Angiotensin II induces apoptosis in renal proximaltubular cells - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：MadhuBhaskaran, KrishnaReddy, NeetuRadhakrishanan, NicholasFranki, GuohuaDing,  Pravin C.Singhal作者单位：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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      【摘要】1 s1 ~, E4 I% G$ k& k2 q
   ANG II hasbeen demonstrated to play a role in the progression of tubulointerstialinjury. We studied the direct effect of ANG II on apoptosis ofcultured rat renal proximal tubular epithelial cells (RPTECs). ANG IIpromoted 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 apoptosisin a dose-dependent manner. ANG II also enhanced RPTEC expression ofFas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuatedANG II-induced RPTEC apoptosis. In addition, ANG II increasedRPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT 1 ) and type 2 (AT 2 ) receptor blockersinhibited ANG II-induced RPTEC apoptosis. SB-202190, aninhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor alsoattenuated 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 ANGII-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted theeffect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT 1 andAT 2 receptors through the generation of TGF-, followedby the transcription of cell death genes such as Fas, FasL, and Bax.Modulation of tubular cell expression of HO-1 has an inverserelationship with the ANG II-induced tubular cell apoptosis. ! }* e# Y" J! y: V9 ~
      【关键词】 Bax Bcl Fas Fas ligand proximal tubular epithelial cells heme oxygenase
   INTRODUCTION

ANG II HAS BEEN DEMONSTRATED to contribute to the progression of renal injurythrough its hemodynamic effects ( 35, 36 ). These effectsare confirmed by blocking its production and receptor sites ( 15, 16, 20 ). However, apart from its hemodynamic effects, the directeffects of ANG II on kidney cells are being increasingly recognized( 2, 6, 9, 25, 27 ). It has been demonstrated that inaddition to circulating ANG II, tissue (intrarenal) generation of ANGII is also important for its net effect ( 35 ).
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Tubulointerstitial lesions have been demonstrated to correlate with theprogression of renal failure ( 5, 11, 18, 21, 26 ), thussuggesting 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 andprogression of renal lesions in both human renal diseases andexperimental animal models of human immunodeficiency virus-associatednephropathy, renal ablation, and ureteric obstruction ( 16, 33, 36 ). Interestingly, in these conditions, elevated blood ANG IIlevels have been reported. Moreover, modalities, which inhibit theproduction of ANG II, have been demonstrated to slow the progression ofrenal lesions ( 15, 16, 20 ).

The effect of ANG II on the growth of proximal tubular cells has beenevaluated 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. Theseinvestigators elegantly delineated the ANG II-induced downstream signaltransduction pathway ( 12 ). Weerackody et al.( 34 ) showed that ANG II-induced tubular cell hypertrophy(protein synthesis) was inhibited by pertussis toxin and losartan butnot by PD-123319. These findings suggest that ANG II type 1 (AT 1 ) rather than ANG II type 2 (AT 2 ) receptorsmay be contributing to ANG II-induced tubular cell hypertrophy.Recently, Cao et al. ( 7 ) demonstrated that infusion of ANGII in rats enhanced the number of both PCNA- andtransferase-dUTP-nick-end labeling (TUNEL)-positive cells in proximaltubules. These investigators suggest that ANG II triggers bothproliferation and apoptosis of proximal tubular cells. Similar findings were reported by Aizawa et al. ( 2 ) in ratproximal tubular cells. Interestingly, these investigators havedemonstrated that agents which induce tubular cell heme oxygenase(HO)-1 expression also provide protection from the growth modulatoryeffects of ANG II; conversely, agents that inhibit tubular cell HO-1expression promote the growth modulatory effect of ANG II.
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Increased cellular expression of HO-1 has been considered to be amarker of oxidative stress ( 24 ). In various animalexperimental models of oxidative injury, induction of HO-1 has beendemonstrated to provide protection from ongoing injury ( 8, 10, 17, 23, 31 ). In addition to the model of ANG II-induced tubularcell injury, enhanced tubular cell HO-1 expression has been shown to confer protection against cisplatin-induced tubular cell injury ( 16, 28 ).
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In the present study, we evaluated the effect of ANG II on theapoptosis of cultured rat proximal tubular cells. We alsostudied the molecular mechanisms involved and the possible relationship between HO-1 expression and ANG II-induced tubular cell apoptosis.

MATERIALS AND METHODS7 E  M$ \; z; h! X: `
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Renal Proximal Tubular Epithelial Cell Cultures, Y* W6 p( y6 _+ s; v

Rat proximal renal tubular epithelial cells (NRK-52E) wereobtained from American Type Culture Collection, Rockville, MD. Cellswere grown in DMEM (GIBCO, Grand Island, NY) containing 2%penicillin-streptomycin, 1% HEPES, 1.5 g NaHCO 3, 2 mM L -glutamine, 1 mM sodium pyruvate, and 10% FCS. In theexperimental protocols, cells were incubated in media containing 1% FCS." L" d7 H/ g: p" r- C
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Apoptotic Studies- h" \& t! p+ `5 E
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Rat and human renal proximal tubular epithelial cells (RPTECs;HK-2) were treated under control and experimental conditions for theindicated times. At the end of incubation period, cells were stainedwith H-33342 and propidium iodide and evaluated for apoptosisas described previously ( 29, 30 ). In these studies, observers were blinded to experimental conditions.' M7 |) s& o' [

Detection of tubular cell apoptosis by gelelectrophoresis. This is a simple method that is specific for isolation and confirmationof DNA fragments from apoptotic cells ( 14 ). Because this method only picks up DNA fragments, one will not visualize anyloading of samples that do not contain DNA fragments. RPTECs weretreated under control and experimental conditions as indicated, and DNAwas extracted and electrophoresed as described previously ( 29, 30 ).7 m4 G8 F1 m. Z" U: I
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Superoxide Assay
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Equal numbers of RPTECs were plated in 100-mm petri dishes andgrown to subconfluence. The cells were washed twice with normal salineand incubated in serum- and phenol red-free media containing eitherbuffer, 10 8 M ANG II, or 1 µg/ml anti-TGF- antibodyor ANG II anti-TGF- antibody at 37°C for a 2-h period.Supernatants were collected into precooled microcentrifuge test tubesat 0, 30, 45, 60, and 120 min. A superoxide assay was subsequentlycarried out. In brief, 50 µl of each supenatant were pipetted into a96-well plate, kept on ice, and mixed with 100 µl of ferricytochrome c (160 µM final concentration, ICN Biomedical, Costa Mesa,CA) diluted with HBSS (GIBCO). Incubation was carried out at 37°C for45, 90, and 150 min, and optical density was read at 550 nm. Resultsare expressed in arbitrary units, and experiments were repeated fourtimes, each in triplicate.: A5 P+ }8 t2 l* K1 q8 z

H 2 O 2 Assay
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Equal numbers of RPTECs were plated in 100-mm petri dishes andgrown to subconfluence. The cells were washed twice with normal salineand incubated in serum- and phenol red-free media containing eitherbuffer, 10 8 M ANG II, or 1 µg/ml anti-TGF- antibodyor ANG II anti-TGF- antibody at 37°C for a 2-h period.Supernatants were collected into precooled microcentrifuge test tubesat 0, 30, 45, 60, and 120 min. An H 2 O 2 assaywas carried out. In brief, 50 µl of each supernatant were pipettedinto a 96-well plate, kept on ice, and mixed with 100 µl of phenolred washing solution containing 140 mM NaCl, 10 mM potassium phosphatebuffer (pH 7.0), 5.5 mM dextrose, 0.1 g/l phenol red, 8.5 units/mlhorseradish peroxidase, and 100 ng/ml phorbol myrisate acetate.Incubation was carried out at 37°C for 45, 90, and 150 min andterminated by the addition of 10 µl of 1 M NaOH. Calorimetric readingwas done in an ELISA microplate reader at 620 nm. Values are plottedagainst a standard curve generated from known concentrations ofH 2 O 2. Results are expressed in arbitrary units,and experiments were repeated four times, each in triplicate.

Protein Extraction and Western Blot Analysis

RPTECs were treated under control and experimental conditions asindicated. At the end of the incubation period, the cells were washedthree 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 a21-gauge needle, into a microcentrifuge tube. The cell lysates werecentrifuged at 15,000 g for 30 min at 4°C. The supernatantwas analyzed for total protein content. Twenty micrograms of proteinwere heated at 100°C for 10 min, loaded, and separated on a 12% PAGEgel under nonreducing conditions. The proteins were electrotransferredto a nitrocellulose membrane in transfer buffer containing 48 mMTris · HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C overnight. Nonspecific binding to the membrane wasblocked for 1 h at room temperature with blocking buffer (0.5% BSA in PBS with 0.1% Tween 20). The membrane was then incubated for16 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 CruzBiotechnology, 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 incubationfor 1 h at room temperature with the appropriate secondaryantibody in blocking buffer. Signals were visualized by an enhancedchemiluminescence detection kit (Pierce) after exposure to X-ray film(Eastman Kodak, Rochester, NY) ( 29, 30 ). To determineloading, blots were stripped and reprobed for -actin. Quantitativedensitometry was performed on the identified bands by using acomputer-based measurement system (IS-1000 Digital Imaging System,Alpha Innotech, San Leandro, CA).

Statistical Analysis

Statistical analysis was performed using GraphPad Instatsoftware. A Newman-Keuls multiple comparison test was used, and P values were calculated.

RESULTS* p/ p% h# J* h- D# K$ H
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Studies Pertaining to Apoptosis& ]! j- }0 H* F" o
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To study the dose-response effect of ANG II on RPTECapoptosis, equal numbers of cells (10,000 cells/well, 24-wellplates) were incubated in media containing either buffer (control) or 10 12 to 10 6 M ANG II for 16 h at37°C. Subsequently, cells were evaluated for apoptosis andnecrosis. Four series of experiments were carried out. ANG II inducedapoptosis in RPTECs in a dose- and time-dependent manner (Fig. 1, A and B ). ANG IIinduced a mild degree of necrosis in tubular cells (control, 1.2 ± 0.8%; 10 8 M ANG II, 3.5 ± 1.5%;10 6 M ANG II, 4.5 ± 1.8% necrosed cells/field).

Fig. 1. A : dose-response effect of ANG II on ratproximal tubular epithelial cell (RPTEC) apoptosis. Equal nos.of cells were incubated in media containing either buffer (control) or10 12 and 10 6 M ANG II for 16 h. At theend of the incubation period, cells were stained for apoptosisand necrosis. Values are means ± SE of 4 sets of experiments,each carried out in triplicate. * P 12 M ANG II. ** P 10 M ANG II. *** P 8 M ANG II. B : timecourse effect of ANG II on RPTEC apoptosis. Equal nos. of cellswere incubated in media containing either buffer (control) or10 6 M ANG II for variable time periods (6, 12, 18, and24 h). At the end of the incubation period, cells were stainedwith H-33342 and propidium iodide. The percentage of live, apoptosed,and necrosed cells was recorded. Values are means ± SE of 4 series of experiments, each carried out in triplicate.* P P P P C : representative gel showing the effect of ANG II ontubular cell apoptosis. Equal nos. of RPTECs were incubated inmedia containing either buffer (control) or 10 8 and10 6 M ANG II for 16 h. At the end of the incubationperiod, cells were lysed, and DNA was extracted and run on gelelectrophoresis. Lane 1, molecular marker; lane2, control; lanes 3 and 4, 10 8 and 10 6 M ANG II-treated cells, respectively.( \( R9 g, {$ M8 P; z% }
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To confirm the occurrence of apoptosis, equal numbers of RPTECswere incubated in 100-mm petri dishes with media containing eitherbuffer (control) or 10 8 and 10 6 M ANG IIfor 16 h. Subsequently, DNA was isolated and electrophoresed. Ingel electrophoresis, DNA isolated from ANG II-treated RPTECs showed aclassic ladder pattern, thus confirming the occurrence ofapoptosis (Fig. 1 C ).

To evaluate whether this effect of ANG II was species specific, weevaluated the effect of ANG II on human proximal tubular cells (HK-2).Equal numbers of cells were incubated in media containing either bufferor 10 8 and 10 6 M ANG II for 16 h.Subsequently, cells were stained for apoptosis. ANG II promotedHK-2 cell apoptosis (control, 1.5 ± 0.5%;10 8 M ANG II, 15.5 ± 1.2%; 10 6 M ANGII, 25.5 ± 2.0%; P the apoptotic effect of ANG II is not species specific.3 w% v# r0 U- `8 `% Z! d6 L: ]+ P
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Studies Pertaining to AT 1 and AT 2 Receptors( p! s  v! i8 _/ |9 \% p

To evaluate the role of AT 1 and AT 2 receptors in the induction of apoptosis, equal numbers ofRPTECs were incubated in media containing vehicle (control), theAT 1 inhibitor losartan (10 6 M, Sigma, St.Louis, MO), or the AT 2 inhibitor PD-123319(10 6 M, Sigma) with or without 10 8 or10 7 M ANG II for 16 h. Subsequently, cells werestained for apoptosis. Four series of experiments were carriedout. As shown in Fig. 2 A, bothlosartan and PD-123319 inhibited ANG II-induced RPTEC apoptosis. This effect of ANG II receptor blockers was alsoconfirmed by DNA gel electrophoresis (Fig. 2 B ).0 |* [1 ~8 ~: r2 y$ e, G
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Fig. 2. A : effect of ANG II type 1 (AT 1 )and type 2 (AT 2 )-receptor antagonists on ANG II-inducedtubular cell apoptosis. Equal nos. of RPTECs were incubated inmedia containing vehicle (control), 10 6 M losartan (LOS),or 10 6 M PD-123319 (PD) with or without 10 8 or 10 7 M ANG II for 16 h. At the end of theincubation period, cells were stained for apoptosis. Values aremeans ± SE of 4 sets of experiments, each carried out intriplicate. * P P B : representativegel showing the effect of ANG II on tubular cell DNA fragmentation.Equal nos. of RPTECs were incubated in media containing either buffer(control) or 10 8 or 10 6 M ANG II with orwithout losartan or PD-123319 (cells were pretreated with eitherlosartan or PD-123319 for 60 min before addition of ANG II) for 16 h. At the end of the incubation period, cells were lysed, and DNA wasextracted and run on gel electrophoresis. Lanes 1 and 7, molecular markers; lane 2, control; lanes 3 and 4, 10 8 and10 6 M ANG II-treated cells, respectively; lanes5 and 6, 10 8 M ANG II losartan and10 8 M ANG II PD-123319, respectively. C :representative gel showing the effect of anti-transforming growthfactor (TGF)- antibody on ANG II-induced tubular cell DNAfragmentation. Equal nos. of RPTECs were incubated in media containingeither buffer (control), 10 8 M ANG II, or anti-TGF- antibody ANG II for 16 h. Subsequently, DNA was extracted and runon gel electrophoresis. Lane 1, molecular marker; lane2, control; lanes 3 and 4, 10 8 M ANG II- and anti-TGF- antibody ANG II-treated cells, respectively. D : effect of anti-TGF- antibody (Ab) on ANG II-inducedapoptosis. Equal nos. of cells were incubated in mediacontaining vehicle (control; CONT) or 1 µg/ml anti-TGF- antibody(Pharmingen) with or without 10 8 or 10 6 MANG II for 16 h. At the end of the incubation period, cells werestained with H-33342 and propidium iodide. The percentage of live,apoptosed, and necrosed cells was recorded. Values are means ± SEof 4 series of experiments, each carried out in triplicate.* P P 8 M ANG IIwithout anti-TGF- antibody. *** P 6 M ANG II without anti-TGF- antibody.+ ?5 u$ \9 y8 p: P  u0 ?$ ?
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Studies Pertaining to the Role of TGF-  x# N1 S1 s2 x. ]: l( Y

To determine whether the effect of ANG II is mediated throughTGF-, equal numbers of cells were incubated in media containing either vehicle (control) or 1 µg/ml anti-TGF- antibody (Santa CruzBiotechnology) with or without 10 8 M ANG II for 16 h. Subsequently, cells were prepared for DNA fragmentation assay. Inparallel experiments, equal numbers of cells were treated with eithervehicle (control) or 1 µg/ml anti-TGF- antibody with or without10 8 or 10 6 M ANG II for 16 h.Subsequently, cells were stained for apoptosis. Four sets ofexperiments were carried out. As shown in Fig. 2, C and D, anti-TGF- antibody inhibited ANG II-induced RPTEC apoptosis.
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To evaluate the effect of TGF- 1 on RPTEC apoptosis,equal numbers of RPTECs were incubated in media containing eitherbuffer (control) or variable concentrations of TGF- 1 (50-2,000pg/ml; Collaborative Biomedical Products, Bedford, MA) for 16 h.Subsequently, cells were stained for apoptosis. Eight sets ofexperiments were carried out. TGF- 1 triggered RPTECapoptosis in a dose-dependent manner (Fig. 3 A ).4 c. F: S) Q- D0 ]- l0 ^. G4 h; s

Fig. 3. A : effect of TGF- in ANG II-induced RPTECapoptosis. Equal nos. of RPTECs were incubated in mediacontaining either buffer (control) or variable concentrations ofTGF- 1 (50, 500, 1,000, 2,000 pg/ml) for 16 h. Subsequently,cells were stained with H-33342 and propidium iodide. The percentage oflive, apoptosed, and necrosed cells was recorded. Values are means ± SE of 8 series of experiments, each carried out in triplicate.* P 1.** P 1. *** P 1. B : effect of anti-TGF- antibody on TGF- -induced RPTEC apoptosis. Equal nos. ofRPTECs were incubated in media containing either buffer, TGF- 1 (TGF;10 ng/ml), anti-TGF- antibody (A-TGF-ab; 1 µg/ml), or anti-TGF- antibody TGF- for 16 h. Subsequently, cells were assayed forapoptosis. * P  A-TGF-ab. C : effect of anti-Fasligand (FasL) antibody in ANG II-induced tubular cellapoptosis. Equal nos. of RPTECs were incubated in mediacontaining buffer (control) or 10 8 or 10 6 MANG II with or without anti FasL antibody (anti-FasL-ab; 1 µg/ml) for16 h. At the end of the incubation period, cells were stained withH-33342 and propidium iodide. The percentage of live, apoptosed, andnecrosed cells was recorded. Values are means ± SE of 4 sets ofexperiments, each carried out in triplicate. * P D :effect of anti-FasL antibody in TGF- -induced tubular cellapoptosis. Equal nos. of RPTECs were incubated in mediacontaining buffer (control) or TGF- 1 (TGF-b; 10 ng/ml) with orwithout 1 µg/ml A-FasL-ab for 16 h. At the end of the incubationperiod, cells were stained with H-33342 and propidium iodide. Thepercentage of live, apoptosed, and necrosed cells was recorded. Valuesare means ± SE of 3 series of experiments, each carried out intriplicate. * P% K. L% r- P0 t# G& Z$ V
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To establish a causal relationship between TGF- and theoccurrence of RPTEC apoptosis, we studied the effect ofanti-TGF- antibody on TGF- -induced tubular cellapoptosis. Equal numbers of RPTECs were incubated in mediacontaining either buffer, 10 ng/ml TGF- 1, 1 µg/mlanti-TGF- 1 antibody, or anti-TGF- antibody TGF- 1 for16 h. Subsequently, cells were assayed for apoptosis.Anti-TGF- antibody attenuated the proapoptotic effect of TGF- (Fig. 3 B ).8 u# y# d' h2 p4 R8 l4 ~0 y
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Studies Pertaining to the Role of FasL

To evaluate the contribution of the FasL pathway in ANG II-inducedtubular cell apoptosis, equal numbers of RPTECs were incubated in media containing buffer (control) or 10 8 or10 6 M ANG II with or without anti-FasL antibody (1 µg/ml, mouse monoclonal antibody, Pharmingen) for 16 h.Subsequently, cells were stained for apoptosis. As shown inFig. 3 C, anti-FasL antibody partially attenuated the effectof ANG II on tubular cell apoptosis.
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To evaluate the contribution of the FasL pathway in TGF- -inducedtubular 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. 3 D, anti-FasL antibody attenuated the effect of TGF- ontubular cell apoptosis.

Studies Pertaining to the Role of Oxidative Stress

To determine the role of oxidative stress in ANG II-induced RPTECapoptosis, we studied the effect of antioxidants such as diphenyleneiodonium chloride (DPI; Sigma), ascorbic acid (AA), and N -acetyl cysteine (NAC). Equal numbers of RPTECs wereincubated in media containing buffer (control), 10 µM DPI, 100 µMAA, or 50 µM NAC with or without 10 8 M ANG II for24 h. At the end of the incubation period, cells were assayed forapoptosis. DPI, NAC, and AA inhibited ( P the effect of ANG II (Fig. 4 A ). These findings indicatethat oxidative stress plays a causal role in ANG II-induced RPTECapoptosis.  Q6 E2 a$ U& R

Fig. 4. A : effect of antioxidants on ANG II-inducedRPTEC apoptosis. Equal nos. of RPTECs were incubated in mediacontaining buffer (control), 10 µM diphenyleneiodonium chloride(DPI), 100 µM ascorbic acid (AA), or 50 µM N -acetylcisteine (NAC) with or without 10 8 M ANG II for 24 h. At the end of the incubation period, cells were assayed forapoptosis. Values are means ± SE of 3 sets ofexperiments, each carried out in triplicate. * P B : effect of ANG II and anti-TGF- antibody onRPTEC superoxide generation. Equal nos. of cells were incubated inmedia containing either buffer or ANG II for 120 min. Supernatants werecollected at 0, 30, 45, 60, 90, and 120 min and assayed for superoxide.The generation of superoxide is shown at 45 min. Values are means ± SE of 3 sets of experiments, each carried out in triplicate.* P antibody ANG II. C : effect of ANG II on RPTEC hydrogenperoxide generation. Equal nos. of cells were incubated in mediacontaining either buffer or ANG II for 120 min. Supernatants werecollected at 0, 30, 45, 60, 90, and 120 min and assayed forH 2 O 2. The generation ofH 2 O 2 is shown at 45 min. Values are means ± SE of 3 sets of experiments, each carried out in triplicate.* P antibody ANG II.
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To evaluate the role of ANG II and TGF- on the generation ofreactive oxygen species (ROS), such as superoxide and hydrogen peroxide, we measured the production of superoxide andH 2 O 2 by RPTECs under control and ANGII-stimulated states in the presence or absence of anti-TGF- antibody. Equal numbers of cells were incubated in media containingeither buffer or ANG II for 120 min. Supernatants were collected at 0, 30, 45, 60, 90, and 120 min and assayed for superoxide and hydrogenperoxide. As shown in Fig. 4, B and C, ANG IIpromoted RPTEC generation of both superoxide and hydrogen peroxide at45 min. The generation of ROS plateaued at 120 min (data on 60, 90, and120 min are not shown). However, anti-TGF- antibody inhibited theeffect of ANG II.4 C* I5 ~) f; ~( x0 y# }; x- G
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To determine the effect of HO-1 preinduction on ANG II-inducedtubular 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 mediacontaining either vehicle (control) or 10 8 M ANG II for16 h and then stained for apoptosis. Pretreatment withhemin as well as curcumin rendered partial protection against theproapoptotic effect of ANG II on tubular cells (Fig. 5 A ).

Fig. 5. A : effect of heme oxygenase (HO)-1inducers on ANG II-induced tubular cell apoptosis. Equal nos.of RPTECs were pretreated in media containing buffer (control), 5 µMhemin, or 15 µM curcumin for 4 h. Subsequently, cells wereincubated in media containing vehicle (control) or 10 8 MANG II for 16 h, followed by staining of cells forapoptosis. Values are means ± SE of 4 series ofexperiments. * P B : effect of HO-1 inhibitionon ANG II-induced tubular cell apoptosis. Equal nos. of RPTECs(10,000 cells/well) were pretreated in media containing either buffer(control) or zinc protoporphyrin (ZnP; 100 µM) for 4 h, followedby incubation in media containing vehicle (control) or10 8 M ANG II for 16 h, followed by staining of cellsfor apoptosis. Values are means ± SE of 4 sets ofexperiments. * P P C : role ofpreinduction vs. simultaneous induction of HO-1 on ANG II-inducedtubular cell apoptosis. Equal nos. of RPTECs were preincubatedin media containing either buffer or 5 µM hemin for 4 h,followed by incubation in either buffer or 10 8 M ANG IIfor 16 h. In parallel studies, equal nos. of RPTECs were incubatedwith either buffer or 5 µM hemin 10 8 M ANG II for16 h. Subsequently, cells were assayed for apoptosis.* P D : role of HO-1in curcumin-induced modulation of ANG II effect. Equal nos. of RPTECswere incubated in media containing either buffer (C), 10 8 M ANG II, 15 µM curcumin (Cur), 50 µM ZnP, Cur ZnP, or Cur ANG II,Cur ZnP, or Cur ZnP ANG II for 16 h. Subsequently, cells wereassayed for apoptosis. Values are means ± SE of 4 sets ofexperiments, each carried out in triplicate. * P P
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To determine the effect of HO-1 inhibition, equal numbers of RPTECswere pretreated in media containing either buffer (control) or zincprotoporphyrin (ZnP; 100 µM, Sigma) for 4 h, followed byincubation in media containing vehicle (control) or 10 8 MANG II for 16 h. Subsequently, cells were stained forapoptosis. As shown in Fig. 5 B, ZnP accentuated theproapoptotic effect of ANG II on RPTECs.+ m& g) r" k# z- p+ `/ f/ |* k

To determine the importance of the timing of HO-1 induction, weevaluated the effect of ANG II on tubular cells that were eitherpretreated 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 10 8 M ANG II for 16 h. In parallelstudies, RPTECs were incubated with either buffer or 5 µMhemin 10 8 M ANG II for 16 h. Subsequently, cellswere assayed for apoptosis. As shown in Fig. 5 C,pretreatment of ANG II-treated cells with hemin (HO-1 preinduction)attenuated the apoptotic effect of ANG II. However, simultaneoustreatment of tubular cells with ANG II and hemin did not prevent theapoptotic effect of ANG II. These studies suggest that onlypreinduction of HO-1 is beneficial to modulate the apoptotic effectof ANG II.

Because curcumin has been demonstrated to induce multiple effects,including the inhibition of JNK, binding of activator protein-1, andHO-1 expression, it may be important to evaluate the mechanism involvedin curcumin-induced modulation of the ANG II effect. Equal numbers ofRPTECs were incubated in media containing either buffer,10 8 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. ZnPattenuated the antiapoptotic effect of curcumin on ANG II-treatedcells (Fig. 5 D ). These findings suggest that thecurcumin-mediated effect on ANG II-treated cells may have been inducedthrough HO-1 expression.5 ~' k" Q/ x* f! V% |6 T

Studies Pertaining to p38 MAPK Activation
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To evaluate the role of p38 MAPK in ANG II-induced RPTECapoptosis, we studied the effect of a selective p38 MAPKinhibitor, SB-202190 (Calbiochem, La Jolla, CA), on ANG II-inducedRPTEC apoptosis. Equal numbers of cells were incubated in mediacontaining either buffer (control) or 5 µM SB-202190 for 1 h. Atthe end of the incubation period, cells were reincubated in mediacontaining vehicle (control) or 10 8 or 10 6 M ANG II for 16 h. Subsequently, cells were stained forapoptosis. As shown in Fig. 6 A, SB-202190 partiallyinhibited the effect of ANG II on RPTEC apoptosis.# m) F' y" c# e8 A/ [3 n/ E$ _
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Fig. 6. A : effect of SB-202190 on ANG II-induced RPTECapoptosis. Equal nos. of cells were incubated in mediacontaining either buffer (control) or 5 µM SB-202190 for 1 h.Subsequently, cells were reincubated in media containing vehicle(control) or 10 8 or 10 6 M ANG II for16 h, followed by staining of cells for apoptosis. Valuesare means ± SE of 4 series of experiments, each carried out intriplicate. * P P B : effect of caspase-3inhibitor (C3i) on ANG II-induced tubular cell apoptosis. Equalnos. of RPTECs were incubated in media containing either buffer(control) or 10 8 and 10 6 M ANG II with orwithout 5 µM C3i for 16 h, followed by staining of cells forapoptosis. Values are means ± SE of 4 sets ofexperiments. * P P 6 M ANG II.
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Studies Pertaining to Cell Death Pathways" K4 z6 z! f3 r0 H- _
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To determine the role of caspase-3 in ANG II-induced tubular cellapoptosis, equal numbers of cells were incubated in media containing either buffer (control), or 10 8 and10 6 M ANG II with or without caspase-3 inhibitor (5 µM, no. 218750, Calbiochem) for 16 h. Subsequently, cellswere stained for apoptosis. Four series of experiments werecarried out. Caspase-3 inhibitor attenuated the proapoptotic effectof ANG II (Fig. 6 B ).
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To determine the role of cell death proteins (Bax, Fas, FasL) andcell survival protein (Bcl-2) in ANG II-induced RPTECapoptosis, equal numbers of tubular cells were incubated inmedia containing either buffer (control), or 10 8 and10 6 M ANG II for 16 h. Subsequently, protein wasextracted, and Western blots were prepared and probed for Bax, Bcl-2,Fas, and FasL. Tubular cells showed an increased ( P in response to ANG II treatment compared withcontrol (Fig. 7 B, depicted in the form of Fas/actinratios). A representative gel is shown inFig. 7 A. Similarly, ANG II-treated cells showed increased expression of FasL compared with control (Fig. 7 D, depictedin the form of FasL/actin ratios). A representative gel showing the effect of ANG II on FasL expression is shown in Fig. 7 C.1 s$ Y+ f/ V8 Q" Z

Fig. 7. Effect of ANG II on tubular cell Fas and FasL expression. Equalnos. of RPTECs were incubated in media containing either buffer(control) or 10 8 and 10 6 M ANG II for16 h. Subsequently, cells were harvested, protein was extracted,and blots were probed for Fas and FasL. A : representativegel showing the effect of 10 8 and 10 6 M ANGII on RPTEC expression of Fas. B : cumulative data from3 sets of experiments showing the effect of ANG II on tubular cell Fasexpression. * P P 8 M ANG II. C : representative gel showing the effect of10 8 and 10 6 M ANG II on RPTEC expression ofFasL. D : cumulative data from 3 series of experimentsshowing the effect of ANG II on tubular cell FasL expression.* P P 8 M ANG II.! ^5 F- y+ g5 g0 Z
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ANG II enhanced RPTEC expression of Bax (Fig. 8 B, depictedin the form of Bax/actin ratios). Arepresentative gel showing the effect of ANG II on RPTEC expression isshown in Fig. 8 A. On the other hand, ANG II decreased RPTECexpression of Bcl-2. (Fig. 8 D, shown in the form ofBcl-2/actin ratios). A representative gel showing the effect of ANG IIon RPTEC expression of Bcl-2 is shown in Fig. 8 C.1 o9 C, U8 C4 J" f' d4 w) F
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Fig. 8. Effect of ANG II on tubular cell Bax and Bcl-2 expression. Equalnos. of RPTECs were incubated in media containing either buffer(control) or 10 8 and 10 6 M ANG II for16 h. Subsequently, cells were harvested, protein was extracted,and blots were probed for Bax and Bcl-2. A : representativegel showing the effect of ANG II on tubular cell expression of Bax. B : cumulative data from 3 sets of experiments showing theeffect of ANG II on tubular cell Bax expression. * P P 8 M ANG II. C : representativegel showing the effect of ANG II on RPTEC expression of Bcl-2. D : cumulative data from 3 series of experiments showing theeffect of ANG II on tubular cell Bcl-2 expression. * P P 8 M ANG II.- J( @; y0 q! N9 t$ }
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Studies Pertaining to Tubular Cell HO-1 Expression

To study the effect of ANG II on RPTEC HO-1 induction, equalnumbers of cells were incubated in six-well plates with media containing either buffer (control) or 10 8 and10 6 M ANG II for 16 h. Subsequently, protein wasextracted, and Western blots were generated and probed for HO-1. ANG IIpromoted RPTEC expression of HO-1 (Fig. 9 B ). A representative gelshowing the effect of ANG II on tubular cell HO-1 expression is shownin Fig. 9 A.6 w2 C: c+ z; c

Fig. 9. A : effect of ANG II on RPTEC HO-1 induction. Equal nos.of cells were incubated in media containing either buffer (control) or10 8 and 10 6 M ANG II for 16 h.Subsequently, Western blots were generated and probed for HO-1. B : cumulative data from 3 series of experiments showing theeffect of ANG II on tubular cell HO-1 expression. * P P 8 M ANG II. C : effect ofA-TGF-ab on ANG II-induced HO-1 expression. Equal nos. of cells weretreated with either buffer (control) or 10 8 and10 6 M ANG II with or without 10 ng/ml A-TGF-ab for16 h. Subsequently, Western blots were generated and probed forHO-1. D : cumulative data from 3 series of experimentsshowing the effect of A-TGF-ab on ANG II-induced tubular cell HO-1expression. * P 8 M ANG II. ** P 6 M ANG II. E : effect of curcumin and hemin on HO-1 exspression. Equalnos. of RPTECs were incubated in media containing either buffer(control), 5 µM hemin, 15 µM curcumin, or hemin curcumin for16 h. Subsequently, cells were prepared for Western blottingand probed for HO-1. F : cumulative data from 3 sets ofexperiments showing the effect of hemin and curcumin on tubular cellHO-1 expression. * P P P- c+ y* `+ M, Z% @# ~# l* F( |

To determine the role of TGF- in ANG II-induced HO-1 expression,equal numbers of cells were treated with either buffer (control) or10 8 and 10 6 M ANG II with or withoutanti-TGF- antibody for 16 h. Subsequently, protein wasextracted, and Western blots were generated and probed for HO-1. Asshown in Fig. 9 D, the effect of ANG II on RPTEC was inhibited by anti-TGF- antibody. A representative gel showing theeffect of anti-TGF- antibody on ANG II-induced HO-1 expression isshown in Fig. 9 C. 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, equalnumbers of RPTECs were incubated in media containing either buffer(control), 5 µM hemin, 15 µM curcumin, or hemin curcumin for16 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 cellHO-1 expression (Fig. 9, E and F ).$ e+ F: V3 r) I* Y; u% v! j( p! C
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DISCUSSION

The present study demonstrates that ANG II promotes tubular cellapoptosis. Because anti-TGF- antibody attenuated this effect and TGF- promoted tubular cell apoptosis, it appears thatANG II-induced tubular cell apoptosis may be mediated throughTGF-. Both AT 1 and AT 2 receptor antagonistspartially blocked ANG II-induced tubular cell apoptosis. ANG IIalso promoted the expression of HO-1 and tubular cell expression ofFasL, whereas anti-FasL antibody inhibited the effect of ANG II as wellas TGF- on tubular cell apoptosis. ANG II not only promotedtubular cell expression of Bax but also inhibited the expression ofBcl-2. Pretreatment of tubular cells with inducers of HO-1 (hemin andcurcumin) attenuated the response of ANG II, thus suggesting thatpreinduction of HO-1 expression perhaps provides protection against ANGII-induced tubular cell injury. On the other hand, pretreatment ofRPTECs with ZnP, an inhibitor of HO-1 activity, exacerbated theproapoptotic effect of ANG II. Only pretreatment and not thesimultaneous treatment of tubular cells with hemin provided protectionagainst the proapoptotic effect of ANG II.* ^& D1 l4 l- T  f# ^/ D! \
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Fas and the FasL pathway have been reported to mediateapoptosis in the kidney ( 19 ). Theseproapoptotic factors have been found to be attenuated by ANG IIinhibition ( 32 ). In addition, captopril, anangiotensin-converting enzyme inhibitor, has been shown to inhibitFas-induced apoptosis and FasL expression in human activatedperipheral T cells ( 22 ). Our finding of enhanced expression of tubular Fas and FasL is consistent with theseobservations. Moreover, inhibition of ANG II-induced tubular cellapoptosis by anti-FasL antibody established a causalrelationship. We also observed the alteration in the expression of Baxand Bcl-2 in ANG II-treated cells, which may have tilted the balancetoward apoptosis. In the present study, there was apparentvariability in -actin expression in control and ANG II-treatedtubular cells. This may have partly contributed to the alteredBax/actin ratio. Aizawa et al. ( 2 ) found increasedexpression of Bax in the kidneys of ANG II-infused rats without anydecrease in Bcl-2. Because these investigators studied Bcl-2 expressionin whole kidney lysates, their finding may not necessarily representBcl-2 expression by proximal tubular cells.2 L' i( {; G  E' A

AT 1 receptors are responsible for most of the reportedactions of ANG II ( 25 ). AT 2 receptors arepredominantly expressed during fetal development and are considered tobe critical for ontogenesis. Expression of AT 2 receptorsdeclines rapidly after cessation of developmental apoptosis.Conversely, Cao et al. ( 7 ) demonstrated that proximaltubular cells in adult rats express AT 2 receptors. Theseinvestigators demonstrated that infusion of ANG II for 14 days in8-wk-old rats induced the proliferation and apoptosis ofproximal tubular cells. The administration of the AT 2 antagonist PD-123319 or the AT 1 antagonist valsartan was associated with attenuation of the increase in both PCNA- and TUNEL-positive cells after ANG II infusion. These findings suggest thatboth AT 1 and AT 2 receptors are involved in bothproliferation and apoptotic processes in proximal tubular cells. Inthe present study, both the AT 1 antagonist (losartan) andAT 2 antagonist (PD-123319) inhibited ANG II-inducedapoptosis of tubular cells. These findings suggest thatdownstream signaling mediated by both AT 1 andAT 2 receptors is needed for the activation of ANGII-induced tubular cell apoptosis.* h, W8 p2 K2 u* C. n) Q9 U

Haugen et al. ( 13 ) studied the occurrence of oxidativestress in two models of hypertension, i.e., ANG II- and DOCAsalt-treated rats. However, only ANG II-treated rats showed evidence ofrenal oxidative stress in the form of lipid peroxidation, proteincarbonyl content, and induction of HO-1 ( 13 ). Theseinvestigators further localized the induction of proximal tubular cellHO-1 in ANG II-treated animals ( 13 ). Because DOCAsalt-treated rats did not show renal oxidative stress despite havingidentical levels of blood pressure, these investigators suggested thatANG II induces oxidative stress independently of its hemodynamiceffects. Similarly, Aizawa et al. ( 3 ), in two models ofhypertension, i.e., ANG II- and norepinephrine-infused rats, showedthat only ANG II-infused rats developed a decrease in glomerularfiltration rate and proteinuria. Interestingly, tubular cell HO-1 wasupregulated only in ANG II-treated rats (not in norepinephrine-treatedrats). Pretreatment of rats with HO-1 expression modulators, i.e.,hemin, an inducer, and ZnP, an inhibitor, modulated not only tubularcell HO-1 expression but also proteinuria (hemin decreased and ZnPincreased ANG II-induced proteinuria) ( 3 ). Theseinvestigators also studied the occurrence of apoptosis andproliferation of tubular cells in these models ( 2 ). OnlyANG II-infused rats showed increased numbers of both PCNA- andTUNEL-positive cells. Pretreatment of these rats with HO-1 inducers andinhibitors modulated the severity of ANG II-induced tubular cellapoptosis and proliferation. Moreover, only ANG II-treated ratsshowed tubular cell upregulation of HO-1 and Bax expression. The effectof ANG II partially persisted despite normalization of blood pressurewith hydralazine, again suggesting a nonhemodynamic effect of ANG II.The in vitro observations in the present study are consistent with thein vivo findings of Aizawa et al. ( 2 ).% l. c2 ]- u) Q5 f) {

In the present study, ANG II promoted HO-1 expression, thus indicatingthe occurrence of oxidative stress. Because anitoxidants such as DPI,NAC, and AA inhibited the proapoptotic effect of ANG II, it appearsthat ANG II-induced tubular cell apoptosis is mediated throughoxidative stress. Moreover, enhanced production of superoxide andH 2 O 2 by tubular cells in response to ANG II further delineates the molecular mechanism involed. Both superoxide andH 2 O 2 have been demonstrated to promote HO-1expression in a variety of cells ( 1 ). Thus it appears thatANG II-induced HO-1 expression may be mediated through the generationof superoxide and H 2 O 2 by tubular cells.
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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 acuteoxidative stress, of which it is a marker. On the other hand, ifpreinduced, it may act as part of the armamentarium against incominginjury. Preinduction of HO-1 may generate enough antioxidants toneutralize the effect of oxidative stress. Therefore, preinduction ofHO-1 has been used as a tool in preventing damage in various models ofoxidative injury ( 8, 10, 17, 23, 31 ). Because ANG IIinduces oxidative stress, it is likely to promote HO-1 expression.Because ANG II could induce tubular cell injury despite ongoingexpression of HO-1, it appears that expression of HO-1 may have been afutile attempt to contain oxidative stress. This hypothesis is furthersupported by our data showing that the proapoptotic effect of ANGII was attenuated in tubular cells pretreated with hemin but not intubular cells that were treated simultaneously with hemin. On the otherhand, ZnP, an inhibitor of HO-1 activity, enhanced thepropapoptotic effect of ANG II. These findings suggest that ANGII-induced HO-1 expression may be providing at best limited protectionagainst ANG II-induced oxidative stress.% z) A0 ?  s, \3 l4 h

We conclude that ANG II induces proximal renal tubular cellapoptosis through the generation of TGF- and ROS, p38 MAPKphosphorylation, expression of Fas, FasL, and Bax, and activation ofcaspase 3. This effect of ANG II seems to be mediated by bothAT 1 and AT 2 receptors. Modulation of tubularcell HO-1 expression inversely affects ANG II-induced tubular cellapoptosis. These findings may provide a basis for a hypothesisthat nonhemodynamic effects of ANG II may be playing a role in thedevelopment and progression of tubular cell injury in conditionsassociated with elevated levels of ANG II.6 Z1 b, `: v2 J; ?: d# v  Q
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ACKNOWLEDGEMENTS
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This work was supported by National Institutes of Health GrantRO1-DA-12111. A portion of this work was presented at the 33rd annualmeeting of the American Society of Nephrology (October 2000, Toronto,Ontario, Canada).
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