Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF-β-Induced Epithelial-Mesenchymal Transition
Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF--Induced Epithelial- Mesenchymal Transition
Lining Jia 0 1
Xiaotao Ma 0 1
Baosong Gui 0 1
Heng Ge 0 1
Li Wang 0 1
Yan Ou 0 1
Lifang Tian 0 1
Zhao Chen 0 1
Zhaoyang Duan 0 1
Jin Han 0 1
Rongguo Fu 0 1
0 Department of Nephropathy, The Second Affiliated Hospital of Xi'an Jiaotong University , Xi'an , China
1 Academic Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES
Competing Interests: The authors have declared
that no competing interests exist.
This study was to investigate whether sorafenib can inhibit the progression of renal fibrosis
and to study the possible mechanisms of this effect.
Eight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were
intragastrically administered sorafenib, while control and sham groups were administered
vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-1 and sorafenib for 24 or 48
hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each
group. The expression of -SMA and E-cadherin in kidney tissue and NRK-52E cells were
performed using immunohistochemistry and immunofluorescence. The apoptosis rate of
NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3
and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis.
HE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory
cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared
with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of
fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with
vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the
cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib
group compared with the vehicle-treated-UUO group (p<0.05). Compared with the
vehicletreated-UUO and TGF--stimulated NRK-52E groups, the expression of a-SMA and
E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the
sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with
sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the
vehicle-treated UUO and TGF--stimulated NRK-52E groups, the ratio of p-Smad3 to
Smad3 decreased in the sorafenib-treated groups (p<0.05).
Our results suggest that sorafenib may useful for the treatment of renal fibrosis through the
suppression of TGF-/Smad3-induced EMT signaling.
Renal fibrosis is the final outcome of many chronic kidney diseases (CKDs) . Activated
myofibroblasts and epithelial-mesenchymal transition (EMT) play essential roles in the
pathogenesis of renal fibrosis . However, the regulatory mechanisms of renal fibrosis processes are not
fully understood, and there is currently no effective treatment.
There is increasing evidence that the transforming growth factor (TGF)- pathway is a
potent moderator of progressive renal fibrosis. EMT is also known to be involved in various
physiological and pathological states, including organ fibrosis . TGF- triggers EMT primarily
via a Smad-dependent mechanism . Smad2/3 is phosphorylated by Smad4 and then
translocates to the nucleus, where it regulates the transcription of the target genes responsible for
EMT . Therefore, we hypothesized that inhibiting the TGF-/Smad pathway would slow or
reverse the process of renal fibrosis.
Sorafenib, a multi-kinase inhibitor, was initially approved for use in humans with renal
cancer and liver cancer . Sorafenib significantly inhibits epithelial cancer cell proliferation and
EMT . Furthermore, sorafenib is known to target both Raf and several tyrosine kinases,
including vascular endothelial growth factor R2 (VEGF-R2), platelet-derived growth factor
(PDGF) receptor, and VEGF receptor , and to regulate receptor tyrosine kinase pathways in
adjacent stromal cells, including myofibroblasts and endothelial cells . Interestingly,
myofibroblast activation and endothelial cell proliferation contribute to matrix production and
vascular sclerosis during renal fibrosis. In addition, previous studies have demonstrated that
sorafenib has potential action in the treatment of liver and lung fibrosis [10, 11, 12, 13]. Thus,
sorafenib may be able to ameliorate renal fibrosis through inhibition of TGF--induced EMT.
This study aimed to explore the therapeutic potential and possible mechanisms of action of
sorafenib in renal fibrosis. Therefore, we examined the effects of sorafenib on TGF--mediated
EMT in NRK-52E cells in vitro and in a rat model of UUO renal fibrosis.
Materials and Methods
Reagents and Antibodies
Sorafenib (Nexavar, BAY43-9006) is manufactured by Bayer Pharmaceuticals (West Haven,
CT, USA). Recombinant human TGF-1 was purchased from R&D Systems (Minneapolis,
MN, USA), and primary antibodies against Smad3 and p-Smad3 were purchased from Cell
Signaling Technology (Beverly, MA, USA). The rabbit monoclonal antibodies against a-SMA and
E-cadherin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the monoclonal
anti--actin antibody was purchased from Sigma Chemical Company (St. Louis, MO, USA).
Establishment of the UUO Model
Forty male Sprague-Dawley rats were used in this study and were given free access to water
and food throughout the experiments. The rats were acclimatized for at least 1 week prior to
the experiments. The UUO model was established in groups of eight rats (males, 8 weeks of
age, 162 to 202 g body weight) by left ureteral ligation. Eight normal sham rats were used as
The rats were divided into 5 groups (eight rats per group). The experimental rats were
administered sorafenib intragastrically (20, 40, 80 mg/kg d), and the UUO and sham group rats were
administered vehicle for 14 or 21 days. Kidney tissue samples were collected for HE and
Masson staining. The protein expression of a-SMA and E-cadherin in kidney tissue was assessed by
immunohistochemistry and immunochemistry. The expression of Smad3 and phosphorylated
Smad3 in kidney tissue was detected by western blot analysis. All of the experimental protocols
described in this study were approved by the local committee for animal use and care (Animal
Care Committee of Xi'an Jiaotong University).
NRK-52E cells were cultured in Dulbeccos modified Eagles medium (DMEM) and 10% fetal
bovine serum at 37C in 5% CO2. The cells were divided into the following six groups: control
group, TGF- (5 ng/mL) group, sorafenib group (10 umol/L), and TGF- (5 ng/mL) co-treated
with sorafenib (1, 5 and 10 umol/L) groups. NRK-52E cells were treated in vitro with TGF-1
and sorafenib for 24 or 48 hours.
HE staining, Masson staining, Immunohistochemistry, and
Immunofluorescence of Kidney Tissue
The tissue was stained with hematoxylin, and ponceau red liquid dye acid complex was then
administered, followed by soaking in 1% phosphomolybdic acid solution. The tissue was then
directly stained with aniline blue liquid and 1% acetic acid, followed by dehydration with a
series of differing ethanol concentrations. Staining showed collagen fibers in blue and cytoplasm
The renal tissue sections were fixed for 10 min in acetone and immersed in 0.3% H2O2 for
30 min to quench endogenous peroxidase activity. The sections were incubated at room
temperature with an optimal dilution of a-SMA and E-cadherin monoclonal antibodies for 1 hour.
After the sections were washed with phosphate-buffered saline (PBS), anti-rabbit secondary
antibody was added for 30 min. The sections were then washed with PBS, incubated with an
ABC kit for 30 min, developed with 3,3-diaminobenzidine (DAB), and counterstained with
hematoxylin. The IPP6 was assessed to quantify the expression of a-SMA and E-cadherin in
the kidney tissue.
HE and Masson staining as well as immunostaining intensity were scored, and the scoring
criteria were as follows: 10 high-power fields (x200) were randomly selected and photographed
in each group. None, mild, moderate and severe involvement were scored as 0, 1, 2, or 3
according to the degree and extent of tubular degeneration and necrosis, tubular atrophy,
inflammatory cell infiltration and fibrosis. The blue area of collagen by Masson staining, which
represents the extent of the lesion, was calculated. The dyed area was measured to calculate the
average optical density in the immunostaining intensity scores.
After proteins were isolated from the kidney tissues or cells, proteins were transferred to
membranes, and the membranes were incubated overnight at 4C with rabbit anti-rat Smad3 and
pSmad3 monoclonal antibodies. After the membranes were washed repeatedly with Tris buffer
containing 0.1% Tween-20 (TBST), they were incubated with secondary antibodies. The blots
were assessed by the enhanced chemiluminescence method. The Smad3 and p-Smad3 levels
were normalized to -actin as an internal control. The expression of Smad3 and
phosphorylated Smad3 in NRK-52E cells and kidney tissue was assessed by western blot analysis.
Flow Cytometry Analysis
NRK-52E cells from each group were collected and stained with FITC-labeled Annexin V and
propidium iodide (PI). The apoptosis rate of NRK-52E cells was determined by flow cytometry
analysis (FCM; Becton Dickinson, San Jose, CA).
Statistical analysis was performed using SPSS software, version 19.0. The data are expressed as
the means standard deviations (SDs). The statistical significance of differences was calculated
using the t-test and one-way analysis of variance (ANOVA), and p 0.05 was considered
Sorafenib Attenuates Kidney Tissue Injury in UUO
HE staining demonstrated that compared with the UUO group, renal interstitial fibrosis,
tubular atrophy, and inflammatory cell infiltration were significantly decreased in the UUO group
treated with sorafenib (p<0.05), especially the medium-dose sorafenib group (Fig. 1A,
p<0.01). The results of Masson staining showed that there was less collagen deposition around
the renal tubules and that the area of fibrosis in the sorafenib-treated-UUO groups was
significantly decreased compared with the vehicle-treated-UUO group (Fig. 1B, p<0.01). In addition,
in the group treated with the middle dose of sorafenib, the size of the kidney was not
significantly increased; the cortex of the kidney was thicker and had a richer blood supply compared
with the vehicle-treated-UUO group (Fig. 1C, p<0.05).
Sorafenib Inhibits Kidney Tissue Fibrosis
Immunohistochemistry and immunofluorescence demonstrated that E-cadherin was mainly
expressed in the nuclei and interstitium. The expression of a-SMA and E-cadherin increased
and decreased, respectively, in the kidneys of the UUO group compared with the sham group.
These changes were partly reversed after treatment with sorafenib. These changes were most
obvious in the group treated with the middle dose (p<0.05, Fig. 2).
To assess whether sorafenib inhibits TGF--induced EMT in renal tubule epithelial cells, we
used rat NRK-52E cells as an in vitro model system for assessing EMT. E-cadherin (red) and
aSMA (red) were expressed in the cytoplasm of NRK-52E cells. Compared with TGF-1
stimulation, E-cadherin and a-SMA expression were increased and decreased, respectively, in the
sorafenib treatment groups, especially in the group receiving the 5 M sorafenib dose (p<0.05,
Fig. 3A). The apoptotic rates were measured by flow cytometry analysis. The apoptotic rate of
Fig 1. HE, Masson staining of UUO kidney tissue. (A) HE staining demonstrated that compared with the
vehicle-treated-UUO, kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration were
decreased in the sorafenib-treated-UUO groups (p<0.05), especially in the medium-dose sorafenib group
(p<0.01). (B) The results of Masson staining showed that there was less collagen deposition around the renal
tubules and that the area of fibrosis in the UUO group treated with the middle dose of sorafenib was
significantly decreased compared with the vehicle-treated-UUO group and sham group (p<0.01). (C) The
size of the kidney was not significantly increased and the cortex of the kidney was thicker and had a richer
blood supply in the sorafenib middle-dose group compared with the control group (p<0.05).
Fig 2. Immunohistochemical and immunofluorescence staining of E-cadherin and a-SMA protein in kidney tissue. (A-D) Immunohistochemistry and
immunofluorescence demonstrated that E-cadherin was expressed in the nuclei and interstitium. Compared with the vehicle-treated-UUO group, the
expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys of sorafenib-treated groups (p<0.05).
Sorafenib Inhibits the Expression of Smad3 and Phosphorylated Smad3
in Kidney Tissue and NRK-52E Cells
Western blotting was performed to assess the protein levels of p-Smad3 and Smad3 in kidney
tissue and NRK-52E cells, -actin was used as a loading control. Compared with the
vehicletreated UUO and TGF-1-stimulated NRK-52E cells groups, the ratio of p-Smad3 to Smad3
was decreased in the sorafenib treatment groups (p<0.05, Figs. 4 and 5).
The present study demonstrated that sorafenib ameliorated kidney fibrosis in a UUO model
and inhibited EMT and apoptosis induced by TGF- in NRK-52E cells in vitro. Our data are
consistent with those of Chen et al. [12, 13], who found that sorafenib inhibits TGF--induced
EMT and apoptosis in mouse hepatocytes. In recent years, an increasing number of studies
have examined kinase inhibitor amelioration of organ fibrosis [14, 15, 16]. Zhang et al. 
proposed that sorafenib inhibits both TGF-1-induced EMT through a possible epigenetic
mechanism and the subsequent epigenetic switching of relevant genes that are critical for EMT
in human lung epithelial cells. Jin et al.  found that knockout of homeodomain-interacting
Fig 3. Immunofluorescence staining of E-cadherin and a-SMA protein and analysis of apoptosis in NRK-52E cells. (A) The expression of E-cadherin
was increased, and the expression of a-SMA was decreased in the sorafenib-treated group relative to the TGF-1-stimulated group (p<0.05). (B) The
apoptotic rates were measured by flow cytometry. The rate of apoptosis in the NRK-52E cells began to significantly increase after 24 h of TGF-1 stimulation.
Sorafenib effectively reduced apoptosis in the TGF-1-stimulated groups (p<0.05).
protein kinase 2 (HIPK2) improves renal function and attenuates proteinuria and kidney
fibrosis in animal models of kidney fibrosis. Recent studies have also suggested that serine protease
inhibitors may provide a new class of therapeutic drugs for the treatment of renal fibrosis
through the suppression of TGF- signaling [19, 20, 21]. Consistent with these studies, we
Fig 4. Expression of Smad3 and phosphorylated Smad3 in NRK-52E cells. (A, B) Western blotting was performed to detect the protein levels of
pSmad3 and Smad3, and beta-actin was used as a loading control. The ratio of p-Smad3 to Smad3 was decreased in the sorafenib-treated groups relative to
the TGF-1-stimulated group in a dose-dependent manner (p<0.05).
Fig 5. Expression of Smad3 and phosphorylated Smad3 in kidney tissue. (A, B) Sorafenib-treated groups compared with the vehicle-treated UUO
group; the ratio of p-Smad3 to Smad3 was decreased in a dose-dependent manner (p<0.05).
demonstrated that sorafenib reduced the rate of apoptosis and EMT in TGF--induced
NRK52E cells and ameliorated renal fibrosis in a UUO model, suggesting a potential and novel use
of the drug in the treatment of renal fibrosis.
In 2005, sorafenib became the first FDA-approved oral agent for the treatment of patients
with advanced hepatocellular and renal cell carcinomas . Previous reports largely focused on
the role of sorafenib in tumors and apoptosis via blocking receptor tyrosine kinases. In this
study, we discovered a novel action of sorafenib, namely, it significantly suppressed
TGF--induced EMT and apoptosis in NRK-52E cells and attenuated renal fibrosis in a UUO model.
The available evidence has shown that TGF- is the major mediator of progressive renal
fibrosis, largely via the Smad-dependent pathway. However, the exact molecular mechanisms
underlying the link between TGF- and disease progression in kidney fibrosis have remained
elusive. These findings provide the first evidence that sorafenib ameliorates renal fibrosis
through a TGF--mediated, Smad-dependent mechanism .
However, several limitations of our study must be acknowledged. This investigation was
primarily limited to the TGF-/Smad pathway; other detailed underlying molecular mechanisms
should be explored in future studies. In addition, the details of how sorafenib inhibits kidney
fibrosis should also be further explored in future studies. In summary, we found that sorafenib is
a potential therapeutic agent for the treatment of kidney fibrosis through inhibition of the
Conceived and designed the experiments: LJ. Performed the experiments: XM LT ZC.
Analyzed the data: LW YO. Contributed reagents/materials/analysis tools: HG JH. Wrote the
paper: LJ. Revised the manuscript: BG RF.
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