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

liangxiubin@njmu.edu.cn

& Rongshan Li

rongshanli13@163.com

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.

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