet was solubilized by sonication in PBS-6 M urea.
Immunocytochemistry of kidney
sections. Adult Sprague-Dawley rats (70-100 days
old) were anesthetized with pentobarbital sodium (6.5 mg/100 g body
wt). Anesthetized rats were perfused retrogradely through the aorta for
3 min each with PBS-2% paraformaldehyde and subsequently with PBS-18%
sucrose at a pressure of 200-220 mmHg. Newborn Sprague-Dawley rats
were anesthetized with ether and perfused with PBS-2% paraformaldehyde
through the left ventricle at a pressure of 180-200 mmHg, before
the kidneys were removed and immersed for 2 h in PBS-18% sucrose.
Kidneys were snap-frozen in isopentane cooled with liquid nitrogen and
stored at 
80°C until further use. Kidneys were sectioned at
6- to 8-µm thickness, air-dried for 30 min, and blocked for 2 h at
room temperature in PBS-2% BSA-0.1% Triton X-100. The
(NH4)2SO4-precipitated
anti-Kid-1 antibody 5D12 was applied at a dilution of 1:50-1:100
in PBS-2% BSA, after which the section was first incubated for 2 h at
room temperature before being stored overnight at 4°C. The next
morning, sections were washed three times with PBS and incubated 1 h at room temperature with the secondary antibody [Cy3-coupled rat anti-mouse IgG from Dianova (Hamburg, Germany) diluted 1:300]. The sections were washed again three times with PBS and then mounted. When necessary, nuclei were stained after the third wash by incubating the sections for 3 min in Hoechst stain 33258 (Sigma) at a
concentration of 10 µg/ml.
Double-labeling was performed with a sheep anti-human Tamm-Horsfall
protein antibody (diluted 1:200; Biotrend, Köln, Germany), which
was applied simultaneously with the murine monoclonal anti-Kid-1 antibody 5D12. The primary antibodies were detected with
FITC-conjugated anti-sheep Ig antibody (diluted 1:80; Sigma) and
Cy3-conjugated anti-mouse Ig antibody (Dianova). For preabsorption
experiments, the primary antibody was incubated 2 h at room temperature
with 100 ng of GST or a GST/Kid-1N (GST fused to amino
acids 1-195 of the rat Kid-1 protein) fusion protein per 1 µl of
antibody solution before being applied to the tissue section.
Alkaline phosphatase histochemistry.
After incubation with the secondary antibody and the ensuing three
washes, the sections were incubated 15 min at room temperature (or
until an appropriate color development occurred) in 0.3 mM nitro blue
tetrazolium chloride and 0.3 mM 5-bromo-4-chloro-3-indolyl phosphate
(4-toluidine salt) in 0.2 M Tris · HCl buffer, pH
9.5. The reaction was stopped by immersing the sections for 10 min in
distilled water, after which the sections were fixed again in 4%
paraformaldehyde (in PBS) for 10 min, rinsed in water, and mounted.
Animal models of polycystic kidney disease and Eker
rat model of hereditary renal cell carcinoma. The
Han:SPRD (cy/+) rat, a model for
autosomal dominant polycystic kidney disease (PKD) (9, 30); the
C57BL/6J
(cpk/cpk)
mouse, a model for autosomal recessive PKD (12, 13, 28); and the Eker
rat model of hereditary renal cell carcinoma (11, 36) have been
characterized in detail in previous reports. Animals were maintained as
inbred colonies in the University of Kansas Medical Center Animal Care
Facility [Han:SPRD (cy/+) rats
and C57BL/6J
(cpk/cpk)
mice], the Animal Care Facility of the Medical Research Center in
Mannheim [Han:SPRD (cy/+)
rats], or the Chemical Industry Institute of Toxicology in
Research Triangle Park, NC (Eker rats).
Cell culture. Transformed rat kidney
epithelial (TRKE) cell lines were established and characterized as
described previously (19, 35). In brief, immortal rat kidney epithelial
cell lines were created by exposure of primary cultures to the chemical
mutagen N-methyl-N'-nitro-N-nitrosoguanidine.
TRKE-8, in addition to being immortalized, forms adenocarcinomas in
nude mice. Cell lines TRKE-4, TRKE-5, TRKE-7, and TRKE-8 were used in
the experiments described in this report. Cells were harvested either
at logarithmic growth or after having reached confluency.
Preparation of RNA. By use of a
modification of the technique of Chomczynski and Sacchi (6), RNA was
isolated from right kidneys of Han:SPRD rats as previously described
(8, 9). Briefly, right kidneys were homogenized in GTC solution (4 M
guanidine thiocyanate, 25 mM trisodium citrate, 0.1 M
-mercaptoethanol, 0.1% antifoam A, pH 7.0) using a Polytron tissue
homogenizer. GTC homogenates were treated by sequential addition of 2 M
sodium acetate (pH 4), phenol, and chloroform with vortexing after each addition. After centrifugation, the aqueous layer was transferred to a
fresh tube, and RNA was precipitated with isopropanol. RNA was pelleted
by centrifugation, redissolved, chloroform extracted, and ethanol
precipitated. RNA was again pelleted by centrifugation, redissolved,
and quantitated by spectrophotometry. After a final ethanol
precipitation, RNA was stored at 20°C until use.
Generation of 32P-labeled DNA probes.
Human full-length glyceraldehyde-3-phosphate dehydrogenase (a gift of
M. Alexander-Bridges, Massachusetts General Hospital) was random primed
according to standard protocols (1). Unincorporated nucleotides were
removed by running the reaction over a spin column (Bio-Rad, Hercules, CA).
Radiolabeled single-stranded antisense DNA was prepared as described by
Sturzl and Roth (33). PCR buffer (final concentration 50 mM KCl; 10 mM
Tris · HCl, pH 8.4; 2.5 mM
MgCl2); 250 µM dATP, dGTP, and
dTTP; 6.3 µM dCTP; 5 µl
-32P-dCTP (10 µCi/µl, 3,000 Ci/mmol); 250 ng primer; 2 U AmpliTaq polymerase; and 200 ng linearized
template pBlue/Kid-1() (containing nucleotides 1-897 of the
rat Kid-1 cDNA) (38) were combined in a total volume of 10 µl. Fifty
microliters of mineral oil was added to each reaction tube. After 40 cycles of 30 s at 95°C, 1 min at 50°C, 2 min at 72°C, and a
final extension of 10 min at 72°C, unincorporated nucleotides were
removed over a spin column (Bio-Rad).
Northern blot and hybridization. RNA
samples were run on agarose gels and blotted onto Nylon membranes
according to standard protocols (1). Blots were prehybridized in
2× SDE (10× SDE is 1 M NaCl; 500 mM sodium phosphate, pH
7.0; 25 mM EDTA), 5% SDS, 100 µg/ml yeast tRNA, and 100 µg/ml
denatured salmon sperm DNA at 55°C for 5 h (38). Then, 2.5 × 105 cpm of
random-primed or single-stranded DNA probe per milliliter of
hybridization buffer was added, and the hybridization was continued overnight at the same temperature. The following morning, the blots
were washed three times for 15 min each at room temperature with
2× SSC, two times for 10 min each at 65°C with 2× SSC,
and two times for 5 min each at 65°C with 0.1× SSC (this last
wash was carried out only when probe and RNA were from the same species).
RNase protection assay. T3/T7 buffer
(final concentrations: 20 mM Tris · HCl, pH 7.5; 3 mM
MgCl2; 5 mM DTT; 2 mM spermidine); 400 µM ATP, GTP, and UTP; 40 U RNasin; 10 µl of
-32P-CTP (10 µCi/µl); 0.5 µg of linearized template Z5.9zf() (containing nucleotides
552-897 of the rat Kid-1 cDNA) (38); and 10 U of T3 or T7 RNA
polymerase were combined in a total volume of 20 µl. After 60 min at
37°C, 2 µl of 10 mg/ml tRNA and 10 U of DNase I (RNase free) were
added, and the incubation continued for 15 min at 37°C.
Unincorporated nucleotides were removed over a spin column. The RNA
probe was precipitated in the presence of ethanol, and the pellet was
dissolved in 4:1 (vol:vol) formamide:hybridization mix (final
concentration 40 mM PIPES, pH 6.4; 400 mM NaCl; 1 mM EDTA) to achieve a
probe concentration of 1.5 × 104 cpm/µl. Lyophilized sample
RNA was brought up in 30 µl of probe RNA and denatured 5 min at
85°C. Hybridization was carried out overnight at 45°C. The next
morning, 350 µl RNase digestion buffer (10 mM
Tris · HCl, pH 7.5; 300 mM NaCl; 5 mM EDTA; 40 µg/ml RNase A, 2 µg/ml RNase T1) were added and free probe digested
for 45 min at 30°C. RNase was inactivated by incubating for 15 min
at 37°C after addition of 10 µl of 20% SDS and 5 µl of 10 mg/ml proteinase K. Finally, the reaction was phenolized and the
supernatant was ethanol precipitated and analyzed on a sequencing gel
(1).
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RESULTS |
Characterization of the monoclonal anti-rat Kid-1
antibody 5D12. To characterize the endogenous Kid-1
protein, we generated a monoclonal antibody by immunizing BALB/c mice
with a recombinant rat Kid-1 peptide (amino acids 72-173), lacking
the highly conserved KRAB and zinc finger domains, to minimize the
possibility of cross-reactivity of an anti-Kid-1 antibody with other
zinc finger proteins. One hybridoma, 5D12, produced an antibody of the
IgG1-
class that was tested on a Western blot with various
bacterially expressed GST/Kid-1 fusion proteins (Fig.
1, A and
C). Whereas proteins containing
amino acids 53-195 were recognized by the 5D12 antibody (Fig.
1B, lanes
4 and 5), a peptide
containing amino acids 81-195 was not (Fig.
1B, lane
3). GST alone or the zinc finger domain of Kid-1 did
not react with the antibody (Fig. 1B,
lanes 2 and 6, respectively). This detection
pattern demonstrates the specificity of the anti-Kid-1 antibody. Since
the peptide used to generate the antibody included only amino acids
72-173, the amino acids recognized by the antibody 5D12 likely include
those at position 72-81 of Kid-1.
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Fig. 1.
Specificity of the anti-Kid-1 antibody 5D12. Fusion proteins between
glutathione S-transferase (GST) and
various portions of Kid-1 (A shows
bacterial extracts with different fusion proteins on a
Coomassie-stained gel; C describes the
various constructs aligned with the domain structure of Kid-1) were
tested on a Western blot for their reactivity with the anti-Kid-1
antibody 5D12. Only 2 of the fusion proteins reacted with antibody 5D12
(B), attesting to the specificity of
the antibody and narrowing down the epitope that is recognized by this
monoclonal antibody to a region close to the Krüppel-associated
box (KRAB)-B domain between amino acids 53 and 81 (molecular weight
markers shown at left). Multiple
bands seen with both constructs on the Western blot probably correspond
to partially degraded Kid-1 fusion proteins. In COS-7 cells transiently
expressing Kid-1, only a single band can be detected in the insoluble
nuclear pellet fraction (D). ni, Not
induced; C, detergent-soluble proteins; N, soluble nuclear proteins; P,
insoluble nuclear proteins; ZF, zinc finger encoding region of the cDNA
(e.g., ZF1-4 encodes first 4 zinc fingers of Kid-1); A, KRAB-A
encoding region; B, KRAB-B encoding region (see Ref. 38).
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The 5D12 antibody was further characterized using COS-7 cells
transiently transfected with an HA epitope-tagged, full-length Kid-1
protein. Two days after transfection, the cells were harvested and
lysed with nonionic detergent, and nuclear extracts were prepared by a
protocol using 420 mM NaCl. Western blot analysis with the 5D12
monoclonal anti-Kid-1 antibody yielded a single band in the pellet
fraction remaining after the extraction with 420 mM NaCl, indicating
that the Kid-1 protein adhered tightly to as-yet-unidentified nuclear
structures (Fig. 1D).
Mock-transfected cells yielded no signal, again attesting to the
specificity of the anti-Kid-1 antibody 5D12.
Expression of the Kid-1 protein in adult and newborn
rat kidneys. In a previous report (38), we described
the temporal expression pattern of the Kid-1 mRNA during renal
development and in the recovery phase after ischemic acute renal
failure. Having demonstrated the specificity of the anti-Kid-1
antibody, we then set out to determine the spatial expression pattern
of the Kid-1 protein in the rat kidney. Staining of kidney sections
from adult rats with the anti-Kid-1 antibody 5D12 resulted in the
obvious nuclear staining of tubular profiles in the cortex and the
outer stripe of the outer medulla. This nuclear staining could be
competed out by preabsorbing the antibody with an excess of a GST/Kid-1 fusion protein, whereas preabsorption with GST alone had no effect (data not shown). To define more precisely which part of the nephron expresses the Kid-1 protein, we performed histochemical staining for
alkaline phosphatase, a marker for proximal tubules, on the same tissue
section. Tubular profiles with alkaline phosphatase activity also
showed a strong expression of the Kid-1 protein, thus
demonstrating that Kid-1 is expressed in the S1, S2, and S3
segments of proximal tubules (Figs. 2 and
3). In addition, some faint nuclear
staining was seen in tubular profiles not showing alkaline
phosphatase activity. Such profiles were identified as thick
ascending limbs by staining with an anti-Tamm-Horsfall
protein antibody, and as collecting ducts (Fig. 3).
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Fig. 2.
Immunohistochemical analysis of the expression of Kid-1 in the cortex
of adult rat kidneys. Staining of cortical rat kidney sections with the
anti-Kid-1 antibody 5D12 resulted in a strong nuclear labeling of many
tubular profiles (A). These profiles
could be identified as proximal tubules by histochemical staining for
alkaline phosphatase (B). G,
glomerulus; pt, proximal tubule. Magnification, ×185.
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Fig. 3.
Immunohistochemical analysis of expression of Kid-1 in the outer stripe
of adult rat kidneys. Staining of the outer stripe of adult rat kidneys
with the anti-Kid-1 antibody 5D12 shows prominent nuclear staining in
the S3 segment of the proximal tubule
(A), as demonstrated by simultaneous
histochemical staining for alkaline phosphatase activity
(B). There was faint nuclear
staining in collecting ducts and thick ascending limbs. The identity of
the thick ascending limbs was confirmed by the simultaneous application
of an anti-Tamm-Horsfall protein antibody
(C). pt, Proximal tubule; tal, thick
ascending limb; cd, collecting duct. Magnification, ×190.
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At time of birth, the rat kidney is not completely developed,
containing a rim of metanephrogenic mesenchyme immediately beneath the
capsule and early metanephrogenic forms such as comma- and S-shaped
bodies deeper toward the medulla (22). In the kidney of the newborn rat
only background staining could be detected with the anti-Kid-1 antibody
below the capsule, whereas there was a more pronounced nuclear staining
toward the medulla, which suggested that the Kid-1 protein was
expressed in the tubule at latter stages of nephron development (Fig.
4A).
Alkaline phosphatase activity has been described to increase
progressively during postnatal renal development and can therefore
serve as a marker for the differentiation state of proximal tubules
(5). The histochemical appearance of alkaline phosphatase activity
(Fig. 4B) and Kid-1 immunoreactivity
(Fig. 4A) correlated well,
indicating that the expression of the Kid-1 protein occurred in the
late stages of differentiation of the proximal tubule. Comparison of
immunohistochemical staining for Kid-1 (Fig.
4A) and hematoxylin and eosin
staining (Fig. 4C) on the same
section demonstrated that other structures, such as the ureter stem,
reacted only weakly and the ureteric bud, the condensed mesenchyme, and
comma- and S-shaped bodies did not react at all with the anti-Kid-1
antibody.
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Fig. 4.
Expression of Kid-1 in the kidney of the newborn rat. Kidney sections
from newborn rats were subjected to immunohistochemistry with the
anti-Kid-1 antibody 5D12 (A) and to
histochemistry for alkaline phosphatase
(B). To clearly demonstrate the
morphology of the various developmental stages, the same section was
subsequently stained with hematoxylin and eosin
(C). Kid-1 is strongly expressed in
those profiles, which are also alkaline phosphatase positive, only
weakly in the stem of the invading ureter (arrowheads), and not at all
in ureteric buds (arrows) and earlier stages of nephron development
such as S-shaped bodies (S). Magnification, ×185.
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Expression of Kid-1 mRNA and protein in polycystic
kidneys from rat and mouse. The Han:SPRD
(cy/+) rat is a model for autosomal dominant polycystic kidney disease. The cysts in the Han:SPRD (cy/+) rat originate predominantly
from the proximal tubule (26). We analyzed the expression of the Kid-1
mRNA and protein in this model of polycystic kidney disease. When
polycystic kidneys from 3-, 8-, and 24-wk-old male rats were studied,
Kid-1 mRNA levels in kidneys from heterozygous
(cy/+) rats were lower than those in
kidneys from age-matched, wild-type (+/+) controls (Fig.
5). In polycystic kidneys from female
Han:SPRD rats, however, no marked difference could be seen between the
Kid-1 mRNA levels of heterozygous (cy/+) animals and those of wild-type
animals at any age (Fig. 5).
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Fig. 5.
Kid-1 mRNA levels are low in polycystic kidney disease in the Han:SPRD
(cy/+) rat. Northern blots with 20 µg of total RNA from Han:SPRD (cy/+)
and (+/+) rat kidneys were hybridized with PCR-generated
single-stranded Kid-1 antisense probe. Random-primed
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA served as control
for loading of RNA. Whereas Kid-1 mRNA levels in male Han:SPRD
(cy/+) rat kidneys were lower than
those in kidneys from wild-type (+/+) animals at 3, 8, and 24 wk of
age, no difference could be detected at any age when kidneys from
female (cy/+) animals were compared
with those from age-matched, wild-type (+/+) animals.
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These observations were extended by analyzing the distribution of the
Kid-1 protein in cyst wall cells of male Han:SPRD
(cy/+) rats using
immunocytochemical staining with the anti-Kid-1 antibody 5D12.
Alkaline phosphatase served as a marker for the differentiation state of cyst wall epithelia. Many cyst-lining cells showed no alkaline
phosphatase activity, as demonstrated previously (26) (Fig.
6A).
Staining with the anti-Kid-1 antibody was decreased in the cyst
epithelial cells, but was lost completely only in a subpopulation of
cyst wall cells (Fig. 6B). Thus in
many cysts the Kid-1 protein was still present when alkaline
phosphatase staining was absent, although in these cyst-lining cells
the Kid-1 expression level was decreased relative to cells in noncystic proximal tubules or cyst wall cells that still expressed alkaline phosphatase.
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Fig. 6.
Expression of the Kid-1 protein in cyst wall cells of a polycystic
kidney from a 38-day old male Han:SPRD
(cy/+) rat. Alkaline phosphatase
expression, as demonstrated by histochemistry, is markedly decreased in
large cysts, but not in smaller cysts and in noncystic proximal tubule
cells (A). The Kid-1 protein,
detected with the monoclonal anti-Kid-1 antibody 5D12 and subsequent
incubation with a Cy3-labeled secondary antibody
(B), is expressed strongly in
nondilated proximal tubules and cysts that still expressed alkaline
phosphatase markedly (arrows), but only weakly in cyst wall cells
lacking alkaline phosphatase activity (arrowheads). The same section
was also stained with the DNA-binding dye Hoechst 33258 to identify
nuclei (C). Magnification,
×185.
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To determine whether our findings were specific to polycystic kidneys
of the Han:SPRD (cy/+) rat, we also
evaluated the expression of Kid-1 mRNA by Northern blot analysis in
polycystic kidneys from C57BL/6J
(cpk/cpk)
mice, a model for autosomal recessive PKD (Fig.
7). Compared with unaffected (+/+)
littermates, the levels of Kid-1 mRNA were markedly decreased, results
similar to our observations in polycystic kidneys from Han:SPRD
(cy/+) rats.
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Fig. 7.
Kid-1 mRNA is low in advanced stages of polycystic kidney disease in
the C57BL/6J
(cpk/cpk)
mouse kidney. A Northern blot with 5 µg of poly(A)-selected RNA from
kidneys of 3-wk-old C57BL/6J
(cpk/cpk)
mice was hybridized with PCR-generated, single-stranded Kid-1 antisense
probe. Random-primed GAPDH cDNA served as control for equal loading of
RNA. Homozygous (cpk/cpk) animals express much
less Kid-1 mRNA in their kidneys than do their normal (+/+) litter
mates.
|
|
Expression of Kid-1 mRNA in rat renal cell carcinomas
and transformed rat renal epithelial cell lines. If
decreased Kid-1 expression is a common feature of the dedifferentiated
proximal tubule cell, then one might expect Kid-1 expression to be
suppressed in renal cell carcinoma and transformed renal epithelial
cell lines, too. The expression of Kid-1 mRNA was examined in three different Eker renal cell carcinomas (Fig.
8). Eker tumors are derived from rats
carrying a mutation in the Tsc-2 tumor
suppressor gene (20, 41), which predisposes them to renal cell
carcinomas (11). In all three samples of renal cell carcinomas
obtained, the mRNA levels of Kid-1 were lower than in the normal adult
kidney tissue taken from the same rat kidney. Kid-1 mRNA levels were also low in several transformed rat kidney epithelial cell lines. Interestingly, the expression level of Kid-1 mRNA in the tumor-forming TRKE-8 cell line was the lowest in the four cell lines examined (Fig.
8). TRKE-8 is the only one of these cell lines found to have
deletions in p15INK4B and
p16INK4, two members of a tumor
suppressor cyclin-dependent kinase-4 family (19). No
differences were found between cells in logarithmic growth and at
confluence in any of the cell lines.
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Fig. 8.
Expression of Kid-1 in rat Eker renal cell carcinomas (RCC) and
transformed epithelial cell lines. Ten micrograms each of total RNA
from 1 sample of normal adult rat kidney (NK) and 3 samples of
different Eker renal cell carcinomas were analyzed in a RNase
protection assay with in vitro-transcribed anti-sense Z5.9zf().
The protected band is shown in each lane. In all 3 cases of renal cell
carcinomas, intensity of the band was considerably lower than in the
normal adult rat kidney. RNA from 4 permanent rat kidney epithelial
cell lines, TRKE-4, TRKE-5, TRKE-7, and TRKE-8, was harvested either at
logarithmic growth (L) or at confluency (C). Five (TRKE-5) or 10 µg
(TRKE-4, -7, -8) of total RNA was hybridized to anti-sense
Z5.9zf() in a RNase protection assay.
|
|
| |
DISCUSSION |
Expression of Kid-1 in the adult and newborn rat
kidney. In the adult rat kidney, the Kid-1 protein is
strongly expressed in all segments of the proximal tubule, a
localization confirmed by simultaneous histochemical analysis of
alkaline phosphatase activity. By the same measure, Kid-1 appears at a
relatively advanced stage of nephron development, that is, shortly
after the S-shaped body stage at a time when alkaline phosphatase
activity is also expressed. Kid-1 is a transcriptional repressor (38),
and it can therefore be hypothesized that Kid-1 shuts off genes that are dispensable or even inhibitory for the establishment of the phenotype of the proximal tubule. Alternatively, similar to the hypothesis set forth for the transcriptional repressor
REST/neuron-restrictive silencer factor, which is expressed
ubiquitously outside the nervous system and shuts off neuron-specific
genes (7, 31), Kid-1 may shut off genes that are expressed in other
nephron segments, such as the thin limb and distal tubule of the
nephron. This expression pattern of Kid-1 is similar to the one
described for the transcription factors HNF-1 (27) and AP-2 (25).
The HNF-1 protein appears to be necessary for the transcription of
certain genes in proximal tubular cells. An inactivation of the
HNF-1 gene results in a renal Fanconi
syndrome caused by a dysfunction of the proximal tubule, because target
genes of HNF-1 are no longer
transcribed at sufficiently high levels (27). Whether Kid-1 is one of
those target genes has yet to be evaluated.
Expression of Kid-1 in kidney disease.
After renal injury, Kid-1 mRNA levels decline rapidly and transiently
(38) at a time when the surviving tubular cells dedifferentiate and
divide (37). Kidney Kid-1 mRNA levels increase with time after birth
and are highest in the adult rat (38). These findings suggest that
transcription of the Kid-1 gene
and/or the stability of the transcript are decreased in cells
that are less differentiated. The expression pattern of the Kid-1
protein during nephron development is consistent with this concept. In
addition, our results with polycystic kidneys, renal cell carcinomas,
and immortal rat kidney epithelial cell lines lend more support to this
hypothesis. Both polycystic kidneys and renal cell carcinomas are
characterized by a lower degree of cellular differentiation, higher
mitotic rates, and lower Kid-1 mRNA levels than the adult normal
kidney. It is interesting that in the polycystic kidneys of
heterozygous female Han:SPRD (cy/+) rats, in which the polycystic phenotype is milder than in male (cy/+) rats (14), the mRNA levels of
Kid-1 are the same as in age-matched, wild-type animals.
Similar to human autosomal dominant PKD, only a subset of nephrons in
the Han:SPRD (cy/+) rat become
cystically dilated (26). Thus Northern blot analysis, in which total
kidney RNA is examined, underestimates the marked decrease of Kid-1
protein expression in cyst-lining cells demonstrated by
immunohistochemical staining. The loss of Kid-1 protein expression
occurred in the same cells in which there was loss of alkaline
phosphatase activity, which is consistent with the appearance of Kid-1
late during nephron development when the epithelial cells are more
differentiated. During nephron development, Kid-1 protein appears
approximately at the same time as alkaline phosphatase activity; in
cyst wall epithelia, Kid-1 disappears after or at the same time as the
loss of alkaline phosphatase. Dedifferentiation in cyst wall cells in
kidneys from the Han:SPRD (cy/+) rat
likely is a graded process and appears first to be associated with a
loss of expression of alkaline phosphatase and decrease in Kid-1
protein, followed by further loss of the Kid-1 protein.
The pattern of Kid-1 mRNA expression in polycystic kidneys is not
specific to the Han:SPRD (cy/+) rat
model of autosomal dominant PKD, because a similar pattern can be seen
in polycystic kidneys from the C57BL/6J
(cpk/cpk)
mouse, a model for autosomal recessive PKD. Kid-1 mRNA levels were much
lower in kidneys from homozygous (cpk/cpk)
mice than in kidneys from control animals of the same age. The affected
gene in Han:SPRD rats is located on rat chromosome 5 (2), whereas in
the
(cpk/cpk)
model the affected gene lies on mouse chromosome 12 (10). Both loci are
different from the locus of the Kid-1
gene [chromosome 10 in the rat (40) and chromosome 11 in the
mouse (32)], so that a mutation in the
Kid-1 gene cannot be the cause of the
disease in either animal model. It is possible that the decrease of
Kid-1 expression contributes to a pattern of epithelial
dedifferentiation common to both models. Because Kid-1 is a
transcriptional repressor, it is likely that a regulated gene is
repressed when Kid-1 is expressed. When Kid-1 expression is suppressed
there may be enhanced production of a protein, which then may
contribute to the cystic phenotype.
In transformed epithelial cell lines from the rat kidney, mRNA levels
of Kid-1 are low. This observation is consistent with the hypothesis
that rapidly dividing and dedifferentiated cells have downregulated the
expression of Kid-1. We could, however, see no difference between
cells in a logarithmic growth state or at confluency. The mechanisms,
however, by which growth arrest and differentiation are brought about
probably share certain features, but also are different in other
aspects. Growing these cell lines to confluence may not have been
sufficient to achieve a level of differentiation associated with
increases in Kid-1 mRNA levels.
In summary, Kid-1 is expressed prominently in the proximal tubule.
Levels of Kid-1 increase during nephron development and are markedly
reduced in two animal models of PKD, in rat renal cell carcinomas, and
in TRKE cell lines. Each of the latter conditions is characterized by a
proliferative phenotype with a low level of differentiation. We propose
that this decreased Kid-1 expression may contribute to the derepression
of genes, which in turn may contribute to the abnormal cyst phenotype
of PKD and perhaps the dedifferentiated and highly proliferative state
of renal tumor cells and transformed cell lines.
| |
ACKNOWLEDGEMENTS |
We acknowledge the skillful introduction to hybridoma technology by
Peter Mundel and the kind gifts of materials from Jack Dixon (pGEX-KG)
and John Kyriakis (pMT3), without which this work would not have been
possible. Michael Kramer kindly established the isotype of the
hybridoma 5D12. The expert photographic work of Ingrid Ertel and
graphics of Rolf Nonnenmacher are gratefully acknowledged. We also
thank Karen Dellovo and Alessandro Alessandrini for help with the figures.
| |
FOOTNOTES |
The experiments described were made possible by financial support from
the Deutsche Forschungsgemeinschaft to R. Witzgall (Wi 1042/2-1
and Wi 1042/2-2) and National Institutes of Health MERIT Award
DK-39773 to J. V. Bonventre.
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: R. Witzgall, University of Heidelberg,
Institute of Anatomy and Cell Biology I, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
Received 13 March 1998; accepted in final form 3 September 1998.
| |
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Abstract
Introduction
