Author for correspondence: Sonia Alves Gouvêa, Departamento de Ciências Fisiológicas, Centro de Ciências da Saúde da UFES, Avenida Marechal Campos, 1456, 29040-577 Vitória, ES, Brazil (fax +55 27 3335 7330, e-mails firstname.lastname@example.org; email@example.com).
Abstract: Raloxifene is a selective oestrogen receptor modulator that has been approved for the prevention and treatment of osteoporosis in post-menopausal women. Studies have revealed several effects of raloxifene on the cardiovascular system, which might contribute to the blood pressure regulatory mechanisms, particularly in the systemic arterial hypertension. Therefore, the aim of this study was to investigate the effects of raloxifene on the blood pressure, renal excretion of water and Na+ and plasma nitrite/nitrate levels in 2-kidney-1-clip (2K1C) hypertensive female rats. The groups were as follows: hypertensive (2K1C), hypertensive ovariectomized (2K1C + OVX) and hypertensive ovariectomized treated with raloxifene (2K1C + OVX + R). Seven days after the surgery that produced menopause, 2K1C hypertension was produced in anaesthetized animals. Seven days after the clip application, the rats were put into metabolic cages to allow for the measurement of water ingestion and diuresis, and raloxifene was administered (2 mg/kg/day i.p., for 7 more days). We found a large reduction (p <0.01) in mean arterial pressure (197 ± 6 to 164 ± 2 mmHg), an increase in renal excretion of sodium and water (p <0.05) and an increase in plasma levels of nitrite/nitrate in 2K1C + OVX + R animals, when compared with the 2K1C (23.4 ± 1 versus 14 ± 0.5 nmol/mL; p <0.01, respectively). These findings suggest that raloxifene exerted its antihypertensive effect, at least in part, by improving the renal excretion of sodium and water.
In addition to its known effects on the reproductive system, nervous system and kidney [1–4], oestrogen exerts haemodynamic, metabolic and vascular effects that can explain the cardioprotection  seen in pre-menopausal women and in women who receive oestrogen replacement therapy for the relief of post-menopausal symptoms .
Hormone replacement therapy (HRT) with oestrogen (alone or combined with a progestin) is usually prescribed for the relief of post-menopausal symptoms. In contrast to the increased risk of developing endometrial cancer , epidemiological data indicate that the use of oestrogen in women after menopause is associated with up to a 50% reduction in the incidence of cardiovascular diseases .
The 2-kidney-1-clip (2K1C) hypertension model in rodents and stenosis of the renal artery in humans share pathophysiological mechanisms because they both primarily stimulate the renin-angiotensin system (RAS) [9,10], suggesting that the 2K1C model is relevant to clinical studies of hypertension. It is known that several humoral factors, including gonadal hormones, affect blood pressure in hypertensive patients . For example, selective oestrogen receptor modulators (SERMs) such as raloxifene have several pharmacological effects on steroid hormone receptors, including both agonist and antagonist actions. Whether a SERM acts as an agonist or an antagonist depends on both the expression and spatial conformation of the α-oestrogen receptor (ER) and β-ER . In addition, the local availability of co-factor proteins, which can be activators or repressors, also helps to determine SERM agonist and antagonist actions on ERs in different tissues and organs [11–13].
Raloxifene acts as an ER antagonist in bone and reduces osteoclastic activity; it is used to treat osteoporosis for this reason. The drug also acts as an ER antagonist in the breast and uterus. These antagonist properties have inspired studies of its use in the treatment of advanced breast cancer [12,13]. In the cardiovascular system, SERMs have several interesting effects, including reduction in plasma lipids, improvement in endothelial NO production and consequently blood pressure reduction [12–15]. In the kidney, raloxifene reduces the mesangial expansion and fibronectin accumulation in experimental models of type 2 diabetes mellitus . Raloxifene evaluation in trial analysis of the multiple outcomes was associated with significantly fewer kidney-related adverse events compared with placebo, showing safe and renoprotective treatment . Furthermore, in healthy post-menopausal women, raloxifene enhanced flow-mediated vasodilation [17–20], increased plasma nitric oxide (NO) concentrations  and decreased plasma endothelin-1 levels.
Several factors are implicated in the development of hypertension , including endothelial dysfunction and reduced nitric oxide (NO) production, the importance of which in the modulation of vascular tone is unquestionable. NO promotes vasodilation and causes several other physiological and pathophysiological effects  in various systems, particularly in the cardiovascular system. Of particular relevance to the cardiovascular and renal effects of NO are its ability to reduce platelet aggregation, its vascular antiproliferative effect and its modulation of glomerular filtration and negative inotropic effect . Studies have shown that increased formation of NO may be an important component in the reversal of renovascular hypertension (1K1C) and increased renal perfusion because it facilitates and promotes cardiac and systemic vasodilation . Therefore, it is necessary to clarify the effect of raloxifene on blood pressure under hypertensive conditions to understand the mechanisms involved in the SERM-induced antihypertensive effect in this experimental hypertension model (2K1C).
All surgical procedures and experimental protocols were in accordance with the Biomedical Research Guidelines for the Care and Use of Laboratory Animals, as stated by the Federation of Brazilian Societies of Experimental Biology (FeSBE).
Two-month-old female Wistar rats from our breeding stock, weighing 180–190 g, were randomly allocated into three experimental groups (N =8 per group): 2-kidney-1-clip (2K1C), ovariectomized + 2K1C (2K1C + OVX) and raloxifene-treated (2K1C + OVX + R). Prior the ovariectomy, all animals were cycling normally and were in the oestrous phase. Ovariectomy was performed under general anaesthesia (chloral hydrate, 40 mg/kg, i.p.). In brief, a peritoneal incision was made to expose the uterine tube, and the ovaries were removed. Then, the abdominal wall was surgically closed. Seven days after the ovariectomy, the animals were anaesthetized again, and a silver clip (ID 0.2 mm) was applied around the left renal artery. During both surgeries, the animals received an appropriate dose of antibiotic (2.5% enrofloxacin, 0.1 mL per rat, i.m.). Seven days after the clip insertion, the animals were put into metabolic cages maintained at 22–24°C under a 12-hr light: dark cycle for 1 week. The animals were maintained on a standard rat chow (Purina, Evialis) without alteration in nitrite/nitrate concentration. At the same time, the animals from the third experimental group (2K1C + OVX + R) were treated with raloxifene (Lilly Laboratories, Sao Paulo, Brazil), at a dose of 2 mg/kg/day, i.p., for 7 days. Water ingestion and urinary volume were measured daily. Urinary sodium content was determined by flame photometry (mod. 943; Instrumental Laboratories, São Paulo, Brazil) and expressed as mEq/day .
At the end of the experimental protocol (after 21 days), the rats were anaesthetized with ketamine and xylazine (50 and 10 mg/kg, i.p.). For the direct measurement of mean arterial pressure (MAP), a polyethylene catheter (PE-50 attached to PE-10 tubing) was inserted into the femoral artery and tunneled to the dorsal neck region, connected to a pressure transducer (Spectramed P23XL, Astro-Med, Rockland, MA, USA). The data were digitally stored and analysed using the software Acknowledge for Windows, Biopac Inc (Biopac MP100, Santa Barbara, CA, USA). This direct measurement of MAP was performed in conscious animals 24 hr after the artery catheterization. At the same time, the left femoral vein was cannulated to allow blood collection later.
Soon after the MAP determination, blood was collected from the venous cannula and centrifuged at 1000 × g for 15 min. at 4°C. Then, the plasma was transferred into 10 kDa cut-off interruption filters (Centricon 10) and centrifuged at 11,000 × g for 1 hr, at 4°C. The filtered material was kept in a freezer until the fluorescence measurement was performed. The concentrations of end products of nitric oxide (NO), i.e. nitrites + nitrates, were determined using the Fluorometric Nitrate/Nitrite Assay Kit (Cayman Chemical Company, MI, USA). In this method , the enzyme nitrate reductase converts nitrate to nitrite, which reacts with 2,3-amino-phtalenne in alkaline medium and emits fluorescence. All assays were performed in duplicate, and the nitrite levels in the samples were measured in a Hitachi F-2000 fluorometer (Tokyo, Japan) at wavelengths of 375 nm for excitation and 415 nm for emission.
At the end of the experimental procedures, the rats were killed by exsanguinations, and the kidneys were removed and weighed. Organ weight was expressed as grams of wet tissue per 100 g body weight.
Statistical analysis. Unless otherwise stated, results are presented as mean ± S.E.M. Statistical significance was determined using anova followed by Tukey’s tests with the level of significance set at p <0.05.
The ovariectomy reduced significantly the oestradiol and progesterone plasma concentrations when compared with the controls animals (19 ± 2; 22 ± 2 and 40 ± 6; 58 ± 4 pg/mL, respectively).
As expected, rats from the 2K1C group showed a high MAP (165 ± 3 mmHg; fig. 1), even when treated with raloxifene (168 ± 3 mmHg, data not shown). The hypertensive ooforectomized rats (2K1C + OVX) showed a further increase in MAP (197 ± 6 mmHg, p <0.05). However, when raloxifene was added (group 2K1C + OVX + R), MAP levels (164 ± 2 mmHg) were found to be almost identical to those found in the 2K1C animals (fig. 1).
Renal excretion of Na+ (Table 1) was reduced in the 2K1C + OVX when compared with intact hypertensive (2K1C) rats p <0.01. On the other hand, the addition of raloxifene to the ovariectomized rats (i.e. the 2K1C + OVX + R group) caused an increase in urinary Na+ excretion p <0.01 when compared with 2K1C + OVX groups.
Table 1. The effects of raloxifene (R) administration on sodium (Na+) excretion 14 days after two-kidney, one-clip renovascular (2K1C) surgery to induce hypertension.
2K1C(n = 8)
2K1C + OVX(n = 8)
2K1C + OVX + R (n = 8)
Data are means ± S.E.M.
Rats received raloxifene (2 mg/day/kg) intraperitoneally for 7 days after clip placement.
*p <0.01 compared with 2K1C; **p <0.01 compared with 2K1C + OVX (Tukey’s test).
1.134 ± 0.054
0.755 ± 0.030*
1.247 ± 0.076**
Water ingestion (fig. 2A) was similar among the three experimental groups (2K1C, 2K1C + OVX, 2K1C + OVX + R: 32.5 ± 0.9; 32.1 ± 0.8; and 31 ± 1 mL/day, respectively). Nevertheless, mean urinary volume (fig. 2B) was higher in the 2K1C + OVX + R animals compared with the 2K1C and 2K1C + OVX groups (16 ± 2 versus 13.9 ± 1 versus 8.7 ± 0.8 mL/day, respectively, p <0.01).
At the end of the experimental period (after 21 days), the body weights of the animals were similar among the three experimental groups (2K1C, 170 ± 11 g; 2K1C + OVX, 195 ± 15 g; 2K1C + OVX + R, 183 ± 7 g). The clipped (left) kidney was lighter in the non-treated animals when compared with the raloxifene-treated rats (1.69 ± 0.15 mg/g versus 2.66 ± 0.33 mg/g, respectively, p <0.01). The right (non-clipped) kidney weights (in mg/g b.w.) of the groups were as follows: 2K1C, 4.51 ± 0.26; 2K1C + OVX, 5.21 ± 0.11; and 2K1C + OVX + R, 4.72 ± 0.09. These results confirm that the intact kidney displayed a compensatory increase in weight. Furthermore, the right kidney weight in the ovariectomized non-treated rats was slightly greater than in the other groups (p <0.05).
Fig. 3 shows that animals from the 2K1C + OVX group had lower plasma concentrations of nitrate/nitrite when compared with the intact hypertensive animals (2K1C; 14 ± 0.5 versus 23.4 ± 1 nmol/mL, respectively, p <0.01). Raloxifene treatment (group 2K1C + OVX + R) caused a significant increase in nitric oxide production, as estimated by the nitrate/nitrite plasma concentration (29.2 ± 1.7 nmol/mL; p <0.01).
The results of this study show that raloxifene reduced the blood pressure in animals with early-stage renovascular 2K1C hypertension model, which is consistent with the cardioprotective effects of oestrogen that have been shown previously [26–28]. The reduction in the arterial hypertension was accompanied by increased Na+ and water renal excretion and plasma of nitrite/nitrate.
In the 2K1C model of hypertension, the pressure reduction that occurs at the post-stenosis region activates the juxtaglomerular apparatus that activates the RAS, which is ultimately responsible for vasoconstriction, hydro-saline retention, mitogenic effects in the blood vessels and the heart and sympathetic activation [9,29]. In response to the reduction in perfusion pressure, there is an increase in renin production and release by the ischaemic kidney, which increases the levels of angiotensin I in the bloodstream and initiates the hypertensive process [30,31].
Urinary sodium concentration was measured because urinary retention is thought to be linked to increases in blood pressure. The results showed that the 2K1C + OVX animals had lower urinary sodium excretion when compared with the 2K1C group, suggesting that ovariectomy induces Na+ retention, which contributes to the increase in blood pressure observed in this particular group. The 2K1C + OVX + R group showed a significant increase in urinary excretion of Na+ when compared with the other groups; this natriuretic effect reduces blood volume and therefore reduces blood pressure.
Several studies have demonstrated an important role for oestrogen in regulating the balance of salt and water . Oestrogen has direct effects on renal tubules  and causes an increase in the expression of the α- and β-ER . Oestrogen also has indirect effects; it modulates RAS, which decreases plasma renin and angiotensin-converting enzyme activities . Therefore, raloxifene could be acting, similarly to oestrogen, in the kidney via mechanisms that cause natriuresis and therefore a reduction in blood pressure.
A decrease in the wet weight of the left kidney was expected because of the unilateral renal artery stenosis in this hypertension model [34,35]. Ovariectomized animals treated with raloxifene had higher kidney weights when compared with the untreated groups. These data suggest that HRT with raloxifene improved left renal blood flow, possibly because this SERM promoted an improvement in renal perfusion. In fact, studies have shown that raloxifene increases coronary and uterine blood flow via endothelial mechanisms, specifically the release of NO . On the other hand, the contra-lateral (right) kidney excretes the excess Na+ imposed by the clipped kidney , which promotes greater glomerular filtration and thus explains the observed increase in wet weight of the right kidney.
With respect to the pathophysiology of renovascular hypertension, it is accepted that in addition to increases in angiotensin II formation, there is a contribution from endothelial dysfunction. This endothelial dysfunction may occur because of a deficiency in endogenous NO or free radicals, resulting in oxidative stress in the vessel wall, which accounts for the decreased levels of NO . This study aimed to investigate a possible correlation between the cardiovascular effects of raloxifene and the formation of circulating NO, which was used as an indicator of the contribution of vascular endothelium to the effects of raloxifene. The fact that the 2K1C + OVX + R group showed a significant increase in plasma nitrate/nitrite (a surrogate of NO production) compared with other experimental groups is consistent with this hypothesis.
Studies have shown that increased formation of NO may be an important component of the reversal of 2K1C renovascular hypertension because it facilitates renal and cardiac reperfusion and promotes systemic vasodilation [9,38]. Probably, the main limitation of the present study was not performed measures of urinary and renal levels of nitrite/nitrate. However, it has been shown that raloxifene in vitro relaxes the coronary artery by acting at oestrogen receptors and promotes the release of NO , suggesting that its effects depend on the subtype of oestrogen receptor and its action on the vascular endothelium.
Another finding suggests that 6 months of raloxifene treatment affects endothelial function and improves flow-mediated endothelium-dependent vasodilation of the brachial artery . A recent clinical study found no antihypertensive effect in women chronically treated with raloxifene [40,41]. However, we must consider the small number of women in this study, the lack of control over the stage and type of hypertension and the dose of raloxifene. Raloxifene is currently prescribed to women for the treatment and prevention of osteoporosis. These patients may derive additional benefits from this drug in terms of cardiovascular protection secondary to improved endothelial function .
To summarize, the present study demonstrates that HRT with raloxifene reduces blood pressure in menopausal rats with renovascular hypertension. This reduction in MAP occurs, at least partly, because of effects such as increased renal excretion of sodium and water and indirectly increased plasma NO.