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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
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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.
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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).
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. 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.
DISCUSSION
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.
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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.
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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
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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.
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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
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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-
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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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