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. 2008 Aug 13;295(4):F1158–F1165. doi: 10.1152/ajprenal.00057.2008

Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells

Silvia Kelsen 1,*, Bijal J Patel 1,*, Lawson B Parker 1, Trinity Vera 1, John M Rimoldi 2, Rama S V Gadepalli 2, Heather A Drummond 1, David E Stec 1
PMCID: PMC2576158  PMID: 18701634

Abstract

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 μM) or hemin (50 μM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10−9 M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5 ± 5 to 136 ± 18 relative fluorescence units (RFU)/μm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64 ± 5, 64 ± 8, and 41 ± 4 RFU/μm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 μM) before exposure to ANG II. DHE fluorescence averaged 80 ± 7 RFU/μm2 after incubation with ANG II and was significantly decreased to 55 ± 7 and 53 ± 4 RFU/μm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

Keywords: bilirubin, carbon monoxide, carbon monoxide-releasing molecule A1

recent studies have demonstrated the importance of superoxide generation in the development and maintenance of angiotensin II (ANG II)-dependent hypertension (1, 14, 35). ANG II can increase the production of superoxide by direct activation of NADPH oxidase (6, 7). In the kidney, ANG II-mediated increases in superoxide production have been demonstrated to mediate renal vasoconstriction, cortical hypoxia, and reduced efficiency of O2 usage for sodium transport (10, 13, 31). Increased superoxide production in the thick ascending loop of Henle (TALH) has been reported to stimulate sodium reabsorption by decreasing the levels of nitric oxide (NO) (19, 20). ANG II-mediated increases in superoxide production in the TALH have also been reported to limit NO availability to vasa recta, which could lead to decreases in medullary blood flow, blunting of pressure natriuresis, and hypertension (16). In line with this hypothesis, enhanced expression of NADPH oxidase and increased superoxide production in the medulla have been demonstrated to cause hypertension in normal rats and may contribute to salt-sensitive hypertension in Dahl salt-sensitive rats (17, 26).

Heme oxygenase (HO)-1 is an important cellular defense protein that is induced by a variety of stimulants including hypoxia and heavy metals. In the rat kidney, HO-1 is mainly expressed in the renal medulla, where it plays an important role in maintaining the blood flow to the renal medulla (36). Increases in HO-1 levels have also been shown to be protective against oxidant damage in tissues such as brain and liver (2, 9). In agreement with these observations, induction of HO-1 in TALH cells was found to limit ANG II-induced oxidative damage (21).

While increases in HO-1 levels have been found to limit oxidative damage, the mechanism responsible for this protection is unknown. HO-1 metabolites carbon monoxide (CO) and bilirubin have antioxidant properties that may protect cells against excessive superoxide production. Bilirubin is an antioxidant molecule that can decrease superoxide production by direct actions as well as inhibition of NAD(P)H oxidase activity (11, 12, 24). CO also can inhibit NAD(P)H oxidase by interactions with the heme-containing subunits (25). We previously reported (29) that induction of HO-1 was associated with a decrease in ANG II-mediated superoxide production in the renal medulla of the mouse. The goal of this study was to determine whether induction of HO-1 could attenuate ANG II-mediated superoxide production in cultured TALH cells and to determine which metabolite of HO-1 may be responsible for this response.

METHODS

Cell culture.

Mouse (m)TALH cells were derived from those originally described by Wolf et al. (32) and kindly provided by Dr. Christopher Y. Lu (Univ. of Texas Southwestern Medical Center, Dallas, TX). Identity of the cells was confirmed by immunohistochemistry for two specific TALH markers, Tamm-Horsfall protein (THP) and sodium-potassium-2 chloride transporter (NKCC). Cells (20,000) were plated on collagen-coated glass slides. All of the cells stained positive for both of these TALH-specific markers (Fig. 1). mTALH cells were grown and maintained in a medium consisting of 89% Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and a 1% penicillin-streptomycin antibiotic (Pen/Strep). For all experiments, cells were treated with serum-free medium. The serum-free medium consisted of 98.6% DMEM, 0.4% FBS, and 1% Pen/Strep. Cells were plated on collagen-coated eight-well glass slides at a density of 20,000 cells/well, incubated at 37°C, and used when they reached 70–80% confluence.

Fig. 1.

Fig. 1.

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Immunohistochemistry for thick ascending loop of Henle (TALH) cell-specific markers in cultured mouse (m)TALH cells. Cells were incubated with or without antibody (Ab) to Tamm-Horsfall protein (THP; B) or sodium-potassium-2 chloride transporter (NKCC; A). Positive staining was observed in all of the cells.

Chemical and genetic induction of HO-1 in mTALH cells.

Seventy to eighty percent confluent mTALH cells were treated with a 10 μM solution of cobalt protoporphyrin (CoPP, Frontier Scientific, Logan, UT) or 50 μM hemin (Sigma-Aldrich, St. Louis, MO) diluted in the cell culture medium. After incubation for 24 h, the cells were collected and protein lysates were obtained for Western blot analysis for the HO-1 enzyme. mTALH cells were also transfected with a plasmid expressing the human HO-1 isoform under the control of the cytomegalovirus promoter. The plasmid vector without the HO-1 cDNA insert served as a control. Transfections were performed with 5 μg of DNA mixed with Lipofectamine 2000 reagent (1-to-2 ratio) diluted in Opti-MEM medium. The cells were incubated with DNA-Lipofectamine mixture for 4 h at 37°C before the medium was replaced with standard medium. Cells were then incubated with ANG II (10−9 M, Bachem, King of Prussia, PA) for 1 h at 37°C and processed for dihydroethidium (DHE) staining as described below. Transfection efficiency was 80–90% in preliminary experiments using plasmids expressing green fluorescent protein (data not shown). Small interfering RNA (siRNA) directed against mouse HO-1 consisted of a mixed pool of siRNA purchased from a commercial source (ON-TARGETplus SMARTpool, Dharmacon, Chicago, IL). As a control, a nontargeting, RNAi silencing complex (RISC)-activating siRNA was used (Non-Targeting siRNA no. 2, Dharmacon). siRNAs were resuspended as 20 μM stock solutions according to manufacturer's guidelines. Transfection was performed on 30–40% confluent cells with Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA) diluted in Opti-MEM medium according to the manufacturer's protocol. Cells were incubated with siRNA for 4 h at 37°C, after which time the siRNA-Opti-MEM medium was removed and replaced with growth medium. Cells were used in experiments 48 h after transfection.

Measurement of HO activity.

HO assay was performed on lysates prepared from control and CoPP- and hemin-treated TALH cells as previously described (28). The reactions were incubated for 1 h at 37°C in the dark. The formed bilirubin was extracted with chloroform, and the change in optical density (ΔOD) at 464–530 nm was measured with an extinction coefficient of 40 mM/cm for bilirubin. HO activity was expressed as picomoles of bilirubin formed per hour per milligram of total protein.

CORM-A1, bilirubin, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one treatment of mTALH cells.

These experiments were also performed with eight-well slides, in which the mTALH cells were exposed to 100 μM concentrations of CO-releasing molecule (CORM)-A1, inactive CORM-A1 (iCORM-A1), or bilirubin acutely for 1 h. CORM-A1 was prepared as previously described (18). iCORM-A1 was prepared by dissolving CORM-A1 in 0.1 M HCl while bubbling nitrogen into the mixture for 10 min; then the pH was adjusted to 7.0 with 0.1 M NaOH. To block soluble guanylyl cyclase (sGC), cells were treated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 30 μM) for 1 h before the addition of CORM-A1. After 1-h incubation, the reagents were removed and the cells were exposed to ANG II (10−9 M) for 1 h, after which time the cells were processed for DHE staining as described below.

Dihydroethidium staining.

DHE (5 μM, Molecular Probes, Eugene, OR) was added to cells seeded on eight-well collagen-coated glass slides. Slides were then incubated at 37°C for 30 min in the dark. The cells were then fixed in formaldehyde and mounted, and a coverslip was placed over the slide. DHE fluorescence was measured with confocal microscopy performed on a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems, Exton, PA). Fluorescence was measured at excitation of 490 nm with emission at 560 and 590 nm as described previously (34). Data were collected from 50 cells obtained from 5 random fields with a ×40 objective lens. Fluorescence intensity at 560 nm was then normalized for cell size and analyzed as relative fluorescence units (RFU)/cell area (μm2).

2-Dihydroxyethidium fluorescence.

Superoxide production in cultured mTALH cells was also measured by 2-dihydroxyethidium fluorescence as previously described (26). Cell lysates (20 μg) were incubated with DHE (10 μM), salmon sperm DNA (0.5 mg/ml), and NADPH (100 μM) in a 100-μl volume at 37°C for 30 min. The increase in oxy-ethidium fluorescence was then measured at an excitation of 485 nm and an emission of 570 nm with a dual-beam spectrophotometer. Samples were assayed in triplicate, and the values for each sample were averaged and combined to get an average for each group. Data are expressed as the normalized fluorescence of each sample per microgram of protein used in the assay.

Western blot analysis.

Western blots were performed on cell lysates. Samples of 30 μg of protein were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA) for 5 min, electrophoresed on 7.5% SDS-polyacrylamide gels, and blotted onto nitrocellulose membranes. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 2 h at room temperature and then incubated with mouse anti-HO-1 monoclonal antibody (1:2,000, StressGen, Vancouver, BC, Canada) as well as a rabbit anti-β-actin antibody (1:5,000, BD Gentest, San Jose, CA) overnight at 4°C. The membranes were then incubated with Alexa 680-conjugated goat anti-mouse IgG (1:2,000, Molecular Probes) and IRDye 800-conjugated goat anti-rabbit IgG (1:2,000, Rockland, Gilbertsville, PA) for 1 h at room temperature. The membranes were visualized with an Odyssey infrared imager (LI-COR), which allows for the simultaneous detection of two proteins. Densitometry analysis was performed with Odyssey software (LI-COR). Levels of HO-1 protein are expressed as the ratio of HO-1 to β-actin for each sample.

Immunohistochemistry.

Immunohistochemistry was performed on formalin-fixed cells. Cells were incubated overnight with primary antibodies for THP (anti-human THP, 1:150; Biomedical Technologies, Stoughton, MA) or NKCC (anti-rabbit NKCC; 5 μg/ml, Aviva Systems Biology, San Diego, CA). Antibody labeling was visualized with fluorescent-labeled secondary antibodies. All samples were examined by fluorescent confocal microscopy (Leica Microsystems).

Assays for cell viability.

Cell viability was determined by lactate dehydrogenase (LDH) release as well as Trypan blue exclusion. LDH release was determined in cells treated with hemin, bilirubin, or CORM-A1. Cells were treated with hemin for 16 h and with bilirubin and CORM-A1 for 1 h. LDH was measured spectrophotometrically at a wavelength of 490 nm, with background absorbance measured at 690 nm. LDH released into the medium is presented as a percentage of total LDH measured. Cell viability was also determined by Trypan blue exclusion. Cells were treated with either CoPP or hemin for 16 h or bilirubin or CORM-A1 for 1 h. Cells were then incubated with 0.4% Trypan blue for 10 min, and the total number of cells as well as the blue cells were counted. The percent viable cells are the total number of non-dye-containing cells divided by the total number of cells. Experiments for both assays were performed in triplicate.

Statistics.

Mean ± SE values are presented. Significant differences between mean values were determined by unpaired t-test or with the use of an ANOVA followed by a post hoc test (Dunnett's). A P <0.05 was considered to be significant.

RESULTS

HO-1 inducers, metabolites, and viability of mTALH cells.

Viability of mTALH cells to hemin, CoPP, bilirubin, and CORM-A1 was determined by measuring LDH release as well as Trypan blue exclusion. Hemin treatment (50 μM) resulted in significant increase in LDH release in mTALH cells, whereas treatment with bilirubin or CORM-A1 (100 μM) had no effect on LDH release (Fig. 2A). Next, we determined cell survival after treatment with hemin (50 μM), CoPP (10 μM), or bilirubin or CORM-A1 (100 μM), using Trypan blue exclusion. Of the four compounds tested, only CORM-A1 had a significant effect on the viability of mTALH cells (Fig. 2B).

Fig. 2.

Fig. 2.

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A: lactate dehydrogenase (LDH) release in mTALH cells treated with hemin (50 μM), bilirubin (100 μM), or CO-releasing molecule (CORM)-A1 (100 μM). Cells were incubated as described in methods. *P < 0.05 vs. control; n = 3. B: cell survival as measured by Trypan blue exclusion in cells treated with cobalt protoporphyrin (CoPP, 10 μM), hemin (50 μM), bilirubin (100 μM), or CORM-A1 (100 μM). Cells were incubated as described in methods. *P < 0.05 vs. control; n = 3.

HO expression and activity in mTALH cells.

Western blot analysis of cell lysates was performed in order to determine the levels of HO-1 and HO-2 protein expression in the mTALH cell line under basal conditions and in response to two known inducers of HO-1, hemin and CoPP. HO-1 protein was detected in the control mTALH cells. However, treatment with 5 and 10 μM CoPP or 50 and 100 μM hemin caused a significant induction of HO-1 (Fig. 3, A and B) but not in HO-2 (Fig. 3C). These results demonstrate that the HO-1 isoform could be significantly increased in mTALH cells by the inducers of HO, hemin and CoPP. Hemin and CoPP treatment also resulted in significant increase in HO activity in TALH cells (Fig. 3D).

Fig. 3.

Fig. 3.

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A: representative Western blots of heme oxygenase (HO)-1, HO-2, and β-actin levels in control and CoPP- and hemin-treated mTALH cells. B and C: quantification of HO-1 (B) and HO-2 (C) in mTALH cells. CoPP and hemin treatment resulted in a significant increase in HO-1 protein levels in mTALH. C, control. D: HO activity in cells treated with hemin (50 μM) or COPP (10 μM) *P < 0.05 vs. control; n = 4.

Chemical induction of HO-1 decreases ANG II-mediated superoxide production in mTALH cells.

Two fluorescence-based methods were used to examine the effect of HO-1 induction on ANG II-mediated superoxide production in cultured mTALH cells. ANG II treatment (10−9 M) resulted in a greater than threefold increase in superoxide production compared with control cells measured by DHE staining and confocal microscopy (Fig. 4). Pretreatment of the mTALH cells with either CoPP (10 μM) or hemin (50 μM) significantly reduced ANG II-mediated superoxide production (Fig. 4).

Fig. 4.

Fig. 4.

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A: chemical induction of HO-1 with CoPP or hemin (50 μM) decreases angiotensin II (ANG II)-mediated superoxide production in mTALH cells. B: representative confocal images of dihydroethidium (DHE) fluorescence in each of the treatment groups. *P < 0.05 vs. ANG II, †P < 0.05 vs. control; n = 3.

Next, we examined superoxide production with 2-dihydroxyethidium fluorescence. In accordance with the results obtained by DHE staining, ANG II treatment of mTALH cells resulted in a significant increase in superoxide compared with control cells (Fig. 5). Hemin pretreatment by itself resulted in a slight increase in superoxide production that was not statistically different from control. However, hemin pretreatment was able to normalize superoxide production to control levels in ANG II-treated cells (Fig. 5).

Fig. 5.

Fig. 5.

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Superoxide production in control and hemin-, ANG II-, and ANG II + hemin-treated mTALH cells. Superoxide production was measured with 2-dihydroxyethidium fluorescence as outlined in methods. Hemin treatment decreased ANG II-induced superoxide production. *P < 0.05 vs. control; n = 6.

Overexpression of HO-1 decreases ANG II-mediated superoxide production in mTALH cells.

Experiments were performed in which mTALH cells were transiently transfected with a plasmid expressing the human HO-1 isoform (HHO-1). Transfection of mTALH with the HHO-1 plasmid resulted in a significant increase in HO-1 protein 48 h after transfection (Fig. 6A). Treatment of mTALH cells with ANG II resulted in a significant increase in superoxide production (Fig. 6, B and C). Transfection of mTALH cells with the HHO-1-containing plasmid resulted in a significant attenuation of ANG II-mediated superoxide production (Fig. 6, B and C). This effect was specific for HHO-1, because transfection with an empty plasmid had no effect on ANG II-mediated superoxide production (Fig. 6, B and C).

Fig. 6.

Fig. 6.

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A: representative Western blot of mTALH cells transfected with a plasmid overexpressing human (H)HO-1. C, control; T, transfected. B: expression of human HO-1 decreases ANG II-induced superoxide production in mTALH cells. CMV, cytomegalovirus. C: representative confocal images of DHE fluorescence in each of the treatment groups. *P < 0.05 vs. ANG II-treated cells; n = 3.

HO metabolites bilirubin and CO decrease ANG II-induced superoxide generation in mTALH cells.

To determine the role of the HO metabolites bilirubin and CO in the attenuation of superoxide generation by HO-1 induction, experiments were performed in which mTALH cells were exposed to specific increases in bilirubin or CO before treatment with ANG II. CO levels were increased with the specific CO donor CORM-A1. Bilirubin and CORM-A1 treatment were both able to decrease ANG II-mediated superoxide production in mTALH cells (Fig. 7). Treatment of mTALH cells with iCORM-A1 did not have any significant effect on ANG II-mediated superoxide production.

Fig. 7.

Fig. 7.

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A: bilirubin and CORM-A1 treatment decreases ANG II-mediated superoxide production in mTALH cells. iCORM-A1, inactive CORM-A1. B: representative confocal images of DHE fluorescence in each of the treatment groups. *P < 0.05 vs. ANG II-treated cells; n = 3.

Soluble guanylyl cyclase activation does not mediate antioxidant effects of CO in mTALH cells.

To determine the role of sGC in mediating the antioxidant actions of CO, we performed studies in the presence of the sGC inhibitor ODQ. Cells were treated with ODQ before exposure to CORM-A1 and ANG II. CORM-A1 treatment resulted in a decrease in ANG II-mediated superoxide production, which was not altered by ODQ treatment (Fig. 8). ODQ treatment by itself resulted in a decrease in ANG II-mediated superoxide production; however, this decrease in superoxide production was not statistically different from treatment with ANG II alone (Fig. 8).

Fig. 8.

Fig. 8.

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Soluble guanylyl cyclase (sGC) inhibition, CO, and ANG II-mediated superoxide production in mTALH cells. Inhibition of sGC activity with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 μM) did not have any effect on the reduction of ANG II-mediated superoxide production in mTALH cells. *P < 0.05 vs. ANG II-treated cells; n = 5.

Inhibition of HO-1 protein with siRNA reverses antioxidant effects of hemin treatment in mTALH cells.

To test the role of HO-1 induction in the antioxidant actions of hemin treatment, experiments were performed in the presence of siRNA directed against HO-1. Treatment of mTALH with a mixed pool of siRNAs directed against mouse HO-1 (100 nM) resulted in significant decrease in hemin-induced induction of HO-1 (Fig. 9A). Next, experiments were performed in which ANG II-mediated superoxide production was measured in mTALH pretreated with hemin, hemin + nontargeting siRNA, and hemin + HO-1 siRNA. Hemin treatment alone was found to decrease ANG II-mediated superoxide production; however, this effect was abolished in cells treated with siRNA directed against HO-1 (Fig. 9B). In fact, treatment with HO-1 siRNA resulted in an enhancement of ANG II-mediated superoxide production (Fig. 9B). Treatment with the nontargeting siRNA had no effect on the ability of hemin to decrease ANG II-mediated superoxide production in mTALH cells (Fig. 9B).

Fig. 9.

Fig. 9.

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A: representative Western blot of mTALH cells transfected with HO-1 small interfering RNA (siRNA) before treatment with hemin. Treatment with 100 nM siRNA resulted in a significant decrease in the levels of HO-1 protein after hemin treatment. *P < 0.05 vs. hemin-treated cells; n = 4. NT, nontargeting siRNA. B: inhibition of HO-1 protein with siRNA attenuates antioxidant actions of hemin. *Significantly decreased (P < 0.05) vs. ANG II-treated cells, n = 3; †significantly increased (P < 0.05) vs. ANG II-treated cells, n = 3.

DISCUSSION

The results of the present study demonstrate that induction of HO-1 either chemically or genetically is able to attenuate ANG II-mediated superoxide production in mTALH cells. Moreover, inhibition of HO-1 protein synthesis with siRNAs was able to completely block the antioxidant actions of HO-1 induction with hemin and resulted in enhancement of ANG II-mediated superoxide production in mTALH cells. These results are in agreement with previous studies reporting that induction of HO-1 decreases ANG II-mediated oxidative damage in endothelial and TALH cells (21, 33). These results are also supported by our recent study (29) in which we found that induction of HO-1 reduced ANG II-mediated superoxide production in the renal medulla. While these studies support an antioxidant role for HO-1 in the TALH, the specific role of the primary metabolites of HO, bilirubin and CO, in this response was studied in further detail. One reason for using a cell culture-based approach was to perform experiments to separate out the antioxidant actions of CO and bilirubin by selectively increasing each of the individual metabolites and determining their effect on ANG II-mediated superoxide production.

In the present study, mTALH cells were incubated with bilirubin or CORM-A1, which is a newly described CO donor (18). The results of the present study show that increases in bilirubin or CO alone are capable of reducing ANG II-mediated superoxide production in mTALH cells. Bilirubin has been reported to be a potent antioxidant capable of both scavenging superoxide ion as well as inhibiting NAD(P)H oxidase directly (11, 24). Recent studies have also found that increases in bilirubin are able to decrease ANG II-mediated superoxide production in vascular smooth muscle cells (3). These results, coupled with the results from the present study, clearly demonstrate the important role of increases in bilirubin in mediating the antioxidant action of HO-1 induction.

There is some potential for cellular toxicity with the use of inducers of HO-1 such as hemin and with CO (5). In the present study, we found that use of hemin increased LDH release; however, we did not find any significant affect of hemin treatment on cell viability as measured by Trypan blue exclusion. However, while we did not observe any affect of CORM-A1 on LDH release, we did find a small but significant effect of CORM-A1 on cell survival. We did not test whether lower concentrations of CORM-A1 are effective in blocking ANG II-mediated superoxide production in TALH cells, but it is possible that lower concentrations of CORM-A1 may be more beneficial to cell survival.

The results from the present study not only emphasize the importance of bilirubin generation in the antioxidant actions of HO-1 induction but reveal an important role for increases in CO in mediating this response in mTALH cells. Our studies clearly show that increases in CO with the specific CO-releasing molecule CORM-A1 are able to attenuate ANG II-mediated superoxide production in mTALH cells. CO as an antioxidant is an emerging concept. Recent studies have found that CO can inhibit NAD(P)H oxidase activity in both vascular smooth muscle cells and macrophages (22, 25). It is believed that CO inhibits NAD(P)H oxidase activity through interaction with the heme moiety in cytochrome b558, which is a component of the gp91phox subunit of NAD(P)H oxidase. While it has been thought that CO directly inhibits NAD(P)H oxidase activity (22, 25), the role of sGC activation in this response had not been previously determined. CO, like NO, signals in cells via activation of sGC, which increases intracellular cyclic GMP (cGMP) levels (4). The increase in cGMP levels mediates the vasodilatory effects of CO on vascular smooth muscle cells through modification of potassium channels (30). The results of our present study demonstrate that inhibition of sGC with ODQ had no effect on the decrease in ANG II-induced superoxide production by increased CO in mTALH cells. This observation provides further support for CO acting directly to inhibit NAD(P)H oxidase activity in mTALH cells. We did observe some decrease in ANG II-mediated superoxide production with ODQ treatment. Since previous studies have demonstrated that ANG II decreases sGC levels (15), it is not likely that this pathway is involved in ANG II-induced superoxide generation. The slight decrease in ANG II-mediated superoxide production with ODQ observed in the present study may simply be reflective of a small direct antioxidant effect of ODQ. This possibility needs to be tested in future studies.

Superoxide anion is an important regulator of both vascular and tubular function in the renal medulla. Increased superoxide produced in the TALH can react with NO and limit its diffusion to the vasa recta, thereby increasing the susceptibility of the vasa recta to vasoconstrictors (16). This could result in decreases in renal medullary blood flow and sodium excretion. Superoxide anion has also been demonstrated to increase sodium reabsorption in the TALH by direct stimulation of the NKCC transporter as well as by reducing NO levels in the TALH (8, 20, 27). Both of these factors may play an important role in the development and maintenance of ANG II-dependent hypertension. We recently demonstrated (29) that induction of HO-1 was associated with decreased superoxide production and lower blood pressure in ANG II hypertensive mice. We hypothesize that increases in both bilirubin and CO generation in the renal medulla mediate the blood pressure-lowering actions of HO-1 induction via improvement in renal pressure natriuresis. The role for a renal mechanism is supported by the observation that systemic induction of HO-1 does not improve vascular function in ANG II hypertensive mice despite lowering of blood pressure (23). The results of the present study further support the antioxidant role of HO-1 and its primary metabolites, bilirubin and CO, in the TALH. However, more detailed in vivo studies are needed in order to specifically test the role of alterations in medullary superoxide production in the development and maintenance of ANG II hypertension.

In summary, induction of HO-1 either chemically or genetically was able to attenuate ANG II-mediated increases in superoxide production in mTALH cells. Increases in either bilirubin or CO alone were also able to significantly decrease ANG II-mediated superoxide production in mTALH cells. CO decreases ANG II-mediated superoxide production via a mechanism that does not involve activation of sGC in mTALH cells. These data provide additional support for the important antioxidant role of HO-1 induction in the TALH.

GRANTS

This work was supported by grants from the American Heart Association (0755330B, D. E. Stec) as well as the National Heart, Lung, and Blood Institute [PO1-HL-5197 and HL-086996 (to H. A. Drummond)].

Acknowledgments

The authors thank Dr. Christopher Y. Lu, University of Texas Southwestern Medical Center, Dallas, TX, for the gift of the mouse thick ascending loop of Henle cells and Dr. Nader G. Abraham, New York Medical College, Valhalla, NY, for the gift of the human heme oxygenase-1 plasmid.

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.

REFERENCES

  • 1.Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol 285: R117–R124, 2003. [DOI] [PubMed] [Google Scholar]
  • 2.Chen K, Gunter K, Maines MD. Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death. J Neurochem 75: 304–313, 2000. [DOI] [PubMed] [Google Scholar]
  • 3.Datla SR, Dusting GJ, Mori TA, Taylor CJ, Croft KD, Jiang F. Induction of heme oxygenase-1 in vivo suppresses NADPH oxidase derived oxidative stress. Hypertension 50: 636–642, 2007. [DOI] [PubMed] [Google Scholar]
  • 4.Furchgott RF, Jothianandan D. Endothelium-dependent and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light. Blood Vessels 28: 52–61, 1991. [DOI] [PubMed] [Google Scholar]
  • 5.Gonzalez-Michaca L, Farrugia G, Croatt AJ, Alam J, Nath KA. Heme: a determinant of life and death in renal tubular epithelial cells. Am J Physiol Renal Physiol 286: F370–F377, 2004. [DOI] [PubMed] [Google Scholar]
  • 6.Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141–1148, 1994. [DOI] [PubMed] [Google Scholar]
  • 7.Hanna IR, Taniyama Y, Szocs K, Rocic P, Griendling KK. NAD(P)H oxidase-derived reactive oxygen species as mediators of angiotensin II signaling. Antioxid Redox Signal 4: 899–914, 2002. [DOI] [PubMed] [Google Scholar]
  • 8.Juncos R, Garvin JL. Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. Am J Physiol Renal Physiol 288: F982–F987, 2005. [DOI] [PubMed] [Google Scholar]
  • 9.Kato H, Amersi F, Buelow R, Melinek J, Coito AJ, Ke B, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation. Am J Transplant 1: 121–128, 2001. [PubMed] [Google Scholar]
  • 10.Kopkan L, Castillo A, Navar LG, Majid DS. Enhanced superoxide generation modulates renal function in ANG II-induced hypertensive rats. Am J Physiol Renal Physiol 290: F80–F86, 2006. [DOI] [PubMed] [Google Scholar]
  • 11.Lanone S, Bloc S, Foresti R, Almolki A, Taille C, Callebert J, Conti M, Goven D, Aubier M, Dureuil B, El Benna J, Motterlini R, Boczkowski J. Bilirubin decreases NOS2 expression via inhibition of NAD(P)H oxidase: implications for protection against endotoxic shock in rats. FASEB J 19: 1890–1892, 2005. [DOI] [PubMed] [Google Scholar]
  • 12.Llesuy SF, Tomaro ML. Heme oxygenase and oxidative stress. Evidence of involvement of bilirubin as physiological protector against oxidative damage. Biochim Biophys Acta 1223: 9–14, 1994. [DOI] [PubMed] [Google Scholar]
  • 13.Lopez B, Salom MG, Arregui B, Valero F, Fenoy FJ. Role of superoxide in modulating the renal effects of angiotensin II. Hypertension 42: 1150–1156, 2003. [DOI] [PubMed] [Google Scholar]
  • 14.Modlinger P, Chabrashvili T, Gill PS, Mendonca M, Harrison DG, Griendling KK, Li M, Raggio J, Wellstein A, Chen Y, Welch WJ, Wilcox CS. RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response. Hypertension 47: 238–244, 2006. [DOI] [PubMed] [Google Scholar]
  • 15.Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res 90: E58–E65, 2002. [DOI] [PubMed] [Google Scholar]
  • 16.Mori T, Cowley AW Jr. Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla. Hypertension 42: 588–593, 2003. [DOI] [PubMed] [Google Scholar]
  • 17.Mori T, O'Connor PM, Abe M, Cowley AW Jr. Enhanced superoxide production in renal outer medulla of Dahl salt-sensitive rats reduces nitric oxide tubular-vascular cross-talk. Hypertension 49: 1336–1341, 2007. [DOI] [PubMed] [Google Scholar]
  • 18.Motterlini R, Sawle P, Hammad J, Bains S, Alberto R, Foresti R, Green CJ. CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J 19: 284–286, 2005. [DOI] [PubMed] [Google Scholar]
  • 19.Ortiz PA, Garvin JL. Interaction of O2− and NO in the thick ascending limb. Hypertension 39: 591–596, 2002. [DOI] [PubMed] [Google Scholar]
  • 20.Ortiz PA, Garvin JL. Superoxide stimulates NaCl absorption by the thick ascending limb. Am J Physiol Renal Physiol 283: F957–F962, 2002. [DOI] [PubMed] [Google Scholar]
  • 21.Quan S, Yang L, Shnouda S, Schwartzman ML, Nasjletti A, Goodman AI, Abraham NG. Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury. Kidney Int 65: 1628–1639, 2004. [DOI] [PubMed] [Google Scholar]
  • 22.Srisook K, Han SS, Choi HS, Li MH, Ueda H, Kim C, Cha YN. CO from enhanced HO activity or from CORM-2 inhibits both O2− and NO production and downregulates HO-1 expression in LPS-stimulated macrophages. Biochem Pharmacol 71: 307–318, 2006. [DOI] [PubMed] [Google Scholar]
  • 23.Stec DE, Vera T, McLemore GR Jr, Kelsen S, Rimoldi JM, Gadepalli RS, Ryan MJ. Heme oxygenase-1 induction does not improve vascular relaxation in angiotensin II hypertensive mice. Am J Hypertens 21: 189–193, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 235: 1043–1046, 1987. [DOI] [PubMed] [Google Scholar]
  • 25.Taille C, El Benna J, Lanone S, Boczkowski J, Motterlini R. Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle. J Biol Chem 280: 25350–25360, 2005. [DOI] [PubMed] [Google Scholar]
  • 26.Taylor NE, Glocka P, Liang M, Cowley AW Jr. NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats. Hypertension 47: 692–698, 2006. [DOI] [PubMed] [Google Scholar]
  • 27.Varela M, Herrera M, Garvin JL. Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2− and is diminished by a high-salt diet. Am J Physiol Renal Physiol 287: F224–F230, 2004. [DOI] [PubMed] [Google Scholar]
  • 28.Vera T, Henegar JR, Drummond HA, Rimoldi JM, Stec DE. Protective effect of carbon monoxide-releasing compounds in ischemia-induced acute renal failure. J Am Soc Nephrol 16: 950–958, 2005. [DOI] [PubMed] [Google Scholar]
  • 29.Vera T, Kelsen S, Yanes LL, Reckelhoff JF, Stec DE. HO-1 induction lowers blood pressure and superoxide production in the renal medulla of angiotensin II hypertensive mice. Am J Physiol Regul Integr Comp Physiol 292: R1472–R1478, 2007. [DOI] [PubMed] [Google Scholar]
  • 30.Wang R, Wu L. The chemical modification of KCa channels by carbon monoxide in vascular smooth muscle cells. J Biol Chem 272: 8222–8226, 1997. [DOI] [PubMed] [Google Scholar]
  • 31.Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol Heart Circ Physiol 288: H22–H28, 2005. [DOI] [PubMed] [Google Scholar]
  • 32.Wolf G, Ziyadeh FN, Helmchen U, Zahner G, Schroeder R, Stahl RA. ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop. Am J Physiol Renal Fluid Electrolyte Physiol 268: F940–F947, 1995. [DOI] [PubMed] [Google Scholar]
  • 33.Yang L, Quan S, Abraham NG. Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury. Am J Physiol Lung Cell Mol Physiol 277: L127–L133, 1999. [DOI] [PubMed] [Google Scholar]
  • 34.Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vasquez-Vivar J, Kalyanaraman B. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl Acad Sci USA 102: 5727–5732, 2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Zimmerman MC, Lazartigues E, Sharma RV, Davisson RL. Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system. Circ Res 95: 210–216, 2004. [DOI] [PubMed] [Google Scholar]
  • 36.Zou AP, Billington H, Su N, Cowley AW Jr. Expression and actions of heme oxygenase in the renal medulla of rats. Hypertension 35: 342–347, 2000. [DOI] [PubMed] [Google Scholar]

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