Establishing the Role of Angiotensin-Converting Enzyme in Renal Function and Blood Pressure Control Through the Analysis of Genetically Modified Mice

Review

Establishing the Role of Angiotensin-Converting Enzyme in Renal Function and Blood Pressure Control through the Analysis of Genetically Modified Mice

Kenneth E. Bernstein, Hong D. Xiao, Jon W. Adams, Kristen Frenzel, Ping Li, Xiao Z. Shen, Justin M. Cole, and Sebastien Fuchs Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia J Am Soc Nephrol 16: 583–591, 2005. doi: 10.1681/ASN.2004080693

ngiotensin II is a vasoconstrictor and a hypertensive serum and urinary potassium concentrations. They are anemic,

peptide that binds to the AT1 receptor and, through and they present with a renal lesion characterized by renal A both direct and indirect mechanisms, induces salt medullary thinning associated with expansion of the renal pel- reabsorption. Also, angiotensin II is thought to be a profibrotic vis. ACE knockout mice also have defective male fertility as a and proproliferative peptide; abundant evidence now suggests result of the absence of the testis isozyme of ACE. Published that angiotensin II can stimulate cytokines and oxygen radicals, work suggests that this reproductive defect is not found in inducing an inflammatory response (1,2). Indeed, so much has angiotensinogen knockout mice, suggesting an important sub- been written about angiotensin-converting enzyme (ACE) and strate for ACE other than angiotensin I (10). angiotensin II that it is legitimate to ask what new can be found concerning actions of the renin-angiotensin system (RAS). This review shows that, rather than being a subject without novelty, Knockout Mice: Advantages and the ability to genetically manipulate mice gives scientists the Disadvantages power to investigate the many physiologic roles of angiotensin Although knockout mice are a wonderful methodologic ad- II with a level of understanding not previously available. At vance, this technique has some disadvantages. For instance, the last, scientific tools are available to test some of the hypotheses complete lack of protein production is different from a human and dogmas concerning RAS action. Perhaps you are not con- who takes a pharmaceutical to inhibit that protein. Indeed, the vinced and think that, in a system that has seen the publication more one works with knockout mice, the more one is aware of angiotensinogen, renin, and ACE knockout mice, how could that the total genetic abrogation of a protein represents a truly even the powerful technology of mouse genetic targeting pro- extreme phenotype. The most common human diseases typi- vide novel insight? Answering precisely this question is the cally do not result from the complete absence of a particular subject of the review. protein. The technique of creating a knockout mouse is now widely The power of targeted recombination in embryonic stem cells appreciated (3). Cultured embryonic stem cells are manipulated extends beyond the ability to create a simple knockout model. by targeted homologous recombination to induce virtually any For example, the research group of Smithies and colleagues (5) genetic change that can be imagined. The most common use of used gene targeting to create a series of genetically altered mice this method has been to eliminate a gene that, in a mice that are that bear zero to four copies of the angiotensinogen gene. This homozygous for the mutation, results in a knockout animal. In celebrated study used gene targeting to either eliminate or such models, no protein is made during the entire embryonic duplicate the angiotensinogen gene and showed that there was and extraembryonic life of the animal. The resulting phenotype a gene dosage effect in which every additional copy of the is often dramatic and at any rate gives great insight into the angiotensinogen gene beyond one was responsible for an functional role of a protein as assessed in a mammal that is not 8-mmHg rise in BP. These data, combined with human studies, fundamentally different from humans. For example, mice that suggest that genetic variability in angiotensinogen can account for some genetic differences in human BP (11,12). lack angiotensinogen, ACE, or all AT1 receptors have a similar phenotype (4–9). These animals show a reduction of systolic BP In addition to gene elimination or duplication, targeted ho- in excess of 35 mmHg. They are unable to produce a concen- mologous recombination can create precise mutations within a trated urine much above 1000 mOsm/L. They have elevated protein. For example, our group used such an approach to study ACE. This enzyme is a single polypeptide chain but contains two zinc binding sites and two separate and indepen-

Published online ahead of print. Publication date available at www.jasn.org. dent catalytic domains (13). Although angiotensin I is a good substrate for both of the two catalytic sites of ACE, other Address correspondence to: Dr. Kenneth Bernstein, Department of Pathology, Emory University, Room 7107A WMB, 101 Woodruff Circle, Atlanta, GA 30322. peptide substrates show distinct preferences for only one of the Phone: 404-727-3134; Fax: 404-712-9625; E-mail: [email protected] ACE catalytic sites. An example is the ACE substrate acetyl-

SDKP that has been implicated as an antifibrotic and a bone ination of that protein. This particular model allowed study of marrow–suppressive peptide (14,15). Acetyl-SDKP is effec- the physiologic effects of acetyl-SDKP accumulation in a setting tively hydrolyzed only by the N-terminal catalytic domain of free of the secondary effects of low BP, and ultimately it dis- ACE; ACE inhibition is associated with an elevation in blood proved the suggested hypothesis for the actions of acetyl- levels of this peptide (16). As discussed, ACE null (knockout) SDKP. Even with this study, it remains unclear why animals animals have anemia characterized by hematocrits approxi- that lack all ACE (ACE knockout mice) are anemic. Most likely, mately 20% lower than that in wild-type mice. One hypothesis the anemia reflects the inability to generate angiotensin II (as suggested that the anemia was a consequence of acetyl-SDKP opposed to another peptide) because the infusion of angioten- build-up in the absence of ACE activity. To test this, our group sin II into ACE null mice is associated with correction of the introduced point mutations into the ACE gene that eliminated anemia (18). the ability of the N-terminal ACE catalytic domain to bind zinc and thus rendered this domain catalytically inactive (Figure 1) Selected Tissue Expression of ACE (17). The result was a mouse model called ACE 7/7, which If one views ACE null mice as expressing an extreme phe- makes normal amounts of an altered ACE protein that has only notype, then it would be useful to have mouse models with a one catalytic domain. In the absence of the N-terminal domain, less severe genetic change. One approach might be to selec- these animals have elevated serum levels of acetyl-SDKP but a tively eliminate ACE expression in a particular organ such as normal BP as a result of the ability of the unaltered C-terminal the kidney (Figure 2). This is technologically feasible using gene ACE catalytic domain to effectively hydrolyze angiotensin I. targeting but probably would not yield a phenotype in the Study of these mice found that they had no evidence of anemia. resulting mice. ACE is widely distributed in animal tissues; Thus, this is an example in which genetic targeting in mice large amounts of ACE are made by vascular endothelium, the produced an altered protein, as opposed to the complete elim- kidney, areas of the gut, and activated macrophages and in

Figure 2. (A) In a wt mouse, ACE is widely distributed. In contrast, a knockout mouse that is null for all ACE expression represents an extreme phenotype in that this animal makes no Figure 1. (A) In a wild-type animal, angiotensin-converting ACE during its entire lifetime. One way to approach a mouse enzyme (ACE) is a single polypeptide chain with two zinc (Zn) model with selective expression of ACE is to eliminate geneti- binding sites and two catalytic domains. The ACE.7 mutation cally the protein from a particular organ such as the kidney. introduced point mutations into the ACE gene that changed the However, given the wide distribution of ACE, such a model amino acid sequence of the N-terminal Zn binding site from seemed unlikely to give a phenotype. The approach that our HEMGH to KEMGK. This new sequence is unable to bind zinc, group has adopted is to functionally replace the endogenous eliminating catalytic activity in the N-terminal domain. ACE ACE promoter with another, tissue-specific promoter. In this 7/7 mice are homozygous for the ACE.7 mutation. The ACE way, expression of ACE is directed to the small subset of tissues made by these mice has catalytic activity only in the C-terminal that recognize the new promoter. (B) The genetic construct used domain. (B) A rabbit anti-ACE antibody was used in Western to create the ACE.3 mutation. Arrows indicate the endogenous blot analysis to evaluate ACE protein expression in wild-type somatic and testis promoters. The wt ACE allele was modified (wt), ACE 7/7, and ACE 1/7 mice. ACE 1/7 animals are com- by the insertion of a neomycin cassette followed by the albumin pound heterozygotes in which the 1 allele is null for ACE promoter between the endogenous somatic ACE promoter and expression. ACE 7/7 mice express ACE levels equivalent to wt the transcribed portions of the gene. In this way, the effects of animals. In contrast, ACE 1/7 mice have reduced ACE protein the endogenous ACE promoter are blocked and control of the expression. gene is directed by the albumin promoter. J Am Soc Nephrol 16: 583–591, 2005 ACE and Genetically Modified Mice 585 parts of the brain. Given the highly regulated expression of renin, it seemed unlikely that the elimination of ACE expression in one target organ would have much effect. Our group has selected a different approach: To create a mouse model devoid of ACE activity, except for selected organs engineered to express this protein (Figure 2). Models of this type can be created by genetic manipulation of the promoter region of the ACE gene (the portion of the gene that regulates tissue and temporal expression). For example, consider the mouse model that is termed ACE.3, in which genetic engineering was used to position the albumin promoter in place of the natural ACE promoter (Figure 2B) (19). In these mice, ACE expression is controlled not by the ACE promoter but by the engineered albumin promoter, leading to ACE protein expression by hepatocytes within the liver. Because the coding portions of the ACE gene were not modified, the protein was transported to the hepatocyte cell surface, similar to its typical localization in endothelium. However, as the albumin promoter is active only in hepatocytes (and not in endothelium), this model resulted in mice with very restricted ACE expression. For example, wild- type mice normally produce large amounts of ACE in the lung as a result of the high content of endothelium within this tissue (Figure 3). In contrast, ACE.3 mice produced no ACE in the lung or by any endothelium throughout the mouse. Only in the kidney was there aberrant low-level recognition of the modified ACE promoter, leading to renal ACE levels approximately Figure 3. Tissue distribution of ACE in ACE 3/3 mice. Tissues 15% those of wild-type mice. The low-level ACE found in from a wt mouse (ϩ/ϩ) and an ACE 3/3 mouse (Ϫ/Ϫ) were kidney was present in the proximal tubule and probably re- fixed in formalin and prepared by immunostaining with a sulted from the use of a man-made albumin promoter to control rabbit polyclonal anti-ACE antibody. ACE is indicated as a ACE expression. However, even in the kidney, vascular expres- brown pigment. (A and B) Sections of liver and show that, sion of ACE was not present. Thus, a mouse model in which the whereas a wt mouse makes very little ACE in this tissue (A), the widespread distribution of ACE in endothelium and epithe- ACE 3/3 mouse expresses abundant hepatic ACE (B) and lo- lium was replaced by localized ACE expression on the surface calizes the protein on the hepatocyte cell surface (B, insert). In contrast, wt mice have abundant ACE in the lung (C), but ACE of hepatocytes was created. 8/8 mice express no ACE in lung (D). In the kidney, wt mice One advantage of this approach is that compound heterozy- make abundant ACE in the straight portion of the proximal gous mice can easily be created through simple breeding. For tubule (E). In ACE 3/3 mice, renal ACE is reduced to approx- instance, our group refers to ACE knockout mice as ACE.1; a imately 14% that of wt levels (F). In a wt mouse, an abundant homozygous knockout mouse has an ACE genotype called source of ACE is vascular endothelium (G, arrow) and the ACE 1/1 to indicate that both ACE alleles contain the ACE.1 vascular adventitia. As seen in H, ACE 3/3 mice have no mutation (Table 1). These animals are null for all ACE expres- vascular ACE expression by either endothelium or the adven- sion. Likewise, a mouse homozygous for the ACE mutation titia. These data show that ACE expression in ACE 3/3 mice that targets expression to the liver has a genotype termed ACE was genetically transferred from the endothelium and the kid- 3/3. A mouse with point mutations that inactivate the ACE ney to the liver. N-terminal catalytic domain is termed ACE 7/7. These num- bers merely refer to the order in which the mouse models were created, but they are useful in signifying the ACE genotype. For 1). Rather than discuss each model individually, the major instance, an ACE heterozygous mouse with one ACE.1 allele conclusions obtained from the combined data are presented and one wild-type allele is termed wt/1. If this animal is mated below. with an ACE 3/3 mouse, then half of the offspring will have the genotype ACE 1/3. In these mice, the “1” ACE allele is null, Blood Pressure whereas the “3” ACE allele directs ACE expression to the liver. Mice that lack all ACE have a systolic BP of approximately 73 Because of the null allele, ACE 1/3 mice have half of the mmHg as opposed to a wild-type mouse with a systolic BP of hepatic, renal, and plasma levels of ACE found in ACE 3/3 approximately 110 mmHg (Figure 4). This marked reduction in mice (20). As compared with wild-type mice, ACE 1/3 mice the absence of all ACE belies that mice (and probably humans) have only 7% normal renal ACE activity. are very tolerant of marked changes in tissue patterns and This promoter-swapping technique was used to create a se- overall levels of ACE expression. For example, ACE 1/7 mice, ries of mice with restricted patterns of ACE expression (Table which express only half of normal levels of an ACE protein that 586 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 583–591, 2005

Table 1. Important characteristics of mouse lines with genetic changes to the ACE genea

Urine Male Mutation Genotype Description Distribution of ACE BP Renal Development Concentration Hematocrit Fertility

Wild-type wt/wt Two normal ACE alleles Vascular endothelium (lung), Normal Normal Normal Normal Normal kidney, gut, brain, plasma, testis ACE.1 1/1 Null for all ACE No ACE Low Medullar and papillary Dilute Low Low dysplasia ACE.2 2/2 Stop codon in ACE gene No tissue ACE, 34% plasma Low Normal renal papilla Dilute Low Low ACE activity ACE.3 3/3 Albumin promoter Hepatocytes11, kidney Normal Normal Normal Normal Normal controlling ACE (14% of wild type), plasma, testis ACE 1/3 1/3 Compound heterozygote Half the expression of 3/3 Normal Normal Normal Normal Normal ACE.4 4/4 KAP promoter controlling Testis Low Medullar and papillary Dilute Low Normal ACE dysplasia ACE.5 5/5 ␥-GT promoter controlling Ͻ6% kidney ACE, testis Low Most kidney present Dilute Low ND ACE medullar and papillary dysplasia ACE.7 7/7 N-terminal catalytic domain Same as wild type Normal Normal Normal Normal Normal inactivated ACE 1/7 1/7 Compound heterozygote Half the expression of 7/7 Normal Normal Normal Normal ND ACE.8 8/8␣␣-MHC promoter Heart11, lung (40% of wild Nearly Normal Normal Normal Normal controlling ACE type), plasma (50% of wild normal type), testis ACE.10 10/10 c-fms promoter controlling Macrophages11, plasma, In progress ACE testis ACE.11 11/11 THP promoter controlling Similar to ACE.4 Low Medullar and papillary Dilute Low ND ACE dysplasia

aACE, angiotensin-converting enzyme; ND, not done. We measured serum angiotensin II levels in several of these lines. As would be anticipated, low BP is associated with very low plasma levels of angiotensin II. Most mouse models with normal BP (e.g., ACE 7/7 and ACE 8/8) have plasma angiotensin II levels that are not significantly different from wild-type mice. However, ACE 1/3 mice have a normal BP but also have threefold wild-type levels of plasma angiotensin II. Normal aldosterone levels in this model suggest that these mice may have normal end-organ concentrations of angiotensin II. lacks enzymatic activity in one of the two ACE catalytic do- liver or the heart are sufficient for renal compensation and mains (the N-terminal domain), have a normal BP (17). ACE maintenance of normal BP. Only when ACE activity falls to 1/3 mice, animals with no endothelial expression of ACE and very low levels can the RAS not achieve compensation. These renal levels approximately 7% those of wild-type mice, also conclusions apply to otherwise healthy animals that are main- have a normal BP (20). Even an animals that are engineered to tained in a relatively disease- and stress-free laboratory envi- express ACE only in myocardium and lung smooth muscle ronment. (ACE 8/8) present with a BP not much different from that of wild-type mice (21). The ability to tolerate wide changes in ACE Role of Bradykinin activity was postulated through a computer-based analysis of In addition to producing angiotensin II, ACE is thought to BP control conducted by Smithies et al. (22). This group pre- play a major role in the degradation of the vasodilator brady- dicted that as ACE levels diminished, compensatory upregula- kinin. To study this, we bred ACE knockout mice with a dif- tion of renin and angiotensin I levels would lead to homeostatic ferent line of mice that lack the bradykinin B2 receptor to maintenance of angiotensin II (and BP) until such a point as generate double-knockout mice (23). It is the B2 receptor that is ACE activity fell to such low levels as to overwhelm the kid- thought to mediate the cardiovascular effects of bradykinin ney’s ability to compensate. This conclusion is highly consistent (24). The experimental design was to compare mice that lack with what our group observed experimentally. In the ACE 1/3 only ACE with the double-knockout mice that lack both ACE mouse, normal BP is maintained by an elevation of plasma and the bradykinin B2 receptor. The double-knockout mice had renin, plasma angiotensin I, and even plasma angiotensin II a phenotype identical to single ACE knockout mice (very low concentrations. ACE 1/3 mice have normal concentrations of BP, underdevelopment of renal medulla, and inability to con- serum and urinary aldosterone. These data suggest that al- centrate urine). Although this study was somewhat incomplete though the plasma levels of angiotensin II are elevated, the in that it did not account for bradykinin B1 receptors, it sug- end-organ level of angiotensin II is precisely regulated to main- gests that the main pathologic defect in ACE null mice is a lack tain a normal BP. More than just changes in ACE activity, of angiotensin II and not a surplus of bradykinin. studies of genetically manipulated mice show that animals are remarkably tolerant of different tissue patterns of ACE expres- Local versus Systemic Angiotensin II sion. In total, this work suggests that the proper way to view What about the question of local versus systemic production ACE is to apply the concept of “total body load” of ACE. As of angiotensin II? This discussion reflects the presence of vari- total body load diminishes, the kidney compensates through ous components of the RAS throughout the body. In turn, this increased production of renin. In a mouse, even modest total has engendered a lively discussion as to the importance of local body loads of ACE positioned in tissues as diverse as either the production of angiotensin II (tissue based) versus the systemic J Am Soc Nephrol 16: 583–591, 2005 ACE and Genetically Modified Mice 587

generation of angiotensin II is that, at least for BP control, both of these systems contribute to normal regulation. In a normal animal, it is the final concentration of angiotensin II that is regulated; this peptide originates from both systemic and local production. If local production is genetically diminished, then systemic production is upregulated.

Renal ACE In the kidney, ACE is associated with vascular endothelium and proximal tubular epithelium, particularly the distal (S3) portion of the proximal tubule. We now have examined two strains of mice with very low or no renal ACE. This is the ACE 1/3 mouse (7% normal renal ACE) and the ACE 8/8 mouse (no renal ACE) (20,21). In both strains, deprivation of water for 24 h resulted in urinary concentrations in excess of 3000 mOsm/L, which was indistinguishable from wild-type mice. Perhaps this is not surprising given that these animals retain a functional RAS with the ability to stimulate aldosterone production. How- Figure 4. (A) BP was measured in conscious mice using a ever, it does indicate that the ACE within the kidney plays no computer-controlled tail-cuff system that determines systolic irreplaceable physiologic role, at least as measured by renal BP (40). Mice were trained in the machine for several days concentrating ability. before data were collected. The final data point for each mouse We studied ACE 1/3 mice after a 2-wk diet that totally lacked ϩ is the average of 80 measurements taken over 4 d. ,wtACE NaCl (21). In the absence of all exogenous salt, mice maximally allele; other strain designations are explained in Table 1. (B) retained salt. Nonetheless, there is a small loss of urinary salt Plasma renin activity was measured by assessing the conver- and a consequent small reduction of BP. A typical wild-type sion of rat angiotensinogen to angiotensin I during a normal mouse will reduce systolic BP by 5 mmHg or less. Under the salt diet and during salt deprivation. The designation 3/wt refers to a compound heterozygous mouse with one ACE allele extreme stress of 2 wk without salt, we did observe a difference targeting expression to the liver (the “3” allele) and one wt ACE between the ACE 1/3 mice and wild-type animals: There was a allele. These mice are phenotypically similar to wt mice. ACE small but significant additional loss of urinary sodium by the 1/3 are compound heterozygotes with one ACE null allele (the ACE 1/3 mice. This resulted in a greater reduction of systolic “1” allele) and one ACE 3 allele. Even with a normal salt diet, BP (approximately 10 mmHg). Nonetheless, it was remarkable these mice upregulate renin activity to maintain a normal BP. In that ACE 1/3 mice were able to tolerate easily this regimen and the absence of dietary salt, all animals increase renin expres- to maintain systolic BP in excess of 90 mmHg. An explanation sion. ACE 1/3 mice continue to maintain BP by a marked for the remarkable behavior of the ACE 1/3 mice is the eleva- increase of plasma renin activity. tion of plasma renin levels (Figure 4B). These data show that, under basal conditions, the ACE 1/3 mice have an elevated renin level as compared with wild-type mice (it is this that generation of the peptide (25,26). Examining data from all of maintains normal BP). In response to total salt deprivation, the our models gives insight into this controversy. First, in both RAS is activated and these mice respond with much higher humans and mice, the majority of ACE activity is physically plasma renin levels than control animals. Thus, the ACE 1/3 associated with endothelium and other tissues such as tubular mice dramatically demonstrate that, given a normal kidney and epithelium in the kidney. Tissue ACE is such a large percentage its compensatory ability, animals can tolerate a marked change of the total body load that it is responsible for the majority of in both the quantity and the tissue expression patterns of ACE. the conversion of angiotensin I to angiotensin II. However, total Under conditions of salt deprivation, the ACE 1/3 mice main- ACE levels are not in great excess, as indicated by ACE wt/1 tain substantial homeostasis; it is only under extreme condi- mice with roughly 60% wild-type ACE levels. Even this rela- tions that the selective lack of ACE expression by endothelium tively modest reduction of ACE necessitates increased angio- and within the kidney seems at all deleterious. We end this tensin I production to maintain normal plasma levels of angio- portion of the discussion with a caveat that our experiments tensin II and normal BP (18). In fact, animals can easily were performed with young mice that were held under labo- maintain normal BP in the complete absence of all endothelial ratory conditions. Clearly, the human response to the chronic or renal expression of ACE. This is because, under basal con- injuries that are typical of old age are different paradigms. ditions, there is both a tissue-fixed and a circulating pool of Although the ACE 1/3 mice have not yet been tested in these ACE. As the endothelial generation of angiotensin II dimin- paradigms, they do underline the amazing ability of the RAS to ishes, the kidney is able to compensate through upregulation of compensate given normal renal function. In a sense, these data circulating renin, angiotensin I, and thus angiotensin II gener- support the idea that abnormalities in human BP must be ation. Thus, our view of the controversy of local versus systemic associated with some type of renal dysfunction. 588 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 583–591, 2005

Renal Angiotensin II will provide insight into the local, renal generation of angio- Mice with altered tissue patterns of ACE expression provide tensin II and the renal and systemic effects of this peptide. insight into the organ-specific generation and function of an- My group prepared three mouse lines that were engineered giotensin peptides. In fact, an evaluation of angiotensin peptide such that control of the ACE gene was under promoters that levels was recently performed in mice that genetically lack ACE were previously described as specifically active in the kidney. and in wild-type mice that were exposed to the ACE inhibitor These are the ␥ glutamyl transpeptidase (␥-GT), the kidney lisinopril (27). These data proved that, in the mouse, ACE is the androgen regulated protein (KAP), and the Tamm-Horsfall predominant pathway leading to angiotensin II formation. ACE promoters. Although published literature supported the use of is responsible for at least 90% of the conversion of angiotensin these promoters in targeting gene expression to the kidney, our I to angiotensin II in the blood, kidney, heart, lung, and brain experience was very different (30–35). The KAP and Tamm- and at least 77% in the adrenal. In fact, evaluation of angioten- Horsfall promoters were inactive and gave rise to mice that sin II peptide levels in the kidneys of ACE null mice showed were similar in BP and renal structure to the ACE null mice that this peptide was reduced by Ͼ97% as compared with previously discussed in this article. Even an ACE 8/Tamm- wild-type mice. These data suggest that, at least in the mouse, Horsfall heterozygous animal, in which the “8” allele targets chymase-like enzymes do not play an important role in angio- ACE to the heart and ensures normal renal tubular develop- tensin II formation. These data also completely refute the study ment, lacked renal expression of ACE (data not published). The by Wei et al. (28) reporting similar angiotensin I and angioten- ␥-GT promoter did lead to small levels of ACE expression sin II levels in the organs of ACE knockout mice as compared within the kidney, but these levels were only approximately 6% with control animals. Angiotensin II is difficult to isolate under the normal ACE expression present in wild-type kidneys. The conditions that prevent the artificial conversion of angiotensin combination of low renal expression and the lack of ACE I (present in large amounts in ACE null mice) into angiotensin expression in other organs of the animal resulted in the ␥-GT II. Our group took particular care to homogenize freshly iso- mice having a low BP equivalent to that of ACE null animals lated organs in 4 mol/L guanidine thiocyanate to prevent this (data not published). artifact. Data from our group documenting markedly reduced In one manner, these mice were different from an animal tissue angiotensin II levels in ACE knockout mice seems very absolutely null for all ACE expression. These new strains of consistent with the striking reduction of BP measured by all mice continued to express the testis isoform of ACE. Because of groups who have studied ACE null mice (6,7). this, the mice were fully fertile, despite low BP. This compares An interesting aspect of the kidney is that wild-type mice to ACE null mice, lacking testis ACE, in which fertility is have renal levels of angiotensin II that are significantly higher markedly reduced in male mice. Our group now routinely uses than those found in blood. This has engendered the idea that mice that we term ACE 4/4 (homozygous for the mutation in renal angiotensin II levels are the result of de novo formation in which the KAP promoter controls ACE expression) as a substi- the kidney (29). Our data argue against de novo formation as tute for ACE 1/1 null mice (36). Nonetheless, the lack of reliable being the major source of renal angiotensin II. ACE 8/8 mice renal-specific promoters was a disappointment and a source of have no ACE in renal tissue, instead having ACE within the frustration for our group. The development of tools for reliably heart and the blood. Despite no renal ACE, the kidney concen- expressing proteins selectively within the kidney is important. tration of angiotensin II averaged 130 fmol/g. This was Ͼ16 The lack of reliable renal-specific promoters will hinder the times higher than the concentration observed in the plasma and genetic manipulation of mice and the physiologic evaluation of even exceeded the level found in the lung of a wild-type mouse. protein expression in the kidney. The high residual angiotensin II peptide concentrations present in renal tissue that totally lacks tissue-bound ACE suggests that ACE and the Heart a significant percentage of total renal angiotensin II peptide The role of ACE and angiotensin II within the heart has been levels must be due to absorption of the peptide from the blood. the subject of much discussion. Some authors have suggested that the local generation of angiotensin II within cardiac tissue may be deleterious, promoting pathologic development of car- ACE Selectively Expressed in Kidney diac fibrosis (37,38). To investigate this question, we made a Our group has worked hard to create a mouse with ACE mouse in which the cardiac-specific ␣-myosin heavy chain expression restricted to the kidney. It is our belief that the promoter was positioned to control the ACE gene (21). Our evaluation of this model or even mouse lines with ACE expres- hope was that this animal would make ACE selectively in sion limited to different portions of the nephron will be infor- cardiac tissue, thus focusing production of angiotensin II spe- mative as to the special role of the kidney in the maintenance of cifically within the heart. In fact, precisely this occurred in these BP and electrolyte balance. Although animal models previously mice called ACE 8/8. Whereas wild-type mice have virtually no discussed indicate that renal concentrating ability is retained in ACE expression by the myocardium, the ACE 8/8 animals have the absence of renal ACE, it is important to remember that these extensive ACE enzyme located on the surface of cardiac myo- models have other compensating sources of ACE. Wild-type cytes in both the atria and the ventricles. Ironically, vascular mice do contain considerable ACE in the brush border of the endothelium within the heart is completely absent of ACE proximal tubule and in renal vascular endothelium. Our hope expression. Although the ␣-myosin heavy chain promoter is is that animals with ACE expression restricted to the kidney often thought to be specific for the heart, we found that the J Am Soc Nephrol 16: 583–591, 2005 ACE and Genetically Modified Mice 589 promoter also induced an unusual expression pattern in the to our surprise, the ventricles of ACE 8/8 mice were in many lung, where some vascular smooth muscle expressed ACE. In ways normal. For example, histologic staining to identify col- addition, there was a patchy distribution of ACE within the lagen detected no increase of ventricular fibrosis. Both echocar- lung parenchyma with the result that the lungs of ACE 8/8 diography and intraventricular catheterization (with a Millar mice have approximately 40% normal ACE activity. In contrast catheter) failed to show any marked abnormalities of ventric- to the lung, ACE 8/8 mice totally lack renal expression of ACE; ular function despite greater than a fourfold increase of cardiac these animals are null for all renal epithelial and vascular angiotensin II. Thus, this model questions the concept of an expression. Indeed, endothelium throughout the animal pro- intrinsically deleterious effect of angiotensin II for myocardial duce no ACE. function. We carefully evaluated cardiac levels of angiotensin II. Our In contrast to the myocardial findings, the ACE 8/8 mice hope was that these animals would increase the cardiac con- developed atrial enlargement between 2 and 3 wk of age centration of angiotensin II, and, in fact, we found levels of the through mechanisms that are under intense study. Although peptide that were 4.3-fold greater than those of wild-type mice. work continues to help us understand the phenotype of these Although the BP of the mice was near normal and renal con- mice, the findings in the ACE.8 model underscore that genetic centrating ability was indistinguishable from wild-type mice, manipulation of mice is a powerful method to test accepted the ACE 8/8 animals definitely were not normal; the death rate concepts of pathology and disease etiology. For example, there of the mice was markedly increased such that only 64% of the is an extensive literature implicating angiotensin II as proin- animals were alive 66 d after birth (as opposed to 100% survival flammatory for disease processes such as vascular injury and for wild-type mice). The increased mortality in this model was even atherosclerosis (2,39). Our group is preparing mice that the result of two unusual findings: A marked increase in size of overexpress ACE by macrophages. Our hope is to use this and the atria and marked cardiac electrical abnormalities character- other mouse models to quantify precisely the role of angioten- ized by low voltage and atrial fibrillation (Figure 5). However, sin II in vascular injury.

Conclusion Our group has created mouse models in which ACE expression is limited to certain small subsets of tissue. This was in response to the realization that a total ACE null mouse represents an extreme phenotype that, although very interesting, is intrinsically limited. Our new mouse models are examples of an approach to manipulate gene promoters and restrict/re- arrange protein expression. Some of what we found was con- trary to previously held concepts of tissue-specific RAS expression. This emphasizes the power of our approach and of genetic manipulation of the mouse as a tool to dissect the complex role of the RAS. We hope that these mouse models will be a resource for many other laboratories interested in the physiology and pathology of renal and cardiac disease.

Acknowledgments This work was supported by grants from the National Institutes of Health (DK39777, DK51445, and DK55503). H.D.X. is supported by a fellowship from the Georgia affiliate of the National Kidney Founda- tion. K.F. is supported by a postdoctoral fellowship from the National Institutes of Health (F32 DK65410). S.F. is supported by a postdoctoral fellowship from the National Kidney Foundation. We acknowledge the help of Dr. Mario Capecchi, Department of Genetics, University of Utah, and Dr. Pierre Corvol, College de France, Paris, France.

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