Maintenance of Normal Blood Pressure and Renal Functions Are Independent Effects of Angiotensin-converting Enzyme
2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês
10.1074/jbc.m302347200
ISSN1083-351X
AutoresSean P. Kessler, P. Senanayake, Thomas S. Scheidemantel, Janette Gomos, Theresa Rowe, Ganes C. Sen,
Tópico(s)Hormonal Regulation and Hypertension
ResumoAngiotensin-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. 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. Angiotensin-converting enzyme (ACE) 1The abbreviations used are: ACE, angiotensin-converting enzyme; sACE, somatic ACE; gACE, germinal Ace; RAS, renin angiotensin system; Ts, Tie-somatic ACE; Ggt, γ-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. is one of several vital proteins that regulate hemodynamic homeostasis through the renin-angiotensin system (RAS) (1Soffer R.L. Soffer R.L. Biochemical Regulation of Blood Pressure. Wiley-Interscience, New York1981: 123-164Google Scholar). 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 (2Kim H.S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (582) Google Scholar, 3Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar, 4Esther Jr., C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Invest. 1996; 74: 953-965PubMed Google Scholar, 5Sharp M.G. Fettes D. Brooker G. Clark A.F. Peters J. Fleming S. Mullins J.J. Hypertension. 1996; 28: 1126-1131Crossref PubMed Scopus (73) Google Scholar, 6Tsuchida S. Matsusaka T. Chen X. Okubo S. Niimura F. Nishimura H. Fogo A. Utsunomiya H. Inagami T. Ichikawa I. J. Clin. Invest. 1998; 101: 755-760Crossref PubMed Scopus (293) Google Scholar). 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 (7Corvol P. Williams T.A. Soubrier F. Methods Enzymol. 1995; 248: 283-305Crossref PubMed Scopus (229) Google Scholar). 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 (8Mastrobattista J.M. Semin. Perinatol. 1997; 21: 124-134Crossref PubMed Scopus (46) Google Scholar). 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 (9Cornell M.J. Williams T.A. Lamango N.S. Coates D. Corvol P. Soubrier F. Hoheisel J. Lehrach H. Isaac R.E. J. Biol. Chem. 1995; 270: 13613-13619Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Williams T.A. Michaud A. Houard X. Chauvet M.T. Soubrier F. Corvol P. Biochem. J. 1996; 318: 125-131Crossref PubMed Scopus (63) Google Scholar). 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 (11Hubert C. Houot A.M. Corvol P. Soubrier F. J. Biol. Chem. 1991; 266: 15377-15383Abstract Full Text PDF PubMed Google Scholar, 12Kumar R.S. Thekkumkara T.J. Sen G.C. J. Biol. Chem. 1991; 266: 3854-3862Abstract Full Text PDF PubMed Google Scholar, 13Thekkumkara T.J. Livingston W.d. Kumar R.S. Sen G.C. Nucleic Acids Res. 1992; 20: 683-687Crossref PubMed Scopus (49) Google Scholar). 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 (14El-Dorry H.A. Bull H.G. Iwata K. Thornberry N.A. Cordes E.H. Soffer R.L. J. Biol. Chem. 1982; 257: 14128-14133Abstract Full Text PDF PubMed Google Scholar, 15Ehlers M.R. Chen Y.N. Riordan J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1009-1013Crossref PubMed Scopus (78) Google Scholar, 16Sen I. Samanta H. Livingston W.D. Sen G.C. J. Biol. Chem. 1991; 266: 21985-21990Abstract Full Text PDF PubMed Google Scholar, 17Wei L. Alhenc-Gelas F. Soubrier F. Michaud A. Corvol P. Clauser E. J. Biol. Chem. 1991; 266: 5540-5546Abstract Full Text PDF PubMed Google Scholar). A plasma soluble form of the sACE protein is also produced by the regulated action of a membrane-associated cleavage-secretion process (18Beldent V. Michaud A. Bonnefoy C. Chauvet M.T. Corvol P. J. Biol. Chem. 1995; 270: 28962-28969Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Oppong S.Y. Hooper N.M. Biochem. J. 1993; 292: 597-603Crossref PubMed Scopus (96) Google Scholar, 20Sadhukhan R. Sen G.C. Ramchandran R. Sen I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 138-143Crossref PubMed Scopus (63) Google Scholar, 21Santhamma K.R. Sen I. J. Biol. Chem. 2000; 275: 23253-23258Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 (22Sen G.C. Thekkumkara T.J. Kumar R.S. J. Cardiovasc. Pharmacol. 1990; 16: 14-18Crossref Scopus (11) Google Scholar, 23Williams T.A. Corvol P. Soubrier F. J. Biol. Chem. 1994; 269: 29430-29434Abstract Full Text PDF PubMed Google Scholar). 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 (16Sen I. Samanta H. Livingston W.D. Sen G.C. J. Biol. Chem. 1991; 266: 21985-21990Abstract Full Text PDF PubMed Google Scholar, 24Isaac R.E. Williams T.A. Sajid M. Corvol P. Coates D. Biochem. J. 1997; 328: 587-591Crossref PubMed Scopus (23) Google Scholar, 25Williams T.A. Barnes K. Kenny A.J. Turner A.J. Hooper N.M. Biochem. J. 1992; 288: 875-881Crossref PubMed Scopus (43) Google Scholar). 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 (26Ehlers M.R. Riordan J.F. Biochemistry. 1991; 30: 7118-7126Crossref PubMed Scopus (135) Google Scholar, 27Rousseau A. Michaud A. Chauvet M.T. Lenfant M. Corvol P. J. Biol. Chem. 1995; 270: 3656-3661Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The full repertoire of physiological functions of ACE has been revealed by examining the defects of Ace-/- mice (3Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar, 4Esther Jr., C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Invest. 1996; 74: 953-965PubMed Google Scholar, 28Tian B. Meng Q.C. Chen Y.F. Krege J.H. Smithies O. Oparil S. Hypertension. 1997; 30: 128-133Crossref PubMed Scopus (48) Google Scholar). 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 (29Hagaman J.R. Moyer J.S. Bachman E.S. Sibony M. Magyar P.L. Welch J.E. Smithies O. Krege J.H. O'Brien D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2552-2557Crossref PubMed Scopus (302) Google Scholar). 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 (30Cole J. Ertoy D. Lin H. Sutliff R.L. Ezan E. Guyene T.T. Capecchi M. Corvol P. Bernstein K.E. J. Clin. Invest. 2000; 106: 1391-1398Crossref PubMed Scopus (138) Google Scholar). 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 (4Esther Jr., C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Invest. 1996; 74: 953-965PubMed Google Scholar). Because of the lack of Ang II production by ACE, these mice lack tubuloglomerular feedback response, as well (31Traynor T. Yang T. Huang Y.G. Krege J.H. Briggs J.P. Smithies O. Schnermann J. Am. J. Physiol. 1999; 276: F751-F757PubMed Google Scholar). 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 (7Corvol P. Williams T.A. Soubrier F. Methods Enzymol. 1995; 248: 283-305Crossref PubMed Scopus (229) Google Scholar, 32Friedland J. Setton C. Silverstein E. Biochem. Biophys. Res. Commun. 1978; 83: 843-849Crossref PubMed Scopus (106) Google Scholar, 33Strittmatter S.M. Snyder S.H. Neuroscience. 1987; 21: 407-420Crossref PubMed Scopus (43) Google Scholar). Germinal ACE is expressed exclusively in maturing sperm cells (34Langford K.G. Zhou Y. Russell L.D. Wilcox J.N. Bernstein K.E. Biol. Reprod. 1993; 48: 1210-1218Crossref PubMed Scopus (76) Google Scholar). 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 (35Ramaraj P. Kessler S.P. Colmenares C. Sen G.C. J. Clin. Invest. 1998; 102: 371-378Crossref PubMed Scopus (86) Google Scholar) 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 (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (37Kessler S.P. Gomos J.B. Scheidemantel T.S. Rowe T.M. Smith H.L. Sen G.C. J. Biol. Chem. 2002; 277: 4271-4276Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). 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. Transgene Construction—The 808-bp mouse receptor tyrosine kinase (Tie-1) promoter (38Korhonen J. Lahtinen I. Halmekyto M. Alhonen L. Janne J. Dumont D. Alitalo K. Blood. 1995; 86: 1828-1835Crossref PubMed Google Scholar) 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 γ-glutamyl transpeptidase (Ggt) promoter (39Sepulveda A.R. Huang S.L. Lebovitz R.M. Lieberman M.W. J. Biol. Chem. 1997; 272: 11959-11967Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) 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 (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) 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 (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (3Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar, 36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (16Sen I. Samanta H. Livingston W.D. Sen G.C. J. Biol. Chem. 1991; 266: 21985-21990Abstract Full Text PDF PubMed Google Scholar, 36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (40Senanayake P.S. Smeby R.R. Martins A.S. Moriguchi A. Kumagai H. Ganten D. Brosnihan K.B. Peptides. 1998; 19: 1685-1694Crossref PubMed Scopus (27) Google Scholar). 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 × 5 min) Clear-Rite; (2 × 3 min) Clear-Rite; (2 × 1 min) 100% Flex; (1 × 1 min) 95% Flex; (1 × 1 min) 80% Flex; (1 × 1 min) H2O; (1 × 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 (20Sadhukhan R. Sen G.C. Ramchandran R. Sen I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 138-143Crossref PubMed Scopus (63) Google Scholar), 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 (3Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar), for ten generations with Ace+/+ FVB female mice (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (37Kessler S.P. Gomos J.B. Scheidemantel T.S. Rowe T.M. Smith H.L. Sen G.C. J. Biol. Chem. 2002; 277: 4271-4276Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). 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). 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 (3Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar). 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 (36Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 38Korhonen J. Lahtinen I. Halmekyto M. Alhonen L. Janne J. Dumont D. Alitalo K. Blood. 1995; 86: 1828-1835Crossref PubMed Google Scholar, 39Sepulveda A.R. Huang S.L. Lebovitz R.M. Lieberman M.W. J. Biol. Chem. 1997; 272: 11959-11967Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) 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. 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). 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 (30Cole J. Ertoy D. Lin H. Sutliff R.L. Ezan E. Guyene T.T. Capecchi M. Corvol P. Bernstein K.E. J. Clin. Invest. 2000; 106: 1391-1398Crossref PubMed Sco
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