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Blood pressure variability, baroreflex sensitivity and organ damage in spontaneously hypertensive rats treated with various antihypertensive drugs
European Journal of Pharmacology 543 (2006) 77 – 82 www.elsevier.com/locate/ejphar
Blood pressure variability, baroreflex sensitivity and organ damage in spontaneously hypertensive rats treated with various antihypertensive drugs He-Hui Xie, Fu-Ming Shen, Xiao-Fei Zhang, Yuan-Ying Jiang, Ding-Feng Su ⁎ Department of Pharmacology, Second Military Medical University, 325 Guo He Road, Shanghai 200433, China Received 27 November 2005; received in revised form 16 May 2006; accepted 18 May 2006 Available online 2 June 2006
Abstract Besides blood pressure, blood pressure variability and baroreflex sensitivity maybe important factors determining organ damage in hypertension. This study was designed to investigate the effects of various antihypertensive drugs on blood pressure and blood pressure variability reductions, baroreflex sensitivity, and target organ damage in spontaneously hypertensive rats (SHR). The dose is 20 mg/kg/day for atenolol, and 10 mg/kg/day for nifedipine, irbesartan and hydrochlorothiazide. We used relatively low doses of drugs to avoid a very remarkable normalization of blood pressure in the treatment, which would make it much difficult to distinguish the contribution of blood pressure variability and baroreflex sensitivity to organ protection from that of blood pressure. Drugs at the aforementioned doses were mixed into rat chow. SHR were treated for 4 months. Blood pressure was then continuously recorded for 24 h. After the determination of baroreflex sensitivity, rats were killed for organdamage evaluation. It was found that long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide all markedly reduced blood pressure variability, enhanced baroreflex sensitivity, and produced significant organ protection. Compared with blood pressure level, blood pressure variability and baroreflex sensitivity values showed a much closer or similar relationship with organ-damage parameters in every treatment group of rats. Multiple-regression analysis showed that the decrease in left ventricular hypertrophy, the decrease in aortic hypertrophy and the amelioration in renal lesion were all most closely correlated with the increase in baroreflex sensitivity and the decrease in systolic blood pressure variability. In conclusion, long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide produced organ protection in SHR. Besides the blood pressure reduction, the decrease in blood pressure variability and the restoration of baroreflex sensitivity may contribute to this organ protection. © 2006 Elsevier B.V. All rights reserved. Keywords: Hypertension; Antihypertensive drug; End-organ damage; Blood pressure variability; Baroreflex sensitivity
1. Introduction Complications associated with hypertension, including stroke, heart failure and renal failure, are often lethal. End-organ damage, including arteriosclerosis, renal lesion, occurs during early phase of these complications. Therefore, it is important to prevent or reduce end-organ damage in the treatment of hypertension (Su and Miao, 2005; Parati et al., 1987). It is well known that blood pressure (BP) level is an important determinant for the end-organ damage in hypertensive patients or hypertensive animals. How⁎ Corresponding author. Tel.: +86 21 2507 0323; fax: +86 21 6549 3951. E-mail address: [email protected] (D.-F. Su). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.05.034
ever, blood pressure level is certainly not the unique determinant for end-organ damage. Recently, it has been proposed that blood pressure variability (BPV) and baroreflex sensitivity (BRS) maybe two important factors determining organ damage in hypertension (Su and Miao, 2001, 2005; Parati et al., 1987; Parati and Mancia, 2001; Sleight, 1997). This implies that antihypertensive treatment should aim at not only reducing BP values but also reducing blood pressure variability and enhancing baroreflex sensitivity. Our previous studies showed that blood pressure variability played an important role in the organ protection of nitrendipine in spontaneously hypertensive rats (SHR) (Liu et al., 2003). In this study, the effect of baroreflex sensitivity was not observed. In addition, with respect to the other antihypertensive drugs,
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limited information is available about the role of blood pressure variability and baroreflex sensitivity in their organ protective effects during long-term treatment. Irbesartan (an angiotensin AT1 receptor antagonist), hydrochlorothiazide (a diuretic), atenolol (a β-adrenoceptor antagonist) and nifedipine (a calcium channel antagonist), are four different types of widely used antihypertensives. The present work was designed to investigate the effects of long-term treatment with these four kinds of antihypertensive drugs on blood pressure variability, baroreflex sensitivity and end-organ damage in SHR. Employment of four antihypertensive drugs from different classes in the study would make the findings universal and of more significance. 2. Materials and methods 2.1. Animals and chemicals Male SHR with an age of 18 weeks were provided by the animal center of our university. The rats were housed with controlled temperature (23–25 °C) and lighting (8:00–20:00 light, 20:00–8:00 dark) and with free access to food and tap water. All the animals used in this work received humane care in compliance with institutional animal care guidelines. Antihypertensive drugs used in this study are as follows: nifedipine (Nanjing Pharmaceutical Co. Ltd, China), hydrochlorothiazide (Shanghai Xinyi Pharmaceutical Co. Ltd, China), atenolol (Shanghai Second Pharmaceutical Co. Ltd, China) and irbesartan (Jiangsu Hengrui Pharmaceutical Co. Ltd, China). 2.2. Drug administration Studies were performed in five groups of SHR. Atenolol, nifedipine, irbesartan or hydrochlorothiazide was mixed in the rat chow. The consumption of rat chow containing drugs was determined previously. The content of drugs in the rat chow was calculated according to the chow consumption, and the ingested doses of drugs were approximately 20 mg/kg/day for atenolol and 10 mg/kg/day for nifedipine, irbesartan and hydrochlorothiazide. The control SHR group received the same diet without any drugs. We used relatively low doses of drugs to avoid a very remarkable normalization of BP in the treatment, which would make it much difficult to distinguish the contribution of blood pressure variability and baroreflex sensitivity to organ protection from that of blood pressure. After 4 months of drug administration, BP was recorded during 24 h, and then blood pressure variability was calculated and baroreflex sensitivity was determined in conscious freely moving rats. Histopathological examinations were performed after blood pressure recording and baroreflex sensitivity studies. 2.3. Blood pressure measurement Systolic BP (SBP), diastolic BP (DBP) and heart period (HP) of rats were continuously recorded using a previously described technique (Xie et al., 2003; Norman et al., 1981). Briefly, rats were anesthetized with a combination of ketamine (40 mg/kg) and diazepam (6 mg/kg). A floating polyethylene catheter was
inserted into the lower abdominal aorta via the left femoral artery for blood pressure measurement, and another catheter was placed into the left femoral vein for intravenous injection. The catheters were exteriorized through the interscapular skin. After a 3-day recovery period, the animals were placed for blood pressure recording in individual cylindrical cages containing food and water. The aortic catheter was connected to a blood pressure transducer via a rotating swivel that allowed the animals to move freely in the cage. After about 14-h habituation, the blood pressure signal was digitized by a microcomputer. Blood pressure recordings were edited to remove artifacts that might have affected an accurate assessment of blood pressure variability. The criteria used for data editing are as follows: a heartbeat with the blood pressure value 40% greater or lower over the contiguous heartbeat was discarded. Other artifacts such as temporarily low pulse pressure were removed manually. SBP and HP values from every heartbeat were determined on line. The mean values and standard deviation of these parameters during a period of 24 h were calculated. The standard deviation was defined as the quantitative parameter of BPV, i.e. systolic BPV (SBPV) and HP variability (HPV). These parameters were also determined over several other time periods, including 1-h daytime period (14:00– 15:00), 1-h nighttime period (2:00–3:00), 12-h daytime period (8:00–20:00), and 12-h nighttime period (20:00–8:00). 2.4. Baroreflex sensitivity measurement To determine the function of arterial baroreflex in conscious rats, the methods widely used are derived from that of Smyth firstly applied for humans (Smyth et al., 1969). The principle of this method is to measure the prolongation of HP in response to an elevation of BP. With some modifications, this method was used in conscious rats (Fu et al., 2004; Xie et al., 2005). A bolus injection of phenylephrine was used to induce an elevation of BP. The dose of phenylephrine was adjusted to raise SBP between 20 and 40 mmHg. HP was plotted against SBP for linear regression analysis and the slope of SBP-HP was expressed as BRS (ms/mmHg). 2.5. Morphological examination Morphological examinations were performed after blood pressure recording and baroreflex sensitivity studies. The animal was weighed and killed by decapitation. The thoracic and peritoneal cavities were immediately opened. The right kidney, aorta and heart were excised and rinsed in cold physiological saline. The right kidney was blotted. The left ventricle was isolated, blotted, and weighed. At the same time, the aorta was cleaned of adhering fat and connective tissue. Just below the branch of the left subclavicular artery, a 30-mm-long segment of thoracic aorta was harvested, blotted, and weighed. Ratios of left ventricular weight to body weight (LVW/BW) and aortic weight to the length of aorta (AW/length) were calculated (Hayakawa and Raij, 1997). Histopathological observation was also carried out with our conventional method (Miao et al., 2001). Briefly, immediately after gross detection, all samples of kidneys were immersed in formalin solution for more than 1
H.-H. Xie et al. / European Journal of Pharmacology 543 (2006) 77–82 Table 1 Effects of long-term treatment with antihypertensive drugs on hemodynamics in spontaneously hypertensive rats Con (n = 12) Nif (n = 9) SBP (mmHg) SBPV (mmHg) SBPV-d12 (mmHg) SBPV-n12 (mmHg) SBPV-d1 (mmHg) SBPV-n1 (mmHg) HP (ms) HPV (ms) HPV-d12 (ms) HPV-n12 (ms) HPV-d1 (ms) HPV-n1 (ms) BRS (ms/ mmHg)
Ate (n = 12) Irb (n = 9)
Hyd (n = 9)
189 ± 3.1
179 ± 4.0a
172 ± 4.2b
186 ± 1.4
176 ± 4.8a
15.7 ± 0.8
12.6 ± 0.6b
12.1 ± 0.7b
12.5 ± 0.2b
12.1 ± 0.6b
14.4 ± 0.6
11.7 ± 0.7b
10.1 ± 0.7b
11.5 ± 0.5b
11.5 ± 0.6b
15.2 ± 0.8
12.0 ± 1.0a
10.6 ± 0.9b
12.6 ± 0.5a
11.9 ± 0.4b
11.4 ± 0.8
8.6 ± 0.6a
8.5 ± 0.7b
8.7 ± 0.3b
8.7 ± 0.4b
12.4 ± 1.0
9.1 ± 0.5b
8.9 ± 1.1a
9.7 ± 0.5a
9.2 ± 0.3b
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2.7. Statistical analysis Data are expressed as mean ± S.E.M. Comparisons among groups were made by analysis of variance (ANOVA) followed by Duncan test. The relationships between hemodynamic parameters and pathological parameters were analyzed by classic univariate correlation analysis. Stepwise multiple-regression analysis was performed to study the independent effect of hemodynamic parameters on organ damage. F to enter and F to remove were set to P < 0.05 and P > 0.10 respectively. P < 0.05 was considered statistically significant. Statistical analysis was performed by using software SPSS 11.0.0. 3. Results
161 ± 4.2 30.1 ± 2.3 28.9 ± 2.6
154 ± 4.3 26.5 ± 1.0 25.3 ± 0.9
177 ± 7.7 32.2 ± 1.4 33.4 ± 2.0
156 ± 5.5 25.6 ± 2.6 25.0 ± 2.2
152 ± 3.5 25.0 ± 1.2 25.8 ± 1.5
29.3 ± 2.7
25.9 ± 0.7
32.5 ± 2.2
25.5 ± 2.1
25.4 ± 1.0
29.6 ± 2.4
26.8 ± 0.8
33.3 ± 1.9
25.1 ± 2.4
25.1 ± 1.3
30.2 ± 3.0
27.1 ± 1.1
31.8 ± 2.1
25.3 ± 2.5
27.1 ± 1.1
0.29 ± 0.06
0.50 ± 0.07a 0.55 ± 0.05b 0.50 ± 0.05a 0.59 ± 0.05b
Values are mean ± S.E.M. aP < 0.05; bP < 0.01 vs. Con. Con, control; Nif, nifedipine; Ate, atenolol; Irb, irbesartan; Hyd, hydrochlorothiazide; SBP, systolic blood pressure; HP, heart period; SBPV, systolic blood pressure variability over 24-h period; SBPV-d12, SBPVover 12-h daytime period; SBPV-n12, SBPVover 12-h nighttime period; SBPV-d1, SBPV over 1-h daytime period; SBPV-n1, SBPV over 1-h nighttime period; HPV, heart period variability; HPV-d12, HPV over 12-h daytime period; HPV-n12, HPV over 12-h nighttime period; HPV-d1, HPV over 1-h daytime period; HPV-n1, HPV over 1-h nighttime period; BRS, baroreflex sensitivity.
week, dehydrated in ethanol, cleared in dimethylbenzene and embedded in paraffin. Then the 5-μm-thick sections were prepared and stained with hematoxylin and eosin for light microscopic evaluation. 2.6. Glomerulosclerosis score For the semiquantitative evaluation of glomerulular damage, the glomerulosclerosis score (GSS) was defined as previously described (Kimula et al., 1991). Under light microscopy, approximately 50 glomeruli from the outer cortex and the same number of glomeruli from the inner cortex for each kidney were graded according to the degree of sclerosis: 0, if no mesangial expansion; 1, if mild mesangial expansion (less than 30% of a glomerular area); 2, if moderate mesangial expansion (30–60% of a glomerular area); 3, if marked mesangial expansion (more than 60% of a glomerular area); and 4, if the sclerosis was global. This was performed by one observer in a blind fashion using coded slides. A weighted composite sclerosis score was then calculated for each kidney according to the following formula: glomerulosclerosis score =[1× (number of grade 1 glomeruli) + 2 × (number of grade 2 glomeruli) + 3 ×(number of grade 3 glomeruli) +4 ×(number of grade 4 glomeruli)] ×100/(number of glomeruli observed).
3.1. Effects of 4 long-term treatments on hemodynamic parameters in SHR Long-term treatment with nifedipine, atenolol or hydrochlorothiazide all markedly decreased blood pressure and blood pressure variability, and enhanced BRS in SHR. Blood pressure was reduced by 5–9% while the magnitudes of blood pressure variability reduction and baroreflex sensitivity enhancement are relatively greater (about 20% and 72–103% respectively). Longterm treatment with irbesartan (10 mg/kg/day) had no obvious effect on blood pressure level, but significantly decreased blood pressure variability and enhanced baroreflex sensitivity in SHR. No obvious change was found in heart period and heart period variability in any treatment group (Table 1). 3.2. Effects of 4 long-term treatments on organ damages in SHR Among pathological parameters studied, some representative parameters are shown in Table 2. They are left ventricular weight/ body weight (reflecting left ventricular hypertrophy), aortic weight/length (reflecting aortic hypertrophy) and glomerulosclerosis score (reflecting renal damage). It was found that longterm treatment with irbesartan, which had no effect on blood pressure levels, significantly decreased left ventricular weight/ body weight (−10%, P < 0.01), aortic weight/length (−14%, P < 0.01) and glomerulosclerosis score (−15%, P < 0.01) in SHR. Significant decrease in all the three pathological parameters was
Table 2 Effects of long-term treatment with antihypertensive drugs on pathological changes in ventricles, kidneys and aortae in spontaneously hypertensive rats Co (n = 12) Nif (n = 9)
Ate (n = 12) Irb (n = 9)
Hyd (n = 9)
LVW/BW 3.64 ± 0.09 3.23 ± 0.07a 3.19 ± 0.09a 3.28 ± 0.1a 3.18 ± 0.11a (mg/g) AW/length 1.51 ± 0.03 1.30 ± 0.02a 1.15 ± 0.05a 1.30 ± 0.03a 1.19 ± 0.03a (mg/mm) GSS 60 ± 2.4 51 ± 2.2a 46 ± 1.7a 51 ± 1.5a 46 ± 1.8a Values are mean ± S.E.M. aP < 0.01 vs. Con. Con, control; Nif, nifedipine; Ate, atenolol; Irb, irbesartan; Hyd, hydrochlorothiazide; LVW, left ventricular weight; BW, body weight; AW, aortic weight; GSS, glomerulosclerosis score.
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Table 3 Linear regression coefficient (r) between hemodynamic parameters and organ damages in all animals included in the study (n = 51) LVW/BW SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 HP HPV HPV-d12 HPV-n12 HPV-d1 HPV-n1 BRS
a
0.350 0.583b 0.579b 0.578b 0.589b 0.522b 0.003 0.161 0.151 0.156 0.114 0.232 − 0.601b
AW/length b
0.529 0.669b 0.623b 0.668b 0.551b 0.626b − 0.076 − 0.063 − 0.126 − 0.167 − 0.151 − 0.090 − 0.639b
GSS b
0.403 0.617b 0.640b 0.626b 0.535b 0.524b 0.046 0.023 −0.091 −0.055 −0.041 −0.088 −0.684b
Table 4 Linear regression coefficient (r) between hemodynamic parameters and organ damages in spontaneously hypertensive rats Groups
n
Con + Nif
21
Con + Ate
P < 0.05; bP < 0.01. See Tables 1 and 2 for abbreviations.
3.3. Relationships between hemodynamic parameters and organ damages in SHR Con + Hyd
GSS
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.400 0.455a 0.355 0.392 0.635b 0.469a − 0.457a
0.456a 0.545a 0.379 0.462a 0.469a 0.462a − 0.636b
0.359 0.549a 0.529a 0.508a 0.438a 0.489a − 0.620b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.417a 0.565b 0.673b 0.656b 0.677b 0.562b − 0.558b
0.619b 0.705b 0.753b 0.747b 0.565b 0.620b − 0.712b
0.530b 0.615b 0.707b 0.753b 0.669b 0.629b − 0.847b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.150 0.479a 0.500a 0.378 0.570b 0.408 − 0.260
0.055 0.554b 0.476a 0.490a 0.468a 0.448a − 0.581b
0.248 0.554b 0.414 0.616b 0.545a 0.611b − 0.761b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.374 0.644b 0.610b 0.482a 0.484a 0.417 − 0.562b
0.475a 0.637b 0.545a 0.594b 0.456a 0.510a − 0.685b
0.474a 0.640b 0.583b 0.672b 0.599b 0.660b − 0.820b
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also found in nifedipine-, atenolol- or hydrochlorothiazide-treated rats.
When all the SHRs employed in the present study were pooled as a whole (n = 51) for linear regression analysis, relationships between hemodynamic parameters and organ damages are shown in Table 3. It was found that all the three pathological parameters studied were negatively related to baroreflex sensitivity and positively related to blood pressure and blood pressure variability, but not related to heart period
AW/length
24
a
Con + Irb
LVW/BW
21
P < 0.05; bP < 0.01. See Tables 1 and 2 for abbreviations.
a
and heart period variability. Important correlations are shown in Fig. 1. The relationships between hemodynamic parameters and organ damages were also analyzed in every treatment group (Table 4). Considering the fact that the number of rats in each treatment group was limited (n = 9 or 12), nifedipine-, atenolol-, irbesartan- or hydrochlorothiazide-treated rats were respectively pooled together with control SHRs as a whole for linear regression analysis. It was found that:
Fig. 1. Examples of correlation between hemodynamic parameters and pathological parameters in all animals included in the study. n = 51. See Tables 1 and 2 for abbreviations.
1) A positive relationship between systolic blood pressure and aortic weight/length was found in Con + Nif, Conl + Ate and Con + Hyd rats but not in Con + Irb rats, and systolic blood pressure was positively correlated with glomerulosclerosis score in Con + Ate and Con + Hyd rats but not in Con + Nif and Con + Irb rats, whereas the positive relationship between systolic blood pressure and left ventricular weight/body weight was only found in Con + Ate rats. 2) A positive correlation between blood pressure variability and organ damage was found in all the four groups of rats, with the exception that left ventricular weight/body weight was not related to SBPV-d12 and SBPV-n12 in Con + Nif rats, to SBPV-n12 and SBPV-n1 in Con + Irb rats, and to SBPV-n1 in Con + Hyd rats, and SBPV-d12 was not correlated with
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aortic weight/length in Con + Nif rats and with glomerulosclerosis score in Con + Irb rats. 3) Baroreflex sensitivity was negatively correlated with all the three pathological parameters in every group of rats. 4) Either heart period or heart period variability was not correlated with all the three pathological parameters in any group of rats. As aforementioned, compared with blood pressure level, blood pressure variability and baroreflex sensitivity values showed a much closer or similar relationship with pathological parameters in every group of rats. Furthermore, when all the SHRs employed in the present study were pooled as a whole, the relative dependencies of organ damage on hemodynamic parameters were assessed by stepwise multiple-regression analysis. Left ventricular weight was independently associated with lower baroreflex sensitivity (β=−0.395, P<0.01; where β is the standardized partial regressive coefficient) and higher SBPV-d1 (β=0.367, P<0.01). Aortic weight/length was independently associated with higher SBPV (β=0.441, P<0.01) and lower baroreflex sensitivity (β=−0.361, P<0.01). Glomerulosclerosis score was independently associated with lower baroreflex sensitivity (β=−0.467, P<0.01) and higher SBPV-d12 (β=0.355, P<0.01). 4. Discussion The present work clearly demonstrated that atenolol, nifedipine, irbesartan or hydrochlorothiazide all significantly reduced blood pressure variability, enhanced baroreflex sensitivity and produced organ protection, and both the decrease in blood pressure variability and the enhancement of baroreflex sensitivity might contribute to this organ protection. It is well known that a high blood pressure level induces organ damage and that decreasing blood pressure level can prevent organ damage. But blood pressure level is really not the unique determinant for hypertensive organ damage. Clinical observations suggested that blood pressure variability was related to organ damages in hypertensive patients (Parati et al., 1987; Mancia et al., 2000; Sander et al., 2000). In animal studies, a series of studies have been carried out on this subject. It was found that: (1) blood pressure variability was related to end-organ damage in aged SHR (Su and Miao, 2001); (2) increased blood pressure variability alone, without hypertension, could also induce organ damage in Sprague–Dawley normotensive rats (Su and Miao, 2001, 2005; Xie et al., 2003; Miao et al., 2001); (3) a decrease in blood pressure variability contributed importantly to the organ protection induced by long-term treatment with nitrendipine (Liu et al., 2003). Accordingly, it seems very important to emphasize the role of blood pressure variability reduction in antihypertensive therapy. In the present work, compared with blood pressure level, blood pressure variability showed a much closer or similar relationship with organ-damage parameters in every treatment group of rats, and multiple-regression analysis showed that the organdamage parameters studied were all independently associated with the reduction in blood pressure variability. Therefore,
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decrease in blood pressure variability may contribute to the organ protection of drugs in SHR. Accumulative evidences from recent studies suggest that four mechanisms contribute to high blood pressure variability-induced organ damage, including arterial endothelial lesion, tissue angiotensin II elevation, tissue inflammatory response and cardiomyocyte apoptosis (Su and Miao, 2005). Accordingly, the possible mechanisms by which reduced blood pressure variability could protect organs from the damage might be attributed to blocking or reducing the abovementioned four events. Arterial baroreflex dysfunction is another feature of hypertension. This impairment is mainly the result of elevated blood pressure level (Parmer et al., 1990, 1992; Brown, 1980). Therefore, baroreflex sensitivity will be less reduced when blood pressure level is lowered by an antihypertensive drug. In accordance with this view, the present study showed that longterm treatment with atenolol, nifedipine, or hydrochlorothiazide decreased blood pressure and increased baroreflex sensitivity at the same time. However, treatment with irbesartan significantly enhanced baroreflex sensitivity but had no obvious effect on blood pressure level in SHR. This implies that this enhancement of baroreflex sensitivity was not attributable to the normalization of blood pressure level. Irbesartan, an AT1 receptor antagonist, might act at AT1 receptors in the nucleus of the solitary tract level to enhance the ABR function in SHRs (Matsumura et al., 1998). In addition, irbesartan might also have a presynaptic effect. It should be noted that baroreflex sensitivity measured in the present work provides rather limited insight into baroreflex function and does not provide insight into the vascular regulation. However, baroreflex sensitivity measured with this technique may mainly reflect the vagal component of baroreflex and is important in the pathology of cardiovascular diseases (La Rovere et al., 1998; Mortara et al., 1997; Cai et al., 2005). Our previous study proposed that baroreflex sensitivity was one of the independent variables related to end-organ damage in hypertension (Shan et al., 1999). In the present work, baroreflex sensitivity showed a much closer relationship with organdamage parameters than blood pressure level in every treatment group of rats, and multiple-regression analysis showed that the organ-damage parameters studied were all independently associated with the increase in baroreflex sensitivity. In our previous studies, it was found that an interrupt of baroreflex by sinoaortic denervation might produce organ damages including cardiac and vascular remodeling, cardiac and renal lesions, etc (Miao et al., 2001). Accordingly, the enhancement of baroreflex function might be one of the major mechanisms for the organ protection produced by long-term treatment with antihypertensive drugs in SHR. However, it is not clear whether the enhancement of baroreflex sensitivity is the cause of the organ protection or is the result of the reduced organ damage by different drugs according to the present work. A longitudinal study in hypertensive patients or hypertensive rats will be necessary to elucidate this interesting question. The present work showed that irbesartan produced organ protective action independent of its blood pressure-lowering effect. In Con + Irb rats, all the three pathological parameters
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were significantly related to systolic blood pressure variability but not to blood pressure level, and both aortic weight/length and glomerulosclerosis score were markedly correlated with baroreflex sensitivity. Accordingly, in addition to the known non-hemodynamic mechanisms for AT1 receptor antagonists (such as cellular growth inhibition, etc), the present work might show another two possible mechanisms for this blood pressureindependent organ protection of irbesartan: the reduction of blood pressure variability and the enhancement of baroreflex sensitivity. In conclusion, long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide produced organ protection in SHR. Besides the blood pressure reduction, the decrease in blood pressure variability and the restoration of baroreflex sensitivity may contribute to this organ protection. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30330650), the High Tech Research and Development (863) Program of China (2002 AA2Z346C). References Brown, A.M., 1980. Receptors under pressure: an update on baroreceptors. Circ. Res. 46, 1–10. Cai, G.J., Miao, C.Y., Xie, H.H., Lu, L.H., Su, D.F., 2005. Arterial baroreflex dysfunction promotes atherosclerosis in rats. Atherosclerosis 183, 41–47. Fu, Y.J., Shu, H., Miao, C.Y., Wang, M.W., Su, D.F., 2004. Restoration of baroreflex function by ketanserin is not blood pressure dependent in conscious freely moving rats. J. Hypertens. 22, 1165–1172. Hayakawa, H., Raij, L., 1997. The link among nitric oxide synthase activity, endothelial function, and aortic and ventricular hypertrophy in hypertension. Hypertension 29, 235–241. Kimula, K., Tojo, A., Matsuoka, H., Sugimoto, T., 1991. Renel arteriolar diameters in spontaneously hypertensive rats: vascular cast study. Hypertension 18, 101–110. La Rovere, M.T., Bigger Jr., J.T., Marcus, F.I., Mortara, A., Schwartz, P.J., 1998. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. ATRAMI (autonomic Tone and Reflexes After Myocardial Infarction) Investigators. Lancet 351, 478–484. Liu, J.G., Xu, L.P., Chu, Z.X., Miao, C.Y., Su, D.F., 2003. Contribution of blood pressure variability to the effect of nitrendipine on end-organ damage in spontaneously hypertensive rats. J. Hypertens. 21, 1961–1967.
Mancia, G., Omboni, S., Parati, G., 2000. The importance of blood pressure variability in hypertension. Blood Press. Monit. 5 (Suppl 1), S9–S15. Matsumura, K., Averill, D.B., Ferrario, C.M., 1998. Angiotensin II acts at AT1 receptors in the nucleus of the solitary tract to attenuate the baroreceptor reflex. Am. J. Physiol. 275, R1611–R1619. Miao, C.Y., Tao, X., Gong, K., Zhang, S.H., Chu, Z.X., Su, D.F., 2001. Aterial remodeling in chronic sinoaortic-denervated rats. J. Cardiovasc. Pharmacol. 37, 6–15. Mortara, A., La Rovere, M.T., Pinna, G.D., Prpa, A., Maestri, R., Febo, O., Pozzoli, M., Opasich, C., Tavazzi, L., 1997. Arterial baroreflex modulation of heart rate in chronic heart failure: clinical and hemodynamic correlates and prognostic implications. Circulation 96, 3450–3458. Norman Jr., R.A., Coleman, T.G., Dent, A.C., 1981. Continuous monitoring of arterial pressure indicates sinoaortic denervated rats are not hypertensive. Hypertension 3, 119–125. Parati, G., Mancia, G., 2001. Blood pressure variability as a risk factor. Blood Press. Monit. 6, 341–347. Parati, G., Pomidossi, G., Albini, F., Malaspina, D., Mancia, G., 1987. Relationship of 24-hour blood pressure mean and variability to severity of target organ damage in hypertension. J. Hypertens. 5, 93–98. Parmer, R.J., Cervenka, J.H., Stone, R.A., O'Connor, D.T., 1990. Autonomic function in hypertension: are there racial difference? Circulation 81, 1305–1311. Parmer, R.J., Cervenka, J.H., Stone, R.A., 1992. Baroreflex sensitivity and heredity in essential hypertension. Circulation 85, 497–503. Sander, D., Kukla, C., Klingelhofer, J., Winbeck, K., Conrad, B., 2000. Relationship between circadian blood pressure patterns and progression of early carotid atherosclerosis: a 3-year follow-up study. Circulation 102, 1536–1541. Shan, Z.Z., Dai, S.M., Su, D.F., 1999. Relationship between baroreceptor reflex function and end-organ damage in spontaneously hypertensive rats. Am. J. Physiol. 277, H1200–H1206. Sleight, P., 1997. The importance of autonomic nervous system in health and disease. Aust. N.Z. J. Med. 27, 467–473. Smyth, H.S., Sleight, P., Pickering, G.W., 1969. Reflex regulation of arterial pressure during sleep in man: a quantitative method of assessing baroreflex sensitivity. Cir. Res. 24, 109–121. Su, D.F., Miao, C.Y., 2001. Blood pressure variability and organ damage. Clin. Exp. Pharmacol. Physiol. 28, 709–715. Su, D.F., Miao, C.Y., 2005. Reduction of blood pressure variability: a new strategy for the treatment of hypertension. Trends. Pharmacol. Sci. 26, 388–390. Xie, H.H., Miao, C.Y., Liu, J.G., Su, D.F., 2003. Effects of long-term treatment with candesartan on organ damages in sinoaortic denervated rats. J. Cardiovasc. Pharmacol. 41, 325–331. Xie, H.H., Miao, C.Y., Jiang, Y.Y., Su, D.F., 2005. Synergism of atenolol and nitrendipine on hemodynamic amelioration and organ protection in hypertensive rats. J. Hypertens. 23, 193–201.
Blood pressure variability, baroreflex sensitivity and organ damage in spontaneously hypertensive rats treated with various antihypertensive drugs He-Hui Xie, Fu-Ming Shen, Xiao-Fei Zhang, Yuan-Ying Jiang, Ding-Feng Su ⁎ Department of Pharmacology, Second Military Medical University, 325 Guo He Road, Shanghai 200433, China Received 27 November 2005; received in revised form 16 May 2006; accepted 18 May 2006 Available online 2 June 2006
Abstract Besides blood pressure, blood pressure variability and baroreflex sensitivity maybe important factors determining organ damage in hypertension. This study was designed to investigate the effects of various antihypertensive drugs on blood pressure and blood pressure variability reductions, baroreflex sensitivity, and target organ damage in spontaneously hypertensive rats (SHR). The dose is 20 mg/kg/day for atenolol, and 10 mg/kg/day for nifedipine, irbesartan and hydrochlorothiazide. We used relatively low doses of drugs to avoid a very remarkable normalization of blood pressure in the treatment, which would make it much difficult to distinguish the contribution of blood pressure variability and baroreflex sensitivity to organ protection from that of blood pressure. Drugs at the aforementioned doses were mixed into rat chow. SHR were treated for 4 months. Blood pressure was then continuously recorded for 24 h. After the determination of baroreflex sensitivity, rats were killed for organdamage evaluation. It was found that long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide all markedly reduced blood pressure variability, enhanced baroreflex sensitivity, and produced significant organ protection. Compared with blood pressure level, blood pressure variability and baroreflex sensitivity values showed a much closer or similar relationship with organ-damage parameters in every treatment group of rats. Multiple-regression analysis showed that the decrease in left ventricular hypertrophy, the decrease in aortic hypertrophy and the amelioration in renal lesion were all most closely correlated with the increase in baroreflex sensitivity and the decrease in systolic blood pressure variability. In conclusion, long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide produced organ protection in SHR. Besides the blood pressure reduction, the decrease in blood pressure variability and the restoration of baroreflex sensitivity may contribute to this organ protection. © 2006 Elsevier B.V. All rights reserved. Keywords: Hypertension; Antihypertensive drug; End-organ damage; Blood pressure variability; Baroreflex sensitivity
1. Introduction Complications associated with hypertension, including stroke, heart failure and renal failure, are often lethal. End-organ damage, including arteriosclerosis, renal lesion, occurs during early phase of these complications. Therefore, it is important to prevent or reduce end-organ damage in the treatment of hypertension (Su and Miao, 2005; Parati et al., 1987). It is well known that blood pressure (BP) level is an important determinant for the end-organ damage in hypertensive patients or hypertensive animals. How⁎ Corresponding author. Tel.: +86 21 2507 0323; fax: +86 21 6549 3951. E-mail address: [email protected] (D.-F. Su). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.05.034
ever, blood pressure level is certainly not the unique determinant for end-organ damage. Recently, it has been proposed that blood pressure variability (BPV) and baroreflex sensitivity (BRS) maybe two important factors determining organ damage in hypertension (Su and Miao, 2001, 2005; Parati et al., 1987; Parati and Mancia, 2001; Sleight, 1997). This implies that antihypertensive treatment should aim at not only reducing BP values but also reducing blood pressure variability and enhancing baroreflex sensitivity. Our previous studies showed that blood pressure variability played an important role in the organ protection of nitrendipine in spontaneously hypertensive rats (SHR) (Liu et al., 2003). In this study, the effect of baroreflex sensitivity was not observed. In addition, with respect to the other antihypertensive drugs,
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limited information is available about the role of blood pressure variability and baroreflex sensitivity in their organ protective effects during long-term treatment. Irbesartan (an angiotensin AT1 receptor antagonist), hydrochlorothiazide (a diuretic), atenolol (a β-adrenoceptor antagonist) and nifedipine (a calcium channel antagonist), are four different types of widely used antihypertensives. The present work was designed to investigate the effects of long-term treatment with these four kinds of antihypertensive drugs on blood pressure variability, baroreflex sensitivity and end-organ damage in SHR. Employment of four antihypertensive drugs from different classes in the study would make the findings universal and of more significance. 2. Materials and methods 2.1. Animals and chemicals Male SHR with an age of 18 weeks were provided by the animal center of our university. The rats were housed with controlled temperature (23–25 °C) and lighting (8:00–20:00 light, 20:00–8:00 dark) and with free access to food and tap water. All the animals used in this work received humane care in compliance with institutional animal care guidelines. Antihypertensive drugs used in this study are as follows: nifedipine (Nanjing Pharmaceutical Co. Ltd, China), hydrochlorothiazide (Shanghai Xinyi Pharmaceutical Co. Ltd, China), atenolol (Shanghai Second Pharmaceutical Co. Ltd, China) and irbesartan (Jiangsu Hengrui Pharmaceutical Co. Ltd, China). 2.2. Drug administration Studies were performed in five groups of SHR. Atenolol, nifedipine, irbesartan or hydrochlorothiazide was mixed in the rat chow. The consumption of rat chow containing drugs was determined previously. The content of drugs in the rat chow was calculated according to the chow consumption, and the ingested doses of drugs were approximately 20 mg/kg/day for atenolol and 10 mg/kg/day for nifedipine, irbesartan and hydrochlorothiazide. The control SHR group received the same diet without any drugs. We used relatively low doses of drugs to avoid a very remarkable normalization of BP in the treatment, which would make it much difficult to distinguish the contribution of blood pressure variability and baroreflex sensitivity to organ protection from that of blood pressure. After 4 months of drug administration, BP was recorded during 24 h, and then blood pressure variability was calculated and baroreflex sensitivity was determined in conscious freely moving rats. Histopathological examinations were performed after blood pressure recording and baroreflex sensitivity studies. 2.3. Blood pressure measurement Systolic BP (SBP), diastolic BP (DBP) and heart period (HP) of rats were continuously recorded using a previously described technique (Xie et al., 2003; Norman et al., 1981). Briefly, rats were anesthetized with a combination of ketamine (40 mg/kg) and diazepam (6 mg/kg). A floating polyethylene catheter was
inserted into the lower abdominal aorta via the left femoral artery for blood pressure measurement, and another catheter was placed into the left femoral vein for intravenous injection. The catheters were exteriorized through the interscapular skin. After a 3-day recovery period, the animals were placed for blood pressure recording in individual cylindrical cages containing food and water. The aortic catheter was connected to a blood pressure transducer via a rotating swivel that allowed the animals to move freely in the cage. After about 14-h habituation, the blood pressure signal was digitized by a microcomputer. Blood pressure recordings were edited to remove artifacts that might have affected an accurate assessment of blood pressure variability. The criteria used for data editing are as follows: a heartbeat with the blood pressure value 40% greater or lower over the contiguous heartbeat was discarded. Other artifacts such as temporarily low pulse pressure were removed manually. SBP and HP values from every heartbeat were determined on line. The mean values and standard deviation of these parameters during a period of 24 h were calculated. The standard deviation was defined as the quantitative parameter of BPV, i.e. systolic BPV (SBPV) and HP variability (HPV). These parameters were also determined over several other time periods, including 1-h daytime period (14:00– 15:00), 1-h nighttime period (2:00–3:00), 12-h daytime period (8:00–20:00), and 12-h nighttime period (20:00–8:00). 2.4. Baroreflex sensitivity measurement To determine the function of arterial baroreflex in conscious rats, the methods widely used are derived from that of Smyth firstly applied for humans (Smyth et al., 1969). The principle of this method is to measure the prolongation of HP in response to an elevation of BP. With some modifications, this method was used in conscious rats (Fu et al., 2004; Xie et al., 2005). A bolus injection of phenylephrine was used to induce an elevation of BP. The dose of phenylephrine was adjusted to raise SBP between 20 and 40 mmHg. HP was plotted against SBP for linear regression analysis and the slope of SBP-HP was expressed as BRS (ms/mmHg). 2.5. Morphological examination Morphological examinations were performed after blood pressure recording and baroreflex sensitivity studies. The animal was weighed and killed by decapitation. The thoracic and peritoneal cavities were immediately opened. The right kidney, aorta and heart were excised and rinsed in cold physiological saline. The right kidney was blotted. The left ventricle was isolated, blotted, and weighed. At the same time, the aorta was cleaned of adhering fat and connective tissue. Just below the branch of the left subclavicular artery, a 30-mm-long segment of thoracic aorta was harvested, blotted, and weighed. Ratios of left ventricular weight to body weight (LVW/BW) and aortic weight to the length of aorta (AW/length) were calculated (Hayakawa and Raij, 1997). Histopathological observation was also carried out with our conventional method (Miao et al., 2001). Briefly, immediately after gross detection, all samples of kidneys were immersed in formalin solution for more than 1
H.-H. Xie et al. / European Journal of Pharmacology 543 (2006) 77–82 Table 1 Effects of long-term treatment with antihypertensive drugs on hemodynamics in spontaneously hypertensive rats Con (n = 12) Nif (n = 9) SBP (mmHg) SBPV (mmHg) SBPV-d12 (mmHg) SBPV-n12 (mmHg) SBPV-d1 (mmHg) SBPV-n1 (mmHg) HP (ms) HPV (ms) HPV-d12 (ms) HPV-n12 (ms) HPV-d1 (ms) HPV-n1 (ms) BRS (ms/ mmHg)
Ate (n = 12) Irb (n = 9)
Hyd (n = 9)
189 ± 3.1
179 ± 4.0a
172 ± 4.2b
186 ± 1.4
176 ± 4.8a
15.7 ± 0.8
12.6 ± 0.6b
12.1 ± 0.7b
12.5 ± 0.2b
12.1 ± 0.6b
14.4 ± 0.6
11.7 ± 0.7b
10.1 ± 0.7b
11.5 ± 0.5b
11.5 ± 0.6b
15.2 ± 0.8
12.0 ± 1.0a
10.6 ± 0.9b
12.6 ± 0.5a
11.9 ± 0.4b
11.4 ± 0.8
8.6 ± 0.6a
8.5 ± 0.7b
8.7 ± 0.3b
8.7 ± 0.4b
12.4 ± 1.0
9.1 ± 0.5b
8.9 ± 1.1a
9.7 ± 0.5a
9.2 ± 0.3b
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2.7. Statistical analysis Data are expressed as mean ± S.E.M. Comparisons among groups were made by analysis of variance (ANOVA) followed by Duncan test. The relationships between hemodynamic parameters and pathological parameters were analyzed by classic univariate correlation analysis. Stepwise multiple-regression analysis was performed to study the independent effect of hemodynamic parameters on organ damage. F to enter and F to remove were set to P < 0.05 and P > 0.10 respectively. P < 0.05 was considered statistically significant. Statistical analysis was performed by using software SPSS 11.0.0. 3. Results
161 ± 4.2 30.1 ± 2.3 28.9 ± 2.6
154 ± 4.3 26.5 ± 1.0 25.3 ± 0.9
177 ± 7.7 32.2 ± 1.4 33.4 ± 2.0
156 ± 5.5 25.6 ± 2.6 25.0 ± 2.2
152 ± 3.5 25.0 ± 1.2 25.8 ± 1.5
29.3 ± 2.7
25.9 ± 0.7
32.5 ± 2.2
25.5 ± 2.1
25.4 ± 1.0
29.6 ± 2.4
26.8 ± 0.8
33.3 ± 1.9
25.1 ± 2.4
25.1 ± 1.3
30.2 ± 3.0
27.1 ± 1.1
31.8 ± 2.1
25.3 ± 2.5
27.1 ± 1.1
0.29 ± 0.06
0.50 ± 0.07a 0.55 ± 0.05b 0.50 ± 0.05a 0.59 ± 0.05b
Values are mean ± S.E.M. aP < 0.05; bP < 0.01 vs. Con. Con, control; Nif, nifedipine; Ate, atenolol; Irb, irbesartan; Hyd, hydrochlorothiazide; SBP, systolic blood pressure; HP, heart period; SBPV, systolic blood pressure variability over 24-h period; SBPV-d12, SBPVover 12-h daytime period; SBPV-n12, SBPVover 12-h nighttime period; SBPV-d1, SBPV over 1-h daytime period; SBPV-n1, SBPV over 1-h nighttime period; HPV, heart period variability; HPV-d12, HPV over 12-h daytime period; HPV-n12, HPV over 12-h nighttime period; HPV-d1, HPV over 1-h daytime period; HPV-n1, HPV over 1-h nighttime period; BRS, baroreflex sensitivity.
week, dehydrated in ethanol, cleared in dimethylbenzene and embedded in paraffin. Then the 5-μm-thick sections were prepared and stained with hematoxylin and eosin for light microscopic evaluation. 2.6. Glomerulosclerosis score For the semiquantitative evaluation of glomerulular damage, the glomerulosclerosis score (GSS) was defined as previously described (Kimula et al., 1991). Under light microscopy, approximately 50 glomeruli from the outer cortex and the same number of glomeruli from the inner cortex for each kidney were graded according to the degree of sclerosis: 0, if no mesangial expansion; 1, if mild mesangial expansion (less than 30% of a glomerular area); 2, if moderate mesangial expansion (30–60% of a glomerular area); 3, if marked mesangial expansion (more than 60% of a glomerular area); and 4, if the sclerosis was global. This was performed by one observer in a blind fashion using coded slides. A weighted composite sclerosis score was then calculated for each kidney according to the following formula: glomerulosclerosis score =[1× (number of grade 1 glomeruli) + 2 × (number of grade 2 glomeruli) + 3 ×(number of grade 3 glomeruli) +4 ×(number of grade 4 glomeruli)] ×100/(number of glomeruli observed).
3.1. Effects of 4 long-term treatments on hemodynamic parameters in SHR Long-term treatment with nifedipine, atenolol or hydrochlorothiazide all markedly decreased blood pressure and blood pressure variability, and enhanced BRS in SHR. Blood pressure was reduced by 5–9% while the magnitudes of blood pressure variability reduction and baroreflex sensitivity enhancement are relatively greater (about 20% and 72–103% respectively). Longterm treatment with irbesartan (10 mg/kg/day) had no obvious effect on blood pressure level, but significantly decreased blood pressure variability and enhanced baroreflex sensitivity in SHR. No obvious change was found in heart period and heart period variability in any treatment group (Table 1). 3.2. Effects of 4 long-term treatments on organ damages in SHR Among pathological parameters studied, some representative parameters are shown in Table 2. They are left ventricular weight/ body weight (reflecting left ventricular hypertrophy), aortic weight/length (reflecting aortic hypertrophy) and glomerulosclerosis score (reflecting renal damage). It was found that longterm treatment with irbesartan, which had no effect on blood pressure levels, significantly decreased left ventricular weight/ body weight (−10%, P < 0.01), aortic weight/length (−14%, P < 0.01) and glomerulosclerosis score (−15%, P < 0.01) in SHR. Significant decrease in all the three pathological parameters was
Table 2 Effects of long-term treatment with antihypertensive drugs on pathological changes in ventricles, kidneys and aortae in spontaneously hypertensive rats Co (n = 12) Nif (n = 9)
Ate (n = 12) Irb (n = 9)
Hyd (n = 9)
LVW/BW 3.64 ± 0.09 3.23 ± 0.07a 3.19 ± 0.09a 3.28 ± 0.1a 3.18 ± 0.11a (mg/g) AW/length 1.51 ± 0.03 1.30 ± 0.02a 1.15 ± 0.05a 1.30 ± 0.03a 1.19 ± 0.03a (mg/mm) GSS 60 ± 2.4 51 ± 2.2a 46 ± 1.7a 51 ± 1.5a 46 ± 1.8a Values are mean ± S.E.M. aP < 0.01 vs. Con. Con, control; Nif, nifedipine; Ate, atenolol; Irb, irbesartan; Hyd, hydrochlorothiazide; LVW, left ventricular weight; BW, body weight; AW, aortic weight; GSS, glomerulosclerosis score.
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Table 3 Linear regression coefficient (r) between hemodynamic parameters and organ damages in all animals included in the study (n = 51) LVW/BW SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 HP HPV HPV-d12 HPV-n12 HPV-d1 HPV-n1 BRS
a
0.350 0.583b 0.579b 0.578b 0.589b 0.522b 0.003 0.161 0.151 0.156 0.114 0.232 − 0.601b
AW/length b
0.529 0.669b 0.623b 0.668b 0.551b 0.626b − 0.076 − 0.063 − 0.126 − 0.167 − 0.151 − 0.090 − 0.639b
GSS b
0.403 0.617b 0.640b 0.626b 0.535b 0.524b 0.046 0.023 −0.091 −0.055 −0.041 −0.088 −0.684b
Table 4 Linear regression coefficient (r) between hemodynamic parameters and organ damages in spontaneously hypertensive rats Groups
n
Con + Nif
21
Con + Ate
P < 0.05; bP < 0.01. See Tables 1 and 2 for abbreviations.
3.3. Relationships between hemodynamic parameters and organ damages in SHR Con + Hyd
GSS
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.400 0.455a 0.355 0.392 0.635b 0.469a − 0.457a
0.456a 0.545a 0.379 0.462a 0.469a 0.462a − 0.636b
0.359 0.549a 0.529a 0.508a 0.438a 0.489a − 0.620b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.417a 0.565b 0.673b 0.656b 0.677b 0.562b − 0.558b
0.619b 0.705b 0.753b 0.747b 0.565b 0.620b − 0.712b
0.530b 0.615b 0.707b 0.753b 0.669b 0.629b − 0.847b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.150 0.479a 0.500a 0.378 0.570b 0.408 − 0.260
0.055 0.554b 0.476a 0.490a 0.468a 0.448a − 0.581b
0.248 0.554b 0.414 0.616b 0.545a 0.611b − 0.761b
SBP SBPV SBPV-d12 SBPV-n12 SBPV-d1 SBPV-n1 BRS
0.374 0.644b 0.610b 0.482a 0.484a 0.417 − 0.562b
0.475a 0.637b 0.545a 0.594b 0.456a 0.510a − 0.685b
0.474a 0.640b 0.583b 0.672b 0.599b 0.660b − 0.820b
21
also found in nifedipine-, atenolol- or hydrochlorothiazide-treated rats.
When all the SHRs employed in the present study were pooled as a whole (n = 51) for linear regression analysis, relationships between hemodynamic parameters and organ damages are shown in Table 3. It was found that all the three pathological parameters studied were negatively related to baroreflex sensitivity and positively related to blood pressure and blood pressure variability, but not related to heart period
AW/length
24
a
Con + Irb
LVW/BW
21
P < 0.05; bP < 0.01. See Tables 1 and 2 for abbreviations.
a
and heart period variability. Important correlations are shown in Fig. 1. The relationships between hemodynamic parameters and organ damages were also analyzed in every treatment group (Table 4). Considering the fact that the number of rats in each treatment group was limited (n = 9 or 12), nifedipine-, atenolol-, irbesartan- or hydrochlorothiazide-treated rats were respectively pooled together with control SHRs as a whole for linear regression analysis. It was found that:
Fig. 1. Examples of correlation between hemodynamic parameters and pathological parameters in all animals included in the study. n = 51. See Tables 1 and 2 for abbreviations.
1) A positive relationship between systolic blood pressure and aortic weight/length was found in Con + Nif, Conl + Ate and Con + Hyd rats but not in Con + Irb rats, and systolic blood pressure was positively correlated with glomerulosclerosis score in Con + Ate and Con + Hyd rats but not in Con + Nif and Con + Irb rats, whereas the positive relationship between systolic blood pressure and left ventricular weight/body weight was only found in Con + Ate rats. 2) A positive correlation between blood pressure variability and organ damage was found in all the four groups of rats, with the exception that left ventricular weight/body weight was not related to SBPV-d12 and SBPV-n12 in Con + Nif rats, to SBPV-n12 and SBPV-n1 in Con + Irb rats, and to SBPV-n1 in Con + Hyd rats, and SBPV-d12 was not correlated with
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aortic weight/length in Con + Nif rats and with glomerulosclerosis score in Con + Irb rats. 3) Baroreflex sensitivity was negatively correlated with all the three pathological parameters in every group of rats. 4) Either heart period or heart period variability was not correlated with all the three pathological parameters in any group of rats. As aforementioned, compared with blood pressure level, blood pressure variability and baroreflex sensitivity values showed a much closer or similar relationship with pathological parameters in every group of rats. Furthermore, when all the SHRs employed in the present study were pooled as a whole, the relative dependencies of organ damage on hemodynamic parameters were assessed by stepwise multiple-regression analysis. Left ventricular weight was independently associated with lower baroreflex sensitivity (β=−0.395, P<0.01; where β is the standardized partial regressive coefficient) and higher SBPV-d1 (β=0.367, P<0.01). Aortic weight/length was independently associated with higher SBPV (β=0.441, P<0.01) and lower baroreflex sensitivity (β=−0.361, P<0.01). Glomerulosclerosis score was independently associated with lower baroreflex sensitivity (β=−0.467, P<0.01) and higher SBPV-d12 (β=0.355, P<0.01). 4. Discussion The present work clearly demonstrated that atenolol, nifedipine, irbesartan or hydrochlorothiazide all significantly reduced blood pressure variability, enhanced baroreflex sensitivity and produced organ protection, and both the decrease in blood pressure variability and the enhancement of baroreflex sensitivity might contribute to this organ protection. It is well known that a high blood pressure level induces organ damage and that decreasing blood pressure level can prevent organ damage. But blood pressure level is really not the unique determinant for hypertensive organ damage. Clinical observations suggested that blood pressure variability was related to organ damages in hypertensive patients (Parati et al., 1987; Mancia et al., 2000; Sander et al., 2000). In animal studies, a series of studies have been carried out on this subject. It was found that: (1) blood pressure variability was related to end-organ damage in aged SHR (Su and Miao, 2001); (2) increased blood pressure variability alone, without hypertension, could also induce organ damage in Sprague–Dawley normotensive rats (Su and Miao, 2001, 2005; Xie et al., 2003; Miao et al., 2001); (3) a decrease in blood pressure variability contributed importantly to the organ protection induced by long-term treatment with nitrendipine (Liu et al., 2003). Accordingly, it seems very important to emphasize the role of blood pressure variability reduction in antihypertensive therapy. In the present work, compared with blood pressure level, blood pressure variability showed a much closer or similar relationship with organ-damage parameters in every treatment group of rats, and multiple-regression analysis showed that the organdamage parameters studied were all independently associated with the reduction in blood pressure variability. Therefore,
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decrease in blood pressure variability may contribute to the organ protection of drugs in SHR. Accumulative evidences from recent studies suggest that four mechanisms contribute to high blood pressure variability-induced organ damage, including arterial endothelial lesion, tissue angiotensin II elevation, tissue inflammatory response and cardiomyocyte apoptosis (Su and Miao, 2005). Accordingly, the possible mechanisms by which reduced blood pressure variability could protect organs from the damage might be attributed to blocking or reducing the abovementioned four events. Arterial baroreflex dysfunction is another feature of hypertension. This impairment is mainly the result of elevated blood pressure level (Parmer et al., 1990, 1992; Brown, 1980). Therefore, baroreflex sensitivity will be less reduced when blood pressure level is lowered by an antihypertensive drug. In accordance with this view, the present study showed that longterm treatment with atenolol, nifedipine, or hydrochlorothiazide decreased blood pressure and increased baroreflex sensitivity at the same time. However, treatment with irbesartan significantly enhanced baroreflex sensitivity but had no obvious effect on blood pressure level in SHR. This implies that this enhancement of baroreflex sensitivity was not attributable to the normalization of blood pressure level. Irbesartan, an AT1 receptor antagonist, might act at AT1 receptors in the nucleus of the solitary tract level to enhance the ABR function in SHRs (Matsumura et al., 1998). In addition, irbesartan might also have a presynaptic effect. It should be noted that baroreflex sensitivity measured in the present work provides rather limited insight into baroreflex function and does not provide insight into the vascular regulation. However, baroreflex sensitivity measured with this technique may mainly reflect the vagal component of baroreflex and is important in the pathology of cardiovascular diseases (La Rovere et al., 1998; Mortara et al., 1997; Cai et al., 2005). Our previous study proposed that baroreflex sensitivity was one of the independent variables related to end-organ damage in hypertension (Shan et al., 1999). In the present work, baroreflex sensitivity showed a much closer relationship with organdamage parameters than blood pressure level in every treatment group of rats, and multiple-regression analysis showed that the organ-damage parameters studied were all independently associated with the increase in baroreflex sensitivity. In our previous studies, it was found that an interrupt of baroreflex by sinoaortic denervation might produce organ damages including cardiac and vascular remodeling, cardiac and renal lesions, etc (Miao et al., 2001). Accordingly, the enhancement of baroreflex function might be one of the major mechanisms for the organ protection produced by long-term treatment with antihypertensive drugs in SHR. However, it is not clear whether the enhancement of baroreflex sensitivity is the cause of the organ protection or is the result of the reduced organ damage by different drugs according to the present work. A longitudinal study in hypertensive patients or hypertensive rats will be necessary to elucidate this interesting question. The present work showed that irbesartan produced organ protective action independent of its blood pressure-lowering effect. In Con + Irb rats, all the three pathological parameters
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were significantly related to systolic blood pressure variability but not to blood pressure level, and both aortic weight/length and glomerulosclerosis score were markedly correlated with baroreflex sensitivity. Accordingly, in addition to the known non-hemodynamic mechanisms for AT1 receptor antagonists (such as cellular growth inhibition, etc), the present work might show another two possible mechanisms for this blood pressureindependent organ protection of irbesartan: the reduction of blood pressure variability and the enhancement of baroreflex sensitivity. In conclusion, long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide produced organ protection in SHR. Besides the blood pressure reduction, the decrease in blood pressure variability and the restoration of baroreflex sensitivity may contribute to this organ protection. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30330650), the High Tech Research and Development (863) Program of China (2002 AA2Z346C). References Brown, A.M., 1980. Receptors under pressure: an update on baroreceptors. Circ. Res. 46, 1–10. Cai, G.J., Miao, C.Y., Xie, H.H., Lu, L.H., Su, D.F., 2005. Arterial baroreflex dysfunction promotes atherosclerosis in rats. Atherosclerosis 183, 41–47. Fu, Y.J., Shu, H., Miao, C.Y., Wang, M.W., Su, D.F., 2004. Restoration of baroreflex function by ketanserin is not blood pressure dependent in conscious freely moving rats. J. Hypertens. 22, 1165–1172. Hayakawa, H., Raij, L., 1997. The link among nitric oxide synthase activity, endothelial function, and aortic and ventricular hypertrophy in hypertension. Hypertension 29, 235–241. Kimula, K., Tojo, A., Matsuoka, H., Sugimoto, T., 1991. Renel arteriolar diameters in spontaneously hypertensive rats: vascular cast study. Hypertension 18, 101–110. La Rovere, M.T., Bigger Jr., J.T., Marcus, F.I., Mortara, A., Schwartz, P.J., 1998. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. ATRAMI (autonomic Tone and Reflexes After Myocardial Infarction) Investigators. Lancet 351, 478–484. Liu, J.G., Xu, L.P., Chu, Z.X., Miao, C.Y., Su, D.F., 2003. Contribution of blood pressure variability to the effect of nitrendipine on end-organ damage in spontaneously hypertensive rats. J. Hypertens. 21, 1961–1967.
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