Effects of dietary magnesium on blood pressure and vascular lesions in hypertensive rats

Pathology (1994), 26, pp. 365-369

EFFECTS OF DIETARY MAGNESIUM ON BLOOD PRESSURE AND VASCULAR LESIONS IN HYPERTENSIVE RATS DENNIS T. LIU, STEVEN W. TURNER, MIN-XIA W ANG* AND JUDITH

A.

WHITWORTH

Department ofMedicine, St George Hospital, University of New South Wales, *Department of Immunology, Sf George Hospital

Summary The present study examines the effects of dietary magnesium on the development of hypertension and hypertensive vascular lesions in deoxycorticosterone acetate and salt induced (DOCAsalt) and 2 kidney one clip (2K1 C) hypertensive as well as normotensive control rats. Animals received a regular (0.12% Mg), high (0.4% Mg) or low (0.03% Mg) magnesium diet for 6 wks. Dietary magnesium did not alter the growth or blood pressure in control, DOCA-salt and 2K1 C rats even though the plasma magnesium concentration was significantly altered by the diets (ANOVA, p<0.05 in control, DOCA-salt and 2K1C, respectively). Dietary magnesium did not alter the urinary potassium excretion, plasma sodium, potassium, total calcium concentration and plasma renin activity in any group, while the high magnesium diet significantly increased the urinary sodium excretion in DOCA-salt (p < 0.05) but not in control and 2K1 C rats when compared with the regular magnesium diet. In histological studies, dietary magnesium did not alter the percentage media area of intramyocardial arteries, or glomerular and renal arterial and arteriolar lesions in DOCA-salt and 2K1 C rats. This study suggests that moderate alterations of dietary magnesium do not modify blood pressure in normotensive control, DOCA-salt and 2K1C hypertensive rats, nor do they modify vascular disease in these 2 hypertensive models. Key words: Dietary magnesium, DOCA-salt, hypertension, rats, two kidney one clip, vascular lesions. Accepted 17 May, 1994

INTRODUCTION

Dietary magnesium intake has been associated inversely with blood pressure in human subjects. 1 In the International Cooperative Cardiovascular Diseases and Alimentary Comparison Study, urinary magnesium excretion was negatively and independently associated with blood pressure in populations from 20 countries. 2 The effects of dietary magnesium on experimental hypertension are unclear. Some studies suggest that dietary magnesium may play a role in the pathogenesis of hypertension' and hypertensive vascular diseases, however, the mechanism is unknown. Dietary magnesium supplementation to 0.75070 has been shown to attenuate the development of hypertension in DOCA-salt rats 3 and spontaneously hypertensive rats (SHR).4.5 A beneficial effect of magnesium supplementation has been found in

renal and vascular lesions in SHR while a low magnesium diet aggravated end organ damage. 6 - 8 However, Evans et a1. 9 failed to find any effect of magnesiumsupplementation on blood pressure in SHR, moreover, Ganguli et al. 10 showed that a high magnesium diet accelerated the rise of blood pressure and increased stroke mortality in stroke prone SHR (SHRsp). In the present study, 3 levels of magnesium in the diet, regular, high and low, were given to normotensive, DOCA-salt and 2KIC hypertensive rats to study the effect of dietary magnesium on blood pressure and hypertensive vascular lesions. We chose to use moderate magnesium supplementation and restriction rather than doses which are unlikely to be achieved in the clinical setting. MATERIALS AND METHODS Experimental protocol This study was approved by the Animal Care and Ethics Committee, the University of New South Wales. Male Sprague-Dawley rats (Animal resource, Western Australia) with initial body weight 150-180 g were used and housed in a climate controlled, 12 hr light cycle room, with free access to food and demineralized water. Four days were allowed for acclimatization after arrival, followed by 3 control days for determination of baseline body weight, metabolic balance, and tail cuff blood pressure. DOCA-saJt rats underwent a left uninephrectomy and were injected subcutaneously with deoxycorticosterone acetate (Sigma, 30 mg/ kg body weight) twice weekly and offered 1010 sodium chloride solution to drink. Two kidney one clip hypertension was induced in rats by constricting the left renal artery with a 0.22 mm internal diameter silver clip and the contralateral kidney was untouched. After operation, rats were subdivided into a further 3 groups given either regular (0.12010 Mg), high (0.4010 Mg) or low (0.03010 Mg) magnesium diet. Numbers of animals in each of the 3 diet groups were as follows: normotensive controls (n = 10 in each group), DOCA-salt (regular Mg: n = 15 until wk 5, n 12 at wk 6; high Mg: n = 15 until wk 3, n = 13 at wk 4, n= 12 at wk 5 & 6; low Mg: n= 15 until wk 4, n= 13 at wk 5 & 6) and 2KIC (regular Mg: n 12 until wk 5, n = 11 at wk 6; high Mg: n = 12 until wk 5, n = 10 at wk 6; low Mg: n = 14 until wk 5, n = 13 at wk 6). After 7 days recovery, systolic blood pressure was measured in conscious, prewarmed rats by the tail cuff method once a week. The equipment comprised an occluding cuff, Pneumatic pulse transducer (Narco Biosystems, Inc., Houston, Texas, USA), Programmed ElectroSphygmomanometer PE-300 (Narco Biosystems, Inc., Houston, Texas, USA), MPIOO data acquisition system (Biopac System, Inc., Goleta, CA 93117, USA) and Macintosh computer . Several consecutive cycles were performed and the mean of last 3 recordings, among which there was not more than to mmHg difference, was taken as the systolic

366

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LIU et al.

blood pressure. aody weight was measured, followed by 24 hr water and food intake and urinary volume measurement in metabolic cage once weekly. Urine samples were collected for determination of urinary electrolyte concentration. After 6 wks, intra-arterial blood pressure was measured. Rats,were anesthetized with intraperitoneal nembutal injection (60 mg/kg), and the left carotid artery was cannulated with clear vinyl tubing which was connected to a physiological pressure transducer (Bentley Trantec, Inc., 17502 Armstrong Avenue, Irvine, California 92714, USA). Direct blood pressure was read from a cardiac monitor (Pressure Monitor BP-I, World Precision Instruments, Sarasota, FL, USA). Blood samples were collected for estimation of plasma sodium, potassium, calcium, magnesium concentrations and plasma renin activity. Heart and kidney were quickly removed and immersed, fixed in mercuric formalin and stained with Haematoxylin and Eosin (H&E) and Masson's trichrome method for histological studies. Analytical techniques Urinary and plasma sodium and potassium concentrations were measured by flame photometry (Technicon Auto-analyser, Technicon Instrument Co., Tarrytown, NY, USA). Plasma total calcium concentration was measured by colorimetry (Technicon Auto-analyser, Technicon Instrument Co., Tarrytown, NY, USA). Urjnary and plasma magnesium concentrations were measured by colorimetry (Merkotest 3338 magnesium kits, E. Merck, Postfach 4119, D-6100 Darmstadt I, Germany). Plasma renin activity was determined by the rate of angiotensin I production as estimated by radioimmunoassay. 11 Histological assessment The slides were independently graded by 2 observers who were unaware of their origin. For heart sections, morphometric measurements were made using a Hewlett Packard 9874 digitizer attached to a pre-programmed Hewlett Packard 9825A ealculator (Hewlett Packard, Fort Collins, Colorado, USA). Transverse sections of arteries and arterioles with circular outline were examined. The total vessel area was quantified by tracing the perimeter of the media/adventitia boundary or external elastic lamina, where visible, with automatic quantification of the area contained within these lines. The lumen area was determined from the perimeter of the internal elastic lamina, with the media calculated by subtraetion (total area lumen area). The media area was expressed as a percentage of the total area. 12 For renal sections, interlobular arteries in the right kidney were graded by the method described by Hewitson et al 13 according to severity of damage to the arterial tree. Grade I - no abnormality (score = 1). Grade II - slight thickening of the media (score: 2). Grade HI - deposition of fibrinoid material in focal area, with a moderate thickening of the media (score = 3). Grade IV - medial thickening with extensive deposition of fibrinoid material around the whole circumference (score = 4). Grade V - fibrinoid necrosis and/or cellular hyperplasia with narrowing of the arterial lumen and/ or thrombus formation (score = 5). The number of vessels in each grade was expressed as a percentage of total vessels. The vascular score for each animal was calculated according to the formula: Vascular score = OJo grade I x 1 + OJo grade II x 2 + OJo grade III x 3 + OJograde IV x 4 + OJo grade V x 5. The number of glomeruli were recorded for each renal section. Glomerular damage was assessed according to the presence of fibrin and/ or crescent formation (score = I) and expressed as a percentage of total glomeruli for each section. Statistical analysis Values were expressed as mean ± SEM. For repeated measurements (blood pressure, body weight, urine volume and urine electrolytes), results were analysed using repeated measures analysis of variance to test the effects of diet and time. Significance of within-subject factors was examined by the method of Greenhouse and Geisser. 14 If there was, a significant diet effect, an unpaired t-test with correction for multiple comparisons was used to compare the 3 diet groups against each other

15

at each time point. Nonparametric data were expressed as median and range. Statistical significance was tested using the rank sum test.

RESULTS

Baseline blood pressure, body weight, urine volume and urinary sodium, potassium and magnesium excretion were not different between diet groups in control, DOCA-salt or 2KI C rats. Systolic blood pressure and body weight Dietary magnesium had no significant effect on tail cuff blood pressure in control, DOCA-saIt and 2KIC rats by repeated measures analysis of variance; mean systolic blood pressure among different diet groups in control, DOCA-salt or 2KIC rats were not significantly different at any time point (Fig. I). Direct blood pressure measured by intra-arterial cannulation did not differ between each diet group (Tables I, 2 and 3). Dietary magnesium did not alter body weight in control, DOCA-salt and 2KIC rats in the study (Tables I, 2, and 3). Metabolic studies Dietary magnesium had no effect on urinary sodium and potassium excretion in control (Table 1) and 2KIC rats (Table 3). In DOCA,salt rats, urinary sodium excretion was significantly increased by the high magnesium diet, while urinary sodium and potassium excretion in the low magnesium rats and urinary potassium excretion in the high magnesium rats were comparable to rats on the regular diet (Table 2). Urinary magnesium excretion was significantly increased by the high magnesium diet in control, DOCA-salt and 2KIC rats (Tables 1,2 and 3). Plasma chemistries In control, DOCA-salt and 2KIC rats, plasma sodium, potassium, total calcium concentration and plasma renin activity were not altered by dietary magnesium while plasma magnesium concentration was significantly increased by the high magnesium diet, and decreased by the low magnesium diet (Tables I, 2 and 3). Histological assessment (Table 4) Percentage media area of intramyocardial arteries was significantly increased in DOCA-salt (p <0.01) and 2KIC rats (p < 0.05) as compared to control rats. Dietary magnesium had no effect on the percentage media area of intramyocardial arteries in each group. In control rats on the 3 different magnesium diets, kidney sections appeared normal. Extensive glomerular fibrosis, hyalinosis and sclerosis, vascular hyperplasia and fibrinoid necrosis were seen in DO CA-salt and 2KIC rats (p < 0.05 compared with control). Neither the percentage of glomerular lesions nor the renal arterial and arteriolar lesion score was altered by dietary magnesium in DOCAsalt and 2KIC rats. DISCUSSION

The present study has shown that moderate dietary magnesium supplementation or restriction did not alter blood pressure in normotensive control, DOCA-salt

DIETARY MAGNESIUM AND VASCULAR LESIONS

120

TABLE 1 Direct blood pressure (SBP), body weight (BW), urine and plasma chemistries at wk 6 in control rats

controls

Regular Mg

100

bD

80

El El

160

::z::

BW (g) SBP (mmHg) Urinary electrolytes (mmollday) Na K Mg Plasma (mmollL) Na K Total Ca Mg PRA (ng Al/mLlhr)

DOCA-salt

4)

s-

.,., ~

4)

's-

120

c..

'" 0 0

co ~

.,;..,0

..0

367

n=1O 372±1 118±2

High Mg

Low Mg

n=lO 371±2 118±2

n=lO 382± 11 117±3

1.2±0.1 O.7±O.1 O.04±0.OI

1.3 ±0.1 O.8±O.1 O.O6±O.Ol*

1.l±0.1 O.9±O.1 O.O2±O.OO2

139± 111 5.0±0.2 2.25±O.O5 0.86±0.02 11 ±3

139±1 4.5±O.2 2.19±O.O3 0.93±O.05* 8±2

138± 1 4.8±O.2 2.21 ±0.04 0.74±O.04* 13±4

Mean±s.e.m., *p<0.05 vs regular Mg diet. All animals survived until the end of experiment.

80 180

2KIC

Cl)

1.30

80

o

2

.3

4

5

6

Time (weeks) o regular Mg diet. • high Mg diet. v low Mg diet. Fig. 1 Mean systolic blood pressure in the 3 diet groups for normotensive control (n = 10 in each group), DOCA-salt (regular Mg: n = 15 until wk 5, n = 12 at wk 6; high Mg: n = 15 until wk 3, n = 13 at wk 4, n = 12 at wk 5 & 6; low Mg: n 15 until wk 4, n = 13 at wk 5 & 6) and 2KIC (regular Mg: n = 12 until wk 5, n = 11 at wk 6; high Mg: n 12 until wk 5, n = 13 at wk 6; low Mg: n = 14 until wk 5, n = 13 at wk 6) rats. Values are expressed as mean±s.e.m.

hypertensive or 2KI C hypertensive rats, even though the urinary and plasma magnesium were significantly altered. Variations in dietary magnesium also did not modify the histological changes in either DO CA-salt or 2KIC hypertensive rats. Some studies reported that dietery magnesium supplementation above 0.75070 lowered blood pressure in both DOCA-salt rats and SHR when compared to a 0.21 % magnesium diet. 3,4,5 The present study demonstrated that dietary magnesium at levels of 0.03,0.12, and 0.4% had no significant effect on blood pressure in normotensive, DOCA-salt, or 2KIC hypertensive rats. The level of magnesium content in the high magnesium diet used in the present study was lower than in previous reports, but plasma and urinary magnesium were significantly altered by the diets. Similar observations were made in SHR. Evans et al. 9 used 3 levels of magnesium diet similar to that used in the present study (0.01,0.05, and 0.4% Mg), and failed to show any effect on blood pressure even though the serum magnesium concentration was significantly altered. The absence of any effect of dietary magnesium on blood pressure in this study and that of Evans et al. 9 suggests that moderate modification of dietary magnesium has only little, if any, effect on blood pressure, even though magnesium homeostasis is significantly altered.

Hattori et al 16 reported that there was a positive correlation between blood pressure and intra-erythrocyte sodium content in DOCA-salt hypertensive rats, and a high level of magnesium supplementation significantly reduced intra-erythrocyte sodium, and thereby reduced blood pressure. In Hattori's study,16 plasma sodium concentration was not changed by magnesium supplementation although urinary sodium excretion was not reported. In the present study, intra-erythrocyte sodium content was not measured, and plasma sodium concentration was not significantly altered by diet, but urinary sodium excretion was significantly increased by high magnesium diet. Possibly, magnesium supplementation at a very high level is able to exert a hypotensive effect through alterations in sodium handling at a cellular level. In SHR, a low magnesium diet was reported to aggravate renal and myocardial tissue calcification and gross renal lesions. 6.7 Magnesium supplementation in the diet' or drinking water 8 modified the hypertensive vascular lesions and end renal damage without lowering the blood pressure. There may be an important role of dietary TABLE 2

Direct blood pressure (SBP), body weight (BW), urine and plasma chemistries at wk 6 in DOCA-treated rats

BW (g) SBP (mmHg) Urinary electrolytes (mmollday) Na K Mg Plasma (mmoI/L) Na K Total Ca Mg PRA (ng AIImllhr)

Regular Mg

High Mg

LowMg

n=12 311 ± 11 162±4

n=12 312±13 165±3

n=13 310± 12 159±5

14.1±2 l.3±0.2 0.11 ±0.02

18.3 ± 2* 1O.9±2 1.6±O.1 1.3 ±0.3 0.24 ± 0.02*' 0.03 ± 0.01 '*##

145±3 3.8±0.2 2.32±0.05 0.75±O.OI 0.24±0.O2

150±3 3.5±0.2 2.31 ±O.OS 0.S3±0.02* 0.27 ±0.04

14S±2 4.1 ±0.3 2.25±0.04 0.68 ± 0.04*# O.26±O.OS

Mean±s.e.m., *p<0.05, **p
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Pathology (1994), 26, October

LIU et al.

TABLE 3 Direct blood pressure (SBP), body weight (BW), urine and plasma chemistries at wk 6 in 2KIC rats Regular Mg BW (g) SBP (mmHg) Urinary electrolytes (mmollday) Na K Mg Plasma (mmoIlL) Na K Total Ca Mg PRA (ng/mLlhr)

High Mg

LowMg

n=l1 323± 10 178±6

n= 10 320± 11 182±5

1.8 ±O.l 1.4±0.1 0.03 ±0.01

1.8 ±0.2 1.2±0.2 0.1 ±0.03* 0.03±0.01

n=13 322 12 185

1.6 ±0.2

1.3 ±0.3

136±1 135±2 136 I 4.8±0.2 4.7±0.1 5.1 ±0.5 2.24±0.02 2.32 ± 0.03 2.30±0.02 0.82±0.02 0.87±0.02* 0.75±0.04*# 38±6 44±8 34±9

Mean±s.e.m., *p<0.05 vs regular diet; #p<0.05 vs high Mg diet. Data from animals surviving until the end of the experiment. calcium in the protective effect of dietary magnesium supplementation, as shown in Evans's study.7 A high magnesium diet only exerted its beneficial effect on kidney damage when the animals were also offered a high calcium diet. Suzuki et aP also found that magnesium supplementation attenuated the development of DOCAsalt hypertension, which was associated with a decrease in cytosolic free calcium concentration. In contrast, Ganguli et apo found that a high magnesium diet (0.26010 vs 0.085%) appeared to accelerate the rise of blood pressure in stoke-prone SHR. In the present study, different levels of magnesium in the diet did not alter vascular histomorphologicaI changes in either DOCA-saIt or 2KIC hypertensive rats. Typical abnormalities such as fibrinoid deposition in the renal arteriole wall and glomeruli, renal arteriolar fibrinoid necrosiS and cellular hyperplasia were consistently developed in all hypertensive groups. There is no conclusive explanation for the differences in outcome in the studies mentioned above. However, the interaction with other cations, especially calcium, in the diet cannot be neglected. In previous studies, we demonstrated that moderate alteration of other dietary cations had significant effects on hypertensive vascular lesions. Moderate decrease of dietary sodium intake attenuated glomerular and vascular TABLE 4 Histological assessments in DOCA-SALT and 2KIC rats 0/0 Media Score % GL mean ± sem median (range) median (range)

REGULAR DIET Control (n = 10) DOCA-salt (n = 12) (n = 11) 2KIC HIGH Mg DIET (n = 10) Control DOCA-salt (n = 12) (n= 10) 2KIC LOW Mg DIET Control (n = 10) DOCA-saJt (n= 13) 2KIC (n = 13)

64±2 69±2 71 ±3

131 (122-148) 237 (178-342) 229 (173-321)

0(0-0) I I (0-46) 18 (0-36)

65± I 68± I 69±2

129 (120-140) 235 (146-264) 241 (164-348)

0(0-0) 17 (0-29) 23 (0-41)

64±1 71±1 68±6

126 (120-136) 252 (128-297) 224 (196-296)

0(0-0) 20 (0-45) 25 (0-33)

% media percentage of media area in intramyocardial arterIes; Score - renal arterial lesions score; Percentage of glomerular lesions.

damage 17 while moderate decrease of calcium intake aggravated renal vascular lesions and high calcium diet reduced intramyocardial arterial hypertrophy18 in hypertensive rats. We also found that a moderate high potassium diet significantly reduced glomerular and renal arterial and arteriolar lesions and intramyocardial arterial wall thickening but did not alter the blood pressure in 2KIC rats.19 As discussed above, a very high magnesium intake significantly altered sodium 16 and calcium3 status, and reduced blood pressure in hypertensive rats. 16.3 The absence of effect of a moderate alteration of magnesium intake on blood pressure and vascular lesions in the present study, as well as the finding of Evans et aF thaJ the protective effect of a moderate high magnesium intake against renal damage was dependent on a simultaneously high calcium intake, suggest that a primary effect of dietary magnesium on blood pressure and vascular lesions is unlikely, however, when the alteration of magnesium intake is sufficient to change body sodium and calcium balance, effects on blood pressure and vascular lesions may be seen. Results from the present study suggest that moderate alterations of dietary magnesium do not modify blood pressure in normotensive, DOCA-salt and 2KIC hypertensive rats, nor do they modify vascular disease in these 2 experimental hypertensive models. ACKNOWLEDGEMENTS This study was supported by a General Development Grant from the University of New South Wales. We are grateful to Professor Priscilla Kincaid-Smith and Mr lan Birchall of the Department of Pathology at the University of Melbourne for preparing the histological slides. Address for correspondence: J .A. W.,

Department of Medicine, St George Hospital, University of New South Wales, Kogarah, NSW 2217.

Referen.ces 1. loffres MR, Reed DM, Yano K. Relationship of magnesium intake and other dietary factors to blood pressure: the Honolulu heart study. Amer J Clin Nutr 1987; 45: 469-75. 2. Yamori Y, Nara Y, Mizushima S et al. International cooperative study on the relationship between dietary factors and blood pressure: a report from the Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study. J Cardiovasc Pharmacol 1990; 16(suppl 8): S43-S47. 3. Suzuki H, Sano H, Fukuzaki H. Decreased cytosolic free calcium concentration in lymphocytes of magnesium-supplemented DOCAsalt hypertensive rats. Clin Exp Hypertens 1989; All: 487-500. 4. Wolf JP, Luthringer C, Berthelot A et al. Blood pressure and plasma renin activity after magnesium supplementation in the spontaneously hypertensive rat: a study during developing and established hypertension. Magnesium 1987; 6: 243-8. 5. Luthringer C, Trouvin JH, Laurant P et al. Effect of high magnesium intake on central catecholamines in spontaneously hypertensive rat. Magnes Res 1988; 1: 163-7. 6. Chrysant SG, Ganousis L, Chrysabt C. Hemodynamic and metabolic effects of hypomagnesemia in spontaneously hypertensive rats. Cardiology 1988; 75: 81-9. 7. Evans GH, Weaver CM, Harrington DD et al. Association of magnesium deficiency with the blood pressure-lowering effects of calcium. J Hypertens 1990; 8: 327-37. 8. Saito N. The suppressive effect of MgC12 on vascular lesions in SHRSP. Clin Exp Hypertens 1990; A12: 672.

DIETARY MAGNESIUM AND VASCULAR LESIONS

9. Evans OH, Weaver CM, Harrington DD et al. Dietary Mg does not affect blood pressure in spontaneously hypertensive rats. Clin Exp Hypertens 1989; All: 619-32. 10. Oanguli M, Tobian L, Sugimoto T. High magnesium diets increase blood pressure and enhance stroke mortality in hypertensive SHRsp rats. Am J Hypertens 1989; 2: 780-83. 11. Johnston Cl, Mendelsohn F, Casley D. Plasma renin determination employing a radioimmunoassay for angiotensin I. Proc Aust Soc Med Res 1969; 2: 271-2. 12. Whitworth JA, Hewitson TD, Wilson RS et al. ACTH hypertension in the rat: Haemodynamic, metabolic and morphologic characteristics. J Hypertens 1990; 8:' 27-36. 13. Hewitson T, Kamitsuji H, Whitworth JA et al. A comparison of the vascular and glomerular changes in aortic-ligature and DOCA-salt hypertension. Clin Exp Pharmacol Physiol 1989; 16: 641-5.

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14. Greenhouse SW, Geisser S. On method in the analysis of profile data. Psychometrika 1959; 24: 95-112. 15. Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing. Stat Med 19'90; 9: 811-18. 16. Hattori K, Sano H, Kubota Y et al. The effect of Ca and Mg supplementation and the role of the opioidergic system on the development of DO CA-salt hypertension. Am J Hypertens 1991; 4: 72-5. 17. Liu DT, Birchall I, Kincaid-Smith P et al. Effect of dietary sodium chloride on the development of renal glomerular and vascular lesions in hypertensive rats. Clin Exp Pharmacol Physiol1993; 20: 763-72. 18. Liu DT, Birchall I, Hewitson T et al. Effect of dietary calcium on the development of hypertension and hypertensive vascular lesions in DOCA-salt and 2KIC rats. J Hypertens 1994; 12: 145-53. 19. Liu DT, Wang MX, Kincaid-Smith P et al. The effects of dietary potassium on vascular and glomerular lesions in hypertensive rats. Clin Exp Hypertens 1994; 16: 391-414.