original article

http://www.kidney-international.org
& 2006 International Society of Nephrology

Peroxisome proliferator-activated receptor-gamma

agonist is protective in podocyte injury-associated
sclerosis
H-C Yang1,2, L-J Ma1, J Ma2,3 and AB Fogo1
1

Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; 2Department of Nephrology, Huashan
Hospital, Shanghai Medical School, Fudan University, Shanghai, China and 3Department of Pediatrics, Vanderbilt University Medical
Center, Nashville, Tennessee, USA

We have previously observed increased expression of

peroxisome proliferator-activated receptor gamma (PPARc) in
podocytes in both rat and human sclerotic conditions. The
aim of the present study was to investigate whether
activation of PPARc can attenuate podocyte injury-associated
glomerulosclerosis in vivo. Puromycin aminonucleoside
nephropathy was induced in SpragueDawley rats. The
animals then either received no further treatment (control
group (CONT)); or the PPARc agonist, pioglitazone (Pio)
starting at week 0 (P0); or Pio starting at week 6 (P6), with
sacrifice at week 12. At week 12, urinary protein excretion
and systolic blood pressure were similar in the three groups.
Glomerular filtration rate and glomerulosclerosis were
decreased in CONT and P0 at week 12, but preserved in P6
rats. PPARc expression in CONT at 12 weeks was increased in
podocytes and in mesangial WT-1 cells in segmentally
sclerotic glomeruli, with less Wilms tumor 1 (WT-1) staining.
In P6 rats, mesangial WT-1 staining was lessened, but
podocyte staining was strongly accentuated. Delayed
treatment with Pio partially restored podocyte staining and
tended to decrease the ratio of proliferating cell nuclear
antigen-positive to apoptotic cells in glomeruli. Both
treatment groups showed significantly reduced infiltrating
glomerular macrophages and plasminogen activator
inhibitor-1 mRNA expression in cortex, with no change in
transforming growth factor-b1 and tissue inhibitor of
metalloproteinase-1 mRNA. Pio also decreased renal cortical
angiopoietin-like protein 4 expression to almost 20%
of CONT group, associated with increased vascular
endothelial-derived growth factor expression in glomeruli.
We conclude that treatment with PPARc agonist has
protective effects on progression of glomerulosclerosis.
Kidney International (2006) 69, 17561764. doi:10.1038/sj.ki.5000336;
published online 5 April 2006
Correspondence: AB Fogo, MCN C3310, Department of Pathology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
E-mail: agnes.fogo@vanderbilt.edu
Received 31 August 2005; revised 9 November 2005; accepted 18
November 2005; published online 5 April 2006
1756

KEYWORDS: PPARg; podocytes; angiopoietin-like protein 4; VEGF; PAI-1

Peroxisome proliferator-activated receptor gamma (PPARg)

is a member of the steroid/thyroid nuclear hormone receptor
superfamily of ligand-activated transcription factors. It forms
a heterodimer with the retinoid X receptor-a and binds to
common consensus response elements called PPAR response
elements consisting of a direct repeat of two hexanucleotides
spaced by one nucleotide.1 PPARg can also repress gene
transcription by interfering with nuclear factor-kB, a signal
transducer and activator of transcription, and activator
protein-1 signaling pathways in a DNA-binding independent
manner.2 PPARg plays important roles in lipid and glucose
metabolism and adipocyte differentiation. PPARg is also
expressed in macrophages and vascular smooth muscle cells,
and plays important roles in the pathogenesis of atherosclerosis.
PPARg agonists have beneficial effects in models of
diabetic disease and in hyperlipidemia.3,4 Troglitazone, one
of the thiazolidinediones that are specific synthetic ligands
for PPARg, also ameliorates the progression of glomerulosclerosis in the nondiabetic model of 5/6 nephrectomy. These
beneficial effects were independent of insulin/glucose effects
and were associated with regulation of glomerular cell proliferation, hypertrophy, and decreased plasminogen activator
inhibitor-1 (PAI-1) and transforming growth factor-beta
(TGF-b) expression.5
Focal segmental glomerular sclerosis (FSGS) is a lesion
common to chronic renal failure. Podocyte injury is
postulated to be the primary event in primary FSGS.6 We
have previously observed increased expression of PPARg in
podocytes in both rat and human sclerotic conditions in vivo.
We postulate that this PPARg increase could be counterregulatory and a response to podocyte injury. Further, our
recent in vitro data showed that PPARg activation protects
against puromycin aminonucleoside (PAN)-induced apoptosis and necrosis of podocytes (Kanjanabuch et al. J Am Soc
Nephrol 2003; 14: 93A (abstract)).
Kidney International (2006) 69, 17561764

original article

H-C Yang et al.: PPARc and sclerosis

We therefore evaluated the effects of pioglitazone (Pio), a

PPARg agonist, on PAN-induced sclerosis, a model of
podocyte injury-induced FSGS, and possible mechanisms
for its actions.

PPARg agonist before PAN had sclerosis similar to control

(2.6270.09, P NS vs CONT). Effects on tubulointerstitial
fibrosis paralleled glomerulosclerosis.
Glomerular PPARc expression

Renal morphological changes

Systolic BP (mm Hg)

At 12 weeks, glomerulosclerosis, tubular atrophy, dilation,

and interstitial fibrosis were present in all rats. Rats receiving
delayed PPARg agonist starting after PAN showed significant
amelioration of the development of glomerulosclerosis
compared with control (SI CONT 2.9470.13 vs P6
2.3370.09, Po0.05; Figure 3a and b). In contrast, rats given

200
150
100

P0
P6
CONT

50
0

W0

W4
Time

W8

Figure 1 | SBP. PAN injection increased SBP significantly compared to

baseline in all groups. Pio did not affect blood pressure (P0, early
treatment; P6, delayed treatment; CONT, control).
Kidney International (2006) 69, 17561764

Glomerular proliferation and apoptosis

Immunostaining for proliferating cell nuclear antigen

(PCNA) and apoptosis was negative in the normal rat
glomerulus (data not shown). In CONT rats, both PCNAand apoptosis-positive cells were present in glomeruli and
tubules. Glomerular visceral and parietal epithelial cells,
and cells in the mesangial area and capillary loops, consistent
with mesangial and endothelial cells, respectively, showed

a
24 h urine protein (mg/24 h)

Body weights increased similarly over time in all groups,

although less rapidly after PAN (at 12 weeks, P0 group
275713, P6 284714, control (CONT) 29077 g, PNS).
Systolic blood pressure (SBP) was increased significantly
in untreated control rats by week 4 (139.078.0 vs baseline
110.073.4 mmHg, Po0.05) and continued to increase until
8 weeks (144.073.9 mmHg) (Figure 1). Interestingly, PPARg
agonist, either delayed or early, had no effect on systolic
blood pressure (at 8 weeks, P0 148.974.0, P6 147.97
2.7 mmHg, PNS vs CONT).
Urinary protein excretion was dramatically increased in
CONT rats by week 4 (474.1735.7 vs baseline 24.174.4 mg/
24 h, Po0.05) and 8 (550.0757.8 mg/24 h), and tapered by
12 weeks (361.0783.0 mg/24 h). Delayed PPARg agonist,
given after PAN, did not affect the course of proteinuria
(week 0 24.872.5, week 4 587.6757.7, week 8 549.3743.6,
week 12 352.2758.1, P NS vs CONT). In contrast, PPARg
agonist given starting at time of uninephrectomy (Nx),
before PAN, increased proteinuria at 4 weeks (743.57
50.9 mg/24 h) and 8 weeks (777.2745.8 mg/24 h), with no
difference vs CONT by 12 weeks (377.0766.8 mg/24 h;
Figure 2a).
Glomerular filtration rate (GFR) was decreased in CONT
and P0 groups by week 12. Rats with delayed PPARg agonist
(P6) had higher GFR than CONT (CONT 0.02470.005, P0
0.03270.004, P6 0.08270.026 ml/min/100 g Bwt (body
weight), P6 vs CONT, Po0.05; Figure 2b). This trend
remained the same when GFR rate was not corrected for Bwt
(CONT 6.8971.60, P0 9.0571.37, P6 26.1878.43 ml/min).

PPARg immunostaining was intense in the transitional

epithelium of the ureter, which served as a positive internal
control. PPARg was expressed diffusely in proximal tubules
and collecting ducts in normal rat kidneys. PPARg expression
in all PAN-treated rats at week 12 was decreased in proximal
tubules, collecting ducts, and in transitional epithelium.
PPARg expression in PAN-treated rats at 12 weeks was also
present in podocytes and in mesangial cells in segmentally
sclerotic glomeruli, with occasional parietal epithelial cell
staining. In rats treated with delayed PPARg agonist,
mesangial staining was lessened, but podocyte staining was
strongly accentuated compared to control or rats with
pretreatment with PPARg agonist, correlating with less severe
overall sclerosis (Figure 4b). Double immunostaining for
both Wilms tumor 1 (WT-1) and PPARg confirmed the
podocyte location of PPARg after PAN injury (Figure 4a).

900
800
700
600
500
400
300
200
100
0

P0
P6
CONT

W0

b
GFR (ml/min per 100 g BW)

RESULTS
Body and renal functional changes

W4

W8

W12

P = NS
0.12
0.10
0.08

P < 0.05
P0
P6
CONT

0.06
0.04
0.02
0.00

Figure 2 | Urinary protein excretion and renal function.

(a) Pretreatment with PPARg agonist significantly increased urine
protein at weeks 4 and 8. At week 12, there were no differences
among groups. (b) Post-treatment PPARg agonist, but not
pretreatment, improved renal function.
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original article

H-C Yang et al.: PPARc and sclerosis

P0

P6

CONT

PPAR

P = NS
Sclerosis index (SI)

3.50

WT1

PPAR
+WT1

P < 0.05

Normal

CONT

P0

P6

3.00
2.50
2.00
1.50

P0
P6
CONT

1.00
0.50
0.00

Figure 3 | Histological changes. (a) Rats with delayed PPARg agonist

treatment had less glomerulosclerosis at week 12 than time-matched
CONT and pretreatment groups (PAS; original magnification
100).
(b) Sclerosis index was decreased in the post-treatment PPARg
agonist group, with no change in pretreatment rats compared to
control.

occasional positive staining. The ratio of PCNA-positive to

apoptotic cells in glomeruli showed a numerical trend to
decrease in PPARg-treated groups compared with CONT,
reflecting a shift in balance of proliferation vs apoptosis (P0
5.9572.12, P6 5.6172.25, vs CONT 13.3976.06, P NS;
Figure 5b).
The normal rat glomerulus showed on average about 10
WT-1-positive cells per glomerular cross-section (data not
shown). PAN decreased this index of differentiated podocyte.
Treatment with PPARg agonist partially restored podocyte
WT-1 staining (P0 1.670.20, P6 1.270.18, vs CONT
0.770.11, Po0.05; Figure 5a).

Figure 4 | PPARc expression in kidney. (a) At week 12 after

puromycin injection in CONT, PPARg (red) was expressed on
podocytes (WT-1, green) (original magnification
400). (b) In normal
rat kidneys, PPARg protein was expressed primarily within proximal
tubules, collecting ducts, and in transitional epithelium (original
magnification
100). PPARg expression in all PAN groups was
decreased in proximal tubules, collecting ducts, and in transitional
epithelium. PPARg expression in CONT PAN rats at 12 weeks was
present in podocytes and in mesangial cells in segmentally sclerotic
glomeruli, with occasional parietal epithelial cell staining. In delayed
PPARg agonist treatment rats (P6; original magnification
400),
mesangial staining was lessened, but podocyte staining was strongly
accentuated, correlating with less severe overall sclerosis, compared
to pretreatment rats (P0, original magnification
400) or CONT
(original magnification
400).

Modulation of glomerular macrophages

No macrophages were seen in normal kidney glomeruli.

Macrophages were increased in glomeruli of CONT at week
12 (macrophages/glomerulus: CONT 11.570.7; Figure 6).
Pre- or post-treatment with PPARg agonist significantly
reduced infiltrating glomerular macrophages (P0 7.371.02,
P6 5.070.53, vs CONT, Po0.05, respectively; Figure 6).
TIMP-1, TGF-b and PAI-1 expression

Kidney PAI-1 mRNA expression was increased in CONT at

week 12, and PPARg agonist treatment decreased this
expression (P0 0.2170.02, P6 0.2670.03, vs CONT
0.4070.05, Po0.05; Figure 7a). In situ hybridization showed
that PAI-1 glomerular mRNA expression in particular was
reduced by PPARg agonist (Figure 7b). PPARg agonist
resulted in a trend to decrease renal mRNA levels for TGFb1 and tissue inhibitor of metalloproteinase-1 (TIMP-1)
(TGF-b1: P0 0.6770.01, P6 0.7070.06, vs CONT 0.767
0.04, P NS; TIMP-1: P0 0.9870.07, P6 0.8970.03, vs CONT
1.0370.06, P NS; Figure 7a).
1758

Alteration of glomerular endothelial cells

Pretreatment with PPARg agonist did not significantly

increase CD31 staining, an endothelial marker in glomeruli
(P0 1.3970.09, vs CONT 1.0670.13, P NS). In contrast,
delayed PPARg agonist treatment significantly increased
glomerular endothelial cell CD31 (P6 1.6770.09, vs CONT,
Po0.05; Figure 8).
Vascular endothelial-derived growth factor (VEGF)-A and
Flk-1 mRNA expression in whole renal cortex was not
different in the three groups (VEGF-A: P0 0.9670.18, P6
1.6570.71, vs CONT 1.4270.39, P NS; Flk-1: P0 1.5470.30,
P6 1.9670.06, vs CONT 1.6570.30, P NS; Figure 9a).
Glomerular VEGF immunostaining increased in PPARgtreated groups (P0 2.5170.28, P6 2.5270.25, vs CONT
1.7070.26, Po0.05; Figure 9b).
We therefore next examined expression of angiopoietinlike protein 4 (Aglp4), as it is modulated by PPARg and may
interfere with angiogenesis. PPARg agonist, especially early
treatment, decreased renal cortical Aglp4 mRNA expression
Kidney International (2006) 69, 17561764

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H-C Yang et al.: PPARc and sclerosis

WT1-positive cell/glomerulus

PCNA-positive/apoptotic cells

P < 0.05
P < 0.05

2.0
1.5

P0
P6
CONT

1.0

P0

P6

CONT

TIMP-1

TGF-

0.5

PAI-1
0.0

GAPDH
P = NS

25.0
20.0
15.0

P0
P6
CONT

P = NS

1.2

P = NS
P = NS

P = NS
P = NS

P < 0.05
P < 0.05

1.0
P0
P6
CONT

0.8
10.0

0.6
5.0

0.4

0.0

0.2

Figure 5 | Podocyte number and glomerular macrophages.

(a) Pretreatment with PPARg agonist maintained podocyte number,
with intermediate podocyte number in post-treatment rats vs
marked decrease in CONT. (b) Both pre- and post-treatment PPARg
agonist groups showed a trend to inhibit cell proliferation in
glomeruli, with numerically decreased PCNA vs apoptosis ratio.

ED-positive cell/glomerulus

P < 0.05
14.0
12.0
10.0

P0
P6
CONT

P < 0.05

8.0

0.0

P0

TGF-

TIMP-1

P6

PAI-1

CONT

Figure 7 | Renal mRNA expression of TIMP-1, TGF-b, and PAI-1.

(a) There was no significant change of expression of TIMP-1 or TGFb
in renal cortex. PPARg agonist treatment markedly decreased whole
kidney cortex expression of PAI-1 mRNA. (b) PAI-1 mRNA was
expressed in glomeruli, tubules, and interstitium. Both pretreatment
and delayed treatment with PPARg agonist reduced overall PAI-1
expression at week 12 (original magnification
200).

6.0
4.0
2.0
0.0

Figure 6 | Macrophage infiltration in glomeruli. ED1-positive

macrophages were decreased in PPARg-treated vs control groups.

dramatically vs CONT (P0 0.1870.05, P6 0.3970.09, vs

CONT 1.0770.16, Po0.05; Figure 10) (key data summarized
in Table 1).
DISCUSSION

Chronic kidney diseases show progressive glomerulosclerosis

and tubulointerstitial fibrosis regardless of the primary insult.
The podocyte is a common initial target of injury. In this
study, we have demonstrated that PPARg agonist alone can
ameliorate the progression of podocyte injury-associated
FSGS, in part by effects on podocytes and endothelial cells.
PAN is a direct epithelial cell toxin that can reduce
podocyte number by apoptosis or by detaching cells from the
GBM.7 Loss of podocytes is postulated to be a major
underlying factor in progressive sclerosis. We found that
podocytes normally show very low expression of PPARg.
Kidney International (2006) 69, 17561764

After injury, PPARg expression was upregulated. Treatment

with exogenous PPARg agonist maintained podocyte morphology and restored synaptopodin after puromycin injury
in vitro in our previous studies. We therefore postulate that
this increase of PPARg is counter-regulatory and promotes
podocyte healing and repair.
PPARg agonist also is an important regulator of
inflammation.8 We observed that effective PPARg agonist
treatment reduced glomerular macrophages. Possible mechanisms of decreased glomerular macrophages in response
to PPARg include inhibition of expression of vascular cell
adhesion molecule-1 and intercellular adhesion molecule-1
in endothelial cells, which could result in significantly
reduced monocyte/macrophage homing.9,10 Further, PPARg
can inhibit macrophage proliferation by reducing macrophage colony-stimulating factor or inhibiting tumor necrosis
factor-alpha, interleukin-6 and interleukin-1b secretion11
and inducible nitric oxide synthase, or matrix metalloproteinase-9 secretion by macrophages.12
PPARg may also have direct effects on key extracellular
matrix regulators.13 We observed decreased TGF-b and PAI-1
when PPARg ameliorated development of sclerosis in the
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H-C Yang et al.: PPARc and sclerosis

hypertensive 5/6 nephrectomy model.5 Of note, the PAN

model has minimal hypertension and a different phenotype
of sclerosis than the remnant kidney model, with more multifocal sclerosis and adhesions in PAN, likely reflecting different
initial mechanisms of injury. We speculate that TGF-b
increases in the remnant kidney model, decreased by PPARg
agonist, likely reflect the more serious hypertensive injury in
that model vs PAN. In this study, amelioration of glomerulosclerosis by PPARg agonist was linked to downregulated
PAI-1 expression. PAI-1, a member of the superfamily of

serine protease inhibitors, is a major physiological inhibitor

of tissue-type plasminogen activator and urokinase-type
plasminogen activator (PA).14 These PAs activate plasminogen to plasmin and promote fibrinolysis as well as
proteolysis. Upregulation of PAI-1 can thus inhibit proteolysis of extracellular matrix, leading to matrix accumulation
and sclerosis. PPARg agonist also decreased basal and tumor
necrosis factor-alpha-stimulated PAI-1 expression in human
umbilical vein endothelial cells.15 PPARg agonist may inhibit
gene transcription by antagonizing the activities of activator
protein-1 and nuclear factor-kB.2,16 The PAI-1 promoter
contains motifs for both these transcription factors. Thus, we
speculate that downregulation of PAI-1 mRNA expression
may be mediated via a PPARg effect on activator protein-1and/or nuclear factor-kB-mediated transcriptional activity
of PAI-1.
Recent evidence has shown that endothelial damage is an
important component of various sclerosing glomerular
diseases, in addition to the well-known role of podocyte
injury. Loss of both glomerular endothelial cells and
peritubular capillaries has been described in the remnant
kidney, age-related sclerosis, and a progressive glomerulonephritis model.1719 In our study, decreased endothelial cells,
assessed by CD31, correlated with sclerosis and glomerular
capillary loss. Treatment with PPARg agonist was linked to
higher CD31 expression. Possible mechanisms include
decreased severity of initial injury, or subsequent repair and
angiogenesis. Endothelial cell growth is regulated mainly by
VEGF and angiopoietin, both expressed in the podocyte.
Effects of injury and intervention on VEGF and related
molecules may thus be especially evident in the PAN model,
which initially primarily affects the podocyte. VEGF expression is increased in bladder cancer cells by PPARg via
transcriptional activation of the VEGF promoter through an
indirect mechanism.20 PPARg agonist also increased VEGF
expression in smooth muscle cells,21 endothelial cells,22 and
osteoblasts by amplifying SAPK/JNK activation.23 Thus,

a
P0

P6

CONT

P = NS
2.0

P < 0.05

1.8
P0
P6
CONT

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

Figure 8 | CD31 staining. (a) At week 12, glomerulosclerosis was

decreased in post-treatment but not pretreatment PPARg agonist
group, compared to CONT. Decreased sclerosis was accompanied by
more CD31 expression in post-treatment PPARg rats (anti-CD31;
original magnification
200). (b) CD31 (04 scale) expression was
significantly increased in post-treated PPARg agonist rats, with only a
numerical increase in pretreatment PPARg rats.

P0

P6

CONT

b
P0

P6

CONT

VEGF
-Actin
Flk-1
P < 0.05

-Actin
3.0

P = NS
P = NS
P0
P6
CONT

VEGF

Flk-1

VEGF-positive
cells/glomerulus

2.5
2.0
1.5
1.0
0.5
0.0

P = NS
P = NS

2.5
2.0

P < 0.05
P0
P6
CONT

1.5
1.0
0.5
0.0

Figure 9 | VEGF and Flk-1 expression. (a) There was no significant change in any group in expression of VEGF or Flk-1 from whole kidney
cortex homogenates (Western, protein expressed relative to b-actin). (b) VEGF was expressed on tubular, endothelial cells, macrophages, and
podocytes. In glomeruli, both pre- and post-treatment PPARg agonist rats had more VEGF-positive cells compared to control (anti-VEGF
antibody; original magnification
200).
1760

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H-C Yang et al.: PPARc and sclerosis

PPARg agonist could stimulate angiogenic progenitor cells

differentiating to endothelial cells.24 The PPARg angiopoietinrelated gene, also called Aglp4, is a target of PPARa or PPARg
and hypoxia-inducible factor-1a, 25 and hypoxia-inducible
factor-1 affects its action.26 Both VEGF-induced angiogenesis
in vivo and vascular leakiness were significantly inhibited by
Aglp4.27 In our study, increased Aglp4 observed in PAN rats
may have been induced by hypoxia related to sclerosis. We
showed that PPARg agonist treatment protected glomerular
capillaries both by increasing podocyte VEGF expression and
by decreasing Aglp4, thus promoting open capillary loops.
An additional unexpected finding in this study was the
striking difference between rats receiving PPARg agonist prePAN vs post-PAN. Although rats pretreated with PPARg
agonist showed increased podocyte PPARg expression,
maintained podocyte number, and decreased glomerular
macrophages and PAI-1, these changes did not protect the
kidney. Interestingly, effects on proteinuria at 12 weeks did
not mirror effects on sclerosis, as we have also observed in the
rat remnant kidney treated with angiotensin receptor
blocker.28 These observations in rat models are somewhat
at variance with observations in humans with chronic kidney
disease, where response of proteinuria to treatment has
generally, but not always, correlated with effects on GFR, and
presumably sclerosis.
Mechanisms of the lack of effect of pretreatment include
the possibility that pretreatment may exhaust endogenous
P < 0.05
Aglp4/GAPDH mRNA

1.4
1.2
1.0

P0
P6
CONT

P < 0.05

0.8
0.6
0.4
0.2
0.0

Figure 10 | Renal cortical mRNA expression of Aglp4. Aglp4 was

significantly reduced in PPARg-treated groups compared to control.

PPARg ligands and/or decrease the response to endogenous

PPARg, without decreasing overall PPARg activity. Endogenous ligands include 15-deoxy-12,14-PGJ2, (15d-PGJ2), lysophosphatidic acid, conjugated linoleic acid and azelaoylphosphocholine.29 Nitrolinoleic acid is a newly described
endogenous agonist of PPARg, found at concentrations of
around 500 nM in healthy people, thus exceeding the Ki for
PPARy, which is around 133 nM. Nitrolinoleic acid is a
significantly more robust PPARg ligand than other reported
endogenous PPARg ligands, and its activity is similar or
exceeds that of several thiazolidinediones.29 If pretreatment
specifically affected some or all of these endogenous PPARg
ligands, then effects on injury may have been modulated.
Of note, pretreatment was still effective in decreasing
glomerular macrophages and PAI-1, although sclerosis was
not affected, which suggests that at least some PPARg
activation occurred. Pretreatment may aggravate the injury in
the early injury stage, contrasting beneficial effect in the late
stage. PPARg agonists inhibit the proliferative response of
endothelial cells to growth factors, including VEGF, as well as
the mRNA expression of VEGF receptors 1 and 2.30
Thiazolidinediones and the endogenous PPARg ligand 15dPGJ2 suppressed in a dose-dependent manner human
umbilical vein endothelial cell differentiation into capillary
tubes in vitro.31 Active PPARg can also arrest the cell cycle at
G1.32 Further, PPARg activation can induce apoptosis in
renal cell carcinoma cells.33 Additional studies will be needed
to examine the differential time-specific interactions of
PPARg and podocyte injury on sclerosis.
In summary, PPARg activation in vivo in a podocyte
injury-associated FSGS model leads to beneficial effects.
These beneficial effects are related to regulation of podocyte
apoptosis, decreased macrophage infiltration and PAI-1
expression, and protection of glomerular capillary loss by
decreasing Aglp4 and increasing VEGF expression. Our
results further indicate that there are key functional and
morphological differences in ultimate responses to PPARg
agonist given before, or after, podocyte injury, with
protection against sclerosis only occurring in the latter. The

Table 1 | Summary of effects of early vs delayed treatment with PPARc agonist in PAN nephropathy

Urinary protein
GFR
SI
WT-1
ED1
PCNA+/apoptosis cells
PAI-1
CD31
VEGF
Aglp4

CONT

Pretreatment

Delayed treatment

361.0783.0
0.02470.005
2.9470.13
0.770.11
11.570.7
13.3976.06
0.4070.05
1.0670.13
1.7070.26
1.0770.16

352.2758.1
0.03270.004
2.6270.09
1.670.20*
7.371.02*
5.9572.12
0.2170.02*
1.3970.09
2.5170.28*
0.1870.05*

377.0766.8
0.08270.026*
2.3370.09*
1.270.18*
5.070.53*
5.6172.25
0.2670.03*
1.6770.09*
2.5270.25*
0.3970.09*

Aglp4, angiopoietin-like protein 4; PAI-1, plasminogen activator inhibitor-1; PCNA, proliferating cell nuclear antigen; SI, sclerosis index; VEGF, vascular endothelial-derived
growth factor; WT-1, Wilms tumor 1.
Pretreatment did not protect against PAN-induced injury, whereas delayed PPARg agonist did. The delayed treatment group had more CD31 and Aglp4 expression,
restoration towards normal WT-1, and increased CD31, and less macrophages than pretreatment.
*Po0.05 vs control.

Kidney International (2006) 69, 17561764

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original article

beneficial effects of delayed time point of intervention are

clinically relevant, as therapies are initiated in humans
following the initial insult. Our results thus suggest that
PPARg agonists can provide a novel approach with
therapeutic potential in podocyte injury-associated FSGS.
MATERIALS AND METHODS
Experimental design and animals
Adult male SpragueDawley rats (250300 g; Charles River, Nashville, TN, USA) were studied. Rats were housed under normal
conditions with a 12-h light/dark cycle at 701F with 40% humidity
and 12 air exchanges per hour and received standard rat powdered
diet (Purina Rodent 5001 meal, 23.4% protein, 4.5% fat, 6.0%
fiber, 0.40% sodium; Tusculum Feed Center, Nashville, TN, USA)
and tap water. All rats underwent nephrectomy (Nx). Rats were
divided into three groups: control group (CONT, N 10);
pretreatment Pio from time of Nx before PAN (P0, N 10,
10 mg/kg Bwt/day mixed with powdered chow); or Pio in the same
dose, starting from week 6 after Nx (P6, N 10). PAN was given
intraperitoneally at weeks 2 (100 mg/kg Bwt), 6, 7, and 8 (40 mg/kg
Bwt). The dose of Pio was chosen based on its efficacy in correcting
abnormalities in diabetes.34 The amount of Pio in chow was
adjusted based on body weight and food consumption to maintain
steady dose. Animals were killed at 12 weeks. Kidneys were harvested
for analysis of morphologic and molecular parameters.
Functional assessments
Systolic blood pressure and 24-h urinary protein were assessed at
weeks 0, 4, 8, and 12. SBP was measured using tail-cuff
plethysmography in unanesthetized prewarmed trained rats at
ambient temperature of 291C. Animals were placed in metabolic
cages for 24 h for urine collection, and urine protein was measured
by the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules,
CA, USA). Serum and urine creatinine were measured by Vitros
CREA slides (Johnson & Johnson Clinical Diagnostics Inc.,
Rochester, NY, USA).
Morphological assessment
Kidney tissue from rats was immersion fixed in 4% paraformaldehyde/phosphate-buffered saline solution and routinely processed,
and 4 m sections were stained with periodic acid-Schiff and Massons
trichrome. A semiquantitative score (sclerosis index) was used to
evaluate the degree of glomerulosclerosis. The severity of sclerosis
for each glomerulus was graded from 0 to 4 : 0 no lesion; 1
sclerosis of o25% of the glomerulus, 2 , 3 , and 4 for 2550,
5075, and 475% sclerosis, respectively. A whole kidney average
sclerosis index was obtained by averaging scores from all glomeruli
on one section. Tubulointerstitial fibrosis was evaluated qualitatively
on Massons trichrome-stained sections. All sections were examined
without knowledge of the treatment protocol.
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were 4 mm deparaffinized and hydrated. For VEGF, sections were fixed in Bouins
solution at 371C for 1 h and microwaved in 0.01 mol/l sodium
citrate (pH 6.0) 3
5 min. For WT-1, PCNA, and ED1, sections
were microwaved in 0.01 mol/l sodium citrate (pH 6.0) 2
5 min.
For CD31, sections were digested by 0.1% trypsin at 371C for
20 min. Endogenous peroxidase was quenched with 3% hydrogen
peroxidase for 10 min and slides then exposed to Power Block
1762

H-C Yang et al.: PPARc and sclerosis

(BioGenex Laboratories, San Ramon, CA, USA) for 30 min. The

primary antibodies used were mouse anti-rat VEGF (1:20; Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-rat WT-1
(1:800; Santa Cruz), mouse anti-human PCNA (1:200; Dako
Corporation, Carpinteria, CA, USA), rabbit anti-rat CD31 (1:200;
BD Pharmingen, San Jose, CA, USA), and mouse anti-rat ED1 (1:50;
Dako). Sections were incubated overnight at 41C. Immunoperoxidase staining was performed with the Vectastatin ABC kit (Vector
Laboratories, Burlingame, CA, USA) with diaminobenzidine as a
chromogen. Hematoxylin was used as a counterstain. Apoptotic cells
were detected by the ApopTag plus peroxidase in situ apoptosis
detection kit (Chemicon International, Temecula, CA, USA).
Double staining for WT-1 and PPARg was carried out as follows:
frozen sections were air-dried and fixed in 4% PFA for 5 min,
incubated with rabbit anti-mouse WT-1 (1:100, Santa Cruz
Biotechnology Inc.) at 371C for 1 h, and anti-rabbit fluorescein
isothiocyanate (1:50; Vector Laboratories) at room temperature for
30 min. Tissue was then blocked with the MOMTM Mouse Ig
Blocking Reagent (MOM kit, Vector Laboratories) for 1 h. PPARg
immunostaining was performed with primary mouse monoclonal
antibodies to human PPARg (Santa Cruz Biotechnology Inc.),
followed by Texas red conjugated rabbit anti-mouse IgG (Dako).
The number of PCNA-, apoptosis-, WT-1-, ED1-, or VEGFpositive nuclei per glomerulus was counted, and localization
assessed based on cell location and morphology. CD31 glomerular
expression was evaluated by a semiquantitative score: 0 represents
no positive lesion; 1 represents positive area at o25% of the
glomerular capillary, whereas 2 , 3 , and 4 represent positive
area of 2550, 5075, and 475% of the glomerular capillary,
respectively. Negative control slides treated with nonspecific antisera
instead of primary antibody showed no staining. All sections were
examined without knowledge of the treatment protocol.
In situ hybridization
35
S-labeled sense and antisense riboprobes for PAI-1 were prepared
and in situ hybridization was performed as previously described.5
Negative control in situ hybridizations with sense probes showed no
specific signal.
Northern blot hybridization
Northern blot was performed as previously described.35 Total RNA
from kidney cortex was extracted by Trizol reagent (Life Technologies, Grand Island, NY, USA). Briefly, 20 mg of total RNA was
loaded and fractionated by electrophoresis in 1% agarose gel and
transferred to a nylon membrane. The blots were hybridized with
mouse PAI-1 (365 bp), mouse TGF-b1 (974 bp), and mouse TIMP-1
cDNA probes, and expression normalized to rat glyceraldehyde-3phosphate dehydrogenase.
Quantitative real-time PCR
Reverse transcription was performed using the TaqMan Reverse
Transcription Kit (Applied Biosystems, Branchburg, NJ, USA) in a
total reaction volume of 80 ml (2 mg total RNA, 5 mM MgCl2, 1 mM
dNTPs, 1 U/ml RNase inhibitor, 2.5 mM random hexamers, and 2.5 U/
ml MuLV-RT) for 30 min at 421C. Quantitative real-time PCR was
performed in a total reaction volume of 25 ml using iQTM SYBR
Green Supermix (Biorad, Hercules, CA, USA), 1 ml cDNA, and
500 nM forward and reverse primers (Aglp4 forward 50 -TCCT
CATCCTGCCTAAGTTCTC, reverse 50 -GTGCCGCTCTCGTTTA
CCTC). Quantitative real-time PCR was carried out using the
Kidney International (2006) 69, 17561764

original article

H-C Yang et al.: PPARc and sclerosis

iCycler iQ Multi-Color Real Time PCR Detection System (Bio-Rad

Laboratories) with the following cycling parameters: polymerase
activation for 3 min at 951C and amplification for 40 cycles of 20 s at
951C, 20 s at 601C, and 40 s at 721C. Following amplification, a final
melting curve was obtained for each sample by cooling the reaction
mixture to 551C and then heating to 951C at 0.21C/s. Experimental
cycle threshold values were normalized to glyceraldehyde-3phosphate dehydrogenase measured on the same plate, and fold
differences in gene expression were determined using the 2  DDCt
method.36
Western blot
Frozen kidney tissues were transferred in RIPA plus buffer containing 150 mmol/l NaCl, 50 mmol/l Tris-HCl, pH 7.5, 5 mmol/l
ethylenediamine tetraacetic acid, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 100 mg/ml phenylmethyl
sulfonyl fluoride, 1:100 phosphatase inhibitor cocktail I, 1:100
phosphatase inhibitor cocktail II (Sigma, Sigma chemical co., St
Louis, MO, USA), and 1:100 proteinase inhibitor cocktail tablet
(Roche Diagnostics GmbH, Mannheim, Germany). Tissue samples
were homogenized in 1.5 ml of homogenizer (Pellet Pestle Dips;
Kontes Glass Company, Vineland, NJ, USA) on ice, and centrifuged
at 13 000 r.p.m. for 15 min at 41C. The protein concentration was
measured using the Dc Protein Assay kit (Bio-Rad). Sixty
microgram of protein from samples was loaded and separated by
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
transferred onto a 0.2 mmol/l nitrocellulose membrane. VEGF was
detected with a specific rabbit anti-VEGF polyclonal antibody (Santa
Cruz Biotechnology Inc.). Flk-1 was detected by rabbit anti-Flk-1
polyclonal antibody, 1:200, overnight at 41C (Santa Cruz Biotechnology Inc.). Anti-rabbit IgG secondary antibody was added after
washing and incubated at room temperature for 1 h. Protein bands
on Western blots were visualized by ECL Plus (Amersham,
Arlington Heights, IL, USA). The membranes were stripped with
100 mmol/l b-mercaptoethanol, 2% sodium dodecyl sulfate, and
62.5 mmol/l Tris-HCL, pH 6.7. b-actin was detected using mouse
anti-b-actin polyclonal antibody (Sigma). Expression was quantified
as the ratio of specific message to b-Actin.
Statistical analysis
Results are expressed as mean7s.e.m. Statistical difference was
assessed by analysis of variance followed by unpaired t-test as
appropriate. Nonparametric data were compared by MannWhitney
U-test. A P-value o0.05 was considered to be significant.

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