Department of Medicine, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040
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ABSTRACT |
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ANG II has
been demonstrated to play a role in the progression of tubulointerstial
injury. We studied the direct effect of ANG II on apoptosis of
cultured rat renal proximal tubular epithelial cells (RPTECs). ANG II
promoted RPTEC apoptosis in a dose- and time-dependent manner.
This effect of ANG II was attenuated by anti-transforming growth factor
(TGF)- antibody. Moreover, TGF-
triggered RPTEC apoptosis
in a dose-dependent manner. ANG II also enhanced RPTEC expression of
Fas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuated
ANG II-induced RPTEC apoptosis. In addition, ANG II increased
RPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT1) and type 2 (AT2) receptor blockers
inhibited ANG II-induced RPTEC apoptosis. SB-202190, an
inhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor also
attenuated ANG II-induced RPTEC apoptosis. ANG II enhanced RPTEC heme oxygenase (HO)-1 expression. Interestingly, pretreatment with hemin as well as curcumin (inducers of HO-1) inhibited the ANG
II-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted the
effect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT1 and
AT2 receptors through the generation of TGF-
, followed
by the transcription of cell death genes such as Fas, FasL, and Bax.
Modulation of tubular cell expression of HO-1 has an inverse
relationship with the ANG II-induced tubular cell apoptosis.
Bax; Bcl-2; Fas; Fas ligand; proximal tubular epithelial cells; heme oxygenase-1
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INTRODUCTION |
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ANG II HAS BEEN DEMONSTRATED to contribute to the progression of renal injury through its hemodynamic effects (35, 36). These effects are confirmed by blocking its production and receptor sites (15, 16, 20). However, apart from its hemodynamic effects, the direct effects of ANG II on kidney cells are being increasingly recognized (2, 6, 9, 25, 27). It has been demonstrated that in addition to circulating ANG II, tissue (intrarenal) generation of ANG II is also important for its net effect (35).
Tubulointerstitial lesions have been demonstrated to correlate with the
progression of renal failure (5, 11, 18, 21, 26), thus
suggesting their contribution to the progression of renal failure
(11). Transforming growth factor (TGF)-, a fibrogenic cytokine, has been shown to play a role in the inception and
progression of renal lesions in both human renal diseases and
experimental animal models of human immunodeficiency virus-associated
nephropathy, renal ablation, and ureteric obstruction (16, 33,
36). Interestingly, in these conditions, elevated blood ANG II
levels have been reported. Moreover, modalities, which inhibit the
production of ANG II, have been demonstrated to slow the progression of
renal lesions (15, 16, 20).
The effect of ANG II on the growth of proximal tubular cells has been evaluated in both in vivo and in vitro studies (4, 32). Wolf et al. (12, 35) in their pioneer studies demonstrated that ANG II induced the hypertrophy of cultured tubular cells. These investigators elegantly delineated the ANG II-induced downstream signal transduction pathway (12). Weerackody et al. (34) showed that ANG II-induced tubular cell hypertrophy (protein synthesis) was inhibited by pertussis toxin and losartan but not by PD-123319. These findings suggest that ANG II type 1 (AT1) rather than ANG II type 2 (AT2) receptors may be contributing to ANG II-induced tubular cell hypertrophy. Recently, Cao et al. (7) demonstrated that infusion of ANG II in rats enhanced the number of both PCNA- and transferase-dUTP-nick-end labeling (TUNEL)-positive cells in proximal tubules. These investigators suggest that ANG II triggers both proliferation and apoptosis of proximal tubular cells. Similar findings were reported by Aizawa et al. (2) in rat proximal tubular cells. Interestingly, these investigators have demonstrated that agents which induce tubular cell heme oxygenase (HO)-1 expression also provide protection from the growth modulatory effects of ANG II; conversely, agents that inhibit tubular cell HO-1 expression promote the growth modulatory effect of ANG II.
Increased cellular expression of HO-1 has been considered to be a marker of oxidative stress (24). In various animal experimental models of oxidative injury, induction of HO-1 has been demonstrated to provide protection from ongoing injury (8, 10, 17, 23, 31). In addition to the model of ANG II-induced tubular cell injury, enhanced tubular cell HO-1 expression has been shown to confer protection against cisplatin-induced tubular cell injury (16, 28).
In the present study, we evaluated the effect of ANG II on the apoptosis of cultured rat proximal tubular cells. We also studied the molecular mechanisms involved and the possible relationship between HO-1 expression and ANG II-induced tubular cell apoptosis.
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MATERIALS AND METHODS |
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Renal Proximal Tubular Epithelial Cell Cultures
Rat proximal renal tubular epithelial cells (NRK-52E) were obtained from American Type Culture Collection, Rockville, MD. Cells were grown in DMEM (GIBCO, Grand Island, NY) containing 2% penicillin-streptomycin, 1% HEPES, 1.5 g NaHCO3, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% FCS. In the experimental protocols, cells were incubated in media containing 1% FCS.Apoptotic Studies
Rat and human renal proximal tubular epithelial cells (RPTECs; HK-2) were treated under control and experimental conditions for the indicated times. At the end of incubation period, cells were stained with H-33342 and propidium iodide and evaluated for apoptosis as described previously (29, 30). In these studies, observers were blinded to experimental conditions.Detection of tubular cell apoptosis by gel electrophoresis. This is a simple method that is specific for isolation and confirmation of DNA fragments from apoptotic cells (14). Because this method only picks up DNA fragments, one will not visualize any loading of samples that do not contain DNA fragments. RPTECs were treated under control and experimental conditions as indicated, and DNA was extracted and electrophoresed as described previously (29, 30).
Superoxide Assay
Equal numbers of RPTECs were plated in 100-mm petri dishes and grown to subconfluence. The cells were washed twice with normal saline and incubated in serum- and phenol red-free media containing either buffer, 10H2O2 Assay
Equal numbers of RPTECs were plated in 100-mm petri dishes and grown to subconfluence. The cells were washed twice with normal saline and incubated in serum- and phenol red-free media containing either buffer, 10Protein Extraction and Western Blot Analysis
RPTECs were treated under control and experimental conditions as indicated. At the end of the incubation period, the cells were washed three times with PBS, scraped into a modified RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 0.1% SDS, 10 µl of protease inhibitor cocktail/ml of buffer, and 100 µg/ml of PMSF) and transferred, using a syringe fitted with a 21-gauge needle, into a microcentrifuge tube. The cell lysates were centrifuged at 15,000 g for 30 min at 4°C. The supernatant was analyzed for total protein content. Twenty micrograms of protein were heated at 100°C for 10 min, loaded, and separated on a 12% PAGE gel under nonreducing conditions. The proteins were electrotransferred to a nitrocellulose membrane in transfer buffer containing 48 mM Tris · HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C overnight. Nonspecific binding to the membrane was blocked for 1 h at room temperature with blocking buffer (0.5% BSA in PBS with 0.1% Tween 20). The membrane was then incubated for 16 h at 4°C with primary antibodies [mouse monoclonal anti-Bax antibody, Pharmingen, San Diego, CA; goat polyclonal anti-Bcl-2 antibody, rabbit polyclonal anti-Fas antibody, Santa Cruz Biotechnology, Santa Cruz, CA; mouse monoclonal anti-Fas ligand (FasL) antibody, Pharmingen; and mouse monoclonal anti-HO-1 antibody, Stressgen, Victoria, BC] in blocking buffer, followed by incubation for 1 h at room temperature with the appropriate secondary antibody in blocking buffer. Signals were visualized by an enhanced chemiluminescence detection kit (Pierce) after exposure to X-ray film (Eastman Kodak, Rochester, NY) (29, 30). To determine loading, blots were stripped and reprobed forStatistical Analysis
Statistical analysis was performed using GraphPad Instat software. A Newman-Keuls multiple comparison test was used, and P values were calculated. ![]() |
RESULTS |
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Studies Pertaining to Apoptosis
To study the dose-response effect of ANG II on RPTEC apoptosis, equal numbers of cells (10,000 cells/well, 24-well plates) were incubated in media containing either buffer (control) or 10
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To confirm the occurrence of apoptosis, equal numbers of RPTECs
were incubated in 100-mm petri dishes with media containing either
buffer (control) or 108 and 10
6 M ANG II
for 16 h. Subsequently, DNA was isolated and electrophoresed. In
gel electrophoresis, DNA isolated from ANG II-treated RPTECs showed a
classic ladder pattern, thus confirming the occurrence of
apoptosis (Fig. 1C).
To evaluate whether this effect of ANG II was species specific, we
evaluated the effect of ANG II on human proximal tubular cells (HK-2).
Equal numbers of cells were incubated in media containing either buffer
or 108 and 10
6 M ANG II for 16 h.
Subsequently, cells were stained for apoptosis. ANG II promoted
HK-2 cell apoptosis (control, 1.5 ± 0.5%;
10
8 M ANG II, 15.5 ± 1.2%; 10
6 M ANG
II, 25.5 ± 2.0%; P < 0.001). These findings
suggest that the apoptotic effect of ANG II is not species specific.
Studies Pertaining to AT1 and AT2 Receptors
To evaluate the role of AT1 and AT2 receptors in the induction of apoptosis, equal numbers of RPTECs were incubated in media containing vehicle (control), the AT1 inhibitor losartan (10
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Studies Pertaining to the Role of TGF-
To evaluate the effect of TGF-1 on RPTEC apoptosis,
equal numbers of RPTECs were incubated in media containing either
buffer (control) or variable concentrations of TGF-
1 (50-2,000
pg/ml; Collaborative Biomedical Products, Bedford, MA) for 16 h.
Subsequently, cells were stained for apoptosis. Eight sets of
experiments were carried out. TGF-
1 triggered RPTEC
apoptosis in a dose-dependent manner (Fig.
3A).
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To establish a causal relationship between TGF- and the
occurrence of RPTEC apoptosis, we studied the effect of
anti-TGF-
antibody on TGF-
-induced tubular cell
apoptosis. Equal numbers of RPTECs were incubated in media
containing either buffer, 10 ng/ml TGF-
1, 1 µg/ml
anti-TGF-
1 antibody, or anti-TGF-
antibody+TGF-
1 for
16 h. Subsequently, cells were assayed for apoptosis.
Anti-TGF-
antibody attenuated the proapoptotic effect of TGF-
(Fig. 3B).
Studies Pertaining to the Role of FasL
To evaluate the contribution of the FasL pathway in ANG II-induced tubular cell apoptosis, equal numbers of RPTECs were incubated in media containing buffer (control) or 10To evaluate the contribution of the FasL pathway in TGF--induced
tubular cell apoptosis, equal numbers of RPTECs were incubated in media containing buffer (control) or 10 ng/ml TGF-
with or without 1 µg/ml anti-FasL antibody for 16 h. Subsequently, the cells were stained for apoptosis. As shown in Fig.
3D, anti-FasL antibody attenuated the effect of TGF-
on
tubular cell apoptosis.
Studies Pertaining to the Role of Oxidative Stress
To determine the role of oxidative stress in ANG II-induced RPTEC apoptosis, we studied the effect of antioxidants such as diphenyleneiodonium chloride (DPI; Sigma), ascorbic acid (AA), and N-acetyl cysteine (NAC). Equal numbers of RPTECs were incubated in media containing buffer (control), 10 µM DPI, 100 µM AA, or 50 µM NAC with or without 10
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To evaluate the role of ANG II and TGF- on the generation of
reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, we measured the production of superoxide and
H2O2 by RPTECs under control and ANG
II-stimulated states in the presence or absence of anti-TGF-
antibody. Equal numbers of cells were incubated in media containing
either buffer or ANG II for 120 min. Supernatants were collected at 0, 30, 45, 60, 90, and 120 min and assayed for superoxide and hydrogen
peroxide. As shown in Fig. 4, B and C, ANG II
promoted RPTEC generation of both superoxide and hydrogen peroxide at
45 min. The generation of ROS plateaued at 120 min (data on 60, 90, and
120 min are not shown). However, anti-TGF-
antibody inhibited the
effect of ANG II.
To determine the effect of HO-1 preinduction on ANG II-induced
tubular cell apoptosis, equal numbers of cells were pretreated in media containing buffer (control), 5 µM hemin, or 15 µM curcumin (Sigma) for 4 h. Subsequently, cells were incubated in media
containing either vehicle (control) or 108 M ANG II for
16 h and then stained for apoptosis. Pretreatment with
hemin as well as curcumin rendered partial protection against the
proapoptotic effect of ANG II on tubular cells (Fig.
5A).
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To determine the effect of HO-1 inhibition, equal numbers of RPTECs
were pretreated in media containing either buffer (control) or zinc
protoporphyrin (ZnP; 100 µM, Sigma) for 4 h, followed by
incubation in media containing vehicle (control) or 108 M
ANG II for 16 h. Subsequently, cells were stained for
apoptosis. As shown in Fig. 5B, ZnP accentuated the
proapoptotic effect of ANG II on RPTECs.
To determine the importance of the timing of HO-1 induction, we
evaluated the effect of ANG II on tubular cells that were either
pretreated with hemin (preinduction) or treated simultaneously. Equal numbers of RPTECs were preincubated in media containing either buffer or 5 µM hemin for 4 h, followed by incubation in either buffer or 108 M ANG II for 16 h. In parallel
studies, RPTECs were incubated with either buffer or 5 µM
hemin+10
8 M ANG II for 16 h. Subsequently, cells
were assayed for apoptosis. As shown in Fig. 5C,
pretreatment of ANG II-treated cells with hemin (HO-1 preinduction)
attenuated the apoptotic effect of ANG II. However, simultaneous
treatment of tubular cells with ANG II and hemin did not prevent the
apoptotic effect of ANG II. These studies suggest that only
preinduction of HO-1 is beneficial to modulate the apoptotic effect
of ANG II.
Because curcumin has been demonstrated to induce multiple effects,
including the inhibition of JNK, binding of activator protein-1, and
HO-1 expression, it may be important to evaluate the mechanism involved
in curcumin-induced modulation of the ANG II effect. Equal numbers of
RPTECs were incubated in media containing either buffer,
108 M ANG II, 15 µM curcumin, 50 µM ZnP,
curcumin+ANG II, curcumin+ZnP, or curcumin+ZnP+ANG II for 16 h. Subsequently, cells were assayed for apoptosis. ZnP
attenuated the antiapoptotic effect of curcumin on ANG II-treated
cells (Fig. 5D). These findings suggest that the
curcumin-mediated effect on ANG II-treated cells may have been induced
through HO-1 expression.
Studies Pertaining to p38 MAPK Activation
To evaluate the role of p38 MAPK in ANG II-induced RPTEC apoptosis, we studied the effect of a selective p38 MAPK inhibitor, SB-202190 (Calbiochem, La Jolla, CA), on ANG II-induced RPTEC apoptosis. Equal numbers of cells were incubated in media containing either buffer (control) or 5 µM SB-202190 for 1 h. At the end of the incubation period, cells were reincubated in media containing vehicle (control) or 10
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Studies Pertaining to Cell Death Pathways
To determine the role of caspase-3 in ANG II-induced tubular cell apoptosis, equal numbers of cells were incubated in media containing either buffer (control), or 10To determine the role of cell death proteins (Bax, Fas, FasL) and
cell survival protein (Bcl-2) in ANG II-induced RPTEC
apoptosis, equal numbers of tubular cells were incubated in
media containing either buffer (control), or 108 and
10
6 M ANG II for 16 h. Subsequently, protein was
extracted, and Western blots were prepared and probed for Bax, Bcl-2,
Fas, and FasL. Tubular cells showed an increased (P < 0.001) expression of Fas in response to ANG II treatment compared with
control (Fig. 7B, depicted in the form of Fas/actin
ratios). A representative gel is shown in
Fig. 7A. Similarly, ANG II-treated cells showed increased expression of FasL compared with control (Fig. 7D, depicted
in the form of FasL/actin ratios). A representative gel showing the effect of ANG II on FasL expression is shown in Fig. 7C.
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ANG II enhanced RPTEC expression of Bax (Fig. 8B, depicted
in the form of Bax/actin ratios). A
representative gel showing the effect of ANG II on RPTEC expression is
shown in Fig. 8A. On the other hand, ANG II decreased RPTEC
expression of Bcl-2. (Fig. 8D, shown in the form of
Bcl-2/actin ratios). A representative gel showing the effect of ANG II
on RPTEC expression of Bcl-2 is shown in Fig. 8C.
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Studies Pertaining to Tubular Cell HO-1 Expression
To study the effect of ANG II on RPTEC HO-1 induction, equal numbers of cells were incubated in six-well plates with media containing either buffer (control) or 10
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To determine the role of TGF- in ANG II-induced HO-1 expression,
equal numbers of cells were treated with either buffer (control) or
10
8 and 10
6 M ANG II with or without
anti-TGF-
antibody for 16 h. Subsequently, protein was
extracted, and Western blots were generated and probed for HO-1. As
shown in Fig. 9D, the effect of ANG II on RPTEC was inhibited by anti-TGF-
antibody. A representative gel showing the
effect of anti-TGF-
antibody on ANG II-induced HO-1 expression is
shown in Fig. 9C. These studies suggest that ANG II-induced tubular cell-HO-1 induction may be mediated through TGF-
.
To determine the effect of curcumin and hemin on HO-1 expression, equal numbers of RPTECs were incubated in media containing either buffer (control), 5 µM hemin, 15 µM curcumin, or hemin+curcumin for 16 h. Subsequently, cells were prepared for Western blot analysis and probed for HO-1. Both curcumin and hemin promoted HO-1 expression. However, curcumin did not enhance the effect of hemin on tubular cell HO-1 expression (Fig. 9, E and F).
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DISCUSSION |
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The present study demonstrates that ANG II promotes tubular cell
apoptosis. Because anti-TGF- antibody attenuated this effect and TGF-
promoted tubular cell apoptosis, it appears that
ANG II-induced tubular cell apoptosis may be mediated through
TGF-
. Both AT1 and AT2 receptor antagonists
partially blocked ANG II-induced tubular cell apoptosis. ANG II
also promoted the expression of HO-1 and tubular cell expression of
FasL, whereas anti-FasL antibody inhibited the effect of ANG II as well
as TGF-
on tubular cell apoptosis. ANG II not only promoted
tubular cell expression of Bax but also inhibited the expression of
Bcl-2. Pretreatment of tubular cells with inducers of HO-1 (hemin and
curcumin) attenuated the response of ANG II, thus suggesting that
preinduction of HO-1 expression perhaps provides protection against ANG
II-induced tubular cell injury. On the other hand, pretreatment of
RPTECs with ZnP, an inhibitor of HO-1 activity, exacerbated the
proapoptotic effect of ANG II. Only pretreatment and not the
simultaneous treatment of tubular cells with hemin provided protection
against the proapoptotic effect of ANG II.
Fas and the FasL pathway have been reported to mediate
apoptosis in the kidney (19). These
proapoptotic factors have been found to be attenuated by ANG II
inhibition (32). In addition, captopril, an
angiotensin-converting enzyme inhibitor, has been shown to inhibit
Fas-induced apoptosis and FasL expression in human activated
peripheral T cells (22). Our finding of enhanced expression of tubular Fas and FasL is consistent with these
observations. Moreover, inhibition of ANG II-induced tubular cell
apoptosis by anti-FasL antibody established a causal
relationship. We also observed the alteration in the expression of Bax
and Bcl-2 in ANG II-treated cells, which may have tilted the balance
toward apoptosis. In the present study, there was apparent
variability in -actin expression in control and ANG II-treated
tubular cells. This may have partly contributed to the altered
Bax/actin ratio. Aizawa et al. (2) found increased
expression of Bax in the kidneys of ANG II-infused rats without any
decrease in Bcl-2. Because these investigators studied Bcl-2 expression
in whole kidney lysates, their finding may not necessarily represent
Bcl-2 expression by proximal tubular cells.
AT1 receptors are responsible for most of the reported actions of ANG II (25). AT2 receptors are predominantly expressed during fetal development and are considered to be critical for ontogenesis. Expression of AT2 receptors declines rapidly after cessation of developmental apoptosis. Conversely, Cao et al. (7) demonstrated that proximal tubular cells in adult rats express AT2 receptors. These investigators demonstrated that infusion of ANG II for 14 days in 8-wk-old rats induced the proliferation and apoptosis of proximal tubular cells. The administration of the AT2 antagonist PD-123319 or the AT1 antagonist valsartan was associated with attenuation of the increase in both PCNA- and TUNEL-positive cells after ANG II infusion. These findings suggest that both AT1 and AT2 receptors are involved in both proliferation and apoptotic processes in proximal tubular cells. In the present study, both the AT1 antagonist (losartan) and AT2 antagonist (PD-123319) inhibited ANG II-induced apoptosis of tubular cells. These findings suggest that downstream signaling mediated by both AT1 and AT2 receptors is needed for the activation of ANG II-induced tubular cell apoptosis.
Haugen et al. (13) studied the occurrence of oxidative stress in two models of hypertension, i.e., ANG II- and DOCA salt-treated rats. However, only ANG II-treated rats showed evidence of renal oxidative stress in the form of lipid peroxidation, protein carbonyl content, and induction of HO-1 (13). These investigators further localized the induction of proximal tubular cell HO-1 in ANG II-treated animals (13). Because DOCA salt-treated rats did not show renal oxidative stress despite having identical levels of blood pressure, these investigators suggested that ANG II induces oxidative stress independently of its hemodynamic effects. Similarly, Aizawa et al. (3), in two models of hypertension, i.e., ANG II- and norepinephrine-infused rats, showed that only ANG II-infused rats developed a decrease in glomerular filtration rate and proteinuria. Interestingly, tubular cell HO-1 was upregulated only in ANG II-treated rats (not in norepinephrine-treated rats). Pretreatment of rats with HO-1 expression modulators, i.e., hemin, an inducer, and ZnP, an inhibitor, modulated not only tubular cell HO-1 expression but also proteinuria (hemin decreased and ZnP increased ANG II-induced proteinuria) (3). These investigators also studied the occurrence of apoptosis and proliferation of tubular cells in these models (2). Only ANG II-infused rats showed increased numbers of both PCNA- and TUNEL-positive cells. Pretreatment of these rats with HO-1 inducers and inhibitors modulated the severity of ANG II-induced tubular cell apoptosis and proliferation. Moreover, only ANG II-treated rats showed tubular cell upregulation of HO-1 and Bax expression. The effect of ANG II partially persisted despite normalization of blood pressure with hydralazine, again suggesting a nonhemodynamic effect of ANG II. The in vitro observations in the present study are consistent with the in vivo findings of Aizawa et al. (2).
In the present study, ANG II promoted HO-1 expression, thus indicating the occurrence of oxidative stress. Because anitoxidants such as DPI, NAC, and AA inhibited the proapoptotic effect of ANG II, it appears that ANG II-induced tubular cell apoptosis is mediated through oxidative stress. Moreover, enhanced production of superoxide and H2O2 by tubular cells in response to ANG II further delineates the molecular mechanism involed. Both superoxide and H2O2 have been demonstrated to promote HO-1 expression in a variety of cells (1). Thus it appears that ANG II-induced HO-1 expression may be mediated through the generation of superoxide and H2O2 by tubular cells.
Induction of HO-1 may be looked at from two perspectives. On one hand, this may occur as a cellular reflex response to ongoing or acute oxidative stress, of which it is a marker. On the other hand, if preinduced, it may act as part of the armamentarium against incoming injury. Preinduction of HO-1 may generate enough antioxidants to neutralize the effect of oxidative stress. Therefore, preinduction of HO-1 has been used as a tool in preventing damage in various models of oxidative injury (8, 10, 17, 23, 31). Because ANG II induces oxidative stress, it is likely to promote HO-1 expression. Because ANG II could induce tubular cell injury despite ongoing expression of HO-1, it appears that expression of HO-1 may have been a futile attempt to contain oxidative stress. This hypothesis is further supported by our data showing that the proapoptotic effect of ANG II was attenuated in tubular cells pretreated with hemin but not in tubular cells that were treated simultaneously with hemin. On the other hand, ZnP, an inhibitor of HO-1 activity, enhanced the propapoptotic effect of ANG II. These findings suggest that ANG II-induced HO-1 expression may be providing at best limited protection against ANG II-induced oxidative stress.
We conclude that ANG II induces proximal renal tubular cell
apoptosis through the generation of TGF- and ROS, p38 MAPK
phosphorylation, expression of Fas, FasL, and Bax, and activation of
caspase 3. This effect of ANG II seems to be mediated by both
AT1 and AT2 receptors. Modulation of tubular
cell HO-1 expression inversely affects ANG II-induced tubular cell
apoptosis. These findings may provide a basis for a hypothesis
that nonhemodynamic effects of ANG II may be playing a role in the
development and progression of tubular cell injury in conditions
associated with elevated levels of ANG II.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant RO1-DA-12111. A portion of this work was presented at the 33rd annual meeting of the American Society of Nephrology (October 2000, Toronto, Ontario, Canada).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. C. Singhal, Div. of Kidney Diseases and Hypertension, Long Island Jewish Medical Ctr., New Hyde Park, NY 11040 (E-mail: singhal{at}lij.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00246.2002
Received 8 July 2002; accepted in final form 2 December 2002.
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