Role of oxidative stress and heme oxygenase activity in morphine-induced glomeru - 《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 13:48 |只看该作者 |倒序浏览 |打印

作者：Jaimita Patel, Nagarathna Manjappa, Rajani Bhat, Pavni Mehrotra, Madhu Bhaskaran,  Pravin C. Singhal作者单位：Immunology and Inflammation Center for Excellence, North Shore-Long Island Jewish Research Institute, and Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, New York 11040
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      【摘要】
   Opiate addiction has been reported to contribute to the progression of renal injury. In addition, opiate addiction is a major risk factor for the development of human immunodeficiency virus-associated nephropathy. In the present study, we evaluated the effects of morphine, an active metabolite of heroin, on glomerular epithelial cell (GEC) growth and the involved molecular mechanism. At lower concentrations, morphine promoted GEC proliferation; however, at higher concentrations, morphine triggered apoptosis. Antioxidants inhibited morphine-induced proliferation as well as apoptosis. Similarly, free radical scavengers prevented morphine-induced GEC proliferation and apoptosis. Because proliferative and proapoptotic effects of morphine were inhibited by free radical scavengers as well as antioxidants, it appears that these effects of morphine are mediated through oxidative stress. Hemin, an inducer of heme oxygenase (HO) activity, inhibited GEC proliferation and promoted GEC apoptosis under basal and morphine-stimulated conditions. On the other hand, zinc protoporphyrin, an inhibitor of HO activity, promoted GEC proliferation and inhibited GEC apoptosis under basal as well as morphine-stimulated conditions. These findings suggest that HO activity is directly related to GEC apoptosis and inversely related to GEC proliferation. Morphine, de novo, had bimodal effects on HO activity: lower concentrations increased and higher concentrations decreased HO activity. It appears that HO activity may be modifying morphine-induced GEC growth.
      【关键词】 human immunodeficiency virusassociated nephropathy focal glomerulosclerosis opiate
   CLINICAL STUDIES SUGGEST that opiate addicts are at increased risk for progressive chronic renal failure ( 2, 12, 16, 17, 35 ). Intravenous opiate addiction has also been considered a risk factor for the development of human immunodeficiency (HIV)-associated nephropathy ( 8, 38 ). In in vitro studies, there is ample evidence that drugs modulate renal cell function ( 30, 44, 47 ). However, the molecular mechanism involved in opiate-induced progressive chronic renal injury is not clear.

Classic lesions of focal glomerulosclerosis (FGS) are usually initiated with mesangial cell hyperplasia and hypertrophy of glomerular epithelial cells (GECs) ( 36 ), whereas patients with HIV-associated nephropathy often have a variant type of FGS, i.e., collapsing glomerulopathy, which manifests in the form of GEC hyperplasia ( 6 ). Nevertheless, in the course of the disease, the classic and variant forms show loss of mesangial and epithelial cells ( 18 ). We asked whether morphine, a metabolite of heroin, might be contributing to this peculiar form of epithelial cell injury.
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An altered balance between forces promoting survival and those promoting death usually determines the fate of a cell. Loss of glomerular cells has been suggested to be an underlying mechanism for the development of glomerulosclerosis ( 21, 39, 40 ). Opiate addiction has been implicated in the progression of renal lesions, and research has demonstrated that opiate addicts develop FGS. There is no clear evidence that opiates have a role in the development of renal lesions. Nevertheless, in in vitro studies, morphine has been shown to induce the apoptosis of fibroblasts, mesangial cells, and macrophages ( 44, 45, 47 ). In the present study, we evaluated the direct effect of morphine on the growth of cultured GECs.8 r2 D. [2 J# m1 D6 a
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Morphine has been demonstrated to stimulate the production of superoxide by macrophages and mesangial cells ( 41, 46 ). We recently reported that morphine also promotes macrophage heme oxygenase (HO) activity ( 34 ). HO-1 expression is considered a biological marker of oxidative stress ( 29 ). Microsomal HO is the rate-limiting enzyme for heme degradation in mammals ( 50 ). HO cleaves heme into biliverdin and releases free iron and carbon monoxide. Two isoforms have been identified showing differences in regulation and localization ( 11, 42, 50 ).* d* a" Q0 V$ A+ g: i
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In the present study, we examined the effect of morphine on GEC proliferation and apoptosis. In addition, we evaluated the role of oxidative stress in morphine-induced modulation of GEC growth. We also examined the role of HO activity in morphine-induced GEC growth.: S( S: q* k) J& P

MATERIALS AND METHODS
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GECs. Rat GECs (RGECs) were kindly provided by Prof. B. S. Kasinath (Texas Health Science Center, San Antonio, TX), and human GECs (HGECs) were a gift from Dr. G. Ding (Pennsylvania State University College of Medicine, Hershey, PA). GECs were identified by their characteristic polygonal shape and cobblestone appearance at confluence, sensitivity to low doses of puromycin aminonucleoside, positive staining for heparan sulfate proteoglycan, vimentin, and WT-1, and negative staining for factor VIII ( 14, 15 ). GECs were grown in 100-mm plastic petri dishes containing 5 ml of incubation medium, including RPMI 1640 medium (GIBCO, Grand Island, NY), fetal calf serum (heat inactivated; GIBCO), penicillin (50 U/ml; GIBCO), and streptomycin sulfate (50 µg/ml; GIBCO). Culture dishes were kept in a 95% air-5% CO 2 environment at 37°C.

Proximal renal tubular epithelial cell culture. Rat proximal renal tubular epithelial cells (NRK 52 E) were obtained from American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco's modified Eagle's medium (GIBCO) containing 2% penicillin-streptomycin, 1% HEPES, 1.5 g of NaHCO 3, 2 mM L -glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum.& h+ ?' f# m2 f" A; Q  N6 W
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Proliferation studies. Equal numbers of RGECs were plated in 24-well plates and grown to semiconfluence, washed in PBS, and incubated in serum-free RPMI 1640 medium containing 0.5% bovine serum albumin and 1% insulin, transferrin, and selenium solution (GIBCO) for 72 h to arrest growth. Subsequently, cells were washed and reincubated in medium containing vehicle (control) or variable concentrations of morphine (10 -20 -10 -6 M) for 48 h. At the end of the incubation period, cells were trypsinized and counted in a hemocytometer. Four series of experiments were carried out, each in triplicate.1 u( I* q+ p% L) S# v1 x; X

Thiazolyl blue assay. Equal numbers of RGECs were plated in 96-well plates and grown to semiconfluence followed by growth arrest (see Proliferation studies ). Subsequently, cells were washed and reincubated in medium containing vehicle (control) or 10 -18 -10 -6 M morphine for 48 h. At the end of the scheduled incubation period, control and morphine-treated cells were treated with 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [thiazolyl blue (MTT), 5 mg/ml; Sigma, St. Louis, MO] and incubated for 4 h at 37°C in the incubator. After 4 h, medium and MTT were aspirated from the wells, and the formazan crystals were dissolved in 100 µl of 0.04 N HCl in isopropanol. The absorbance was recorded in an ELISA reader at 550 nm, with 620 nm as the reference wavelength. The wells that contained only medium and 10 µl of MTT were used as the blank for the plate reader. Four series of experiments were carried out.

To determine whether there is any species-specific difference, equal numbers of growth-arrested HGECs were incubated in medium containing vehicle (control) or 10 -20 -10 -6 M morphine for 48 h. At the end of the incubation period, cells were trypsinized and counted in a hemocytometer. Four sets of experiments were carried out, each set in triplicate.0 q: H. ^$ m, D0 \

To determine the role of HO activity, equal numbers of growth-arrested RGECs were incubated in medium containing vehicle, hemin, or zinc protoporphyrin (ZnP) for 16 h and reincubated in medium containing buffer alone or 10 -14 M morphine for 48 h. Subsequently, cells were trypsinized and counted using a hemocytometer.5 b) @" `- E6 k0 |/ E

Apoptosis studies. For morphological evaluation of GECs, cells were stained with H-33342 (Molecular Probes, Portland, OR) and propidium iodide (Sigma). H-33342 stains the nuclei of live cells and identifies apoptotic cells by increased fluorescence. Double staining by these two agents provides the percentage of live, apoptotic, and necrotic cells ( 45 ). Cells were prepared under control and experimental conditions. At the end of the incubation period, cells were treated with H-33342 (1.0 µg/ml) for 7 min at 37°C. Subsequently, propidium iodide (1.0 µg/ml final concentration) was added to each well. Cells were incubated with the dyes for 10 min on ice, protected from light, and then examined under ultraviolet light. The percentage of live, apoptotic, and necrosed cells was recorded in eight random fields by two observers unaware of the experimental conditions.
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To confirm the effect of morphine on RGEC apoptosis, RGECs treated under control and experimental conditions were assayed by the TdT-mediated dUTP nick end labeling method (kit supplied by Roche Applied Science, Indianapolis, IN) ( 28 )., B$ f; W- z5 e7 x2 g1 C

DNA fragmentation assay: gel electrophoresis. Gel electrophoresis is a simple method that is specific for isolation and confirmation of DNA fragments from apoptotic cells ( 23 ). Because this method only picks up DNA fragments, loading of samples that do not contain DNA fragments will not be visualized. Equal numbers (10 8 cells/petri dish) of RGECs were prepared under control and experimental conditions. At the end of the incubation period, cells were centrifuged at 1,600 g for 10 min at room temperature, and the pellets were resuspended in DNA lysis buffer (1% NP-40 in 20 mM EDTA and 50 mM Tris · HCl, pH 7.5, 10 µl/10 6 cells). After centrifugation, the supernatant was collected, and the extraction was repeated. SDS in a final concentration of 1% was added to the supernatants before the samples were treated with RNase A (5 µg/µl final concentration) at 56°C and digested with proteinase K (Promega, Madison, WI) for 2 h at 37°C. After addition of 0.5 volume of 10 M ammonium acetate, the DNA was precipitated with 2.5 volumes of ethanol, dissolved in gel loading buffer, and separated by electrophoresis on 1.6% agarose gels.

To evaluate the role of HO activity, equal numbers of RGECs were incubated in medium containing vehicle, hemin, or ZnP for 16 h, reincubated in medium containing buffer alone or 10 -6 M morphine for 16 h, and assayed for apoptosis. Three sets of experiments were carried out., E, B5 @$ O; o: ~& l

Superoxide assay. Equal numbers of RGECs 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 medium containing buffer or 10 -14 -10 -6 M morphine at 37°C for 2 h. Supernatants were collected at 0, 30, 45, 60, and 120 min into precooled microcentrifuge test tubes, and a superoxide assay was carried out. Briefly, 50 µl of each supernatant were pipetted into a 96-well plate, kept on ice, and mixed with 100 µl of cytochrome c (160 µM final concentration; ICN Biomedicals, Costa Mesa, CA) diluted with Hanks' balanced salt solution (GIBCO). Incubation was carried out at 37°C for 45, 90, and 150 min, and optical density was read at 550 nm ( 28 ). Results are expressed in arbitrary units, and experiments were repeated four times, each in triplicate.

Measurement of HO enzyme activity. HO activity was measured by the bilirubin generation method ( 34 ). Briefly, RGECs grown to confluence (in tissue flasks) were incubated in medium containing buffer or 10 -14 -10 -4 M morphine for 16 h. At the end of the incubation period, cells were washed, scraped, and centrifuged (1,000 g for 10 min at 4°C). The cell pellet was suspended in MgCl 2 (2 mM)-phosphate (100 mM) buffer (pH 7.4) and sonicated on ice before centrifugation at 18,800 g for 10 min at 4°C. The supernatant was added to the reaction mixture (400 µl) containing rat liver cytosol (2 mg), hemin (20 µM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 U), and NADPH (0.8 mM) for 1 hat 37°C in the dark. The formed bilirubin was extracted with chloroform, and the change in optical density between 464 and 530 nm was measured (extinction coefficient, 40 mM -1 · cm -1 for bilirubin). HO activity is expressed as picomoles of bilirubin formed per microgram of GEC protein per 60 min., b4 b& P7 Q1 l7 N" Y( m2 `5 r

Protein extraction and Western blot analysis. RGECs 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 in a modified RIPA buffer (1 x PBS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 0.1% SDS, 10 µl of protease inhibitor cocktail/ml of buffer, and 100 µg of PMSF/ml of buffer), and transferred with a syringe fitted with a 21-gauge needle to 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% polyacrylamide 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% bovine serum albumin in PBS with 0.1% Tween 20). The membrane was then incubated for 16 h at 4°C with rabbit polyclonal anti-HO-2 (1:5,000 dilution; Stressgen, Victoria, BC, Canada) in blocking buffer and then incubated for 1 h at room temperature with the 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).

Statistical analysis. For comparison of mean values between groups, an unpaired t -test was used. To compare values between multiple groups, analysis of variance was applied, and a Newman-Keuls multiple range test was used to calculate a P value. Values are means ± SE, except where otherwise indicated. Statistical significance was defined as P
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Proliferation studies. To determine the effect of morphine on GEC proliferation, equal numbers of growth-arrested RGECs were incubated in medium containing buffer (control) or 10 -20 -10 -6 M morphine for 48 h. Subsequently, cells were harvested and counted. As shown in Fig. 1 A, morphine had a bimodal effect on GEC growth. At lower concentrations, morphine stimulated GEC proliferation, whereas, at higher concentrations, morphine suppressed GEC growth.; M- @( k- c& L* b

Fig. 1. A : effect of morphine on rat glomerular epithelial cell (RGEC) proliferation. Equal numbers of growth-arrested RGECs were incubated in medium containing vehicle (control) or 10 -20 -10 -6 M morphine for 48 h. At the end of the incubation period, cells were harvested and counted in a hemocytometer. Values are means ± SE from 4 series of experiments. * P -20 -10 -18 M morphine, and 10 -14 -10 -6 M morphine. ** P -20 -10 -18 M morphine, and 10 -10 M morphine; *** P -8 -10 -6 M morphine; **** P -20 -10 -18 M morphine. B : effect of morphine on human glomerular epithelial cell (HGEC) proliferation. Equal numbers of growth-arrested HGECs were incubated in medium containing vehicle (control) or 10 -20 -10 -6 M morphine for 48 h. At the end of the incubation period, cells were harvested and counted in a hemocytometer. Values are means ± SE from 4 series of experiments. * P -10 M morphine; ** P -20 -10 -16 M morphine, and 10 -10 -10 -6 M morphine; *** P -14 M morphine; a P -20 -10 -16 M morphine; b P c P% `: a& t. r/ X3 i" {
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To evaluate whether this effect of morphine is species specific, we repeated the experiment using HGECs. Morphine exhibited a bimodal effect similar to the effect on RGECs ( Fig. 1 B ). It stimulated HGEC proliferation at lower concentrations but suppressed HGEC proliferation at higher concentrations.& c+ j" }' B: F# J6 d3 _

To confirm the effect of morphine on GECs, equal numbers of growth-arrested RGECs were treated with vehicle (control) or 10 -18 -10 -6 M morphine for 48 h and then subjected to MTT assay. As shown in Fig. 2 A, 10 -18 -10 -12 M morphine promoted GEC proliferation; however, 10 -8 -10 -6 M morphine suppressed GEC growth.

Fig. 2. A : effect of morphine on RGEC growth determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Equal numbers of growth-arrested RGECs were treated with vehicle (control) or 10 -18 -10 -6 M morphine for 48 h and then subjected to MTT assay. Values are means ± SE from 4 series of experiments, each carried out in triplicate. * P -16 -10 -6 M morphine; ** P -18 M morphine, 10 -14 M morphine, and 10 -10 -10 -6 M morphine; *** P -18 -10 -16 M morphine, and 10 -12 -10 -6 M morphine; a P -18 -10 -14 M morphine, and 10 -10 -10 -6 M morphine; b P -18 -10 -12 and 10 -8 -10 -6 M morphine; c P -18 -10 -10 M morphine, and 10 -6 M morphine; d P -18 -10 -10 M morphine. B : representative micrographs of control ( a ) and morphine-treated ( b ) RGECs. Equal numbers of cells were incubated in medium containing buffer or 10 -6 M morphine for 18 h and then stained with H-33342 and propidium iodide. Apoptosed cells show bright fluorescence (arrows). C : effect of morphine on DNA fragmentation in RGECs. Equal numbers of RGECs were incubated in medium containing vehicle (control) or 10 -8 and 10 -6 M morphine for 24 h. Subsequently, cells were washed, and DNA was extracted and subjected to electrophoresis. Lanes 1 and 2, molecular marker and control, respectively; lanes 3 and 4, 10 -8 and 10 -6 M morphine, respectively. Morphine (10 -8 and 10 -6 M) induced DNA fragmentation in a ladder form.3 V, [; J2 w: F- Y2 |+ c1 Y5 X

To determine whether morphine has similar effects on proximal tubular cells, equal numbers of growth-arrested cultured proximal tubular cells were incubated in medium containing buffer (control) or 10 -14 -10 -4 M morphine for 72 h. At the end of the incubation period, cells were trypsinized and counted. As shown in Table 1, at higher concentrations, morphine suppressed the growth of tubular cells.4 N* o8 i$ ?* Y% Y7 q  {8 p
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Table 1. Efféct of morphine on proximal tubular cell growth

Apoptosis studies. To evaluate the mechanism of morphine-induced growth suppression, equal numbers of RGECs were incubated in medium containing buffer or 10 -6 M morphine for 16 h. Subsequently, cells were assayed for apoptosis using H-33342. Morphine promoted GEC apoptosis: 2.1 ± 0.5 and 19.8 ± 1.8 apoptosed cells/field in control and morphine-treated cells, respectively ( P Fig. 2 B. Apoptosed cells show condensed nuclei with bright fluorescence. Similar results were obtained by the TdT-mediated dUTP nick end labeling assay (data not shown).& d! g. G8 M# l" e1 Z

To confirm the effect of morphine on GEC apoptosis, equal numbers of RGECs were incubated in medium containing buffer or 10 -8 -10 -6 M morphine for 16 h. Subsequently, cells were harvested, and DNA was extracted and subjected to electrophoresis. As shown in Fig. 2 C, morphine-treated cells showed multiple integers of 180 bp in a ladder pattern.
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Role of oxidative stress. To determine the role of oxidative stress, we evaluated the effect of antioxidants on morphine-induced GEC apoptosis. Equal numbers of RGECs were incubated in medium containing buffer, diphenyleneiodonium iodide (10 µM), ascorbic acid (100 µM), or N -acetyl cysteine (50 µM) with or without 10 -8 M morphine for 24 h. Subsequently, cells were assayed for apoptosis. As shown in Fig. 3 A, diphenyleneiodonium iodide, ascorbic acid, and N -acetyl cysteine inhibited morphine-induced GEC apoptosis.0 _1 f, A( Q+ v6 K) o: m0 C9 t6 w6 N
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Fig. 3. A : effect of antioxidants on morphine-induced RGEC apoptosis. Equal numbers of RGECs were incubated in medium containing buffer (control), diphenyleneiodonium iodide (DPI, 10 µM), ascorbic acid (AA, 100 µM), or N -acetyl cysteine (NAC, 50 µM) with or without 10 -8 M morphine (Mor) for 24 h. Subsequently, cells were stained with H-33342 and propidium iodide. Values are means ± SE from 4 series of experiments, each carried out in triplicate. * P B : effect of free radical scavengers on morphine-induced RGEC apoptosis. Equal numbers of RGECs were incubated in medium containing buffer (control), SOD (50 µM), catalase (Cat, 2,000 U/ml), or dimethylthiourea (DMTU, 10 µM) with or without 10 -8 M morphine for 24 h and stained with H-33342 and propidium iodide. Values are means ± SE from 4 series of experiments, each carried out in triplicate. * P C : effect of morphine on GEC superoxide production. Equal numbers of RGECs were incubated in serum-free medium containing buffer or 10 -14, 10 -12, 10 -8, and 10 -6 M morphine for 2 h. Aliquots of supernatants were collected at 15, 30, 45, 60, and 120 min. Subsequently, superoxide concentration was measured. Values are means ± SE from 4 sets of experiments at 45 min. * P ** P -14 M morphine.2 ~  o4 j$ m3 w) Y
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To confirm the effect of oxidative stress in morphine-induced GEC injury, we evaluated the effect of free radical scavengers. Equal numbers of RGECs were incubated in medium containing buffer (control), SOD (50 µM), catalase (2,000 U/ml), or dimethylthiourea (10 µM) with or without 10 -8 M morphine for 24 h. Subsequently, cells were assayed for apoptosis. As shown in Fig. 3 B, SOD and catalase partially inhibited the effect of morphine.

To further confirm the role of oxidative stress, we evaluated the effect of morphine on superoxide production by GECs. Equal numbers of RGECs were incubated in serum-free medium containing buffer or 10 -14, 10 -12, 10 -8, and 10 -6 M morphine for 2 h. Aliquots of supernatants were collected at 15, 30, 45, 60, and 120 min. Subsequently, superoxide concentration was measured. Morphine stimulated GEC production of superoxide at 45-120 min. The effect of morphine on production of superoxide at 60 min is shown in Fig. 3 C.$ t" @3 i4 V) e) d# P- c/ E3 _
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To determine the effect of morphine on GEC HO activity, equal numbers of GECs were incubated in medium containing buffer or 10 -14 -10 -6 M morphine for 16 h. Subsequently, cells were harvested, and HO activity was measured. As shown in Fig. 4 A, morphine stimulated HO activity at lower concentrations; however, 10 -6 M morphine suppressed HO activity.

Fig. 4. A : effect of morphine on GEC heme oxygenase (HO) activity. Equal numbers of GECs were incubated in medium containing buffer or 10 -16 -10 -6 M morphine for 16 h and harvested, and HO activity was measured. Values are means ± SE from 4 series of experiments. * P a P b P -16 M morphine; c P -16 -10 -8 M morphine. B : effect of morphine on HO-2 expression in GECs. Equal numbers of RGECs were incubated in medium containing buffer or 10 -20 -10 -6 M morphine for 16 h. At the end of the incubation period, cells were washed, protein was extracted, and Western blots were prepared and probed for HO-2. To confirm equal protein loading, blots were stripped and reprobed for -actin. Top : expression of HO-2 by control (C) and morphine-treated cells. Bottom : expression of actin under similar conditions. C : effect of HO-1 activity on morphine-induced RGEC proliferation. Equal numbers of growth-arrested RGECs were incubated in medium containing vehicle, hemin (5 µM), or zinc protoporphyrin (ZnP, 50 µM) for 16 h and then reincubated in medium containing buffer alone or 10 -14 M morphine for 48 h. Subsequently, cells were trypsinized and counted. Values are means ± SE from 4 sets of experiments. * P ** P *** P **** P D : effect of HO activity on morphine-induced RGEC apoptosis. Equal numbers of RGECs were incubated in medium containing vehicle, hemin (5 µM), or ZnP (50 µM) for 16 h and reincubated in medium containing buffer alone or 10 -6 M morphine for 16 h. Subsequently, cells were evaluated for apoptosis. Values are means ± SE from 4 sets of experiments. * P ** P *** P
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To evaluate whether morphine-induced alteration of HO-2 induction might have also contributed to HO activity, equal numbers of RGECs were incubated in medium containing buffer or 10 -20 -10 -6 M morphine for 16 h. Subsequently, cells were harvested, protein was extracted, and Western blots were prepared and probed for HO-2. To confirm equal protein loading, blots were stripped and reprobed for -actin. As shown Fig. 4 B, morphine did not modulate GEC expression of HO-2.
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To determine the role of HO activity in morphine-induced RGEC proliferation, equal numbers of growth-arrested RGECs were incubated in medium containing vehicle, hemin (5 µM), or ZnP (50 µM) for 16 h and then reincubated in medium containing buffer alone or 10 -14 M morphine for 48 h, and cells were counted. As shown in Fig. 4 C, morphine promoted GEC growth. Hemin, an inducer of HO activity, decreased GEC proliferation, whereas ZnP, an inhibitor of HO activity, increased GEC proliferation. Hemin also attenuated morphine-induced GEC proliferation. On the other hand, ZnP accentuated the mitogenic effect of morphine on GECs.

To confirm the role of HO-1 induction in GEC proliferation, we evaluated the effect of curcumin (a known inducer of HO-1) on HIV transactivator (Tat)-induced GEC proliferation ( 7, 10 ). Equal numbers of growth-arrested GECs were incubated in medium containing buffer (control), curcumin (15 µM), Tat (2 ng/ml), or curcumin Tat for 48 h and then subjected to MTT assay. As shown in Table 2, Tat promoted GEC proliferation. However, curcumin attenuated GEC proliferation under basal and Tat-stimulated states.
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Table 2. Effect of curcumin on Tat-induced GEC proliferation# [" K, n/ b9 ^; I, a6 \: H
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To determine the effect of HO activity on morphine-induced RGEC apoptosis, equal numbers of RGECs were incubated in medium containing vehicle, hemin (5 µM), or ZnP (50 µM) for 16 h and then reincubated in medium containing buffer alone or 10 -6 M morphine for 16 h. Subsequently, cells were evaluated for apoptosis. As shown in Fig. 4 D, morphine promoted GEC apoptosis. Hemin promoted GEC apoptosis under basal and morphine-stimulated states. However, ZnP attenuated morphine-induced GEC apoptosis.
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DISCUSSION, ^" M! l+ p0 F* w! \! S( a# o9 j1 B

The present study demonstrates that, at lower concentrations, morphine promotes GEC proliferation; however, at higher concentrations, morphine triggers the apoptosis of GECs. Because proliferative and proapoptotic effects of morphine were inhibited by free radical scavengers as well as antioxidants, it appears that these effects of morphine are mediated through oxidative stress. Hemin, an inducer of HO activity, inhibited GEC proliferation but promoted GEC apoptosis under basal and morphine-stimulated states. On the other hand, ZnP, an inhibitor of HO activity, promoted GEC proliferation and inhibited GEC apoptosis under basal as well as morphine-stimulated conditions. HO activity in GECs was stimulated by morphine at lower concentrations and suppressed by morphine at higher concentrations.

Superoxide has been demonstrated to stimulate MAP kinases in smooth muscle cells ( 1 ). Similarly, in rat neuronal cells, MAP kinase is stimulated by superoxide ( 24 ). In renal tubular cells, reactive oxygen species activate p44 MAP kinase as well as p38 MAP kinase ( 20 ). In the present study, at lower concentrations, morphine stimulated a moderate amount of superoxide production. However, at higher concentrations, morphine promoted a greater amount of superoxide production. It appears that, at lower concentrations (low-dose morphine-induced), superoxide stimulates GEC proliferation, perhaps through the activation of p44 MAP kinase, whereas, at higher concentrations (high-dose morphine-induced), superoxide promotes apoptosis via p38 MAP kinase activation. However, this hypothesis needs to be tested in future studies.8 x5 ?5 {2 ?& {- U$ w( Q

HO-2 is a constitutive enzyme that is localized primarily in vasculature, brain, and testis ( 22, 31, 43 ). HO-3 exhibits 90% homology to HO-2, but it lacks significant catalytic activity and only functions as a heme-regulatory protein ( 31 ). In the present study, morphine did not modulate GEC expression of HO-2. Thus it appears that morphine-induced alteration in GEC HO activity may be representing the status of HO-1 induction. These findings are consistent with those of other investigators ( 22 )." Q7 V. K/ x; b" f
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HO-1 is an inducible form previously known as heat shock protein-32 ( 25 ). It is an integral part of the antioxidant response element of cells. It is upregulated by a variety of factors, including hypoxia, hyperoxia, heat shock, cytokines, heavy metals, H 2 O 2, ultraviolet irradiation, and its substrate heme ( 4, 9, 26, 27, 32, 52 ). Because heme synthesis and degradation are essential for cytochromes and other heme-containing enzymes such as catalase and nitric oxide synthase, HO-1 is expressed in many cells. Moreover, heme is prooxidant and induces cytotoxicity ( 5 ). Thus expression of HO-1 after oxidative stress may be a protective response. Furthermore, the products of heme cleavage (bilirubin and biliverdin) may act as antioxidants ( 3, 33 ). Many investigators have used preinduction of HO-1 to prevent cellular injury in various models of inflammation ( 4, 19, 48 ). In the present study, morphine showed a bimodal effect on HO activity in GECs. HO activity was stimulated by morphine at lower concentrations and suppressed by morphine at higher concentrations. At lower concentrations, morphine also promoted GEC proliferation. Is there any relation between morphine-induced GEC proliferation and elevated HO activity? To evaluate this aspect, we examined the effect of an inhibitor of HO activity on morphine-induced GEC proliferation. ZnP, an inhibitor of HO activity, promoted GEC proliferation under basal and morphine-stimulated conditions. Thus it appears that, at lower concentrations, morphine-induced HO activity may be a negative-feedback phenomenon to contain the morphine-induced GEC proliferation.

Oxidative stress induced by generation of free radicals (e.g., superoxide and H 2 O 2 ) is a major inciting mechanism of renal injury, leading to a cascade of events resulting in renal cell damage followed by renal cell proliferation, fibrosis, and, ultimately, glomerulosclerosis ( 13, 37 ). In in vitro studies, H 2 O 2, at various concentrations, induced mouse mesangial cell apoptosis ( 49 ). However, the role of reactive oxygen species in the development of GEC proliferation and apoptosis has not been investigated previously. The present study clearly demonstrates the role of oxidative stress in the growth of GECs. Morphine showed a bimodal effect on GEC growth. At lower concentrations, morphine stimulated GEC proliferation, whereas, at higher concentrations, it promoted GEC apoptosis. However, both of the effects of morphine were inhibited by antioxidants.+ I9 a) A5 f/ w' y' g

Morphine promoted GEC apoptosis at higher concentrations. However, at higher concentrations, morphine suppressed HO activity. Because hemin, an inducer of HO activity, promoted GEC apoptosis under basal as well as morphine-stimulated conditions, it appears that morphine-induced suppression of HO activity may have been an attempt to contain the proapoptotic effect of morphine.
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We conclude that morphine has a bimodal effect on GEC growth. This effect of morphine is mediated through oxidative stress. HO activity modulates GEC growth under basal and morphine-stimulated conditions.
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DISCLOSURES- J* [. ^# c6 L/ [: e% s

This work was supported by National Institute on Drug Abuse Grant RO1 DA-12111.

ACKNOWLEDGMENTS7 x% I9 |. u8 H* x' r6 E

Part of this study was presented at the 33rd Annual Meeting of the American Society of Nephrology, Philadelphia, PA, October 2002.
      【参考文献】3 Y6 t' i8 m- T
Baas AS and Berk BC. Differential activation of mitogen protein-activated kinases by H 2 O 2 and in vascular smooth muscle cells. Circ Res 77: 29-36, 1995.
' d5 {8 J& G. w3 G9 [$ c  _
  b+ m+ p3 Q: b! l6 K2 U

Baldwin DS, Gallo GR, and Neugarten J. Drug abuse with narcotics and other agents. In: Diseases of the Kidney (5th ed.), edited by Schrier RW and Gottschalk CW. Boston, MA: Little Brown, 1993.

Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton W, and Vercellotti GM. Ferritin: a cytoprotective antioxidant stratagem of endothelium. J Biol Chem 267: 18148-18153, 1992., g% ?1 L3 F- U# [
$ A3 P# k7 l2 d0 {
! S0 g3 s- m0 S, q# G

Balla G, Vercellotti GM, Muller-Eberhard U, Eaton J, and Jacob HS. Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species. Lab Invest 64: 648-655, 1991.
$ e, ~" ?* O( Y

Balla J, Jacob HS, Balla G, Nath K, Eaton JW, and Vercllotti GM. Endothelial cell-heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage. Proc Natl Acad Sci USA 90: 9285-9289, 1993.

Barisoni L, Kriz W, Mundel P, and D'Agati V. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. J Am Soc Nephrol 10: 51-61, 1999.
; c5 _) I' G% g; T  d/ `: E2 h

Bhaskaran M, Reddy K, Radhakrishanan N, Franki N, Ding G, and Singhal PC. Angiotensin II induces apoptosis in renal proximal tubular cells. Am J Physiol Renal Physiol 284: F955-F965, 2003.2 c% r: b+ J# T" L* w9 V
3 Z) m& e+ f8 |- H; [

Bourgoignie JJ. Renal complications of human immunodeficiency virus type 1. Kidney Int 37: 1571-1584, 1990.3 M  Y4 L9 \  U1 T  b
! z, i, `$ U$ e7 r9 `2 l
, b6 _5 p# n- R7 X

Cantoni L, Rossi C, Rizzardini M, Gadina M, and Ghezzi P. Interleukin-1 and tumor necrosis factor induce hepatic heme oxygenase. Feedback regulation by glucocorticoids. Biochem J 279: 891-894, 1991.5 A" F9 Q" n" @8 U9 d

Conaldi PG, Bottelli A, Baj A, Serra C, Fiore L, Federico G, Bussolati B, and Camussi G. Human immunodeficiency virus-1 Tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells. Am J Pathol 161: 53-61, 2002./ w6 A3 w& y% b: P: T# q/ j
& p4 ?2 x: P) |0 T1 o

Cruse I and Maines MD. Evidence suggesting that the two forms of heme oxygenase are products of different genes. J Biol Chem 263: 3348-3353, 1988., H& j! _: _$ Q6 m9 N

' \: B9 \# Y% k
Cunningham EE, Brentjens JR, Zielezny MA, Andres GA, and Venuto RC. Heroin nephropathy. A clinicopathologic and epidemiologic study. Am J Med 68: 47-53, 1980.

Dijkhorst-Oei LT, Stroes ES, Koomans HA, and Rabelink TJ. Acute simultaneous stimulation of nitric oxide and oxygen radicals by angiotensin II in humans in vivo. J Cardiovasc Pharmacol 33: 420-424, 1999.( h% r1 h! I6 X8 g8 S
* \$ [5 M8 p8 k6 |. e4 t1 h

Ding G, Reddy K, Kapasi AA, Franki N, Gibbons N, Kasinath BS, and Singhal PC. Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol 283: F173-F180, 2002.0 ]% b+ h, y: f3 K5 s
- B& I) B+ j" o4 H
4 Y9 _* R0 c' x9 ?1 m/ o% P

Ding G, van Goor H, Ricardo SD, Orlowski JM, and Diamond JR. Oxidized LDL stimulates the expression of TGF- and fibronectin in human glomerular epithelial cells. Kidney Int 51: 147-154, 1997.) e5 l+ y, |% \" p3 U/ _
" R9 b, O6 L4 D, R" N/ I  R

Dunea G, Arruda JL, Bakir AA, Share DS, and Smith EC. Role of cocaine in end-stage renal disease in some hypertensive African Americans. Am J Nephrol 15: 5-9, 1995.
- [6 a( s, K0 A
6 \' t/ C% @) S% F) _5 K

Fogo A, Superdock KR, and Atkinson JB. Severe arteriosclerosis in the kidney of cocaine addict. Am J Kidney Dis 20: 513-515, 1992.
( ^% y8 d% C1 W4 l2 \
) \  w6 @  _% A" S

Fogo AB. Progression and potential regression of glomerulosclerosis. Kidney Int 59: 804-819, 2001.  p; b! ?% j' i* ~8 V) I5 T

/ ]  Z0 j& C# A, i7 O: f$ ]
7 j6 m/ \& |2 B/ _6 Z1 N
Hancock WW, Buelow R, Sayegh MH, and Turka LA. Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat Med 4: 1392-1396, 1998.
: D8 Q/ O, t1 n8 B1 j5 `! ~* V

Hanken T, Schroeder R, Zahner G, Stahal RAK, and Wolf G. Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27 kipi : role in angiotensin II-mediated hypertrophy of proximal tubular cells. J Am Soc Nephrol 11: 1387-1397, 2000." w5 K! K/ U5 g" }0 d4 ]6 _
3 Q* J7 a+ W+ j6 `9 e3 a
0 W) h2 S% r9 n9 m8 n

Hattori T, Shindo S, and Kawamura H. Apoptosis and expression of Bax protein and Fas antigen in glomeruli of a remnant-kidney model. Nephron 79: 186-191, 1998.
$ ^: ^6 x. B  z. `2 G  ~  C9 t

/ }* M1 y4 m- |% b, K& c
Haugen EN, Croat AJ, and Nath KA. Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int 58: 144-152, 2000.: J, O+ ~& b. e7 k, I6 ^$ U

Herrmann H, Lorenz HM, Voll R, Grunke M, Woith W, and Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22: 5506-5507, 1994.
7 ?3 @. e! Z$ T. {2 b  ^! K9 N8 S

5 [" T1 i. O5 A9 z
Kanterewicz BI, Knapp LT, and Klann E. Stimulation of p42 and p44 mitogen-activated protein kinases by reactive oxygen species and nitric oxide in hippocampus. J Neurochem 70: 1009-1016, 1998.# s& s* b9 c1 d5 T% `8 L3 j
) Z; y. a2 [8 F5 z: \

) D- P* j' \" y; l
Keyse SM and Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 86: 99-103, 1989.
6 B; |' p) y5 C/ U* i0 w0 Y

Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, and Wiggert B. Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-. J Cell Physiol 159: 371-378, 1994.
8 A% V$ ^& i( h
5 {$ g5 N; Z% l( V, c" f4 {
$ T2 G( [6 l, w: a( p  X
Lee PJ, Alam J, Sylvester SL, Inamdar N, Otterbein L, and Choi AM. Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury. Am J Respir Cell Mol Biol 14: 556-568, 1996.3 f9 e; k7 @7 r* @  [
9 R+ q( v4 n" C% e5 h$ ?
6 F$ R7 e2 O* O/ S; x0 I! D

Lodha S, Dani D, Mehta R, Bhasakaran M, Reddy K, Ding G, and Singhal PC. Angiotensin II-induced mesangial cell apoptosis: role of oxidative stress. Mol Med 8: 830-840, 2002.

Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms and clinical implications. FASEB J 2: 2557-2568, 1988.

Mattana J, Gibbons N, and Singhal PC. Cocaine interacts with macrophages to modulate mesangial cell proliferation. J Exp Pharmacol Ther 271: 311-318, 1994.
! N* f# n+ g* T; p! a" I: L5 L. E
( c/ u# E' r0 v

McCoubrey WK Jr, Huang TJ, and Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247: 725-732, 1997.
& r; A1 o8 f0 r  t* L7 t; I) b  c8 z2 @
+ v& ?( u9 V  f* I5 d
* _- n& J; l3 T$ h
Morita T, Perrella MA, Lee ME, and Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc Natl Acad Sci USA 92: 1475-1479, 1995.

& ~- \- N4 S8 ]9 E9 {3 |% R: Y

Nath KA. Heme oxygenase-1: a redoubtable response that limits reperfusion injury in the transplanted adipose liver. J Clin Invest 104: 1485-1486, 1999.
. ?  f# D( h) w. k

. `0 H3 p" S+ h: C; C2 Y
Patel K, Bhaskaran M, Dani D, Reddy K, and Singhal PC. Role of heme oxygenase (HO)-1 in morphine-modulated apoptosis and migration of macrophages. J Infect Dis 187: 47-54, 2003.! p$ y, T* w' k) _# k

Perneger TV, Klag MJ, and Whelton PK. Recreational drug use: a neglected risk factor for end-stage renal disease. Am J Kidney Dis 38: 49-56, 2001.
) I# p0 c! J- o2 V% ?3 K8 L
; _8 S$ u* l- n/ s& C% |8 s, ^7 H2 D

Pesce CM, Striker U, Peten E, Elliot S, and Striker GE. Glomerulosclerosis at both early and late stage is associated with cell turnover in mice transgenic for growth hormone. Lab Invest 65: 601-605, 1991.

Rajgopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Grienaling KK, and Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest 97: 1916-1923, 1996.. C6 H- B/ d# |% V
0 S2 l9 e5 ]+ q1 C' ^

/ v! @, [" Z: P
Rao TKS, Filppone EJ, Nicastri AD, Landesman H, Frank E, Chen K, and Friedman EA. Associated focal segmental glomerulosclerosis in the acquired immunodeficiency syndrome. N Engl J Med 310: 664-673, 1984.
8 w! j" K# U, C" j. T/ f
" m/ S4 g8 a; ?( n

Shankland SJ, Floege J, Thomas SE, Nangaku M, Hugo C, Pippin J, Henne K, Hockenberry DM, Johnson RJ, and Couser WG. Cyclin kinase inhibitors are increased during experimental membranous nephropathy: potential role in limiting glomerular epithelial cell proliferation in vitro. Kidney Int 52: 404-413, 1997.
* ^0 t9 z' I( g# }
8 p) I% [& c" k6 U; w, ^* a" W

Shankland SJ and Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. Am J Physiol Renal Physiol 278: F515-F529, 2000." t1 O& ?' X  u# P! [8 ?
' h7 B5 l7 j" g

Sharp BM, Keane WF, Suh HJ, Gekker G, Tsukayama D, and Peterson PK. Opioid peptides stimulate production by human polymorphonuclear leukocytes and macrophages. Endocrinology 117: 793-795, 1985.

* }* r  z% J+ _, k( E7 k4 \& a
  W, T- p0 g+ H: f) L+ T2 x, J
Shibahara S, Muller M, and Taguchi H. Transcriptional control of rat heme oxygenase by heat shock. J Biol Chem 262: 12889-12892, 1987.
3 L$ [, X* c% u2 p2 P: `

7 N3 r; `& Z. C4 }2 d8 b$ _: ?
Shibahara S, Yoshizawa M, Suzuki H, Takeda K, Meguro K, and Endo K. Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J Biochem 113: 214-218, 1993.

  Q$ t$ c- R5 n5 L( h6 p- m

Singhal PC, Gibbons N, and Abramovici M. Long-term effects of morphine on mesangial cell proliferation and matrix synthesis. Kidney Int 41: 1560-1570, 1992.

9 B6 k  i) z+ M! G) A
Singhal PC, Kapasi AA, Franki N, and Reddy K. Morphine-induced macrophage apoptosis: the role of TGF-. Immunology 100: 57-62, 2000.
4 F9 S2 O% ?4 `; {! D

! l8 r$ X* t9 G6 S! Y% N, a8 O% |
Singhal PC, Pamarthi M, Shah R, Chnadra D, and Gibbons N. Morphine stimulates superoxide formation by glomerular mesangial cells. Inflammation 18: 293-299, 1994.3 p" y2 S4 o$ ?8 b, P- l1 R

Singhal PC, Sharma P, Sanwal V, Prasad A, Kapasi A, Franki N, Reddy K, and Gibbons N. Morphine modulates proliferation of kidney fibroblasts. Kidney Int 53: 350-357, 1998.1 |# m9 u: x- D3 |# I% U) R

$ K5 D0 ?! z) O' ]6 L6 Z
6 q% J+ R) t% {. z1 R
Soares MP, Lin Y, Anrather J, Csizmadia E, Takigami K, Sato K, Grey ST, Colvin RB, Choi AM, Poss KD, and Bach FH. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med 4: 1073-1077, 1998.- C& z* M' v$ @# J
8 I1 G. `0 o7 {% w! T! T- N; M$ Y
; _$ \" s. p; l9 F2 t. ]+ H
2 l* o! e/ H8 u5 s. G
Sugiyama H, Kashihara N, Yamasaki Y, Sekikawa T, Okamoto K, Kanao K, Maeshima Y, Makino H, and Ota Z. Reactive oxygen species induce apoptosis in cultured mesangial cells (Abstract). J Am Soc Nephrol 6: 796, 1994.

1 z. k9 s5 j$ o2 ]

Tenhunen R, Marver HS, and Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem 244: 6388-6394, 1969.; N2 |/ ^) X( C8 u" C

1 D4 m* E9 _7 M0 D# P

Willis D, Moore AR, Frederick R, and Willoughby DA. Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat Med 2: 87-90, 1996.

Yoshida T, Biro P, Cohen T, Muller RM, and Shibahara S. Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur J Biochem 171: 457-461, 1998.

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