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Physiological Insights from Genetic Manipulation of the Renin-Angiotensin System

David E. Stec and Curt D. Sigmund

D. E. Stec and C. D. Sigmund are in the Departments of Internal Medicine and Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242.

	Abstract

 
The renin-angiotensin system is one of the most widely studied endocrine systems. It has an important role in the regulation of normal homeostasis, and disturbances in this system may be important in numerous pathological states. This review will focus on the major insights and important questions raised from gene targeting of this system.

	Introduction

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One of the most widely recognized functions of the renin-angiotensin system (RAS) is its role in the regulation of arterial pressure. Angiotensin II (ANG II), the effector hormone of the RAS, regulates blood pressure by exerting both direct and indirect effects on a variety of cardiovascular tissues, including blood vessels, brain, and kidney. ANG II is generated by the serial cleavage of angiotensinogen (AGT) first by renin and then by angiotensin-converting enzyme (ACE). ANG II exerts its effects via two classes of receptors, AT₁ and AT₂, with most of its classic actions mediated through the AT₁ receptor. Two subtypes of AT₁ receptor, AT_1A and AT_1B, that cannot be distinguished pharmacologically exist in rodents. Most of our current understanding of the RAS has come from pharmacological studies using inhibitors; however, the emergence of gene targeting has allowed us to dissect this system with a targeted specificity not previously possible.

	Blood pressure regulation

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The first gene-targeting studies on the RAS were performed on the renin substrate AGT (6). AGT is synthesized in a wide variety of tissues, including the brain, kidney, and liver. Plasma levels are due to synthesis of the AGT in the liver, and gene variants that are associated with altered levels of AGT in the plasma have been correlated with human essential hypertension. In studies of mice with 1–4 copies of the AGT gene, blood pressure was increased by 8 mmHg per gene copy, with plasma AGT levels 35% (1 copy), 124% (3 copies), and 145% (4 copies) of wild type. This data supports the view that chronic alterations in plasma levels of AGT are able to affect blood pressure even in the presence of normal blood pressure regulatory mechanisms. Blood pressure data in AGT knockout mice has been difficult to obtain because of the lethality associated with altered renovascular development in these mice. However, the few measurements obtained indicated that blood pressure is dramatically decreased and dependent on sodium intake.

Gene knockouts have also been generated for ACE and renin (7, 15). ACE knockout mice have blood pressure that is reduced by 15–20 mmHg from wild-type controls. As expected, ACE knockout mice fail to exhibit a pressor response to infusion of angiotensin I (ANG I) and exhibit an enhanced depressor response to infusions of bradykinin. The same renovascular pathologies that are present in AGT knockouts are also found in ACE knockout mice. The experimental situation is more complicated when considering renin because mice exhibit two genotypes at the renin locus. Some inbred strains, such as C57BL/6 and BALB/c, contain one renin gene (Ren-1^c), and other strains, such as 129 and DBA, contain two renin genes (Ren-1^d and Ren-2). All three mouse renin genes are highly homologous but produce distinct proteins having different glycosylation potentials. Targeted disruption of all of the renin genes has been reported, with the major cardiovascular phenotype being a decrease in blood pressure in Ren-1^c homozygous knockout mice. These mice also lack detectable levels of plasma renin activity and ANG I; however, levels of ANG II were not measured in these mice (15). Juxtaglomerular (JG) cells in Ren-1^d knockout mice lack secretory granules, suggesting differential sorting of renin-1 and renin-2 in the secretory pathway and perhaps a proactive role for renin in defining the "epithelioid" character of the JG cell.

Most of the classic cardiovascular effects of ANG II have been attributed to the AT₁ receptor. In rodents, there are two unique AT₁ receptor subtypes, AT_1A and AT_1B, both of which are blocked by the general AT₁ receptor antagonist losartan. Since these receptor subtypes cannot be distinguished pharmacologically, gene-targeting studies have been critical in defining the role of each subtype in vivo. Mice with a targeted disruption of the AT_1A receptor exhibit a significant decrease in blood pressure that is further lowered by losartan treatment (9). These mice also exhibit a markedly reduced, but not completely abolished, pressor response to ANG II infusion. These findings suggest a potential role for the AT_1B receptor in the regulation of blood pressure in the absence of the AT_1A receptor. Recently, we reported that the pressor response to centrally administered ANG II can be fully attributed to the AT_1A receptor, whereas the dipsogenic response to central ANG II administration is mediated by the AT_1B receptor (4). This is the first report describing a distinct and separate function for the AT_1B receptor.

The other major class of ANG II receptor is the AT₂ receptor. This receptor is believed to be involved in pathways that oppose the actions of the AT₁ receptor such as vasodilatation and apoptosis. Much of what is known about the function of the AT₂ receptor has come from gene deletion studies of the AT₂ receptor and from studies in mice lacking both the AT_1A and AT_1B receptors. Disruption of the AT₂ receptor was originally reported to have no effect on basal blood pressure; however, recent studies have detected an increase in blood pressure in mice lacking this gene (5, 12). Although the effect of AT₂ receptor deletion is controversial, its role in the regulation of blood pressure may be important during blockade of the AT₁ receptor. Studies in mice lacking both AT₁ receptors have demonstrated that administration of captopril increases blood pressure, presumably because it prevents ANG II binding the AT₂ receptor (10). However, infusion of ANG II into these mice did not lower blood pressure as might be expected, and duplication of this effect with an AT₂ receptor antagonist has yet to be reported. Transgenic mice in which the AT₂ receptor is overexpressed specifically in vascular smooth muscle cells (VSMC) have been generated (14). Chronic infusion of ANG II into these mice fails to elicit a pressor response unless accompanied by blockade of bradykinin type 2 receptor or nitric oxide synthase. This was associated with increased cGMP production in aortic explants and a markedly diminished ANG II-induced constriction, both of which were dependent on an intact endothelium.

Recently, a significant body of evidence has demonstrated that organs such as the kidney, heart, and brain contain a local tissue RAS that plays a significant role in the control of organ function and blood pressure regulation. To test whether alterations in the intrarenal RAS system are sufficient to affect blood pressure, we created a model in which the human AGT gene is specifically overexpressed in the proximal tubule of the kidney (a site where AGT is normally expressed in the kidney) (Fig. 1A). Human AGT protein was elevated in urine but absent from plasma, suggesting apical secretion of the protein into the lumen. When crossed with mice carrying the human renin gene, the resultant double transgenic mice were hypertensive (2) (Fig. 1B). Interestingly, the hypertension displayed by the double transgenic mice was not associated with any increase in the plasma levels of ANG II (Fig. 1C). Moreover, acute intravenous infusion of losartan was unable to normalize pressure in these mice, further suggesting that the observed hypertension was due to increase in intrarenal (perhaps proximal tubular) levels of ANG II (Fig. 1D). These results provide experimental proof that alterations in intrarenal RAS function may cause elevated arterial pressure without affecting the endocrine RAS.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Physiological analysis of the kidney androgen-regulated protein (KAP)-hAGT model. A: schematic model of the proximal tubule showing expression and luminal secretion of angiotensinogen (AGT). Luminal AGT is cleaved by renin derived from the filtrate, transport across the epithelial cells from the interstitium, or from direct expression and released from proximal tubule cells. Angiotensin-converting enzyme (ACE) is abundantly expressed in brush border membranes, and the apical surface contains angiotensin II (ANG II) receptors. B: basal blood pressure was recorded in male control mice (C), kidney-specific double transgenic mice (K), and systemic double transgenic mice (S). *P < 0.01 vs. control. C: plasma ANG II was measured in control (C), male (M), and testosterone-treated female (FT) kidney-specific double transgenic mice as well as systemic double transgenic mice (S) *P < 0.0001 vs. control. D: peak decrease in mean arterial pressure (MAP) in response to 10 mg/kg losartan was recorded in male control mice (C), kidney-specific double transgenic mice (K), and systemic double transgenic mice (S). *P < 0.01 vs. control.

	Renal function

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Further examination of renal hemodynamics in AT_1A knockout mice failed to find any differences in GFR and RPF compared with wild-type mice under basal conditions or in response to volume expansion. This is in contrast to studies performed on AGT knockout mice in which autoregulation of GFR was found to be absent in response to changes in sodium intake (8). These data suggest that in AT_1A knockout mice compensation by either AT_1B or AT₂ receptor may allow for the maintenance of normal renal hemodynamics in the face of lower blood pressure. However, in the AGT knockout mouse, these receptors are not able to compensate for the loss of ANG II and GFR becomes more dependent on blood pressure.

The RAS is upregulated in the kidney in response to a low sodium diet and in pathological states such as hemorrhagic shock and heart failure, and it stimulates sodium reabsorption in the proximal tubule by both direct and indirect effects. The AT_1A receptor is believed to be the primary receptor responsible for the vascular and tubular actions of the RAS in the kidney. Loss of either AGT or the AT_1A receptor results in chronic loss of extracellular volume and lowering of blood pressure (1). Volume expansion is able to increase blood pressure in AT_1A receptor-deficient mice to levels observed in control mice. Both AGT and AT_1A/AT_1B deficient mice exhibit a deficiency in the ability to concentrate urine, which is markedly exaggerated when sodium intake is reduced. Part of this effect may be related to the severe atrophy of the renal papilla observed in these mice.

The relationship between the RAS, renal perfusion pressure, and sodium excretion (pressure natriuresis) has been extensively examined in both rats and dogs. The RAS is believed to be a key player in this relationship via its actions on proximal tubular sodium reabsorption and renal vascular tone. Although pressure natriuresis studies have not yet been performed in AGT-, AT_1A-, or AT_1A/AT_1B-deficient mice, one would expect this relationship to be shifted to the left to normalize sodium and water excretion given the lower arterial pressures observed in these mice. Pressure natriuresis studies have been performed in AT₂ receptor knockout mice (5). The results of this study indicate that the relationship is shifted to the right such that AT₂ receptor knockout mice require higher renal perfusion pressures to excrete similar amounts of sodium to wild-type mice. This result is in contrast with pharmacological studies performed in rats, which demonstrated a leftward shift in response to inhibition of AT₂ receptors. AT₂ receptor knockout mice also failed to exhibit increases in medullary blood flow in response to the increase in renal perfusion pressure. However, it is unclear whether these effects are directly related to lack of AT₂ receptors in the renal vasculature or compensation by another regulatory system.

	Renal development

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One of the most interesting areas to arise from gene targeting the RAS has been the role of the RAS in renal development. ANG II has a widely recognized growth and proliferative effect in VSMC, mesangial cells, and renal tubular cells that is mediated via AT₁ receptors. Mice in which the AGT or ACE gene has been disrupted are born in expected numbers, but most mice die before 3 wk of age. Death in these mice is associated with a variety of vascular and tubular pathologies that are surprisingly restricted to the kidney. These include thickening of arterial walls, cortical inflammation, and hypoplasia of the inner medulla. Interestingly, the degree of the renal pathology and frequency of lethality differs between the two AGT knockout strains previously described, perhaps due to difference in genetic background between the strains. Systemic expression of both human renin and human AGT is able to rescue the renal and lethal phenotypes when bred onto the AGT knockout background (3). However, when the expression of the human AGT gene is restricted to the proximal tubule, expression of the human transgenes is not able to rescue the lethal phenotype (Ding and Sigmund, unpublished observations). This suggests that loss of tubular ANG II is not the cause of lethality and that a vascular-derived source of ANG II in the kidney may be essential to prevent the lethality observed in AGT knockout mice. Whether this renal vascular source of ANG II can be derived from the plasma alone or requires synthesis directly by renal blood vessels has yet to be determined.

The vascular and tubular pathologies found in AGT and ACE knockout mice are not exhibited to the same extent in mice lacking only the AT_1A receptor but are recapitulated in mice that lack both the AT_1A and AT_1B receptors (10). This is an interesting finding given that the vast majority of AT₁ receptors in the adult kidney are of the AT_1A subtype. It is possible that the expression of AT_1B receptors in early development is able to compensate for and limit the abnormalities in renal development; however, the expression pattern of the AT_1B receptor in the kidney of AT_1A knockout mice has yet to be reported.

	Future directions

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Clearly, the greatest limitation in the study of RAS knockout mice is the lethal phenotype that is exhibited shortly after birth. Although these mice have revealed a role for the RAS in the developing kidney, cardiovascular phenotyping of these mice has been more difficult. Also, since components of the RAS are expressed in a wide variety of tissue and cell types that all have important roles in cardiovascular regulation, it is difficult to specifically address the contribution of various tissue RAS in mice that have a global loss of gene function. Recently, it has become feasible to perform tissue-specific deletion of a gene via the use of specific recombinases. The Cre-loxP system from bacteriophage P1 has become a popular means of performing tissue-specific deletions of genes in vivo (Fig. 2A). We have incorporated this system to dissect the role of the various tissue RAS in a model of ANG II-dependent hypertension (13). By placing loxP sites around the ANG II coding regions of the human AGT gene, we have been able to perform tissue-specific deletion of this gene in the liver via adenoviral delivery of the cre recombinase (Fig. 2, B and C). The generation of transgenic mice harboring the cre recombinase in specific tissues and cell types will allow for a more detailed dissection of the RAS in the future (Table 1). Finally, using temporally controlled promoters, one will be able to inactivate genes of the RAS at any time during development. Hopefully, this will bypass the lethal phenotypes and allow for a more thorough examination of the role of the various tissue RAS in cardiovascular regulation. Moreover, the use of targeted homologous recombination in which genes can be placed in the mouse genome at identical sites in a single copy will allow for the determination of the functional significance of gene variants (or single nucleotide polymorphisms) of the RAS in an in vivo model system.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Liver-specific knockout of a floxed human AGT transgene with the use of the Cre-loxP system. A: cre recombinase causes an intramolecular recombination event between 2 loxP sites. In the hypothetical 4-exon gene shown, loxP sites (black arrowheads) have been engineered surrounding exon 3, which encodes a critical portion of the hypothetical protein. Exons are denoted by boxes and are numbered. In the presence of cre recombinase, an intramolecular recombination results in the deletion of exons 3 and 1 of the loxP sites. This occurs when the loxP sites are oriented in the same relative direction as shown. B: levels of circulating human AGT protein (as %control) before (day 0) or 1-5 days after infection with adenovirus expressing ß-galactosidase (AdßGal) (gray bars) or adenovirus expressing cre recombinase (Adcre) (filled and crosshatched bars). The filled and crosshatched bars represent two different lines of human AGTflox mice. C: pressor response after human renin infusion (as %control) before (day 0), 5 days, and 7 days after Adcre infection. Human renin infusion elicited an ~30 mmHg pressor response. There was no change in the pressor response to human renin in mice infected with AdßGal.

	References

Top Introduction Blood pressure regulation Renal function Renal development Future directions References

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