Extract
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Introduction
Tubulointerstitial fibrosis (TIF) is a common final
pathway leading to end-stage renal disease, irrespective of the
nature of the initial renal injury. The process of TIF involves
the loss of renal tubuli and the accumulation of extracellular
matrix (ECM) proteins. Recent studies have highlighted the
role of tubular epithelial cells in mediating TIF through
epithelial_mesenchymal transition (EMT); the latter is
characterized by the loss expression of E-cadherin and the
increasing expression of α-smooth muscle actin
(α-SMA)[1]. The selective blockade of tubular EMT, due to the preservation
of tubular basement membrane integrity in
tPA_/_ mice, protects the kidney from developing fibrotic lesions after
obstructive injury[2]. These observations documented the
crucial importance of tubular EMT in the onset and progression
of chronic renal fibrosis which eventually results in
end-stage renal failure.
Tubular EMT is regulated by numerous growth factors
and hormones in different ways. Among them, transforming
growth factor-β1 (TGF-β1) is of most importance
[3]. Li et al suggested that a linear pathway might exist that couples
TGF-β1, Smad signaling, the integrin-linked kinase (ILK), and
tubular EMT[4].
The ILK, an intracellular serine/threonine protein
kinase that interacts with the cytoplasmic domains of
β-integrins and numerous cytoskeleton-associated
proteins[5], has been shown to be involved in the regulation of a number of
integrin-mediated processes, including cell adhesion, cell
phenotypic changes, gene expression, and ECM
deposition[6]. Recent studies have implicated ILK dysregulation in the
development of several chronic glomerular diseases. For
instance, Kretzler et al identified the ILK as a candidate
downstream effector of proteinuria in patients with congenital
nephritic syndrome[7]. The abundant expression of the ILK
is associated with progressive focal segmental
glomerulosclerosis[8]. Likewise, analyses of glomerular mesangial cells
and human kidney tissues suggest that the ILK is involved
in mesangial matrix expansion in response to hyperglycemia
in diabetic nephropathy[9]. However, questions remain as to
whether the dysregulation of the ILK is also implicated in
chronic renal interstitial fibrosis, specifically, how ILK
induction leads to the activation of matrix-producing
fibroblasts in the fibrotic kidney.
Recent studies have suggested that the activation of the
intrarenal renin angiotensin system (RAS) might play an
important role in progressive renal
fibrosis[10]. Angiotensin II (Ang II) is not only served as a vasoactive agent, but also as
a true cytokine with an active role in renal
pathology[11]. The activation of the Angiotensin II type 1 (AT1) receptor
results in vasoconstriction, stimulation of growth, and the
activation of fibroblasts and
myocytes[12], thereby largely mediating the fibrogenesis process. Irbesartan, a new type 1
Ang II receptor blocker (ARB), has been proven to be renal
protective in both diabetic and non-diabetic
nephropathy[13]. Although its renal protective role through the reduction
of proteinuria and blood pressure has been noted, more
attention has been paid to its mechanism on renal structural
remodeling, and an important point of note is the influence
of Ang II on renal tubular EMT. The purpose of this study
was to investigate the influence of irbesartan on the
expression of recently recognized ILK and its relationship with
EMT in mice with unilateral ureteral obstruction (UUO).
Materials and methods
Animal model Male CD-1 mice weighing 18_25 g (10
weeks old) were obtained from the Institute of Experimental
Animals, Nanjing Jinling Hospital, Nanjing University
(Nanjing, China). The mice were randomly divided into 3
groups: control (sham operation, n=20), UUO
(n=40), and UUO with irbesartan treatment
(UUO+irbesartan, n=40). UUO was performed using an established procedure as
described in a previous study[14]. Irbesartan was given at a
dose of 50 mg/kg body weight per day by gavage. The
experimental animals in the control group received the same
volume of vehicle (0.9% saline solution). The mice were
killed on d 1, 3, 7, and 14, respectively, and the kidneys were
removed for pathological study. The kidneys for the
immunohistochemical study were fixed in 10% phosphate
buffered formalin, followed by paraffin embedding. The tissues
for the protein and mRNA extractions were snap-frozen in
liquid nitrogen and stored at -80 °C.
Light microscopic study The paraffin-embedded
sections (3 µm) were stained with Masson trichrome. Each of
the 30 random, non-overlapping high power fields (magnification ×400) was transferred to the screen of a
computer. The percentage fractional area of interstitial
fibrosis was determined by taking the number of grid points
staining for the blue fibrotic tissue in the interstitium
(excluding areas within glomeruli and tubules) using Image
Pro Plus software (Media Cybernetics, San Diego, CA,
America)
Immunohistochemistry The sections were placed into
xylene to remove the paraffin wax and hydrated in graded
ethanol. The immunohistochemical detection for the ILK,
E-cadherin, and α-SMA was performed according to the
streptavidin-biotin immunoperoxidase method SP method (SP
kit, Maixin Biotechnology, Fuzhou, China). Briefly, the
tissue sections were incubated with pepsin for 5 min to expose
the antigen and then endogenous peroxidase activity was
blocked for 30 min at room temperature. After washing the
sections in phosphate-buffered saline (PBS) 3 times, the
sections were then incubated with the primary antibody (rabbit
polyclonal anti-ILK [1:100], anti-E-cadherin [1:100], and
anti-α-SMA [1:100]) overnight at 4 °C. Each section was washed
3 times in PBS and then incubated with the second antibody
for 1 h at room temperature and then reagent D for 30 min.
The end compounds reacted with the AEC
(3-amino-9-ethy1-carbazole) or DAB (Diaminobenzidin) reagents. The slides
were counterstained with hematoxylin and mounted. As a
negative control, the primary antibody was replaced with
PBS. The positive-stained area was calculated using Image
Pro Plus software described earlier and then averaged.
Western blot analysis 50 mg renal tissues from each
group were homogenized using an electric tissue grinder in
0.5 mL RIPA buffer [50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L
EGTA, 140 mmol/L NaCl, and 1.0% Nonidet P-40], containing
1 µg/mL leupeptin and aprotinin, 1 mmol/L sodium fluoride,
0.1 mmol/L sodium orthovanadate, and 1.0 mmol/L
phenyl-methylsulfonyl fluoride. The protein concentrations of all
the tissue extracts were determined using the bicinchoninic
acid BCA protein assay kit (Pierce, Rockford, IL, America).
The samples were heated at 100 °C for approximately 3_4 min
before loading and separated on precast 10% SDS_PAGE.
After the proteins were electrotransferred to a PVDF
(polyvinylidene difluoride) membrane, non-specific binding
to the membrane was blocked for 1 h at room temperature
with 5% BSA. The membranes were then incubated for 2 h at
room temperature with various primary Ab (anti-ILK antibody,
Cell Science, USA; dilution of 1:500) in blocking buffer
containing 5% milk at the dilutions specified by the
manufac-turers. Following extensive washing in TBS buffer, the
membranes were incubated with HRP (Peroxidase,
Horseradish)-conjugated secondary Ab (1:10000) for 1.5 h at room
temperature in 5% non-fat milk dissolved in TBS. The
membranes were then washed with TBS buffer and the signals
were visualized using the ECL(enhanced chemilumine-scene)
system (KangChen KC-420,Shanghai, China) as recommended by the manufacturer. Images were analyzed by
Image-J software (NCBI ).
Extraction of total RNA and semiquantitative real-time
PCR analysis The total RNA was isolated with the Trizol
1-step method from renal tissues, dissolved in DEPC (diethyl
pyrocarbonate) -treated water, quantitated by spectrometry
at 260 nm, and stored at -80 °C until assay. One microgram of
total RNA from each sample was used for reverse
transcription (RT) using the TaKaRa SYBR RT-PCR kit (TaKaRa
Biotechnology, Dalian, Jiangsu, China) with 1×
M-MLV(Moloney Murine Leukemia Virus) buffer,
deoxy-ribonucleoside triphosphate (dNTP) mixture (0.5 mmol/L), oligo dT primer
(25 pmol), M-MLV RTase (50 U), RNase inhibitor (10 U), and
RNase-free water up to 10 µL at 42 °C for 15 min, 99 °C for 5
min, and then 5 °C for 5 min. For the quantification of mRNA
for connective tissue growth factor CTGF, the ILK,
E-cadherin, α-SMA, and fibronectin, LightCycler-FastStart
DNA Master SYBR Green I system was used (GeneAmp ,
Foster City, CA, USA). Sequences of the mouse primer pairs
are listed in Table 1. The PCR reaction mixture was filled up
with SYBR premix Ex Taq (TaKaRa Biotechnology, Dalian,
Jiangsu, China) and distilled water to a final volume of 20 uL.
PCR reactions were carried out in a real-time PCR cycler
(LightCycler) and analyzed using Roche Molecular Biochemicals LightCycler software version 3.5 (Roche
Diagnostics, Roche Molecular Biochemicals, Mannheim,
Germany). The program was optimized and performed
finally as denaturation at 95 °C for 10 min, followed by 40
cycles of amplification (Table 1). The temperature ramp rate
was 20 °C/s. At the end of each extension step, the
fluorescence of each sample was measured to allow the
quantification of the PCR product. After completion of the PCR, the
melting curve of the product was measured by temperature
gradient from 60 to 95 °C at 0.2 °C/s with continuous
fluorescence monitoring to produce a melting profile of
the primers. The amount of PCR products was normalized with GAPDH
to determine the relative expression ratios for each mRNA in
relation to the control group called time zero. Target gene=
2_ΔΔCt×Control, ΔΔCt=(Cttarget
gene_Ctreference gene)treat
group_(Cttarget gene_Ctreference
gene)control group RT-PCR experiments were
repeated 3 times under identical conditions to verify the
results.
Statistical analysis All the data were shown as mean±SD
and analyzed by one-way ANOVA using SPSS software
version 11.5 (SPSS, Chicago, IL, USA). P-values <0.05 were
accepted as statistically significant.
Results
Influence of irbesartan on renal TIF in mice with UUO
It was shown by Masson staining that there was no
histological abnormality of the kidneys in the sham group.
Cellular swelling could be detected in part of the tubular epithelial
cells 1 d after the surgery in UUO mice. At d 3, vacuole
degeneration, tubular expansion, and leucocyte infiltration
could be detected. Tubular atrophy, more infiltration of
leucocytes, the expanded tubular interstitial volume, and the
over accumulation of the extracellular matrix occurred 7 d
after the surgery. Fibrosis could be detected in the renal
interstitial area at d 7 and was more severe at d 14 when
almost all of the tubules were destroyed. Irbesartan could
significantly inhibit renal pathological changes and TIF at
different time-points (Figure 1).
Influence of irbesartan on the expression of the ILK
To unravel the mechanism by which irbesartan inhibits tubular
EMT, we investigated the effects of blockade of Ang II on
the ILK expression. The ILK was mainly expressed in the
podocytes of the control group (Figure 2A); a significant
increase in the protein and mRNA expressions were detected
in mice kidneys with UUO 1 d after the surgery reaching a
peak at d 7 (Figures 2B, 2D, 3; Table 2). When severe renal
TIF was formed at d 14, both the protein and mRNA
expressions of the ILK appeared to decline compared to that at d 7
(Figures 3C, 4; Table 3). Irbesartan could significantly
inhibit the expression of the ILK at the protein and mRNA
levels (Figures 2C, 2D, 3; Table 2).
Influence of irbesartan on renal tubular EMT
It was shown that a positive reaction to the antibody
of α-SMA was detected exclusively in vascular smooth muscle cells in
the control group (Figure 4A, 4D). In the mice in the d 3
group, immunostaining demonstrated that de
novo α-SMA was expressed in renal tubular epithelial cells (TEC) (Figure
4B). As the disease progressed, the number of
α-SMA-positive cells increased, which were mainly expressed in the
fibrotic area (Figure 4E). The mRNA expression of
α-SMA detected by real-time PCR was continuously upregulated
from d 3 onwards (Table 3). As shown in Figure 4G,
E-cadherin, an epithelial marker, was expressed in almost all of
the epithelial cells within glomeruli and tubuli. Three days
after UUO, the expression of E-cadherin significantly
decreased, and was continuously downregulated with
lesion progression (Figure 4H, 4K; Table 3). Irbesartan could
significantly inhibit the expression of α-SMA and the loss of
E-cadherin at different time-points. One of the cellular
consequences of EMT is the production of massive interstitial
matrix components. Along with this, we examined the
effects of irbesartan on fibronectin expression in the kidneys.
A real-time PCR analysis revealed that the fibronectin mRNA
expression significantly increased from d 3 onwards, and
irbesartan could significantly abrogate this effect (Table 3).
Correlation between the ILK and EMT markers
To clarify the involvement of the ILK in the development of
EMT, we examined the correlation between various proteins.
Among the parameters tested by immunohistochemistry from
d 1 to d 7 after the surgery, the protein expression of the ILK
was positively correlated with that of α-SMA
(r=0.88, P<0.01), but negatively with E-cadherin (r=-0.87,
P<0.01).
Discussion
A number of studies have shown that the activation of
the RAS plays an important role in mediating the
progression of renal diseases. As the main effector of the RAS, Ang
II exerts its vasoconstricting effect predominantly on the
post-glomerular arterioles, thereby increasing glomerular
hydraulic pressure and promoting glomerular ultrafiltration,
which may contribute to the onset and progression of chronic
renal damage[13]. Furthermore, several lines of evidence have
demonstrated that Ang II might also accelerate renal damage
by its non-hemodynamic effects[14], such as increasing the
production of reactive oxygen species, the upregulation of
cytokines, cell adhesion molecules, and profibrotic growth
factors leading to renal fibrosis. Ang II acts through its
binding to 2 specific receptors, AT1 and
AT2[12]. AT1 is responsible for most of the pathophysiological actions of
Ang II. The ARB are highly effective and well-tolerated
antihypertensive medications, and recent clinical trials have
shown that these agents have renoprotective effects
beyond lowering blood pressure. Irbesartan is a new type of
ARB. Two important studies that included more than 1500
patients with type 2 diabetes and overt nephropathy
demonstrated that irbesartan reduced the relative risk of
micro-albuminuria progression to macroproteinuria compared to
amlodipine, but its exact mechanism for renoprotection is
still uncertain.
Fibroblasts are the principal effectors in the
development of TIF. Under pathological conditions, they can
rapidly proliferate, produce interstitial collagens, and
contribute to the progression of tissue
fibrosis[1]. Interstitial fibroblasts are much more heterogeneous than expected and they
might come from interstitial resident fibroblasts, circulating
fibrocytes, EMT-derived fibroblasts, and bone marrow-derived fibroblasts[15]. Among them, the most plausible
explanation for the replenishment of fibroblasts during
fibrogenesis is EMT. The strongest support for a
pathogenetic role of EMT is derived from in vivo
observations using bone marrow chimeras and transgenic reporter mice in which
fibroblasts were specifically labeled with green fluorescent
protein. Taking this approach, Iwano et
al[16] showed that during renal fibrogenesis, interstitial kidney fibroblasts are
derived in small numbers from bone marrow and in large
numbers from local EMT. Such an accumulation of EMT-derived
fibroblasts associated with tubular decondensation and
atrophy is considered to be a key determinant of renal fibrosis
during chronic injury.
The feature of EMT is cell loss of epithelial adhesion
properties and de novo expression of α-SMA and actin
reorganization; disruption of tubular basement membrane;
and enhanced cell migration and
invasion[17]. In this study, we found that the expression of E-cadherin for both mRNA
and protein significantly decreased from d 3 onwards after
the surgery in the kidneys of mice with UUO. Under normal
conditions, tubular epithelial cells are tightly connected to
each other to form an integrated epithelial sheet through
various cell adhesion mechanisms. E-cadherin, the
well-characterized adhesion receptor found within adherens type
junctions, plays an essential role in maintaining the
structural integrity of renal epithelia and its polarization. The
suppression of E-cadherin expression is regarded as a key
step that precedes other major events during tubular EMT
and will presumably lead to the destabilization of epithelial
sheet integrity, making cells ready to lose polarity, to
dissociate from their neighbors, and migrate. α-SMA, the
hallmark of myofibroblasts, may provide a structural foundation
not only for defining the morphology of the transformed
cells, but also for them to migrate, invade, and even acquire
the capacity for contractility. α-SMA-positive TEC are of a
stable, terminally-differentiated, irreversible, transformed cell
type[18]. Positive staining for α-SMA was detected
exclusively in vascular smooth muscle cells in the control group.
At d 3 after the surgery, de novo α-SMA protein expression
could be detected in renal TEC and the mRNA expression of
α-SMA, detected by real-time PCR, was continuously
upregulated from d 3 onwards. The de novo expression of
α-SMA and the loss of E-cadherin indicate that the
mesenchymal program gene is turned on when the epithelial program
gene has been switched off.
Another hallmark for tubular EMT is that the tubular
epithelial cells begins to overproduce ECM components and
properly assembles in the extracellular compartment, leading
to excessive accumulation of ECM, causing massive tissue
fibrosis as seen in diseased kidneys. The present study
showed that the mRNA expression of fibronectin significantly
increased from d 3 onwards. Irbesartan significantly
inhibited the expression of α-SMA and fibronectin and reversed
the loss of E-cadherin, suggesting that irbesartan could
reverse tubular EMT. EMT is regulated by many factors, such
as TGF-β[19], Ang II, the epidermal growth factor, fibroblast
growth factor-2[20], and reactive oxygen
species[21,22], among which TGF-β is believed to be of most importance. Liu
et al demonstrated that Ang II acts as a strong promoter that
dramatically potentiates the ability of TGF-β1 to induce EMT
in tubular epithelial cells[23]. We recently demonstrated that
Ang II potentiated tubular cells developing EMT via the
production of CTGF[24]. In the present study, we also
demonstrated that the AT1 receptor antagonist irbesartan
significantly inhibited tubular EMT, but the mechanism of this
process is currently under investigation. The ILK is an
intracellular serine/threonine protein kinase that interacts with
the cytoplasmic domains of β-integrins and numerous other
cytoskeleton-associated proteins. Differential regulation of
the ILK has been observed during the pathogenesis of
glomerular disease and TIF. In outside_in signaling, ILK
mediates the response of renal cells to alterations in the matrix
and growth factor environments. In inside_out signaling,
ILK transduces inflammatory and oxidative stress responses
into decreased matrix attachments[25]. The downstream
signaling of ILK activates the Wnt pathway with a switch
towards a proliferative, mesenchymal
phenotype[26]. In this process, ILK influences the actin cytoskeleton, resulting in
shape change and focal adhesion dysfunction observed in
podocyte failure and TIF. Recent studies found that
TGF-β could induce the Smad-dependent expression of ILK in
tubular epithelial cells. ILK plays a critical role in
TGF-β-induced EMT[8]. Hepatocyte growth factor, a well-known
antifibrotic factor, was also shown to have an antifibrotic
effect through the inhibition of ILK and the blockade of EMT.
However, the relationship between Ang II and ILK in the
process of EMT has not been addressed previously. In this
study, we first demonstrated that irbesartan could
significantly inhibit the expression of ILK both at the mRNA and
protein levels, and the protein expression of ILK is
positively correlated with that of α-SMA, but negatively
correlated with that of E-cadherin. It is therefore suggested that
there might be an independent pathway of Ang II-ILK in
mediating EMT.
Taken together, our findings shed new light on the
mechanism underlying Ang II in mediating TIF. Irbesartan exerted a
renal protective effect in mice with UUO through inhibiting
renal tubular EMT by downregulating the expression of ILK.
These data provide further evidence for preventing the
development of TIF by blocking the intrarenal RAS.
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