Maintenance of Normal Blood Pressure and Renal Functions Are Independent Effects of Angiotensin-converting Enzyme*

Sean P. Kessler {ddagger}, Preenie deS. Senanayake §, Thomas S. Scheidemantel {ddagger}, Janette B. Gomos {ddagger}, Theresa M. Rowe {ddagger} and Ganes C. Sen {ddagger} 

From the Departments of {ddagger}Molecular Biology, Lerner Research Institute and §Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, March 6, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin-converting enzyme (ACE) is expressed in many tissues, including vasculature and renal proximal tubules, and its genetic ablation in mice causes abnormal renal structure and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by its expression in specific tissues, we generated different mouse strains, each expressing ACE in only one tissue. Here, we report the properties of two such strains of mice that express ACE either in vascular endothelial cells or in renal proximal tubules. Because of the natural cleavage secretion process, both groups also have ACE in the serum. Both groups were as healthy as wild-type mice, having normal kidney structure and fluid homeostasis, though males remained sterile, because they lack ACE expression in sperm. Despite equivalent serum ACE and angiotensin II levels and renal functions, only the group that expressed ACE in vascular endothelial cells had normal blood pressure. Expression of ACE, either in renal proximal tubules or in vasculature, is sufficient for maintaining normal kidney functions. However, for maintaining blood pressure, ACE must be expressed in vascular endothelial cells. These results also demonstrate that ACE-mediated blood pressure maintenance can be dissociated from its role in maintaining renal structure and functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin-converting enzyme (ACE)1 is one of several vital proteins that regulate hemodynamic homeostasis through the renin-angiotensin system (RAS) (1). The importance of each member, including ACE, in the RAS has been demonstrated by individual gene ablation via gene disruption technology. Angiotensinogen, AT1A and AT1B receptor, and ACE-deficient mice all share a common phenotype, namely, hypotension, arterial hypertrophy, interstitial fibrosis, renal atrophy, and growth retardation leading to decreased vigor and pre-mature mortality. All of these mutant mice fail to concentrate their urine (2, 3, 4, 5, 6).

ACE plays a pivotal role within the RAS in that it cleaves angiotensin I (Ang I) to produce angiotensin II (Ang II), the vasoactive peptide. ACE also inactivates bradykinin, a vasodilator peptide (7). Although therapeutic management of hypertension routinely includes a regimen of inhibitors prescribed to block ACE enzymatic activity, elimination of ACE production entirely leads to the aforementioned, severely abnormal phenotype. In fact, utilization of ACE inhibitors is contraindicated during pregnancy because of adverse development of fetal renal structures reminiscent of kidney abnormalities observed in Ace-/- mice (8). The conservation of ACE and ACE-like proteins from Drosophila to mammals and expression in diverse tissue types within any one organism makes it clear that the relevance of ACE extends beyond a mere physiological importance to that of physiological prerequisite for survival of a species (9, 10).

The ACE gene encodes two structurally related isozymes, somatic ACE and germinal ACE, that are expressed in specific cell types because of alternate choice of transcription initiation and alternate splicing patterns (11, 12, 13). Both the 140-kDa somatic ACE (sACE) and 70-kDa germinal (gACE) isoforms possess unique N-terminal domains yet share identical C-terminal domains that anchor these type I ectoproteins in the plasma membrane (14, 15, 16, 17). A plasma soluble form of the sACE protein is also produced by the regulated action of a membrane-associated cleavage-secretion process (18, 19, 20, 21). There is a 67% identity between the sACE N-domain and C-domains, inclusive of their common zinc-binding (His-Glu-X-X-His) active site motifs (22, 23). This accounts for the fact that the N-domain of sACE and the identical C-domains of sACE and gACE all cleave Ang I to produce Ang II (16, 24, 25). In vitro assays have indicated that there are domain-distinct and therefore isoform-specific substrate preferences for both ACE enzymes. The sACE N-domain cleaves LHRH 30 times faster and the hematopoietic peptide NH2-acetyl-Ser-Gly-Lys-Pro (AcSDKP) 40 times faster than the C-terminal active site (26, 27).

The full repertoire of physiological functions of ACE has been revealed by examining the defects of Ace-/- mice (3, 4, 28). In addition to suffering from low blood pressure, the male Ace-/- mice are sterile. Although there is no defect in sperm number, morphology, or motility, Ace-/- males sire no or a very small number of pups. Sperm lacking ACE are defective in transport within the oviduct and in binding to zonae pellucidae (29). However, the relevant substrate for ACE activity in sperm action has not been identified yet. ACE also has a role in erythropoiesis. The null mice have normocytic anemia associated with elevated plasma erythropoietin levels (30). The renal defects of Ace-/- mice are manifested both structurally and functionally. Cortical thinning, focal areas of atrophy, renal vascular changes, and localized tubular obstructions are present in the kidneys of these mice. They are defective in concentrating urine with a higher urine output of a lower osmolality (4). Because of the lack of Ang II production by ACE, these mice lack tubuloglomerular feedback response, as well (31).

The locations of sACE and gACE expression in the body are distinctly different. Somatic ACE is expressed in vascular endothelial cells, kidney proximal tubules, intestinal brush border cells, macrophages, monocytes, and Leydig cells of the testes (7, 32, 33). Germinal ACE is expressed exclusively in maturing sperm cells (34). We have been examining the specific physiological roles played by ACE expressed in specific tissues. For this purpose, we generated new strains of mice in which expression of transgenic ACE was driven by tissue-specific transcriptional promoters. These mice were interbred with Ace-/- mice to produce experimental mice whose physiological characterization causally connected ACE expression in one tissue with restoration of a specific Ace-/- deficiency. Using this approach, we demonstrated previously (35) that transgenic gACE expression in maturing sperm alone restores male fertility without curing other problems of Ace-/- mice. However, sACE cannot substitute for gACE, demonstrating that the two isozymes are not interchangeable for fertility functions (36). In another transgenic line with ectopic expression of germinal ACE in the serum, normal health and renal functions were restored without concomitant restoration of blood pressure (37).

In the current study, we have generated one mouse strain that expresses transgenic sACE in vascular endothelial cells and a second mouse strain that expresses transgenic sACE in renal proximal tubule cells. In addition to tissue-bound sACE in the targeted tissue, both groups also have transgenic sACE in the serum. In direct contrast to ACE knockout mice, both corresponding experimental transgenic mice of Ace-/- background were as healthy as wild-type mice. Both transgenic strains exhibited normal renal structure and function, though all male mice remained sterile. Despite wild-type levels of transgenic sACE and high levels of Ang II in the serum of both groups, only the mice expressing sACE in the vascular endothelial cells had normal blood pressure.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgene Construction—The 808-bp mouse receptor tyrosine kinase (Tie-1) promoter (38) was cloned by PCR using the "mousetieS" sense primer, 5'gggcaagctttcttaagacatgcaactcg 3', and the "mousetieAS" antisense primer, 5'gggggatccgggcccggggtcagttgc 3', into the pGEM3 vector. The 433-bp mouse {gamma}-glutamyl transpeptidase (Ggt) promoter (39) was cloned by PCR using the "mouseggtS" sense primer, 5'gggcaagcttagatctaagctatgtctagtgc 3', and the "mouseggtAS" antisense primer, 5'gggggatccggcaagaggtcagctaagg 3', into the pGEM 3 vector. Both sequences were confirmed by Cleveland Genomics and tested for function by cloning 5' to the luciferase gene in the pGL2 Basic vector (Promega). Luciferase activity, following transfection into HT1080 and opossum kidney cells, was performed as described (36). The Tie-sACE construct (plasmid AP008) and the Ggt-sACE construct (plasmid AP007) were created by replacing the phosphoglycerate kinase-2 (PGK2) promoter in the PGK2-somatic ACE-BGHpA construct (36) with the functional Tie-1 or Ggt promoters. The 5582-bp Tie-sACE-BGHpA (Ts) transgene and the 5207-bp Ggt-sACE-BGHpA (Gs) transgene were released from plasmids AP008 and AP007, respectively, by SpeI and AsnI digestion. The transgenes were purified from agarose gel using a Gene Clean system (Bio101) prior to microinjection into FVB zygotes by the Cleveland Clinic Foundation Transgenic Core Facility utilizing standard techniques.

Southern Blot Hybridization—Southern blot genotyping was performed as described previously (36). Heterozygosity or homozygosity of the transgene was determined by normalizing the transgene Imagequant value to the endogenous mouse Ace gene value in the same genomic DNA sample. The endogenous Ace genotype is determined by the presence of a wild-type 6.4-kB SacI genomic fragment or the disrupted 8.4-kB SacI (Ace null) genomic fragment (3, 36).

ACE Enzyme Assay—The standard ACE enzyme assay, which measures ACE cleavage of the Ang I analog Hip-His-Leu, was performed by incubating 50 µg of total protein extract from Ace+/+, Ace-/-, and experimental Ace-/-, Ts+/+, and Ace-/-, Gs+/- adult mouse kidney or serum. All tissues were homogenized in ACE lysis buffer as described previously (16, 36). All tissues originated from five age-matched FVB strain adult mice.

Angiotensin II and Angiotensin I Measurements—For each genotype, the blood from four adult mice of the same sex and age were pooled to achieve a 1-ml plasma sample. Duplicate plasma pools from each genotype were extracted for the measurement of Ang II and Ang I levels as described previously (40). The results are the average of duplicate pools ± S.E.

Histology and Immunohistochemistry—Age-matched, adult organs were paraffin embedded, cross-sectioned at 5-µm thickness, and hematoxylin- and eosin-stained by the Histology Core (Lerner Research Institute, Cleveland, OH). Immunohistochemistry was performed following de-paraffinization by dipping in the following solutions (Richard-Allen): (1 x 5 min) Clear-Rite; (2 x 3 min) Clear-Rite; (2 x 1 min) 100% Flex; (1 x 1 min) 95% Flex; (1 x 1 min) 80% Flex; (1 x 1 min) H2O; (1 x 5 min) phosphate-buffered saline (PBS). Slides were incubated in 10 mM sodium citrate, pH 6.0, for 30 min at 25 °C and then returned to PBS. The slides were blocked for 2 h at 25 °C in PBS + 10% horse serum + 0.3% Triton X-100 (blocking buffer). The anti-rabbit sACE antibody (20), which weakly binds with mouse ACE in this procedure, diluted 1:1000 in blocking buffer was applied to the slides in a humid chamber for 16 h at 4 C. Following washes in PBS + 0.3% Triton X-100 (PBST), anti-goat-fluorescein isothiocyanate (Santa Cruz Biotechnology, Inc.) was applied to each section for 2 h in the dark at 25 °C. Following washes in PBST, VectaShield (Vector Laboratories) diluted 1:1 in PBS was applied. All stained slides were visualized with a Leica digital fluorescent microscope and Adobe Photoshop software.

Establishment of Transgenic Lines and Male Fertility Tests—Adult FVB Ts transgenic founder mice (Ace+/+, Ts+/-) and Gs transgenic founder mice (Ace+/+, Gs+/-) were mated with Ace+/- FVB male mice to generate Ace+/-, Ts+/-, and Ace+/-, Gs+/- mice. Male Ace+/- FVB strain mice were generated by back-crossing the Ace null allele, originally developed in c57Bl/6 strain mice (3), for ten generations with Ace+/+ FVB female mice (36). Interbreeding between male and female Ace+/-, Ts+/- mice was performed to generate the Ace-/-, Ts+/+ experimental mice. Interbreeding between Ace+/-, Gs+/- male and female mice was performed to generate Ace-/-, Gs+/- experimental mice. Genotyping of all mice was performed by Southern blotting as described above.

For fertility comparison, the number of pups sired from each mating above was noted. Fertility testing of all experimental adult males was then conducted by mating three Ace-/- Ts+/- males, three Ace-/- Ts+/+ males, three Ace+/+, Ts+/+ males, three Ace-/-, Gs+/- line E males, and Ace-/-, Gs+/- line 3200 males with a total of six wild-type adult FVB strain females (Jackson Laboratories) for 10 days, the equivalent to two complete estrous cycles. Each mating consisted of two females per male. Females were observed for plugs. If no pups were produced within 22 days from experimental male removal, the same females were mated with adult Ace+/+ FVB males for 10 days. The number of pups per litter was noted. As a control, the previously described Ace-/-, PGK2-gACE adult male FVB mice, which express gACE on their sperm alone, were mated with wild-type FVB mice, and the number of pups sired was also recorded.

Water Uptake, Urine Output, and Osmolality Measurement—Age-matched, adult mice of the following genotypes (Ace+/+, Ace-/-, Ace-/-, Ts+/+, Ace-/-, Gs+/- line E, and Ace-/-, Gs+/- line 3200) were individually placed in a Nalgene metabolic cage supplied with powdered standard chow and water ad libitum. Daily (24 h) water consumption and urine produced was measured for 5 consecutive days for each mouse. Urine osmolality was measured for each mouse using the Osmette A (Precision Instruments, Inc., Natick, MA) freezing point osmometer according to the manufacturers instructions. Triplicate readings were performed on the urine collected from five mice of each genotype to determine an average osmolality value.

Blood Pressure Measurement—The non-invasive computerized RTBP007 Tail cuff blood pressure system for mice (Harvard Apparatus, Holliston, MA) was used to obtain systolic blood pressure (37). The mice were housed separately, fed autoclaved chow with water ad libitum, and maintained on a 12-hour light/dark cycle. Each adult mouse was trained for 4 days to acclimate them to the apparatus and restraint. Training included handling the mice, warming them to 30 °C, and restraining in a darkened restraint. Unrecorded measurements were taken on day two and day three of training. All measurements and training was performed on consecutive days between 12:00 and 3:00 p.m. each day. Computer-recorded measurements were then taken for 3–5 consecutive days following training. A minimum of 10 blood pressure readings per mouse per day were used to calculate the average daily blood pressure for each FVB mouse. The average blood pressure for each mouse was then calculated by averaging the daily blood pressure of each mouse over the 3 to 5 consecutive days of readings. Final mean blood pressure for each genotype was calculated on a minimum of two mice (Ace-/-, Gs+/- line 3200 females) and a maximum of 14 mice (Ace-/-, Ts+/+ females).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Experimental Mice—The experimental mice were generated by crossing Ace+/- mice with the appropriate transgenic mice. All mice were of the FVB strain to which the Ace null genotype had been back-crossed by us for at least ten generations from the Ace-/- C57Bl/6 mice obtained from the Smithies Laboratory (3). The transgene consisted of rabbit sACE cDNA, a polyadenylation and splicing cassette from the bovine growth hormone gene, and either the 808-bp murine Tie-1 promoter or the 433-bp murine Ggt promoter, which were shown previously (36, 38, 39) to direct expression to the vascular endothelial cells or renal proximal tubule cells, respectively. Both promoters were cloned by PCR using published sequences. The Ts transgene and the Gs transgene were assembled as shown in Fig. 1A and tested in vitro for transgene expression by coupled transcription-translation and by transfecting into opossum kidney cells. ACE activity assay of extracts prepared from transfected cells confirmed synthesis of the sACE protein from both promoters (data not shown). The SpeI-AsnI fragments of the Ts and Gs transgenes (Fig. 1A) were independently microinjected into pronuclei of FVB zygotes and then implanted in the uteri of pseudopregnant mothers. Tail DNA of resulting pups was digested with SacI and analyzed by Southern blotting using a 405-bp rabbit sACE cDNA fragment as the probe (shown as Southern probe in Fig. 1A). Four lines carried the Ts transgene and five lines carried the Gs transgene (Fig. 1B). Founder mice were crossed with Ace+/- FVB mice, and the progenies were genotyped by Southern blot analysis. All of the lines transmitted the transgene to their progenies. Thus, four transgenic Ace+/-, Ts+/- lines (I, J, K, and L) were established, and five transgenic Ace+/-, Gs+/- lines (E, F, G, H, and 3200) were established.



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FIG. 1.
Generation of experimental mice. The Ts, Gs, and rabbit sACE cDNA Southern probe (black bar) were designed as shown in A. The four FVB strain founder Ts mice, designated as lines I, J, K, and L and the five FVB founder Gs mice, designated as lines E, F, G, H, and 3200 in B were each mated with an FVB Ace+/- mouse to transmit their transgene to their progeny. C, Western blot with anti-rabbit sACE showing sACE expression in lung of Ts lines and kidney of Gs lines. D, Southern blot genotype of Ace-/-, Ts+/+ and Ace-/-, Gs+/- experimental mice. In all Southern blot genotypes shown, the genomic DNA cut with SacI yields a 3.7-kB transgenic band, a 6.6-kB native Ace allelic band, and an 8.4-kB disrupted Ace allelic band.

 

Because the Ts transgene was expected to be expressed in vascular endothelial cells, we first examined its expression in the lung, a highly vascularized tissue and the richest source of natural sACE. Western blotting of lung extracts of the four transgenic lines with our anti-(rabbit sACE)-specific antibody revealed that only line I, but not J, K, and L, expressed large quantities of transgenic sACE, though line L expressed a larger cross-reactive protein (Fig. 1C). Transgenic expression was also detected in heart, kidney, liver, and serum (data not shown). In like manner, the Gs transgene was expected to be expressed in kidney. Western blotting of kidney extracts showed that only line E and line 3200 mice expressed transgenic sACE in the kidney (Fig. 1C). Lines F, G, and H did not express the Gs transgene in kidney or in any other tissue. Because lines I, E, and 3200 expressed the correct molecular weight, enzymatically active sACE in the organs tested, mice heterozygous for both the Ace allele and the Ts or Gs transgenic allele in these lines were interbred to produce the experimental Ace-/-, Ts+/+ or Ace-/-, Gs+/- mice that were utilized for the remainder of this study. No Gs+/+ line could be produced with either Wt or Ace-/- background, but one copy of the transgene in the Gs+/- mice was sufficient to produce enough ACE. In this Southern blot analysis, the transgene produced a 3.7-kB fragment, the resident Ace gene produced a 6.6-kB fragment, and the disrupted ACE allele produced an 8.4-kB fragment. The probe recognized both the rabbit transgene and the endogenous mouse Ace gene (Fig. 1D).

Expression Profile of the Transgene—To further characterize the experimental mice, ACE enzymatic activities were measured in the kidney and serum. Though the level of ACE activity in the experimental Gs line E kidney was 5-fold greater than Gs line 3200 and slightly greater than that observed in the Ts line kidney, all three groups had significantly lower renal ACE activity when compared with wild-type mice (Fig. 2A). On the other hand, the serum ACE levels of the experimental Ts and Gs line E mice were higher than that observed in the Ace+/+ mice (Fig. 2B). Line 3200 had 17% of wild-type levels of sACE in the serum (Fig. 2B).



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FIG. 2.
ACE activity assay. Age-matched, adult kidneys (A) and serum (B) were prepared from Ace+/+, Ace-/-, Ts+/+, and Ace-/-, Gs+/- mice as described under "Experimental Procedures." Kidney protein extract (12.5 µg) or serum (1 µl) was assayed for ACE enzyme activity, measured as nanomoles of His-Leu liberated from the Ang I analog Hip-His-Leu per min, in a Beckman spectrofluorometer. Each bar represents the average value from duplicate samples from five mice of the same genotype. Error bars indicate the standard deviation between the individual data points.

 

To determine whether the ACE in the serum of the experimental mice could produce Ang II, the level of both Ang I and Ang II was measured in Ace+/+, Ace-/- and experimental mice. The data presented in Fig. 3, A and B are the averages of the measurements obtained from pooled samples ± S.E. The Ang II values for the Ace+/+ and Ace-/- FVB mice were comparable with those reported for other strains of mice (30). Although the Ang II levels in all three strains of experimental mice were above that of Ace-/- mice, the Ang II level was still less than the level observed in Ace+/+ mice. The experimental mice with Ang II levels closest to wild-type levels were the Gs line E group (Fig. 3A). The Ang I levels were complementary to the Ang II levels; the mice with higher Ang II level had a lower Ang I level (Fig. 3B). There was no significant difference in Ang I or Ang II levels between sexes of the same genotype.



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FIG. 3.
Plasma Ang II and Ang I measurements. One ml of plasma pooled from four adult mice of the same age, sex, and genotype was assayed for Ang II (panel A) and Ang I (panel B) levels as described under "Experimental Procedures." Each data point is the average of duplicate measurements (pg/ml plasma) from two independent pools (±S.E.).

 

For identifying the transgene-expressing cell type in a tissue, immunohistochemistry was performed on thin sections using an anti-rabbit sACE antibody. Because this antibody also recognizes mouse ACE, albeit weakly, in immunohistochemistry assays, all analyses were performed with organs from transgenic Ace-/- mice. As shown in Fig. 4A, the Ts transgene was expressed in brain, heart, liver, lung, kidney, and testes. In all tissues, expression was restricted to the vascular endothelial cell lining the lumen of blood vessels. Expression of sACE in the Gs (line E) mice occurred in the renal proximal tubule S1 region immediately adjacent to the Bowman's capsule (Fig. 4B) whereas expression of sACE in the Gs (line 3200) was in the S2-S3 region of the proximal tubule, which is the natural location of sACE expression (Fig. 4B). The proximal tubules and blood vessels of the Ace-/- mice were devoid of ACE protein (Fig. 4B).



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FIG. 4.
Immunohistochemistry of ACE expression. Organs were removed from Ace-/-, Ts+/+ adult mice (panel A), paraffin-embedded and sectioned at 3-µm thickness. Following paraffin removal, slides were probed with anti-rabbit sACE antiserum and anti-goat fluorescein isothiocyanate. Slides were mounted with VectaShield and viewed with a Leica fluorescent microscope at x40 magnification. The vessel luminal walls in all organs appear brightly stained. Panel B shows wild-type (Ace+/+), Ace-/-, and Ace-/-, Gs+/- (lines E and 3200) adult kidney sections prepared as in panel A except they are at x20 magnification. The proximal tubule (PT) cells in Ace+/+ and experimental Gs mice are brightly stained whereas the glomerulus (G) is unstained.

 

We paid special attention to the expression profile of the transgene in the blood vessels as shown in Fig. 5. Expression of the transgene in the Ts experimental mice was restricted to the vascular endothelial cells (Fig. 5B, e). There was no expression in the other vascular cell types or in epithelial cells of the proximal tubules in the Ts mice. The vessels of line 3200 mice were devoid of any sACE protein (Fig. 5C). However, the Gs (line E) mice expressed sACE in the adventitia and media layers of the blood vessel (Fig. 5D, A and M). However, no signal was detected in the vascular endothelial cells (Fig. 5D, e) of the Gs (line E) mice. These results clearly demonstrated the non-overlapping cell type-specific expression pattern of sACE in the Ts and Gs mice. In addition, the immunohistochemical staining of wild-type, Ts, and Gs kidneys supports the observed renal ACE activity level in each strain (see Fig. 2A).



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FIG. 5.
Immunohistochemistry of blood vessels. Age-matched, adult kidneys from wild-type Ace+/+ (panel A), experimental Ace-/-, Ts+/+ (panel B), experimental Ace-/-, Gs+/- line 3200 (panel C), and experimental Ace-/-, Gs+/- line E (panel D) mice were prepared and stained as described for Fig. 4. The blood vessel lumen (L), the endothelium (e), the media (M), and the adventitia (A) are noted with arrows.

 

Physiological Properties of the Experimental Mice—Ace-/- FVB mice have poor general health. Fewer than the expected number of pups of this genotype are born, and they are runted in the early weeks of their lives. Only 13% live by the end of 4 weeks of age, and all die by the age of 30–35 weeks. In all experimental transgenic Ace-/- mice, the above defects were corrected. Their longevity and general health were indistinguishable from those of the wild-type mice.

A major phenotype of Ace-/- mice is male sterility (3, 4, 35). This phenotype was not corrected in the experimental male mice. The average number of pups born per litter of Ace+/+ mice was nine, whereas, no pups were sired by the Ace-/- male mice (Fig. 6). Although transgenic expression of sACE in the vasculature of Ts or Gs Ace+/+ male mice did not adversely affect fertility, it did not rescue Ace-/- male fertility. In contrast, as reported previously (36), expression of transgenic gACE in the sperm of Ace-/- FVB mice restored its fertility (Fig. 6).



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FIG. 6.
Fertility assessment of experimental mice. Three adult male FVB mice of the indicated genotype were mated with six FVB wild-type females, and the number of progeny per litter is noted (•). The reported average litter size for wild-type (Ace+/+) FVB mice is annotated as {diamondsuit}. The number of matings when no pups were produced is noted as n. Pg, PGK2-gACE-BGHpA transgene.

 

Kidneys of Ace-/- FVB mice have dramatic structural defects; the kidneys of older mice accumulate large amounts of fluid and are anatomically enlarged. Internally, the cortex becomes thinner, and a large cavity appears in the center (Fig. 7B). This is accompanied by a thickening of the arterial vessel wall (Fig. 7B, V). The structural defects were completely cured in all experimental mouse strains. Their kidneys were similar to those of Wt mice, and their arterial walls were not hyperplastic (Fig. 7, compare C, D, and E with A).



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FIG. 7.
Renal histology. Hematoxylin- and eosin-stained slides from 2-month-old kidneys of Ace+/+ mice reveal normal structure and vessel thickness (arrow + V) in panel A. Panel B shows the perforation and thickened renal blood vessel in the Ace-/- mice. The absence of the kidney perforation and restoration of normal renal vessel thickness are shown in the experimental Ace-/-, Ts+/+ (panel C), Ace-/-, Gs+/- line 3200 (panel D), and Ace-/-, Gs+/- line E (panel E). All kidneys were isolated from age-matched adult FVB strain mice and photographed at x0.6 (whole cross-section) or x40 (blood vessel) with a digital Leica light microscope.

 

Restoration of normal kidney structure by transgenic expression in the Ace-/- mice is paralleled by corresponding restoration of fluid homeostasis. Water uptake was more than doubled (Fig. 8B), and urine output was six times higher (Fig. 8A) in Ace-/- mice as compared with the Wt mice. In the experimental mice, both parameters were restored to normal (Fig. 8, A and B). Urine osmolality, which is decreased by about 63% in the Ace-/- mice, was similarly restored to normal levels in the Ts and Gs experimental mice (data not shown). These results demonstrated that transgenic expression of sACE, not only in the proximal tubule of the Gs mice, but also in the vasculature of the Ts mice, without a concomitant expression in the proximal tubules, is sufficient for maintaining normal kidney structure and function.



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FIG. 8.
Renal function analysis. Urine output produced in a 24-h period from Ace+/+, Ace-/-, Ace-/-, Ts+/+, and Ace-/-, Gs+/- FVB mice is reported in milliliters in panel A. Volume of water, in milliliters, consumed during the same 24-h period is noted in B. The average volume from a minimum of five age-matched adult mice over a period of 5 days is represented by each bar.

 

The most well recognized physiological role of ACE is in maintaining normal blood pressure. To examine the effects of transgenic expression on this parameter, we used the computerized tail-cuff method (41) for measuring systolic blood pressures of wild-type, null, and experimental mice. As shown in Fig. 9, male and female mice of the FVB strain have very similar blood pressure levels. Loss of ACE expression in the Ace-/- mice caused a considerable drop in the blood pressure in both sexes, although the drop was slightly greater in female mice. Most importantly, blood pressures returned to normal levels in the experimental Ts male and female mice. Surprisingly, this was not the case for the line E or line 3200 Gs male and female mice, thus demonstrating a major difference in the phenotypes of the Ts and the Gs transgenic mice.



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FIG. 9.
Systolic blood pressure measurements. The non-invasive, computerized tail cuff method was used to determine the systolic blood pressure of adult male and female Ace+/+, Ace-/-, Ace-/-, Ts+/+, and Ace-/-, Gs+/- FVB strain mice. The average blood pressure (in mm Hg) for each genotype, separated by sex, was calculated on the average daily blood pressure over a 3–5-day reading period for the number of mice, indicated as n. A minimum of ten readings per day was used to calculate the average daily blood pressure. Error bars reflect the S.D. blood pressures of all of the mice within a genotype group. The mean blood pressures were as follows: Ace+/+, male (M) = 115.8 and female (F) = 116.3; Ace-/-, M = 91.3 and F = 87.5; Ace-/-, Ts+/+, M = 116.2 and F = 114.0; Ace-/-, Gs+/- line E, M = 90.2 and F = 87.8; and Ace-/-, Gs+/- line 3200, M = 88.0 and F = 81.0.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We are interested in determining the physiological need for ACE expression in multiple tissues of the body. Such a need is not obvious if the primary or the sole function of ACE is to regulate blood pressure by producing Ang II in the circulation. However, the observation that Ace-/- mice have many defects, in addition to low blood pressure, suggested that local actions of ACE in many tissues may be required for normal health (3, 4, 42). Moreover, because some of these defects, such as anemia and male sterility, are not manifested in mice lacking other components of the RAS, ACE must mediate some physiological functions by cleaving substrates other than Ang I (2, 30). The above considerations provided the impetus for studying tissue-specific functions of ACE. Another aspect of ACE, namely, the evolutionary conservation of the structurally related isozymes with similar enzymatic activity, presents an intriguing question regarding their physiological equivalence (9, 10).

To address these issues, we have been generating different strains of genetically modified mice, designed to express only one isozyme of ACE in only one tissue. For this purpose, first a transgenic mouse strain is produced in which a tissue-specific promoter drives the expression of the ACE isozyme in only one tissue. This mouse is then interbred with Ace+/- mice to produce the experimental mice. Because many relevant properties of the mouse, such as blood pressure, litter size, and renal function, vary considerably among different strains (43), it was imperative that all of our studies are restricted to one pure strain of mouse. For the sake of experimental convenience, we chose the FVB strain. This strain is hardy, and the animals are larger than some other strains. Their litter size is high, and it is convenient to produce transgenic mice of this strain. To use this strain, we needed to transfer the Ace-/- genotype to this strain, as well. This was achieved by repeated back-crossing of a C57Bl/6 Ace+/- mouse with FVB mice.

Using the above approach, we have demonstrated previously (35) that expression of gACE in spermatocytes is sufficient for restoring male fertility of Ace-/- mice. As anticipated, those mice, however, still suffer from kidney problems and low blood pressure because of the lack of ACE expression in somatic tissues. In another study, we expressed enzymatically active sACE, instead of gACE, in sperm. Surprisingly, these male mice were sterile demonstrating that sACE is non-functional in the context of male reproduction, even when expressed in the relevant cell type (36). In still another study, we generated mice that exhibited high levels of germinal ACE in the serum in addition to the sperm. These mice were as healthy as wild-type mice, demonstrating normal renal structure and function without concomitant restoration of normal blood pressure (37).

In the current study, we have developed two different strains of transgenic mice, designed to express sACE exclusively in the vascular endothelial cells (Ts mice) or the renal proximal tubule cells (Gs mice). The promoters used for this study came from the Tie-1 gene and the Ggt gene of the mouse. Natural expression of Tie is restricted to the large and small blood vessels, and in previous transgenic studies, this promoter has been shown to direct expression of transgenes to vascular endothelial cells (38). In a like manner, the Ggt promoter was selected, because it was shown previously (39) to drive a reporter gene exclusively in renal proximal tubule epithelial cells of a mouse.

In our experimental Ts mice, transgenic sACE was expressed in many tissues, but the expression was totally localized to the vascular endothelium (see Fig. 4A and Fig. 5B). Most notable was the distinct expression profile of the transgene in the kidney. Unlike the expression of the endogenous ACE in both the vascular endothelial (Fig. 5A) and proximal tubular cells (Fig. 4B) of Wt kidney, expression of the transgene in the Ts mice was restricted to the blood vessel endothelial cells (Fig. 5B). The Gs line 3200 experimental group expressed sACE as expected. Somatic ACE expression was restricted to the brush border of renal proximal tubules (Fig. 4B) and not in the vasculature (Fig. 5C). However, the expression pattern in the experimental Gs line E was very different from what was expected. Though sACE was expressed in intracellular renal proximal tubule epithelial cells, the location was restricted to the S1 region, which is more proximal to the Bowman's capsule than that which is found in Wt mice (Fig. 4B). This group also showed expression in the adventitia and media layers of blood vessels, but no expression was observed in the endothelial cell (Fig. 5D). This group also had greater than Wt levels of sACE in their serum (Fig. 2B), which, though unexpected, proved very useful for this comparative study.

Three major physiological effects of ACE were monitored in the Ts and Gs experimental mice: male fertility, kidney structure, and function and blood pressure. As anticipated, the experimental Ts and Gs male mice were sterile despite sACE expression in serum and Ts strain testis vasculature (Fig. 4A). This observation further demonstrated the total dissociation of the somatic functions of sACE from the reproductive functions of gACE. In the FVB strain, absence of ACE expression lowered the blood pressure of the females more than the males, but the blood pressures of both sexes of the Ts experimental mice were in the normal range (Fig. 9). Thus, the presence of sACE in the vascular endothelium was sufficient for restoring the blood pressure defect. It should be pointed out, however, that there was abundant ACE in the serum of the Ts experimental mice, as well (Fig. 2B). Soluble ACE present in the serum originates from cell-bound ACE by natural cleavage secretion (21). However, the line E Gs mouse strain also had serum ACE levels comparable with the Ts experimental group. This allowed us to distinguish between the functions of endothelium-associated ACE and serum ACE in this study. The blood pressure of the Gs line E mice remained as low as Ace-/- mice (Fig. 9), although their serum ACE activity was comparable with that of the Ts line and higher than the activity in Ace+/+ mice (Fig. 2B). This result correlated with our previous observation in our line 400 PGK2-germinal ACE and line 800 PGK2-germinal ACE mouse strains expressing transgenic germinal ACE, both of which had low blood pressure despite high serum gACE activity, and normal renal structure and function (37). It is also worth noting that low level sACE expression in the renal proximal tubules of Gs line 3200 mice (Fig. 2A) or in the serum (Fig. 2B) was sufficient to restore renal structure and function (see Fig. 7D and Fig. 8A, respectively).

One of the most interesting conclusions resulting from this study is the dissociation of blood pressure regulation and correct renal structure/functions. The gross structural defects of the Ace-/- kidney, including the thickening of the arterial wall, were cured by the presence of sACE in the serum (Ts mice). Thus, ACE expression in the proximal tubules does not appear to be critical for any of these physiological properties. Functionally, the fluid homeostasis of the experimental animals was normal; both the high water intake and the high urine outputs of the Ace-/- mice were lowered to normal levels in Ts and Gs mice. The lowered urine output was accompanied by an increase in the osmolality of urine, confirming the restoration of normal urine concentration function. Thus, it appears that ACE expression in the kidney proximal tubules is dispensable for maintaining normal kidney functions as long as ACE is present in the serum.

In contrast to the effects on kidney functions, maintenance of normal blood pressure in our experimental mice was strictly dependent on the location of ACE expression. Normal blood pressure was restored only in the Ts mice, but not the Gs mice, despite the fact that line E Gs mice had serum ACE levels similar to those of Ts mice (Fig. 2B). Our observation does not support the conclusions of a recent study (44) that serum ACE levels determine blood pressure. The reasons for the noted difference are not obvious. However, the strains of the mice used in the two studies were different, indicating that genetic backgrounds may play a role. Moreover, the experimental mice in the other study expressed an approximately 100-fold higher level of ACE in the liver as compared with the Wt mice. It was suggested that blood pressure was restored in their mice, because circulating Ang II levels were restored to Wt levels. The data presented in Fig. 3A indicate that the level of Ang II in the plasma does not correlate with blood pressure. The experimental Gs line E had higher Ang II levels than the experimental Ts mice; however, unlike the Ts mice, their blood pressure was low (Fig. 9). Our data indicate that the sACE-mediated production of Ang II on or near the vascular endothelial cells is more critical for correcting blood pressure than circulating Ang II levels. Our results also suggest that endothelial cell-bound ACE is critically important for maintaining blood pressure. Ang II produced by circulating sACE is not sufficient for this purpose.

The results presented here clearly showed that expression of ACE in the endothelial cells of the vasculature is critical for blood pressure maintenance. This need could not be satisfied in the Gs line E mice, even though ACE was expressed in other cell types of their vasculature but not in endothelial cells.


    FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant HL-48258 (to G. C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Molecular Biology, NC20, Lerner Research Inst., 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-0636; Fax: 216-444-0513; E-mail: seng{at}ccf.org.

1 The abbreviations used are: ACE, angiotensin-converting enzyme; sACE, somatic ACE; gACE, germinal Ace; RAS, renin angiotensin system; Ts, Tie-somatic ACE; Ggt, {gamma}-glutamyl transpeptidase; Gs, Ggt-somatic ACE; PGK2, phosphoglycerate kinase 2; Wt, wild-type; PT, proximal tubule; V, vessel; Ang I, angiotensin I; Ang II, angiotensin II; PBS, phosphate-buffered saline; BGHpA, bovine growth hormone polyA. Back


    ACKNOWLEDGMENTS
 
We thank Paulette Zavacky, David Young, Heather Smith, and John Boros for expert technical assistance and Valerie Stewart and Clemencia Colmenares for helpful discussions. We also thank Indira Sen and Kizhakkekara Santhamma for the anti-ACE antibody and assistance with ACE enzyme assays.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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