910 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
Pkd1 regulates immortalized proliferation
of renal tubular epithelial cells through
p53 induction and JNK activation
Saori Nishio,1,2 Masahiko Hatano,2 Michio Nagata,3 Shigeo Horie,4
Takao Koike,1 Takeshi Tokuhisa,2 and Toshio Mochizuki1
1Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 2Department of Developmental Genetics (H2),
Graduate School of Medicine, Chiba University, Chiba, Japan. 3Department of Pathology, Institute of Basic Medical Sciences,
University of Tsukuba, Tsukuba, Japan. 4Department of Urology, Teikyo University School of Medicine, Tokyo, Japan.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic dis-
order and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The
cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD–/–) renal tubular
epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1–/– ES cells
and Pkd1+/+ morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the
kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1–/– and
Pkd1+/+ renal tubular epithelial cells in the early stages of cystogenesis. Pkd1–/– cyst epithelial cells changed in
shape from cuboidal to flat and replaced Pkd1+/+ cyst epithelial cells lost by JNK-mediated apoptosis in interme-
diate stages. In late-stage cysts, Pkd1–/– cells continued immortalized proliferation with downregulation of p53.
These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortal-
ized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic
fibroblasts from Pkd1–/– mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular
epithelial cells through the induction of p53 and activation of JNK.
Autosomal dominant polycystic kidney disease (ADPKD) is the
most common human monogenic genetic disorder and is charac-
terized by progressive bilateral renal enlargement with numerous
cysts and fibrosis in the renal parenchyma. It is often accompanied
by extra-renal manifestations, such as hypertension, intracranial
aneurysms, and hepatic and pancreatic cysts (1). The disease is pro-
gressive, and many patients develop renal insufficiency in the fifth
and sixth decades of life. Cystogenesis has been studied by micro-
dissection of ADPKD kidneys. The initial event in cyst formation
is believed to be the dilatation and “out-pocketing” of tubules.
The cysts arise from any segment of one nephron and maintain
continuity with the “parental” nephron (2). Fully developed cysts
are apparently isolated from the “parental” nephron and expand
through the accumulation of cyst fluid (3).
The PKD1 gene (encoding polycystin-1) (4) and the PKD2 gene
(encoding polycystin-2) (5) have been identified by positional
cloning as being the genes responsible for ADPKD. Loss of het-
erozygosity or second somatic mutations at the PKD1 or PKD2
loci have been reported in cystic epithelia from ADPKD patients
(6–10). Several lines of mice in which the Pkd1 or Pkd2 gene was
targeted show similar phenotypes. Although heterozygous knock-
out mice develop renal and hepatic cysts later in life (after age 16
months) (11), those mice do not fully recapitulate the severity of
human ADPKD. Homozygous knockout mice die in utero and
develop severely polycystic kidneys (12–16). Interestingly, com-
pound heterozygous Pkd2WS25/– mice, which carry a unique Pkd2
allele that is prone to genomic rearrangement leading to a null
allele, develop severely polycystic kidneys during adulthood and
thus resemble the ADPKD phenotypes (12). These model animals
suggested that a “2-hit” mechanism at either the PKD1 or PKD2
gene explains the late onset of the disease as well as some of the
variation in clinical symptoms (17, 18).
The molecular mechanisms of the cyst formation of Pkd-defi-
cient (Pkd–/–) renal tubular epithelial cells have been studied
extensively. Polycystin-1 and polycystin-2 are localized in the pri-
mary cilium of renal tubular epithelial cells (19). The relationship
between cystogenesis and the disruption of cilia has been reported
(20, 21). Although polycystin-2 in node monocilia contributes to
the development of left-right asymmetry (22), polycystin-1 and
polycystin-2 in the primary cilium transduce the extracellular
mechanical stimulus induced by urinary flow into increases in
cytosolic Ca2+, which may regulate renal tube size (19, 23).
The cyst epithelial cells of ADPKD kidneys have a high mitotic
rate in vitro (24) and in vivo, as detected by immunostaining for
proliferating cell nuclear antigen (PCNA) (25), c-Myc, and Ki-67
(26). Their high mitotic rate has also been supported by the fol-
lowing results. First, expression of growth factors such as EGF and
their receptors increases in ADPKD cysts (3, 27). Second, cAMP
stimulates the in vitro proliferation of ADPKD cyst epithelium
and cyst growth (28, 29). Third, overexpression of the Pkd1 gene
in a cell line induced cell cycle arrest at the G0/G1 phase with
upregulation of p21 through activation of the JAK-STAT pathway
(30). Thus, the proliferation of a PKD–/– cyst epithelial cell might
explain the cystogenesis of ADPKD kidneys. However, polycystin-1
Nonstandard abbreviations used: ADPKD, autosomal dominant polycystic kidney
disease; DBA, Dolichos biflorus agglutinin; LZ, LacZ; MEF, mouse embryonic fibroblast;
p-, phosphorylated; PCNA, proliferating cell nuclear antigen.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 115:910–918 (2005).
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
and polycystin-2 can be detected in some of the cyst epithelial cells
of ADPKD kidneys (31–36). These results suggest a contribution
of normal renal tubular epithelial cells to cystogenesis.
The cystogenesis of ADPKD kidneys cannot be fully reproduced
in the kidneys of Pkd1–/– mice, because these mice die in utero and
their renal tubular epithelial cells are not mosaic for Pkd1–/– and
normal cells, as are ADPKD kidneys. In an attempt to establish an
animal model for human ADPKD, we generated chimeric mice by
an aggregation method using Pkd1–/– ES cells and normal morulae
from LacZ+ (LZ+) ROSA26 mice (37). We show here that chime-
ric mice with a low degree of chimerism survived for more than
1 month and had multiple cysts not only in the kidneys but also
in the liver and pancreas, suggesting this may be a feasible model
for human ADPKD. Surprisingly, both Pkd1–/– and wild-type (LZ+)
epithelial cells were involved in early cystogenesis in kidneys of the
chimeric mice. We discuss here the molecular mechanisms of the
cystogenesis of Pkd1–/– and Pkd1+/+ renal tubular epithelial cells.
Cystogenesis of Pkd1–/–/LZ+ chimeric mice. We generated mice carry-
ing a mutation in the Pkd1 gene using standard gene-targeting
procedures by replacing exons 2–6 with the neomycin-resistance
gene (Figure 1A). Homozygous mutant (Pkd1–/–) mice died in
utero with severely polycystic kidneys and cardiac abnormali-
ties (data not shown), similar to previous descriptions (14, 15).
A second targeting vector with the hygromycin-resistance gene
(Figure 1B) was transfected into heterozygous (Pkd1+/–) ES cells
to obtain Pkd1–/– ES cells. Each gene targeting was confirmed by
Southern blot (Figure 1, C and D). Then, we generated chimeric
mice composed of mixtures of Pkd1–/– and wild-type cells. To
monitor cells derived from Pkd1–/– ES cells in chimeric mice, we
used morulae from LZ+ ROSA26 mice. Four independently tar-
geted Pkd1–/– ES clones were aggregated with ROSA26 morulae
to generate Pkd1–/–/LZ+ chimeric mice.
Several Pkd1–/–/LZ+ mice survived beyond 1 month of age, and
their survival closely depended on the degree of chimerism, as esti-
mated by coat color. When the contribution of Pkd1–/– ES cells to
coat color was more than 30%, the chimeric mice either died in
utero or died by P7 with severely polycystic kidneys. Pkd1–/–/LZ+
mice with a lower contribution (less than 10%) of Pkd1–/– ES cells
to their coat color survived beyond 1 month of age. Renal cysts
were detected in all the Pkd1–/–/LZ+ mice examined (n = 90). When
we compared chimerism and cyst formation in P7 Pkd1–/–/LZ+ kid-
neys, the incidence of cysts roughly correlated with the degree of
chimerism (Figure 2, A and B). Pkd1–/–/LZ+ kidneys were enlarged
due to scattered tubular cysts observed in both the cortex and the
outer medulla. These cysts occupied roughly 20–90% of the cut
surface of the kidneys, in parallel with the degree of chimerism. A
P60 Pkd1–/–/LZ+ mouse had bilateral enlarged kidneys deformed by
many cysts and often accompanied by hemorrhage (Figure 2C). Cut
surfaces of the kidney showed little renal parenchyma (Figure 2D).
This mouse also exhibited hepatic and pancreatic cysts. These
pathological findings in Pkd1–/–/LZ+ mice with low degree of chi-
merism were similar to those of human ADPKD.
To examine initial cyst formation in kidneys of Pkd1–/–/LZ+
and Pkd1–/– mice, we microdissected a single nephron from the
kidneys of those mice at E17.5. As shown in Figure 2E, multi-
ple “out-pocketing” cysts were observed in all segments of the
nephron from Pkd1–/–/LZ+ mice, whereas cysts in the nephrons
from Pkd1–/– mice were confined mainly to the distal tubule.
Surprisingly, the cyst epithelia in chimeric mice were composed
of not only Pkd1–/– cells but also LZ+ wild-type cells, as detected
by β-gal staining (Figure 2F). Histochemical examination also
showed the presence of LZ+ wild-type cells in the cyst epithelia of
Pkd1–/–/LZ+ kidneys (Figure 2G).
Dedifferentiation of cyst epithelial cells in Pkd1–/–/LZ+ mice. Cystogen-
esis in kidneys of Pkd1–/–/LZ+ mice with low degree of chimerism
was analyzed histologically between P1 and P30. At the early stage
(P1), small cysts were numerous and their cyst epithelia were com-
posed of many LZ+ cells and some Pkd1–/– cells (Figure 3A). At the
late stage (P30), individual cysts were enlarged and most of the
cyst epithelia were composed of Pkd1–/– cells. Similar histological
findings were observed in the livers of Pkd1–/–/LZ+ mice (data not
shown). Morphological analysis of cyst epithelial cells at the early
stage of cystogenesis demonstrated that many of the cyst epithelial
Pkd1–/– and LZ+ cells were cuboidal in shape (Figure 3B). Although
the shape of LZ+ cyst epithelial cells was still cuboidal at the inter-
mediate stage of cystogenesis, many Pkd1–/– cyst epithelial cells
changed their shape from cuboidal to flat (Figure 3C), suggesting
that flat cyst epithelial cells are dedifferentiated.
Generation of Pkd1–/– ES cells. (A and B) Genomic organization of 2 targeting vectors. Exons are depicted as filled boxes. The targeting vectors
were designed to replace a DNA segment of exons 2–6 by a neomycin-resistance gene cassette (neo) (A) or a hygromycin-resistance gene
cassette (hyg) (B). EGFP, gene encoding enhanced GFP. (C and D) Southern blots of genomic DNA derived from ES clones. Purified DNA
was digested with EcoRV and bands were detected by a probe, as described in Methods. Fragments corresponding to wild-type (15.1 kb) and
targeted (7.7 kb and 8.3 kb) alleles are shown. +/+, wild-type; –/–, Pkd1–/–.
912 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
To examine dedifferentiation of flat cyst epithelial cells, we
examined expression of polycystin-2 and acetylated tubulin as a
marker of primary cilia in the cyst epithelial cells of Pkd1–/–/LZ+
kidneys. All of the cyst epithelial cells expressed polycystin-2 regard-
less of morphological changes and Pkd1 expression (Figure 4A),
and both LZ+ and Pkd1–/– (LZ–) cyst epithelial cells manifested cilia
(Figure 4B). However, some of the cyst epithelial cells had lost
expression of Dolichos biflorus agglutinin (DBA) lectins (Figure 4C)
and Na-K ATPase (Figure 4D). The loss of expression did not cor-
relate with loss of the Pkd1 gene in cyst epithelial cells. The mean
cell height of cyst epithelial cells with or without Na-K ATPase
was lower than that of normal epithelial cells (P < 0.001), and the
cell height of cyst epithelial cells without Na-K ATPase
was slightly lower than that of cyst epithelial cells with
Na-K ATPase (P = 0.029) (Figure 4E), indicating a ten-
dency of correlation between the dedifferentiation and
the flat shape of cyst epithelial cells.
Proliferation and apoptosis of cyst epithelial cells.
Immunohistochemistry of cyst epithelial cells in Pkd1–/–/LZ+
kidneys revealed that LZ+ cells occasionally showed focal
hyperplastic features (Figure 5A) such as micropolyps,
as observed in human ADPKD. Some of cuboidal cyst
epithelial cells were accompanied by PCNA expression
(Figure 5B). We investigated expression of the cell cycle
regulators p21 and p53 in Pkd1–/–/LZ+ kidneys by West-
ern blot. Although very low expression of p21 has been
reported in the whole body of Pkd1–/– embryos at E15.5
(30), the amount of p21 in the kidneys of Pkd1–/– embry-
os at E16.5 and Pkd1–/–/LZ+ mice 1 month of age was
slightly less than that in wild-type mice (Figure 5C). Sta-
tistical analysis of the amount of p21 in 4 independent
experiments indicated a significant difference between
wild-type kidneys and Pkd1–/– kidneys (P = 0.016) but no
difference between wild-type kidneys and Pkd1–/–/LZ+
kidneys (P = 0.107). Interestingly, the amount of p53 in
Pkd1–/– (P = 0.003) and Pkd1–/–/LZ+ (P = 0.044) kidneys
was reduced compared with that in wild-type kidneys.
Indeed, the amount of p53 decreased in the cuboidal cyst
epithelial cells as well as in the flat cyst epithelial cells of
Pkd1–/–/LZ+ kidneys (Figure 5D).
To examine the proliferation of cyst epithelial cells in
vitro, we cultured microdissected single nephrons with
cysts from Pkd1–/–/LZ+ kidneys in collagen gel with 10%
FCS. Cells in cystically dilated parts of the nephrons rapid-
ly proliferated in a sheet-like fashion within 18 hours (Fig-
ure 5, E–G). Although the great majority were Pkd1–/– cells,
some LZ+ cyst epithelial cells proliferated. This significant
proliferation of Pkd1–/– and LZ+ cyst epithelial cells was
sustained by FCS, as a less significant proliferation was
observed in collagen gel without FCS (data not shown).
Cyst epithelia at the early stage of cystogenesis were
composed of cuboidal Pkd1–/– and LZ+ cells in Pkd1–/–/LZ+
kidneys, then flat Pkd1–/– cells became dominant in
cyst epithelia at the intermediate stage. As there were
many apoptotic cells present in Pkd1–/–/LZ+ kidneys at
the intermediate stage (data not shown), the cuboidal
LZ+ cells in the cyst epithelia might have been dead
due to apoptosis and then filled with flat Pkd1–/– cells.
Indeed, TUNEL staining of the cyst epithelial cells in
Pkd1–/–/LZ+ kidneys revealed scattered TUNEL-positive
cells (Figure 6A). Apoptosis in LZ+ cyst epithelial cells was 3- to
4-fold larger than that in Pkd1–/– cyst epithelial cells (Figure 6B).
Electron microscopic analysis of the cyst epithelia showed occa-
sional apoptotic figures in cuboidal cells overlaid by neighboring
cells (Figure 6C). In addition, flat cells overlaid several degenerated
cells that were detached from the tubular basement membrane
(Figure 6D), suggesting rearrangement by flat Pkd1–/– cells.
Signaling pathways in relation to proliferation or apoptosis of cyst epithelial
cells. Signaling pathways related to cell proliferation were analyzed
in the kidneys of Pkd1–/– and Pkd1–/–/LZ+ mice. Phosphorylated
EGFR (p-EGFR) detected in the cyst epithelial cells of Pkd1–/–/LZ+
kidneys was significantly greater than in those of wild-type kidneys
Pkd1–/–/LZ+ mice as an animal model of human ADPKD. (A) Appearance of a
P7 Pkd1–/–/LZ+ mouse with an intermediate chimeric rate. The chimeric rate
was estimated by coat color. (B) Kidneys of P7 Pkd1–/–/LZ+ mice. Low and Mid
(intermediate) indicate the chimeric rate as estimated by coat color: Low, less
than 10%; Mid, 10% to approximately 30%. (C) Kidneys of a P60 Pkd1–/–/LZ+
mouse. Black arrowheads indicate hemorrhagic cysts; white arrowhead indicates
pancreatic cysts. (D) Cross sections of kidney (Kid), liver, and pancreas (Panc)
of a P60 Pkd1–/–/LZ+ mouse. Approximately 90% of the renal parenchyma is
occupied by large cysts (PAS staining). Liver and pancreas show numerous
cysts (H&E). Original magnification, ×2 (kidney) and ×2.5 (liver and pancreas).
(E) Single nephrons of Pkd1–/–/LZ+, Pkd1–/–, and wild-type mice at E17.5. Mul-
tiple “out-pocketing” cysts are present in all segments of the nephron from the
Pkd1–/–/LZ+ mouse. Cystic dilation begins at the distal tubule of the nephron of
the Pkd1–/– mouse. Scale bar: 100 μm. (F) Staining of a microdissected tubule
with β-gal. A cystic fragment of the Pkd1–/–/LZ+ mouse was composed of Pkd1+/+
(blue; LZ+) and Pkd1–/– (white; LZ–) cells. Scale bar: 100 μm. (G) Histochemical
analysis of the kidney of a Pkd1–/–/LZ+ mouse at E17.5 with β-gal. The cyst (*)
began at tubules involving Pkd1–/– (LZ–) and LZ+ cells. Some tubules composed
of LZ– cells (black arrowheads) showed no cystic dilatation. Counterstaining:
Nuclear Fast Red. Original magnification, ×400.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
(data not shown). To elucidate the downstream signaling path-
way of growth factors, we analyzed the amount of activated MAP
kinases in kidneys using Western blot and immunohistochemistry.
Although the amount of p-ERK in Pkd1–/– and Pkd1–/–/LZ+ kid-
neys was not different from that in wild-type kidneys (Figure 7A),
expression of p-ERK was significantly more in the cyst epithelial
cells of Pkd1–/–/LZ+ kidneys regardless of their shape, cuboidal
(Figure 7B) or flat (data not shown).
As for signaling pathways related to apoptosis, the amount
of p-JNK was more in Pkd1–/–/LZ+ kidneys (P = 0.046) but
less in Pkd1–/– kidneys (P = 0.002) than in wild-type kidneys.
Immunohistochemistry revealed that p-JNK expression was
increased in the cuboidal cyst epithelial cells rather than in the
flat ones of Pkd1–/–/LZ+ kidneys, suggesting that LZ+ cyst epithe-
lial cells with p-JNK expression induce apoptosis. In contrast,
the amount of p-p38 in Pkd1–/–/LZ+ and Pkd1–/– kidneys was
similar to that in wild-type kidneys. Furthermore, the amount of
p-Akt, an apoptotic inhibitory signal, in both Pkd1–/– (P = 0.008)
and Pkd1–/–/LZ+ (P = 0.037) kidneys was more than that in
wild-type kidneys. Indeed, p-Akt expression was significantly
increased in both cuboidal (data not shown) and flat cyst epi-
thelial cells. The amount of Bcl-XL in Pkd1–/–/LZ+ kidneys was
clearly less than that in wild-type kidneys (P = 0.032), whereas
that in Pkd1–/– kidneys was similar to that in wild-type kidneys.
The amount of Bcl-2 and Bax in Pkd1–/–/LZ+ and Pkd1–/– kidneys
was similar to that in wild-type kidneys (P = 0.224 and 0.821,
respectively). These findings suggest that LZ+ cyst epithelial cells
are more apoptotic than are Pkd1–/– cyst epithelial cells.
Immortalized growth of Pkd1–/– mouse embryonic fibroblasts. Most
cyst epithelial cells in Pkd1–/–/LZ+ kidneys at the late stage of
cystogenesis were Pkd1–/– cells, and these grew very well in col-
lagen gel, suggesting a relationship between loss of Pkd1 and cell
immortalization. Because the 3T3 culture protocol of mouse
embryonic fibroblasts (MEFs) is one of the well characterized
experimental models of cell immortalization (38) and because
normal MEFs express Pkd1 (data not shown), we cultured Pkd1–/–
MEFs according to the 3T3 protocol. Wild-type MEFs entered a
characteristic cell cycle arrest known as cell senescence after pas-
sages 8–9 and immortalized cells (2 of 21 wells) appeared stochas-
tically and eventually overtook the senescent cells (Figure 8A).
In contrast, immortalized cells appeared in a large number of
wells (21 of 24 wells) in Pkd1–/– MEF cultures, thereby suggesting
a high incidence of immortalization.
Because the amount of the cell cycle regulator p16 increases in
MEFs at the senescence stage (39), we analyzed expression of the
cell cycle regulators p16, p21, and p53 in Pkd1–/– MEFs using West-
ern blot. The amount of these proteins was similar in Pkd1–/– and
wild-type MEFs until passage 8, whereas the amount of p16 clear-
ly increased in both Pkd1–/– and wild-type MEFs after passage 8
(Figure 8B). However, the amount of p53 in Pkd1–/– MEFs at pas-
Histochemical analysis of Pkd1–/–/LZ+ kidneys. Kidneys of
Pkd1–/–/LZ+ mice were stained with β-gal and counterstained
with Nuclear Fast Red. (A) Kidneys of Pkd1–/–/LZ+ mice with
the low chimeric rate at P1, P17, and P30. At the early stage
(P1), cyst epithelia were composed of Pkd1–/– (LZ–) and LZ+
cells. At the late stage (P30), individual cysts were enlarged
and most cyst epithelia were composed of Pkd1–/– (LZ–) cells.
Original magnification, ×200. (B) Kidney of a P3 Pkd1–/–/LZ+
mouse with the intermediate chimeric rate. At the early stage
of cystogenesis, both Pkd1–/– (LZ–) and LZ+ cyst epithelial cells
are cuboidal in shape. Original magnification, ×400. (C) Kidney
of a P8 Pkd1–/–/LZ+ mouse with the low chimeric rate. Cyst epi-
thelia are composed of flat Pkd1–/– (LZ–) cells and cuboidal LZ+
cells. Pkd1–/– (LZ–) cyst epithelial cells changed their shape
from cuboidal to flat. Original magnification, ×200.
Dedifferentiation of Pkd1–/– cyst epithelial cells.
(A–D) A kidney from a P8 Pkd1–/–/LZ+ mouse was
stained with anti–polycystin-2 (red, polycystin-2;
blue, DAPI) (A), anti–acetylated tubulin (red, acety-
lated tubulin; green, β-gal; blue, DAPI) (B), anti-DBA
(C) or anti–Na-K ATPase (D). (Right panels: A, C,
and D) The same section was stained with β-gal
and counterstained with Nuclear Fast Red. White
and black arrowheads indicate the same epithelial
cells. Original magnification, ×400. (B) Left and right
panels indicate stainings in cuboidal and flat cyst
epithelia, respectively. (E) Relationship between
cell height and Na-K ATPase expression in the cyst
epithelial cells shown in D. Each symbol indicates
a cyst epithelial cell (Pkd1–/–/LZ+) or normal tubular
epithelial cell (WT).
914 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
sage 30 (P = 0.001) but not that in wild-type MEFs at passage 13
(P = 0.636) was clearly less than that in Pkd1–/– and wild-type MEFs
at passage 1. The amount of activated MAP kinases in MEFs was
examined further. The amount of p-ERK in Pkd1–/– MEFs at passage
30 was slightly more than that in wild-type MEFs at passage 13.
Although the amount of p-JNK (P = 0.032) and p-p38 (P = 0.038)
increased in wild-type MEFs from passage 8 to passage 13, it was
stable in Pkd1–/– MEFs until passage 30. The amount of p-Akt in
Pkd1–/– MEFs was also stable until passage 30. Furthermore, the
amount of Bcl-2 family proteins was similar in Pkd1–/– and wild-
type MEFs at passages 30 and 13, respectively (data not shown).
In the present study, we developed Pkd1–/–/LZ+ chimeric mice.
The pathological findings in Pkd1–/–/LZ+ kidneys were similar to
those in human ADPKD kidneys. Therefore, Pkd1–/–/LZ+ mice, like
Pkd2WS25/– mice (12), are a feasible model for human ADPKD. As
intragenic recombination events in Pkd2WS25/– mice occurred grad-
ually and postnatally, as in human ADPKD, whereas Pkd1–/–/LZ+
mice have Pkd1–/– cells by inheritance, cyst formation in Pkd1–/–/LZ+
kidneys progressed more rapidly than that in Pkd2WS25/– and
human ADPKD kidneys. However, with Pkd1–/–/LZ+ mice, we
have the advantage of distinguishing Pkd1–/– cells from normal
(LZ+) cells and of monitoring the contribution
of Pkd1–/– cells in cystogenesis. Analyses of the
cyst epithelial cells in Pkd1–/–/LZ+ kidneys can
help us to understand the in vivo effect of poly-
cystin-1 on cystogenesis.
Proliferation of normal tubular epithelial cells
in early cystogenesis. Cystogenesis in human
ADPKD has been proposed as being a mono-
clonal proliferation of PKD1- or PKD2-defi-
cient epithelial cells (6–10). However, we found
that the cystic epithelium at the early stage of
cystogenesis was composed of both Pkd1–/– and
LZ+ wild-type cells. This finding is supported
by results showing that expression of polycys-
tin-1 and polycystin-2 was detected in most
cultured cells derived from ADPKD kidneys
(40). We stress that the strong expression of
polycystin-1 and polycystin-2 on cystic epi-
thelia in ADPKD kidneys (31–36) may reflect
involvement of normal cyst epithelial cells in
the cystogenesis of human ADPKD.
The initial cystogenesis in the kidney with
Pkd1–/– tubular epithelial cells requires stimula-
tion, as some of the tubules with Pkd1–/– epithe-
lial cells occasionally had no cystic dilatation and
metanephric culture of organs harvested from
Pkd1–/– mice at E13.5 failed to show cyst develop-
ment (data not shown). It has been suggested that
urinary flow promotes nephron development, in
particular, tubular elongation with cell differen-
tiation (41). During nephron development, it is
hypothesized that the renal tubular diameter is
maintained at the proper size (23) and that the
primary cilium affects the maintenance of the
tubular diameter by its mechanosensor function
(19, 20). Cilia structure and polycystin-2 expres-
sion were manifested in the cyst epithelial cells
of Pkd1–/–/LZ+ kidneys. Thus, we surmise that polycystin-1 in the
primary cilium is required for further inhibition of the proliferation
of tubular epithelial cells to maintain their proper size. A Pkd1–/– epi-
thelial cell, which is missing negative regulatory signals from poly-
cystin-1, continuously proliferates, and this proliferation induces a
“compensatory” proliferation of the surrounding normal epithelial
cells in an attempt to re-establish appropriate tubular diameter and
structure. This proliferation of tubular epithelial cells accounts for
early cyst formation in human ADPKD.
Proliferation of Pkd1–/– cyst epithelial cells. EGFR (14, 27), cAMP
(28, 29), Wnt/β-catenin (42), and p21 (30) have all been linked to
the proliferation of cyst epithelial cells. However, the relationship
between activation of these molecules and PKD deficiency is not
clear, as those studies assumed that only PKD–/– cells proliferated
in human ADPKD. Although downregulation of p21 expression in
whole embryos of Pkd1–/– mice has been suggested to be involved in
the proliferation of cyst epithelial cells (30) and we reproduced this
downregulation in Pkd1–/– kidneys, p21 expression in Pkd1–/–/LZ+
kidneys revealed only a slight decrease. The expression of p53 was
significantly decreased in the kidneys of Pkd1–/– and Pkd1–/–/LZ+
mice. These results support the findings that p53 expression
is decreased in human embryonic kidney 293 cells with loss of
polycystin-1 activity (43) and is also slightly decreased in human
Proliferation of Pkd1–/– cyst epithelial cells. (A) A kidney from a P17 Pkd1–/–/LZ+ mouse
was stained with β-gal and counterstained with Nuclear Fast Red. LZ+ epithelial cells
occasionally showed focal hyperplastic features such as micropolyps. Original magnifica-
tion, ×400. (B) A kidney froma P12 Pkd1–/–/LZ+ mouse was stained with an anti-PCNA.
Some cuboidal LZ+ cyst epithelial cells were accompanied by PCNA expression. Original
magnification, ×400. (C) Expression of p21 and p53 in the kidneys of Pkd1–/– mice at
E16.5 and Pkd1–/–/LZ+ mice 1 month of age. The amount of p21 and p53 in kidneys was
examined using Western blot. Actin was used as a loading control for protein. Data pre-
sented are 1 representative of 4 independent experiments. (D) Kidneys of P12 Pkd1–/–/LZ+
and P12 wild-type mice were stained with anti-p53. Expression of p53 was detected in
the flat epithelial cells (white arrowhead) but was significantly decreased in the cuboidal
cyst epithelial cells (black arrowheads) of the Pkd1–/–/LZ+ mouse. Original magnification,
×400. (E–G) The proliferation of cyst epithelial cells in vitro. A single nephron isolated by
microdissection from the kidney of a Pkd1–/–/LZ+ mouse at E17.5 was cultured in collagen
gel for 18 hours. (F and G) Higher magnifications of the boxed area above. Both LZ+ and
Pkd1–/– (LZ–) cells proliferated. Scale bars: 100 μm.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
ADPKD kidneys compared with normal kidneys (26). p53 inhibits
cell cycle by induction of p21 (44), and polycystin-1 inhibits Cdk2
activity by upregulation of p21 through the activation of JAK2
(30). Thus, the decrease in p53 in addition to the lack of activa-
tion of the JAK-STAT pathway in Pkd1 deficiency may compound
the decrease in p21 expression. Because expression of p53, among
the cell cycle regulators examined, was affected most strongly in
both Pkd1–/– cyst epithelial cells and immortalized Pkd1–/– MEFs,
polycystin-1 may regulate the growth of renal tubular epithelial
cells through induction of p53.
Pkd1–/– cyst epithelial cells were not transplantable in nude mice
(data not shown). Isolated early cysts from Pkd1–/–/LZ+ kidneys
exhibited significant proliferation in vitro with 10% FCS, whereas
the proliferation was stunted without FCS (data not shown), sug-
gesting that proliferation of Pkd1–/– cyst epithelial cells is not auton-
omous, as in neoplasms, but instead is growth factor dependent.
Among many growth factors and their receptors related with cys-
togenesis, strong EGFR expression was observed on cystic epithelia
of Pkd1–/– kidneys (14). Interestingly, EGFR expression increased
on both cuboidal and flat cyst epithelial cells in Pkd1–/–/LZ+
kidneys (data not shown). This was supported by the finding of
scattered activation of the ERK pathway in both cuboidal and flat
cyst epithelial cells in Pkd1–/–/LZ+ kidneys.
Dedifferentiation of Pkd1–/– cyst epithelial cells. The cyst epithelia in
human ADPKD are composed of cuboidal cells such as normal
renal epithelial cells and flat cells (24). A similar phenomenon was
noted in Pkd2WS25/– kidneys. These 2 morphologically different cells
constitute the cyst epithelium at the early and intermediate stages
of cystogenesis (45). We also detected 2 kinds of cyst epithelial cells
in Pkd1–/–/LZ+ kidneys. Both Pkd1–/– and LZ+ cyst epithelial cells
were cuboidal in shape at the early stage of cystogenesis, and some
Pkd1–/– cyst epithelial cells changed their shape to flat at the inter-
mediate stage. As LZ+ cyst epithelial cells are nearly cuboidal, Pkd1
deficiency is related to the morphological change.
Most flat cyst epithelial cells are negative for nephron segment
markers and Na-K ATPase (45), suggesting that the morphological
transition of Pkd1–/– cyst epithelial cells is accompanied by loss of
functional phenotype. However, expression of renal tubular markers
within single cysts in Pkd1–/–/LZ+ kidneys was discontinuous. Loss
of expression was also detected in the cuboidal cyst epithelial cells at
the early stage of cystogenesis. Although there is a tendency for cor-
relation between loss of Na-K ATPase expression and flat shape of
cyst epithelial cells, the morphological change of cyst epithelial cells
is not completely correlated with the loss of tubular markers.
The cell adhesion molecules E-cadherin and β-catenin are bound
to polycystin-1 and polycystin-2 (42), and E-cadherin expression
decreases in Pkd1–/– kidneys (14). Thus, Pkd1 deficiency may change
the polarity of cyst epithelial cells by affecting cell adhesion or
Apoptosis of cyst epithelial cells. (A) Apoptosis of cyst epithelial cells
in the kidney of a P8 Pkd1–/–/LZ+ mouse was detected by the TUNEL
assay. The same section was stained with β-gal and counterstained
with Nuclear Fast Red. Arrowheads indicate TUNEL-positive cells. Origi-
nal magnifications, ×400. (B) Summary of results shown in A. Each
graph represents the percentage of TUNEL-positive cells in LZ+ (black
bars) and in LZ– (Pkd1–/–) cyst epithelial cells (white bars), respectively.
The mean and SD are from 9 independent mice. *P < 0.05. (C and D)
Electron microscopic analysis of the cyst epithelium of a Pkd1–/–/LZ+
kidney. (C) Occasional apoptotic cells (black arrowheads) are overlaid
by neighboring cells. (D) Flat cells (white arrowheads) overlay several
degenerated cells that are detached from the tubular basement mem-
brane (black arrowheads). Original magnification, ×1,500.
Signaling pathways related to proliferation or apoptosis in
cyst epithelial cells. (A) Expression of signal transducers in
the kidneys of Pkd1–/– mice at E16.5 and Pkd1–/–/LZ+ mice
1 month of age was analyzed using Western blot. Actin was
used as a loading control for protein. Data presented are 1
representative of 4 independent experiments. (B) A kidney
from a P8 Pkd1–/–/LZ+ mouse was stained with anti–p-ERK,
anti–p-JNK, or anti–p-Akt. The same section was stained with
β-gal and counterstained with Nuclear Fast Red. Black and
white arrowheads indicate the same epithelial cells. Original
916 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
cytoskeletal organization. However, the localization and intensity
of both E-cadherin and β-catenin in some cyst epithelial cells were
similar to those in normal renal tubular cells at the intermediate
stage of cystogenesis (data not shown). These results indicate that
the morphological change in cyst epithelial cells is not due to loss
of tubular markers or to repression of cell adhesion molecules. As
Pkd1–/– cyst epithelial cells cultured in collagen gel sometimes made
tubules in the gel (data not shown), outgrowing Pkd1–/– cyst epi-
thelial cells retain some functions of renal tubular epithelial cells.
Further studies will be done to elucidate the cause of the morpho-
logical change and the dedifferentiation (loss of tubular markers)
of cyst epithelial cells initiated by deficiency in the Pkd1 gene.
Apoptosis on normal (LZ+) cyst epithelial cells. Apoptosis has been fre-
quently observed in non-dilated and cystic tubuli and glomeruli
in ADPKD kidneys, whereas it is extremely rare in normal kidneys
(46). In addition, the increased rate of growth in cyst epithelial cells
is accompanied by an increased rate of apoptosis in human ADPKD
(26). Of note, our study showed that apoptotic cells were present
mainly in cuboidal epithelial cells in Pkd1–/–/LZ+ kidneys. Elec-
tron microscopy revealed characteristic apoptotic features among
cuboidal cyst epithelium, which was covered by flat cells. Apoptotic
cells were lost from cyst epithelium and neighboring flat cells lined
tubular lumina. These findings suggest net replacement of cuboi-
dal LZ+ epithelial cells by flat Pkd1–/– epithelial cells.
Expression of p-JNK increased in Pkd1–/–/LZ+ kidneys but not
in Pkd1–/– kidneys. In contrast, Bcl-XL expression was decreased in
Pkd1–/–/LZ+ kidneys but not in Pkd1–/– kidneys. Although p-Akt
expression was significantly increased in both cuboidal and flat
cyst epithelial cells, cuboidal cyst epithelial cells are more apoptotic
than are flat cyst epithelial cells. The 3T3 cell
cultures using Pkd1–/– MEFs also demon-
strated that expression of p-JNK and p-p38
was increased in wild-type MEFs at the cell
senescence stage (after passage 13). However,
this expression did not increase in immortal-
ized Pkd1–/– MEFs until passage 30. As poly-
cystin-1 triggers activation of JNK but not
that of p38 (47), flat Pkd1–/– epithelial cells
and Pkd1–/– MEFs in the 3T3 culture escape
apoptosis mediated by activation of JNK.
These immortalized flat Pkd1–/– epithelial
cells slowly spread to form large cysts.
A model of cystogenesis. We developed chime-
ric mice by aggregation of Pkd1–/– ES cells and
Pkd1+/+ morulae of LZ+ ROSA26 mice. These
mice are a unique mouse model for human ADPKD. In Pkd1–/–/LZ+
kidneys, sporadic Pkd1–/– epithelial cells deteriorated the entire
tubular integrity by the proliferation of both Pkd1–/– and normal
(LZ+) epithelial cells at the early stage of cystogenesis (Figure 9).
When tubular epithelial cells, including Pkd1–/– epithelial cells,
receive stimulation, both Pkd1–/– and normal tubular epithelial
cells proliferate to expand the tubular size. The Pkd1–/– tubular epi-
thelial cells lack negative signals for proliferation by polycystin-1
and continue to proliferate. Although surrounding normal tubu-
lar epithelial cells also proliferate to retain both the round shape
and diameter of the tubule, normal epithelial cells are gradually
lost by JNK-mediated apoptosis at the intermediate stage. Some
Pkd1–/– tubular epithelial cells change shape from cuboidal to flat
(dedifferentiation), and the flat Pkd1–/– epithelial cells grow in an
immortalized fashion to form large cysts in the kidney at the late
stage of cystogenesis. As p53 expression and JNK activation were
very low in flat Pkd1–/– cyst epithelial cells, polycystin-1 plays a role
in the prevention of immortalized proliferation of renal tubular
epithelial cells via p53 induction and JNK activation.
Generation of Pkd1–/– mice. Murine Pkd1 genomic clones were obtained by
screening a 129/Sv mouse genomic library (14). R1 ES cells were transfect-
ed with linearized Pkd1 neomycin-targeting vectors by electroporation and
were subjected to positive and negative selection for 14 days using G418
and diphtheria toxin. Approximately 134 clones were examined using
Southern blot, and homologous recombination was detected in 18 clones.
One independent targeted ES clone was used to generate chimeric mice
using the aggregation method (48). DNA from tail tissue of agouti pups
Outgrowing Pkd1–/– MEFs. (A) The 3T3-type culture
of MEFs from Pkd1–/– and wild-type mice. (B) Signal-
ing pathways related to proliferation or apoptosis in
Pkd1–/– MEFs. Expression of signal transducers and
cell cycle regulators was analyzed using Western
blot. Actin was used as a loading control for protein.
Data presented are 1 representative of 4 indepen-
A model schema of the cystogenesis in ADPKD. The germline mutation of 1 allele of the Pkd1
gene is present in all tubular epithelial cells.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
obtained by mating chimeric mice with C57BL/6 mice (Japan SLC) was
analyzed using Southern blot. Homozygous mutant pups were generated
by intercrossing of heterozygous mutant mice. All procedures conformed
to the Chiba University Resolution on Use of Animals in Research and
were approved by the Institutional Animal Care and Use Committee of the
Graduate School of Medicine, Chiba University (Chiba, Japan).
Generation of Pkd1–/– ES cells and Pkd1–/–/LZ+ chimeric mice. One of the Pkd1-
targeted ES clones was transfected by electroporation with linearized Pkd1
hygromycin targeting vectors to generate Pkd1–/– ES cells. Approximately
17 ES clones were examined by Southern blot, and 4 independent Pkd1–/–
ES clones were obtained. Those Pkd1–/– ES cells were aggregated with mor-
ulae of ROSA26 mice with the exogenous LacZ gene (a gift from H. Koseki,
RIKEN Research Center for Allergy and Immunology, Yokohama, Japan)
to generate Pkd1–/–/LZ+ chimeric mice.
Southern blot. Genotyping was done by digestion of genomic DNA (10 μg)
with EcoRV, Southern transfer, and hybridization with a 1.3-kb DNA probe
that was external to the targeting vector. The probe was labeled with digoxi-
genin (Roche Diagnostics) using PCR. The probe detected the wild-type allele
as a 15.1-kb fragment and the mutant alleles as 7.7-kb and 8.3-kb fragments.
Histology and immunohistochemistry. Tissues were fixed in 10% phos-
phate-buffered formalin and were embedded in paraffin. Sections
(3 μm thick) were stained with H&E according to standard protocols. For
immunohistochemistry, after deparaffinization through a graded xylene
and ethanol series, sections were washed in PBS (pH 7.4) and were treated
for 15 minutes with 0.3% hydrogen peroxide in methanol. After blocking,
sections were stained with the following antibodies: anti-p53, anti–p-EGFR
(Santa Cruz Biotechnology Inc.), and anti-PCNA (Sigma-Aldrich). For
immunofluorescence, frozen section were stained with YCC2 (anti–polycys-
tin-2; a kind gift from Y. Cai, Yale University, New Haven, Connecticut, USA),
anti–β-gal (Chemicon International Inc.), anti–Na-K ATPase (Upstate), anti–
acetylated tubulin, anti-DBA, anti–lectin Lotus tetragonolobus (Sigma-Aldrich),
anti–p-ERK, anti–p-Akt, or anti–p-JNK (Cell signaling Technology Inc.). Pho-
tomicrographs were obtained using a microscope (Carl Zeiss International).
β-gal staining of kidneys. Kidneys were fixed for 30 minutes at 4°C in 2.7%
formaldehyde, 0.02% NP-40, and 0.2% glutaraldehyde in PBS (pH 7.4) and
were washed. Processing was carried out through a graded series of sucrose
concentrations from 15% to 30% in PBS at 4°C for 5–12 hours for each
step. Kidneys were then embedded in OCT (Tissue-Tek) and were frozen in
2-methyl-butane submerged in liquid nitrogen. Sections (3 μm thick) were
then prepared, mounted on slides, and washed in PBS for 5 minutes, and were
subsequently stained at 37°C overnight in X-gal solution (1 mg/ml X-gal
in DMSO, 2 mM MgCl2, 20 mM potassium ferricyanide, 20 mM potassium
ferrocyanide, and 0.02 % NP-40 in PBS). Sections were counterstained with
Nuclear Fast Red (Trevigen Inc.).
Microdissection of nephron segments. Nephrons were isolated from the kid-
neys of wild-type, Pkd1–/–, and Pkd1–/–/LZ+ mice at E17.5. Microdissection
of tubules was done in PBS under a stereomicroscope (2).
Western blot. Kidneys were sonicated in Tris lysis buffer (20 mM Tris-HCl,
150 mM NaCl, 100 mM NaF, 1 mM EDTA, 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride, 1.5 nM aprotinin, and 10 nM leu-
peptin). Proteins were separated by SDS-PAGE and were transferred to
polyvinylidene difluoride membranes (Millipore). Membranes were blocked
with nonfat dry milk (Yukijirushi) and were incubated with the following
antibodies: anti-p53, anti-p21, anti-ERK, anti–Bcl-XL, anti-Bax, anti-p16,
anti-actin (Santa Cruz Biotechnology Inc.), anti-Akt, anti–p-Akt, anti–
p-ERK, anti-p38, anti–p-p38 (Cell Signaling Technology Inc.), anti-JNK,
anti–p-JNK (BD Biosciences–Pharmingen), or anti–Bcl-2 (R&D Systems).
The filters were washed with TBS/0.1% Triton-X, and immunoreactive
bands were visualized by enhanced chemiluminescence.
In vitro culture of microdissected tubules. Microdissection of tubules was
done in L-15 medium (Sigma-Aldrich) followed by culture at 37°C in 5%
CO2 in collagen gel (Neutral Solution, DMEM Culture Medium; Koken) in
DMEM supplemented with 10% FCS (Sigma-Aldrich).
TUNEL assay. Animals were perfused with a solution of 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Organs were dis-
sected and were post-fixed overnight with 4% paraformaldehyde. The tis-
sues were equilibrated with 20% sucrose and were cut into sections 3 μm
in thickness on a cryostat. The TUNEL assay was carried as described
with slight modification (49). The tailing reaction was carried out for
1 hour at 37°C in TdT buffer in the presence of dUTP-biotin (The Meb-
stain Apoptosis kit, Medical & Biological Laboratories). Signals were
visualized using Avidin-Rohdamine (Vector Laboratories). Sections were
counterstained with DAPI (Molecular Probes).
Electron microscopy. Specimens were fixed in formalin followed by 2% glu-
taraldehyde, were post-fixed with 1% osmium tetroxide, and were embed-
ded in epoxy resin mixture. Ultrathin sections were mounted on grids,
stained with uranyl acetate–lead citrate, and observed under a transmis-
sion electron microscope (Hitachi).
Cell culture. MEFs were established from Pkd1–/– embryos (E13.5).
Heads and livers were removed from embryos, and the remaining embry-
onic tissues were trypsinized at 37°C for 30 minutes. The disrupted tis-
sues were plated in DMEM supplemented with 10% FCS (Sigma-Aldrich)
and were cultured at 37°C in 5% CO2. The 3T3-type serial MEF cultiva-
tion was done as described (38). Briefly, 3 × 105 cells were plated on
a 6-cm well; 3 days later, the total number of cells was counted, and
3 × 105 cells were plated on a separate well. The cumulative increase in
cell number was calculated according to the formula Log(Nf / Ni)/Log2,
where Ni is the initial number of cells plated and Nf is the final number
of cells counted after 3 days.
Statistical analysis. Data presented represents the mean ± SD of more
than 3 independent experiments. Statistical analysis was performed
using an unpaired Student’s t test. P values of less than 0.05 were con-
sidered to be significant.
We are grateful to H. Koseki and S. Somlo for discussions and to
Y. Cai for providing YCC2 (anti–polycystin-2). We also thank L.
Fujimura, H. Satake, K. Hanaoka, J. Usui, S. Horita, and H. Haza-
wa for skillful technical assistance; N. Kakinuma for secretarial
services; and M. Ohara for language assistance. This work was
supported in part by Grants-in-Aid from the Ministry of Educa-
tion, Science, Technology, Sports and Culture of Japan, a grant
from the Inamori Foundation (to T. Mochizuki), and a grant
from Sankyo Foundation of Life Science (to T. Mochizuki).
Received for publication July 28, 2004, and accepted in revised
form January 11, 2005.
Address correspondence to: Toshio Mochizuki, Department of Med-
icine II, Hokkaido University Graduate School of Medicine, Kita 15,
Nishi 7, Kita-ku, Sapporo 060-8638, Japan. Phone: 81-11-716-1161;
Fax: 81-11-706-7710; E-mail: email@example.com.
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