George M. O'Brien Kidney and Urological Diseases Center, Renal Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, 63110
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ABSTRACT |
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Tight regulation
of the rates of cell proliferation and apoptosis is critical for normal
nephrogenesis. Nephrogenesis is profoundly affected by the loss of
bcl-2 expression. Bcl-2-deficient (bcl-2 /
) mice are born
with renal hypoplasia and succumb to renal failure secondary to renal
multicystic disease. Cell-cell and cell-matrix interactions impact
tissue architecture by modulating cell proliferation, migration,
differentiation, and apoptosis. E-cadherin mediates calcium-dependent
homotypic cell-cell interactions that are stabilized by its association
with catenins and the actin cytoskeleton. The contribution of altered
cell-cell interactions to renal cystic disease has not been delineated.
Cystic kidneys from bcl-2
/
mice displayed nuclear
localization of
-catenin and loss of apical brush border actin
staining. The protein levels of
-catenin,
-catenin, actin, and
E-cadherin were not altered in cystic kidneys compared with normal
kidneys. Therefore, an altered distribution of
-catenin and actin,
in kidneys from bcl-2
/
mice, may indicate improper
cell-cell interactions interfering with renal maturation and
contributing to renal cyst formation.
-catenin; adherens junctions; actin; renal multicystic disease
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INTRODUCTION |
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RENAL CYSTIC DISEASE (RCD) includes both autosomal dominant and recessive forms, as well as acquired forms. RCD has profound clinical implications and affects a significant number of individuals in the United States (9). Abnormalities in kidney development, growth, differentiation, and apoptosis precede and play a causative role in RCD (3, 4, 7, 8, 10, 22, 23). However, the precise manner in which abnormalities in these processes are causative of RCD is not completely understood.
Proper regulation of apoptosis is an absolute requirement for normal
nephrogenesis (6, 18, 21). B-cell lymphoma/leukemia-2 (bcl-2) is a
novel protooncogene that inhibits apoptosis in a variety of settings
(6). Apoptosis within the developing kidney occurs in areas where bcl-2
is not expressed (5). The loss of bcl-2 expression has a profound
effect on nephrogenesis. Transgenic mice that manifest a loss of
function mutation for bcl-2 (bcl-2 /
mice) complete
embryonic development but are born with renal hypoplasia, which results
from excessive apoptosis of the metanephric blastema during kidney
formation (19). Eventually these animals succumb to renal failure
secondary to RCD (19, 21). Significant cyst formation in kidneys of the
bcl-2
/
mice coincides with kidney maturation. Cyst
formation occurs together with hyperproliferation of tubular epithelium
and increased apoptosis relative to that observed in wild-type (bcl-2
+/+) mice (18).
Cell-cell and cell-matrix interactions impact cell proliferation, migration, differentiation, and apoptosis. Cell adhesion mechanisms determine tissue architecture and are responsible for cell assembly and connection to the internal cytoskeleton (12). Cadherins involved in homotypic interactions mediating cell-cell adhesion colocalize to sites of cell-cell contact with actin-associated junctions (12).
Cadherin-mediated cell-cell adhesion is necessary for establishment and
maintenance of epithelial cell polarity and tight junctions. Several
cadherins are expressed in the kidney including E-cadherin, K-cadherin,
N-cadherin, and cadherin-11. The interactions of
E-cadherin are well documented particularly in the kidney and in Madin-Darby canine kidney (MDCK) cells. Cell adhesions mediated by
E-cadherin require intracellular attachment to the actin cytoskeleton. This is accomplished through E-cadherin's cytoplasmic interaction with
catenins. E-cadherin complexes with -catenin through its cytoplasmic
domain. Cadherin-catenin complexes are formed when
-catenin binds to
-catenin.
-Catenin then can link these cadherin-catenin complexes to the actin cytoskeleton (12). Catenins also act as general
linkers or adapters by interacting with transmembrane or cytoplasmic
proteins. For example,
-catenin associates with the adenomatous
polyposis coli (APC) tumor suppressor protein, epidermal growth factor
receptor, and the tight junction protein zonula occluden-1 protein (2,
11, 13). However, the contribution of these interactions during renal
maturation or renal multicystic disease has not been delineated.
This study examines the expression and distribution of -catenin,
-catenin, actin, and E-cadherin, which participate in establishment and maintenance of cell-cell adhesion and actin cytoskeletal
organization in postnatal kidneys from bcl-2 +/+ and bcl-2
/
mice. Disruption of the cadherin/catenin complex
organization occurred in kidneys from bcl-2
/
mice. An
altered distribution of
-catenin and loss of brush border actin
staining were observed after significant cyst formation had occurred.
Therefore, the aberrant distribution of proteins that participate in
cell-cell adhesion accompany and may precipitate renal cyst formation
in the absence of bcl-2.
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METHODS |
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Animal breeding. Bcl-2 heterozygote
animals were interbred. The genotypes of the offspring were determined
by PCR analysis. The Neo primers
(5'-GCTCTTCAGCAATATCACGG-3' and
5'-GGAGAGGCTATTCGGCTATG-3' ) yielded a 650-bp fragment, and
the bcl-2 primers
(5'-CTTTGTGGAACTGTACGGCCCCAGCATGCG-3' and
5'-ACAGCCTGCAGCTTTGTTTCATGGTACATC-3') yielded a 215-bp
fragment. Thus the wild-type animals would
have only the bcl-2 fragment, the heterozygote animals would have both
fragments, and the bcl-2 /
animals would have only the
Neo fragment.
Processing of kidneys for histological studies and
immunohistochemistry. Kidneys were surgically removed
from mice, placed in OCT compound (VWR Scientific, St.
Louis, MO) and rapidly frozen. Sections of 7 µm each were placed on
polylysine coated slides (Sigma, St. Louis, MO). In experiments
examining the expression of E-cadherin, -catenin, and
-catenin,
the sections were fixed in cold acetone, washed in PBS,
and incubated in PBS blocking buffer (PBS containing 1%
bovine serum albumin, 0.3% Triton X-100, and 0.2% skim milk powder)
for 15 min. The sections were then incubated with rabbit polyclonal
antibodies to
-catenin (1:500, Sigma) or
-catenin (1:600, Sigma)
or rat monoclonal antibodies to uvomorulin (1:600, Sigma) and
Lotus tetragonobolus agglutinin (LTA;
Vector Labs, Burlingame, CA) overnight at 4°C. The sections were
stained with LTA as a marker for proximal tubules (PT) as well as the
apical membrane. The sections were incubated with appropriate
indocarbocyanine (CY3)-labeled secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Some of the sections stained with
-catenin were incubated with Hoechst 33258 (1:1,000; Sigma) in PBS
for 15 min and rinsed in PBS. In addition, for DNA synthesis studies,
sequential kidney sections from mice injected with
5-bromo-2'-deoxyuridine (BrdU) were stained with anti-BrdU as
previously described (18) or anti-
-catenin and LTA. To examine actin
expression, the sections were fixed in 3% paraformaldehyde on ice for
20 min, washed in PBS, incubated in PBS-blocking buffer, and incubated
in PBS-blocking buffer containing tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)-labeled phalloidin (1: 150) (Sigma) for 2 h
at room temperature. The slides were then photographed.
Protein lysate preparation and Western blot
analysis. Kidneys were homogenized and sonicated in a
buffer containing 142.5 mM KCl, 5 mM
MgCl2, 10 mM HEPES, pH 7.5, 1%
Nonidet P-40, 2 mM orthovanadate, 2 mM sodium fluoride,
and complete protease inhibitor cocktail (Boehringer Mannheim,
Indianapolis, IN). The protein concentration was determined utilizing a
Bio-Rad DC protein assay. Twenty micrograms of total protein lysate was
electrophoresed in a 4-20% polyacrylamide gel and transferred to
a Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights,
IL). The membranes were blocked in TBST (20 mM Tris, pH 7.6, 137 mM
NaCl, and 0.05% Tween) containing 3% bovine serum albumin and 3%
nondairy creamer for 1 h at room temperature. The membranes were then
incubated with rabbit polyclonal antibodies to -catenin (1:4,000;
Sigma), rabbit polyclonal antibodies to
-catenin (1:4,000; Sigma),
rat monoclonal antibodies to uvomorulin (1:4,000; Sigma), or mouse monoclonal antibodies to actin (1:500; Sigma) overnight at 4°C. The
membranes were then washed with TBST, incubated with the appropriate secondary antibody (Pierce, Rockford, IL), washed with TBST, and developed with ECL (Amersham).
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RESULTS |
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Altered organization of actin in absence of
bcl-2. The organization of cadherin/catenin complexes
was examined in kidneys from bcl-2 +/+ and bcl-2 /
mice
to determine whether disruption of these interactions occurred during
renal cyst formation. The distribution of actin was examined at birth,
prior to cyst formation, and later, postnatal day
21 (P21), when
significant cyst formation is noted in kidneys from bcl-2
/
mice (18). Actin staining of kidneys from newborn
(P0) bcl-2 +/+ and bcl-2
/
mice was similar. In the nephrogenic zone (capsule
noted by arrows), apical actin staining was noted in PT and
condensates, whereas actin staining on the periphery of cells was noted
in interstitium (Fig. 1, A and
B). Kidneys from
P21 bcl-2 +/+ mice displayed apical brush border actin staining in PT (Fig.
1C, arrowhead) and on the
periphery of individual cells in distal tubules and
collecting duct. However, the distribution of actin in kidneys from
P21 bcl-2
/
mice was
altered. Cystic PT (Fig.
1D), as well as cystic distal tubules and collecting duct, displayed a loss of apical brush border
actin staining (Fig. 1D, solid arrow)
although some noncystic PT did retain apical brush border actin
staining (open arrow, Fig. 1D).
Peripheral actin staining of cells in cystic tubules was never
observed. Thus the altered actin distribution observed in cystic
kidneys may influence interactions of other proteins that are linked to
the actin cytoskeleton.
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Altered organization and nuclear localization of
-catenin accompanies cyst formation.
Cadherin-catenin complexes attach to the actin cytoskeleton. These
interactions are essential for stabilization of adherens junctions and
communication from the outside of the cell to the inside. E-cadherin
complexes with
-catenin through its cytoplasmic domain (12). Thus
altered expression or distribution of
-catenin could affect adherens
junction integrity. The distribution of
-catenin was examined in
kidneys from bcl-2 +/+ and bcl-2
/
mice.
-Catenin was
expressed in all nephron segments. Kidneys from newborn bcl-2 +/+ and
bcl-2
/
mice demonstrated similar staining for
-catenin with intense immunoreactivity on basolateral membranes and
at sites of cell-cell contacts (Fig. 2,
A and
B). Kidneys from
P21 bcl-2 +/+ mice demonstrated
-catenin immunoreactivity at sites of cell-cell contacts (Fig.
2C). The distribution of
-catenin
within nephron segments from bcl-2
/
mice
remained similar to that of bcl-2 +/+ littermates until significant
cyst formation was observed. At P21,
cystic kidneys from bcl-2
/
mice demonstrate nuclear
-catenin staining. The arrow in Fig. 2D indicates nuclear
-catenin
staining in a collecting duct from a kidney of a
P21 bcl-2
/
mouse, which
can be compared with the collecting duct in a kidney from a normal
littermate (arrow, Fig. 2C). As
illustrated in Fig. 3,
A and
B, nuclear localization was confirmed
by incubating slides stained with
-catenin with Hoechst 33258. Furthermore, nuclear localization of
-catenin was typically observed
in tubules in kidneys from bcl-2
/
mice that were
hyperproliferative. The arrow in Fig.
3C points to several nuclei that stain
positively for
-catenin. Staining of a sequential section for
incorporation of BrdU demonstrates that many cells within
this PT and in the same area in which nuclear
-catenin staining was
noted (arrow, Fig. 3D)
were undergoing DNA synthesis. However, DNA synthesis did occur in
areas where nuclear
-catenin staining was not observed. In kidneys
from normal P21 mice, rare nuclei
stained positively for BrdU (data not shown), similar to our previous report (18).
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Distribution of -catenin.
-Catenin is another component of adherens junctions. It links
cadherin-catenin complexes to the actin cytoskeleton and is detected at
sites of cell-cell contacts (12).
-Catenin was expressed in all
nephron segments. Strong basolateral membrane staining for
-catenin
was observed in kidneys from P0 bcl-2
+/+ and bcl-2
/
mice (Fig. 4,
A and
B). The pattern of expression and
distribution of
-catenin was similar in kidneys from
P0 and
P21 bcl-2 +/+ and bcl-2
/
mice.
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Distribution of E-cadherin. E-cadherin
is involved in homotypic interactions mediating cell-cell adhesion and
localizes to sites of cell-cell contacts with actin-associated
junctions. Cell adhesion mediated by E-cadherin requires
intracellular attachment to the actin cytoskeleton, which is
accomplished through interactions with catenins (12). E-cadherin was
expressed in most nephron segments, except glomeruli, in kidneys
from bcl-2 +/+ and bcl-2 /
mice. Kidneys from
bcl-2 +/+ and bcl-2
/
mice demonstrated a
similar pattern of expression and distribution at
P0 and
P21 (Fig.
5).
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Levels of adherens junction proteins were not altered
during cyst formation. Western blot analysis was
utilized to determine the levels of -catenin,
-catenin, actin,
and E-cadherin in kidney lysates. Protein lysates were prepared from
kidneys of P7 and P21 bcl-2 +/+ and bcl-2
/
mice. Examination of protein levels at
P7 determined levels prior to kidney
maturation and prior to significant renal cyst formation (bcl-2
/
), whereas examination at
P21 determined levels following kidney
maturation and after significant renal cyst formation was observed
(bcl-2
/
). Illustrated in Fig. 6 is a
representative Western blot. The level of
-catenin,
-catenin,
actin, and E-cadherin was unchanged between comparable kidney protein
lysates from bcl-2 +/+ and bcl-2
/
mice at
P7 or
P21 (Fig. 6). Although the level of
actin did increase in lysates from mature bcl-2 +/+ and bcl-2
/
kidneys, it occurred to a similar extent (Fig. 6).
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DISCUSSION |
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Bcl-2 /
mice are born with oligomeganephronic
hypoplasia and succumb to renal failure secondary to multicystic renal
disease (19, 21). Pronounced renal cyst formation in bcl-2
/
mice is noted when renal maturation should be completed
(18). The expression of
-catenin,
-catenin, actin, and E-cadherin
was examined to determine whether altered expression and/or
organization occurred during renal cyst formation. The distribution of
actin and
-catenin was altered when significant cyst formation was noted following renal maturation in the bcl-2
/
mice.
Piepenhagen and Nelson (14) have observed that
-catenin and
-catenin are ubiquitously expressed along the nephron, similar to
the distribution of E-cadherin. However, E-cadherin, unlike
-catenin
and
-catenin, is not expressed in glomeruli or the initial proximal
tubular segment (14, 15). The studies presented here demonstrated expression in the same nephron segments, both in kidneys from normal
and bcl-2
/
mice, as observed by Piepenhagen and
co-workers (14, 15). In contrast, the distribution of actin and
-catenin was altered within nephron segments of kidneys from bcl-2
/
mice.
The pathways underlying cyst formation remain a mystery. Numerous
abnormalities including altered proliferative and apoptotic capacities,
epithelial membrane polarity, and extracellular matrix and fluid
secretion have been identified (1, 3, 10, 17, 23). Investigators have
drawn parallels between a renal cystic phenotype and a benign tumor or
epithelial cells that never fully differentiate (9). In both cases, RCD
is compared with states that are proliferative and motile. Perhaps in
kidneys from the bcl-2 /
mice, adherens junctions do not
form properly, disrupting renal maturation and resulting in renal
multicystic disease.
The positioning of cells into organs during morphogenesis relies on
proper regulation of cell-cell interactions and formation of adherens
junctions. Cadherin/catenin complexes play a central role in these
interactions. In the kidney, E-cadherin-mediated contacts may generate
a basal level of cell-cell adhesion with additional strength to
cell-cell contacts in the distal nephron being provided by desmosomal
junctions (15). In addition, studies with MDCK cells demonstrate an
essential role for dynamic -catenin-APC protein interactions in the
regulation of cell migration during epithelial tubulogenesis (16). Thus
disruption of the E-cadherin/catenin complex could have a profound
effect on kidney development or renal maturation.
Cystic kidneys from bcl-2 /
mice display nuclear
-catenin staining, loss of apical brush border actin staining, but
normal
-catenin staining. In humans, adenomatous polyps demonstrate nuclear
-catenin staining with a concomitant downregulation of membrane staining as well a reduced expression of
E-cadherin. However,
-catenin staining was not
altered in these polyps (20). These alterations are very similar to
those observed during renal cyst formation in the bcl-2
/
mice.
-Catenin functions in signal transduction initiated by wingless/Wnt
and is localized to the nucleus upon activation of this pathway (2,
11).
-Catenin binds to the transcription factors Tcf and Lef-1 and
tumor suppressor APC (2, 11). The binding to the latter may impact
migration and tubulogenesis (16). Thus it is consistent that nuclear
localization of
-catenin in cystic tubules correlates with an
increased proliferative capacity of cells within these
tubules. Therefore, an altered distribution of these
proteins may lead to disruption of cadherin/catenin complexes and
cell-cell adhesion and precipitate renal cyst formation. However, whether these changes are the direct or indirect result of the loss of
bcl-2 requires further investigation.
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ACKNOWLEDGEMENTS |
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I thank Dr. Adrianna Dusso and Dr. Marc Hammerman for critical reading of the manuscript, Dr. Nader Sheibani for insightful discussions, and Dr. Kevin Ho for computer graphic expertise.
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FOOTNOTES |
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This research was funded by the Polycystic Kidney Research Foundation. I am funded by a Scientist Development Grant from the American Heart Association.
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: C. M. Sorenson, Renal Division Box 8126, Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110.
Received 21 July 1998; accepted in final form 22 October 1998.
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