The rat Pkd2 protein assumes distinct subcellular distributions
in different organs
Nicholas
Obermüller1,
A. Rachel
Gallagher2,
Yiqiang
Cai3,
Nikolaus
Gassler4,
Norbert
Gretz1,
Stefan
Somlo3, and
Ralph
Witzgall2
1 Medical Research Center, Klinikum Mannheim,
University of Heidelberg, 68167 Mannheim;
2 Institute for Anatomy and Cell Biology I,
University of Heidelberg, 69120 Heidelberg, Germany;
3 Section of Nephrology, Yale University School of
Medicine, New Haven, Connecticut 06520-8029; and
4 Department of Pathology, University of Heidelberg,
69120 Heidelberg, Germany
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ABSTRACT |
Mutations in the
PKD2 gene account for ~15% of all cases of
autosomal-dominant polycystic kidney disease. In the present study the
cellular distribution of the Pkd2 protein was investigated by
immunohistochemistry in different rat organs. Although the Pkd2 protein
showed a widespread expression, a strikingly different distribution of
the protein was observed between individual organs. Whereas in renal
distal tubules and in striated ducts of salivary glands a
basal-to-basolateral distribution of Pkd2 was found, a punctate
cytoplasmic location was detected in the adrenal gland, ovary, cornea,
and smooth muscle cells of blood vessels. Interestingly, in the adrenal
gland and ovary, the rat Pkd2 protein was more heavily
N-glycosylated than in the kidney and salivary gland. These
results suggest that Pkd2 accomplishes its functions by interacting
with proteins located in different cellular compartments. The
extrarenal expression pattern of the Pkd2 protein hints at other
candidate sites of disease manifestations in patients carrying PKD2 mutations.
polycystic kidney disease; immunohistochemistry; endoplasmic
reticulum
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INTRODUCTION |
THE MOLECULAR BASIS for the vast majority of cases of
autosomal-dominant polycystic kidney disease (ADPKD) has been
traced back to mutations in two principally responsible genes,
PKD1 (20-22) and PKD2 (14). Linkage studies show
that mutations in PKD1 account for ~85% of all ADPKD cases,
whereas most of the remaining forms are likely due to PKD2
mutations (18). Very rare genetic cases of ADPKD are caused by as yet
unmapped genes (4). So far, most studies have concentrated on
PKD1, whereas only a few studies have dealt with PKD2.
The PKD2 gene encodes a 968-amino acid protein with 6 putative
membrane-spanning domains (14). Interestingly, PKD2 shows homology to
PKD1 and to a subclass of voltage-gated calcium channel subunits
(~25% in both cases). More recent investigations have identified
another gene which is structurally closely related to
PKD2, therefore named PKDL (15) and
PKD2L (28). The functional role of PKD2, PKDL/PKD2L, as well as
that of PKD1 remains to be established, although the understanding of
the molecular pathogenesis has been increased by the generation of mice
carrying a mutant exon adjacent to the wild-type exon in the murine
Pkd2 locus (27). Intragenic recombination between
the mutant and wild-type sequences led to the loss of the wild-type
allele and to the development of polycystic kidneys involving
predominantly distal tubules and collecting ducts. By the use of a
specific antibody it was shown that in these polycystic kidneys,
cyst-lining cells did not express the Pkd2 protein any longer (27).
This observation, together with a recent report on human polycystic
kidneys (12), supports the concept that both Pkd2 alleles must
carry mutations in order to cause cyst formation, thus arguing for the
two-hit/loss-of-function hypothesis.
In the same study, immunocytochemical analysis of the Pkd2 protein in
the mouse kidney revealed a basal-to-basolateral staining pattern in
distal tubular cells and collecting ducts (27). However, no
investigations have been conducted so far in other organs and in other
species, although both the human PKD2 mRNA and its mouse homolog are
expressed in a variety of organs (14, 17, 29). The current
investigation was undertaken to determine the tissue and cellular
expression of the Pkd2 protein in different rat organs. We were able to
show that, in addition to its prominent expression in renal distal
tubules, the Pkd2 protein is also strongly expressed in striated ducts
of the salivary glands, displaying a basal-to-basolateral staining
pattern comparable to that observed in the kidney. A completely
different expression pattern of the Pkd2 protein was noticed in other
organs, e.g., the adrenal cortex, where a punctate cytoplasmic staining
was detected.
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MATERIALS AND METHODS |
Tissue preparation for immunohistochemistry. Adult male and
normal cycling female Sprague-Dawley rats (70-100 days of age) were anaesthetized by an intraperitoneal injection of pentobarbital. Animals were then perfused retrogradely through the distal abdominal aorta with 4% paraformaldehyde in PBS for 4 min at a pressure level of
180-200 mmHg. Thereafter organs were removed, sliced, and immersed
in the same fixative overnight at 4°C. To obtain kidneys from
newborn rats and from adult BALB/c mice (70 to 90 days of age), animals
were treated similarly, except that perfusion-fixation was conducted
via the left ventricle. Tissue blocks from human organs, obtained from
surgical specimens, were immersion-fixed in 4% paraformaldehyde in PBS
for 24 h. All the tissues were subsequently embedded in
paraffin. Four-micrometer-thick paraffin sections were prepared for
histological examination by immunohistochemistry and hematoxylin and
eosin (H & E) staining.
Immunohistochemistry. Deparaffinized sections were equilibrated
in PBS and incubated with blocking solution (2% BSA in PBS) for 30 min
at room temperature. The rabbit polyclonal anti-PKD2 antibody YCC2,
raised against a glutathione S-transferase (GST)-PKD2 fusion
protein containing amino acids 687-962 of human PKD2 (27), was
applied at a dilution of 1:400 in blocking solution for 2 h at room
temperature and subsequently overnight at 4°C. The next morning,
sections were rinsed twice for 10 min each in PBS and incubated with a
Cy3-coupled secondary antibody (Dianova, Hamburg, Germany) for 1 h at
room temperature. After washing in PBS, sections were mounted in
bicarbonate-buffered glycerol pH 8.6. For double-labeling experiments,
the anti-PKD2 antibody was applied together with one of the following
antibodies: mouse monoclonal anti-calbindin D28k (catalog
no. C 8666; Sigma, Deisenhofen, Germany) diluted 1:500, or sheep
polyclonal anti-Tamm-Horsfall glycoprotein (catalog no. 8595-0054;
Biotrend, Cologne, Germany) diluted 1:300. Signal detection was carried
out using FITC- and Cy3-labeled secondary antibodies (Dianova and
Sigma). To facilitate the detailed morphological analysis of Pkd2
expression in various cellular structures, slides were subjected to H & E staining after photographing the immunofluorescence results.
In control experiments, primary antibodies were replaced by PBS;
otherwise, sections were processed as described. No specific staining
was obtained under these conditions. As a further control, the primary
antibody was preabsorbed for 30 min with either GST (2 ng/µl) or the
GST-PKD2 fusion protein (4 ng/µl) used to generate the polyclonal
antibody (these concentrations correspond to approximately equimolar
amounts of both proteins). After the preabsorption step, the antibody
was applied to the sections; otherwise, the staining protocol was
followed as described in the previous paragraph.
Preparation of membrane proteins from rat organs and PNGase
treatment. Membrane proteins were prepared from whole organs
(adrenal glands, kidneys, ovaries, and salivary glands) of adult female Sprague-Dawley rats. Fresh tissues were added to 4 vol of a
homogenization buffer containing 25 mM Tris · HCl, pH
7.4, 20 mM sucrose, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and
1 µg/ml of leupeptin, then homogenized with a motor-driven Teflon
pestle (Dounce homogenizer). The homogenized tissue samples were first
centrifuged for 15 min at 6,000 g and 4°C, then the
supernatant was transferred to a fresh tube and centrifuged for 15 min
at 10,000 g and 4°C. After the second centrifugation step,
the supernatant was centrifuged for 60 min at 100,000 g and
4°C. The resulting pellet represented the membrane fraction and was
resuspended in the homogenization buffer. The protein concentration was
determined according to the method of Bradford (3) using the Protein
Assay kit from Bio-Rad (Munich, Germany) and BSA as a standard.
Fifty micrograms of membrane protein were digested with 500 U of
peptide:N-glycosidase F (PNGase F) (New England
Biolabs, Schwalbach, Germany) under nondenaturing conditions at
37°C overnight.
Western blotting. Membrane protein preparations with and
without PNGase treatment were combined with a reducing SDS sample buffer and incubated for 15 min at 37°C. After centrifugation for
15 min at 14,000 rpm, the supernatant was separated on a 7% SDS-polyacrylamide gel and transferred in 25 mM
Tris · HCl, pH 8.3, 192 mM glycine, and 20% methanol
to a polyvinylidene difluoride (PVDF) membrane (Immobilon from
Millipore, Eschborn, Germany). Prestained molecular weight markers were
used (New England Biolabs). The membrane was blocked overnight in 5%
low-fat dry milk powder, 0.5% Tween 20, and PBS and then exposed to
primary antiserum (YCC2 anti-PKD2 antibody as described above, diluted
1:4,000 in blocking solution) for 2 h at room temperature. After two
washes with 0.5% Tween 20 and PBS and two washes with 5% low-fat dry
milk powder, 0.5% Tween 20, and PBS, the membrane was incubated with
horseradish peroxidase-linked anti-rabbit IgG secondary antibody
(Cappel, Eppelheim, Germany) for 1 h. Antigen-antibody complexes were
visualized using a chemiluminescence detection kit (NEN Life Science,
Cologne, Germany).
Processing of images. Black-and-white photographs were taken
both from immunofluorescent- and H & E-stained specimens.
Photographs and X-ray films from Western blots were scanned with a
Nikon Coolscan LS-2000 using the Silverfast 4.1 software (LaserSoft,
Kiel, Germany) and then processed with Photoshop 4.0 (Adobe Systems,
San Jose, CA).
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RESULTS |
Immunohistochemical localization of the Pkd2 protein in the
kidney. The distribution of the Pkd2 protein in the rat kidney is
shown at low-power magnification in Fig. 1.
Pkd2 is expressed in tubular profiles from the cortex through the inner
stripe of the outer medulla. In the cortex, labeling was found in the
medullary rays as well as in the cortical labyrinth. In addition to
profiles with a continuous labeling, some cortical profiles displayed a discontinuous staining pattern. To more precisely determine the distribution of the Pkd2 protein along the nephron, double
immunofluorescence labeling with specific markers as well as structural
characteristics were used. Pkd2 expression in thick ascending limb
cells of the outer stripe was demonstrated using an antibody against
the Tamm-Horsfall glycoprotein as a marker for this nephron segment
(10). Double immunostaining with both antibodies revealed expression of
Pkd2 in thick ascending limb profiles. Neighboring proximal tubules did
not exhibit any detectable immunostaining for Pkd2 (Fig.
2, A and B). To assess the
expression of Pkd2 in distal convoluted tubules and connecting tubules,
double immunohistochemistry with antibodies against Pkd2 and calbindin
D28k was performed (Fig. 2, C and D).
Calbindin, a Ca2+-binding protein, is strongly expressed in
connecting tubule cells, whereas its expression in distal convoluted
tubule cells is only weak (13). Figure 2, C and D,
shows many tubular profiles intensely stained for Pkd2 and calbindin
D28k, indicating coexpression in connecting tubule cells,
whereas intermingling intercalated cells, known to be unreactive for
calbindin D28k, were also unreactive with the anti-PKD2
antibody. Other profiles were stained homogeneously for Pkd2, but not
for calbindin D28k, therefore probably representing distal
convoluted tubule profiles (Fig. 2, C and D). To
confirm the expression of the Pkd2 protein in distal convoluted tubule profiles and to see whether macula densa cells express this protein, the end portion of the thick ascending limb and its transition to the
distal convoluted tubule were investigated more closely. Cells of the
macula densa, which lie within the end portion of the cortical thick
ascending limb, were consistently unreactive with the anti-PKD2
antibody, whereas surrounding thick ascending limb cells were heavily
stained (Figs. 2C and 3, A and C). Expression of the Pkd2 protein was also demonstrated in profiles of the distal convolute, which were identified by their onset shortly beyond the
macula densa, the increased height of the cells, and their apical
nuclei. Figure 3, C and D,
shows the Pkd2-negative macula densa cells, followed by continuous
labeling of tubular cells reaching far into the distal convoluted
tubule. In all Pkd2-expressing cells, a basal staining pattern was
observed. This is emphasized by a high-power micrograph of
a thick ascending limb profile in the inner stripe (Fig.
4, A and B), where the Pkd2
immunoreactivity appears to be associated with the lateral
interdigitations of a cell. Pkd2 immunoreactivity was also detected as
a thin basal stripe in collecting duct profiles of the cortex and outer
stripe (Fig. 4, C and D), but the signal in collecting
ducts was much more difficult to detect. Although we cannot present
direct evidence for expression of Pkd2 in principal cells of collecting
ducts because of the faint signal, we are inclined to assume that
intercalated cells do not express Pkd2, since they were already
unreactive with the anti-PKD2 antibody in the connecting tubule. In
kidneys of neonatal rats, the Pkd2 protein again was found on the basal side of distal tubules, whereas the subcapsular region, containing the
structures of the nephrogenic zone, was unreactive (data not shown).

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Fig. 1.
Overview of Pkd2 protein expression in the adult rat kidney.
Immunofluorescence labeling of tubules is seen in the cortex and outer
medulla, but not in the inner medulla. Magnification, ×32.
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Fig. 2.
Expression of the Pkd2 protein in distal segments of the nephron in rat
kidney. Paraffin sections of rat kidneys were stained by double
immunofluorescence with the anti-PKD2 antibody YCC2 (A and
C) and an anti-Tamm-Horsfall protein antibody (B) or
anti-calbindin D28k antibody (D). In A,
Pkd2 protein expression corresponds exactly to the Tamm-Horsfall
protein-expressing thick ascending limb profiles (B) of the
outer stripe. Connecting tubule profiles, recognized in D by
the expression of calbindin D28k, also express the Pkd2
protein (C). Coexpression occurs in connecting tubule cells,
but not in intercalated cells (the latter are unreactive with both
antibodies, arrowheads in C and D). Asterisks in
C and D mark putative distal convoluted tubule
profiles, which also express Pkd2 protein but are nearly unreactive
with the anti-calbindin D28k antibody on paraffin sections.
Arrows in C and D delimit the Pkd2-negative macula
densa in an otherwise stained thick ascending limb profile. G,
glomerulus. Magnifications, ×220 (A and B) and
×175 (C and D).
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Fig. 3.
Pkd2 protein expression at the transition from thick ascending limb to
distal convoluted tubule in rat kidney. Sections were first stained by
immunofluorescence with the anti-PKD2 antibody (A and
C) and subsequently with hematoxylin and eosin (H & E)
(B and D). Pkd2 protein is expressed in thick ascending
limb cells but not in macula densa cells, which can easily be
visualized by the close apposition of the nuclei. Border between
immunolabeled thick ascending limb and unreactive macula densa cells is
indicated by arrows. In C and D, a Pkd2-expressing
thick ascending limb is followed by the distal convoluted tubule after
having passed its parent glomerulus. Magnifications, ×400
(A and B) and ×245 (C and D).
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Fig. 4.
High-power magnification of the distribution of the Pkd2 protein in rat
kidney. Immunofluorescence labeling is shown in A and
C. Interference phase-contrast views of the same sections are
seen in B and D. Basal aspect of thick ascending limb
cells (probably corresponding to lateral interdigitations) is strongly
labeled with the anti-PKD2 antibody (A), and also collecting
duct cells from the outer stripe are stained on their basal side with
the anti-PKD2 antibody (arrowheads in C and D). To
clearly show the discrete basal staining in collecting duct cells, a
longer exposure was needed, which also increased the staining in thick
ascending limb profile located above collecting duct (C).
Magnifications, ×675 (A and B) and ×806
(C and D).
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The expression pattern of the Pkd2 protein in the rat kidney was then
compared with the distribution in human and mouse kidneys. A low-power
magnification of the border between the inner stripe and the inner
medulla illustrates that the immunofluorescence pattern seen in both
species is strikingly similar to that obtained in the rat kidney (Fig.
5, A and B). Accordingly,
in the cortex of all three species, distal tubules are heavily labeled
by the anti-PKD2 antibody (data not shown). Moreover, the distinct
basal distribution is virtually identical in all three species (compare Fig. 5, C and D, with Fig. 4A). As in the rat,
a faint basal staining for PKD2 was noted in individual collecting duct
cells of the human kidney (Fig. 5F).

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Fig. 5.
Expression of the Pkd2 protein in mouse (A, C, and
E) and human (B, D, and F) kidneys.
Immunofluorescence labeling of thick ascending limb profiles at border
between outer and inner medulla are seen both in mouse (A) and
human (B) kidneys. High-power magnification from thick
ascending limb profiles of the inner stripe again reveals a basal
staining pattern (C, D, and F) (also compare
corresponding phase-contrast view of C in E). In
F, a collecting duct (CD), located in vicinity of thick
ascending limb profiles, is also weakly stained. Magnifications,
×118 (A), ×47 (B), ×480
(C-E), and ×275 (F).
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In addition to the kidney, the following organs were examined: adrenal
gland, aorta, bladder, brain, epididymis, esophagus, eye, heart, small
and large intestine, liver, lung, lymph nodes, mammary gland, ovary,
pancreas, prostate, salivary glands, seminal vesicle, skeletal muscle,
skin, spinal cord, spleen, stomach, testis, thymus, trachea, and
uterus. Consistent specific staining could only be detected in the
adrenal gland, blood vessels, the eye, ovary, and salivary gland and
will be described in the following paragraphs.
Localization of the Pkd2 protein in salivary glands.
Immunohistochemical analysis revealed that the Pkd2 protein is
abundantly expressed in striated ducts of the rat submandibular and
sublingual glands. As seen in Fig. 6, Pkd2
displays a basal-to-basolateral distribution within striated duct
cells. Heavy staining was detected in intra- as well as in interlobular
striated ducts of both glands; acinar cells and intercalated ducts were
not stained with the anti-PKD2 antibody. Immunohistochemical staining
of paraffin sections from human submandibular glands confirmed the
abundance of the PKD2 protein in cells of smaller and larger striated
ducts as seen in the overview of Fig. 7. In
addition to the basal staining in the kidney, a lateral distribution of
PKD2 was noticed (Fig. 7, B and D). In the human
parotid gland, a similar cellular and subcellular distribution was
found (data not shown).

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Fig. 6.
Expression of the Pkd2 protein in rat salivary glands. Sections were
first stained by immunofluorescence with anti-PKD2 antibody (A,
C, and E) and subsequently with H & E (B,
D, and F). Pkd2 immunoreactivity is seen in a
basal-to-basolateral pattern in intralobular striated ducts of both the
serous submandibular gland (A and B) and the mucous
sublingual gland (C and D). In E and F,
a Pkd2-expressing interlobular striated duct of sublingual gland is
shown. Magnifications, ×285 (A and B) and
×180 (C-F).
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Fig. 7.
Expression of PKD2 protein in human submandibular gland. Sections were
first stained by immunofluorescence with anti-PKD2 antibody (A,
B, and D) and subsequently with H & E (C and
E). Strong immunoreactivity is exclusively found in numerous
striated duct profiles (A-C). In D, basal and
lateral distribution of PKD2 is shown at a high
magnification, and arrows point to lateral cell borders.
Magnifications, ×37 (A), ×150 (B and
C), and ×460 (D and E).
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Localization of the Pkd2 protein in the adrenal gland. Pkd2
protein-containing cells were found in different layers of the adrenal
cortex as shown in Fig. 8. The Pkd2 protein
was detectable in the outermost parenchymal cells of the zona
glomerulosa. Intensive staining with the anti-PKD2 antibody was also
observed in cells of the zona fasciculata, and again the labeling was
confined to only a subpopulation of cells. Although most heavily
labeled cells could be detected in the outer part of this layer,
Pkd2-positive cells were also found closer to the zona reticularis
(Fig. 8, A and B). No labeling was found in the adrenal
medulla. The cellular distribution of the Pkd2 protein is demonstrated
by high-power magnification in Fig. 8C, where a fine punctate
staining within the entire cytoplasm of zona fasciculata cells can be
seen.

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Fig. 8.
Expression of the Pkd2 protein in rat adrenal gland. Sections were
first stained by immunofluorescence with the anti-PKD2 antibody
(A and C) and subsequently with H & E (B and
D). An overview of adrenal cortex (A and B)
demonstrates that in the zona glomerulosa only the outer portion of
cells expresses Pkd2 (arrowheads point to the capsule). Columns of the
zona fasciculata also contain numerous Pkd2 protein-expressing cells. A
high-power magnification of the zona fasciculata (C and
D) demonstrates the punctate cytoplasmic distribution of Pkd2.
Note that the Pkd2-expressing cells appear lighter than the surrounding
cells and tend to have a more euchromatic nucleus
(D). Magnifications, ×100 (A and B) and
×590 (C and D).
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Localization of the Pkd2 protein in the ovary. Prominent
immunoreactivity for Pkd2 could be detected in granulosa cells of corpora lutea (Fig. 9). As in the adrenal
cortex, a punctate cytoplasmic staining pattern was noticed. The
portion of Pkd2-expressing granulosa cells varied when individual
corpora lutea of a section were examined, which probably reflects the
different developmental stages of the corpora lutea. No Pkd2
immunoreactivity was found in granulosa cells of developing follicles.

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Fig. 9.
Expression of the Pkd2 protein in rat ovary. Section was first stained
by immunofluorescence with anti-PKD2 antibody (A) and
subsequently with H & E (B). High-power magnification of a
corpus luteum demonstrates a punctate cytoplasmic labeling of granulosa
cells (A), whereas smaller fibroblasts are not stained.
Arrowheads in A and B mark the border between the
Pkd2-expressing cells of the corpus luteum and the unreactive
surrounding stroma. Magnification, ×190.
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Localization of the Pkd2 protein in vascular structures.
Arteries of the muscular type revealed labeling of the smooth muscle cell layers as demonstrated in Fig. 10,
where several arteries branching into a salivary gland are shown. A
high-power view shows a punctate to striped staining pattern. Although
clearly detectable, it should be emphasized that the staining intensity
was less pronounced than in the striated duct profiles. Arteries of the
elastic type such as the aorta were unreactive with the anti-Pkd2
antibody.

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Fig. 10.
Expression of the Pkd2 protein in rat vasculature. Section was first
stained by immunofluorescence with anti-PKD2 antibody (A) and
subsequently with H & E (B). A fine punctate
immunohistochemical labeling (A) is detected in cytoplasm of
smooth muscle cells of 3 muscular arteries in submandibular gland.
Magnification, ×235.
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Localization of the Pkd2 protein in the eye. Sagittal paraffin
sections of the rat eye were subjected to immunohistochemical analysis.
Intense labeling occurred exclusively in the basal cell layer of the
corneal epithelium, demonstrating again a punctate cytoplasmic
distribution of the protein (Fig. 11).
The staining intensity for Pkd2 appeared to vary between different
basal cells. Other structures, notably the retina, were not found to
express Pkd2.

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Fig. 11.
Expression of the Pkd2 protein in rat cornea. Section was first stained
by immunofluorescence with anti-PKD2 antibody (A) and
subsequently with H & E (B). Only the basal cell layer of the
corneal epithelium expresses Pkd2 (A). Border between basal
cell layer and stroma is indicated by arrowheads in A and
B. Magnification, ×245.
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Western blot analysis of Pkd2 protein expression. Because the
subcellular distribution of the Pkd2 protein varied strikingly between
different organs (basal/basolateral in kidney and salivary gland vs.
punctate cytoplasmic in adrenal gland, ovary, smooth muscle cells, and
the eye), we wanted to rule out a nonspecific staining in the different
organs. Preabsorption of the anti-PKD2 antibody with the GST-PKD2
fusion protein used to generate the antibody abolished both the
basolateral staining in the kidney and salivary gland and the punctate
staining in the adrenal gland, ovary, and blood vessels, whereas after
preabsorbing the antibody with GST alone, the respective staining
patterns were still present (data not shown). Furthermore, membrane
proteins were prepared from the adrenal gland, kidney, ovary, and
salivary gland and subjected to Western blot analysis with the
anti-PKD2 antibody. Somewhat surprisingly, the Pkd2 protein from the
rat adrenal gland and ovary displayed a slower mobility than the Pkd2
protein from the rat kidney and salivary gland (Fig.
12, left). To examine whether a
different degree of N-glycosylation accounted for that
difference, the membrane protein preparation was digested with PNGase
F, a glycosidase that cleaves between the asparagine of
the peptide backbone and the first sugar residue. Treatment with PNGase
F resulted in an equal mobility of the rat Pkd2 protein from all four
organs (Fig. 12, right).

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Fig. 12.
Western blot analysis. Fifty micrograms of membrane proteins from rat
adrenal gland (A), ovary (O), kidney (K), and salivary gland (S) were
separated under reducing and denaturing conditions on a 7%
polyacrylamide gel and then analyzed by Western blot with the anti-PKD2
antibody YCC2 at a dilution of 1:4,000. Whereas without PNGase F
treatment the Pkd2 protein from the adrenal gland and the ovary
displayed a slower mobility than the Pkd2 protein from the kidney and
salivary gland, treatment with PNGase F resulted in an equal mobility
of Pkd2 protein from all 4 organs, indicating a different degree of
N-glycosylation in the various organs.
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DISCUSSION |
In the present study, we were able to determine the distribution of the
Pkd2 protein in different rat and human organs using immunohistochemistry and Western blot analysis.
In the rat kidney, Pkd2 is strongly expressed in cells of the distal
nephron but at much lower levels in collecting duct cells. A comparable
expression of the Pkd2 protein was noted in human and mouse kidneys.
The pronounced Pkd2 staining in the thick ascending limb, distal
convoluted tubule, and connecting tubule contrasted with the faint
labeling in the collecting duct, where lateral interdigitations are
absent and basal infoldings are scarce (11). Interestingly, macula
densa cells lack Pkd2 expression. These cells, which serve as a sensor
of tubular function, do not interdigitate with each other, nor do they
participate in electrolyte absorption. Basal infoldings, however, are
also present in the macula densa (11). The observations made in the
kidney were significantly extended, since we could also document the
expression of Pkd2 in striated ducts of rat and human salivary glands.
This is of particular interest since the salivary glands share some
important functional properties with the kidney (26). The composition of the isosmotic saliva produced by the acinar cells is later modified
by the electrolyte-transporting epithelium of the striated ducts. These
cells, which for example reabsorb sodium and secrete potassium, show
obvious structural similarities to renal thick ascending limb cells. As
in the thick ascending limb, the basal labyrinth with numerous
mitochondria is highly developed in striated duct cells. Comparable to
the kidney, a countercurrent exchange exists between saliva and blood.
Therefore, the comparable staining pattern for Pkd2 in the thick
ascending limb of the kidney and in striated ducts of the salivary
gland suggests that this protein accomplishes a similar function in
both organs.
The basal-to-basolateral distribution of Pkd2 in the kidney and
salivary gland strikingly differed from that seen in the adrenal cortex. There, the Pkd2 protein was located in fine cytoplasmic structures often occupying the entire cytoplasm. It is known that cells
of the adrenal cortex have a well-developed smooth endoplasmic reticulum and contain numerous mitochondria, which represent the compartments of the early and late stages of steroid biosynthesis. The
distribution of Pkd2 in the adrenal cortex, where its expression was
most prominent in the outer zona glomerulosa and the outer zona
fasciculata, corresponds well with the notion that these subregions
exhibit very strong steroidogenic activity for mineralocorticoids and
glucocorticoids, respectively (26). The assumption that Pkd2 is located
in the endoplasmic reticulum is supported by the distribution of Pkd2
in the ovary and by in vitro findings. First, the subcellular
distribution pattern of Pkd2 in the adrenal gland was virtually
identical to that seen in granulosa cells of corpora lutea in the
ovary. These cells show ultrastructural and functional characteristics
similar to cells in the adrenal cortex (both produce steroid hormones)
(26), thus suggesting an analogous subcellular location and function
for Pkd2 in both endocrine cell types. Second, in all permanent cell
lines examined so far, both the endogenous protein and stably expressed
human PKD2 protein were located in the endoplasmic reticulum,
regardless of the tissue from which the cell lines were derived (3a;
and unpublished observations of A. Cedzich, N. Obermüller, and R. Witzgall). Therefore, an additional signal probably exists in vivo that
is responsible for the basal-to-basolateral distribution of Pkd2 in the
kidney and salivary gland. Interestingly, it had been shown previously that the endoplasmic reticulum extends deep down into the lateral interdigitations of renal distal tubular cells, where it appears to
wrap around the mitochondria and comes to lie between these and the
plasma membrane (1, 2). Furthermore, there also is evidence for an
extensive intercellular fibrillar network that connects the lateral
interdigitations and may be responsible for the very regular spacing of
neighboring distal tubular cells (30). If Pkd2 indeed is a subunit of a
channel protein, then it should also be considered that in the adrenal
cortex this protein is part of a structure that functionally links the
endoplasmic reticulum and the mitochondrion. Such a structure-function
relationship has been proposed for the regulation of aldosterone
secretion in bovine adrenal glomerulosa cells (19).
The different distribution patterns of Pkd2 in the kidney and salivary
gland on the one hand and in the adrenal gland and ovary on the other
hand also appear to be reflected in the distinct glycosylation patterns
of Pkd2 in the respective organs. In the adrenal gland and the ovary,
two organs with a punctate distribution of Pkd2, the Pkd2 protein was
more heavily N-glycosylated than in the kidney and salivary
gland, two organs with a basal-to-basolateral distribution of Pkd2. We
do not believe that the immunohistochemical signals and the signals on
the Western blot arise from different proteins, because after a digest
with PNGase F, the detected protein in all four organs displayed the
same mobility. Cross-reactivity with Pkd2L/PkdL also is unlikely,
because the human proteins are only 38% identical in the region of
PKD2 used to generate the antibody [the comparison was done with
the Gap program of the Genetics Computer Group of Wisconsin (5) using a
gap creation penalty of 8 and a gap extension penalty of 2].
Our results may help to recognize other extrarenal symptoms in patients
with mutations in the PKD2 gene. Although several reports of
polycystic disease in the salivary glands have been published (e.g.,
Refs. 6 and 25), a connection to polycystic disease of the kidney
has not been demonstrated yet. It will be interesting to see whether
patients with PKD2 mutations develop cysts in their salivary
glands or present with other dysfunctions in this organ. It might
also be relevant to find out whether PKD2 mutations affect
the endocrine status of patients because of the prominent expression
of the protein in the adrenal gland and in the ovary. Interestingly, a
possible association between abnormal steroid synthesis and cyst
formation has been suggested for the Ke 6 gene in several
murine models of PKD (Ref. 8 and references therein).
Our observation that the Pkd2 protein is also present in the cornea of
the eye is of particular interest, since mutations in a variety of
genes can cause abnormalities in both the eye and the kidney. Somewhat
surprisingly, Pkd2 expression was confined to the basal cell layer of
the corneal epithelium. These cells, which show discrete infoldings on
their lateral sides, are sitting on a prominent basement membrane which
demarcates the epithelium from the stromal compartment of the cornea
(26). However, since we could not detect any expression
of the Pkd2 protein in the epidermal layer of the skin, which also
contains a stratified squamous epithelium, we consider it unlikely that
Pkd2 is involved in cell-matrix interactions in the cornea. This
assumption is also supported by the punctate cytoplasmic distribution
of the Pkd2 protein in the corneal epithelium. In contrast to PKD2, the PKD2L mRNA is expressed in the retina, but not in the cornea (28), suggesting distinct functions for both proteins in different cells.
Apart from the expression in epithelial cells, our data provide clear
evidence for Pkd2 expression in smooth muscle cells of the vasculature,
thus confirming observations by other investigators (16, 23), although
in the article by Ong et al. (16) expression of the human PKD2 protein
was also described in the endothelium of arteries. The expression of
the Pkd2 protein in vascular smooth muscle cells provides a clue about
the pathogenesis of intracranial aneurysms in ADPKD patients (24). It
is tempting to speculate that a mutated PKD2 protein expressed in
smooth muscle cells affects the vascular compliance, especially under
conditions of higher blood pressure levels.
Although by Northern blot analysis the human PKD2 mRNA and its murine
homolog could also be detected in other organs such as the brain,
heart, liver, and pancreas (14, 29), we could not find convincing
evidence for the expression of the rat Pkd2 protein in those organs.
The reason for this discrepancy is not clear, but it may result from
species differences, lack of sensitivity of the immunofluorescence
protocol, and/or posttranscriptional regulation of Pkd2 expression in
those organs, i.e., lack of translation of the mRNA. In contrast to the
well-documented occurrence of cysts in the livers of Pkd2
mutant mice (27) and in the liver and pancreas of ADPKD patients (for
review, see Refs. 7 and 9), we failed to detect Pkd2 protein expression
in bile ducts and pancreatic ducts of the rat. We cannot rule out that
in the pancreas and the liver Pkd2 is expressed at a level below the detection limit; we were able, however, to detect Pkd2 in
intrapancreatic vessels. In a recent article, expression of the human
PKD2 protein was demonstrated in the liver and pancreas (16). The same
article also describes that in the adult kidney the distal convoluted tubules and the collecting ducts were predominantly stained, and an
even more widespread distribution in the fetal kidney was shown (16).
This contrasts with our observations of the strongest expression of the
Pkd2 protein in the distal tubules and connecting tubules and a much
weaker expression in the collecting ducts. Furthermore,
we only detect the rat Pkd2 protein at an advanced stage of nephron
development (unpublished observations of N. Obermüller and R. Witzgall).
In summary, our demonstration of Pkd2 protein expression in different
structures underlines the widespread distribution and potentially very
distinct function of Pkd2 in various organ systems. The
basal-to-basolateral staining pattern observed on the one hand (kidney
and the salivary glands) and the punctate cytoplasmic distribution on
the other hand (adrenal gland, ovary, cornea, blood vessels) raises the
possibility that the Pkd2 protein accomplishes its function in concert
with different proteins. These proteins may be located in the plasma
membrane or in cytoplasmic compartments. Clearly, highly specific
antibodies and immuno-electron microscopy will help to resolve the
issue of the subcellular location of Pkd2 and Pkd1.
 |
ACKNOWLEDGEMENTS |
We thank Wilhelm Kriz for pointing out to us the particular
organization of the endoplasmic reticulum in the nephron. The superb
expertise of Klaus Tiedemann in the histology of adult and embryonic
rat organs is gratefully acknowledged. We are also thankful for the
expert photographic work of Ingrid Ertel and graphics of Rolf
Nonnenmacher and for the great help of Jutta Christophel and Bernd
Schnabel in preparing the tissue sections.
 |
FOOTNOTES |
The study described in this article was made possible by a grant from
the Fritz Thyssen Foundation to R. Witzgall (1997, 2073).
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 and other correspondence: R. Witzgall,
University of Heidelberg, Institute for Anatomy and Cell Biology I, Im
Neuenheimer Feld 307, 69120 Heidelberg, Germany (E-mail:
ralph.witzgall{at}urz.uni-heidelberg.de).
Received 12 March 1999; accepted in final form 6 August 1999.
 |
REFERENCES |
1.
Bergeron, M.,
P. Gaffiero,
and
G. Thiéry.
Segmental variations in the organization of the endoplasmic reticulum of the rat nephron. A stereomicroscopic study.
Cell Tissue Res.
247:
215-225,
1987[Medline].
2.
Bergeron, M.,
and
G. Thiéry.
Three-dimensional characteristics of the endoplasmic reticulum of rat renal tubule cells, an electron microscopy study in thick sections.
Biol. Cell
42:
43-48,
1981.
3.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
3a.
Cai, Y.,
Y. Maeda,
A. Cedzich,
V. E. Torres,
G. Wu,
T. Hayashi,
T. Mochizuki,
J. H. Park,
R. Witzgall,
and
S. Somlo.
Identification and characterization of polycystin-2, the PKD2 gene product.
J. Biol. Chem.
274:
28557-28565,
1999[Abstract/Free Full Text].
4.
Daoust, M. C.,
D. M. Reynolds,
D. G. Bichet,
and
S. Somlo.
Evidence for a third genetic locus for autosomal dominant polycystic kidney disease.
Genomics
25:
733-736,
1995[Medline].
5.
Devereux, J.,
P. Haeberli,
and
O. Smithies.
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res.
12:
387-395,
1984[Abstract].
6.
Dobson, C. M.,
and
H. A. Ellis.
Polycystic disease of the parotid glands: case report of a rare entity and review of the literature.
Histopathology
11:
953-961,
1987[Medline].
7.
Fick, G. M.,
and
P. A. Gabow.
Hereditary and acquired cystic disease of the kidney.
Kidney Int.
46:
951-964,
1994[Medline].
8.
Fomitcheva, J.,
M. E. Baker,
E. Anderson,
G. Y. Lee,
and
N. Aziz.
Characterization of Ke 6, a new 1717
-hydroxysteroid dehydrogenase, and its expression in gonadal tissues.
J. Biol. Chem.
273:
22664-22671,
1998[Abstract/Free Full Text].
9.
Gabow, P. A.
Autosomal dominant polycystic kidney disease.
N. Engl. J. Med.
329:
332-342,
1993[Free Full Text].
10.
Hoyer, J. R.,
S. P. Sisson,
and
R. L. Vernier.
Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney.
Lab. Invest.
41:
168-173,
1979[Medline].
11.
Kaissling, B.,
and
W. Kriz.
Morphology of the loop of Henle, distal tubule, and collecting duct.
In: Handbook of Physiology: Renal Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, part 1, chapt. 3, p. 109-167.
12.
Koptides, M.,
C. Hadjimichael,
P. Koupepidou,
A. Pierides,
and
C. C. Deltas.
Germinal and somatic mutations in the PKD2 gene of renal cysts in autosomal dominant polycystic kidney disease.
Hum. Mol. Genet.
8:
509-513,
1999[Abstract/Free Full Text].
13.
Loffing, J.,
D. Loffing-Cueni,
I. Hegyi,
M. R. Kaplan,
S. C. Hebert,
M. Le Hir,
and
B. Kaissling.
Thiazide treatment of rats provokes apoptosis in distal tubule cells.
Kidney Int.
50:
1180-1190,
1996[Medline].
14.
Mochizuki, T.,
G. Wu,
T. Hayashi,
S. L. Xenophontos,
B. Veldhuisen,
J. J. Saris,
D. M. Reynolds,
Y. Cai,
P. A. Gabow,
A. Pierides,
W. J. Kimberling,
M. H. Breuning,
C. C. Deltas,
D. J. M. Peters,
and
S. Somlo.
PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.
Science
272:
1339-1342,
1996[Abstract].
15.
Nomura, H.,
A. E. Turco,
Y. Pei,
L. Kalaydjieva,
T. Schiavello,
S. Weremowicz,
W. Ji,
C. C. Morton,
M. Meisler,
S. T. Reeders,
and
J. Zhou.
Identification of PKDL, a novel polycystic kidney disease 2-like gene whose murine homologue is deleted in mice with kidney and retinal defects.
J. Biol. Chem.
273:
25967-25973,
1998[Abstract/Free Full Text].
16.
Ong, A. C. M.,
C. J. Ward,
R. J. Butler,
S. Biddolph,
C. Bowker,
R. Torra,
Y. Pei,
and
P. C. Harris.
Coordinate expression of the autosomal dominant polycystic kidney disease proteins, polycystin-2 and polycystin-1, in normal and cystic tissue.
Am. J. Pathol.
154:
1721-1729,
1999[Abstract/Free Full Text].
17.
Pennekamp, P.,
N. Bogdanova,
M. Wilda,
A. Markoff,
H. Hameister,
J. Horst,
and
B. Dworniczak.
Characterization of the murine polycystic kidney disease (Pkd2) gene.
Mamm. Genome
9:
749-752,
1998[Medline].
18.
Peters, D. J. M.,
and
L. A. Sandkuijl.
Genetic heterogeneity of polycystic kidney disease in Europe.
Contrib. Nephrol.
97:
128-139,
1992[Medline].
19.
Rossier, M. F.,
M. M. Burnay,
M. B. Vallotton,
and
A. M. Capponi.
Distinct functions of T- and L-type calcium channels during activation of bovine adrenal glomerulosa cells.
Endocrinology
137:
4817-4826,
1996[Abstract].
20.
The American PKD1 Consortium.
Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease (PKD1) gene predicts the presence of a leucine-rich repeat.
Hum. Mol. Genet.
4:
575-582,
1995[Abstract].
21.
The European Polycystic Kidney Disease Consortium.
The polycystic kidney disease 1 gene encodes a 14 kb transcript, and lies within a duplicated region on chromosome 16.
Cell
77:
881-894,
1994[Medline].
22.
The International Polycystic Kidney Disease Consortium.
Polycystic kidney disease: the complete structure of the PKD1 gene, and its protein.
Cell
81:
289-298,
1995[Medline].
23.
Torres, V. E.,
Y. Cai,
X. Chen,
G. Q. Wu,
K. Cleghorn,
and
S. Somlo.
Vascular expression of PKD2 mRNA and protein (Abstract).
J. Am. Soc. Nephrol.
8:
382,
1997.
24.
van Dijk, M. A.,
P. C. Chang,
D. J. M. Peters,
and
M. H. Breuning.
Intracranial aneurysms in polycystic kidney disease linked to chromosome 4.
J. Am. Soc. Nephrol.
6:
1670-1673,
1995[Abstract].
25.
Wiedemann, H.-R.
Salivary gland disorders and heredity.
Am. J. Med. Genet.
68:
222-224,
1997[Medline].
26.
Williams, P. L.,
L. H. Bannister,
M. M. Berry,
P. Collins,
M. Dyson,
J. E. Dussek,
and
M. W. J. Ferguson.
Gray's Anatomy (38th ed.). New York: Churchill Livingstone, 1995.
27.
Wu, G.,
V. d'Agati,
Y. Cai,
G. Markowitz,
J. H. Park,
D. M. Reynolds,
Y. Maeda,
T. C. Le,
H. Hou, Jr.,
R. Kucherlapati,
W. Edelmann,
and
S. Somlo.
Somatic inactivation of Pkd2 results in polycystic kidney disease.
Cell
93:
177-188,
1998[Medline].
28.
Wu, G.,
T. Hayashi,
J.-H. Park,
M. Dixit,
D. M. Reynolds,
L. Li,
Y. Maeda,
Y. Cai,
M. Coca-Prados,
and
S. Somlo.
Identification of PKD2L, a human PKD2-related gene: Tissue-specific expression and mapping to chromosome 10q25.
Genomics
54:
564-568,
1998[Medline].
29.
Wu, G.,
T. Mochizuki,
T. C. Le,
Y. Cai,
T. Hayashi,
D. M. Reynolds,
and
S. Somlo.
Molecular cloning, cDNA sequence analysis, and chromosomal localization of mouse Pkd2.
Genomics
45:
220-223,
1997[Medline].
30.
Zampighi, G.,
and
M. Kreman.
Intercellular fibrillar skeleton in the basal interdigitations of kidney tubular cells.
J. Membr. Biol.
88:
33-43,
1985[Medline].
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