Osmotically relevant membrane signaling complex: association
between HB-EGF,
1-integrin, and CD9 in mTAL
David
Sheikh-Hamad1,
Keith
Youker2,
Luan D.
Truong3,
Soren
Nielsen4, and
Mark L.
Entman2
1 Renal Section, 2 Cardiovascular Sciences Section,
3 Renal Pathology Laboratory, Baylor College of Medicine,
Houston, Texas 77030; and 4 Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus,
Denmark
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ABSTRACT |
The
integral membrane proteins cluster of differentiation-9 (CD9),
1-integrin, and heparin-binding epidermal growth
factor-like (HB-EGF) exist in association in many cell lines and are
linked to intracellular signaling mechanisms. Two of the proteins (CD9 and
1-integrin) are induced by hypertonicity, suggesting
that their related signaling processes may be relevant to osmotic
stress. The validity of this hypothesis rests upon coexpression and
physical association between these molecules in nephron segments that
are normally exposed to high and variable ambient osmolality. In this work, we show that CD9 and
1-integrin are induced in rat
kidney medulla after dehydration. Immunohistochemistry and
immunoprecipitation studies show that CD9, HB-EGF, and
1-integrin are coexpressed and physically associated in
medullary thick ascending limbs (mTAL), nephron segments that are
normally exposed to high and variable extracellular osmolality. Our
findings are consistent with the existence of a cluster of integral
membrane proteins in mTAL that may initiate or modulate osmotically
relevant signaling pathways.
cluster of differentiation-9; heparin-binding epidermal growth
factor;
1-integrin; osmotic stress; thick ascending
limb; kidney
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INTRODUCTION |
MANY ORGANISMS, FROM
BACTERIA to mammals, adapt to sustained hyperosmotic stress by
the preferential accumulation of protective organic solutes
(36). These compounds do not perturb cellular macromolecules and are considered important for the survival of cells
in hypertonic environment (36). The accumulation of these substances is facilitated by the induction of specific genes, such as
transporters for betaine, inositol, and taurine, as well as the aldose
reductase enzyme, which catalyzes the reduction of
D-glucose to form the organic solute sorbitol
(6, 13, 31, 35).
The induction of these genes is thought to correlate with the
intracellular ionic strength (the sum of Na and K concentrations; see
Ref. 30). However, it remains unclear how changes in ionic strength
regulate gene expression.
We recently reported the cloning of a partial cDNA corresponding to the
membrane protein CD9 and demonstrated its regulation by hypertonicity
in two renal epithelial cell lines of medulla origin, Madin-Darby
canine kidney (MDCK) and the rabbit papillary cells, PAP-HT25
(27). Additional studies in MDCK cells demonstrate that
the adhesion molecule
1-integrin is induced by
hypertonicity and exists in association with cluster of
differentiation-9 (CD9; see Ref. 28). Furthermore, the induction of CD9
and
1-integrin by hypertonicity is attenuated by
supplementation of the culture media with organic osmolytes, suggesting
a physiological role for CD9 and
1-integrin in the
adaptation of kidney cells to osmotic stress (27,
28). Previous work by others demonstrated an association between CD9,
1-integrin, and heparin-binding epidermal
growth factor-like (HB-EGF) in monkey kidney cells (20).
Increased expression of CD9 upregulates the number of functional
cell-surface HB-EGF molecules and their juxtacrine epidermal growth
factor (EGF) receptor stimulatory activity (8,
11). Because CD9, HB-EGF, and
1-integrin
are linked to signal transduction pathways, such as extracellular
regulated kinase (ERK), Jun NH2-terminal kinase (JNK), and
focal adhesion kinase (125FAK; see Refs. 3, 7, 8, 19, 20, 22, 25, 33),
the induction by hypertonicity of CD9 and
1-integrin may
lead to initiation or modulation of CD9-, HB-EGF-, and
1-integrin-related signaling molecules in an
osmolality-dependent manner. The validity of this hypothesis rests upon
coexpression and physical association between these proteins in kidney
structures that are normally exposed to high and variable osmolality.
In these studies, we report that water deprivation, which leads to
increased osmolality in the kidney medulla, upregulates CD9 and
1-integrin, suggesting that the induction of these
molecules by hypertonicity has its in vivo correlate. Furthermore,
immunoprecipitation and immunohistochemistry studies demonstrate
physical association between and coexpression of CD9, HB-EGF, and
1-integrin in cells of the medullary thick ascending
limbs (mTAL). The data are consistent with a role for CD9, HB-EGF, and
1-integrin in initiating or modulating osmotically
relevant signaling pathways in mTAL cells.
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METHODS |
Experimental Animals
Male Sprague-Dawley rats weighing 200 g (Harlan Bioproducts
for Science, Indianapolis, IN) were placed on rat chow and either of
the following: 1) ad libitum tap water or 2) ad
libitum 500 mM sucrose in tap water for water loading. A portion of the
sucrose-fed animals was water deprived, whereas the rest served as
controls. At predetermined times, the animals were killed by lethal
injection of pentobarbital sodium. For urine osmolality determination,
a urine sample was collected by needle aspiration directly from the
bladder after opening the abdominal cavity. Samples of kidney cortex
and medulla were harvested for analysis.
Northern Blot Analyses
Total RNA was isolated using RNazol (Tel-Test, Friendswood, TX;
see Ref. 4). Poly(A)+ RNA was prepared using oligo(dT)
columns (Collaborative Biomedical Products), as previously described
(6). Electrophoresis was performed after loading equal
amounts of Poly(A)+ RNA per lane in a 1% agarose-2.2 M
formaldehyde gel, followed by transfer to Gene Screen membrane (New
England Nuclear; see Ref. 6). Human full-length (2-kb)
-actin cDNA
(Clontech) was labeled with [
-32P]dCTP (Random Primed
DNA Labeling Kit; Boehringer Mannheim, Indianapolis, IN) for use
as probe. Synthetic antisense CD9-specific oligonucleotide (35 NT),
corresponding to base pairs 109-143 of rat CD9 cDNA
(accession no. X76489), and
1-integrin-specific
oligonucleotide (46 nt) corresponding to base pairs
522-567 of rat
1-integrin cDNA (accession no.
U12309) were used as probes (12, 15). The CD9
probe sequence (5'-TCTTG CTCGA AGATG CTCTT GGTCT GAGAG TCGAA-3') is unique to CD9 from different species and is not shared with other known
members of the tetraspan family (TM4SF), whereas the
1-integrin probe sequence (5'-TCCTC ATTTC ATTCA TCAGA
TCCGT CCCAA GACTC TTCAC ATTCT C-3') is in the coding region of
1-integrin and is not shared with other known members of
the integrin family of proteins. Sequence specificity of the probes was
determined using the BLAST search of the National Center for
Biotechnology Information (NCBI) databases. Both oligonucleotide probes
were end-labeled with [
-32P]ATP (5,000 Ci/mmol;
Amersham Life Science, Arlington Heights, IL), as previously described
(16).
-Actin probe was hybridized to the blots
overnight at 42°C in a solution containing 40% formamide, 5× SSC
(1× SSC = 150 mM NaCl and 15 mM trisodium citrate, pH 7.0), 5×
Denhardt's solution [1× Denhardt's = 0.02% (wt/vol)
polyvinylpyrrolidone, 0.2% (wt/vol) BSA, and 0.2% (wt/vol) Ficoll
400], 0.5% SDS, 250 µg/ml salmon sperm DNA, 10 mM Tris (pH 7.5),
and 10% dextran sulfate. The blots were then washed at 65°C as
follows: 1 h in 2× SSC, 0.5% SDS; 1 h in 0.5× SSC, 0.5%
SDS; 1 h in 0.1× SSC, 0.5% SDS. The end-labeled oligonucleotide
probes were hybridized to the blots overnight at 42°C in a solution
as above but containing 100 µg/ml salmon sperm DNA and no formamide.
These blots were then washed at 42°C, as described above for
-actin probe. Autoradiographs were prepared using X-OMAT AR film
(Kodak, Rochester, NY) with an intensifying screen. Bands on Northern
blots were scanned using a UMAX Astra 1200S scanner and were
quantitated using Adobe Photoshop 4 and UTHSCSA Image Tool software.
Band intensities were determined relative to the corresponding
-actin bands.
Antibodies
Because of the inadequacy of commercially available anti-CD9
antibodies for our immunohistochemistry studies, we custom-made polyclonal anti-CD9 antibodies (L355). These were raised in rabbit (Lofstrand, Rockville, MD) against keyhole limpet
hemocyanin-conjugated 15-amino acid synthetic oligopeptide
(CFYKDTYQKLRNKDE; cysteine in the first position was added to
facilitate conjugation of the oligopeptide to resin for affinity
purification of the antibody), which was derived from the putative
major extracellular domain (amino acids 122-135) of rat CD9
protein (accession no. P40241). Sequence analysis of the oligopeptide,
using GCG sequence analysis software (University of Wisconsin Software
Package), predicted high antigenicity and no posttranslational
modification sites. The specificity of the oligopeptide was determined
using the BLASTP+BEAUTY/nr protein search protocol of the National
Center for Biotechnology Information databases. To determine the
specificity of the anti-CD9 antibody, immunoblots carrying L355
immunoprecipitates of kidney medulla protein lysate were reacted with
ALB6 (Immunotech, Westbrook, ME), a known monoclonal anti-CD9 antibody.
The antibody detected a 25-kDa protein, the predicted size for CD9. CD9
was not detected by ALB6 in precipitates of preimmune serum or L355
that was pretreated with the immunizing peptide (Fig.
1A). Polyclonal rabbit
anti-human (3100) and anti-rat (3096) HB-EGF
antibodies (21) were a generous gift from Dr. Michael
Klagsbrun (Surgical Research Department, Children's Hospital, Harvard
Medical School, Boston, MA). Anti-BSC1 bumetanide-sensitive
cotransporter (BSC1 or Na-K-2Cl transporter) antibody was provided by
Dr. Kishore Belamkonda (Division of Nephrology, University of
Cincinnati, Cincinnati, OH; see Ref. 5). K20, a mouse
anti-
1-integrin monoclonal antibody, and ALB6, a mouse anti-CD9 monoclonal antibody, were purchased from Immunotech
(Westbrook, ME). Polyclonal rabbit anti-human
1-integrin
antibody (AB1937) was purchased from Chemicon International (Temecula,
CA). Monoclonal anti-plasma membrane Ca2+-ATPase antibody
(anti-PMCA, no. MA3-914) was purchased from Affinity Bioreagents (Golden, CO).

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Fig. 1.
A: characterization of L355 anti-cluster of
differentiation-9 (CD9) antibodies. Rat outer medulla protein lysates
were immunoprecipitated using L355 anti-CD9 antibodies (lane
3), preimmune serum (lane 1), or L355 that was
pretreated with the immunizing peptide (lane 2).
Precipitates were run on 12% SDS-PAGE, and immunoblots were reacted
with a known anti-CD9 antibody (ALB6). Upper band corresponds to the
glycosylated form of CD9, whereas the lower band corresponds to the
nonglycosylated form. B: association between CD9 and
1-integrin. Rat outer medulla protein lysates were
immunoprecipitated using L355 anti-CD9 antibodies (lane 3),
preimmune serum (lane 1), or L355 that was pretreated with
the immunizing peptide (lane 2). Precipitates were run on
12% SDS-PAGE and immunoblots reacted with
anti- 1-integrin antibodies (AB1937). Ab, antibody.
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Preparation of Kidney Medulla Protein Lysate
Male Sprague-Dawley rats weighing 200 g were killed by
lethal injection of pentobarbital sodium, and samples of kidney cortex and inner and outer medulla were obtained. For preparation of total
cell protein lysate, tissue samples were homogenized for 60 s in
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)
lysis buffer [10 mM HEPES, pH 7.0, 150 mM NaCl, 10 mM CHAPS, 1 µg/ml
leupeptin, and 0.4 mM phenylmethylsulfonyl fluoride (PMSF)] using a
Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and were
left at 4°C for 30 min. Lysates were cleared from insoluble material
by 10 min centrifugation at 3,000 g and 4°C, and the
supernatants were collected. For preparation of membrane protein
lysate, total cell lysates prepared as above were centrifuged at 16,000 g for 20 min. The pellets were then suspended in Triton lysis buffer (50 mM Tris, pH 7.2, 10 mM EDTA, 150 mM NaCl, 1% Triton
X-100, 0.5 mM PMSF, and 1 µg/ml leupeptin). Protein was quantitated
using the bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL)
and was stored at
80°C until used.
In Situ Hybridization
Riboprobe generation and selection.
T7 and SP6 RNA polymerases were used (2 U/µl) on linearized template
(1 µg/20 µl) to generate FITC-conjugated sense and antisense riboprobes, using an Amersham kit as per the manufacturer's
instructions. After ethanol precipitation and washes (two times in 75%
ethanol), the labeled RNA was suspended in 50 µl diethyl
pyrocarbonate-treated water. Probe concentration was estimated by
comparing serial dilutions of the probe with labeled RNA of known
concentration. Probe specificity was verified by Northern blot analysis
following the manufacturer's protocol, as previously described
(37). Calorimetric detection of FITC, using alkaline
phosphatase-conjugated secondary antibody, revealed signal with the
antisense probe but not with the sense probe.
In situ hybridization.
In situ hybridization was performed on 2% paraformaldehyde-embedded
sections, as previously described (37). After sectioning and deparaffinization, the tissue was incubated in 2× SSC buffer for 5 min and again for 1 h. After 1 h prehybridization at room temperature in 100 µl of hybridization solution (50% formamide, 4×
SSC, 1× Denhardt's reagent, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml
yeast tRNA, and 10% dextran sulfate), the probe was added (500 ng/ml),
and the tissue was hybridized overnight at 42°C followed by serial
washes in 2× SSC (1 h at room temperature), 1× SSC (1 h at room
temperature), 0.5× SSC (0.5 h at 37°C), and 0.5× SSC (0.5 h at room
temperature). Detection was carried out using anti-FITC antibody and
nitro blue tetrazolium staining for alkaline phosphatase reaction, as
previously described (37). No signal was detected using
"sense" riboprobe (data not shown).
Immunohistochemistry
Male Sprague-Dawley rats weighing 200 g were
killed by lethal injection of pentobarbital sodium. Coronal sections of
the kidney (2-3 mm thick) were fixed in 2% paraformaldehyde.
Tissue was embedded in paraffin, sectioned, and subjected to dual
immunostaining using FITC-tagged anti-
1-integrin
plus indocarbocyanine (Cy3)-tagged anti-CD9 (L355) or FITC-tagged
anti-
1-integrin plus Cy3-tagged anti-HB-EGF
(3096) antibodies. All antibodies were diluted 1:300 in
blocking solution (Vector Laboratories, Burlingame, CA). For BSC1 and
anti-CD9 antibody (L355) dual staining, a peroxidase-based detection
system was used (Vector Laboratories). First detection (with BSC1)
employed 3-amino-9-ethyl carbazol as substrate, followed by ethanol
washes (to erase the first staining), sequential immunolabeling with
anti-CD9 antibody, and detection using diaminobenzidine as substrate.
Preimmune serum or pretreatment of anti-CD9 (L355) with the immunizing
peptide served as controls for immunostaining with L355, whereas
omission of the primary antibodies served as the negative controls for
HB-EGF,
1-integrin, and BSC1 antibodies.
SDS-PAGE
This method is based on Laemmli (14), with slight
modifications based on Rubinstein et al. (24) and Seehafer
and Shaw (26). Briefly, equal amounts of protein were run
on 12% nonreducing SDS-PAGE. Proteins were transferred overnight at
4°C, 40 volts, on Hybond-enhanced chemiluminescence (ECL) membrane
(Amersham) in Laemmli buffer (25 mM Tris and 52 mM glycine, pH 8.3)
containing 20% methanol. Blots were blocked for 1 h with 5%
dried milk in 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20 (TBST), washed quickly in TBST, and incubated overnight at 4°C with
primary antibody (1:20 dilution for ALB6; 1:50 dilution for K20; and
1:500 dilution for anti-HB-EGF and anti-
1-integrin) in
TBST containing 1% BSA. After a 30-min wash in TBST, blots were
incubated with 1:1,000 dilution of peroxidase-conjugated secondary
antibody in TBST containing 1% BSA. Protein bands were visualized
using the ECL-Plus detection system (Amersham Life Sciences, Little
Chalfont, UK) as per the manufacturer's instructions. Blots designated
for reaction with anti-PMCA were transferred in Laemmli buffer lacking
methanol. Primary antibody dilution was 1:500, and all incubations and
washes were as above, except for substitution of PBS (150 mM NaCl and 50 NaPO4, pH 7.5) for TBST.
Immunoprecipitation and Immunoblotting
Protein lysate (100 µg) was incubated with 5 µg of one of
the following antibodies: anti-CD9 (L355), anti-HB-EGF
(3100), or anti-
1-integrin. The samples
were rocked for 2 h at 4°C in the presence of 30 µl of protein
A and G-agarose (Santa Cruz) followed by 6 min centrifugation at 4°C
and 6,000 g. The immunoprecipitates were then washed three
times in Triton lysis buffer (see above), and the recovered material
was analyzed on 12% SDS-PAGE under nonreducing conditions. Blots were
reacted with one of the following antibodies: anti-CD9 (ALB6),
anti-
1-integrin (AB1937), or anti-HB-EGF (3100). For control experiments, total cell lysates of
outer medulla prepared as above were immunoprecipitated with either
anti-
1-integrin or anti-PMCA. Precipitates were resolved
on 12% SDS-PAGE, and immunoblots were reacted with anti-PMCA antibodies.
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RESULTS |
Regulation of Rat Medulla CD9 and
1-Integrin by
Water Balance
Because of the urinary concentrating mechanism, cells of the
renal medulla are exposed to hyperosmotic milieu. Cells of the renal
cortex on the other hand are not. The expression of osmotically regulated genes in the kidney is typically higher in the medulla than
in the cortex and correlates with the hydration state (6, 13, 18, 27, 32,
35). Thus changes in the expression of CD9 and
1-integrin with variations in medulla osmolality would suggest a physiological role for these proteins in osmoregulation. Therefore, we examined the abundance of CD9 and
1-integrin in rat kidney under conditions of water
loading or deprivation. Rats were placed on either tap water or tap
water containing 500 mM sucrose for 5 days (for hydration). A portion
of the rats on sucrose-supplemented water was then water deprived for
incremental intervals up to 2 days. Urine samples were collected for
osmolality measurement, and kidney cortex and medulla tissues were
analyzed for CD9 and
1-integrin abundance. Results were
compared with those obtained from water-loaded (sucrose in water) rats
or rats maintained on ad libitum tap water. Water loading for 5 days
decreased urine osmolality from 2,150 ± 100 to 600 ± 50 mosmol/kgH2O (Fig. 2) and was
associated with a two- to threefold decrease in the abundance of
medulla CD9 mRNA and protein (Fig. 3).
Water deprivation for 24 h after water loading increased urine
osmolality from 600 ± 50 to 3,100 ± 125 mosmol/kgH2O (Fig. 4) and was
associated with a two- to threefold increase in medulla CD9 mRNA and
protein (Fig. 5A). Similarly,
1-integrin mRNA decreased twofold after water loading
(data not shown). However, maximal induction (3-fold) of
1-integrin mRNA in the medulla after water deprivation
occurred after 10 h (Fig. 5B), concomitant with
increased urine osmolality from 600 ± 50 to 2,100 ± 150 mosmol/kgH2O (Fig. 4). These data are consistent with our
previously published findings in cultured MDCK cells in which peak
induction of
1-integrin mRNA occurred between 6 and
16 h after osmotic stress, whereas that of CD9 occurred after
16-24 h (27, 28). CD9 and
1-integrin mRNAs were detectable at lower levels in the
cortex and showed no variations with water loading or deprivation (data
not shown). There was no equivalent difference in the expression of
-actin mRNA in the medulla under the various experimental
conditions. In conclusion, the expression of CD9 and
1-integrin in the kidney medulla correlates with
extracellular osmolality and suggests a role for these proteins in the
cellular adaptation to osmotic stress.

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Fig. 2.
Urine osmolality after hydration. Male Sprague-Dawley
rats were given either tap water or sucrose-supplemented water (500 mM)
for 5 days. Urine samples were collected by aspiration directly from
the bladder, and osmolality was measured using a vapor-pressure
osmometer. Data represent the means and SE of 4 independent
determinations.
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Fig. 3.
Decreased kidney medulla CD9 mRNA and protein abundance
with water loading. Male Sprague-Dawley rats were given either tap
water (3 rats in each group, lanes 1-3) or
sucrose-supplemented water for 5 days (3 rats in each group,
lanes 4-6). Kidney medulla samples were analyzed for
CD9 mRNA and protein abundance. For Western blots, 40 µg protein were
loaded per lane; for Northern blots, 3 µg of poly(A)+ RNA
were loaded per lane. Top, CD9 protein; middle,
CD9 mRNA; bottom, -actin mRNA.
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Fig. 4.
Increased urine osmolality by water deprivation after
water loading. Male Sprague-Dawley rats were placed on
sucrose-supplemented water for 5 days. At the end of the 5th day
(time 0 point), rats were water deprived for incremental
periods up to 48 h. Urine samples were collected by aspiration
directly from the bladder, and osmolality was measured using a
vapor-pressure osmometer. Data represent the means and SE of 4 independent determinations.
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Fig. 5.
A: modulation of kidney medulla CD9 mRNA and
protein abundance by water balance. Male Sprague-Dawley rats were
placed on sucrose-supplemented water. At the end of the 5th day [water
(W) loaded], rats were water deprived for incremental time points up
to 48 h. Kidney medulla samples were analyzed for CD9 mRNA and
protein abundance. For Western blots, 40 µg protein were loaded per
lane; for Northern blots, 3 µg of poly(A)+ RNA were
loaded per lane. Top, CD9 protein; middle, CD9
mRNA; bottom, -actin mRNA. Experiments were carried out
in triplicate (3 rats in each group): lanes 1-3, water
loading for 5 days; lanes 4-6, water deprivation for
24 h after water loading; lanes 7-9, water
deprivation for 48 h after water loading. B: rat kidney
medulla 1-integrin mRNA abundance after water
deprivation. Rats were water loaded as described in A,
followed by water deprivation for 10 h. Kidney medulla samples
were analyzed for 1-integrin mRNA abundance.
Poly(A)+ RNA (3 µg) was loaded per lane. Top,
1-integrin mRNA; bottom, -actin mRNA.
Experiments were carried out in triplicate (3 rats in each group):
lanes 1-3, water loading for 5 days; lanes
4-6, water deprivation for 10 h after water loading.
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Coexpression of CD9,
1-Integrin, and HB-EGF in mTAL
Different nephron segments have unique cellular elements that are
identified with specific physiological function(s). Identification of
the cellular elements that express CD9,
1-integrin, and
HB-EGF may suggest a function with which the proteins are associated. Therefore, we subjected rat kidney sections to dual immunostaining using a combination of FITC-tagged anti-
1-integrin plus
Cy3-tagged anti-CD9 or FITC-tagged anti-
1-integrin plus
Cy3-tagged anti-HB-EGF antibodies. Regional colocalization of all three
proteins was found in thick ascending limb (mTAL) cells in the inner
stripe of the outer medulla (Fig. 6).
Figure 7 demonstrates that this staining,
as shown for HB-EGF, occurs exclusively in mTAL and has a predominant
basolateral membrane localization. To confirm the mTAL expression of
HB-EGF, CD9, and
1-integrin, we carried out sequential
"dual" immunostaining with anti-BSC1 (Na-K-2Cl cotransporter, a
marker for TAL and macula densa) followed by anti-CD9 (L355). BSC1 and
CD9 are coexpressed in the same tubular structures (Fig.
8), confirming the localization of
HB-EGF, CD9, and
1-integrin to mTAL. We note with
particular interest, the expression of all three proteins in the
cortex, in few tubular elements that are in immediate proximity to
interlobular arteries and veins (Fig. 9).
This expression stands in stark contrast with the negative labeling in
the majority of tubular elements in the cortex. Representative staining
is shown for CD9 (Fig. 10). However, an
identical pattern is seen for HB-EGF and
1-integrin
(data not shown). Staining with BSC1 antibodies confirmed the identity of these structures as thick ascending limbs (Fig. 8). To rule out the
possibility of nonspecific labeling in these structures secondary to
what is known as the "edge effect," we carried out in situ
hybridization using CD9 cDNA as a probe (27). CD9 mRNA expression in these tubular structures is identical to that obtained using immunolabeling (Fig. 11), thus
ruling out nonspecific edge effects. In addition to colocalization of
all three proteins in mTAL, isolated expression of one or co-expression
of only two of the proteins was observed in other nephron segments (see
Table 1 for details). Last, consistent
with the in vivo quantitative studies for CD9 and
1-integrin described earlier and in agreement with the
in situ hybridization studies previously reported by Homma et al.
(10), the highest level of labeling observed for each of
these proteins was in the medulla (outer). We conclude that HB-EGF,
CD9, and
1-integrin are coexpressed in mTAL, a nephron segment that is normally exposed to high and variable osmolality.

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Fig. 6.
Coexpression of CD9, heparin-binding epidermal growth
factor (HB-EGF), and 1-integrin in outer medullary
tubules. Dual immunostaining with FITC-tagged
anti- 1-integrin (A) plus indocarbocyanine
(Cy3)-tagged anti-CD9 (B) or FITC-tagged
anti- 1-integrin (C) plus Cy3-tagged
anti-HB-EGF (D) antibodies. All three proteins are expressed
in the same tubular structures in the inner stripe of outer medulla.
ISOM, inner stripe of outer medulla; IM, inner medulla.
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Fig. 7.
HB-EGF expression localizes to medullary thick ascending
limbs. Basolateral predominance of HB-EGF staining in medullary thick
ascending limbs. TAL, thick ascending limbs.
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Fig. 8.
CD9 is expressed in bumetanide-sensitive cotransporter
(BSC1)-positive tubules. Dual immunostaining with anti-BSC1
(B, D, and F) and L355 anti-CD9
(A, C, and E). A and
B: image captured from the border between the IM and ISOM
showing identical staining pattern in medullary TAL with both
antibodies. At higher magnification, BSC1 staining in the TAL has
apical predominance (F) while CD9 staining is mostly
basolateral (E). In C and D, the image
was captured from the region of interlobular vessels in the cortex.
Identical staining pattern in TAL (arrow) is seen with both antibodies.
GM, glomerulus. In G, control staining with preimmune serum
(L355) shows no labeling.
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Fig. 9.
Coexpression of CD9, HB-EGF, and
1-integrin in cortical TAL. Dual immunostaining with
FITC-tagged anti- 1-integrin (A) plus
Cy3-tagged anti-HB-EGF antibodies (B) or FITC-tagged
anti- 1-integrin (C) plus Cy3-tagged anti-CD9
(D). V, interlobular vein; arrows, TAL.
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Fig. 10.
Representative staining for CD9 in cortical TAL
surrounding interlobular vessels. A, interlobular artery; arrow, TAL.
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Fig. 11.
In situ hybridization using CD9 cDNA. Two percent
paraformaldehyde-fixed and paraffin-embedded kidney sections were
deparaffinized and hybridized with CD9 antisense riboprobe.
A: positive labeling of tubules surrounding interlobular
vessels. B: positive labeling of tubular structures in the
ISOM.
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Physical Association Between CD9,
1-Integrin, and
HB-EGF
To determine whether HB-EGF, CD9, and
1-integrin are physically associated, we performed
immunoprecipitation studies on rat kidney outer medulla protein
lysates using anti-CD9 antibody (L355). Precipitates were resolved on
SDS-PAGE, and immunoblots were reacted consecutively with a known
anti-CD9 antibody (ALB6) and anti-
1-integrin (AB1937).
CD9 (Fig. 1A) and
1-integrin (Fig.
1B) were detected in anti-CD9 (L355) immunoprecipitates but
not in precipitates of preimmune serum or L355 that was pretreated with
the immunizing peptide, suggesting physical association between CD9 and
1-integrin in vivo. In a separate experiment, outer
medulla protein lysates were immunoprecipitated with
anti-
1-integrin antibody (AB1937) or anti-HB-EGF
(3100). Precipitates were resolved on SDS-PAGE, and
immunoblots were reacted with ALB6 (a monoclonal anti-CD9). CD9 is
detected in immunoprecipitates of both antibodies (Fig. 12), suggesting an in vivo association
between CD9 and
1-integrin as well as CD9 and HB-EGF.
Last, outer medulla protein lysates were immunoprecipitated with
anti-
1-integrin antibody (AB1937) or anti-HB-EGF
(3100). Precipitates were resolved on SDS-PAGE, and
immunoblots were reacted consecutively with anti-HB-EGF
(3100) and anti-
1-integrin antibody
(AB1937). HB-EGF was detected in
1-integrin
precipitates, whereas
1-integrin was detected in HB-EGF
precipitates (Fig. 13), suggesting an
in vivo association between HB-EGF and
1-integrin. As a
control experiment, similarly prepared protein lysates were
immunoprecipitated with an antibody directed against the PMCA, which is
not known to associate with
1-integrin or HB-EGF. HB-EGF
and
1-integrin were not detected in anti-PMCA
immunoprecipitates (Fig. 13). It is concluded that CD9, HB-EGF, and
1-integrin are physically associated in vivo in outer
medulla structures. Based on coexpression of these three proteins in
mTAL as discussed above, it is also concluded that HB-EGF, CD9, and
1-integrin are physically associated in mTAL.

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Fig. 12.
CD9-HB-EGF and CD9- 1-integrin
association. Rat outer medulla lysates were immunoprecipitated using
either anti- 1-integrin (lane 1) or
anti-HB-EGF (lane 2). Precipitates were run on 12% SDS-PAGE
and blots reacted with anti-CD9 (ALB6). Band on top
corresponds to the glycosylated form of CD9, whereas the band on
bottom corresponds to the nonglycosylated form.
|
|

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|
Fig. 13.
HB-EGF and 1-integrin association. Rat
outer medulla lysates were immunoprecipitated using one of the
following: anti- 1-integrin (lane 1),
anti-HB-EGF (lane 2), or an irrelevant Ab, anti-plasma
membrane Ca-ATPase (PMCA, lane 3). Precipitates were run on
SDS-PAGE and blots reacted consecutively with
anti- 1-integrin (AB1937) or anti-HB-EGF
(3100).
|
|
 |
DISCUSSION |
Studies in other in vitro systems have suggested that CD9, HB-EGF,
and
1-integrin exist as a complex at the cell membrane (20). Our findings suggest physical association between
and coexpression of CD9, HB-EGF, and
1-integrin in mTAL,
lending support for their existence in vivo as a functional unit.
In addition, the current data extend our previously published tissue
culture findings of CD9 and
1-integrin induction by hypertonicity in MDCK cells (27, 28) to the
kidney medulla. Like CD9, HB-EGF and
1-integrin are
linked to signal transduction pathways and are physically associated;
the induction by hypertonicity of CD9 and
1-integrin
suggests direct relevance of CD9-, HB-EGF-, and
1-integrin-related signaling cascades to osmotic stress. HB-EGF is expressed as a membrane-bound molecule that can activate EGF
receptor in a juxtacrine manner (8). Increased expression of CD9 upregulates the number of functional cell-surface HB-EGF molecules and their juxtacrine EGF receptor stimulatory activity (8, 11). This in turn leads to activation of
ERK and JNK pathways. In parallel, integrin-mediated cell attachment to
extracellular matrix activates mitogen-activated protein kinase and
pp125FAK (3, 19, 33). Osmotic
stress causes immediate shrinkage of cells and raises intracellular
ionic strength. Such changes are likely to lead to physicochemical
alteration(s) in integrin-matrix or integrin-cytoskeletal
interaction(s). A change in the abundance of CD9 and/or
1-integrin, or altered integrin-matrix-cytoskeletal interaction, may in turn initiate HB-EGF-, CD9-, and/or
integrin-mediated signaling. This is conceivably a mechanism by which
the cell can sense osmotic stress and initiate adaptive responses to
it. These adaptive processes may be important for cell survival or
function in a hypertonic environment. The basolateral expression of
1-integrin, HB-EGF, and CD9 in mTAL, coupled with the
basolateral membrane localization of EGF receptors (2,
9) and the suggestion that MDCK cells respond more
efficiently to basolaterally directed hypertonicity, lend further
credence to this hypothesis (34). Indeed, such a scenario
has been suggested by Rosette and Karin (23) in an in
vitro system where clustering of EGF, tumor necrosis factor (TNF), and
interleukin (IL)-1 receptors is induced in osmotically stressed HeLa
cells and provides an incremental activation of the JNK pathway
(23).
Osmotic stress activates ERK, JNK, 125FAK, and p38 kinase
(1, 17, 29, 33).
Activation of three of these kinases in osmotically stressed cells may
be initiated from the HB-EGF/CD9/
1-integrin protein
complex. Does this complex stand alone in this context, and what is the
significance of its localization to TAL? Considering the findings of
Rosette and Karin, it is conceivable that mammalian cells utilize
multiple cell surface molecules to initiate and/or modulate signaling
pathways in osmotically stressed cells. In this setting, TNF/EGF/IL-1
receptors may work in synergy with HB-EGF/CD9/
1-integrin
to initiate or modulate osmotically relevant signaling from the cell
membrane. Of note, cells of the TAL are metabolically more active than
other cells in the medulla. As a result, these cells may be more
susceptible to toxic and metabolic injury. Hence, they may have adapted
unique protective mechanisms to enhance their survival and/or function
in the hypertonic and potentially injurious environment of the kidney medulla.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants AGO
no. 1 R01 DK-55137-01 (to D. Sheikh-Hamad) and HL-42550 (to K. Youker
and M. L. Entman) and junior faculty seed funds provided by Baylor
College of Medicine for D. Sheikh-Hamad.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
D. Sheikh-Hamad, Renal Section, Dept. of Medicine, Baylor College of Medicine, 6535 Fannin St., MS F505, Houston, TX 77030 (E-mail: sheikh{at}bcm.tmc.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. §1734 solely to indicate this fact.
Received 3 February 1999; accepted in final form 20 January 2000.
 |
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