Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice

Abstract

Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg⁻¹·min⁻¹) infused by miniosmotic pumps for 2 wk in female Cyp1b1^+/+ mice did not alter water consumption, urine output, Na⁺ excretion, osmolality, or protein excretion. However, in Cyp1b1^−/− mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na⁺ excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-β in Cyp1b1^−/− but not Cyp1b1^+/+ mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17β-estradiol metabolite 2-methoxyestradiol in Cyp1b1^−/− but not Cyp1b1^+/+ mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17β-estradiol.

it is well established that male subjects are more prone to developing cardiovascular diseases, including hypertension and renal dysfunction, compared with premenopausal female subjects of the same age (31, 42, 53). Sex differences in hypertension and renal disease have also been demonstrated in experimental models, including ANG II- and DOCA-salt-induced hypertension and Dahl salt-sensitive and spontaneously hypertensive rats (SHRs) (18, 37, 43, 47, 61). Female subjects are protected or have a substantially lower increase in blood pressure (BP) and associated cardiovascular and renal dysfunction than do their male counterparts in these models of hypertension (14, 18, 37, 43, 47, 56, 61). Sex differences in the development of hypertension have been attributed to differential effects of sex hormones on the renin-angiotensin system and sex chromosomes (19). Testosterone stimulates the expression of renal angiotensinogen and/or renin mRNA in SHRs (7, 13) and Dahl salt-sensitive rats (63). Estrogen also increases plasma and tissue levels of angiotensinogen and has been shown to either increase (3, 34) or decrease (49) renin levels. In additon, estrogen reduces angiotensin-converting enzyme (ACE) activity in blood and tissues and increases circulating levels of ANG(1–7) (46, 49, 55). Estrogen downregulates expression of the ANG II type 1 (AT₁) receptor but upregulates expression of the ANG II type 2 (AT₂) receptor in the vasculature and kidney; ovariectomy downregulates AT₂ receptor expression, which is prevented by estrogen in SHRs (52). AT₂ receptor blockade in mice minimizes ANG II-induced hypertension (45), and Mas receptor antagonists reverse protection against hypertension in female SHRs (55). Thus, the effect of estrogen to downregulate the prohypertensive components and upregulate the antihypertensive components of the renin-angiotensin system contribute to lower BP in female subjects.

We have previously reported that cytochrome P-450 (CYP)1B1, which is highly expressed in the cardiovascular and renal systems and is capable of metabolizing fatty acids, retinoids, and sex steroids, contributes to the development of hypertension and associated pathogenesis in male mice, mostly by generating ROS (20, 21, 23). However, in female mice, CYP1B1, which metabolizes 17β-estradiol to 2-hydroxyestradiol, which is then converted by catechol-O-methyltransferase (COMT) to 2-methoxyestradiol (2-MeE₂), protects against ANG II-induced hypertension and associated cardiovascular pathophysiological changes, including oxidative stress (22). ANG II, via its actions in the kidney, plays an important role in regulating BP, and increased levels of ANG II promote renal dysfunction and end-organ damage (9, 17, 20, 21, 44). Premenopausal female subjects are protected against the progression of renal disease, and estrogen and some of its metabolites exert renoprotective effects (12, 35). These observations and the demonstration that CYP1B1 is expressed in the kidney led to the hypothesis that CYP1B1 protects against renal dysfunction and end-organ damage associated with ANG II-induced hypertension in female subjects. To test this hypothesis, we investigated the effect of Cyp1b1 gene disruption on the actions of ANG II on renal function and the underlying mechanism(s) in female mice.

MATERIALS AND METHODS

Materials.

ANG II was purchased from Bachem (Torrance, CA), and dihydroethidium (DHE) was from Invitrogen (Carlsbad, CA). CYP1B1 antibody was purchased from BD Biosciences (Franklin Lakes, NJ), and antibodies against α-smooth muscle (α-SMA), 3-nitrotyrosine (3-NT), and transforming growth factor (TGF)-β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RT-PCR primers for ACE, AT_1A receptor, ACE2, AT₂ receptor, and Mas receptor were purchased from Integrated DNA Technologies (Coralville, IA); probes were purchased from Roche Diagnostics (Indianapolis, IN). All other chemicals were purchased from Sigma (St. Louis, MO).

Animals.

All experiments were performed according to protocols approved by our Institutional Animal Care and Use Committee in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Cyp1b1^−/− mice were validated as previously described (4) and backcrossed 10 generations to a C57BL/6 background, and then brother-sister mated to generate a homozygous line. Female C57BL/6 (Cyp1b1^+/+, Jackson Laboratory, Bar Harbor, ME) mice were used as control animals throughout the experiments for comparison of drug effects. All animals were 20–30 g and ∼8 wk of age at the beginning of the experiments. The genotype of Cyp1b1^+/+ and Cyp1b1^−/− mice was routinely assessed in our laboratory by PCR as previously described (6). For PCR analysis, genomic DNA was obtained from tail snips using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) according to the manufacturer's instructions.

Metabolic cage study for analysis of renal function.

Mice were anesthetized with ketamine (87 mg/kg ip) and xylazine (13 mg/kg ip), and micro-osmotic pumps were implanted subcutaneously to infuse ANG II (700 ng·kg⁻¹·min⁻¹) or vehicle (0.9% saline) for 14 days (model 1002, Alzet, Cupertino, CA). Systolic BP was measured twice weekly using a noninvasive tail-cuff method (model XBP 1000, Kent Scientific). The data on systolic BP have been previously reported (22). To assess renal function, these mice were housed on day 12 of ANG II infusion in metabolic cages for 24 h for the measurement of water consumption and separation of urine from fecal material and food waste. Urine was collected in tubes that contained a small volume of mineral oil to prevent evaporation. Animals were euthanized on day 14, and kidneys were collected for biochemical and histological analysis. After the calculation of volume, urine was aliquoted and stored at −80°C for further analysis. Urine was analyzed for osmolality using a Vapro vapor pressure osmometer (model 5520, Wescor, South Logan, UT), protein content by the standard Bradford method, and Na⁺ concentration by a flame photometer (model 443, Instrumentation Laboratory, Lexington, MA). Albumin concentration in urine samples was measured by a mouse albumin ELISA kit (Bethyl Laboratroies, Montgomery, TX) according to the manufacturer's instructions. Creatinine concentration in plasma samples was determined by HPLC/MS/MS at the Mouse Metabolic Phenotyping Center at Yale School of Medicine (New Haven, CT). For plasma collection, animals were anesthetized, and blood was withdrawn directly from the abdominal aorta and transferred to K⁺-EDTA tubes (BD Microtainer, BD Biosciences). Blood was centrifuged at 1,500 g for 15 min at 4°C, and plasma was collected and stored at −80°C until further analysis.

CYP1B1 activity assay.

CYP1B1 activity was determined using the P450-Glo Assay Kit (Promega) as previously described (20). At the completion of the experiments, animals were euthanized as described above, the left ventricle was punctured, and blood was flushed by perfusion with cold saline (3 min). Kidneys were dissected free, cleaned of surrounding tissue, snap frozen in liquid N₂, and stored at −80°C until use. Kidney samples were homogenized in ice-cold 0.1 M potassium phosphate buffer (pH 7.4) using a TissueLyser II (2 × 3 min). After homogenization, samples were centrifuged at 10,000 g for 20 min at 4°C, and the supernatant was collected and stored at −80°C until further use. Protein content in the samples was determined by the Bradford method, and 500 μg protein was added to a reaction mixture containing 20 μM L-CEE substrate and 0.1 M potassium phosphate buffer (pH 7.4) and incubated at 37°C for 10 min. NADPH (final concentration: 100 μM) was added, and the solution was further incubated at 37°C for 45 min. Finally, a 1:1 volume of luciferin detection reagent was added to the samples, and they were mixed for 10 s, after which they were incubated at room temperature for 20 min. Luminescence was measured with a luminometer (model TD-20/20, Turner Designs, Sunnyvale, CA). Potassium phosphate buffer was used as a blank and subtracted from each reading; activity was expressed as relative luminescence units.

Western blot analysis.

Mice were euthanized and kidneys were removed as described above. Kidney samples were homogenized in lysis buffer, and protein content was determined by the Bradford method. Approximately 10 μg protein was loaded and resolved on 8% SDS-polyacrylamide gels and processed for Western blot analysis as previously described (20, 62). Blots were probed with different primary antibodies and the corresponding secondary antibodies, and the intensity of the bands was measured with ImageJ 1.42 software (http://rsb.info.nih.gov/nih-image; NIH). Protein expression of CYP1B1 was calculated as a ratio of expression of β-actin.

Immunohistochemical analysis.

At the completion of the experiments, animals were anesthetized as described above, the carotid artery was cannulated, and animals were perfused with saline (3 min). The kidney was dissected free and placed in OCT compound (Sakura Finetek USA, Torrance, CA). Sections (10 μm) were processed α-SMA (measure of myofibroblasts), TGF-β, and Masson's trichrome staining (collagen deposition) as previously described (33). Additional sections were stained for 3-NT [an indicator of peroxynitrite, the resultant compound of the reaction between nitric oxide (NO) and superoxide] as previously described (2). Stained sections were viewed with an Olympus inverted system microscope (model BX41, Olympus America) and photographed with a SPOT Insight digital camera (model Insight 2MP Firewire, Diagnostic Instruments, Sterling Heights, MI). Staining of TGF-β, collagen, 3-NT, and α-SMA was quantitated by taking the average of three fields from each slide to calculate the mean values for three animals using ImageJ (rsb.info.nih.gov/ij/). Data are presented as the percent area of positive staining.

Measurements of renal mRNA expression of ACE, AT_1A receptor, ACE2, AT₂ receptor, and Mas receptor.

RNA was extracted from snap-frozen kidneys using the TRIzol method (8). Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) with 1 μg total RNA. ACE, AT_1A receptor, ACE2, AT₂ receptor, and Mas receptor expressions were analyzed by real-time RT-PCR. Quantitative real-time PCR was performed in 384-well plates with a LightCycler (LC)480 (Roche) using LC480 Master Mix and a Universal Probe Library (UPL) probe (Roche) at a concentration of 10 μM with a final reaction volume of 10 μl under the following conditions: 95°C for 5 min for activation and 45 cycles of 95°C for 10 s, 60°C for 60 s, and 72°C for 10 s for amplification. After six endogenous control genes were tested, β-actin was used as an endogenous control. The sequences of primers and the relevant probes (UPL) for ACE, AT_1A receptor, ACE2, AT₂ receptor, Mas receptor, and β-actin are shown in Table 1. All samples were analyzed in triplicate. Relative mRNA levels were normalized to the housekeeping gene mRNA in the same sample of cDNA. Expression of ACE, AT_1A receptor, ACE2, AT₂ receptor, and Mas receptor relative to β-actin in each sample was calculated on the basis of the ΔΔC_t method (where Ct is threshold cycle) as previously described (48).

Table 1.

Primer sequences for measuring renal RNA expression from genes associated with the ANG II pathway

	Sequence
Identification	Forward	Reverse	Amplicon Length	UPL Probe Number
AT1A	5′-GGCTTGAGTCCTGGTCCAC-3′	5′-CAGCCATTTTATACCAATCTTTCA-3′	131	41
AT2	5′-GGAGCTCGGAACTGAAAGC-3′	5′-CTGCAGCAACTCCAAATTCTT-3′	131	41
Mas	5′-GCATCGTCTTTCAGCTACTTCTC-3′	5′-TCTGGCAGGATCACAGAGTTT-3′	101	52
ACE	5′-TCTGCTTCCCCAACAAGACT-3′	5′-AGGATGTTGGTGAGTCTGG-3′	61	62
ACE2	5′-TGTAGAACGTACCTTCGCAGAG-3′	5′-GTAGGAAGGGTAGGTATCCATCAA-3′	91	101
β-Actin	5′-AAGGCCAACCGTGAAAAGAT-3′	5′-GTGGTACGACCAGAGGCATAC-3′	110	56

UPL, Universal Probe Library; AT_1A, ANG II type 1A receptor; AT₂, ANG II type 2 receptor; ACE, ANG-converting enzyme.

Measurement of NADPH oxidase activity.

NADPH oxidase activity was determined in tissue homogenates by measuring lucigenin (N,N′-dimethyl-9,9′-biacridinium dinitrate)-enhanced chemiluminescence as previously described (64) with some modifications (20). After anesthesia (described above), the kidney was isolated, cleaned of surrounding tissue, snap frozen in liquid N₂, and stored at −80°C until use. Tissue samples were homogenized (2 × 3 min) in ice-cold lysis buffer containing protease inhibitors (20 mmol/l phosphate buffer, 1 mmol/l EGTA, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 0.5 mmol/l PMSF, and 150 mmol/l sucrose) using a TissueLyser II (Qiagen). After homogenization, samples were sonicated and centrifuged at 10,000 g for 20 min at 4°C, and the supernatant was collected and stored at −80°C until further use. Protein content in the samples was determined by the Bradford method, and equal amounts of protein were combined 1:1 with a reaction mixture containing 5 μmol/l lucigenin (final concentration) and 100 μmol/l NADPH (final concentration). Luminescence was measured every minute for 10 min using a luminometer. Lysis buffer was used as a blank and subtracted from each reading, and activity was expressed as arbitrary units.

Measurement of renal superoxide production.

To measure superoxide production, kidney sections were exposed to DHE following previously described and validated methods (32). Fresh, unfixed kidney and artery samples were placed in OCT compound (Sakura Finetek USA) and frozen at −80°C. Kidney and vascular ring segments were cut into 30-μm sections using a cryostat (model CM1850, Leica Microsystems, Bannockburn, IL) and placed on a glass slide. Sections were incubated in PBS for 30 min at 37°C, and DHE (10 μmol/l) was then topically applied (59). Coverslips were applied, and sections were further incubated at 37°C in a light-protected, humidified chamber for 30 min. Sections were then rinsed in PBS, and fluorescence was detected using a 585-nm filter and an Olympus inverted system microscope (model IX50, Olympus America). Images were photographed with an Olympus digital camera (model DP71, Olympus America) and analyzed using ImageJ (version 1.42).

Measurement of urinary levels of thiobarbituric acid-reactive substances.

Urine levels of thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxidation, were measured using a TBARS assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.

Measurement of renal activity of antioxidant enzymes.

The renal activity of antioxidant enzymes (SOD and catalase) was measured. Total SOD activity was measured in kidney tissue using a SOD assay kit (Cayman Chemical) according to the manufacturer's instructions. Catalase activity was measured in kidney tissue using a Catalase assay kit (Cayman Chemical) according to the manufacturer's instructions.

Measurement of urinary levels of nitrate/nitrite.

Urine levels of nitrate/nitrite (NO_x), an indicator of NO bioavailability, were measured using a NO_x colorimetric assay kit (Cayman Chemical) according to the manufacturer's instructions.

Measurement of urinary levels of 2-MeE₂.

Urine levels of 2-MeE₂ were determined using a 2-MeE₂ enzyme immunoassay kit (Cayman Chemical) according to the manufacturer's instructions.

Statistical analysis.

Data were analyzed by one-way ANOVA followed by a Neuman-Keuls post hoc test or Student's t-test. Values of a minimum of three different experiments are expressed as means ± SE. P values of <0.05 were considered statistically significant.

RESULTS

Infusion of ANG II increased renal CYP1B1 activity and expression in female mice.

CYP1B1 activity and protein expression were increased in kidneys of ANG II-infused Cyp1b1^+/+ mice (Fig. 1, A and B, respectively). CYP1B1 activity and protein expression were absent in Cyp1b1^−/− mice (Fig. 1, A and B, respectively).

Fig. 1.

ANG II-induced hypertension is associated with increased renal cytochrome P-450 (CYP)1B1 activity and expression in female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk, as described in materials and methods. Animals were euthanized, and renal tissue was collected to measure CYP1B1 activity using the P450-Glo assay, as described in materials and methods. A: activity of CYP1B1 expressed as relative luminescence units (RLU). B: CYP1B1 protein expression was measured by Western blot analysis in renal tissue from vehicle- and ANG II-infused Cyp1b1^+/+ and Cyp1b1^−/− mice using ∼10 μg protein for loading, as described in materials and methods. CYP1B1 protein expression was normalized against β-actin, which was used as a loading control. au, Arbitrary units. Data are expressed as means ± SE; n = 3–5 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle.

ANG II-induced hypertension was associated with increased thirst and renal dysfunction in Cyp1b1^−/− but not Cyp1b1^+/+ mice.

Basal water intake and urine output were not different between Cyp1b1^+/+ and Cyp1b1^−/− mice (Table 2). Infusion of ANG II did not alter 24-h water consumption or urine output in Cyp1b1^+/+ mice; however, in Cyp1b1^−/− mice, infusion of ANG II caused a marked increase in daily water consumption and urine output (Table 2). Plasma levels of creatinine were not altered by ANG II in Cyp1b1^+/+ or Cyp1b1^−/− mice. ANG II infusion decreased urine osmolality, increased urinary excretion of Na⁺, and caused proteinuria and albuminuria in Cyp1b1^−/− but not Cyp1b1^+/+ mice (Table 2).

Table 2.

Renal dysfunction caused by infusion of ANG II is observed in Cyp1b1^−/− but not Cyp1b1^+/+ female mice

	Cyp1b1+/+ Mice		Cyp1b1−/− Mice
Parameter	Vehicle	ANG II	Vehicle	ANG II
Water intake, ml/24 h	5.20 ± 0.12	5.40 ± 0.94	5.50 ± 0.42	8.80 ± 0.60*
Urine output, ml/24 h	1.44 ± 0.26	2.46 ± 0.74	1.44 ± 0.37	4.30 ± 0.37*
Plasma creatinine, mg/dl	0.078 ± 0.002	0.077 ± 0.005	0.072 ± 0.002	0.075 ± 0.010
Urinary osmolality, mosM/kg	2720 ± 115	2140 ± 262	2630 ± 79	1281 ± 205*
Urinary Na+ excretion, mmol/24 h	0.045 ± 0.018	0.045 ± 0.021	0.031 ± 0.016	0.099 ± 0.010*
Proteinuria, mg/24 h	2.49 ± 0.47	2.64 ± 0.50	2.60 ± 0.30	6.96 ± 0.55*
Albuminuria, mg/24 h	0.23 ± 0.13	0.30 ± 0.10	0.13 ± 0.03	1.63 ± 0.33*
Body weight, g	20.1 ± 0.3	20.6 ± 1.2	21.0 ± 0.6	22.3 ± 0.7
Food intake, g/24 h	5.03 ± 0.21	4.62 ± 0.84	5.83 ± 0.18	5.31 ± 1.13

Data are expressed as means ± SE; n = 5 for all experiments. Cytochrome P-450 (Cyp)1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk and placed in metabolic cages for 24 h before the experiments were completed. The parameters listed above were determined as described in materials and methods.

^*P < 0.05, vehicle versus the corresponding value from ANG II-infused animals.

Cyp1b1 gene disruption resulted in renal damage caused by infusion of ANG II.

Infusion of ANG II was associated with increased interstitial fibrosis, as indicated by increased staining of interstitial α-SMA and TGF-β in kidneys of Cyp1b1^−/− but not Cyp1b1^+/+ mice (Figs. 2 and 3, respectively). Staining of kidney sections with Masson's trichrome revealed collagen deposition in the interstitial spaces of kidneys from ANG II-treated Cyp1b1^−/− mice; collagen deposition was reduced in Cyp1b1^+/+ mice (Fig. 4).

Fig. 2.

Renal interstitial accumulation of α-smooth muscle actin (α-SMA) caused by ANG II infusion in Cyp1b1^−/− but not Cyp1b1^+/+ female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk, as described in materials and methods. A: ANG II infusion increased renal interstitial staining of α-SMA in Cyp1b1^−/− mice, which was minimized in Cyp1b1^+/+ mice. B: graph showing quantified data. Data are expressed as means ± SE; n = 3 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Fig. 3.

Renal deposition of transforming growth factor (TGF)-β caused by ANG II infusion in Cyp1b1^−/− but not Cyp1b1^+/+ female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk, as described in materials and methods. A: ANG II infusion increased interstitial staining of TGF-β in kidneys of Cyp1b1^−/− but not Cyp1b1^+/+ mice. B: graph showing quantified data. Data are expressed as means ± SE; n = 3 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Fig. 4.

Renal collagen deposition caused by ANG II infusion in Cyp1b1^−/− but not Cyp1b1^+/+ female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk, as described in materials and methods. A: ANG II infusion increased collagen deposition, as revealed by blue Masson's trichrome staining in kidneys of ANG II-treated Cyp1b1^−/− but not Cyp1b1^+/+ mice. B: graph showing quantified data. Data are expressed as means ± SE; n = 3 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Infusion of ANG II increased renal NADPH oxidase activity, superoxide production, and urinary excretion of TBARS in Cyp1b1^−/− but not Cyp1b1^+/+ mice.

Infusion of ANG II was associated with increased renal NADPH oxidase activity in Cyp1b1^−/− but not Cyp1b1^+/+ mice (Fig. 5A). The increase in NADPH oxidase activity in Cyp1b1^−/− mice correlated with an increase in renal superoxide production, as indicated by increased 2-hydroxyethidium fluorescence intensity within the kidney sections, specifically in the glomerulus; there was no increase in 2-hydroxyethidium fluorescence in ANG II-infused Cyp1b1^+/+ mice (Fig. 5, B and C). ANG II infusion was also associated with increased urinary excretion of TBARS, an indicator of lipid peroxidation, in Cyp1b1^−/− but not Cyp1b1^+/+ mice (Fig. 5D).

Fig. 5.

ANG II infusion increased renal NADPH oxidase activity, superoxide production, and urinary excretion of thiobarbituric acid-reactive substances (TBARS) in Cyp1b1^−/− female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk. A: NADPH oxidase activity in renal homogenates. B: superoxide production, as determined by fluorescence intensity of 2-hydroxyethidium, in the kidney. Representative photomicrographs of kidneys from mice in each of the different treatment groups after incubation with dihydroethidium are shown (Arrows indicate glomeruli). C: graph showing quantified data. D: at the completion of the experiments, urine was collected, and TBARS excretion was determined using a commercially available kit. Data are expressed as means ± SE; n = 4–5 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Infusion of ANG II increased renal SOD activity in Cyp1b1^+/+ but not Cyp1b1^−/− mice and decreased renal catalase activity in Cyp1b1^−/− but not Cyp1b1^+/+ mice.

In Cyp1b1^+/+ mice, infusion of ANG II was associated with an increase in total SOD activity in the kidney; this increase was absent in ANG II-infused Cyp1b1^−/− mice (Fig. 6A). In contrast, renal catalase activity was decreased in Cyp1b1^−/− mice infused with ANG II but was unchanged in ANG II-infused Cyp1b1^+/+ mice (Fig. 6).

Fig. 6.

ANG II infusion differentially regulates activities of antioxidant defense enzymes in kidneys of Cyp1b1^+/+ and Cyp1b1^−/− female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk. At the completion of the experiments, renal tissue was collected to measure SOD activity (A) and catalase activity (B) using commercially available kits. Data are expressed as means ± SE; n = 5 for all experiments. *P < 0.05 ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Infusion of ANG II was associated with decreased urinary excretion of NO_x and increased renal expression of 3-NT in Cyp1b1^−/− but not Cyp1b1^+/+ mice.

Urinary excretion of NO_x, an indicator of NO bioavailability, was not altered in Cyp1b1^+/+ mice infused with ANG II; however, in Cyp1b1^−/− mice, infusion of ANG II resulted in a dramatic decrease in urinary excretion of NO_x (Fig. 7A). The decreased urinary excretion of NO_x in Cyp1b1^−/− mice correlated with an increase in 3-NT-positive staining in the kidney, an indicator of peroxynitrite formation, which resulted from the reaction between NO and superoxide; this increase was minimized in Cyp1b1^+/+ mice (Fig. 7, B and C).

Fig. 7.

Infusion of ANG II decreased nitric oxide (NO) bioavailability and increased production of 3-nitrotyrosine (3-NT) in kidneys of Cyp1b1^−/− but not Cyp1b1^+/+ female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk. A: at the completion of the experiments, urine was collected, and excretion of nitrite/nitrate (NO_x) was determined using a commercially available kit. B: increased staining of 3-NT, an indicator of peroxynitrite formation, was observed in kidney sections from ANG II-treated Cyp1b1^−/− mice but not Cyp1b1^+/+ mice. C: graph showing quantified data of 3-NT-positive staining. Data are expressed as means ± SE; n = 3 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

ANG II infusion decreased renal mRNA expression of Mas receptor but not AT_1A receptor, AT₂ receptor, ACE, or ACE2 in Cyp1b1^+/+ mice and decreased expression of AT₂ receptor, Mas receptor, and ACE2 in Cyp1b1^−/− mice.

Cyp1b1 gene disruption did not alter basal mRNA encoding AT_1A receptor, AT₂ receptor, ACE, ACE2, or Mas receptor (Fig. 8, A–E). Infusion of ANG II did not alter mRNAs encoding AT_1A receptor, AT₂ receptor, ACE, or ACE2 compared with basal levels of these mRNAs in Cyp1b1^+/+ and Cyp1b1^−/− mice (Fig. 8, A–D). However, infusion of ANG II decreased mRNA expression of Mas receptor in Cyp1b1^+/+ and Cyp1b1^−/− mice (Fig. 8E). Comparison of the expression of renal mRNA in these two mouse phenotypes showed that, during ANG II infusion, AT₂ receptor mRNA expression and ACE2 mRNA expression were decreased in Cyp1b1^−/− mice compared with Cyp1b1^+/+ mice (Fig. 8, B and D, respectively).

Fig. 8.

Infusion of ANG II is not associated with changes in renal ANG II type 1A (AT_1A) receptor or angiotensin-converting enzyme (ACE)2 expression in Cyp1b1^+/+ and Cyp1b1^−/− mice but decreased ANG II type 2 (AT₂) receptor and ACE2 expression in Cyp1b1^−/− mice and Mas receptor expression in Cyp1b1^+/+ and Cyp1b1^−/− female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk. A–E: renal tissue was collected to measure mRNA expression of AT_1A receptor (A), AT₂ receptor (B), ACE (C), ACE2 (D), and Mas receptor (E), as described in materials and methods. mRNA gene expression was normalized against β-actin, which was used as a housekeeping gene, and calculated on the basis of the ΔΔC_t method (where C_t is threshold cycle). Data are expressed as means ± SE; n = 3 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice.

Measurement of urinary levels of 2-MeE₂.

Basal urinary excretion of 2-MeE₂ was decreased in Cyp1b1^−/− mice (Fig. 9). Infusion of ANG II increased urinary excretion of 2-MeE₂ in Cyp1b1^+/+ but not Cyp1b1^−/− mice (Fig. 9).

Fig. 9.

Infusion of ANG II increased 2-methoxyestradiol (2-MeE₂) production in Cyp1b1^+/+ but not Cyp1b1^−/− female mice. Cyp1b1^+/+ and Cyp1b1^−/− mice were infused with vehicle or ANG II for 2 wk. At the completion of the experiments, urine was collected, and excretion of 2-MeE₂ was determined using a commercially available kit. Data are expressed as means ± SE; n = 4 for all experiments. *P < 0.05, ANG II vs. the corresponding value with vehicle; †P < 0.05, ANG II-infused Cyp1b1^−/− mice vs. ANG II-infused Cyp1b1^+/+ mice; ‡P < 0.05, vehicle-infused Cyp1b1^−/− mice vs. vehicle-infused Cyp1b1^+/+ mice.

DISCUSSION

The major finding of the present study is that CYP1B1, which contributes to renal dysfunction and organ damage in male mice associated with ANG II-induced hypertension (20, 21), had an opposite role in female mice. CYP1B1 protects against ANG II-induced renal dysfunction and end-organ damage in female mice by promoting decreased oxidative stress and increased activity of antioxidant systems, most likely as a result of the effect of 2-MeE₂ from 17β-estradiol. Infusion of ANG II for 2 wk did not alter water intake or urine output or promote renal dysfunction in Cyp1b1^+/+ mice, but in Cyp1b1^−/− mice, ANG II increased water intake, urine output, and Na⁺ excretion, decreased urine osmolality, and caused marked proteinuria and albuminuria. These observations, together with our demonstration that ANG II increased CYP1B1 activity and expression in Cyp1b1^+/+ mice, suggest that CYP1B1 plays a protective role against ANG II-induced renal dysfunction in female mice. Furthermore, the finding that ANG II increased water intake in Cyp1b1^−/− but not Cyp1b1^+/+ mice indicated that Cyp1b1 could also be involved in regulating the central action of ANG II on thirst in female mice. Alternatively, the increased urine output in Cyp1b1^−/− mice could be due to increased water intake. Another interesting finding was that ANG II increased Na⁺ excretion in Cyp1b1^−/− mice, which could be due to an increased effect of ANG II on BP that overrides the direct tubular effects of ANG II. In contrast to female Cyp1b1^−/− mice, in male Cyp1b1^+/+ mice, where ANG II markedly increases BP, it also increased Na⁺ excretion but minimized the increase in both BP and Na⁺ excretion in Cyp1b1^−/− mice (20). Further studies to determine fractional Na⁺ excretion and its time course and total body Na⁺ are required to address the role of CYP1B1 on the actions of ANG II on Na⁺ balance in female and male mice. Our demonstration that ANG II in Cyp1b1^−/− but not Cyp1b1^+/+ mice caused proteinuria and albuminuria, as well as renal fibrosis, as indicated by interstitial α-smooth muscle actin, TGF-β, and collagen accumulation, suggests that CYP1B1 also protects against ANG II-induced organ damage in female mice. The degree of these changes produced by ANG II in Cyp1b1^−/− mice appeared to be insufficient to alter glomerular filtration rate, as indicated by the lack of change in plasma levels of creatinine.

The mechanism by which ANG II produces renal dysfunction and end-organ damage in Cyp1b1^−/− mice could be due to increased BP. Although we cannot exclude this possibility, ANG II may also produce cardiovascular or renal pathophysiological changes independent of increased BP (15, 25, 30, 54). For example, treatment with triple therapy (hydralazine, reserpine, and hydrochlorothiazide) prevents increased BP but not end-organ damage, inflammation, or cellular growth in the kidney of transgenic rats carrying both human renin and angiotensinogen genes (30). ANG II is known to increase production of ROS, which contributes to the development of hypertension and end-organ damage in male rats and mice (20, 21, 23, 41). However, ROS do not appear to contribute to the development of hypertension in various models of experimental hypertension in female subjects (27). The progression of renal disease caused by renal artery pseudoaneurysm, which is minimized in female rats by 17β-estradiol, is associated with decreased NADPH oxidase activity and expression of its subunit p22^phox (24). ANG II infusion does not alter the expression of components of NADPH oxidase or urinary excretion of TBARS and reduces NO_x in the female mouse kidney (50). In the present study, infusion of ANG II did not alter NADPH oxidase activity, production of ROS, or urinary excretion of NO_x, an index of NO production, but increased SOD activity and did not affect catalase activity in Cyp1b1^+/+ mice. However, in Cyp1b1^−/− mice, ANG II infusion increased renal NADPH oxidase activity and ROS production, reduced urinary excretion of NO_x, and increased 3-NT deposition and activity of SOD and catalase. These observations suggest that CYP1B1 plays a protective role in the female kidney against ANG II-induced oxidative stress by increasing the activity of antioxidants and minimizing an increase in the activity of oxidants and consequently preventing end-organ damage.

The protection against ANG II-induced hypertension in female subjects compared with male subjects has been attributed to the action of 17β-estradiol to downregulate AT₁ receptors (10, 45) and upregulate AT₂ receptors (52). Expression of ACE2 and ANG(1–7) levels, which are increased in female SHRs compared with male SHRs, has been shown to be responsible for the lower BP in female SHRs than in male SHRs (55). ANG II, via AT₂ receptor activation, and ANG(1–7), generated via increased ACE2 activity through Mas and/or AT₂ receptors, produce cardiovascular and renoprotective effects and counteract the actions of ANG II mediated via AT₁ receptors (5). Although the renal expression of AT₂ receptors in female rats, including SHRs, is higher than in male rats, infusion of ANG II does not alter their expression (46, 52). In the present study, Cyp1b1 gene deletion did not alter basal mRNA expression of AT_1A receptor, AT₂ receptor, Mas receptor, ACE, or ACE2 compared with Cyp1b1^+/+ mice, indicating that basal expression of these components of the renin-angiotensin system in the kidney is independent of the Cyp1b1 gene. Infusion of ANG II did not alter renal mRNA expression of AT₂ receptor or ACE2 in Cyp1b1^+/+ or Cyp1b1^−/− mice. ANG II produces a small, insignificant decrease in Mas receptor mRNA expression but an increase in protein expression in female SHRs (55). In our study, infusion of ANG II decreased Mas receptor mRNA expression in both Cyp1b1^+/+ and Cyp1b1^−/− mice, suggesting that it is likely to contribute to renal protection against ANG II in Cyp1b1^+/+ mice. We, however, have no explanation for the decreased mRNA expression of Mas receptor in Cyp1b1^−/− mice by ANG II.

In the present study, Cyp1b1 gene disruption abolished both basal and ANG II-induced increases in its activity; however, it did not alter basal renal function or cause kidney damage but allowed ANG II to cause renal dysfunction, oxidative stress, and end-organ damage. Therefore, it appears that CYP1B1 is not required to maintain basal renal function but is required to suppress the deleterious effect of ANG II in female mice. Since CYP1B1 is constitutively expressed, it appears that ANG II stimulates the production of a product that protects against the renal actions of this peptide in Cyp1b1^+/+ mice. The protection against ANG II-induced hypertension in female rats and mice has been attributed to estrogen, which also protects against the progression of renal disease in female subjects (18, 37, 43, 61). Lack of estrogen produced by ovariectomy promotes renal dysfunction, and 17β-estradiol replacement improves renal function and associated pathogenesis in streptozotocin-induced diabetic rats (28).

CYP1B1 metabolizes 17β-estradiol into hydroxyestradiols (OHEs), namely, 2-OHE and 4-OHE, which are subsequently metabolized by COMT into 2-MeE₂ and 4-MeE₂ (40, 65). 2-MeE₂ exerts cardiovascular protective effects (12). Recently, we showed that ANG II stimulates the production of 2-MeE₂, which protects against ANG II-induced hypertension in female Cpy1b1^+/+ mice (22). 17β-Estradiol, 2-OHE, and/or 2-MeE₂ also exert several renoprotective effects, including inhibition of mesangial cell proliferation and synthesis of collagen (11), expression and activity of TGF-β, expression of collagen types I and IV (29, 36, 51), and increased expression of extracellular matrix-degrading metalloproteinase (39). 2-OHE also slows the progression of renal dysfunction in an experimental model of polycystic kidney disease (1). These observations, together with the demonstration that ANG II increased urinary levels of 2-MeE₂ in Cyp1b1^+/+ mice and decreased its level in Cyp1b1^−/− mice, suggest that protection against ANG II-induced renal dysfunction and end-organ damage in Cyp1b1^+/+ female mice is mediated by 2-MeE₂. Since ANG II-induced changes in urinary levels of 2-MeE₂ followed the same pattern in Cyp1b1^−/− and Cyp1b1^+/+ female mice to that observed in plasma levels, the urinary 2-MeE₂ could be derived from extrarenal sources. However, in view of the expression of both CYP1B1 and COMT in the kidney, it is possible that urinary 2-MeE₂ could also be derived from the kidney. Further studies are required to determine the effect of 2-OHE, MeE₂, and other estrogen metabolites (26) on ANG II-induced renal dysfunction and end-organ damage in Cyp1b1^−/− female and Cyp1b1^+/+ male mice and their relationship to oxidative stress and antioxidant systems. Moreover, in view of an essential role of T cells in ANG II-induced hypertension in male mice (16), which is minimized in female mice and enhanced by ovariectomy (38), the contribution of T cells to17β-estradiol metabolites in modulating the actions of ANG II needs to be investigated. Furthermore, in light of the paradoxical effects of estrogen and its CYP1B1-derived metabolite 2-MeE₂ to attenuate (57, 60) and 16α-hydroxyesterone (58) to promote hypoxia-induced pulmonary arterial hypertension, it is not known if Cyp1b1 gene disruption modifies any action of ANG II on the pulmonary circulation and right ventricular function in female mice.

In conclusion, the present study showed that CYP1B1 plays a critical role in female mice in protecting against ANG II-induced renal dysfunction and end-organ damage associated with hypertension by minimizing oxidative stress and by increasing activity of antioxidant systems. These renoprotective effects of CYP1B1 against ANG II in female mice are most likely mediated by the 17β-estradiol metabolite 2-MeE₂. In contrast, in male mice, CYP1B1 contributes to ANG II-induced hypertension (20). Our preliminary observations indicate that in male mice, 6β-hydroxytestosterone, which can be generated by CYP1B1 from testosterone, contributes to ANG II-induced hypertension and its pathogenesis (Pingili A. K. et al., unpublished observations). Recently, it has been demonstrated that, in addition to gonadal hormones, sex chromosome complement plays an important role in the development of ANG II-induced hypertension (19). Therefore, further studies are required to elucidate the relationship between sex chromosome complement and CYP1B1 in the action of ANG II on renal function in both male and female subjects. The present study has important clinical implications: agents that inhibit activity of Cyp1b1 could be detrimental to renal function in female subjects by reducing the production of the Cyp1b1 metabolite 2-OHE and, subsequently, 2-MeE₂, which have renoprotective effects.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-19134-38-39 (to K. U. Malik) and K01-HL-96410-04 (to A. Kanu). J. A. Moore was supported by a summer student fellowship from the American Society of Pharmacology and Experimental Therapeutics.

DISCLAIMER

The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Heart, Lung, and Blood Institute.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: B.L.J. and K.U.M. conception and design of research; B.L.J., J.A.M., A.K.P., A.M.E., and X.R.F. performed experiments; B.L.J., J.A.M., and A.K.P. analyzed data; B.L.J., J.A.M., A.K.P., and K.U.M. interpreted results of experiments; B.L.J. and J.A.M. prepared figures; B.L.J. and K.U.M. drafted manuscript; B.L.J., A.K.P., A.K., F.J.G., and K.U.M. edited and revised manuscript; B.L.J., J.A.M., A.K.P., A.M.E., X.R.F., A.K., F.J.G., and K.U.M. approved final version of manuscript.