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*Division of Hypertension and Vascular Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; Institute of Pharmacology and Toxicology, Lausanne, Switzerland; and Department of Pathology, Centre Médical Universitaire, Geneva, Switzerland.
Correspondence to Dr. Michel Burnier, Division of Hypertension and Vascular Medicine, CHUV, Hôpital Nestlé, 1011 Lausanne, Switzerland. Phone: 0041-21-314-07-50; Fax: 0041-21-314-07-61; E-mail: Michel.Burnier{at}chuv.hospvd.ch
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Materials and Methods |
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BP Measurements
BP and heart rate were recorded intra-arterially, with a computerized data-acquisition system (13). For placement of the intra-arterial catheter, mice were anesthetized via inhalation of 1 to 2% halothane with oxygen. The right carotid artery was exposed for a length of approximately 4 mm. A PE-10 catheter filled with 0.9% NaCl solution containing heparin (300 IU/ml) was inserted into the artery. After ligation, the catheter was subcutaneously tunneled to exit at the back of the neck. Mice were allowed 3 h to recover from the anesthesia and were placed in Plexiglas tubes for partial restriction of their movements. Thirty minutes later, the arterial line was connected to a pressure transducer; BP and heart rate were then monitored every 20 s for 15 to 30 min.
BP Responses to Blockade of Angiotensin II AT1 Receptors
For evaluation of the role of the renin-angiotensin system in maintaining BP in the various mice, the angiotensin II receptor antagonist irbesartan was injected intravenously (1.5 mg/kg, in 50 µl) and changes in BP were measured. For injection of the antagonist, a venous catheter was inserted into the left jugular vein.
In a second set of experiments, one- and two-renin DOCA/salt-treated mice were treated with the angiotensin II receptor antagonist losartan. Losartan was added to the drinking water at a dose of 0.2 mg/ml water. The treatment was started on the first day of administration of DOCA and salt. Control groups received the vehicle. To test the ability of the dose of losartan to block the renin-angiotensin system, exogenous angiotensin II was injected intravenously into DOCA/salt-treated mice receiving the vehicle and losartan. The BP response to exogenous angiotensin II (40 ng/kg, administered intravenously) was markedly blunted by losartan (31 mmHg for the control group and 4.5 mmHg for the losartan-treated group, P < 0.001).
Biochemical Analyses
For measurement of plasma sodium and potassium levels, 300 µl of blood was obtained from the carotid artery (after BP measurements), in a 0.6-ml Multivette tube containing gel/clot activator (Sarstedt, Numbrecht, Germany). Serum and urinary sodium and potassium levels were measured by flame photometry (model 943; Instrumentation Laboratory). PRA and PRC were measured using methods described previously and adapted for small amounts of blood (13,14). Blood was drawn from the arterial catheter after the BP measurements were completed. Studies performed in our laboratory have demonstrated that blood sampling via an intra-arterial catheter generates less stress in the mice then does decapitation or retro-orbital blood sampling. Indeed, plasma norepinephrine levels were sixfold higher with decapitation than with the use of a carotid artery catheter and were almost twofold higher with retro-orbital blood sampling (E. Grouzman, C. Cavadas, D. Grand, M. Moratel, J. F. Aubert, H. R. Brunner, and L. Mazzolai, submitted for publication).
Light Microscopy
Heart samples were fixed in 10% neutral buffered formol and embedded in paraffin. Four-micron sections were used for immunochemical analyses with anti-
-skeletal actin 1 (1:10 dilution in Tris-buffered saline) (15). Immunoperoxidase staining was performed essentially as described previously (16). After staining, sections were observed with a Zeiss Axiophot photomicroscope (Zeiss Vision, Oberkochen, Germany). Images were acquired with a high-sensitivity Photonic Coolview color camera (Zeiss), stored, and printed as described above. Images were subsequently analyzed by using KS400 software (Kontron System; Zeiss), as described previously (15).
Statistical Analyses
All results are presented as means ± SEM. Statistical comparisons between groups were performed with ANOVA, followed by t test or Dunnett’s t test when appropriate. P < 0.05 was considered to be the minimal level of significance.
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Effects of DOCA/Salt in One- and Two-Renin Gene Mice
Table 1 also presents the changes induced by the administration of DOCA and salt. As expected, with the administration of DOCA, marked decreases in amiloride-sensitive rectal PD values were observed in association with the development of hypokalemia in both strains. The administration of DOCA was also associated with the development of alkalosis, with blood pH increasing from 7.30 ± 0.04 to 7.43 ± 0.02 (P = 0.01, n = 5 to 7). In one-renin gene mice, PRA and PRC values were almost totally suppressed by the administration of DOCA/salt but BP was not affected (Figure 3). However, both cardiac and renal hypertrophy developed in those mice. In two-renin gene mice, DOCA induced a further increase in BP and the administration of irbesartan still caused a marked decrease in BP. Neither PRA nor PRC values were reduced by the administration of DOCA and salt (Figure 3). In those mice, DOCA produced an additional increase in cardiac and kidney weight indices. Because -skeletal actin expression increases during cardiac hypertrophy, we evaluated -skeletal actin expression in mouse myocardium with immunohistochemical staining (Figure 5). The area of positive staining was significantly increased in one-renin gene mice treated with DOCA/salt, compared with one-renin gene mice treated with tap water (P 
0.01). Moreover, under all experimental conditions, the sizes of -skeletal actin-positive areas were greater in two-renin gene mice than in the corresponding one-renin gene mice, as demonstrated in Figure 5.
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Discussion |
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An intact renin-angiotensin system is fundamental to the maintenance of basal BP in mice. Thus, several gene-targeting experiments in mice noted that mice lacking genes for angiotensinogen (17), angiotensin-converting enzyme (18), or the angiotensin type 1 receptor (19,20) exhibited lower BP, compared with wild-genotype mice. However, it has been more difficult to determine the relative contributions of the Ren-1d and Ren-2 genes to the maintenance of BP in mice. Bertaux et al. (21) observed no change in BP in mice with a disrupted Ren-1d gene, suggesting that the product of the Ren-2 gene is physiologically active and can compensate for the loss of Ren-1d, supporting BP. In contrast, Clark et al. (22) observed that inactivation of the Ren-1d gene decreased BP in female mice but not in male mice. The latter observation suggested that the Ren-2 protein could not fully compensate for the loss of Ren-1d in female mice, whereas the production of renin was sufficient to compensate for the decrease in active renin and the resulting decrease in BP in male mice, which express larger amounts of Ren-2 in the submaxillary gland. No change in BP was observed in adult mice homozygous for the mutated Ren-2 gene (10). Whereas most previous comparative studies were conducted with strains with different genetic backgrounds, in this study one-renin gene mice of the C57BL/6J strain were crossbred with 129Ola mice, which have two renin genes, to yield one- and two-renin gene mice with the same genetic background.
Our results clearly demonstrated that mice with two renin genes exhibited higher baseline BP. This finding was mainly attributable to overactivity of the renin-angiotensin-aldosterone cascade. Indeed, we observed that mice with two renin genes exhibited 100-fold higher PRA and PRC values. These differences cannot be attributed to more stressful conditions during sampling for two-renin gene mice, which could release great amounts of salivary gland renin, as suggested previously (23). Indeed, we have observed that catheter blood sampling is much less stressful than decapitation or retro-orbital blood sampling and produces the lowest serum catecholamine levels (E. Grouzmann, personal communication). Furthermore, selective blockade of angiotensin type 1 receptors induced a marked decrease in BP in two- but not one-renin gene mice. The amiloride-sensitive rectal PD, which is an indirect indicator of mineralocorticoid activity (11), was significantly more negative in two-renin gene mice receiving tap water, suggesting that aldosterone activity was also increased in those mice. As a direct consequence of the elevated BP, the cardiac weight index was significantly increased in two-renin gene mice. These observations thus demonstrate that the Ren-2 gene is physiologically active in two-renin gene mice and participates in the increase in BP under basal conditions, even when the Ren-1d gene is active. This finding is consistent with the results of recent studies demonstrating that overexpression of the mouse Ren-2 gene induces severe hypertension in rats (24). However, the possibility that linked genes may still be producing strain effects and that some differences may not be attributable exclusively to the presence of the Ren-2 gene cannot be excluded.
The second major finding of these experiments was that the presence of two renin genes markedly increased the sensitivity of the mice to salt. Indeed, in contrast to one-renin gene mice, mice with Ren-1d and Ren-2 genes became hypertensive when 1% NaCl was provided, instead of tap water, as the drinking fluid. The development of salt-sensitive hypertension in two-renin gene mice can most likely be explained by the fact that the renin-angiotensin system was not suppressed by the increased salt intake. Hall et al. (25) demonstrated in dogs that BP became salt-sensitive when the activity of the renin-angiotensin system could not be adapted to the sodium intake. Interestingly, two-renin gene mice receiving 1% salt solution to drink became even more dependent on the activity of the renin-angiotensin system, as demonstrated by the greater decrease in BP induced by AT1 receptor blockade. Whether this is attributable to higher levels of angiotensin II or to upregulation of AT1 receptors cannot be concluded from these studies. However, it is also interesting to note that amiloride-sensitive rectal PD values became less negative with a high-salt diet even in two-renin gene mice, suggesting decreased activity of the epithelial sodium channel in the colon. We previously reported that high angiotensin II levels decrease and AT1 receptor blockade increases amiloride-sensitive rectal PD values in mice (11).
As expected on the basis of the increase in BP, two-renin gene mice receiving salt developed even more cardiac hypertrophy than under normal conditions. However, with respect to renal hypertrophy, increased kidney weight indices were observed for both one- and two-renin gene mice. High dietary salt intake has been demonstrated to induce cardiac hypertrophy and collagen deposition in rats (26,27). With a very high-salt diet (8% NaCl), increased kidney weights have also been documented in rats (28). The effects of salt itself in mice, particularly after uninephrectomy, have not been reported. These results are thus the first to demonstrate that even a modest increase in salt intake induces renal hypertrophy in uninephrectomized mice.
To further investigate the role of renin genes in the development of hypertension in mice, the DOCA/salt model was developed in one- and two-renin gene mice. The deoxycorticosterone/salt hypertension model is a low-renin, salt-dependent model of acquired experimental hypertension that has been extensively used to investigate the epithelial and extraepithelial effects of mineralocorticoids (29). In contrast to previous studies, we administered DOCA subcutaneously, using a homemade delivery system (Figure 2), and not as a pellet. Our system provides a constant release of DOCA for several weeks. The marked decrease in serum potassium levels and the decrease in amiloride-sensitive rectal PD values clearly demonstrated that a hypermineralocorticoid state was achieved with this system. Despite the development of severe hypokalemia, no increase in BP with the administration of DOCA was observed in one-renin gene mice, whereas an additional increase in BP was measured in two-renin gene animals. PRA and PRC values were totally suppressed in one-renin gene mice but were not affected in two-renin gene mice, again suggesting that renin production in the latter mice was not suppressed by salt or DOCA. Moreover, the BP response to AT1 receptor blockade remained greater in two-renin gene mice. This finding may be attributable to upregulation of AT1 receptors or increased production of angiotensin II. Several previous articles suggested that mineralocorticoids could promote upregulation of AT1 receptors (30,31). Our finding would be in accordance with that hypothesis. The reason why one-renin gene mice remain normotensive with the administration of DOCA/salt, despite the development of hypokalemia, is not yet clear. In this respect, mice differ from rats, which also have one renin gene but still develop low-renin hypertension with DOCA/salt (32). Because BP was measured several hours after anesthesia, small changes in BP soon after surgery might have been masked. The lack of changes in BP with the administration of the angiotensin II receptor antagonist losartan suggests that the mice were not particularly stressed. Another possible explanation involves the lower dose of DOCA used in these experiments, although a hypermineralocorticoid state was clearly obtained in our animals. Of note, most reported DOCA/salt-treated mouse models of hypertension have been created with two-renin gene mice (1–3).
The last finding of our study was that DOCA/salt induced cardiac and renal hypertrophy in both one-renin gene normotensive and two-renin gene hypertensive mice. This DOCA-induced hypertrophic response is in agreement with several other reports demonstrating that hypermineralocorticoid states are associated with cardiac hypertrophy and cardiac fibrosis in rodents (26–30). We did not detect myocardial fibrosis with light microscopy. However, two-renin gene mice were characterized by a significant increase in -skeletal actin expression, which is an early marker of the development of myocardial muscle hypertrophy (33–35). The DOCA/salt-treated mouse model constitutes an additional example of the induction of -skeletal actin expression in normotensive animals, as described for mice that overexpressed angiotensinogen exclusively in the heart (36). As mentioned above, there is evidence suggesting that mineralocorticoids could promote the upregulation of AT1 receptors (30,31). Our observation that losartan prevents the development of cardiac hypertrophy in both normotensive one-renin gene and hypertensive two-renin gene mice supports the hypothesis that DOCA-induced cardiac hypertrophy is mediated by activation of angiotensin II AT1 receptors.
In summary, this study demonstrates that the number of renin genes is an important determinant of BP and the responses to salt and DOCA in mice. Therefore, careful characterization of the renin gene background should be performed whenever mice are used to investigate the pathophysiologic features of hypertension. Our data also suggest that the Ren-2 gene, which controls renin production mainly in the submaxillary gland and to a lesser degree in the kidney, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, our data provide further evidence that mineralocorticoids promote cardiac and renal hypertrophy even in the absence of BP changes. Activation of angiotensin II AT1 receptors seems to contribute to the DOCA/salt-induced cardiac hypertrophy.
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-skeletal actin is identified by a specific polyclonal antibody. Circ Res 85: 51e–58e, 1999ENaC knockout heterozygous mice. Kidney Int 59: 2216–2221, 2001[Medline]
-Skeletal muscle actin mRNA’s accumulate in hypertrophied adult rat hearts. Circ Res 59: 551–555, 1986-actin in hearts from normal and hypophysectomized rats. Proc Natl Acad Sci USA 87: 2456–2460, 1990-skeletal actin expression in cardiomyocytes in vitro and in vivo in the absence of hypertension. Differentiation 2002, in pressThis article has been cited by other articles:
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, one-renin gene mice; 
, two-renin gene mice. Bars represent SEM (n = 4 or 5 animals). ***P