Journal of Histochemistry & Cytochemistry 59(9) 864 –874
© The Author(s) 2011
Reprints and permission:
The interactions of cells with the extracellular matrix (ECM)
are critical determinants of the biological processes of the
cells. Through these interactions, the microenvironment
affects cell behavior and the cell fate, such as proliferation,
migration, differentiation, and apoptosis, resulting in mor-
phogenesis and organogenesis (Streuli 2009). The expres-
sions of both ECM proteins and ECM receptors, integrins,
are under temporal and spatial control in the developing kid-
ney (Ekblom 1989; Ekblom et al. 1981; Korhonen et al.
1990; Kreidberg et al. 1996; Kreidberg and Symons 2000;
Muller et al. 1997). In the 1980s, pioneering studies revealed
distinct changes in the expression of ECM accompanied by
nephrogenesis, such as fibronectins (Ekblom et al. 1981),
interstitial collagens (Ekblom 1989), and laminin (Bonadio
et al. 1984). Indeed, these changes might be involved in the
integration of cell behavior during kidney development
(Kanwar et al. 2004; Wallner et al. 1998). Integrins are het-
erodimeric transmembrane glycoproteins consisting of α and
β subunits. In mammals, 18α and 8β subunits form 24 differ-
ent dimers, each of which has different ligand-binding and
-signaling properties (Hynes 2002; Legate et al. 2009). They
© The Author(s) 2010
ura et al.Focal Adhesions in Developing Rat Kidneys
Reprints and permission:
Received for publication December 29, 2010; accepted May 24, 2011.
Presented in part at the 42nd Annual Meeting of the American Society of
Nephrology; San Diego, CA; October 28–November 1, 2009.
Shuji Kondo, Department of Pediatrics, Institute of Health Bioscience,
The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho,
Tokushima, 770-8503, Japan
Expression of Focal Adhesion Proteins in the Developing Rat
Sato Matsuura, Shuji Kondo, Kenichi Suga, Yukiko Kinoshita, Maki Urushihara, and Shoji
Department of Pediatrics, Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima, Japan
Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental
processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase
(ILK), and hydrogen peroxide–inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we
investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that
the protein levels of FAK, p-FAK397, paxillin, p-paxillin118, and Hic-5 were high in embryonic kidneys, while ILK expression
persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK397, paxillin, and
p-paxillin118 were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating
tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells
as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal
cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly
expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth
and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney
development might play a role in morphogenesis and cell differentiation. (J Histochem Cytochem 59:864–874, 2011)
embryonic kidney development, focal adhesion kinase, hydrogen peroxide–inducible clone-5, integrin-linked kinase, paxillin
Focal Adhesions in Developing Rat Kidneys
signal to the cell interior through adhesion complexes, which
are plasmacytoplasmic platforms that are assembled around
the cytoplasmic face of clustered, ECM-associated integrins.
These signals mediate diverse biological functions including
cell polarity, cell migration, and angiogenesis (Damsky and
Ilic 2002; Hynes 2002). In addition to ECM proteins, integ-
rins also exhibit spatiotemporal expression in the develop-
ment of mammalian metanephros (Korhonen et al. 1990;
Kreidberg and Symons 2000).
Focal adhesions are integrin-based adhesion complexes
that provide anchor points for assembly of the cytoskeleton
and control the architecture of the cell. They also control cell
fate and the function of cells by influencing proliferation,
migration, differentiation, and apoptosis. They recruit both
adaptor proteins (paxillin, integrin-linked kinase [ILK], and
hydrogen peroxide–inducible clone-5 [Hic-5]) and enzymes
(focal adhesion kinase [FAK], Src, and Rho family GTPases),
which trigger distal signaling pathways that control cell-fate
decisions (Giancotti and Tarone 2003). Thus, focal adhesions
might affect complicated processes in kidney morphogenesis
by orchestrating different cells such as mesenchymal, endothe-
lial, and epithelial cells (Chatzizacharias et al. 2010; Sorenson
and Sheibani 1999).
The role of focal adhesion proteins in kidney develop-
ment and morphogenesis has not yet been adequately clari-
fied because focal adhesions composed of various proteins
are structurally complicated and all of the upstream and
downstream proteins, ECM, integrins, and signaling mole-
cules are very diverse and intricate. Unfortunately, the mice
lacking focal adhesion proteins including FAK (Ilic et al.
1995), paxillin (Hagel et al. 2002), and ILK (Lange et al.
2009) are lethal in the early stage of the embryonic period.
Knockout mice for ECM and integrins, such as fibronectin
(Romberger 1997), α5 (Yang et al. 1993), β1 (Fassler and
Meyer 1995), α3 (Kreidberg et al. 1996), and α8 integrin
(Muller et al. 1997), also show embryonic fatality or com-
promised kidney morphogenesis. To extend the research on
focal adhesions following the pioneering works on ECM
and integrins (Ekblom 1989; Ekblom et al. 1981; Korhonen
et al. 1990), we examined the expression of four major pro-
teins among focal adhesions, which might be the most
important molecules that regulate ECM and integrin
Materials and Methods
Rabbit polyclonal anti-FAK antibody, rabbit monoclonal
anti–p-FAK397 antibody, mouse monoclonal anti-paxillin
antibody, rabbit anti–p-paxillin118 antibody, and goat poly-
clonal anti-CD31 antibody were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), Invitrogen (Carlsbad, CA),
Transduction Laboratories (Lexington, KY), and Millipore
(Billerica, MA), respectively. Mouse monoclonal anti-ILK
antibody and mouse monoclonal anti–Hic-5 antibody were
purchased from Upstate (Billerica, MA) and BD Transduction
(Franklin Lakes, NJ). Mouse monoclonal anti–α-smooth
muscle actin (α-SMA) 1A4 antibody and mouse monoclonal
anti–proliferating cell nuclear antigen (PCNA) antibody were
purchased from Sigma-Aldrich (St. Louis, MO) and
Calbiochem Merck KGaA (Darmstadt, Germany). Fluorescein
isothiocyanate (FITC)–labeled donkey anti-rabbit IgG
antibody, FITC-labeled donkey anti-mouse IgG antibody,
tetramethylrhodamine isothiocyanate (TRITC)–coupled
donkey anti-mouse IgG antibody, and TRITC-coupled don-
key anti-goat IgG antibody were purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA).
Experimental Animals and Sampling
All procedures and protocols used in this study were approved
by the Institutional Animal Care and Committee of the
University of Tokushima Graduate School. Sprague-Dawley
rats were obtained from Japan SLC (Shizuoka, Japan).
Pregnancy was determined by the detection of a vaginal plug.
Before removal of the embryos, pregnant rats were anesthe-
tized by isoflurane with oxygen gas. Embryos were removed
and decapitated on day 13 (E13), 14 (E14), 15 (E15), 16 (E16),
and 18 (E18) of gestation. E13, E14, and E15 were fixed in
10% neutral buffered formalin. Kidneys from E16 and E18
were harvested and either fixed with 10% neutral buffered
formalin or homogenized in cell lysis buffer containing prote-
ase inhibitors (Cell Signaling Technology; Danvers, MA).
Kidneys from rats 1 (P1), 7 (P7), 21 (P21), and 42 (P42) days
after birth were treated in the same manner.
Whole kidneys from E16, E18, P1, P7, P21, and P42 were
harvested and homogenized using 15 strokes of a motor-
driven Teflon (DuPont; Wilmington, DE) pestle in a tightly
fitted glass tube in cell lysis buffer containing protease
inhibitors (Cell Signaling Technology). After incubation of
samples on ice for 15 minutes, insoluble materials were
removed by centrifugation (10,500 × g, 10 minutes). The
protein content in kidney lysates was measured using a BCA
protein assay kit (Pierce Biotechnology; Rockford, IL).
Protein samples (30 µg) were separated by 12.5% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to nitrocellulose membranes
(Amersham Bioscience; Piscataway, NJ). The membranes
were probed with primary antibodies and then incubated
with horseradish peroxidase–conjugated secondary antibod-
ies. Immunoreactive proteins were detected with an enhanced
chemiluminescence detection system (Amersham Corp.;
Arlington Heights, IL). Bands were quantified by ImageJ
1.33u (National Institutes of Health; Bethesda, MD).
Matsuura et al.
Histology and Immunohistochemistry
To examine the expression and localization of focal adhesion
proteins, paraffin sections (3 µm thick) were deparaffinized
and rehydrated. Endogenous peroxidase activity was
quenched by incubating sections in 2.0% H2O2/methanol for
30 minutes. To unmask antigens, slides were autoclaved at
121C for 10 minutes in 0.01 M citrate buffer (pH 6.0).
The expressions of focal adhesions were detected by an
immunoperoxidase technique as previously described
(Kondo et al. 2006). Briefly, the sections were incubated
with primary antibodies against FAK, p-FAK397, paxillin,
p-paxllin118, Hic-5, and ILK (all antibodies diluted to 1:100)
for 24 hours at 4C. After washing, the sections were
incubated with biotinylated secondary antibodies, avidin-
biotin-peroxidase (ABC Elite) (Vector Laboratories;
Burlingame, CA), and immunoreaction products were devel-
oped using 3,3′-diaminobenzidine (Dojindo; Kumamoto,
Japan). The sections were then counterstained with Mayer’s
hematoxylin (Wako; Tokyo, Japan), dehydrated, and cover-
slipped. Negative control experiments were examined by
omitting primary antibodies or using control IgG.
Double-staining experiments were performed using anti-
FAK antibody or anti–Hic-5 antibody with anti-PCNA anti-
body or anti-CD31 antibody, respectively. The sections
were incubated with an appropriate FITC-labeled second-
ary antibody for anti-FAK antibody or anti–Hic-5 antibody
and an appropriate TRITC-labeled secondary antibody for
anti-PCNA antibody or anti-CD31 antibody.
To recognize the localization of focal adhesions, we
defined various cells in developing kidneys with segment-
specific markers as follows: endothelial cell, CD31; proxi-
mal tubule, Phaseolus vulgaris erythroagglutinin; distal
tubule, calbindin D 28k; collecting duct, aquaporin 2
(Togawa et al. 2011).
Values are expressed as means ± standard deviations (SDs).
Differences were evaluated with the StatMate 3 software
package (ATMS Co. Ltd.; Tokyo, Japan). Comparisons of
variables between groups were performed by one-way
ANOVA and the Dunnett test. All experiments were
repeated at least three times. Values of p < 0.05 were con-
sidered statistically significant.
Western blotting was performed to examine protein levels of
FAK, p-FAK397, paxillin, p-paxillin118, ILK, Hic-5, and α-
SMA in rat kidneys in different stages of development. The
expression levels of FAK and p-FAK397 were upregulated in
embryonic kidneys (E16 and E18) and significantly decreased
after birth. FAK was slightly detected, whereas p-FAK397
was almost absent in P1. They were both undetectable from
P7 to mature kidneys (Fig. 1A). Paxillin and p-paxillin118
were especially high in embryonic kidneys and peaked at
E18 (Fig. 1B). Similar to FAK, they were decreased after
birth and became faint in mature kidneys. Although the lev-
els of FAK and paxillin were very low for detection by
Western blotting, their expression could be observed in the
following immunohistochemical results, which are summa-
rized in Table 1. Hic-5 was strongly expressed around birth
from E16 to P1 and then rapidly decreased and was dimin-
ished in mature kidneys (Fig. 1D). The course of Hic-5
expression was similar to that of α-SMA (Fig. 1E). On the
other hand, the expression of ILK was continuously observed
in all stages of development. An increased level was sus-
tained from E18 to P7, and then this slightly lessened after
P21 (Fig. 1C).
The localization of each protein is summarized in Table 1.
The levels of their expression were graded as follows: –
means negative staining, ± means faint staining, + means
mild staining, and ++ means strong staining by reference to
another previous article (Omori et al. 2000).
Expression of FAK and p-FAK397
FAK was predominantly expressed at the cell membrane of the
ureteric bud and condensed mesenchymal cells around the
ureteric bud and weakly observed in mesenchymal cells (Fig.
2A). In the nephrogenic zone, FAK was expressed in comma-
and S-shaped bodies and in immature glomerular endothelial
and mesangial cells (Fig. 2B). With maturation, FAK expres-
sion was decreased and localized in tubules in mature kidneys
(Fig. 2C). Similar to FAK expression, p-FAK397 was observed
at the cell membrane of the ureteric bud and condensed mes-
enchymal cells in E16 (Fig. 2D). It was expressed in S-shaped
bodies and immature glomerular cells in E18 (Fig. 2E). It was
decreased during the course of development and was not
observed in mature glomeruli (Fig. 2F).
To compare the distribution of FAK with proliferating
cells, we investigated PCNA-positive cells by double-staining
experiments (Fig. 2G and 2H). PCNA was mainly stained in
the ureteric bud and condensed mesenchymal cells around
the ureteric bud, and FAK was expressed on the cell mem-
brane of the PCNA-positive cells in E16 (Fig. 2G). PCNA
was subsequently expressed in comma- and S-shaped bod-
ies and in elongating epithelial cells in the nephrogenic
zone (Fig. 2H). The distribution of PCNA-positive cells
was similar to those of FAK, paxillin, and ILK, which might
be involved in renal growth and morphogenesis. FAK-
positive cells with negative PCNA staining were also
Focal Adhesions in Developing Rat Kidneys
observed in E18 by double staining. These cells seemed to
be mesenchymal cells, immature endothelial cells, and
mesangial cells, which appeared to migrate or invade into
the cleft of S-shaped bodies (Fig. 2H).
Expression of Paxillin and P-paxillin118
The expression and localization of paxillin and p-paxillin118
were similar to those of FAK, and these were detected mainly
in the ureteric bud and weakly in mesenchymal cells in the
embryonic stage (Fig. 3A and 3D). They were then expressed
in comma- and S-shaped bodies and immature glomerular
endothelial and mesangial cells in the nephrogenic zone (Fig.
3B and 3E). With maturation, paxillin expression was
decreased and limited to distal tubular cells (Fig. 3C), while
p-paxillin118 expression on tubules was very weak (Fig. 3F).
Expression of ILK
Although ILK expression was similar to that of FAK and pax-
illin during development, they were very different with matu-
ration. ILK expression was detected in the ureteric bud and
mesenchymal cells from E14 to P1 (Fig. 4A). It was mainly
expressed in immature endothelial and mesangial cells in the
nephrogenic zone (Fig. 4B) and then localized in endothelial
cells and podocytes in mature glomeruli (Fig. 4C).
Expression of Hic-5
Hic-5 was mainly expressed in mesenchymal cells and
immature glomerular endothelial and mesangial cells in
embryonic kidneys (Fig. 5A and 5B). Double-staining
experiments showed that Hic-5 is mainly expressed in
immature mesangial cells and partially co-localized with
CD31 in E18 and P1 (Fig. 5G and 5H). The expression of
Hic-5 was decreased with maturation, was not observed in
glomeruli, and was limited to vascular smooth muscle cells
in mature kidneys (Fig. 5C). To elucidate the relationship
between Hic-5 and myofibroblastic cells, we investigated
cells that were positive for α-SMA (Carey et al. 1992) in
serial sections. α-SMA was detected in mesenchymal cells
in embryonic kidneys (Fig. 5D). It was also localized in
Figure 1. FAK, paxillin, ILK, Hic-5, and α-SMA expression during the development of rat kidneys. FAK, p-FAK397, paxillin, and p-paxillin118
were highly expressed in embryonic kidneys (A and B). ILK expression was detected in embryonic, postnatal, and mature kidneys (C).
Hic-5 and α-SMA were highly expressed in E16 to P1 during development (D and E). Results are shown as means ± standard deviations
from at least three independent experiments. Significant differences versus E16: ‡p < 0.05; †p < 0.01; **p < 0.001; *p < 0.0001.
Matsuura et al.
immature glomerular endothelial and mesangial cells and in
vascular smooth muscle cells in E18 (Fig. 5E). This expres-
sion was then decreased and limited to vascular smooth
muscle cells in mature kidneys (Fig. 5F). The distribution of
α-SMA–positive cells was similar to that of Hic-5, which
might be related to mesenchymal cell development.
This study has four major findings: 1) The expression lev-
els of FAK, p-FAK397, paxillin, p-paxillin118, and Hic-5
were high in the embryonic kidney, whereas the level of
ILK expression was maintained in embryonic, postnatal,
and mature kidneys. 2) FAK, p-FAK397, paxillin, and
p-paxillin118 were strongly expressed in mesenchymal cells
and the ureteric bud. In the nephrogenic zone, they were
detected in elongating epithelial cells in tubules and collect-
ing ducts and in immature glomerular endothelial and
mesangial cells. They were decreased in mature kidneys. 3)
Hic-5 was predominantly expressed in mesenchymal cells
and immature glomerular endothelial and mesangial cells,
similar to α-SMA–positive cells. 4) ILK was expressed
similarly to FAK in developing stages. In contrast, ILK was
strongly expressed in glomerular endothelial cells and
podocytes in mature kidneys.
Kidney development proceeds in sequential steps that
involve the dynamic and accurately controlled program-
ming of cellular events. The induction of the metanephric
mesenchyme by the ureteric bud promotes aggregation of
the condensed mesenchyme around the bud tips. These
Table 1. Expression of Focal Adhesion Proteins in Developing and Adult Rat Kidneys
FAKPaxillin ILK Hic-5
Comma-, S-shaped bodies
Gl epithelial cells
Gl endothelial cells
Ureteric bud branches
Comma-, S-shaped bodies
Gl epithelial cells
Gl endothelial cells
Collecting duct cells
Gl visceral epithelial cells
Gl parietal epithelial cells
Gl endothelial cells
Proximal tubular cells
Distal tubular cells
Collecting duct cells
Note: – = negative staining; ± = faint staining; + = mild staining; ++ = strong staining; Gl = glomerular; VSMC = vascular smooth muscle cells.
Focal Adhesions in Developing Rat Kidneys
aggregates undergo mesenchyme-to-epithelial conversion
to generate the renal vesicles. Meanwhile, the ureteric buds
elongate and reiterate branching and induce new aggregates
at the bud tips. By the S-shaped body stage, the nephron is
patterned along the proximal-distal axis. Invasion of the
proximal cleft by endothelial cells starts the process of glo-
merulogenesis (Dressler 2006). All the cells that take part in
nephrogenesis perform some fundamental activity, includ-
ing proliferation, migration, polarization, and differentia-
tion through these developmental processes, which are
always determined by adhesive interactions between cells
and their local microenvironment (Streuli 2009). In all of
these developmental processes, focal adhesions are consid-
ered to play crucial roles in receiving signals from complex
extracellular environments and in conveying intracellular
signals that lead to changes in cell behavior.
FAK is a 125-kDa nonreceptor and non–membrane pro-
tein tyrosine kinase, which has been identified as a substrate
of viral Src oncogene, and is considered to be related to
tumor growth (Schaller et al. 1992). FAK is phosphorylated
at several tyrosine residues including tyrosine 397 (Tyr397),
the first tyrosine residue of its phosphorylation. Clustering
of integrins facilitates the autophosphorylation of Tyr397,
which increases and regulates the catalytic activity of FAK
(van Nimwegen and van de Water 2007). Through multifac-
eted and diverse molecular connections, FAK can influence
the cytoskeletal organization at structures of cell adhesion
sites to regulate cell movement (Schlaepfer et al. 2004;
Figure 2. Expression of FAK and p-FAK397 during the development of rat kidneys. Expression of FAK and p-FAK397 in E16 was intense at
the cell membrane of the ureteric bud and mesenchymal cells, especially in the condensed mesenchyme (red arrows) (A and D). Around
the nephrogenic zone in E18, FAK and p-FAK397 were expressed in S-shaped bodies (sb) as well as in immature glomerular endothelial
and mesangial cells (black arrows in B and E, which are the serial sections). FAK expression was decreased and localized in tubules in
P42 (C), while p-FAK397 was not detected in mature glomeruli of P42 (F). Double-staining experiments were performed to compare
the expression of FAK (green, FITC) and PCNA-positive cells (red, TRITC) (G and H). FAK was expressed on the cell membrane of the
ureteric bud and condensed mesenchymal cells (red arrows), where the PCNA was positive in E16 (G). In E18, FAK expression was also
positive in cleft-invading endothelial and mesangial cells (arrow head) and mesenchymal cells (*), where the PCNA expression was weak
(H). rm = renal mesenchymal cells; red arrows and “cm” = condensed mesenchymal cells; ub = ureteric bud; u = ureteric bud tip; sb =
S-shaped body; m = immature glomerulus; black arrows in B and E = immature endothelial and mesangial cells; g = glomerulus. Scale bars
(A, B, D, and F) = 25 µm; scale bars (C and F) = 50 µm; scale bars (G and H) = 100 µm.
Matsuura et al.
Sonoda et al. 2000). FAK expression has been investigated
in a variety of human cancers, including primary, meta-
static, and recurrent lesions. These studies suggest that FAK
may play diverse roles including proliferation and migra-
tion in different tumors and/or in different stages of tumor
progression (van Nimwegen and van de Water 2007). In this
study, we found that many FAK and p-FAK397–positive
cells were also positive for PCNA in the ureteric bud and
mesenchymal aggregates during early stages as well as in
elongating epithelial cells in the nephrogenic zone. These
observations suggest that FAK plays a main role in cell pro-
liferation in kidney development. In contrast, FAK expres-
sion was also positive in cleft-invading endothelial cells
and immature mesangial cells. Because FAK also plays a
role in migration as well as proliferation (Sonoda et al.
2000; van Nimwegen and van de Water 2007), we thought
that FAK might also be necessary for immature endothelial
and mesangial cells to migrate inside vesicles to organize
the capillary network of glomeruli. Interestingly, these
migratory processes might be associated with the shift of
Figure 3. Expression of paxillin and p-paxillin118 during the development of rat kidneys. Paxillin and p-paxillin118 expression were similar
to those of FAK and were detected at the cell membrane of the ureteric bud in E16 (A and D are the serial sections). Both were also
observed in immature endothelial (black arrow) and mesangial cells in comma- and S-shaped bodies in the nephrogenic zone of E18 (B
and E). Paxillin was decreased and was limited to distal tubules in P42 (C), while p-paxillin118 expression on tubules was very weak (F).
rm = renal mesenchymal cells; cm = condensed mesenchymal cells; ub = ureteric bud; u = ureteric bud tips; sb = S-shaped body; m =
immature glomerulus; black arrows in B and E = immature endothelial and mesangial cells; dt = distal tubules; g = glomerulus. Scale bars
(A, B, D, and F) = 25 µm; scale bars (C and F) = 50 µm.
Focal Adhesions in Developing Rat Kidneys
laminin composition and the expression of some integrins
including α1 integrin (Abrahamson 2009; Korhonen et al.
1990). In addition, Ma et al. (2010) showed that podocyte-
specific deletion of FAK abrogated the proteinuria and foot
process effacement induced by glomerular injury, and
podocytes isolated from these conditional FAK knockout
mice demonstrated reduced spreading and migration. These
findings also supported that FAK activation might affect
cell motility in unsettled conditions when foot processes by
podocytes separate from the ECM in glomerular basement
membranes. The decision of a cell to either proliferate or
migrate depends on the balance of many stimulatory and
inhibitory factors because the property of FAK might be
determined by the integrin-ECM proteins with which it
interacts (Wozniak et al. 2004).
Paxillin is a 68-kDa protein that contains five leucine-
and aspartate-rich LD motifs, which bind vinculin and FAK.
Paxillin also contains four LIM domains, which are double
zinc-finger motifs that mediate protein-protein interactions.
Similar to FAK, multiple tyrosine, serine, and threonine
phosphorylation sites exist throughout paxillin molecules;
these sites are targeted by a diverse array of kinases that are
activated in response to various adhesion stimuli and by
growth factors, and its phosphorylation plays a vital role in
signaling (Deakin and Turner 2008). Paxillin can be phos-
phorylated by FAK and may potentially be a downstream
component of FAK signaling. Indeed, the localization and
expression of paxillin and p-paxillin118 in developing kid-
neys were similar to those of FAK or p-FAK397. This result
supports the idea that the phosphorylation of paxillin through
the FAK/Src complex is important for paxillin to act as a
docking molecule at focal adhesions and that they have a
common role in cell behavior in development, such as cell
migration and proliferation. This is well supported by the
findings in paxillin- or FAK-deficient mice, which are both
fatal before the onset of kidney morphogenesis (Hagel et al.
2002; Ilic et al. 1995). Although both molecules are dimin-
ished in mature glomeruli, our results showed different
expressions in adult kidneys. FAK was expressed mainly in
the proximal tubule, whereas paxillin was limited to distal
tubules. This indicates that paxillin might play a different
role than FAK in the maintenance of tubular cells.
Hic-5 was first identified in a screen for transforming
growth factor beta1 (TGF-β1) and hydrogen peroxide–
induced genes (Shibanuma et al. 1994). Hic-5 encodes a
55-kDa protein, which has a structure that is very similar to
that of paxillin, and both consist of four LIM proteins and
LD proteins (Shibanuma et al. 1994). Hic-5 expression is
elevated in platelets (Hagmann et al. 1998), cells of mesen-
chymal origin, and also in stromal and smooth muscle tissue
layers (Brunskill et al. 2001; Cai et al. 2005; Yuminamochi
et al. 2003). Brunskill et al. (2001) showed that Hic-5 was
strongly and transiently expressed in the early developing
heart and then in the smooth muscle layer of developing tis-
sues, including the intestinal tract and bronchial airways in
mice. Yuminamochi et al. (2003) showed that there was a
difference in the expression of Hic-5 and paxillin in adult
human tissues. Paxillin expression was widespread and
Figure 4. Expression of ILK during the development of rat kidneys. ILK expression was similar to that of FAK in developing stages. ILK
was expressed in the ureteric bud and mesenchymal cells in E15 (A). In E18, ILK was detected in S-shaped bodies as well as in immature
endothelial and mesangial cells (B). ILK was strongly expressed in podocytes and epithelial cells in P42 (C). rm = renal mesenchymal cells;
cm = condensed mesenchymal cells; u = ureteric bud tips; sb = S-shaped body; m = immature glomerulus; arrow = immature endothelial
and mesangial cells; arrowhead = podocytes; g = glomerulus. Scale bars = 25 µm.
Matsuura et al.
observed in both nonmuscle and muscle tissue, while Hic-5
was limited to muscle tissues, mainly mononuclear smooth
muscle. Our study demonstrated that Hic-5 expression was
very different from paxillin expression. We recognized that
the expression of Hic-5 was limited to mesenchymal cells,
endothelial cells, and mesangial cells in immature glomer-
uli. The expression of both Hic-5 and paxillin was localized
to mesenchymal cells and immature endothelial mesangial
cells in embryonic kidneys, whereas paxillin was mainly
expressed in epithelial cells. This suggests that Hic-5 may
induce or maintain a mesenchymal cell character through
competition with paxillin (Mori et al. 2009). Interestingly,
the expression of Hic-5 coincides with the distribution of
α-SMA–positive cells, suggesting that it may be associated
with mesenchymal phenotypes in each developmental stage
(Carey et al. 1992).
ILK is a 59-kDa serine/threonine kinase that interacts
with cytoplasmic domains of β1 integrins and has been
implicated in the regulation of cell adhesion, proliferation,
and ECM organization (Wu 2001). ILK is activated by β1
integrin–mediated adhesion to the ECM or stimulation with
growth factors such as TGF-β. ILK is considered to func-
tion as the effector of PI3-K signaling, which regulates pro-
tein kinase B/Akt and glycogen synthase kinase-3 activity,
and thereby mediates a wide range of cellular processes. In
the present study, the localization of ILK was similar to that
of FAK and paxillin in embryonic kidneys, while it was
mainly expressed in mature podocytes in adult kidneys.
Along with the results of previous studies, the present find-
ings suggest that ILK plays several roles in development.
First, it might be involved in the proliferation and migration
of epithelial and mesenchymal cells. The concept was
Figure 5. Expression of Hic-5 and α-SMA–positive cells during the development of rat kidneys. Hic-5 was mainly expressed in
mesenchymal cells in E16 but was not detected in the ureteric bud or epithelial cells (A). α-SMA–positive cells were detected in
mesenchymal cells in E16 (D). Distributions of Hic-5–positive cells were similar to α-SMA–positive cells in E16. In E18, Hic-5 was
detected in immature glomerular endothelial and mesangial cells and also in mesenchymal cells (black arrows in B). Most Hic-5 expression
was very similar to the distribution of α-SMA–positive cells in E18 (B and E are the serial sections). In mature kidneys, both expressions
were not observed in glomeruli and were limited to vascular smooth muscle cells (C and F). Double-staining experiments showed that
Hic-5 (green, FITC) and CD31 (red, TRITC) were partially co-localized in immature glomeruli of E18 (yellow arrowhead in G and H). rm
= renal mesenchymal cells; u = ureteric bud tips; ub = ureteric bud; sb = S-shaped body; m = immature glomerulus; black arrows in B and
E = immature endothelial and mesangial cells; g = glomerulus; v = vascular smooth muscle cells; red in G and H = CD31; green in G and
H = Hic-5. Scale bars (A-F) = 25 µm; scale bars (G and H) = 75 µm.
Focal Adhesions in Developing Rat Kidneys
supported in a recent work using conditionally deficient
mice that ILK expression was selectively knocked down in
the ureteric bud (Smeeton et al. 2010). Second, it might be
involved in mesenchyme-to-epithelial conversion, which is
observed when mesenchymal aggregates change to epithe-
lial cells in renal vesicles (Leung-Hagesteijn et al. 2005).
With maturation, its function might switch to the differen-
tiation and maturation of epithelial cells and podocytes (Dai
et al. 2006).
ECM, integrins, and focal adhesions are expressed
diversely in each developmental stage. Focal adhesions
appear to integrate and modulate signals to the cell interior,
which results in accurate cell proliferation, migration, and
differentiation, as well as signal transduction to achieve
normal organ morphogenesis. Because focal adhesions also
mediate signals from growth factors such as TGF-β or glial
cell–derived neurotrophic factor, the expression and activa-
tion of focal adhesions appear to be important for regulating
and coordinating diverse signals by both ECM and growth
factors during kidney development and morphogenesis.
Further studies on the relationship between focal adhesions
and stimulatory extracellular factors are needed to elucidate
the precise mechanisms of renal development and
In conclusion, we demonstrated that the expression of
focal adhesion proteins is temporospatially regulated during
the development of rat kidneys. They might play crucial
roles in renal development and morphogenesis.
The authors are grateful to Naomi Okamoto, Chizuko Yamamoto,
Keita Osumi, Junki Yamajo, and Hiroki Matsumoto for their
excellent technical assistance. They also thank Dr. Christine M.
Sorenson and Dr. Nader Sheibani (University of Wisconsin–
Madison), and Dr. Midori Awazu (Keio University) for their help-
ful suggestions and Dr. Akito Kobayashi (Brigham and Women’s
Hospital) and Dr. Shuta Ishibe (Yale School of Medicine) for
providing helpful discussions.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the authorship and publication of this article.
This work was supported in part by Grants-in-Aid for Scientific
Research (20591277 and 2059127 to S. Kagami and S. Kondo,
respectively). This work was also supported by the Morinaga
Foundation for Health and Nutrition.
Abrahamson DR. 2009. Development of kidney glomerular endo-
thelial cells and their role in basement membrane assembly.
Bonadio JF, Sage H, Cheng F, Bernstein J, Striker GE. 1984. Local-
ization of collagen types IV and V, laminin, and heparan sulfate
proteoglycan to the basal lamina of kidney epithelial cells in
transfilter metanephric culture. Am J Pathol. 116:289–296.
Brunskill EW, Witte DP, Yutzey KE, Potter SS. 2001. Novel cell
lines promote the discovery of genes involved in early heart
development. Dev Biol. 235:507–520.
Cai G, Huang H, Shapiro E, Zhou H, Yeh S, Melamed J, Greco
MA, Lee P. 2005. Expression of androgen receptor associated
protein 55 (ARA55) in the developing human fetal prostate. J
Carey AV, Carey RM, Gomez RA. 1992. Expression of alpha-
smooth muscle actin in the developing kidney vasculature.
Chatzizacharias NA, Kouraklis GP, Theocharis SE. 2010. The role
of focal adhesion kinase in early development. Histol Histo-
Dai C, Stolz DB, Bastacky SI, St-Arnaud R, Wu C, Dedhar S, Liu
Y. 2006. Essential role of integrin-linked kinase in podocyte
biology: bridging the integrin and slit diaphragm signaling. J
Am Soc Nephrol. 17:2164–2175.
Damsky CH, Ilic D. 2002. Integrin signaling: it’s where the action
is. Curr Opin Cell Biol. 14:594–602.
Deakin NO, Turner CE. 2008. Paxillin comes of age. J Cell Sci.
Dressler GR. 2006. The cellular basis of kidney development.
Annu Rev Cell Dev Biol. 22:509–529.
Ekblom P. 1989. Developmentally regulated conversion of mesen-
chyme to epithelium. FASEB J. 3:2141–2150.
Ekblom P, Lehtonen E, Saxen L, Timpl R. 1981. Shift in collagen
type as an early response to induction of the metanephric mes-
enchyme. J Cell Biol. 89:276–283.
Fassler R, Meyer M. 1995. Consequences of lack of beta 1 integrin
gene expression in mice. Genes Dev. 9:1896–1908.
Giancotti FG, Tarone G. 2003. Positional control of cell fate
through joint integrin/receptor protein kinase signaling. Annu
Rev Cell Dev Biol. 19:173–206.
Hagel M, George EL, Kim A, Tamimi R, Opitz SL, Turner CE,
Imamoto A, Thomas SM. 2002. The adaptor protein paxil-
lin is essential for normal development in the mouse and is
a critical transducer of fibronectin signaling. Mol Cell Biol.
Hagmann J, Grob M, Welman A, van Willigen G, Burger MM.
1998. Recruitment of the LIM protein hic-5 to focal contacts
of human platelets. J Cell Sci. 111(Pt 15):2181–2188.
Hynes RO. 2002. Integrins: bidirectional, allosteric signaling
machines. Cell. 110:673–687.
Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N,
Nomura S, Fujimoto J, Okada M, Yamamoto T. 1995. Reduced
cell motility and enhanced focal adhesion contact formation in
cells from FAK-deficient mice. Nature. 377:539–544.
Kanwar YS, Wada J, Lin S, Danesh FR, Chugh SS, Yang Q, Baner-
jee T, Lomasney JW. 2004. Update of extracellular matrix, its
Matsuura et al.
receptors, and cell adhesion molecules in mammalian nephro-
genesis. Am J Physiol Renal Physiol. 286:F202–F215.
Kondo S, Shimizu M, Urushihara M, Tsuchiya K, Yoshizumi M,
Tamaki T, Nishiyama A, Kawachi H, Shimizu F, Quinn MT,
et al. 2006. Addition of the antioxidant probucol to angiotensin
II type I receptor antagonist arrests progressive mesangiop-
roliferative glomerulonephritis in the rat. J Am Soc Nephrol.
Korhonen M, Ylanne J, Laitinen L, Virtanen I. 1990. The alpha
1-alpha 6 subunits of integrins are characteristically expressed
in distinct segments of developing and adult human nephron. J
Cell Biol. 111:1245–1254.
Kreidberg JA, Donovan MJ, Goldstein SL, Rennke H, Shepherd
K, Jones RC, Jaenisch R. 1996. Alpha 3 beta 1 integrin has a
crucial role in kidney and lung organogenesis. Development.
Kreidberg JA, Symons JM. 2000. Integrins in kidney develop-
ment, function, and disease. Am J Physiol Renal Physiol.
Lange A, Wickstrom SA, Jakobson M, Zent R, Sainio K, Fassler R.
2009. Integrin-linked kinase is an adaptor with essential func-
tions during mouse development. Nature. 461:1002–1006.
Legate KR, Wickstrom SA, Fassler R. 2009. Genetic and cell bio-
logical analysis of integrin outside-in signaling. Genes Dev.
Leung-Hagesteijn C, Hu MC, Mahendra AS, Hartwig S, Klamut
HJ, Rosenblum ND, Hannigan GE. 2005. Integrin-linked kinase
mediates bone morphogenetic protein 7-dependent renal epithe-
lial cell morphogenesis. Mol Cell Biol. 25:3648–3657.
Ma H, Togawa A, Soda K, Zhang J, Lee S, Ma M, Yu Z, Ardito
T, Czyzyk J, Diggs L, et al. 2010. Inhibition of podocyte FAK
protects against proteinuria and foot process effacement. J Am
Soc Nephrol. 2:1145–1156.
Mori K, Hirao E, Toya Y, Oshima Y, Ishikawa F, Nose K, Shib-
anuma M. 2009. Competitive nuclear export of cyclin D1 and
Hic-5 regulates anchorage dependence of cell growth and sur-
vival. Mol Biol Cell. 20:218–232.
Muller U, Wang D, Denda S, Meneses JJ, Pedersen RA, Reich-
ardt LF. 1997. Integrin alpha8beta1 is critically important for
epithelial-mesenchymal interactions during kidney morpho-
genesis. Cell. 88:603–613.
Omori S, Hida M, Ishikura K, Kuramochi S, Awazu M. 2000.
Expression of mitogen-activated protein kinase family in rat
renal development. Kidney Int. 58:27–37.
Romberger DJ. 1997. Fibronectin. Int J Biochem Cell Biol.
Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds
AB, Parsons JT. 1992. pp125FAK a structurally distinctive
protein-tyrosine kinase associated with focal adhesions. Proc
Natl Acad Sci U S A. 89:5192–5196.
Schlaepfer DD, Mitra SK, Ilic D. 2004. Control of motile and
invasive cell phenotypes by focal adhesion kinase. Biochim
Biophys Acta. 1692:77–102.
Shibanuma M, Mashimo J, Kuroki T, Nose K. 1994. Characteriza-
tion of the TGF beta 1-inducible hic-5 gene that encodes a
putative novel zinc finger protein and its possible involvement
in cellular senescence. J Biol Chem. 269:26767–26774.
Smeeton J, Zhang X, Bulus N, Mernaugh G, Lange A, Karner
CM, Carroll TJ, Fassler R, Pozzi A, Rosenblum ND, Zent R.
2010. Integrin-linked kinase regulates p38 MAPK-dependent
cell cycle arrest in ureteric bud development. Development.
Sonoda Y, Matsumoto Y, Funakoshi M, Yamamoto D, Hanks SK,
Kasahara T. 2000. Anti-apoptotic role of focal adhesion kinase
(FAK): induction of inhibitor-of-apoptosis proteins and apop-
tosis suppression by the overexpression of FAK in a human
leukemic cell line, HL-60. J Biol Chem. 275:16309–16315.
Sorenson CM, Sheibani N. 1999. Focal adhesion kinase, paxillin,
and bcl-2: analysis of expression, phosphorylation, and asso-
ciation during morphogenesis. Dev Dyn. 215:371–382.
Streuli CH. 2009. Integrins and cell-fate determination. J Cell Sci.
Togawa H, Nakanishi K, Mukaiyama H, Hama T, Shima Y, Sako
M, Miyajima M, Nozu K, Nishii K, Nagao S, et al. 2011. Epi-
thelial-to-mesenchymal transition in cyst lining epithelial cells
in an orthologous PCK rat model of autosomal-recessive poly-
cystic kidney disease. Am J Physiol Renal Physiol. 300:F511–
van Nimwegen MJ, van de Water B. 2007. Focal adhesion kinase:
a potential target in cancer therapy. Biochem Pharmacol.
Wallner EI, Yang Q, Peterson DR, Wada J, Kanwar YS. 1998.
Relevance of extracellular matrix, its receptors, and cell adhe-
sion molecules in mammalian nephrogenesis. Am J Physiol.
Wozniak MA, Modzelewska K, Kwong L, Keely PJ. 2004. Focal
adhesion regulation of cell behavior. Biochim Biophys Acta.
Wu C. 2001. ILK interactions. J Cell Sci. 114:2549–2550.
Yang JT, Rayburn H, Hynes RO. 1993. Embryonic mesodermal
defects in alpha 5 integrin-deficient mice. Development.
Yuminamochi T, Yatomi Y, Osada M, Ohmori T, Ishii Y, Naka-
zawa K, Hosogaya S, Ozaki Y. 2003. Expression of the LIM
proteins paxillin and Hic-5 in human tissues. J Histochem