inhibitory effects of peroxisome proliferator-activated...
TRANSCRIPT
Inhibitory effects of peroxisome proliferator-activated receptor cagonists on collagen IV production in podocytes
Yanjiao Li1,2 • Yachen Shen3 • Min Li3 • Dongming Su4 • Weifeng Xu3 •
Xiubin Liang3 • Rongshan Li1
� Springer Science+Business Media New York 2015
Abstract Peroxisome proliferator-activated receptor-c(PPAR-c) agonists have beneficial effects on the kidney
diseases through preventing microalbuminuria and
glomerulosclerosis. However, the mechanisms underlying
these effects remain to be fully understood. In this study,
we investigate the effects of PPAR-c agonist, rosiglitazone
(Rosi) and pioglitazone (Pio), on collagen IV production in
mouse podocytes. The endogenous expression of PPAR-cwas found in the primary podocytes and can be upregulated
by Rosi and Pio, respectively, detected by RT-PCR and
Western blot. PPAR-c agonist markedly blunted the in-
creasing of collagen IV expression and extraction in
podocytes induced by TGF-b. In contrast, adding PPAR-cantagonist, GW9662, to podocytes largely prevented the
inhibition of collagen IV expression from Pio treatment.
Our data also showed that phosphorylation of Smad2/3
enhanced by TGF-b in a time-dependent manner was sig-
nificantly attenuated by adding Pio. The promoter region of
collagen IV gene contains one putative consensus sequence
of Smad-binding element (SBE) by promoter analysis, Rosi
and Pio significantly ameliorated TGF-b-induced SBE4-
luciferase activity. In conclusion, PPAR-c activation by its
agonist, Rosi or Pio, in vitro directly inhibits collagen IV
expression and synthesis in primary mouse podocytes. The
suppression of collagen IV production was related to the
inhibition of TGF-b-driven phosphorylation of Smad2/3
and decreased response activity of SBEs of collagen IV in
PPAR-c agonist-treated mouse podocytes. This represents
a novel mechanistic support regarding PPAR-c agonists as
podocyte protective agents.
Keywords Podocyte � Peroxisome proliferator-activated
receptor c � Collagen IV � TGF-b � Smad-binding elements
Introduction
Peroxisome proliferator-activated receptor-c (PPAR-c) is amember of the nuclear hormone receptor superfamily.
PPAR-c agonists, known as thiazolidinediones (TZDs),
improve insulin resistance and other metabolic parameters
and are widely used in type 2 diabetic patients over the past
10 years [1, 2].
TZD PPAR-c ligands (troglitazone, rosiglitazone, and
pioglitazone) have been shown not only to lower blood
sugar level through improvement of insulin resistance but
also to ameliorate microalbuminuria in type II diabetic
patients and rats [3–5]. In addition, many studies also
provide evidences that PPAR-c agonists have beneficial
effects on the kidney diseases [6–10]. The beneficial renal
effects were generally thought to be the results of improved
insulin sensitivity induced by TZDs. However, accumu-
lating evidence suggests that TZDs have direct beneficial
effects on glomerulus. A study in non-insulin resistant
diabetic rats showed that troglitazone not only prevented
& Xiubin Liang
& Rongshan Li
1 Department of Nephrology, Shanxi Provincial People’s
Hospital, The Affiliated People’s Hospital of Shanxi Medical
University, Taiyuan 030012, Shanxi, China
2 Department of Endocrinology, The Second Hospital of
Shanxi Medical University, Taiyuan 030001, Shanxi, China
3 Center of Metabolic Disease Research, Nanjing Medical
University, Nanjing 210029, Jiangsu, China
4 Department of Pathology, Nanjing Medical University,
Nanjing 210029, Jiangsu, China
123
DOI 10.1007/s11010-015-2414-2
Mol Cell Biochem (2015) 405:233–241
Received: 12 February 2015 / Accepted: 18 April 2015 / Published online: 29 April 2015
diabetic glomerular hyperfiltration and albuminuria, but
also reduced the expression of extracellular matrix (ECM)
proteins including fibronectin and collagen IV [11].
Moreover, PPAR-c activation has been reported to at-
tenuate the progression of glomerulosclerosis in a non-
diabetic 5/6 nephrectomized animals [12]. These data
strongly suggested that favorable renal effects of TZD
PPAR-c activators in glomerulosclerosis were independent
of metabolic control.
PPAR-c has been identified to present in a variety of
renal tissues, including endothelium, vascular smooth
muscle cells, renal glomerular mesangial cells, endothelial
cells, podocytes, and renal collecting ducts [13–16]. PPAR-
c activation has been found to suppress high-glucose-in-
duced TGF-b expression and TGF-b-mediated collagen
synthesis in cultured mesangial cells [14].
Podocytes are highly specialized cells, which invest the
glomerular capillary endothelium with multiple inter-
digitating foot processes that establish the filtration slit
structure and prevent protein leakage from glomerular
capillaries into urine [17]. There is good evidence that
podocytes are critically involved in abnormal turnover of
the glomerular basement membrane (GBM) and progres-
sive podocyte injury contributes to glomerulosclerosis and
subsequent renal failure [18]. GBM a thin meshwork of
ECM proteins,is an integral part of the glomerular filtration
barrier that is situated between the two cellular compo-
nents, glomerular endothelial cells and podocytes, in the
peripheral capillary wall [19]. GBM are assembled through
an interweaving of collagen IV with laminins, nidogen, and
sulfated proteoglycans [20]. Collagen IV is the most
abundant protein found in basement membranes, compris-
ing about 50 % of total protein mass. Collagen IV of GBM
is produced by and attached to podocytes. Dysregulation of
GBM collagen production produces proteinuria and he-
maturia as seen in Alport’s syndrome and Goodpasture
syndrome clinically [21]. Numerous injurious effectors
such as TGF-b [22, 23], angiotensin II [24] and glucose [25,
26] induce podocytes to express and produce ECM proteins
including collagen to participate in the pathogenesis of
renal injury. Pioglitazone has been previously reported to
ameliorate the progression of glomerulosclerosis in pur-
omycin aminonucleoside (PAN) nephropathy models [27],
associated with increased PPAR-c expression in podocytes.Pioglitazone also protects podocytes from injury with PAN
in vitro, with possible mechanisms only related to de-
creasing PAN-induced apoptosis and necrosis [16]. How-
ever, little is known whether PPAR-c agonist directly
modulates collagen IV production in podocyte. We there-
fore evaluated the effects of PPAR-c agonist on collagen
IV production in mouse podocytes and examined possible
mechanisms for its actions.
Materials and methods
Antibodies and reagents
All reagents were obtained from Sigma-Aldrich (St. Louis,
MO) as otherwise stated. Antibody specific for collagen IV
was purchased from Millipore (Danvers, MA). Secondary
antibodies against mouse or rabbit were obtained from
Amersham Pharmacia Biotech (Piscataway, NJ). Recombi-
nant TGF-b1 was obtained from R & D systems (Min-
neapolis, MN). PPAR-c adenovirus (Ad-PPAR-c), PPAR-c,and SBE-4 luciferase reporters were kindly provided by Dr.
Youfei Guan (Shenzhen University Health Science Center).
Cell culture and treatment
The experiments were performed using primary mouse
podocytes in culture. Primary cultured podocytes were
obtained as previously described [28]. In brief, mouse
kidneys were removed and the renal capsule stripped using
autoclaved surgical instruments. The cortex was isolated
and minced with razor blade, and glomeruli isolated by
sieving (sieve pore size 180 l X2, 75 l X1). Glomeruli
were then suspended in DMEM/Ham’s F-12 (2:1) con-
taining 0.2 lm-filtered 3T3-L1 supernatant, 5 % heat-
inactivated FBS, ITS solution, 100 u/ml penicillin–strep-
tomycin, plated onto collagen type I-coated flask, and in-
cubated at 37 �C, room air with 5 % CO2. After 5 days,
cell colonies began to sprout around the glomeruli. These
cells were characterized as podocytes by detection of the
podocyte specific markers, synaptopodin and Wilms’ tu-
mor 1 (WT-1) by immunofluorescence staining. The
podocytes under the stimulation of 10 ng/ml TGF-b were
treated for 24 h with either PPAR-c agonist, rosiglitazone
(10 lM) or pioglitazone (1 lM) or in the combination of
PPAR-c antagonist, GW9622 (10 lM). The dose of the
mentioned compounds for the cell treatments was referred
from previously published studies [29–32].
Transient transfections of plasmid construct
In vitro transiently transfections with plasmids were per-
formed using Lipofectamine 2000 (Invitrogen) as described
previously [33]. In brief, primary mouse podocytes grown
in 6-well plates were transiently transfected using Lipo-
fectamine 2000 (Invitrogen, Carlsbad, CA) with the indi-
cated luciferase reporter expression plasmids, 4 lg of
cDNA/well. After 24 h, the cells were rinsed with phos-
phate-buffered saline (PBS) and were lysed in RIPA buffer
(50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 % Triton
X-100, 1 % sodium deoxycholate, and 0.1 % SDS). Cell
extracts were used for immunoblot analysis.
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234 Mol Cell Biochem (2015) 405:233–241
RNA expression
Total RNA was extracted from primary mouse podocytes
and reverse transcribed to single-stranded cDNA as de-
scribed previously [28]. Semi-quantitative RT-PCR was
performed to analyze gene expression using 20 pmol of
specific primers for PPAR-c, Collagen IV and b-actin.Primer sequences and PCR conditions were as follows:
PPAR-c sense 50-CCG AAG AAC CAT CCG ATT GA-30
and antisense 50- CGG GAA GGA CTT TAT GTA TGA-30
for 30 cycles with annealing temperature 58 �C, yielding a
271-bp product [34]; Collagen IV 50-GTG CGG TTT GTG
AAG CAC CG-30 and antisense 50- GTT CTT CTC ATG
CAC ACT T-30 for 28 cycles with annealing temperature
54 �C, yielding a 363-bp product [35]; and b-actin sense 50-GAA CCC TAA GGC CAA CCG TGA AAA GAT GAC-
30 and antisense 50-TGA TCT TCA TGG TGC TAG GAG
CCA GAG CAG-30 for 30 cycles with annealing tem-
perature 55 �C, yielding a 700-bp product [28]. The
abundance of each specific mRNA was normalized to b-actin mRNA. Replicate independent experiments were
performed.
Immunocytochemical and immunofluorescence
staining
Primary mouse podocytes cultured on collagen type
I-coated chamber slides were fixed with 2 %
paraformaldehyde and 4 % sucrose in PBS for 5 min at
room temperature followed by permeabilization with 0.3 %
Triton X-100 in PBS for 5 min. After rinsing with PBS,
cells were incubated with 5 % goat serum in PBS for 1 h.
Cells were then incubated with specific antibodies against
synaptopodin (1:200), WT-1 (1:200), PPAR-c (1:200) and
collagen IV (1:100). For immunohistochemical staining,
the Vectastain ABC kit (Vector Laboratories, Burlingame,
CA, USA) with diaminobenzidine as a chromogen. For
immunofluorescence staining, cells were reacted with ei-
ther red Cy3-conjugated donkey anti-mouse IgG or cyanine
FITC-conjugated donkey anti-rabbit IgG (Jackson Im-
munoResearch Laboratories, West Grove, PA) at 37 �C for
1 h. Stained podocytes were mounted with Vectashield
mounting medium (Vector Laboratories, Burlingame, CA)
and viewed with a Carl Zeiss LSM710 microscope equip-
ped with a digital camera (Melville, NY).
Western blot analyses
Equal amounts of protein from podocytes were resolved by
10 % SDS-PAGE and transferred to PVDF membranes. Un-
bound sites were blocked for 1 h at room temperature with
5 % (w/v) skim milk powder in TBST (10 mM Tris pH 8.0,
150 mMNaCl, 0.05 % Tween 20). The blots were incubated
with primary antibodies (anti- PPAR-c, 1:1000; anti-collagenIV, 1:1000; anti-pSmad2/3, 1:1000; anti-Smad2/3, 1:1000; or
anti-b-actin, 1:3000) at room temperature for 2 h. The blots
were thenwashed three times for 10 min eachwith TBST and
incubated for 1 h with 2 lg/ml horseradish peroxidase-con-
jugated secondary antibodies in TBST with 5 % milk, fol-
lowed by three TBST washes. The reactive bands were
visualized with enhanced chemiluminescence (PerkinElmer
Life Sciences, Wellesley, MA) and exposed to X-ray film
(EastmanKodakCo.).b-actin expression provided an internalcontrol. Immunoblot data were scanned and band densities
quantified using ImageJ software.
Measurement of soluble collagen
Podocytes were seeded in DMEM/Ham’s F-12 supple-
mented with 1 % FBS for 24 h before collagen assay. Total
soluble collagen in cell culture supernatants is quantified
using the Sircol collagen assay (Biocolor Co., Westbury,
NY) according to the manufacturer’s instructions. In brief,
1 mL of Sir-ius red dye, an anionic dye that reacts
specifically with basic side chain groups of collagens under
assay conditions, was added to 400 lL of supernatant and
incubated with gentle rotation for 30 min at room tem-
perature. After centrifugation at 12,0009g for 10 min, the
collagen-bound dye was redissolved with 1 mL of 0.5 M
NaOH, and absorbance at 540 nm was measured by en-
zyme-linked immunosorbent assay (MRX; Dynex, Chantil-
ly, VA, USA). The absorbance was directly proportional to
the amount of newly formed collagen in the cell culture
supernatant. The assay was performed in triplicate, and the
mean values of each sample were calculated.
Statistical analysis
Data were obtained from experiments performed 3–4 times
and values were presented as mean ± SEM. p values were
calculated by ANOVA followed by unpaired t test as ap-
propriate. p\ 0.05 was considered to be statistically
significant.
Results
PPAR-c was expressed in primary mouse podocytes
After the glomeruli were isolated from mouse kidneys and
cultured for 5 days, cell colonies began to sprout around
the glomeruli. These cells showed an epithelial mor-
phology with a polyhedral shape (Fig. 1A). Im-
munofluorescence staining with synaptopodin and WT-1
was performed to confirm those cells as podocytes. As
shown in Fig. 1A, there were strongly expressions of
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Mol Cell Biochem (2015) 405:233–241 235
synaptopodin and WT-1, in those differentiated podocytes,
particularly in the cytoplasm and extending toward cell
processes.
The expression of PPAR-c in mouse podocytes was
determined by RT-PCR and Western blotting. PPAR-cwere functionally expressed in mouse podocytes at both
mRMA (Fig. 1B) and protein (Fig. 1C) level. Moreover,
the expression of PPAR-c was significantly increased by
the treatment of its agonist, Rosiglitazone (Rosi 10 lM)
and Pioglitazone (Pio 1 lM), respectively, for 24 h. The
mean data of PPAR-c protein expression are shown in
Fig. 1D. PPAR-c expression in podocytes was also con-
firmed by immunocytochemical staining. Positive signals
of PPAR-c were observed in cytoplasma, nucleus and cell
processes of mouse podocytes (Fig. 1E).
To determine the functional activities of PPAR-c as a
transcription factor in podocytes, the PPAR-c luciferase
reporter plasmid containing a PPAR-c response element
was transfected into the mouse podocyte along with TK
plasmids as a control of transfection efficiency. As shown
in Fig. 1F, the relative level of luciferase activity was
significantly increased by Rosi or Pio treatment.
PPAR-c agonists inhibit the collagen IV production
of podocytes
A large number of evidences indicated that transforming
growth factor b (TGF-b) is a key mediator in kidney fi-
brosis associated with accumulation of ECM [36, 37]. In
our study, TGF-b (10 ng/ml) treatment led to an about 2–3-
a b
c d
PPAR-
ββ-actin
Control Rosi Pio
PPAR-
β-actin
PioControl Rosi
Control Rosi Pio
Rel
ativ
e PP
AR-
γPr
otei
n L
evel
*
*
x 200 x 400x 100
*
*
Rel
ativ
e PP
RE
Luc
ifera
se A
ctiv
ity
Control Rosi Pio
(A)
(E) (F)
(B)
(D)
(C)
Fig. 1 PPAR-c was functionally expressed in primary mouse
podocytes. A Primary mouse podocytes were characterized by
detection of the podocyte specific markers, synaptopodin and Wilms’
tumor 1 (WT-1) by immunofluorescence staining. a Cell colonies
sprout around the glomeruli (9100). b Cells with an epithelial
morphology (9200). c Synaptopodin was detected in podocytes
(9400). d WT-1 was detected in podocytes (9400). B PPAR-cmRNA expression in podocytes. C PPAR-c protein expression in
podocytes treated with Rosi (10 lM) and Pio (1 lM). D Quantitation
of PPAR-c protein expression in C. E Positive signals of PPAR-cwere observed in cytoplasm, nuclear, and cell processes of mouse
podocytes detected by immunocytochemical staining. F The relative
level of PPAR-c luciferase activity induced by Rosi and Pio. Data
represent 3 experiments. Bars are mean ± SE from three independent
experiments, *p\ 0.05 compared with untreated cells
123
236 Mol Cell Biochem (2015) 405:233–241
fold increases in the total collagen production compared
with that in the vehicle-treated cells. Addition of Rosi or
Pio appeared to decrease the collagen production in mouse
podocytes (Fig. 2A). TGF-b also significantly upregulated
the mRNA expressions of Collagen IV in podocytes, Rosi
and Pio treatment markedly decreased its mRNA levels
(Fig. 2B). The mean data of Collagen IV mRNA expres-
sion are shown in Fig. 2C.
Immuno-blots were used to assess podocyte expression
of collagen IV with or without PPAR-c agonist treatments.
As shown in Fig. 2D, PPAR-c agonist, Rosi and Pio, re-
duced collagen IV expression which was increased by
TGF-b stimulation in mouse podocytes. The mean data of
Collagen IV expression are shown in Fig. 2E.
Next, collagen IV expression was determined by im-
munofluorescence staining using a rabbit polyclonal an-
tiserum against collagen IV as the primary antibody. As
shown in Fig. 2F, collagen IV expression was significantly
increased in mouse podocytes stimulated by TGF-b, butPio treatment obviously obliterated collagen IV densities,
which were consistent with biochemical data described
above.
Effects of PPAR-c overexpression and PPAR-cantagonist on collagen IV production in podocytes
To further confirm the regulation of PPAR-c on collagen
IV production of podocyte specifically, the mouse
*
Col
lage
n C
once
ntra
tion
(ug/
ul)
**
Collagen IV
β-actin
Rel
ativ
e C
olla
gen
IV m
RN
A L
evel
* **
β-actin
Collagen IV
**
Rel
ativ
e C
olla
gen
IV P
rote
in L
evel
Control TGF-β TGF-β + Pio
(A)
(C)
(F)
(B)
(D)
(E)
Fig. 2 PPAR-c agonists inhibit
the collagen IV production of
podocytes. A TGF-b (10 ng/ml)
markedly increased collagen
extraction, but Rosi (10 lM) or
Pio (1 lM) decreased the
collagen products in mouse
podocytes. **p\ 0.01
compared with untreated cells;
*p\ 0.05 compared with TGF-
b treatment alone. B TGF-b(10 ng/ml) upregulated the
mRNA expressions of collagen
IV in podocytes, Rosi and Pio
treatment markedly decreased
its mRNA levels.
C Quantitation of collagen IV
mRNA expression in
B. *p\ 0.05. D Rosi and Pio
significantly reduced collagen
IV protein expression which
was increased by TGF-bstimulation in mouse podocytes.
E The mean data of Collagen IV
protein expression in
D. *p\ 0.05. F Collagen IV
expression determined by
immunofluorescence staining
was significantly increased in
mouse podocytes stimulated by
TGF-b, but Pio treatment
obviously obliterated collagen
IV densities. Data represent 3
experiments. Bars are
mean ± SE from three
independent experiments
123
Mol Cell Biochem (2015) 405:233–241 237
podocytes were infected with PPAR-c adenovirus (Ad-
PPAR-c) for 24 h or treated with Pio treatment.
As shown in Fig. 3A, the transfection of podocytes with
Adv-PPAR-c resulted in a about 6-fold increase in PPAR-cexpression in comparison of the transfection of control
adenovirus (Ad-vector). The upregualtion of PPAR-c by
either Pio treatment or Ad-PPAR-c infection ameliorated the
TGF-b-induced collagen IV production of mouse podocytes
for about 2 and 3 times individually, while the empty virus
vector had no effect on collagen IV expression (Fig. 3B).
The data summary of collagen IV expression is shown in
Fig. 3C. The specific significance of PPAR-c activation on
the regulation of collagen IV expression was also evaluated
in primary mouse podocytes treated by PPAR-c antagonist,
GW9662 (10 lM). The increased PPAR-c expression re-
sponsed to Pio treatment was significantly blunted after ex-
posure to GW9662 in podocytes (Fig. 3D).
Compared with the collagen IV production from the
podocytes with TGF-b treatment alone, that was markedly
downregulated by adding Pio that was consistent with the
data described above. However, GW9662 largely abolished
Pio suppression of collagen IV expression under the TGF-btreatment (Fig. 3E). The data summary of Collagen IV
expression is shown in Fig. 3F.
Inhibitory effects of PPAR-c agonists on TGF-b-induced SBE4-luciferase activity
We next investigated potential molecular mechanisms
through which PPAR-c agonist might regulate collagen IV
production in mouse podocytes. TGF-b was known to act
through the downstream small mothers against decapenta-
plegic (Smad) signaling pathway to drive many of the gene
expression changes and pathology observed in renal fi-
brosis [38]. Therefore, mouse podocytes treated with or
without Pio were exposed to 10 ng/ml of TGF-b for var-
ious periods of time (0, 15, and 30 min), the phosphory-
lation of Smad2/3 was determined by western blot. As
shown in Fig. 4A, TGF-b stimulation led to Smad2/3
phosphorylation in a time-dependent manner. However,
PioAd-PPAR-γ
Ad-vector
PPAR-γ
β-actin
- - -+-
-- ---
+
+Pio - +- -
TGF-β + ++ +
Ad-PPAR-γ - -+ -Ad-vector - -- +
β-actin
Collagen IV
Rel
ativ
e C
olla
gen
IV P
rote
in L
evel
TGF-β
Pio
Ad-PPAR-γAd-vector
+ + + +- - + -- - - +- + - -
**
Pio - -+GW 9662 - +-
++
PPAR-γ
β-actin
Pio - -+ +TGF-β + ++ +
GW9662 - +- +
β-actin
Collagen IV
Rel
ativ
e C
olla
gen
IV P
rote
in L
evel
TGF-β
Pio
GW9662
+ + + +- + - +- - + +
**
(A)
(C)
(E)
(B)
(F)
(D)
Fig. 3 Effects of PPAR-coverexpression and PPAR-cantagonist on collagen IV
expression. A Pio (1 lM)
obviously activated PPAR-cexpression, and Ad-PPAR-cinfection dramatically enhanced
PPAR-c expression VS control.
B Collagen IV expression was
significantly decreased in mouse
podocytes infected by Ad-
PPAR-c VS TGF-b stimulation
alone. The empty virus vector
had no effect on collagen IV
expression. C Quantitation of
collagen IV expression in
B. *p\ 0.05 compared TGF-btreatment alone. D The
increased PPAR-c expression
responsed to Pio treatment was
blunted after exposure to
GW9662 in podocytes.
E Collagen IV expression was
downregulated by adding Pio
VS TGF-b treatment alone.
GW9662 largely prevented
suppression of collagen IV
expression by Pio treatment.
F Quantitation of collagen IV
expression in E *p\ 0.05. Data
represent 3 experiments. Bars
are mean ± SE from three
independent experiments
123
238 Mol Cell Biochem (2015) 405:233–241
PPAR-c agonist, Pio, attenuated TGF-b-driven phospho-
rylation of Smad2/3 in the individual time points. The
mean data of Smad2/3 phosphorylation are shown in
Fig. 4B. The promoter analysis indicated that the promoter
of Collagen IV contained Smad-binding elements (SBEs)
(Fig. 4C). Therefore, to determine the effect of PPAR-cagonists on TGF-b-induced transcriptional activation of
Smad response elements, SBE4 luciferase reporters were
transfected into podocytes with TK plasmids as a control of
transfection efficiency. The data demonstrated that TGF-btreatment resulted in significantly enhanced activation of
luciferase. However, the TGF-b-induced SBE4-luciferase
activity was markedly blocked by co-treatment with either
Rosi or Pio (Fig. 4D).
Discussion
Using primary mouse podocytes, this study provides the
convincing evidence that PPAR-c agonists efficiently
suppress collagen IV synthesis through inhibition of TGF-
b-Smad pathway.
We first demonstrated that PPAR-c was functionally
expressed in mouse podocytes and its expression was sig-
nificantly elevated by rosiglitazone and pioglitazone, re-
spectively. PPAR-c agonist markedly blunted the
increasing of collagen IV expression and extraction in
podocytes induced by TGF-b, known as a profibrotic cy-
tokine. In contrast, adding PPAR-c antagonist, GW9662, to
podocytes largely abolished Pio inhibition of collagen IV
expression. These data implicate that enhanced PPAR-cactivation plays a key inhibitory role for collagen IV ex-
pression and extraction in podocyte.
Synthesis and deposition of extracellular matrix ECM
including fibronectin and collagenwithin the glomerulus and
interstitium characterizes renal fibrosis [39, 40]. Many pre-
vious studies demonstrated that PPAR-c agonists had pro-
tective effects on renal fibrosis. Pioglitazone was reported to
ameliorate progression of podocyte injury linked to
glomerulosclerosis and reduce proteinuria in the chronic
PAN-associated focal segmental glomerulosclerosis (FSGS)
model [27]. PPAR-c agonist was also previously demon-
strated to attenuate development of sclerosis in the hyper-
tensive 5/6 nephrectomymodel. And those protective effects
Pio + + +- - -
0 min 15 mins
p-Smad2/3
Smad2/3
TGF-β + + ++ + +
30 minsR
elat
ive
P-Sm
a d2/
3 P
rote
in L
evel
TGF-β
Pio
+ + + + + + + - + - + -
0 min 15 mins 30 mins
*
*
*
ATGTAGACGGATGTT
Smad binding element
Collgen IV
-1595-1610
Rel
ativ
e S
BE
4 L
ucife
rase
Act
ivity
*
**
(A) (C)
(D)(B)
Fig. 4 PPAR-c agonists inhibition of TGF-b-induced SBE4-lu-
ciferase activity. A Mouse podocytes treated with or without Pio
(1 lM) were exposed to 10 ng/ml of TGF-b for the periods of time (0,
15 and 30 min). TGF-b treatment led to Smad2/3 phosphorylation in
a time-dependent manner. Pio attenuated TGF-b-driven phosphory-
lation of Smad2/3 in each single time point. B The mean data of
Smad2/3 phosphorylation in A. *p\ 0.05 compared with TGF-b
treatment alone. C The promoter analysis indicated that the promoter
of Collagen IV contained Smad-binding elements (SBEs). D TGF-b-induced SBE4-luciferase activity was markedly blocked by co-
treatment with either Rosi (10 lM) or Pio (1 lM). *p\ 0.05;
**p\ 0.01 compared with TGF-b treatment alone. Data represent 3
experiments. Bars are mean ± SE from three independent
experiments
123
Mol Cell Biochem (2015) 405:233–241 239
of PPAR-c agonist were linked to downregulated expressionof PAI-1, a major physiological inhibitor of tissue-type
plasminogen activator and urokinase-type plasminogen ac-
tivator (PA) [12]. But the mechanisms underlying this
beneficial process of PPAR-c agonist are incompletely un-
derstood. Since podocytes play a direct role in GBM thick-
ening of renal disease, and collagen IV is the most abundant
constituent of the GBM, the most suitable set of matrix
proteins to assay about favorable renal effects of PPAR-cagonist would be collagen IV. Our current study provides
directly evidence that protective effect of PPAR-c agonists
on podocyte was due to inhibition of collagen IV production,
which contribute to our completely understanding about the
mechanism of PPAR-c protection from podocyte injury.
We next explored potential mechanisms of PPAR-c ago-
nist on collagen production in podocytes. TGF-b represents a
central mediator of fibrogenic process due to its role in in-
creasing ECM production and inhibiting its degradation [38,
41]. Data from in vitro studies clearly demonstrated that TGF-
b can stimulate the biosynthesis of various ECM proteins in-
cluding collagen, fibronectin, and matrix proteoglycans and
induce inhibitors of matrix degrading metalloproteinases and
plasminogen-activated inhibitors [37, 42]. Two cell mem-
brane surface serine/threonine kinase receptors of TGF-b,TbRI, and TbRII, have been found to involve in fibrogenic
TGF-b signaling pathway. TGF-b initially interacted with
TbRII, followed by the recruitment and activation of TbRI.Phosphorylated TbRI is able to activate Smads 2 and 3 [43].
Our data showed that phosphorylation of Smad2/3 enhanced
by TGF-b in time-dependent manner was attenuated by add-
ing Pio, which implied that TGF-b-Smad2/3 pathway was
involved in Pio beneficial effects on collagen IV synthesis in
podocytes. Active Smad2/3 was followed by their subsequent
heteromeric complexes with co-Smad4, which either directly
or through interactions with other transcription factors bind to
SBEs in the target gene TGF-b-regulated [44, 45]. We iden-
tified one putative consensus SBE sequence in the promoter
region of collagen IV gene by analysis of Transcription Ele-
mentSearchingSystem (TESS) and softwarePromoter 2.And
PPAR-c agonist, either Rosi or Pio, significantly ameliorated
TGF-b-induced SBE4-luciferase activity. Additional inves-
tigation is required to further define whether and how acti-
vated Smad2/3 interacted with SBE of collagen IV promoter
as well as other pathways involved.
Conclussions
Taken together, this study showed that PPAR-c activation
by its agonist, Rosi or Pio, in vitro inhibits collagen IV
expression and synthesis in mouse podocytes. The sup-
pression of collagen IV production was related to the in-
hibition of TGF-b-driven phosphorylation of Smad2/3 and
decreased response activity of SBEs of collagen IV in
PPAR-c agonist-treated mouse podocytes. These results
provide a mechanistic support to the clinical evidence re-
garding PPAR-c agonists as podocyte protective agents for
glomerular injury.
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China Grant 31071026, 31271263,
81470040 to X. Liang, 81450033 to R. Li; National Science and
Technology Support Program, China (2011BAI0B01) and Interna-
tional Cooperative Program of Shanxi Province, China (2014081054)
to R. Li.
Conflict of interest No conflicts of interest, financial or otherwise,
are declared by the authors.
References
1. Campbell IW (2005) The clinical significance of PPAR gamma
agonism. Curr Mol Med 5:349–363
2. Bhatia V, Viswanathan P (2006) Insulin resistance and PPAR
insulin sensitizers. Curr Opin Investig Drugs 7:891–897
3. Sarafidis PA, Bakris GL (2006) Protection of the kidney by
thiazolidinediones: an assessment from bench to bedside. Kidney
Int 70:1223–1233
4. Mao Z, Ong AC (2009) Peroxisome proliferator-activated re-
ceptor gamma agonists in kidney disease–future promise, present
fears. Nephron Clin Pract 112:c230–c241
5. Sarafidis PA, Stafylas PC, Georgianos PI, Saratzis AN, Lasaridis
AN (2010) Effect of thiazolidinediones on albuminuria and
proteinuria in diabetes: a meta-analysis. Am J Kidney Dis 55:
835–847
6. Bakris GL, Ruilope LM, McMorn SO, Weston WM, Heise MA,
Freed MI, Porter LE (2006) Rosiglitazone reduces microalbu-
minuria and blood pressure independently of glycemia in type 2
diabetes patients with microalbuminuria. J Hypertens 24:
2047–2055
7. Yang HC, Deleuze S, Zuo Y, Potthoff SA, Ma LJ, Fogo AB
(2009) The PPARgamma agonist pioglitazone ameliorates aging-
related progressive renal injury. J Am Soc Nephrol 20:2380–2388
8. Venegas-Pont M, Sartori-Valinotti JC, Maric C, Racusen LC,
Glover PH, McLemore GR Jr, Jones AV, Reckelhoff JF, Ryan MJ
(2009) Rosiglitazone decreases blood pressure and renal injury in
a female mouse model of systemic lupus erythematosus. Am J
Physiol Regul Integr Comp Physiol 296:R1282–R1289
9. Yotsumoto T, Naitoh T, Kanaki T, Matsuda M, Tsuruzoe N
(2003) A novel peroxisome proliferator-activated receptor
(PPAR) gamma agonist, NIP-222, reduces urinary albumin ex-
cretion in streptozotocin-diabetic mice independent of
PPARgamma activation. Metab, Clin Exp 52:1633–1637
10. Kawai T, Masaki T, Doi S, Arakawa T, Yokoyama Y, Doi T,
Kohno N, Yorioka N (2009) PPAR-gamma agonist attenuates
renal interstitial fibrosis and inflammation through reduction of
TGF-beta. Lab Investig 89:47–58
11. Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T, Kikkawa R
(2000) Thiazolidinedione compounds ameliorate glomerular
dysfunction independent of their insulin-sensitizing action in
diabetic rats. Diabetes 49:1022–1032
12. Ma LJ, Marcantoni C, Linton MF, Fazio S, Fogo AB (2001)
Peroxisome proliferator-activated receptor-gamma agonist
troglitazone protects against nondiabetic glomerulosclerosis in
rats. Kidney Int 59:1899–1910
123
240 Mol Cell Biochem (2015) 405:233–241
13. Guan Y, Zhang Y, Schneider A, Davis L, Breyer RM, Breyer MD
(2001) Peroxisome proliferator-activated receptor-gamma ac-
tivity is associated with renal microvasculature. Am J Physiol
Ren Physiol 281:F1036–F1046
14. Zheng F, Fornoni A, Elliot SJ, Guan Y, Breyer MD, Striker LJ,
Striker GE (2002) Upregulation of type I collagen by TGF-beta in
mesangial cells is blocked by PPARgamma activation. Am J
Physiol Ren Physiol 282:F639–F648
15. Law RE, Goetze S, Xi XP, Jackson S, Kawano Y, Demer L,
Fishbein MC, Meehan WP, Hsueh WA (2000) Expression and
function of PPARgamma in rat and human vascular smooth
muscle cells. Circulation 101:1311–1318
16. Kanjanabuch T, Ma LJ, Chen J, Pozzi A, Guan Y, Mundel P,
Fogo AB (2007) PPAR-gamma agonist protects podocytes from
injury. Kidney Int 71:1232–1239
17. Shirato I, Hishiki T, Tomino Y (2001) Podocyte loss and pro-
gression of diabetic nephropathy. Contrib Nephrol 134:69–73
18. Gassler N, Elger M, Kranzlin B, Kriz W, Gretz N, Hahnel B,
Hosser H, Hartmann I (2001) Podocyte injury underlies the
progression of focal segmental glomerulosclerosis in the fa/fa
Zucker rat. Kidney Int 60:106–116
19. Suh JH, Miner JH (2013) The glomerular basement membrane as
a barrier to albumin. Nat Rev Nephrol 9:470–477
20. Yurchenco PD, Smirnov S, Mathus T (2002) Analysis of base-
ment membrane self-assembly and cellular interactions with na-
tive and recombinant glycoproteins. Methods Cell Biol
69:111–144
21. Hudson BG, Tryggvason K, Sundaramoorthy M, Neilson EG
(2003) Alport’s syndrome, Goodpasture’s syndrome, and type IV
collagen. New Engl J Med 348:2543–2556
22. Wu DT, Bitzer M, Ju W, Mundel P, Bottinger EP (2005) TGF-
beta concentration specifies differential signaling profiles of
growth arrest/differentiation and apoptosis in podocytes. J Am
Soc Nephrol 16:3211–3221
23. Mitu GM, Wang S, Hirschberg R (2007) BMP7 is a podocyte
survival factor and rescues podocytes from diabetic injury. Am J
Physiol Ren Physiol 293:F1641–F1648
24. Liebau MC, Lang D, Bohm J, Endlich N, Bek MJ, Witherden I,
Mathieson PW, Saleem MA, Pavenstadt H, Fischer KG (2006)
Functional expression of the renin-angiotensin system in human
podocytes. Am J Physiol Ren Physiol 290:F710–F719
25. Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M,
Blautzik J, Corat MA, Zeier M, Blessing E (2007) Activated
protein C protects against diabetic nephropathy by inhibiting
endothelial and podocyte apoptosis. Nat Med 13:1349–1358
26. Phillips LM, Wang Y, Dai T, Feldman DL, LaPage J, Adler SG
(2011) The renin inhibitor aliskiren attenuates high-glucose in-
duced extracellular matrix synthesis and prevents apoptosis in
cultured podocytes. Nephron Exp Nephrol 118:e49–e59
27. Yang HC, Ma LJ, Ma J, Fogo AB (2006) Peroxisome prolif-
erator-activated receptor-gamma agonist is protective in podocyte
injury-associated sclerosis. Kidney Int 69:1756–1764
28. Liang XB, Ma LJ, Naito T, Wang Y, Madaio M, Zent R, Pozzi A,
Fogo AB (2006) Angiotensin type 1 receptor blocker restores
podocyte potential to promote glomerular endothelial cell growth.
J Am Soc Nephrol 17:1886–1895
29. Li L, Shen Y, Ding Y, Liu Y, Su D, Liang X (2014) Hrd1 par-
ticipates in the regulation of collagen I synthesis in renal fibrosis.
Mol Cell Biochem 386:35–44
30. Lennon R, Welsh GI, Singh A, Satchell SC, Coward RJ, Tavare
JM, Mathieson PW, Saleem MA (2009) Rosiglitazone enhances
glucose uptake in glomerular podocytes using the glucose
transporter GLUT1. Diabetologia 52:1944–1952
31. Miglio G, Rosa AC, Rattazzi L, Grange C, Camussi G, Fantozzi
R (2012) Protective effects of peroxisome proliferator-activated
receptor agonists on human podocytes: proposed mechanisms of
action. Br J Pharmacol 167:641–653
32. Su HC, Ma CT, Yu BC, Chien YC, Tsai CC, Huang WC, Lin CF,
Chuang YH, Young KC, Wang JN (2012) Glycogen synthase
kinase-3beta regulates anti-inflammatory property of fluoxetine.
Int Immunopharmacol 14:150–156
33. Liang X, Peters KW, Butterworth MB, Frizzell RA (2006) 14-3-3
isoforms are induced by aldosterone and participate in its regula-
tion of epithelial sodium channels. J Biol Chem 281:16323–16332
34. Cha DR, Zhang X, Zhang Y, Wu J, Su D, Han JY, Fang X, Yu B,
Breyer MD, Guan Y (2007) Peroxisome proliferator activated
receptor alpha/gamma dual agonist tesaglitazar attenuates
diabetic nephropathy in db/db mice. Diabetes 56:2036–2045
35. Moridaira K, Morrissey J, Fitzgerald M, Guo G, McCracken R,
Tolley T, Klahr S (2003) ACE inhibition increases expression of
the ETB receptor in kidneys of mice with unilateral obstruction.
Am J Physiol Ren Physiol 284:F209–F217
36. Tamaki K, Okuda S (2009) Role of TGF-beta in the progression
of renal fibrosis. Contrib Nephrol 139:44–65
37. Schnaper HW, Jandeska S, Runyan CE, Hubchak SC, Basu RK,
Curley JF, Smith RD, Hayashida T (2009) TGF-beta signal
transduction in chronic kidney disease. Front Biosci
14:2448–2465
38. Herman-Edelstein M, Weinstein T, Gafter U (2013) TGFbeta1-
dependent podocyte dysfunction. Curr Opin Nephrol Hypertens
22:93–99
39. Liu Y (2006) Renal fibrosis: new insights into the pathogenesis
and therapeutics. Kidney Int 69:213–217
40. Liu Y (2011) Cellular and molecular mechanisms of renal fi-
brosis. Nat Rev Nephrol 7:684–696
41. Bottinger EP, Bitzer M (2002) TGF-beta signaling in renal dis-
ease. J Am Soc Nephrol 13:2600–2610
42. Sharma K, McGowan TA (2000) TGF-beta in diabetic kidney
disease: role of novel signaling pathways. Cytokine Growth
Factor Rev 11:115–123
43. Chipuk JE, Cornelius SC, Pultz NJ, Jorgensen JS, Bonham MJ,
Kim SJ, Danielpour D (2002) The androgen receptor represses
transforming growth factor-beta signaling through interaction
with Smad3. J Biol Chem 277:1240–1248
44. Wrana JL (2000) Regulation of Smad activity. Cell 100:189–192
45. Wrana JL, Attisano L (2000) The Smad pathway. Cytokine
Growth Factor Rev 11:5–13
123
Mol Cell Biochem (2015) 405:233–241 241