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Am J Physiol Renal Physiol 295: F202F214, 2008.

First published April 16, 2008; doi:10.1152/ajprenal.00468.2007.

HSP72 attenuates renal tubular cell apoptosis and interstitial fibrosis

in obstructive nephropathy
Haiping Mao,1* Zhilian Li,1* Yi Zhou,1 Zhijian Li,1 Shougang Zhuang,4 Xin An,1 Baiyu Zhang,1
Wei Chen,1 Jing Nie,1 Zhiyong Wang,2 Steven C. Borkan,2 Yihan Wang,3 and Xueqing Yu1

Department of Nephrology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China; 2Renal Section,
Department of Medicine, Boston Medical Center, Boston University, Boston, Massachusetts; 3Laboratory for Kidney
Pathology, Incorporated, Nashville, Tennessee; and 4Department of Medicine, Brown University School of Medicine,
Providence, Rhode Island
Submitted 9 October 2007; accepted in final form 14 April 2008

Mao H, Li Z, Zhou Y, Li Z, Zhuang S, An X, Zhang B, Chen

W, Nie J, Wang Z, Borkan SC, Wang Y, Yu X. HSP72 attenuates
renal tubular cell apoptosis and interstitial fibrosis in obstructive
nephropathy. Am J Physiol Renal Physiol 295: F202F214, 2008.
First published April 16, 2008; doi:10.1152/ajprenal.00468.2007.
Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and
fibrosis is poorly characterized. In this study, we tested the hypothesis
that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and
in vivo. In rats, orally administered geranylgeranylacetone (GGA), an
agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both
interstitial fibroblast accumulation and collagen I deposition after
unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells,
exposure to TGF-1 induced EMT and apoptosis, major causes of
renal fibrosis and tubular atrophy, respectively. Exposure to a pancaspase inhibitor (ZVAD-FMK) prevented TGF-1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression
in vitro inhibited EMT caused by TGF-1 as indicated by preserving
the E-cadherin expression level and -smooth muscle actin induction.
Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-1-exposed
cells. Taken together, our data indicate that HSP72 ameliorates renal
tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both
renal tubular epithelial cell apoptosis and EMT.
heat stress protein 72 kDa; epithelial-to-mesenchymal transition,
-smooth muscle actin, E-cadherin, transforming growth factor-1
TUBULOINTERSTITIAL FIBROSIS (TIF) is the common final pathway
of diverse forms of chronic renal disease that contributes to
both organ insufficiency and failure. Although a number of
experimental studies have attempted to clarify the pathogenesis
of TIF, its evaluation and management remain poorly defined.
TIF is characterized by leukocytic infiltration, tubular atrophy,
fibroblast proliferation, and the accumulation of extracellular
matrix proteins (3). Transforming growth factor-1 (TGF-1)
is believed to play an important role in TIF by inducing both
epithelial-to-mesenchymal transition (EMT) and apoptosis of
renal tubular cells, which promotes tubular atrophy in many
forms of progressive renal disease (38, 44).

* H. Mao and Z. Li contributed equally to this work.

Address for reprint requests and other correspondence: X. Yu, Dept. of
Nephrology, First Affiliated Hospital, Sun Yat-sen Univ., Guangzhou, China

The extent of tubular apoptosis in animal models of ureteric

obstruction correlates with the severity of tubular injury and
subsequent TIF (5, 23, 28, 50). In contrast, inhibition of initial
tubular cell apoptosis by either neutralizing the activity of
apoptosis-inducing molecules or supplementing with prosurvival factors effectively prevents inflammation and attenuates
progression to fibrosis in the unilateral ureteric obstruction
(UUO) model (24, 36). These data provide evidence for an
apparent interplay between early apoptosis and subsequent
fibrosis, and the apoptosis could be an early event that occurs
before the onset of frank fibrosis. EMT describes a phenotypic
change characterized by the loss of epithelial markers including E-cadherin, the gain of mesenchymal markers such as
-SMA, and epithelial cell migration across damaged tubular
basement membrane to the interstitial space, where cells become activated myofibroblasts (13). The process of EMT
eventually generates extracellular matrix associated with a loss
of viable tubular epithelial cells and nephrons. Although both
EMT and apoptosis contribute to fibrosis and reduction in renal
parenchyma, the cellular mechanisms that regulate apoptosis
and EMT are yet to be fully elucidated.
Heat shock protein 72 kDa (HSP72), a molecular chaperone,
protects renal epithelial cells from acute lethal injury including
necrosis and apoptosis in vivo and in vitro (31, 45, 46) and also
ameliorates sublethal injury by preventing cytoskeletal collapse, by improving cell-cell junction function and by enhancing cell-matrix interaction (7, 19, 21, 42). GGA is a nontoxic
agent that ameliorates acute, ischemic renal injury in the rat by
selectively inducing the expression of HSP72 (35). Furthermore, UUO increases endogenous HSP72 generation in the
kidney of both rats and humans (5, 18). It is still not known
whether an increment of HSP72 in a chronic fibrosis setting is
merely a marker of nonspecific repair mechanisms due to
cellular injury or a potential cytoprotective mechanism that
protects renal epithelial cells from apoptosis, prevents EMT,
and thus facilitates repopulation of injured tubules.
In the present study, we tested the hypothesis that GGAinduced HSP72 expression reduces renal TIF in UUO rats by
inhibiting renal tubular apoptosis and EMT. We find that
selective HSP72 expression reduces EMT in vitro and decreases tubular cell apoptosis, interstitial fibroblast infiltration,
and collagen I deposition in the rat kidney in a model of
ureteric obstruction.
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Reagents and Antibodies

A normal rat kidney proximal tubular epithelial cell line (NRK52E)
was purchased from American Type Culture Collection (Rockville,
MD). Recombinant human TGF-1 and ZVAD-FMK were purchased
from R&D Systems (Minneapolis, MN). GGA was obtained from
Eisai China (Shanghai, China). Scrambled and HSP72-specific small
interfering RNA (siRNA) products were purchased from Shanghai
GenePharma (Shanghai, China), and Lipofectamine 2000 transfection
reagent was purchased from Invitrogen Life Technologies (Paisley,
UK). TdT-mediated dUTP nick end labeling (TUNEL) Assay Kits
(Fluorescent), an annexin V-FITC Apoptosis Detection Kit, protease
inhibitors, and anti-collagen I were obtained from Calbiochem (San
Diego, CA). In addition, the following antibodies were used: anti-Ecadherin antibody (BD Biosciences Pharmingen, San Jose, CA);
anti--SMA and anti-PCNA (DAKO, Cupertino, CA); anti-HSP72,
anti-HSP27, anti-HSP60, and anti-HSP90 (Stressgen Biotechnologies,
Victoria, BC, Canada), anti-GAPDH (Santa Cruz Biotechnology,
Santa Cruz, CA), and anti--actin (Boster, Wuhan, China). Horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidaseconjugated anti-rabbit IgG, and Cy3-conjugated anti-mouse IgG were
obtained from Jackson Immunoresearch (West Grove, PA). All remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Cells and Treatments
NRK52E cells were cultured at 37C in a 5% CO2 atmosphere in
DMEM mixed 1:1 (vol:vol) with F12 medium (Invitrogen-GIBCO,
Carlsbad, CA) supplemented with 10% fetal bovine serum. Cells were
grown to 7080% confluence and subjected to serum deprivation for
24 h before experimental manipulation.
The effects of TGF- on cell phenotype and apoptosis were
induced by adding recombinant TGF-1 (10 ng/ml) to cells for 72 h.
To inhibit the apoptotic signal pathway, cells were pretreated with
various concentrations (25150 M) of ZVAD-FMK, a pan-caspase
inhibitor, for 1 h before and during exposure to TGF-1. All experiments were performed under serum-free conditions.
To increase HSP72 content in vitro, NRK52 cells were treated with
either GGA or adenoviruses containing HSP72 cDNA (Adv-HSP72),
which was kindly provided by Dick Mosser (University of Guelph,
Ontario, Canada). GGA is an emulsion with 5% gum arabic and
0.008% tocopherol. Cells were incubated for 2 h with increasing
concentrations of GGA that varied between 0 and 200 mol/l or with
vehicle at the same concentrations. The time course of HSP72 expression was also studied after administration of 100 mol/l GGA or
the same doses of vehicle in culture medium. Furthermore, NRK52E
cells were coinfected with adenoviruses containing wild-type human
HSP72 and green fluorescent protein (AdvTR5/HSP72-GFP) located on
separate cistrons induced by a tetracyclin-regulated promoter (AdvCMV/
tTA) as described previously (21). Control cells were coinfected with
AdvTR5/GFP and AdvCMV/tTA. Infection efficiency for both adenoviruses was 95% as determined by direct visualization of GFP in cells
infected with adenovirus at multiplicities of infection (MOI) that varied
between 40 and 60. The effect of each adenoviral dose and GGA on
HSP72 expression was assessed by Western blot analysis.
siRNA for HSP72
Incorporation of siRNA targeted against HSP72 was achieved
using Lipofectamine 2000 according to the manufacturers instructions. The cells were transfected for 4 h before replacement of the
transfection medium with DMEM/F12 medium. Initial studies focused on the identification of the target siRNA that would produce the
lowest levels of HSP72 expression in NRK52E cells. Preliminary
results (n 3) showed that silencing was evident at 24, 48, and 72 h
after transfection. Therefore, 24 h after transfection, cells were stimAJP-Renal Physiol VOL


ulated with TGF-1. The effect of HSP72 silencing on EMT was

observed at 72 h after TGF-1 exposure. For determination of the
efficiency of HSP72 knockdown, Western blot analysis for HSP72
was performed. Several concentrations of HSP72 siRNA (10 50 nM)
were used to determine the optimal knockdown concentration.
The experiments were performed with male Sprague-Dawley rats
weighing 200 250 g maintained with free access to water and standard rat food. UUO was performed by using the protocol approved by
Animal Care and Use Committee of the Sun Yat-sen University
(Guangzhou, China). Rats were anesthetized by injection of chloral
hydrate (400 mg/kg ip) and allowed to breathe spontaneously. The left
ureter of each animal was located and double-ligated with 4-0 silk
thread after a midline abdominal incision. Sham-operated rats had
their ureters exposed but not ligated. To induce optimal renal HSP72
expression, the GGA protocol described by Suzuki et al. (35) was
modified by our laboratory, and additional studies on dose- and
time-dependent induction of HSP72 were performed using doses of 0,
200, and 400 to 800 mg/kg daily and time points of 12, 24, and 48 h
after a signal dose of GGA (400 mg/kg). Rats received daily oral
administration with optimal 400 mg/kg GGA, starting 1 day before the
operation and continuing throughout UUO or sham surgery. Rats in
the control group were given the same dose of vehicle. Rats (n 5
each) were killed on day 7 after UUO, and both kidneys were
harvested and were then subjected to the studies described below.
Analysis of Morphology and TIF
Kidney tissues were fixed in 10% phosphate buffer formalin, dehydrated through graded alcohol and xylene, embedded in paraffin, sectioned at 3-m thickness, and then stained with periodic acid-Schiff or
Massons trichrome. Histological examinations were performed in a
blinded manner. A point-counting method was used to evaluate chronic
tubular injury and TIF (40). Briefly, under high magnification (400), 10
nonoverlapping fields from each section of the renal cortex were photographed. A grid containing 117 (13 9) sampling points was superimposed on each photograph. The number of points overlying chronic
tubular injury and TIF was counted and expressed as a percentage of all
sampling points (40). Chronic tubular injury was defined as tubular
atrophy with interstitial fibrosis and infiltrate with/without tubular dilation, tubular cast formation, flattening of tubular epithelial cells, and
thickening of the tubular basement membrane (25).
Immunohistochemical and Immunofluorescence Stainings
All immunostainings were performed on 4-m paraffin-embedded
kidney sections. Antigen retrieval was performed by microwave
treatment. Negative controls were performed with nonimmune mouse
serum substituted for the specific primary antibodies.
In immunohistochemical staining, sections were exposed to 3%
H2O2 for 20 min to destroy endogenous peroxidase activity, rinsed
with PBS once, blocked with 10% nonimmune sheep serum in PBS
for 1 h, incubated with monoclonal mouse anti-HSP72 or PCNA,
respectively, at 4C overnight, rinsed with PBS three times for 5 min
each, and then incubated for 1 h with either sheep or horse horseradish
peroxidase-conjugated anti-mouse IgG. The reaction was stopped by
rinsing with PBS when the color was fully developed using diaminobenzidine for 5 min. The PCNA-positive tubular and interstitial
cells in the cortex were calculated in 10 high-power fields at 400
magnification within the same section of kidney from an individual
animal. Results were expressed as percentage of the total nuclei in cortex.
To detect E-cadherin, -SMA, or collagen I, cells or tissue sections
were incubated with monoclonal mouse anti-E-cadherin, anti--SMA,
and anti-collagen I at 4C overnight followed by Cy3-conjugated
anti-mouse IgG. To identify nuclei, cells or sections were counterstained with 4,6-diamidino-2-phenylindole for 2 min. The positive

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stainings were detected using a laser-scanning confocal microscopy

(Zeiss LSM 510 META, Carl Zeiss). For semiquantitative analysis of
collagen I expression, all cortical fields were scored from 0 to 4 for
each field, with an average for each kidney calculated. The score of
collagen I accumulation was assessed as 0 for 0%, 1 for 25%, 2 for
2550%, 3 for 50 75%, and 4 for 75% of each field occupied by a
collagen I-positive area. Quantification of immunostaining was performed on blinded slides.
Detection of Apoptosis
TUNEL. Apoptosis was quantified in histological sections using a
commercially available in situ Apoptosis Detection Kit according to
the manufacturers instructions. To this end, paraffin-embedded kidney sections were treated with 20 g/ml proteinase K for 20 min at
room temperature. After being washed, slides were incubated with a
TUNEL reaction mixture containing terminal deoxynucleotidyl transferase. Positive staining was identified in the cell nucleus with DNA
fragmentation under fluorescence microscopy and expressed as apoptotic cells per high-power field.
Flow cytometric analysis. Apoptosis in cultured cells was quantified
by flow cytometry after double staining cells with annexin V-FITC and
propidium iodide using Annexin V-FITC Apoptosis Detection Kit according to the manufacturers protocol. Cells positive for annexin-V but
negative for propidium iodide were considered to be apoptotic.

Western Blot Analysis

Kidney cortex and harvested cultured cells were homogenized in
lysis buffer (150 mM NaCl, 10 mM Tris HCl, 5 mM EDTA, 1 mM
EGTA, and 1% Triton X-100) with a protease inhibitor cocktail. The
supernatants of tissue and cell lysates were extracted after centrifugation at 10,000 rpm for 15 min at 4C. The protein concentration was
measured with the Bradford protein assay (Bio-Rad, Hercules, CA).
Equal amounts of protein from lysates were loaded and separated by
10% SDS-PAGE and transferred onto nitrocellulose membrane. The
blots were probed overnight at 4C with primary antibodies respectively directed against proteins including E-cadherin, -SMA, HSP72,
GAPDH, and -actin followed by the respective horseradish peroxidase-linked secondary antibody. Horseradish peroxidase activity was
visualized by an enhanced chemiluminescence system. Densitometric
quantification was performed with the Image analysis program (FluorChemTM 8900, Alpha Inotech).
Statistical Analyses
The data are expressed as means SE. Analysis was performed
with standard statistical software (SPSS for Windows, version 11.0).
Comparison among groups was made with one-way ANOVA followed by the Student-Newman-Keuls test. A P value 0.05 was
considered as statistically significant.

Fig. 1. Induction of heat shock protein (HSP)

expression in vivo and in vitro by geranylgeranylacetone (GGA). A and B: doseand time-dependent expression of HSP 72
kDa (HSP72) after GGA was analyzed by
Western blotting. Graphic presentation is
shown of renal HSP72 protein abundances in
different groups as indicated. Relative HSP72
level is reported after normalization with
GAPDH. Values in baseline were set as 1.0.
Values are means SE; n 5. *P 0.05 vs.
baseline. C: immunohistochemistry in GGAtreated animal revealed HSP72 expression
primarily in the inner stripe of the outer
medulla of normal kidney. Original magnification 100. D: Western blot showed an
increase of HSP72 protein expression in rats
after GGA administration compared with vehicle alone. In contrast, GGA treatment did
not change the protein levels of HSP90,
HSP60, or HSP27. E: quantitative analysis of
the expression levels of HSPs in vehicle
(filled bars) and GGA (open bars) treatment
groups. Relative HSP72 levels were calculated and are expressed as fold-induction over
vehicle after normalization with GAPDH
content. Value in the vehicle was set as 1.0.
Values are means SE; n 5. P 0.05 vs.
vehicle. F: by Western blot analysis, GGA
induced HSP72 overexpression in NRK52E
cells at a dose of 100 mol/l and further
increased HSP72 level at a dose of 200
mol/l GGA. G: with a dose of 200 mol/l
and 2-h exposure, GGA induced a transient
increase in HSP72 expression in NRK52E
cells, which peaked at 2 4 h, followed by a
rapid decline by 6 h.

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GGA Selectively Enhanced Expression of HSP72 In Vivo

and In Vitro
The dose-dependent induction of HSP72 by GGA in the rat
kidney was assessed 24 h after single oral administration of
GGA (Fig. 1A). HSP72 expression increased significantly in a


dose-dependent manner from a starting dose of 200 mg/kg to a

maximal dose of 800 mg/kg. The time-dependent effect of
GGA on the expression of HSP72 was also evaluated by
administration of GGA with a single dose of 400 mg/kg (Fig.
1B). HSP72 expression in kidneys significantly increased from
12 h, peaked at 24 h, and then mildly decreased from the peak
by 48 h. Since HSP72 expression was maximal at 24 h, a dose

Fig. 2. Effect of GGA treatment on renal tubular injury

and interstitial fibrosis after unilateral ureteral obstruction (UUO). A: representative micrographs from different groups with either periodic acid-Schiff (PAS) or
Massons trichrome stain revealed marked increase in
tubulointerstitial fibrosis in UUOVehicle vs. sham
control, and in contrast, there was significantly less
tubulointerstitial fibrosis in UUOGGA. Original magnification 200. B and C: semiquantitative analysis of
relative tubular injury and interstitial fibrosis scores,
respectively. Values are means SE; n 5. *P 0.01
vs. sham. P 0.05 vs. vehicle. D and E: GGA
treatment significantly reduced interstitial collagen I
deposition (arrows) demonstrated by immunofluorescence stain using the antibody directly against collagen
I, and staining scores as indicated. Values are means
SE; n 5. *P 0.01 vs. sham. P 0.05 vs. vehicle.
Original magnification 200.

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of 400 mg/kg was given 24 h before and during the following

experiments. In normal control rats without oral GGA, HSP72
expression located mainly in the inner stripe of the outer
medulla was detected by immunohistochemical staining (data
not shown). Oral administration of GGA markedly enhanced
HSP72, staining in the whole kidney essentially with the same
distribution as in normal kidneys (Fig. 1C). GGA significantly
increased the expression of HSP72, but not HSP90, HSP60, or
HSP27 in the cortex as evaluated by Western blot analysis,
(Fig. 1, D and E). Exposure of NRK52E cells to GGA also
significantly increased HSP72 content in a dose- and timedependent manner (Fig. 1, F and G). HSP72 overexpression
lasted 6 h after transient GGA exposure, whereas TGF-mediated EMT in NRK52E cells occurred between 48 and 72 h
after incubation. Thus we used adenoviruses containing wildtype human HSP72 to increase HSP72 level in vitro and further
observe the effect of HSP72 on TGF--induced renal EMT
over time.
GGA Attenuated Sclerotic Changes
To evaluate the effect of GGA-induced HSP72 expression
on renal fibrosis, tubular injury and interstitial matrix deposition were evaluated at day 7 after UUO. Compared with the
sham-operated kidneys, the obstructed kidneys exhibited a
dramatic tubular dilation and atrophy and widening of interstitial spaces illustrated in periodic acid-Schiff-stained sections.
Obstructed kidneys with vehicle alone demonstrated marked
TIF mostly in cortical areas, indicated by a positive blue color
in Massons trichrome-stained sections. In contrast, obstructed
kidneys from GGA-treated animals had less sclerotic damage
(Fig. 2A). The tubular injury and TIF scores were reduced by
35.2 and 69.6% on day 7 after UUO, respectively (P 0.05;
Fig. 2, AC). Immunofluorescence staining with anti-collagen
I antibody revealed increased accumulation of collagen I in the
interstitium (Fig. 2D), whereas obstructed kidneys with GGAinduced HSP72 expression showed a reduction in the interstitial volume expansion and collagen I accumulation (Fig. 2, D
and E).
GGA Suppressed In Vivo Renal EMT Marked by Retaining
E-Cadherin and Inhibiting -SMA
Myofibroblasts play an important role in interstitial fibrosis.
Therefore, the effects of GGA on the expression of both
E-cadherin, an epithelial adhesion molecule, and -SMA, a
molecular hallmark of mature myofibroblasts, were examined.
Compared with sham-operated animals, the level of HSP72
expression in UUO was elevated in the rat with vehicle alone
and upregulated further in the GGA-treated rats (Fig. 3), which
was associated with preserving E-cadherin expression and
lowering -SMA accumulation in the obstructed kidney at day
7 after UUO (Fig. 4A). Quantitative determination of Western
blot analysis revealed that GGA retained E-cadherin expression by 41.8% and suppressed -SMA expression by 46.5% in
the obstructed kidneys (Fig. 4, B and C). The above-noted
changes in the expression of both E-cadherin and -SMA in
EMT cells were observed and confirmed by immunofluorescence staining of renal tissue after UUO (Fig. 4D). Of note, the
sham group revealed only arterial and arteriolar staining adjacent to the glomerulus, but no interstitial staining (Fig. 4D). In
brief, HSP72 expression induced by GGA significantly reAJP-Renal Physiol VOL

Fig. 3. Effect of GGA on the induction of HSP72 in rat kidney after UUO.
GGA administration increased steady-state content of HSP72 as shown by
Western blot analysis. Numbers (1, 2, and 3) denote each individual animal
within a given group. Values are means SE. *P 0.05 vs. sham. P 0.05
vs. vehicle.

duced EMT associated with UUO (Fig. 4). These findings

suggest that HSP72 targets tubular EMT, a key event in the
pathogenesis of renal interstitial fibrosis.
GGA Reduced Renal Tubular Apoptosis and Proliferation
The protective effects of GGA on cell apoptosis and proliferation in the obstructed kidney at day 7 after UUO were then
assessed by TUNEL staining. Compared with sham UUO,
TUNEL-positive cells were increased in the obstructed kidney
(Fig. 5, A and C). Most TUNEL-positive cells were proximal
tubular epithelial cells, although small numbers of apoptotic
cells were also observed in interstitial and endothelial cells. In
contrast, GGA exposure significantly reduced the number of
TUNEL-positive cells (Fig. 5, A and C). Similarly, GGA
significantly decreased renal PCNA expression in the cortex of
the kidney after UUO (Fig. 5, B and C).
ZVAD-FMK Inhibited TGF-1-Induced Apoptosis But Not
EMT in NRK52E Cells
In the intact animal, GGA exposure attenuated interstitial
matrix protein deposition, cell apoptosis, and cell proliferation.
To examine a potential mechanism by which HSP72 inhibits
EMT, an in vitro model was developed in which NRK52E cells
were exposed to TGF-1, an established inducer of EMT.
Cells treated with TGF-1 underwent both apoptosis (data
not shown) and EMT in both dose- and time-dependent manners (Fig. 6, A and B). Immunofluorescence staining also
confirmed that TGF-1 treatment for 72 h induced a suppression of E-cadherin and a dramatic induction of -SMA (Fig.
6C). These morphological changes are consistent with the
features of EMT. In addition, TGF-1 treatment for 72 h led to
a 36.6% increase in apoptosis (Fig. 7).
To examine whether apoptosis is associated with the occurrence of an EMT, ZVAD-FMK, a cell-permeable pan-caspase
inhibitor, was used. Flow cytometric analysis revealed that
preincubation with 50 M ZVAD-FMK reduced apoptosis by
up to 53.8% in the cells treated with TGF-1 for 72 h (Fig. 7).

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Fig. 4. Effect of GGA on E-cadherin and -smooth muscle actin (SMA) expression after UUO. A: representative Western blot analysis showed the protein levels
of E-cadherin and -SMA from sham, vehicle, and GGA-treated rat kidneys after UUO. Numbers (1, 2, and 3) denote each individual animal within a given
group. UUO showed a virtually complete block of E-cadherin expression and marked induction of -SMA expression. Compared with vehicle treatment, GGA
treatment preserved E-cadherin and lowered -SMA expression in the obstructed kidney. B and C: graphic representation of E-cadherin and -SMA protein levels
in different groups, as indicated after normalization with GAPDH content. Values are means SE. *P 0.05 vs. sham. P 0.05 vs. vehicle. D: representative
pictures of E-cadherin (red staining) and -SMA immunofluorescence (red staining) in different groups as indicated. Original magnification: 200 (E-cadherin)
and 400 (-SMA).

ZVAD-FMK preserved the levels of E-cadherin expression

at the dose of 50 M and above, but not at 25 M (Fig. 8,
A and B). Moreover, ZVAD-FMK did not suppress TGF1-induced expression of -SMA (Fig. 8, A and C), a
hallmark of EMT.
To further confirm the effect of ZVAD-FMK on EMT,
confocal immunofluorescent microscopy was employed to determine whether ZVAD-FMK maintains E-cadherin at the cell
surface and abolishes -SMA expression in TGF-1-treated
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cells. As shown in Fig. 8D, TGF-1 treatment removed Ecadherin from the cell membrane and induced -SMA expression. ZVAD-FMK treatment largely preserved E-cadherin at
the cell periphery following TGF-1 treatment. In contrast,
ZVAD-FMK did not attenuate TGF-1-induced expression of
-SMA. The immunofluorescent images were thus consistent
with the Western blot data, indicating that ZVAD-FMK treatment preserved E-cadherin, but did not inhibit -SMA expression in renal epithelial cells exposed to TGF-1.

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Fig. 5. Effect of GGA on renal tubular cell apoptosis

and proliferation. A and B: number of TdT-mediated
dUTP nick end labeling (TUNEL)-positive cells per
high-power field and percentage of PCNA-positive
cells per high-power field was increased in vehicletreated rats compared with sham-treated rats. GGAtreated rats had significantly less TUNEL-positive
cells per high-power field and PCNA-positive cells
per high-power field compared with vehicle-treated
rats. Values are means SE; n 5. *P 0.05 vs.
sham. P 0.05 vs. vehicle. C: representative images
of TUNEL (green staining, arrows) and PCNA staining (brown staining, arrowheads) in different groups
as indicated. Original magnification 400.

These results indicated that blocking tubular apoptosis via a

caspase-dependent signal pathway could not prevent cells from
TGF-1-induced EMT in culture, suggesting possible separate
underlying mechanism(s) for tubular apoptosis and EMT in
chronic TIF.
HSP72 Inhibited TGF-1-Induced In Vitro EMT
in NRK52E Cells
Our previous studies demonstrated that HSP72 exerted antiapoptotic effects in cultured renal proximal epithelial cells
subjected to ATP depletion (17, 31, 41). Here, we explored
whether HSP72 inhibits EMT in the manner independent of its
antiapoptotic effects. Wild-type HSP72 was selectively expressed before TGF-1 exposure by the infection of NRK52E
cells with adenovirus encoding human HSP72. Adenoviral
infection at 40 and 60 MOI resulted in a significant increase in
steady-state HSP72 protein content (Fig. 9A). Compared with
empty vector control (infected with GFP- and tTA-encoding
adenoviruses), enhanced expression of HSP72 prevented cells
from the TGF-1-induced loss of E-cadherin expression and
from a gain of -SMA expression, as demonstrated by Western
blot analysis (Fig. 9A, lanes 4 and 5). Of note, there are no
significant differences in E-cadherin and -SMA baseline expression levels between normal control and empty vector
control cells (data not shown). In contrast to the results obtained using ZVAD-FMK (Fig. 9A, lane 3), increased HSP72
expression not only preserved E-cadherin expression but also
inhibited -SMA expression. Quantitative determination revealed that overexpression of HSP72 restored E-cadherin content by 47.2% and reduced -SMA expression by 43.8% at 60
MOI of adenoviruses, compared with empty vector control
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(Fig. 9, B and C). This action of HSP72 also was dose

dependent at MOI of adenovirus between 40 and 60.
To determine whether inhibiting TGF-1-induced in vitro
EMT was specifically due to an increase in HSP72 expression,
an HSP72 siRNA knockdown approach was taken. Our studies
showed that endogenous HSP72 was not detectable in
NRK52E cells by Western blotting with a specific anti-HSP72
antibody (Fig. 9, A and E), even though a large amount of
protein (80 g/lane) was loaded. We first increased HSP72
expression by overexpression of an adenoviral vector encoding
human HSP72 in NRK52E cells, and then knocked down
HSP72 expression using siRNA in HSP72-overexpressing
cells. As showed in Fig. 9D, transfection of NRK52E cells with
HSP72-specific siRNA (20 nM) nearly completely abolished
adenovirus-induced HSP72 protein expression, compared with
the same concentration of siRNA controls. Knockdown of
adenovirus-induced HSP72 protein expression blocked protective effects of HSP72 protein overexpression on TGF-1induced EMT (preserving E-cadherin content and inhibiting
-SMA expression) (Fig. 9, EG).
We further investigated the effect of HSP72 siRNA on EMT
using confocal immunofluorescent microscopy. As shown in
Fig. 9H, after TGF-1 treatment for 72 h, both control and
HSP72 siRNA knockdown cells developed EMT, as shown by
decreased E-cadherin expression levels and induction of
-SMA expression. In contrast, HSP72-overexpressing cells
demonstrated resistance to TGF-1-induced EMT. However,
neither the mock transfection nor adenovirus infection control
affected cell morphology and expression of E-cadherin and
-SMA (data not shown). Therefore, these findings support our

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Fig. 6. Dose- and time-dependent effects of transforming growth factor-1 (TGF-1) exposure on
EMT in vitro. A and B: cells were treated with
TGF-1 at different concentrations for 72 h and with
TGF-1 at 10 ng/ml for different times before the
assessment of EMT. E-cadherin and -SMA expression levels were assessed by Western blot analysis.
C: immunofluorescence staining demonstrated that
TGF-1 at 10 ng/ml concentration for 72 h induced
the loss of E-cadherin but the gain of -SMA. Original magnification 400.

hypothesis that HSP72 targets EMT directly by a mechanism(s) independent of its effect on tubular apoptosis.

This study demonstrates that GGA-induced HSP72 expression in the rat kidney after UUO decreased the number of
-SMA-positive myofibroblasts and reduced TIF and collagen
I deposition. In addition, HSP72 expression significantly inhibited tubular cell apoptosis and reduced tubular cell proliferation in obstructed kidneys. In vitro experiments in NRK52E
cells revealed that blocking the caspase-dependent apoptotic
pathway prevented TGF-1-induced apoptosis, but not EMT.
It is of potential significance that HSP72 expression not only
attenuated apoptosis, an established effect of this cytoprotective protein, but also inhibited EMT, a process that contributes
to chronic fibrosis and the progressive loss of organ function.
AJP-Renal Physiol VOL

The effect of HSP72 on EMT could be attributed, at least

partly, to preserving E-cadherin and suppressing -SMA expression both in vivo and in vitro. These results suggest that
enhanced expression of HSP72 attenuates renal tubular apoptosis and TIF caused by ureteric obstruction. Our data shed
new light on the mechanism of HSP72-mediated renoprotection in chronic progressive renal fibrosis.
In both rats and humans, sustained UUO gives rise to tubular
cell injury and death and eventually causes the affected kidney
to become fibrotic (15). In the present study, tubular apoptosis
was markedly increased in the obstructed kidney compared
with the sham-operated kidney. This is important, since renal
tubular apoptosis positively correlates with subsequent tissue
injury as demonstrated by Daemen et al. (4). These investigators reported that inhibition of apoptosis induced by ischemiareperfusion prevented the early onset of not only renal apop-

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Fig. 7. Effect of ZVAD-FMK on TGF-1-induced apoptosis in vitro. NRK52E cells were treated with 10 ng/ml TGF-1 for 72 h in the presence or absence
of ZVAD-FMK (0 150 M). Flow cytometric analysis showed that preincubation with ZVAD-FMK significantly inhibited TGF-1-induced apoptosis by up
to 53.8% at 50 M. Values are means SE; n 3/treatment. *P 0.05 vs. control. P 0.05 vs. TGF-1 treated without ZVAD-FMK.

tosis but also later development of inflammation and organ

dysfunction. Their results indicate that tubular cell apoptosis is
an early event that precedes overt fibrosis (22, 24, 32). According to this paradigm, it is clear that successful inhibition of
initial apoptosis would be expected to limit renal fibrosis.
Previous studies on the protective effects of HSP72 against
various insults have been mainly focused on models of acute
injury. It has been shown that HSP72 can protect a variety of
cells, including renal tubule cells, from thermal, toxic, and
ATP depletion-mediated injury in vitro (31, 35, 47). Enhanced
HSP72 expression also ameliorates myocardial, liver, brain,
and renal damage induced by ischemia-reperfusion, cyclosporin, and endotoxin exposure in rats (9, 10, 26, 35). Our
preliminary results (data not shown), as well as those reported
by others, showed that after ureteric obstruction, increased
HSP72 expression started at day 3, peaked at day 7, and was
maintained over the following 14 days, which paralleled the
progression of renal fibrosis (18, 37). However, endogenous
HSP72 is inadequate to accomplish cytoprotection. In addition,
the underlying mechanism by which HSP72 is elevated after
UUO being not yet known, it is postulated that during stress
HSP72 probably folds proteins into a configuration which can
be refolded to a normal state rapidly after reversal of the injury.
We therefore used a UUO model to evaluate the role of HSP72
in attenuating chronic renal damage.
GGA, an antiulcer drug, has been widely used in the clinical
setting and is a specific inducer of HSP72 in rats (35, 39). GGA
induces HSP72 expression through activation of heat shock
factor 1 (HSF1), expression of HSP72 mRNA, and subsequent
accumulation of HSP72 protein (11, 12, 27). Because GGAAJP-Renal Physiol VOL

induced HSP72 overexpression in vivo peaked at 24 h after

administration of a single oral dose, the rats in our study were
given GGA one day before UUO. Our study demonstrated that
orally administered GGA markedly augmented the expression
of HSP72, but not that of HSP27, HSP60, and HSP90 in the
kidney, confirming its selectivity in our model.
HSP72 may elicit its renoprotective activities by multiple
mechanisms. One relevant finding in our study is that GGAinduced HSP72 expression decreased interstitial volume,
-SMA expression, and collagen I deposition in obstructed
kidneys. Given the fact that increased HSP72 expression can
reduce or slow down fibrogenic processes, we assumed that
delayed administration of GGA after occurrence of UUO
would also be effective. Another interesting observation is that
GGA may exert its beneficial effects by inhibiting tubular
proliferation and apoptosis in the obstructed kidney (Fig. 5).
Because HSP72 is an antiapoptotic and molecular chaperone,
overexpression of HSP72 could limit initial tubular injury and
thereby reduce the requirement of epithelial cell proliferation
during repair. The protective effects of enhanced HSP72 expression on renal interstitial fibrosis and tubular apoptosis after
UUO in rats indicate that HSP72 induction could be of potential therapeutic value for treating chronic kidney diseases in
which both apoptosis and fibrosis have been implicated in
disease progression.
TGF-1 can induce concomitant cell apoptosis and EMT.
TGF-1-induced cell apoptosis is through a caspase-dependent
pathway (14). Loss of E-cadherin on apoptosis has been
described in other studies and is thought to result from the
activation of caspases (8). EMT is also associated with a

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Fig. 8. Effect of ZVAD-FMK treatment on TGF-1-induced epithelial-to-mesenchymal transition (EMT). A: following a 1-h preincubation with 0 150 M
ZVAD-FMK, NRK52E cells were treated with 10 ng/ml TGF-1 for 72 h. ZVAD-FMK preserved E-cadherin expression with concentrations at 50 M and
above, but essentially could not block -SMA expression. B and C: densitometric analysis of the effect of ZVAD-FMK on E-cadherin and -SMA expression,
corrected with GAPDH content. Values are means SE; n 3/treatment. *P 0.05 vs. control. P 0.05 vs. TGF-1-treated cells without ZVAD-FMK.
D: EMT was examined by confocal microscopic images of E-cadherin and -SMA staining in control and ZVAD-FMK treated cells following TGF-1 exposure.
Original magnification 400.

decrease in E-cadherin protein levels. Collectively, both apoptosis and EMT contribute to the decrease or loss of E-cadherin
content. Since TGF-1-induced concomitant apoptosis and
EMT are two mutually exclusive processes, our study demonstrated that preincubation with ZVAD-FMK preserved the
content of E-cadherin, presumably due to its inhibition on the
cleavage of E-cadherin resulting from apoptosis. However,
ZVAD-FMK did not suppress TNF-1-induced expression of
-SMA, the molecular hallmark of myofibroblast transformation and the morphological changes of EMT. This suggests that
TGF-1-induced EMT is independent of its effect on any
AJP-Renal Physiol VOL

caspase-mediated apoptotic response. Recent evidence supports the notion that TGF-1 induces both EMT and apoptosis
via a cell cycle-dependent mechanism in which apoptosis and
EMT occur at G2/M and G1/S phases, respectively (6, 34, 48,
49). In addition, a different signaling mechanism(s) may
underlie tubular EMT and apoptosis in chronic TIF. Signaling molecules, including Smad, Rho-A, p38, and Par-6,
were reported to be involved in the regulation of EMT. On
the other hand, the release of cytochrome c from the mitochondria is a crucial step in the intrinsic pathway of apoptosis in chronic TIF.

295 JULY 2008



Fig. 9. Effect of HSP72 on TGF-1-induced EMT in vitro. HSP72 expression in cells was increased by infection with 2 doses of adenovirus encoding human HSP72
(AdvTR5/HSP70-GFP) in AdvCMV/tTA system, with or without 10 ng/ml TGF-1 or 100 M ZVAD-FMK. Adenovirus without encoding human HSP72
(AdvTR5/GFP) served as a negative control. In addition, cells were transfected with specific HSP72 small interfering RNA (siRNA). Cells treated with control siRNA
were used as a control. A: Western blot showed increase in HSP72 associated with preserving of E-cadherin expression and suppressing of -SMA expression in a
dose-dependent manner in TGF-1-treated cells. B and C: densitometric analysis of the effect of HSP72 expression on E-cadherin and -SMA levels normalized with
GAPDH content in cells treated as described in A. Values are means SE; n 5/treatment. *P 0.01 vs. negative control. P 0.05 vs. TGF-1-treated cells without
either ZVAD-FMK or HSP72 overexpression (bar 2). D: specificity of HSP72 siRNA was examined using transfection agent, control siRNA, and different doses of
HSP72 siRNA. HSP72 expression was analyzed by Western blotting. E: EMT was assessed by Western blot analysis after transfection with HSP72 siRNA. F and
G: E-cadherin and -SMA content were analyzed quantitatively using a densitometer. Values are means SE; n 3/treatment. *P 0.01 vs. negative control. P
0.05 vs. TGF-1-treated cells without either HSP72 overexpression or HSP72 siRNA (bar 2). H: EMT was examined by confocal microscopic images of E-cadherin
and -SMA staining in control, HSP72-overexpressing, and HSP72 siRNA cells following TGF-1 exposure. Original magnification 400.

HSP72 is an inducible cytoprotectant and antiapoptotic protein (1, 20, 33). It plays a pivotal role in cell survival by
interfering with multiple checkpoints in the apoptotic pathway
(2, 43). We employed cultured NRK52E cells in an in vitro
AJP-Renal Physiol VOL

EMT model to examine the renoprotective regulatory mechanisms of HSP72. Compared with the empty vector and exposure to a pan-caspase inhibitor, overexpression of HSP72
markedly inhibited EMT by preserving E-cadherin and pre-

295 JULY 2008


venting -SMA expression. Furthermore, HSP72-mediated

EMT inhibition could be diminished by knockdown of HSP72
using HSP72-specific siRNA. These findings suggest that
HSP72 attenuates TIF after UUO, at least in part, by concurrently inhibiting both tubular EMT and apoptosis. To our
knowledge, the effect of HSP72 on EMT has not yet been
reported, and its underlying mechanism(s) remains unknown.
Since Smad3 can transduce signals of TGF- from the cell
cytosol to the nucleus (29), HSP72 may act as a molecular
chaperone to prevent nuclear translocation of Smad3 (30). In
support of this speculation, Knuesel et al. (16) recently demonstrated an interaction between HSP72 and Smad3 using
coimmunoprecipitation. Thus HSP72 may prevent nuclear
Smad3 translocation and suppress tubular EMT by regulating
the TGF-/smad signaling pathway. Whether HSP72 inhibits
EMT via additional mechanisms, such as TGF-, fibroblast
growth factor, and epidermal growth factor expression requires
further investigation.
In summary, this is the first study to demonstrate that HSP72
significantly attenuates TIF and tubular apoptosis in kidneys
subjected to ureteric obstruction. Although the precise mechanism
remains to be determined, our results showed that HSP72, but not
a pan-caspase inhibitor, inhibited tubular EMT in vitro, in response to TGF-1. This suggests that HSP72 exerts an important
role in chronic TIF by mechanisms independent of its effect on
tubular apoptosis and may be a potential therapeutic agent for
preventing progressive renal fibrosis in humans.










This work was supported by Grant 30671055 from the National Natural
Science Foundation of China and Grant 107087 from the Ministry of Education of the Peoples Republic of China (to H. Mao).
A portion of these studies was presented as an abstract at the 39th annual
meeting of the American Society of Nephrology, November 14 19, 2006,
San Diego, CA.



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