Cardiovascular Research 1992;26:973-977
973
An unsaturated fat diet prevents the increased angiotensin I1 vascular responses in renal artery stenosis Gavin R Norton, Issy E Katzeff, Angela J Woodiwiss, Clive Rosendorff
R
ecent reviews have highlighted the antihypertensive effect of unsaturated fat diets.’” We have shown that unsaturated fat diets lower blood pressure in two kidney, one clip (2K1C) renovascular hyperten~ion.~ Several mechanisms have been proposed to explain the antihypertensive effects of these diets. A blood pressure Of lowering effect Of unsaturated fats in a hypertension induced by angiotensin 115 suggests that these fats may modulate the vascular effects of angiotensin 11. 2K1c renovascular hypertension is a renin-angiotensin dependent model of we have shown that a natriuresis and a decreased blood volume are important in the antihypertensiveeffect Of an unsaturated fat diet in 2K1C to explain One hypertension-4However, it was namely that an elevated plasma renin did not increase total peripheral resistance in 2K1C rats on this diet.4The reactive hyperreninaemiathat occurs in response to in renal artery stenosis normally leads to an increased total peripheral resistance and thus to the return of blood pressures to hypertensive levels.7 It is therefore possible that an attenuated effect Of angiotensin 11 on smooth muscle may contribute to the antihypertensive effect of an
unsaturated fat diet in 2K1C rats. The current study was intended to evaluate possible changes in vascular responsiveness to angiotensin I1 in 2K1C rat fed a diet high in unsaturated fats. Methods
Male Sprague Dawley rats (OLAC, United Kingdom) weighing 250-300 g were used in this study. All experiments were approved by the animal ethics committee of the University of the Witwatersrand. 2KlC renovascular hypertension was induced in two groups of 19 rats after 28 days on a high unsaturated fat diet (“unsaturated”) or a control diet. In addition, two groups of 15 rats received sham operations after 28 days on the unsaturated or the control diets. The diets were continued for eight weeks after surgery. Operations to induce 2KlC hypertension were performed on rats anaesthetised with fentanyl droperidol (0.02 mg fentany[ and 1 mg &operid01 intramuscularly; Janssen Phmaceutica) as previously described.8 The left renal artery was exposed and a 0.25 mm diameter silver clip was placed on the artery. The wound was closed in layers and 22 000 units of procaine penicillin administered intramuscularly. Sham operated rats were subjected to the same procedure except that the clip was not applied. Systolic blood pressure was measured prior to surgery and oFce weekly for eight weeks in all groups as described previously. A random sample of rats from each group was used for systolic blood pressure measurements. Rats were trained in stocks for three days prior to the first measurement of systolic blood pressure in order to allow
University of Witwatersrand Medical School, 7 York Road, Parktown, Johannesberg 2193, South Africa - Department of Physiology: G R Norton, I E Katzeff, C Rosendorff, A J Woodiwiss. Correspondence to Dr Norton.
Downloaded from by guest on June 6, 2016
Objective: The aim was to characterise angiotensin I1 constrictor responses in two kidney, one clip (2K 1C) renal hypertensive rats fed a diet with a high unsaturated fatty acid content. Methods: Two diets with the same total fat (37% by energy; 17% by weight) but different unsaturated fat contents were fed to rats for a three month period. Thirty four Sprague Dawley rats were used per diet group. After one month on the diets, a group of 19 rats in each diet group was operated upon to induce 2K1C hypertension. A separate group of 15 rats within each diet group received sham operations. Systolic blood pressure was measured weekly from prior to surgery to the end of the three month feeding period. At three months, angiotensin I1 constrictor responses were evaluated in the isolated kidney vascular preparation and in intact anaesthetised rats fed the different diets. Phenylephrine constrictor responses were also evaluated in intact anaesthetised rats in order to exclude structural vascular changes accounting for differences in angiotensin 11 constrictor responses. Results: The diet high in unsaturated fats prevented the development of hypertension in 2KlC rats [systolic pressure 134(7) mm Hg at eight weeks] compared to their own preoperative blood pressures [124(3) mm Hg], and to the 2K1C rats fed the control diet [163(7) mm Hg at eight weeks]. The diet high in unsaturated fats did not alter blood pressures in sham operated rats. In isolated perfused kidneys and in anaesthetised 2K1C rats fed the control diet, angiotensin I1 caused a greater vascular response compared to the sham operated groups. The unsaturated fat diet prevented this effect. No differences were found in blood pressure responses to phenylphrine. Conclusions: These data suggest that the antihypertensive effect of a high unsaturated fat diet may in part be due to a depressed responsiveness of vascular smooth muscle to endogenous angiotensin 11. The effect is likely to be due to modulation of angiotensin I1 vascular responses by local vascular changes that cannot be accounted for by structural vascular differences. Cardiovascular Research 1992:26:973-977
974
Norton, Katze8 Woodiwiss, RosendorfS
them to adapt to the procedure. Measurements were taken on prewarmed conscious rats using a tail cuff plethysmographic technique. Each reading was taken at mid-day to avoid diurnal variation and the mean of four measurements was recorded. In a pilot study, there were no significant differences between blood pressures measured by the tail cuff method and by direct intra-arterial recordings in the same rats. Diets The high unsaturated fat ("unsaturated") diet used in these experiments was made up by the addition of 200 g sunflower seed oil to I kg of a 5% fat (by weight) normal rat chow (Epol, South Africa). The control diet was made up in the same way, except that 200 g of hydrogenated coconut oil was added to 1 kg of normal rat chow. The addition of fats to the normal chow reduced the vitamin and mineral concentration by 16%; this was compensated for by supplementation. Similar concentrations of macrominerals, microminerals, vitamins, carbohydrates and proteins were found in the two diets after they were made up in this way. Fats contributed 37% of the total energy in the diets (total energy 4842 kcal.kg-I). The diets were made up once weekly and stored at 4°C. Adequate vitamin E was added to prevent oxidation of fats in the diets. Analysis of the fatty acids in the sunflower seed oil and hydrogenated coconut oil added to the diets showed that no significant changes of the fat concentration occurred on storage. A homogeneous diet preparation was maintained during storage, as mixing of the residual food was repeated on a daily basis. All rats had free access to tap water and food. Total lipids in the diet were extracted according to the method of Folch et allo andtnalysis of fatty acids was performed using gas-liquid chromatography. Table I shows the fatty acid content of the diets analysed after one week of storage at 4°C. The high unsaturated fat diet had a much higher content of monoenoic, n-6 polyenoic, and n-3 polyenoic unsaturated fats and a lower saturated fat content. The fat content of the, control diet is thought to represent that of an average Western diet. In our experiment, we did not attempt to differentiate between the effects of specific unsaturated fats.
Table I
Fat content of diets Control diet
Fatty acids Saturated fats
fgkg-')
Unsaturated ,fat diet fR . k f ' )
1600 18:00
82 30.8 18.3 4.3
Monoenoics
16:l 18:l
0.2 16.5
36
n-6 Polyenoics
18:2 20:4
16.3
103
n-3 Polyenoics
18:3 205 22:6
0.8
12:00
14:00
20:00
Polyunsaturated to saturated fat ratio
0.7
2.5
1.4 14.9 7.0
I .o
0.5
0.9
0.5
0.9 1.2 0.7
0.13
3.9
Whole animal preparation Blood pressure and heart rate responses to infused angiotensin I1 and phenylephrine were monitored in weight matched anaesthetised rats: five animals from each 2K1C diet group and five animals from each sham operated diet group for both vasoactive substances. Catheterisation of the femoral artery (PP50) and vein (PP25) was performed under fentanyl-droperidol anaesthesia. Arterial pressure and heart rates were recorded directly through the femoral artery catheter and infusion of angiotensin I1 and phenylephrine was performed via the femoral vein catheter using a Harvard infusion pump (model 931 A). Pressures were recorded on a Beckman dynograph recorder (type R 5 11A) coupled to a Statham P23AA pressure transducer. Angiotensin I1 (AII, Hypertensin, Ciba) was dissolved in normal saline and infused at 0.02 m1.min-l for each dose. The doses given were 0.08, 0.16, 0.32, 0.63, 1.58, 3.16, and 6.31 nmol.min-I. Phenylephrine (Sigma) was first dissolved in 0.1 NHCl and then made up in four concentrations (10" to w'M) in a 0.1% ascorbate saline solution. These solutions were made up on the day of the experiment and stored at 4°C in the dark. For each molar concentration of phenylephrine, two different infusion rates were used (0.02 m1.min-I and 0.08 m1.rnin-I). At each dose of angiotensin I1 and phenylephrine, blood pressure and heart rate were allowed to stabilise for a 10 min period before recordings were made. Between each infusion of vasoactive agent, blood pressure and heart rate were allowed to return to baseline before the next dose was given. Baseline blood pressure levels did not change between the first and last doses of angiotensin I1 or phenylephrine. Heart rates did not change significantly through baroreceptor stimulation, probably because of the effects of anaesthesia. Infusion of the vehicle of angiotensin 11. at the above rates, did not alter blood pressures or heart rates. Statistical analysis All values presented in the text and in the figures are given as mean(SEM). Differences in systolic blood pressure levels between the diet groups and between 2KlC and sham operated rats were established by a two way analysis of variance with multiple comparisons followed by an unpaired r test with a Bonferroni correction and also by KruskalWallis statistics." Differences in values between diet groups and between 2KIC and sham operated groups in angiotensin I1 and phenylephrine vascular responses were determined using KruskalWallis and unpaired t statistics (Student-Newman-Keuls) with a Bonferroni correcti~n.'~A p value c0.05 was considered to be statistically significant.
Results Figure 1 illustrates the effect of a diet high in unsaturated fats on 2K1C renovascular hypertension. 2K1C rats fed the control diet developed significant hypertension by week one [systolic blood pressure (SBP) = 146(4) mm Hg] compared to their own preoperative blood pressures [SBP = 123(3) mm Hg; p
973
An unsaturated fat diet prevents the increased angiotensin I1 vascular responses in renal artery stenosis Gavin R Norton, Issy E Katzeff, Angela J Woodiwiss, Clive Rosendorff
R
ecent reviews have highlighted the antihypertensive effect of unsaturated fat diets.’” We have shown that unsaturated fat diets lower blood pressure in two kidney, one clip (2K1C) renovascular hyperten~ion.~ Several mechanisms have been proposed to explain the antihypertensive effects of these diets. A blood pressure Of lowering effect Of unsaturated fats in a hypertension induced by angiotensin 115 suggests that these fats may modulate the vascular effects of angiotensin 11. 2K1c renovascular hypertension is a renin-angiotensin dependent model of we have shown that a natriuresis and a decreased blood volume are important in the antihypertensiveeffect Of an unsaturated fat diet in 2K1C to explain One hypertension-4However, it was namely that an elevated plasma renin did not increase total peripheral resistance in 2K1C rats on this diet.4The reactive hyperreninaemiathat occurs in response to in renal artery stenosis normally leads to an increased total peripheral resistance and thus to the return of blood pressures to hypertensive levels.7 It is therefore possible that an attenuated effect Of angiotensin 11 on smooth muscle may contribute to the antihypertensive effect of an
unsaturated fat diet in 2K1C rats. The current study was intended to evaluate possible changes in vascular responsiveness to angiotensin I1 in 2K1C rat fed a diet high in unsaturated fats. Methods
Male Sprague Dawley rats (OLAC, United Kingdom) weighing 250-300 g were used in this study. All experiments were approved by the animal ethics committee of the University of the Witwatersrand. 2KlC renovascular hypertension was induced in two groups of 19 rats after 28 days on a high unsaturated fat diet (“unsaturated”) or a control diet. In addition, two groups of 15 rats received sham operations after 28 days on the unsaturated or the control diets. The diets were continued for eight weeks after surgery. Operations to induce 2KlC hypertension were performed on rats anaesthetised with fentanyl droperidol (0.02 mg fentany[ and 1 mg &operid01 intramuscularly; Janssen Phmaceutica) as previously described.8 The left renal artery was exposed and a 0.25 mm diameter silver clip was placed on the artery. The wound was closed in layers and 22 000 units of procaine penicillin administered intramuscularly. Sham operated rats were subjected to the same procedure except that the clip was not applied. Systolic blood pressure was measured prior to surgery and oFce weekly for eight weeks in all groups as described previously. A random sample of rats from each group was used for systolic blood pressure measurements. Rats were trained in stocks for three days prior to the first measurement of systolic blood pressure in order to allow
University of Witwatersrand Medical School, 7 York Road, Parktown, Johannesberg 2193, South Africa - Department of Physiology: G R Norton, I E Katzeff, C Rosendorff, A J Woodiwiss. Correspondence to Dr Norton.
Downloaded from by guest on June 6, 2016
Objective: The aim was to characterise angiotensin I1 constrictor responses in two kidney, one clip (2K 1C) renal hypertensive rats fed a diet with a high unsaturated fatty acid content. Methods: Two diets with the same total fat (37% by energy; 17% by weight) but different unsaturated fat contents were fed to rats for a three month period. Thirty four Sprague Dawley rats were used per diet group. After one month on the diets, a group of 19 rats in each diet group was operated upon to induce 2K1C hypertension. A separate group of 15 rats within each diet group received sham operations. Systolic blood pressure was measured weekly from prior to surgery to the end of the three month feeding period. At three months, angiotensin I1 constrictor responses were evaluated in the isolated kidney vascular preparation and in intact anaesthetised rats fed the different diets. Phenylephrine constrictor responses were also evaluated in intact anaesthetised rats in order to exclude structural vascular changes accounting for differences in angiotensin 11 constrictor responses. Results: The diet high in unsaturated fats prevented the development of hypertension in 2KlC rats [systolic pressure 134(7) mm Hg at eight weeks] compared to their own preoperative blood pressures [124(3) mm Hg], and to the 2K1C rats fed the control diet [163(7) mm Hg at eight weeks]. The diet high in unsaturated fats did not alter blood pressures in sham operated rats. In isolated perfused kidneys and in anaesthetised 2K1C rats fed the control diet, angiotensin I1 caused a greater vascular response compared to the sham operated groups. The unsaturated fat diet prevented this effect. No differences were found in blood pressure responses to phenylphrine. Conclusions: These data suggest that the antihypertensive effect of a high unsaturated fat diet may in part be due to a depressed responsiveness of vascular smooth muscle to endogenous angiotensin 11. The effect is likely to be due to modulation of angiotensin I1 vascular responses by local vascular changes that cannot be accounted for by structural vascular differences. Cardiovascular Research 1992:26:973-977
974
Norton, Katze8 Woodiwiss, RosendorfS
them to adapt to the procedure. Measurements were taken on prewarmed conscious rats using a tail cuff plethysmographic technique. Each reading was taken at mid-day to avoid diurnal variation and the mean of four measurements was recorded. In a pilot study, there were no significant differences between blood pressures measured by the tail cuff method and by direct intra-arterial recordings in the same rats. Diets The high unsaturated fat ("unsaturated") diet used in these experiments was made up by the addition of 200 g sunflower seed oil to I kg of a 5% fat (by weight) normal rat chow (Epol, South Africa). The control diet was made up in the same way, except that 200 g of hydrogenated coconut oil was added to 1 kg of normal rat chow. The addition of fats to the normal chow reduced the vitamin and mineral concentration by 16%; this was compensated for by supplementation. Similar concentrations of macrominerals, microminerals, vitamins, carbohydrates and proteins were found in the two diets after they were made up in this way. Fats contributed 37% of the total energy in the diets (total energy 4842 kcal.kg-I). The diets were made up once weekly and stored at 4°C. Adequate vitamin E was added to prevent oxidation of fats in the diets. Analysis of the fatty acids in the sunflower seed oil and hydrogenated coconut oil added to the diets showed that no significant changes of the fat concentration occurred on storage. A homogeneous diet preparation was maintained during storage, as mixing of the residual food was repeated on a daily basis. All rats had free access to tap water and food. Total lipids in the diet were extracted according to the method of Folch et allo andtnalysis of fatty acids was performed using gas-liquid chromatography. Table I shows the fatty acid content of the diets analysed after one week of storage at 4°C. The high unsaturated fat diet had a much higher content of monoenoic, n-6 polyenoic, and n-3 polyenoic unsaturated fats and a lower saturated fat content. The fat content of the, control diet is thought to represent that of an average Western diet. In our experiment, we did not attempt to differentiate between the effects of specific unsaturated fats.
Table I
Fat content of diets Control diet
Fatty acids Saturated fats
fgkg-')
Unsaturated ,fat diet fR . k f ' )
1600 18:00
82 30.8 18.3 4.3
Monoenoics
16:l 18:l
0.2 16.5
36
n-6 Polyenoics
18:2 20:4
16.3
103
n-3 Polyenoics
18:3 205 22:6
0.8
12:00
14:00
20:00
Polyunsaturated to saturated fat ratio
0.7
2.5
1.4 14.9 7.0
I .o
0.5
0.9
0.5
0.9 1.2 0.7
0.13
3.9
Whole animal preparation Blood pressure and heart rate responses to infused angiotensin I1 and phenylephrine were monitored in weight matched anaesthetised rats: five animals from each 2K1C diet group and five animals from each sham operated diet group for both vasoactive substances. Catheterisation of the femoral artery (PP50) and vein (PP25) was performed under fentanyl-droperidol anaesthesia. Arterial pressure and heart rates were recorded directly through the femoral artery catheter and infusion of angiotensin I1 and phenylephrine was performed via the femoral vein catheter using a Harvard infusion pump (model 931 A). Pressures were recorded on a Beckman dynograph recorder (type R 5 11A) coupled to a Statham P23AA pressure transducer. Angiotensin I1 (AII, Hypertensin, Ciba) was dissolved in normal saline and infused at 0.02 m1.min-l for each dose. The doses given were 0.08, 0.16, 0.32, 0.63, 1.58, 3.16, and 6.31 nmol.min-I. Phenylephrine (Sigma) was first dissolved in 0.1 NHCl and then made up in four concentrations (10" to w'M) in a 0.1% ascorbate saline solution. These solutions were made up on the day of the experiment and stored at 4°C in the dark. For each molar concentration of phenylephrine, two different infusion rates were used (0.02 m1.min-I and 0.08 m1.rnin-I). At each dose of angiotensin I1 and phenylephrine, blood pressure and heart rate were allowed to stabilise for a 10 min period before recordings were made. Between each infusion of vasoactive agent, blood pressure and heart rate were allowed to return to baseline before the next dose was given. Baseline blood pressure levels did not change between the first and last doses of angiotensin I1 or phenylephrine. Heart rates did not change significantly through baroreceptor stimulation, probably because of the effects of anaesthesia. Infusion of the vehicle of angiotensin 11. at the above rates, did not alter blood pressures or heart rates. Statistical analysis All values presented in the text and in the figures are given as mean(SEM). Differences in systolic blood pressure levels between the diet groups and between 2KlC and sham operated rats were established by a two way analysis of variance with multiple comparisons followed by an unpaired r test with a Bonferroni correction and also by KruskalWallis statistics." Differences in values between diet groups and between 2KIC and sham operated groups in angiotensin I1 and phenylephrine vascular responses were determined using KruskalWallis and unpaired t statistics (Student-Newman-Keuls) with a Bonferroni correcti~n.'~A p value c0.05 was considered to be statistically significant.
Results Figure 1 illustrates the effect of a diet high in unsaturated fats on 2K1C renovascular hypertension. 2K1C rats fed the control diet developed significant hypertension by week one [systolic blood pressure (SBP) = 146(4) mm Hg] compared to their own preoperative blood pressures [SBP = 123(3) mm Hg; p
