Polycystin-1 expression in fetal, adult and autosomal dominant polycystic kidney.

Abstract

The mutation of the PKD1 gene causes autosomal dominant polycystic kidney disease (ADPKD), and the PKD1 gene encodes polycystin-1 (PC-1). PC-1 is thought to be a cell-cell/matrix adhesion receptor molecule at the cell surface that is widely expressed in the kidney. However, there are controversies about the role of PC-1 protein and its expression when using different antibodies to detect it. We used two PC-1 antibodies; C-20 (Santa Cruz, sc-10372) as the C-terminal antibody, and P-15 (Santa Cruz, sc-10307) as the N-terminal antibody. We evaluated the PC-1 expression by performing immunoblotting on the human embryonic kidney (HEK) 293 cells and the renal proximal tubular epithelial cell (RPTEC) lysates. We characterized the expression of PC-1 in the fetal, adult and polycystic kidneys tissues by performing immunohistochemistry. We confirmed the PC-1 expression in the HEK 293 cells and the RPTEC lysates, but the expression was very low. The PC-1 proteins were diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult kidneys, and the PC-1 expression was more prominent in the proximal tubules of the fetal kidney. In the ADPKD kidney, the PC-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells. Our data suggests that the development of the kidney may regulate the expression of PC-1, and an altered PC-1 expression may contribute to cyst formation in ADPKD.

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INTRODUCTION
Autosomal dominant polycystic kidney disease (ADPKD)
is a common inherited disease that affects approximately 1 in
1,000 individuals. The disease’s main clinical manifestations
are bilateral renal cyst formation and hypertension, and the
half of these patients reach end stage renal failure by the age
of 60 yr (1). ADPKD is genetically heterogeneous and most
of the afflicted families have mutations of either PKD1 on
chromosome 16 (-85%) or PKD2 on chromosome 4 (-15%)
(2, 3). PKD1 encodes polycystin-1 (PC-1), which is an inte-
gral membrane protein of 4,302 amino acids with an expect-
ed molecular mass of 462 kDa (4, 5). The functions of the
PC-1 remain unclear. PC-1 is widely expressed in the renal
tubular epithelium and it is thought to be a cell-cell/matrix
receptor molecule at the cell surface. PC-1 and PC-2 may
heterodimerize to form a PC complex and this could func-
tion as the same signaling pathway (6, 7). However, there
are controversies about the PC-1 expression and function when
using different antibodies to study them. The large size and
the low expression of PC-1 have made the study of PC-1 very
difficult. There are also some doubts as to the specificity of
the PC-1 antibodies (8, 9).
As an initial approach towards studying ADPKD and to
obtain more insight into PC-1 expression, we have carried
out immunoblot and immunochemical analyses of the PC-1
expression in the tubular cells of fetal, adult and ADPKD
kidneys.
MATERIALS AND METHODS
Immunoblot
We used human embryonic kidney (HEK) 293 cells and
renal proximal tubular epithelial cellsR (RPTECs) for per-
forming immunoblottting. The cells were grown to 70-90%
confluence and they were then lysed in phosphate buffered
saline that contained 0.5% Nonidet P-40. A large amount of
protein(-400 g each) was separated on 4% SDS-PAGE gel
with or without boiling. The separated proteins were electro-
transferred onto nitrocellulose membranes. The membranes
were blocked with 5% nonfat dry milk for 1 hr and they were
next incubated with primary antibodies diluted at 1:500.
We used two PC-1 antibodies; C-20 (Santa Cruz, sc-10372,
Santa Cruz, CA, U.S.A.) was used as the C-terminal antibody
and P-15 (Santa Cruz, sc-10307, Santa Cruz, CA, U.S.A.) was
used as the N-terminal antibody. After washing, the mem-
Seoung Wan Chae, Eun-Yoon Cho,
Moon Soo Park*, Kyu-Beck Lee
Hyunho Kim
�,
�, Unkyung Kim
�
Departments of Pathology, Pediatrics* and Internal
Medicine
Sungkyunkwan University School of Medicine, Seoul;
Graduate School of Biotechnology
Seoul; Department of Biology
Sciences, Kyungpook National University, Deagu, Korea
�, Kangbuk Samsung Hospital,
�, Korea University,
�, College of Natural
Address for correspondence
Kyu-Beck Lee, M.D.
Department of Internal Medicine, Kangbuk Samsung
Hospital, Sungkyunkwan University School of Medicine,
108 Pyung-dong, Jongro-gu, Seoul 110-746, Korea
Tel : +82.2-2001-2050, Fax : +82.2-2001-2049
E-mail : leekyubeck@yahoo.co.kr
*This study was supported by a Hyoseok Medical
Grant at Kangbuk Samsung Hospital.
425
J Korean Med Sci 2006; 21: 425-9
ISSN 1011-8934
Copyright � The Korean Academy
of Medical Sciences
Polycystin-1 Expression in Fetal, Adult and Autosomal Dominant
Polycystic Kidney
The mutation of the PKD1 gene causes autosomal dominant polycystic kidney dis-
ease (ADPKD), and the PKD1 gene encodes polycystin-1 (PC-1). PC-1 is thought
to be a cell-cell/matrix adhesion receptor molecule at the cell surface that is widely
expressed in the kidney. However, there are controversies about the role of PC-1
protein and its expression when using different antibodies to detect it. We used two
PC-1 antibodies; C-20 (Santa Cruz, sc-10372) as the C-terminal antibody, and P-15
(Santa Cruz, sc-10307) as the N-terminal antibody. We evaluated the PC-1 expres-
sion by performing immunoblotting on the human embryonic kidney (HEK) 293 cells
and the renal proximal tubular epithelial cell (RPTEC) lysates. We characterized the
expression of PC-1 in the fetal, adult and polycystic kidneys tissues by performing
immunohistochemistry. We confirmed the PC-1 expression in the HEK 293 cells
and the RPTEC lysates, but the expression was very low. The PC-1 proteins were
diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult
kidneys, and the PC-1 expression was more prominent in the proximal tubules of
the fetal kidney. In the ADPKD kidney, the PC-1 proteins were heterogenously and
weakly expressed in the tubular or cyst lining epithelial cells. Our data suggests that
the development of the kidney may regulate the expression of PC-1, and an altered
PC-1 expression may contribute to cyst formation in ADPKD.
Key Words : Polycystic Kidney Diseases; Polycystin-1; Polycystic kidney disease 1 protein
Received : 1 September 2005
Accepted : 30 November 2005

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426
S.W. Chae, E.-Y. Cho, M.S. Park, et al.
branes were incubated for 1 hr at room temperature with the
peroxidase-labeled secondary antibody (anti-goat IgG-HRP,
Santa Cruz) at 1:500 dilution. The membrane-bound anti-
bodies were detected by using the enhanced chemilumines-
cence detection system (Amersham Biosciences, Bucks, U.K.).
Immunohistochemistry
Normal fetal kidney was obtained from autopsy (gestation
age: 20 weeks, weight: 305 grams) after spontaneous abortion.
Adult kidneys were obtained from renal tumor nephrectomy
patients (a male 58 yr old and a male 63 yr old). The auto-
somal dominant polycystic kidneys were obtained from PKD1
patients (a male 53 yr old and a male 46 yr old) who had the
typical clinical manifestations during transplant nephrectomy.
All the tissues were embedded in optimal cutting tempera-
ture compound (Sakura Tissue Tek, Torrance, CA, U.S.A.)
and stored at -70℃until further use.
Immunohistochemical staining was performed by using
the streptavidin-biotin peroxidase method. Cryosections 5 m
thick were cut, and the tissues were fixed in acetone at -20℃
for 10 min and then washed with phosphate-buffered saline.
After blocking the endogenous peroxidase activity, the pri-
mary antibodies (C-20 and P-15) were diluted to 1:100 and
the sections were incubated for 1 hr with them at room tem-
perature. After washing, the sections were incubated for 45
min with the secondary antibody (bionylated anti-goat Ig,
Dako, Glostrup, Denmark). The reaction was developed with
3-amino-9-ethylcarbazol (AEC, red stain) or with 3,3′dia-
minobenzidine (DAB, brown stain). The sections were coun-
terstained with using hematoxylin.
RESULTS
Immunoblotting
We confirmed that the C-20 antibody was sensitive for
detecting PC-1 by performing immunoprecipitation (10),
and we confirmed the PC-1 expression in the HEK 293 cells
and in the RPTEC lysates (Fig. 1). However, this was tech-
nically difficult because of the large size of the PC-1 molecule
and its faint expression level.
Immunohistochemistry
In the fetal and adult kidneys, the staining was restricted
to tubular epithelial cells cytoplasm (Fig. 2A to D). The
patterns of expression were diffuse and cytoplasmic at the
proximal tubules, distal tubules and collecting ducts. The
glomerular tufts and interstitium displayed no staining. The
expression in the fetal kidney was more prominent in the
proximal tubules (Fig. 2A, B). In the ADPKD kidney, the
PC-1 proteins were weakly expressed in the tubular or cyst-
lining epithelial cells, as compared to that of the fetal and
adult kidneys. The patterns of expression were heterogenous
and cytoplasmic at the cystic epithelium (Fig. 3). The specific
renal distribution of the PC-1 is summarized in Table 1.
DISCUSSION
The identification of PC-1, which is the major gene mutated
protein seen in ADPKD, is a major step for discovering the
pathogenesis in this common hereditary disease. However,
there’s been some controversy about the PC-1 expression when
using different antibodies to detect it. The difficulties for
studying PC-1 are due to the antibody specificity and the
low levels of PC-1 expression in cells and tissues. Moreover,
two thirds of the PKD1 gene is duplicated on chromosome
16 and there are at least three homologus genes. Our study
showed that PC-1 may be very weakly expressed in the renal
tubular cells, as observed by immunoblotting, and it is widely
expressed in the fetal and adult renal tubular epithelial cells
cytoplasm, as observed by immunochemistry. Before this
study, we confirmed that C-20 antibody was sensitive to detect
the PKD-1 C-terminal by performing immunoprecipitation
293
C-20 P-15
Cell Iysis
RPTEC293
Cell Iysis
RPTEC
Fig. 1. Polycystin-1 in the HEK 293 cell and RPTEC is detected by
using C-20 and P-15 antibodies in 4% SDS-PAGE gel. The poly-
cystin-1 expression is very low.
C20 (C-terminal)P15 (N-terminal)
Fetal kidneyGM -
PT ++
DT +/-
CT +/-
GM -
PT ++
DT +++
CT ++
cyst +
GM -
PT ++
DT +/-
CT +/-
GM -
PT ++
DT +++
CT ++
cyst +
Adult kidney
ADPKD kidney
Table 1. Summaries of the polycystin-1 expressions in the fetal,
adult and autosomal polycystic kidneys
GM, glomerulus; PT, proximal tubule; DT, distal tubule; CT, collecting
duct.

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Polycystin-1 Expression
427
(10). To verify the specificity of the immunohistochemical
staining, we performed immunoblot analysis of the kidney
cells (HEK 293 cells and RPTECs), the kidney tissues (fetal
and adults) and the ADPKD tissues. We were unable to detect
the PC-1 band in the kidney tissues and the ADPKD tissues.
This is suggested that the PC-1 is either not expressed at
detectable levels in normal and ADPKD kidneys, or it is lost
during sample preparation. However, we were able to detect
low levels of PC-1 in the kidney cells. According to the pre-
vious reports (8, 9), many experiments have failed to detect
the PC-1 band. Immunoblotting of PC-1 was technically
difficult because of the large size of the PC-1 molecule and
the faint level of expression.
A number of published papers have described the PC-1
expression in normal renal and cystic tissue. Van Adelsberg
et al. (11) have reported that in the early fetal kidney, PC-1
was localized to the plasma membranes of the ureteric buds
and the S-shaped bodies. However, in the late fetal kidney
and in the ADPKD kidney, the majority of PC-1 staining was
intracellular. They detected several bands in the fetal kidney
and no bands were detected in the adult kidney by immuno-
blotting. Gene et al. (12, 13) found no major differences bet-
Fig. 2. The immunohistochemical expression of polycystin-1 in the fetal (A and B, ×200) and adult (C and D, ×400) kidneys with using
goat polyclonal C-20 (A and C) and P-15 antibodies (B and D). The expression of polycystin-1 shows a diffuse cytoplasmic pattern at the
tubules, and it is negative at the glomerular tufts and interstitium. The expression in the fetal kidney is less prominent in the collecting ducts
and distal tubules than in the proximal tubules.
AB
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428
S.W. Chae, E.-Y. Cho, M.S. Park, et al.
ween human and mouse PC-1 expression. They detected about
a 400 kDa PC-1 band in the fetal and adult mouse and human
kidneys. PC-1 was noted to be localized at the plasma mem-
brane and PC-1 expression was seen in most (-90%) of the
ADPKD cysts. Ong et al. (14) reported that in adult PKD1
tissue, the majority of cysts (-80%) showed PC-1 expression,
although PC-1 staining was absent in a variable, but signif-
icant minority of the cysts (-20%). Leeuwen et al. (15) found
that the mutant Pkd1 mice that had a reduced Pkd1 gene
expression showed polycystic kidney disease. They generat-
eda novel mouse model with a hypomorphic Pkd1allele, and
the pathologic features of this mouse were similar to the human
ADPKD phenotype. Therefore, the reduced PC-1 expression
of the normal allele may lead to ADPKD. Recently, Roitbak
et al. (16) have reported that PC-1 formed a complex with
E-cadherin and -catenin at both the cell membrane and
intracellularly. Enhanced phosphorylation of PC-1 changed
its subcellular localization and its ability to form protein com-
plexes.The plasma membrane expression of PC-1 was dimin-
ished in the ADPKD cells.
There have been different results reported by different re-
search groups. However, researchers have reached some con-
sensus. First, the PC-1 expresssion is temporally and spatially
regulated during renal development. Second, the PC-1 is
Fig. 3. The immunohistochemical expression of polycystin-1 in the autosomal dominant polycystic kidney with using goat polyclonal C-20
(A, ×400 and C, ×1,000) and P-15 antibodies (B, ×200 and D, ×1,000). The ADPKD kidney shows a weak and heterogenous expres-
sion of polycystin-1 in the tubular or cystic lining epithelial cells compared to that of the fetal and adult kidneys.
AB
CD

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expressed in the tubular epithelium and in the ADPKD cyst
epithelium. Third, the subcellular localization of PC-1 is
mainly on the cell surface membrane and it may be changed
in some conditions. Our results showed similar patterns of
PC-1 expression for the fetal, adult and ADPKD kidneys,
but our immunohistochmical study showed a cytoplasmic
staining pattern of PC-1. However, in the HEK 293 cells
grown to 80% confluency, we found that the PC-1 was local-
ized on the cell surface membrane by performing immunoflu-
orescent staining with using C-20 antibody (10). There is a
possible explanation for this result. These PC-1 antibodies are
sensitive, but they are not specific for detecting the PC-1.
The epitope recognized by these antibodies may be not the
full length, but rather, they are only a fraction of the PC-1.
PC-1 is the PKD 1gene translated protein, and it is a 4,302
amino acids glycoprotein with an expected molecular mass
of 460 kDa. Experimental studies have shown that PC-1 is
a highly glycosylated 520 kDa polypeptide that is present
in the plasma membrane (6, 7). By performing amino acid
sequence homology analysis, PC-1 was found to be a mem-
branereceptor that is capable of binding and interacting with
ligands. However, the exact function and binding ligands of
PC-1 are not yet known. Recent research have shown that
PC-1 is also localized to the renal cilia and to the protein-medi-
ated mechanosensation in the primary cilium of the kidney
(17, 18).
In summary, we have found by performing immunoblot-
ting that PC-1 is expressed at a very low level in the renal
tubular cells. However, PC-1 is widely expressed in the fetal
and adult renal tubular epithelial cells cytoplasm, as was
noted upon performing the immunohistochemistry. In the
ADPKD kidney, the PC-1 proteins were heterogenously
and weakly expressed in the tubular or cyst lining epithelial
cells. A reduced PC-1 expression may well be associated with
ADPKD. Our data suggests that the development of kidney
may regulate the expression of PC-1, and the altered PC-1
expression may contribute to cyst formation seen in ADPKD.
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