INTRODUCTION

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MANIPULATION OF GENES in the mouse genome using homologous recombination in embryonic stem cells ("gene targeting") is a powerful experimental tool that has achieved widespread application in a number of scientific disciplines (1). For now, the mouse is the only mammalian species in which gene targeting can been accomplished, because germ line-competent embryonic stem cells have so far only been isolated from mice. Thus, in disciplines such as immunology, a long experience with the mouse as an experimental model facilitated the rapid application and widespread use of gene targeting (28). In contrast, the small body size of the mouse and inexperience using the mouse for whole animal experiments created some impediments to implementing this technology in physiology. However, as these problems have been overcome, gene targeting has proved extremely useful for physiological studies. The series of experiments that have been performed using mouse lines with targeted alterations of genes in the renin-angiotensin system (RAS) are an example of the utility of these genetic approaches in physiological investigations. Conventional transgenic mice and rats with altered RAS gene expression have demonstrated a similar usefulness (35, 54). In this review, I will discuss experiments using mice with targeted alterations of RAS genes, focusing on aspects related to renal development, blood pressure regulation, and kidney function.

	RENIN-ANGIOTENSIN SYSTEM

The role of the RAS in sodium and fluid homeostasis is well recognized (41). Accordingly, the physiology, pharmacology, biochemistry, and molecular biology of this system have been intensely investigated. Recently, gene targeting has been used to study the functions of genes in the RAS. All of the known genes in the RAS have now been disrupted using gene targeting, and mice that are homozygous for these targeted mutations have been generated. In some cases, analysis of these mice has provided confirmation of previous notions regarding the functions of RAS gene products. In other cases, the results have been surprising or have suggested novel physiological roles for RAS genes.

The major effector molecule produced by the RAS is angiotensin II. This octapeptide is produced by a coordinated series of substrate enzyme interactions involving a number of tissues and cell types. In the first step in the pathway, the substrate protein angiotensinogen is cleaved to form the decapeptide angiotensin I by the aspartyl protease renin. In most species, including humans, renin is encoded by a single gene, whereas wild mice and certain strains of laboratory mice possess a second renin gene, which apparently arose through a gene duplication (5). The two mouse renin genes, designated Ren-1 and Ren-2, encode highly homologous proteins, but the pattern and regulation of their expression differ substantially (49).

Following its generation from angiotensin I by the actions of angiotensin converting enzyme (ACE), the biological effects of angiotensin II are mediated by cell surface receptors that belong to the large family of G protein-associated receptors (14, 55). The angiotensin receptors can be divided into two pharmacological classes, type 1 (AT₁) and type 2 (AT₂), on the basis of their differential affinities for various nonpeptide antagonists (55). Studies using these antagonists suggest that most if not all of the classically recognized functions of the RAS are mediated by AT₁ receptors (55). AT₁ receptors from several species have been cloned (18, 36, 45), and two subtypes, designated AT_1A and AT_1B, have been identified in rat (6, 20, 24, 44) and mouse (46). A single report has suggested that AT_1B receptors might also exist in humans (29), but this has not been confirmed in the unpublished work of several independent groups, and the consensus view is that there is no human counterpart of the AT_1B receptor. The physiological functions of the AT₂ receptor have not been clearly defined. However, recent studies have suggested that the AT₂ receptor may function to oppose or modulate the actions of AT₁ receptors with respect to blood pressure and cellular proliferation (16, 17, 33, 37).

	DEVELOPMENTAL EFFECTS OF RAS MUTATIONS

In their first phase, all gene targeting studies are experiments in developmental biology in which one asks whether the induced genetic alteration has consequences for fetal development and survival. As mice with RAS gene disruptions were produced, there was an expectation that disabling genes in this system might have profound effects on development, especially in the kidney. This was based on a number of studies suggesting roles for these genes in fetal development and in early postnatal life. For example, expression of RAS genes is enhanced in a number of fetal tissues (11, 12, 23), and angiotensin receptors are expressed in a tightly regulated program during renal development (13, 15, 25, 56). Since administration of RAS antagonists causes widespread structural and growth abnormalities in the kidney (9, 57), this enhanced expression of RAS genes seemed to predict critical functions related to renal growth and development.

To a limited extent, the initial reports describing the characteristics of mice with targeted inactivation of the angiotensinogen gene (Agt) bore out these predictions. Only a small percentage of angiotensinogen-deficient [Agt (/)] mice survive to weaning, although the expected number of viable Agt (/) mice are present at birth, and the histomorphology of their major organs, including the kidney, was normal (26). Those that live to adulthood develop abnormalities in their kidneys including vascular hypertrophy, focal tubular dropout with interstitial inflammatory infiltrates, and marked atrophy of the renal papilla (26, 38). These vascular abnormalities, which are shown in Fig. 1, are confined to the kidney and are not observed in any other vascular beds (58). This phenotype is also seen in mice that completely lack ACE (7, 30), suggesting that the absence of angiotensin II peptide is critical to its pathogenesis. Abnormal thickening of the walls of renal arterioles is also seen in mice that lack tissue-bound ACE but still posses significant levels of circulating ACE in plasma (8). This suggests that the local generation of angiotensin II plays a role in regulating renal vascular structures. Moreover, expression of human renin and angiotensinogen transgenes in Agt (/) mice can rescue this phenotype (4).

View larger version (163K): [in this window] [in a new window]   Fig. 1.   Renal vascular lesions in angiotensinogen-deficient mice. These photomicrographs of hematoxylin- and eosin-stained sections of kidneys from Agt (/) mice illustrate the vascular lesions that are typically observed in these animals. Marked thickening of the arteriolar wall is seen, often with associated perivascular inflammatory cell infiltrates.

Individual disruption of the Ren-1 or Ren-2 genes does not produce marked developmental abnormalities (48), possibly because of compensation by the residual renin gene. Work is in progress to disrupt both genes simultaneously. However, because these loci are tightly linked (5, 49), this cannot be accomplished through simple breeding but requires a separate targeting strategy. As the absence of renin should be associated with marked impairment of angiotensin II production, a mouse that completely lacks renin may have phenotypic features similar to the Agt and Ace knockouts.

Although angiotensin II appears to play a role promoting neonatal survival and in the maintenance of vascular and papillary structures in the kidney, it is not yet clear which signaling pathways mediate these effects. As most of the known physiological functions of angiotensin II are mediated by the AT₁ class of receptors (55), it was expected that disruption of the AT_1A receptor gene (Agtr1a), the major AT₁ gene in the mouse, might recapitulate the angiotensinogen-deficient phenotype. However, Agtr1a (/) mice on mixed genetic backgrounds mice exhibit only a mild survival disadvantage during early postnatal life, and their renal morphology is normal with the exception of some minor abnormalities of the inner medulla and papilla (19, 34, 40, 53). Mice lacking AT₂ (16, 17) or AT_1B (3) receptors also survive in normal numbers, and their renal morphology is reported to be normal.

The failure to reproduce the Agt (/) phenotype by individual disruption of the known angiotensin receptor genes raises the possibility that there may be an as yet unidentified angiotensin receptor that functions to regulate vascular growth and integrity. Alternatively, the combined absence of signaling through at least two of the known receptor subtypes may be required to reproduce the phenotype associated with the complete absence of angiotensin II. As the genes encoding the individual angiotensin receptors are located on separate chromosomes, they can be combined through simple breeding. Experiments are underway in several laboratories to test this possibility. Finally, preliminary studies suggest that background genes may also influence the expression of this renal phenotype (Oliverio et al., unpublished observations).

	RAS GENES AND BLOOD PRESSURE REGULATION

Consistent with the well-established role of the RAS in blood pressure regulation, many of the RAS gene knockouts affect blood pressure. For example, the complete absence of angiotensinogen (26), ACE (7, 30), or AT_1A receptors (19) results in a reduction in systolic blood pressure of ~20 mmHg, measured by tail cuff in conscious animals. Since the level of blood pressure reduction in Agtr1a (/) mice approaches that seen in Agt and Ace (/) animals, most of the effect of angiotensin II to regulate resting blood pressure in mice seems to be mediated through the AT_1A receptor. In contrast, one line of mice lacking AT₂ receptors was reported to have a modest elevation in resting blood pressure (17), and these animals exhibit enhanced pressor responses following infusions of angiotensin II (16, 17). These observations provide further support for the notion that AT₂ receptors provide negative modulation of AT₁-mediated effects.

Although the complete absence of individual RAS components can have profound effects on blood pressure, more subtle alterations of RAS gene expression may also alter blood pressure regulation. Thus mice that are heterozygous (+/) for null mutations in the Agt (26) or Agtr1a (19) genes have reduced blood pressures, although the level of blood pressure reduction is more modest than in homozygous Agt or Agtr1a (/) animals. Incremental increases in the expression of RAS genes may also affect blood pressure. For example, Jeunemaitre and associates (22) demonstrated linkage between polymorphisms in the AGT gene and elevated blood pressure in humans with essential hypertension. The AGT gene variant-linked hypertension was also associated with elevated levels of plasma angiotensinogen. These authors (22) hypothesized that the elevated levels of angiotensinogen might predispose these patients toward developing hypertension.

To address the question of whether genetically determined elevations of plasma angiotensinogen levels would lead to increased blood pressure, Smithies and Kim (51) developed a novel "gap-repair" gene targeting strategy in which the mouse Agt gene was duplicated along with its surrounding regulatory elements at its native locus. This strategy allows the size of the targeting vector to be substantially smaller that the region to be duplicated. The gaps that are present relative to the target locus are repaired during the process of homologous recombination that mediates gene targeting (see Ref. 51 for a more detailed description). With this approach, mouse lines with three and four functional copies of the Agt gene were produced (26, 51). In these animals, plasma levels of angiotensinogen were increased above normal in proportion to the number of functional Agt gene copies. The three- and four-copy Agt mice with increased plasma angiotensinogen levels had elevated blood pressure. Thus different levels of function of the Agt gene were associated with significant differences in blood pressure. These observations provide direct support for causative effects of variants at the angiotensinogen locus in human hypertension (22).

In contrast, Ace (+/) mice that have a 50% reduction in ACE activity compared with normal do not have reduced blood pressure (7). Likewise, increasing ACE levels by increasing the number of functional Ace gene copies also does not affect blood pressure (31). Relative to angiotensinogen or the AT_1A receptor, this points to a fundamentally different relationship between quantitative changes in ACE activity and blood pressure regulation (50).

In addition to their utility for understanding the functions of the target gene itself, mice with RAS gene knockouts have also proved useful for studying biological effects of other RAS genes. For example, no abnormal phenotype has been identified in Agtr1B (/) mice, and these animals have normal blood pressure (3). Thus the initial analysis of the Agtr1B (/) mice has not provided insights into the specific physiological functions of the AT_1B receptor. However, by studying angiotensin II-mediated responses in Agtr1a (/) mice, in vivo functions of the AT_1B receptor were identified (39).

In these experiments, treatment of Agtr1a (/) mice with enalapril to reduce endogenous angiotensin II production (39) uncovered a pressor response to exogenous angiotensin II. This pressor response was not blocked by pretreatment with the sympatholytic agents hexamethonium or phentolamine but was completely inhibited by the AT₁ receptor antagonists losartan and candesartan, suggesting that it was directly mediated by AT_1B receptors. Chronic administration of losartan to Agtr1a (/) mice caused a further lowering of their already reduced blood pressures, suggesting that in the absence of the AT_1A receptor, the AT_1B receptor may contribute to the regulation of resting blood pressure.

The major difference between the acute effects of angiotensin II in Agtr1a (/) and (+/+) mice was in the magnitude of the pressor response. In wild-type mice, the peak increase in blood pressure following infusion of angiotensin II was significantly greater at each dose of angiotensin II tested and the overall slope of the dose-response curve was higher than in the Agtr1a (/) group (Fig. 2A). However, as shown in Fig. 2B, the shape of the pressure curves following angiotensin II injection were qualitatively similar in the two groups. Accordingly, the simplest explanation for these results is that the differences in responses between Agtr1a (+/+) and (/) mice are likely to be related to differences in receptor number, because AT_1A receptors are much more highly expressed in vascular tissues than AT_1B receptors (2, 10, 21, 24, 27, 32, 43). The similarity in the character of the pressor responses suggests that the signaling-effector mechanisms coupled to vasoconstriction are likely to be quite similar in AT_1A and AT_1B receptors. This hypothesis was further supported by the observation that stimulation of AT_1B receptors by angiotensin II in aortic smooth muscle cells derived from Agtr1a (/) mice induces a brisk increase in intracellular calcium (59).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Pressor effects of angiotensin II (ANG II) in AT1A receptor-deficient mice. A: change in mean arterial blood pressure (MAP) assessed at 120 s after injection of saline and 0.1, 1, and 10 µg/kg of angiotensin II in anesthetized Agtr1a (+/+) (n = 5) and (/) (n = 10) mice that had been pretreated with an angiotensin converting enzyme inhibitor. Data are presented as means ± SE. Open bars represent values in the Agtr1a (+/+) group; striped bars represent values from the Agtr1a (/) group. MAP before treatment was 84.6 ± 3.8 mmHg for the Agtr1a (+/+) group and 69.9 ± 4.5 mmHg for the Agtr1a (/) group (P < 0.04). * P < 0.002 vs. Agtr1a (+/+) saline alone. § P < 0.01 vs. Agtr1a (/). ** P < 0.018 vs. Agtr1a (/) saline alone. B: change in MAP as a percentage of the maximal response in Agtr1a (+/+) and (/) mice expressed over time following injection of 10 µg/kg angiotensin II. MAP immediately preceding the angiotensin II injection is recorded as 0, and the maximal MAP is assigned a value of 100%.

	EFFECTS OF RAS GENE MUTATIONS ON RENAL FUNCTION

Although the presence of severe structural abnormalities in kidneys of Agt and Ace (/) mice reduces the utility of these animals for studies of renal physiology, assessments of various parameters of renal function have been performed in other RAS mutant lines in which kidney structure is normal. The results of these studies are generally consistent with the key role of the RAS in regulating renal hemodynamic and excretory functions. In one such study, we determined the effects of mutations of the Agt locus on glomerular filtration rate (GFR) and renal plasma flow (RPF) in whole kidney clearance studies (26). These studies were performed in mice with one to three functional copies of the Agt gene that had been generated using the combination of conventional and gap-repair gene targeting approaches that were discussed above (52). In these mice, the circulating levels of angiotensinogen are directly proportional to the Agt genotype. Plasma angiotensinogen levels are 35% of normal in mice with one functional Agt gene and 124% of normal in mice with three copies of the Agt gene; normal (100%) is defined by plasma angiotensinogen levels in (+/+) two-copy mice.

As is shown in Fig. 3, GFRs were virtually identical in the three groups. Likewise, RPF was similar in mice with two and three copies of the Agt gene. However, in the Agt (+/) one-copy mice with reduced plasma angiotensinogen levels, RPF was significantly increased. Since blood pressure was reduced in one-copy Agt (+/) mice compared with controls, these findings suggest that their renal vascular resistance is also decreased. Taken together, these results suggest that reduction of circulating angiotensinogen in the range of 35% of normal is sufficient to significantly alter renal hemodynamics. The characteristics of these changes, increased renal blood flow, decreased filtration fraction, and reduced renal vascular resistance, are consistent with reduced angiotensin II effects in the kidney.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Renal function in mice with targeted mutations in the angiotensinogen gene. Clearances of inulin [for glomerular filtration rate (GFR)] and p-aminohippurate [for renal plasma flow (RPF)] measured in F1 mice having one (n = 6), two (n = 9), or three (n = 6) functional copies of the Agt gene. * P < 0.005 vs. Agt two-copy or three-copy groups.

The integrated control of GFR and solute reabsorption is a key element in the kidney's contribution to extracellular fluid homeostasis. Tubuloglomerular feedback (TGF) responses represent a component of this integrated control mechanism. Pharmacological studies have implicated the RAS in controlling TGF, as RAS antagonists generally reduce the magnitude but do not completely abolish TGF responses (42). Recent experiments using Agtr1a (/) mice have further defined the role of the AT_1A receptor in this process (47). With standard micropuncture techniques where stop-flow pressures were determined during changes in loop perfusion rates, TGF responses were found to be virtually absent in Agtr1a (/) mice. This contrasts with the effects of pharmacological RAS antagonists, which generally inhibit TGF by ~50% (42). The observation that TGF responses are absent in Agtr1a (/) mice suggests that, at least in the mouse, the presence of AT_1A receptors is an absolute requirement for the normal TGF response. These studies also demonstrate the feasibility of using complex micropuncture techniques in mice.

	CONCLUSIONS

Top Abstract Introduction Conclusions References

A major strength of gene targeting as an experimental approach is that it provides an avenue for the direct application of precise molecular genetic intervention to the study of complex responses in intact animals. The studies of mice with targeted alterations in RAS genes provide an example of the potential utility of gene targeting in physiology. This work has established a role for angiotensin II in regulating or maintaining renal vascular and papillary structures in the postnatal period. Despite some differences in the RAS system in the mouse compared with other well-studied species such as human and rat, these experiments have provided detail and confirmation of the role of RAS gene products in regulating blood pressure and renal function. Finally, this technology may provide a novel method for understanding how naturally occurring mutations in human RAS genes cause hypertension. Further advances in adapting experimental techniques for acute and chronic experiments in the mouse promise to extend the contributions of gene targeting to physiological investigation.