The effect of minoxidil on salt overload and blood pressure in both normotensive and doca-salt hypertensive rats.
We have previously demonstrated that a decrease in total peripheral resistance (TPR) caused by opening an A-V fistula or chronic administration of minoxidil, a potent peripheral vasodilator, does not decrease mean arterial blood pressure in normotensive rats. Additionally, DOCAsalt hypertension can still develop in the presence of a low TPR, suggesting that changes in TPR are not the primary mechanism by which mean arterial blood pressure is achieved. There is also no correlation between total peripheral resistance and mean arterial blood pressure (16). Numerous previous studies (1, 7-11) have shown that the long-term level of arterial blood pressure occurs where the sodium intake and chronic salt-loading pressure natriuresis curves intersect (equilibrium point). The longterm arterial blood pressure can be changed only by changing one or both of these two factors. If minoxidil treatment results in a decrease in MAP then there must be some change in the chronic salt-loading pressure natriuresis curve.
It has been demonstrated that sodium overload is often an important factor in the pathogenesis of various forms of experimental and clinical hypertension, including essential hypertension (12-15). For instance, a decrease in renal mass (a decrease in number of nephrons) to one-half or one third of normal does not usually change the long-term level of mean arterial blood pressure as long as the ratio of glomerular filtration rate and renal tubular reabsorption are constant. Animals with reduced kidney mass became hypertensive when high salt was administered (13, 14, 1621). Another typical example of sodium dependent hypertension is mineralocorticoid hypertension. A nephrectomized animals that receives mineralocorticoid alone would have only a small increase in blood pressure, but a combination of both mineralocorticoid and high salt results in severe hypertension (13, 22, 23, 24), suggesting that the DOCA-salt hypertensive rats may be particularly sensitive to the overload of salt.
Both experimental and clinical studies have shown that a large change in salt intake (1/10-10 times) in animals or human beings with normal renal function can change the arterial blood pressure no more than 10-20 mmHg. On the other hand, an excessive sodium load can cause a sustained hypertension when sodium excretion by the kidney is somehow compromised. Thus, the role of salt in the pathogenesis of hypertension is determined not only by salt intake but also by the ability of the kidneys to eliminate salt from body (1, 9, 13, 16, 18-21). Therefore, the main objective of the present study was to determine if minoxidil treatment resulted in a decrease in arterial blood pressure in DOCA-salt hypertensive rats, without affecting the renal function curve. We tested the hypothesis that there is a difference between sodium intake and mean arterial blood pressure in both normotensive and DOCA-salt hypertensive rats treated with or without minoxidil.
All experiments were conducted in male Sprague Dawley rats weighing 200-225 grams. The rats were divided into 8 groups: control (n=6), minoxidil (n=7), salt (n=8), salt-minoxidil (n=8), DOCA (n=8), DOCAminoxidil (n=6), DOCA-salt (n=8), and DOCA-salt minoxidil (n=7). In the beginning of the experiments, all of the rats underwent a right nephrectomy during anesthesia with sodium pentobarbital (50 mg/kg i.p.). DOCA pellets (75 mg, Innovative Research of America, Toledo, Ohio, USA) were subcutaneously implanted into the rats in groups of DOCA, DOCA-minoxidil, DOCA-salt and DOCA-salt minoxidil. Minoxidil (Sigma) at a dose of 3 mg/day was given orally to the rats in the salt-minoxidil, DOCA-minoxidil, and DOCA-salt minoxidil groups throughout the experiments (duration:6 weeks). The rats in the salt, salt-minoxidil, DOCA-salt, and DOCA-salt minoxidil groups were given saline ad libitum. All animals were maintained on standard commercial rat diet. The rats were housed individually in animal cages in animal facilities where the temperature and humidity were constant, and they had a 12 hour light cycle from 6 A.M. to 6 P.M..
At the end of the 6 week experimental period, the rats were anesthetized with sodium pentobarbital (50 mg/kg). The carotid artery was cannulated with a polyethylene tubing (Clay-Adams no. 7411, 0.5 mm i.d., 0.97 mm o.d., Parsippany, NJ, USA) for the measurement of mean arterial blood pressure (MAP). The catheter was pre-filled with heparinized saline (1,000 u/ml). MAP was measured for 30 minutes using a Statham pressure transducer (P23ID, Oxnard, CA, USA). Following the MAP measurements, the rats were killed by pentobarbital overdose. Heart and kidneys were removed and weighed.
Renal function curve
In a steady state condition, renal function curve can be depicted by the relationship between the sodium intake and mean arterial blood pressure. Mean arterial pressure was measured at the end of 6 weeks. Sodium intake was measured every 24 hours throughout the experiments. Based on the previous study as well as the time course of sodium intake observed in the present study, sodium intake and arterial blood pressure in the DOCA-salt hypertensive rats reached to plateau after 4 weeks. Therefore, the data for sodium intake from the last week used for the renal function curve. The renal function curve was determined by two points: one point was the intersection between blood pressure and normal salt intake (tap water) in all groups, the other point was the intersection between blood pressure and high salt (saline) intake in all groups.
To determine if there was an interaction between any of the three treatments (salt intake, DOCA, or minoxidil) a [2.sup.3] factorial analysis in a completely randomized design was performed. A statistically significant difference was accepted when p<0.05. Where statistical significance was determined the means grouped according to treatment are presented.
Sodium intake and mean arterial blood pressure
The time course of changes in sodium intake in both normotensive and DOCA-salt hypertensive rats is shown in Figure 1. The data reveal the total of all sources of sodium (0.4% in food, 3.9% in saline) and each data point represents the average value calculated on a weekly basis. The bottom four curves represent the daily sodium intake for the rats in the control, minoxidil, DOCA and DOCA-minoxidil groups (all of these rats drank tap water). The top four curves represent the sodium intake for the rats in the control-salt, control-salt minoxidil, DOCA-salt and DOCA-salt minoxidil groups (all of these rats drank saline).
Sodium intake during the 6th week induced a significant effect of DOCA on the level of salt intake (p<0.01). Additionally, there was an interaction between DOCA and salt to affect salt intake (p<0.01), such that the DOCA-salt animals had a significantly higher salt intake than the DOCA tap animals.
The MAP measured at the end of 6 weeks for the rats in the control group averaged 119 [+ or -] 4 mmHg. MAPs for the rats in the minoxidil, salt and salt-minoxidil groups were 117 [+ or -] 4 mmHg, 111 [+ or -] 3 mmHg, 111 [+ or -] 3 mmHg, respectively. The rats that received DOCA with tap water had a MAP of 139 [+ or -] 8 mmHg (no minoxidil) and 133 [+ or -] 4 mmHg (minoxidil). The MAP in the DOCA-salt animals averaged 160 [+ or -] 5 mmHg (no minoxidil) and 146 [+ or -] 9 mmHg (minoxidil). DOCA treatment alone had a significant effect to increase pressure (p<0.01), and there was a significant interaction between DOCA and salt to increase blood pressure (p<0.01). Minoxidil treatment had no effect on blood pressure (p>0.05). The average data for the treatment groups is presented in Figure 2. The average MAP for all of the tap water no DOCA animals (with and without minoxidil) averaged 117 [+ or -] 3 mmHg (n=13). The average MAP for the tap water, DOCA animals (with and without minoxidil) averaged 136 [+ or -] 5 mmHg (n=14). The average MAP for the saline animals, no DOCA treatment animals (with and without minoxidil) averaged 111 [+ or -] 2 mmHg (n=16). The average MAP for the saline-DOCA animals (with and without minoxidil) averaged 154 [+ or -] 5 mmHg (n=15).
heart weight and kidney weight
The changes in heart weight (HWT), normalized to body weight, are shown in Figure 3. All of the treatments, salt intake, DOCA, and minoxidil, had a significant effect on heart weight, and there were significant interactions between all of the treatment groups. The changes in kidney weight (HWT), normalized to body weight are shown in Figure 4. Both saline and DOCA treatment independently had a significant effect to increase kidney weight (p<0.01). There was also a significant interaction between DOCA and saline treatments to increase kidney weight (p<0.01). Minoxidil treatment had no effect on kidney weight with any of the treatments p>0.05).
The findings of the present study provide additional evidence that the DOCA treated rats are particularly sensitive to an increased salt intake, resulting in develop of hypertension (13, 25, 26). When sodium intake increased from normal salt (tap water) to high salt (0.9% saline) in both control and minoxidil treated normotensive rats, mean arterial blood pressure was not significantly changed. DOCA treatment had a significant effect to increase blood pressure and the addition of saline intake in the DOCA treated animals resulted in a significant interaction, resulting in a larger increase in blood pressure. Additionally, minoxidil treatment had no effect on salt intake or mean arterial pressure in support of our hypothesis that the development of hypertension is not affected by total peripheral resistance. This study shows that sodium intake plays an important role in the determination of the magnitude of rise in mean arterial blood pressure in DOCA-salt hypertensive rats.
Both experimental and clinical studies have shown that a large change of salt intake (1/10-10 times) in animals or human beings with normal renal function can change the arterial blood pressure no more than 10-20 mmHg. On the other hand, an excessive sodium load can cause a sustained hypertension when sodium excretion by the kidney is somehow compromised. Thus, the role of salt in the pathogenesis of hypertension is determined not only by salt intake but also by the ability of the kidneys to eliminate salt from body (2, 8, 13). Therefore, it was the second aim in the present study to determine the renal function curve in both the normal and DOCA-salt hypertensive rats treated with or without minoxidil. The role of renal function in the long-term arterial blood pressure control can be graphically described by a chronic salt-loading pressure natriuresis curve. In the steady state condition, sodium intake is equal to sodium output. Therefore, renal function in the present study was plotted as the relationship between sodium intake and arterial blood pressure (Figure 3).
Several studies have demonstrated that the slope of the normal chronic salt-loading pressure natriuresis curve is very steep. Conversely, the slope of the chronic salt-loading pressure natriuresis curve is depressed when renal mass is reduced. On the other hand, infusion of aldosterone and angiotensin in animals results in a rightward shift of chronic salt-loading pressure natriuresis curve and a suppression of the slope (7, 8, 18). Gross at al (23) reported that DOCA-salt increased blood pressure and rightward shifted pressure natriuresis curve in salt sensitive Sabra rats. But there have been no studies showing the effect of DOCA-salt on the chronic salt-loading pressure natriuresis curve in Sprague Dawley rats during minoxidil treatment. The data presented in the present study have shown that the chronic administration of DOCA-salt in uninephrectomized rats resulted in a shift of pressure and sodium intake relationship to a higher pressure level and a suppression of the slope of this curve as compared to that seen in uninephrectomized control rats (see Figure 3). The possible mechanism responsible for the rightward shift of the chronic salt-loading pressure natriuresis curve in the uninephrectomized DOCA-salt hypertensive rats could be attributed to the increase in sodium reabsorption from renal tubules, or to the increase in renal vascular resistance.
Although increases in renal vascular resistance in DOCA-salt hypertensive rats were observed in the our previous studies (3, 4), the precise portion of the renal vessels responsible for the increases in renal vascular resistance are still unknown. Evidence from the micropuncture studies of Dworkin et al. (16) has shown that afferent arteriolar vascular resistance is increased and efferent arteriolar resistance is decreased in DOCA-salt hypertensive rats. Also, Tojo et al. (27) has measured renal arteriolar diameter using a vascular cast technique and have found that the afferent arterioles were constricted while the efferent arterioles were dilated in DOCA-salt hypertensive rats. This pattern of the resistance changes in the DOCA-salt hypertension may be a regulatory mechanism to protect the glomeruli from hypertensive damage.
It has been demonstrated that a shift of the chronic salt-loading pressure natriuresis curve to a higher pressure level is a common feature of all forms of established hypertension. On the other hand, the shift of the chronic salt-loading pressure natriuresis curve toward normal is always the result of effective antihypertensive therapy. Furthermore, antihypertensive drugs that do not affect chronic salt-loading pressure natriuresis curve will not cause a long-term decrease in arterial blood pressure. The results of the present study provide additional support for the contention that the chronic salt-loading pressure natriuresis curve is the most important determinant in the long-term control of arterial blood pressure and the pathogenesis of hypertension. As shown in Figure 3, the treatment of both normotensive and hypertensive rats with minoxidil did not shift the chronic salt-loading pressure natriuresis curve. This is the effect that would be expected since minoxidil had a preferential effect on peripheral arterioles, and not renal arterioles, as evidenced by a marked decrease in total peripheral resistance but no significant changes in renal vascular resistance (4, 6, 14, 22, 28).
All three treatments had a significant effect to increase heart weight and kidney weight, consistent with reports in the literature. The effect of DOCA and minoxidil on heart weight would be expected as DOCA resulted in an increase in mean arterial pressure and minoxidil treatment resulted in an increase in cardiac output (4). Vaskonen et al. (29) showed, in SHRs, that high salt intake resulted in cardiac and renal hypertrophy independent of any increase in blood pressure. Gu et al. (30) has shown, in cultured myoblasts, that elevated sodium concentration increases cell diameter, volume, and protein content. However for Gu's study it is unclear if the concentrations used in vitro are ever seen in vivo. DOCA has been shown to increase kidney weight in SHRs (31). However, in our studies, we are not sure if the increase in wet kidney weight was due to cellular hypertrophy or an increase in the amount of tubular fluid
In summary, DOCA treatment alone increases blood pressure and the addition of a high salt diet results in a further increase in blood pressure. Chronic administration of the vasodilator minoxidil does not change long-term control of arterial blood pressure in either normotensive rats or hypertensive rats. The correlation between arterial blood pressure and chronic salt loading pressure natriuresis is the primary determinant in the long-term control of arterial blood pressure and the pathogeneses of hypertension.
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Min Huang , Hamed Benghuzzi , Michelle Tucci , and Robert L. Hester 
Departments of Physical Therapy , Diagnostics and Clinical Health Sciences , Orthopedics , and Physiology ; University of Mississippi Medical Center, Jackson, Mississippi 39216-4505
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|Author:||Huang, Min; Benghuzzi, Hamed; Tucci, Michelle; Hester, Robert L.|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Apr 1, 2015|
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