Effect of High Salt Intake in Mutant Mice Lacking Bradykinin-B2 Receptors
Renal kinins release prostaglandins and nitric oxide via the B2 receptor, promoting diuresis and natriuresis; hence, they may also contribute significantly to blood pressure regulation. We hypothesized that mutant mice lacking the gene encoding for the bradykinin-B2 receptor (B2-KO) become hypertensive when placed on a long-term high-salt diet. To test this, B2-KO and control mice were placed on either a normal (0.2%) or high-Na+ diet (3.15% in food plus 1% saline as drinking water) for 8 weeks. Systolic blood pressure was determined during weeks 6 and 8 by a computerized tail-cuff system. At the end of the 8-week period, mice were anesthetized for determination of mean blood pressure, renal blood flow, and renal vascular resistance. In B2-KO mice maintained on high Na+, systolic blood pressure was 15 mm Hg higher than in knockout animals on normal Na+ (P<.01). In contrast, there was no difference in blood pressure in control mice fed either a normal or a high-Na+ diet. Consistent with the systolic blood pressure data, direct mean arterial pressure revealed that B2-KO mice on high Na+ were hypertensive (115±6 in B2-KO on high-Na+ diet versus 79±2.8 in B2-KO on normal Na+, P<.0001); renal blood flow was reduced by 20% (P<.05) and renal vascular resistance was doubled (P<.0001) compared with B2-KO mice on normal Na+. In contrast, control mice on high Na+ were normotensive and tended to have increased renal blood flow and decreased renal vascular resistance compared with control mice on a normal Na+ diet. These findings indicate that kinins play an important role in preventing salt-sensitive hypertension; this may be achieved by maintaining renal blood flow under conditions of high salt intake.
- kallikrein-kinin system
- bradykinin B2 receptor knockout mouse
- mean arterial pressure
- renal blood flow
- renal vascular resistance
- ACh = acetylcholine
- B2-KO = mouse with BK-B2 receptor gene knocked out
- BK = bradykinin
- BP = blood pressure
- MAP = mean arterial pressure
- RBF = renal blood flow
- RVR = renal vascular resistance
- SBP = systolic BP
The mechanisms responsible for the increase of BP in response to high salt intake in salt-sensitive subjects are complex and only partially understood. A complex interaction between neuroendocrine substances, paracrine factors, and the kidney may explain why such people tend to retain salt and develop salt-dependent hypertension. We investigated the role of the kallikrein kinin system in the BP response to high Na+ intake.
The kallikrein-kinin system is an important vasoregulatory component of cardiovascular homeostasis. Kinins are endogenous vasodilators that act as local hormones by activating the release of endothelium-derived relaxing factor and prostaglandins.1,2⇓ They act mainly via two different types of receptors, B1 and B2. Most of the known effects of kinins are mediated by the B2 receptor, which belongs to a family of peptide hormone receptors linked to G proteins.3
In the cardiovascular system, a local kallikrein-kinin system appears to play a role in the protective effect of ACE inhibitors, suppressing neointimal proliferation after endothelial injury, decreasing infarct size after ischemia/ reperfusion, and improving left ventricular function in heart failure models.4–6⇓⇓ In the kidney, the kallikrein-kinin system regulates microcirculation and water and sodium excretion and therefore may play an important role in BP homeostasis.7–9⇓⇓ Moreover, in humans, low urinary kallikrein excretion is a genetic marker associated with a family history of hypertension10; a restriction fragment length polymorphism for the kallikrein gene in spontaneously hypertensive rats is linked to high BP,11 and Brown Norway Katholiek rats, which are kininogen-deficient, are more sensitive to the hypertensive effect of salt and subpressor doses of angiotensin II than their control counterparts.12–14⇓⇓ Taken together, these data have led to the suggestion that blunting of kallikrein-kinin system activity may contribute to the pathophysiology of hypertension.
Using homologous recombination, Borkowski et al15 recently developed a B2-KO mouse. Since we have previously shown that the renal kallikrein-kinin system plays an important role in the regulation of water and sodium excretion,8 we hypothesized that mice lacking the gene encoding for the BK-B2 receptor show a greater hypertensive response to chronic high Na+ intake (salt sensitivity) than do mice with the receptor.
We first obtained dose-response curves for BK, using another endothelium-dependent vasodilator, ACh, as a control, to confirm the absence of B2 receptors in B2-KO mice. Using this mutant model, we then determined the effect of permanent disruption of the kallikrein-kinin system on BP homeostasis and renal hemodynamics under basal conditions and in mice on a high-Na+ diet.
Homozygous B2-KO mice weighing 26 to 30 g were obtained from breeding pairs generated by gene targeting and provided by Fred Hess (Merck Laboratories). The B2-KO homozygous (−/−) breeding pairs were derived from an inbred strain on a 129Sv genetic background. SV129/SvEv mice obtained from Taconic Farms served as controls.
Dose-Response Curves to Confirm the Absence of BK-B2 Receptors
Mice were anesthetized with thiobutabarbital sodium salt (Inactin, 125 μg/g body wt), and modified PE-10 polyethylene catheters (PE-10, Clay-Adams) were placed in the aorta via the carotid artery for injections and in the femoral artery to measure BP. Dose-response curves for bradykinin and acetylcholine (1, 5, 25, and 125 ng/mouse for each drug) were obtained in control and B2-KO mice.
Effect of High-Sodium Diet on BP
Controls and B2-KO mice were fed either a standard diet containing 0.2% Na+ and tap water or a high-sodium diet consisting of 3.15% Na+ in food plus 1% saline as drinking water ad libitum for 8 weeks. SBP was determined during weeks 6 and 8 by tail plethysmography using a newly designed and validated noninvasive computerized tail-cuff system (BP-2000, Visitech Systems).16 The following protocol was applied. Mice were first trained for 7 days; measurements were then recorded daily on 5 consecutive days. Each daily session included 2 sets of 10 measurements; for us to include each set of measurements for an individual mouse, the computer had to successfully identify a BP in at least 6 of the 10 trials within the set. Averaging the data for 5 days, we obtained 1 SBP value per mouse.
After 8 weeks of either a regular or high-Na+ diet, SV129 or B2-KO mice were anesthetized by intraperitoneal injection of 125 mg/kg body wt thiobutabarbital sodium salt (Inactin, RBI) and placed on a heating pad to maintain constant body temperature. Mice were surgically prepared as follows. The left carotid artery was catheterized to measure MAP. The catheter was constructed of PE-50 tubing connected to PE-10, with the latter portion being inserted into the carotid artery. BP was monitored with a Statham pressure transducer (Viggo-Spectramed). The right jugular vein was cannulated with a similar PE-50/PE-10 catheter for constant infusion of saline (≈3 μL/min). After the left kidney was exposed via a midabdominal incision, the renal artery was dissected from the renal vein and fitted with a perivascular 0.5-mm flow probe connected to an ultrasonic flowmeter (Transonic System). The pressure transducer and flowmeter were connected to a chart recorder (Gould) for simultaneous recording of RBF and MAP. After surgery, mice were allowed a 15-minute recovery period, during which BP and RBF were monitored. By this time, MAP and RBF had stabilized, and we began a 15-minute sampling period.
Blood flow to the left kidney was determined directly from the flowmeter and normalized to flow per gram of kidney weight. MAP and RBF were used to calculate RVR. Units of RVR are mm Hg·mL−1·min−1·g kidney wt−1.
After MAP and RBF had been determined, mice were killed and the heart and kidneys excised and weighed. All values were corrected per 10 g body wt.
Values are expressed as mean±SEM. Wilcoxon’s rank sum test was used to evaluate the significance of differences between control and B2-KO mice at each dose of BK. ANOVA for repeated measures was used to analyze the dose-response curve for ACh. A two-way ANOVA was used to assess diet/group interactions in SBP, MAP, RBF, and RVR. Finally, an unpaired Student’s t test was used to assess differences between B2-KO and control tissue weights.
We found that the BP response to BK was abolished in B2-KO mice compared with the controls, whereas the response to ACh (another endothelium-dependent vasodilator) was conserved. The slopes representing the response to ACh were similar in controls and B2-KO mice; however, in the B2-KO mice, the overall response to ACh was shifted to the right (Fig 1).
By weeks 6 and 8 of the diets, SBP was significantly higher in B2-KO mice on high Na+ compared with mutant mice on a regular Na+ diet. On the other hand, SBP in control mice was similar with either normal or high Na+. Two-way ANOVA for group and diet showed a statistically significant SBP interaction between group and diet (P<.01), suggesting salt-sensitive hypertension in B2-KO mice (Fig 2).
The salt sensitivity of B2-KO mice was confirmed by direct MAP measurements (Fig 3). As shown in Table 1, control mice on a high-Na+ diet were normotensive and tended to have increased RBF and decreased RVR compared with controls on normal Na+. In contrast, B2-KO mice on high Na+ were hypertensive; RBF was reduced by ≈20% (Student’s t test, P<.05) and RVR was doubled (Student’s t test, P<.0001) compared with mutant mice on a normal Na+ diet (Figs 4 and 5⇓). Two-way ANOVA for MAP, RBF, and RVR revealed a highly significant interaction between group and diet; in other words, the average difference between normal and high Na+ was significantly different between controls and B2-KO mice, indicating a clear differential effect of salt in terms of MAP and renal hemodynamics between control and knockout mice.
Atrial, left ventricular, right ventricular, total heart, and kidney weights were similar in control and B2-KO mice on normal sodium (Table 2). When B2-KO mice were fed a high-Na+ diet for 8 weeks, heart and kidney weights were significantly greater than in B2-KO mice fed normal Na+ as opposed to the controls, whose heart and kidney weights were similar whether they were fed a normal or high-Na+ diet (Table 2; Fig 6).
Current knowledge suggests that the kallikrein-kinin system participates in the control of water and electrolyte excretion and therefore regulation of BP.1 We studied the response to different vasoactive substances and the effect of chronic high Na+ intake on BP and renal hemodynamics in B2-KO mice. To the best of our knowledge, this is the first physiological study assessing cardiovascular and renal parameters in mice genetically lacking bradykinin B2 receptors.
We first obtained dose-response curves for BK using another endothelium-dependent vasodilator, ACh, as a control to confirm the absence of B2 receptors in B2-KO mice. As expected, the response to BK was completely abolished in B2-KO mice compared with the controls; however, the response to ACh was conserved. Therefore, we were able to confirm the absence of BK-B2 receptors in B2-KO animals pharmacologically.
Bradykinin is known to act mainly via B1 and B2 receptors; most of the known actions of bradykinin are mediated through the B2 receptor, whereas the B1 receptor is activated under certain conditions such as lipopolysaccharide stimulation. Since the response to BK was completely absent in the mutant mice, we confirmed that in mice, the acute vasodepressor effect of BK is mediated exclusively by B2 receptors. Although the slope of the ACh response was similar in the two groups, the curve for the B2-KO mice was shifted to the right compared with the controls, suggesting that the mutant mice may be less sensitive to the effect of endothelium-dependent vasodilators. Although we do not have an explanation for this, one possibility is that lack of bradykinin B2 receptors leads to downregulation of the NO-cGMP pathway, blunting the response to other substances that use the same pathway.
We next determined the effect of chronic high Na+ intake on BP using a new computerized tail-cuff system. Under basal Na+ conditions, SBP was similar in controls and B2-KO mice. This is in accord with previous data suggesting that under basal conditions, the BK-B2 receptor plays only a minor role in BP regulation17; however, we cannot overlook the possibility that B2-KO mice develop compensatory mechanisms to keep BP at normal levels. When mice were fed a high-Na+ diet for 8 weeks, SBP did not change in controls but increased in B2-KO mice compared with their normal-Na+ counterparts. The hypertensive effect of high Na+ on B2-KO mice was confirmed by intra-arterial MAP measurements with the animals under anesthesia, which showed a pattern of BP differences between groups and diets similar to the SBP data.
Control mice on a high-Na+ diet tended to have increased RBF and decreased RVR compared with controls on normal Na+. In contrast, in B2-KO mice on high Na+, RBF was reduced by ≈20% and RVR was doubled compared with mutant mice on a normal Na+ diet. A similar phenomenon is seen in nonmodulators and salt-sensitive hypertensive patients as well as in certain animal models of salt-sensitive hypertension.18,19⇓ In this regard, RBF increases and RVR decreases in response to increased dietary Na+ intake in salt-resistant humans and animals, compared with either no change or decreased RBF and increased RVR in salt-sensitive subjects. This similarity raises the interesting possibility that kinins may be involved in the renal vasodilation observed during high Na+ intake, and hence a functionally normal kallikrein-kinin system may be vital in preventing salt-sensitive hypertension. Thus, one can speculate that the functionally altered kallikrein-kinin system in our mutant mouse model is responsible for the absence of renal vasodilation in response to chronic high Na+ intake, leading to salt-sensitive hypertension; however, since high BP is known to produce vasoconstriction in different vascular beds, we cannot rule out the possibility that the increased RVR and decreased RBF observed in the hypertensive B2-KO mice are secondary to the high BP rather than the cause of the salt-sensitive hypertension. On the other hand, since kinins are reportedly involved in the regulation of tubular water and Na+ reabsorption in the distal nephron,7–9⇓⇓ disruption of the kallikrein-kinin system in B2-KO can result in reduced renal water and Na+ excretion: water and Na+ retention are thereby induced, which might result in hypertension.
The more marked BP response to high Na+ in B2-KO mice seems to be confirmed by heart weight. On a normal Na+ diet, it was similar in both groups; and although high sodium did not affect heart weight in the controls, it significantly increased it in B2-KO mice, suggesting augmented afterload in B2-KO animals on high Na+ compared with controls on the same diet. On the other hand, since B2-KO animals on high-Na+ diets may have retained water and Na+, augmented preload or a combination of preload and afterload may also have participated in the development of cardiac hypertrophy.
Finally, the rise in BP seen in the B2-KO mice was about 20 to 25 mm Hg, which seems modest compared with classic hypertensive models such as Dahl salt-sensitive rats, spontaneously hypertensive rats, or rats with deoxycorticosterone acetate-salt hypertension. Over the range of BP increases, there seem to be species-related differences between rats and mice. Doubly transgenic mice overexpressing both renin and angiotensinogen showed hypertension, with an SBP of ≈125 to 130 mm Hg,20,21⇓ whereas transgenic rats overexpressing renin alone showed marked hypertension (≈210 mm Hg).22 On the other hand, it should be noted that salt sensitivity in classic animal models is not determined by a single abnormal gene.23 For example, Dahl salt-sensitive rats are assumed to carry several genes responsible for hypertension. In contrast, the possible salt sensitivity in our model was achieved by knocking out a single gene. Thus, it is not unusual that the rise in BP was more modest than that in salt-sensitive Dahl rats.
In summary, we have described a new salt-sensitive hypertensive model in mice lacking the gene encoding for the bradykinin B2 receptor. This study suggests that bradykinin acting on the B2 receptor may play an important role in preventing salt-sensitive hypertension.
This work was supported by NIH grant B20212. We thank Fred Hess for providing B2-KO mice.
Published in part in Biochem Biophys Res Commun. 1996;224:625–630
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