Oxidative Stress Contributes to Renovascular Hypertension

articles

nature publishing group

Oxidative Stress Contributes to Renovascular Hypertension Elizabeth B. Oliveira-Sales1, Adriana P. Dugaich1, Bruno A. Carillo1, Nayda P. Abreu2, Mirian A. Boim2, Paulo J. Martins3, Vânia D’Almeida3, Miriam S. Dolnikoff1, Cássia T. Bergamaschi4 and Ruy R. Campos1 Background Oxidative stress is a state in which excess reactive oxygen species (ROS) overwhelm endogenous antioxidant systems. It is known that this state has been involved in the development of hypertension. On the basis of previous data, we hypothesized that overactivity of NAD(P)H oxidase-derived ROS and the lowered activity of CuZnSOD, an endogenous antioxidant within the rostral ventrolateral medulla (RVLM), could contribute to 2K-1C (two-kidney one-clip) hypertension. Moreover, to test the functional significance of whether oxidative stress was involved in the maintenance of sympathetic vasomotor tone and blood pressure in 2K-1C hypertension, we administered Ascorbic Acid (Vit C), an antioxidant, into the RVLM or systemically.

The mean arterial blood pressure, heart rate, and renal sympathetic nerve activity were analyzed. Blood samples were collected and measured using thiobarbituric acid-reactive substances (TBARS).

Methods Experiments were performed in male Wistar rats (6 weeks after renal surgery - Goldblatt hypertension model - 2K-1C). The mRNA expression of NAD(P)H oxidase subunits (p47phox and gp91phox) and CuZnSOD were analyzed in the RVLM using real-time PCR technique.

conclusions The results support the idea that an increase in oxidative stress within the RVLM and systemically plays a major role in maintaining high arterial blood pressure and sympathetic drive in 2K-1C hypertension.

There is increasing evidence to support the hypothesis that reactive oxygen species (ROS) play a role in the pathophysiology of arterial hypertension. The ROS, particularly the superoxide anions (O2−), are important intracellular messengers for Angiotensin II (Ang II) action in the brain. Ang II increases the activity of NAD(P)H oxidase, the major source of O2− in the vasculature, and enhances O2− production, both systemically and in the central nervous system.1,2,3 For instance, intracerebroventricular infusion of NAD(P)H oxidase inhibitor antagonizes the increase in renal sympathetic nerve activity (RSNA) and the pressor response induced by centrally mediated Ang II actions.3,4 In the brain, overexpression of superoxide dismutase (SOD), the enzyme responsible for O2− breakdown, also abolishes the central pressor effect of the octapeptide.5 1Cardiovascular Division, Department of Physiology, Federal University of São Paulo, Santos, São Paulo, Brazil; 2Nephrology Division, Department of Medicine, Federal University of São Paulo, Santos, São Paulo, Brazil; 3Psychobiology Division, Department of Psychobiology, Federal University of São Paulo, Santos São Paulo, Brazil; 4Department of Health Sciences, Federal University of São Paulo, Santos, São Paluo, Brazil. Correspondence: Ruy R. Campos ([email protected])

Received 18 July 2007; first decision 22 August 2007; accepted 1 October 2007. doi:10.1038/ajh.2007.12 © 2008 American Journal of Hypertension, Ltd. 98

Results The mRNA expression of NAD(P)H oxidase subnits (p47phox and gp91pox) was greater in 2K-1C compared to the control group in the RVLM, and CuZnSOD expression was similar in both groups. In the RVLM, Vit C resulted in a fall in arterial pressure and in the sympathetic activity only in the 2K-1C rats. Thiobarbituric acid-reactive substances (TBARS) were significantly greater in 2K-1C rats and the acute infusion of Vit C significantly decreased arterial pressure and renal sympathetic activity in 2K-1C.

Am J Hypertens 2008; 21:98-104 © 2008 American Journal of Hypertension, Ltd.

There is evidence to suggest that the central nervous system is involved in the development and maintenance of arterial hypertension.6 However, the precise mechanisms by which the central nervous system participates in the evolution of arterial hypertension remains unclear and depends on the specific origin and phase of the hypertensive state. It is well known that in the 2K-1C (two-kidney one-clip) Goldblatt hypertension, the rostral ventrolateral medulla (RVLM) is one of the most important regions involved in the hypertension.7 Therefore, the first aim of the present study was to examine whether there is an increase in oxidative stress within the RVLM in 2K-1C hypertension. For this purpose, the NAD(P)H oxidase subunits (p47phox and gp91phox) and CuZnSOD mRNA expression were quantified in the RVLM of 2K-1C hypertensive rats. Tai et al.8 found that in SHR, elevated O2− radical production in the RVLM was associated with increased sympathetic nervous system activity; however, this mechanism had not been tested in a renovascular hypertension model. The second aim of the present study was therefore to examine whether oxidative stress is involved in the maintenance of sympathetic vasomotor tone and hypertension in 2K-1C rats. For this purpose, we injected directly into the RVLM the antioxidant ascorbic acid (vitamin C—Vit C), that acts as a free radical

january 2008 | VOLUME 21 NUMBER 1 | 98-104 | AMERICAN JOURNAL OF HYPERTENSION

Oxidative Stress Contributes to Renovascular Hypertension

scavenger in the brain. Vit C is believed to have critical functions in the brain, including a role as a cofactor of dopamine β-hydroxylase, and is therefore involved in catecholamine biosynthesis. The concentration of Vit C in the brain is tenfold higher in the brain than in other parts of the body, thereby suggesting that there is an active transport of ascorbic acid to the brain.9 However, Vit C crosses the blood-brain barrier only in the form of dehydroascorbic acid—the oxidized form of Vit C—through Glut-1 transporter. In this study, in an attempt to evaluate possible changes in Glut-1 transporter levels within the RVLM of 2K-1C rats, the Glut-1 mRNA expression was quantified in this region of the brain in hypertensive as well as in control rats. Finally, in 2K-1C rats, oxidative stress was indirectly estimated from the levels of thiobarbituric acid-reactive substances (TBARS) in the blood, and the effects of Vit C (administered intravenously) on blood pressure and sympathetic activity were evaluated in renovascular hypertensive rats, so as to address the peripheral contribution of oxidative stress in this experimental model of hypertension. Methods

All experimental procedures were conducted according to the National Institutes of Health guidelines for the use and care of animals, and the study protocol was approved by the Ethics in Research Committee of the Federal University of Sao Paulo School of Medicine (process no. 0662/04). Male Wistar rats (n = 58, 250–300 g) were obtained from the animal care facility of our institution. The animals were housed in group cages, given access to rat chow and water ad libitum and maintained in a temperature-controlled environment (23 °C) on a 12-h light/ dark cycle. The drugs (urethane—Ethil carbamate, phenylefrine, and hexamethonium—Sigma Chemical, St Louis, MO) were all dissolved in saline. The Vit C (Sigma Chemical) and glutamate (Sigma Chemical) were diluted in phosphate-buffered saline (pH 7.4). Phosphate buffered saline (pH 7.4) was injected either directly into the RVLM (100 nl) or intravenously into animals in the control group. Experimental protocols. The study was divided into three series of experiments. In the first series, we compared the p47phox, gp91phox, CuZnSOD, and Glut-1 mRNA expression levels quantified by real-time PCR in the RVLM of control (CT) (n = 5) and 2K-1C rats (n = 5). In the second series of experiments, Vit C (10 nmol/100 nl) or phosphate buffered saline (pH 7.4) was bilaterally microinjected into the RVLM in four groups of rats: (i) CT + Vit C (n = 6); (ii) 2K-1C + Vit C (n = 4); (iii) CT + phosphate-buffered saline (n = 6); (iv) 2K-1C + phosphate-buffered saline (n = 6). In the third series of experiments, we evaluated the acute effects of either Vit C (250 mg/kg)10 diluted in phosphate-buffered saline, or phosphate-buffered saline alone, IV administered using an infusion pump for 6 min. The cardiovascular parameters and RSNA were analyzed up to 30 min after the beginning of the infusion.10 In this series of experiments, the following tests were performed: (i) CT + Vit C (n = 6); (ii) 2K-1C + AMERICAN JOURNAL OF HYPERTENSION | VOLUME 21 NUMBER 1 | january 2008

articles Vit C (n = 6); (iii) CT + phosphate-buffered saline (n = 6); (iv) 2K-1C + phosphate-buffered saline (n = 6). Hypertensive animals. In order to make the animals hypertensive, the left renal artery was partially obstructed with a silver clip of 0.2 mm width and the animals were subjected to the experimental procedures 6 weeks after the surgery.7 The surgical procedure was performed in male Wistar rats (150–180 g). Extraction of RNA from RVLM. The rats were killed and the brains were quickly removed, and sections 100 µm thick were prepared using a cryostat at −7 ± 12 °C. The RVLM was defined according to a rat brain atlas, and the brain region was obtained by using a punch-out technique. The RVLMs were homogenized in 10 µl of 10% TRITON and RNAsin (Invitrogen). In order to avoid contamination with genomic DNA, the RNA samples were treated with DNase (RQ1 Rnase—Free Dnase, Promega). The purity of the RNA was verified by performing PCR on samples not treated with reverse transcriptase. Quantitative real-time PCR. The following primer sequences were used: β-actin, forward (CCT-CTA–TGC-CAA-CACAGT-GC), reverse (ACA-TCT-GCT-GGA–AGG-TGG-AC); p47phox, forward (GTC-TGA-GGG-TGA-AGC-CAT-CG), reverse (CCG-AGA-ACG-CTG-GTG-GAT-GC); gp91phox, forward (AGT-GGT-TCG-CAG-ACC-TGC-TG), reverse (TCT-GGT-GTT-GGG-GTG-TTG-AC); CuZnSOD, forward (TGA-AGG-CGA-GCA-TGG-GTT-CC), reverse (ACA-CCGTCC-TTT-CCA-GCA-GC); and Glut-1, forward (TGG-TCCTCA-GCA-AGG-GGC-TC), reverse (ATC-TGG-ATG-GACGGG-AAC-TC). The sizes of the PCR products amplified using the primers were: β-actin—191 bp; p47phox—211 bp; gp91phox—239 bp; CuZnSOD—165 bp; and Glut-1—190 bp). Real time amplification was obtained using a Rotor Gene 600 (Uniscience). Real-time PCR product accumulation was monitored using the intercalating dye, SYBR Green real-time-PCR Master Mix (Applied Biosystems, CA), which exhibits greater fluorescence upon binding of double-stranded DNA. The relative gene expression was calculated using conditions at the early stages of PCR, when amplification is logarithmic, and can therefore be correlated to the initial copy number of gene transcripts. The reactions were cycled 50 times under the conditions previously determined by conventional PCR. At the end of the PCR, the temperature was increased from 60 to 95 °C at a rate of 2 °C/min, and fluorescence was measured every 15 s in order to construct the melting curve. A non-template control was run with each assay. The relative amount of each mRNA was estimated using a standard curve constructed from serial dilutions of complementary DNA including 1/1, 1/10, and 1/100. Results of five experiments per group are reported as relative expression normalized with the β-actin housekeeping gene (used as an endogenous control), and expressed in arbitrary units. For the purpose of graphic representation, the multiplicity of variation was then determined using the 2−(ΔΔCt) formula according to published protocols11 and following the manufacturer’s recommendations. 99

articles


Analysis of the sympathetic nerve activity. For recording of RSNA, the left renal nerve was retroperitoneally exposed, placed on bipolar silver electrodes and, when the conditions

Statistical analysis. The results are presented as mean values ± SE. The data were evaluated using one-way ANOVA followed by Student-Newman—Keuls method and paired Student’s t-test where appropriate. The level of statistical significance was defined as P < 0.05. a

30

NTS

10

p47phox

STN NA

NA

RVLM

RVLM ION ION CST CST

b

4

1.25

CuZnSOD RVLM

1.00 0.75 0.50 0.25 0.00

gp91phox

c

NTS

STN

*

20

0

b

NAD(P)H subunits RVLM

mRNA expression (Arbitrary units)

a

TBARS. In order to access lipid peroxidation, we analyzed the levels of TBARS in plasma by colorimetric assay (535 nm) modified from Ohkawa et al.13 Briefly, 200 µl of plasma were incubated with 1 ml of thiobarbituric acid (8.6 mg/ml) for 20 min in boiling water, followed by 20 min in an ice bath. Before absorbance measurements, the samples were centrifuged for 6 min at 13,000g to remove debris.


Microinjection procedures. The animals were placed in a stereotaxic frame and an occipital craniotomy was performed to expose the dorsal surface of the brainstem and cerebellum. The RVLM was located 3 mm rostral to the calamus scriptorius, 1.7–1.8 mm lateral to the midline and 3 mm deep from the dorsal medullary surface (bite bar = −11 mm). Bilateral microinjections were administered with the use of glass micropipettes with tip diameters of 10–20 µm connected to a pressure injector. In all the experiments, glutamate was first microinjected into the RVLM to verify that the coordinates had placed the pipette into a functional pressor RVLM site. Microinjections consisted of glutamate (10 nmol) and Vit C (10 nmol) in a volume of 100 nl. At the end of the experiments, 100 nl of Evans blue dye was injected into the RVLM. The animals were killed with an overdose of urethane, and the brainstem was removed and fixed by immersion for at least 72 h in 10% paraformaldehyde solution. Transverse 40 µm frozen sections were cut and mounted. Figure 1 is a representative histological coronal view showing the dye distribution within the RVLM region. When the dye was bilaterally deposited ventral to nucleus ambiguous and lateral to inferior olivary nucleus, this was considered a positive histology.

for nerve recording had been established, the nerve and electrode were covered with paraffin oil. The signal from the renal nerve was displayed on an oscilloscope and the nerve activity was amplified (gain 20,000, Neurolog, Digitimer), filtered with a band-pass filter (50–1,000 Hz), and collected for display and analysis using a PowerLab data acquisition system (ADInstruments). At the end of the experiments, the background noise level was determined after hexamethonium bromide (30 mg/kg, IV) administration. The neural activity was analyzed “off line” using appropriate software (Spike Histogram, ADInstruments). The responses of RSNA to the various stimuli were expressed as the percentage of change compared with the basal value immediately before each test. For this purpose, the raw nerve signal was passed through a spike discriminator (PowerLab) to remove background noise, and then the total nerve activity was computed and expressed in spikes per second from the time point when it changed from basal value to the time point when it returned to basal value. The mean value obtained was compared with the mean value before each test, as reported previously.12 Only experiments in which the level of background noise was confirmed at the end of the experiments following hexamethonium and terminal anesthesia are included in this report.


Surgical instrumentation for the acquisition of the cardiovascular parameters. Six weeks after renal surgery, the rats were anesthetized using ketamine and xylazine (40 and 20 mg/kg, IP, respectively) and fitted with femoral venous and arterial catheters for drug injection and arterial pressure recording, respectively. All experiments were performed >24 h after surgery. On the day of the experiment, arterial blood pressure and heart rate (HR) were first recorded in conscious rats, which were then slowly anesthetized using urethane (1.2–1.4 g/kg IV). The trachea was cannulated for artificial ventilation with oxygen-enriched air. Rectal temperature was maintained at 37 °C by a means of a servo-controlled electric blanket. An adequate depth of anesthesia was monitored by observing blood pressure, HR, and the corneal and pinch reflexes and, if required, additional anesthetic was administered (5% of initial dose).

CT

2K-1C

Glut-1 RVLM

*

3 CT 2K-1C

2 1 0

Figure 1 | Representative histological coronal view of the brain stem. (a) Typical microinjection site in the RVLM evaluated by 100 nl of Evans blue diffusion; (b) schematic representation. CST, corticospinal tract; ION, inferior olivary nucleus; NA, nucleus ambiguous; NTS, nucleus of the tractus solitarii; STN, spinal trigeminal nucleus.

Figure 2 | mRNA expression quantified by real-time PCR. Relative amount of mRNA of (a) p47phox and gp91phox; (b) CuZnSOD; and (c) Glut-1 in the RVLM as compared to mRNA of β-actin in CT and 2K-1C groups. *P < 0.05 in relation to the CT.

100

january 2008 | VOLUME 21 NUMBER 1 | AMERICAN JOURNAL OF HYPERTENSION

CT

2K-1C

articles


0 CT

0

2K-1C

Figure 3 | Basal parameters in control and 2K-1C groups. (a) Comparison of basal mean arterial pressure (MAP) in the 2K-1C and control groups. (b) Comparison of basal renal sympathetic nerve activity (RSNA) in 2K-1C and CT animals. (c) Lipid peroxidation as indicated by plasma TBARS in 2K-1C and CT animals. *P < 0.05 in relation to the CT group.

Results Analysis of the expression of p47phox, gp91phox, CuZnSOD and Glut-1 mRNA in the RVLM of 2K-1C and CT rats

As measured by real-time PCR, the level of p47phox gene expression in the RVLM of 2K-1C rats was significantly higher than that in the CT group (2K-1C, 21.8 ± 3.6 AU vs. CT 1.0 ± 0.1 AU; P < 0.001). Although not significant, the gp91phox expression was higher in the RVLM of 2K-1C rats than in those of CT rats (2K-1C, 4.6 ± 1.8 AU vs. CT, 1.0 ± 0.2 AU; P = 0.07) (Figure 2a). In contrast, there was no difference in the levels of CuZnSOD gene expression between the RVLM of the 2K-1C and CT groups (2K-1C, 0.9 ± 0.01 AU vs. CT, 0.85 ± 0.2 AU; P = 0.7) (Figure 2b). Furthermore, Glut-1 mRNA expression was significantly higher in the RVLM of 2K-1C rats than in those of CT rats (2K-1C, 2.9 ± 0.6 AU vs. CT 1.0 ± 0.2 AU; P