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

Abstract

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) 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. 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Samanta H. Livingston W.D. Sen G.C. J. Biol. Chem. 1991; 266: 21985-21990Abstract Full Text PDF PubMed Google Scholar, R.E. Williams T.A. Corvol P. Coates D. Biochem. J. 1997; PubMed Scopus Google Scholar, T.A. K. Biochem. J. 1992; PubMed Scopus Google Scholar). In have that are and for both ACE The cleaves and the peptide the C-terminal M.R. Riordan J.F. 1991; PubMed Scopus Google Scholar, A. Michaud A. Chauvet M.T. Corvol P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The of physiological functions of ACE has been by the of Ace-/- mice 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, Chen Krege J.H. Smithies O. S. Hypertension. 1997; PubMed Scopus Google Scholar). In to from blood pressure, the male Ace-/- mice are Although is in or Ace-/- males or a of ACE are in within the and in to J.R. Bachman E.S. Smithies O. Krege J.H. O'Brien D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). However, the for ACE in has been ACE also has a role in The mice have plasma levels J. D. H. R.L. E. Capecchi Corvol P. Bernstein K.E. J. Clin. Invest. PubMed Scopus Google Scholar). The renal of Ace-/- mice are both structurally and of atrophy, renal vascular and are in the of these are in urine a urine of a 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 as T. T. Krege J.H. Smithies O. J. J. Google Scholar). The of and expression in the are ACE is expressed in vascular endothelial kidney proximal tubules, and cells of the (7Corvol P. Williams T.A. Soubrier F. Methods Enzymol. 1995; 248: 283-305Crossref PubMed Scopus (229) Google Scholar, J. C. E. Biochem. Res. PubMed Scopus Google Scholar, 21: PubMed Scopus Google Scholar). ACE is expressed in cells Bernstein K.E. Biol. PubMed Scopus Google Scholar). have been the specific physiological by ACE expressed in specific we generated strains of mice in expression of ACE by These mice were Ace-/- mice to produce mice physiological ACE expression in one tissue of a specific Ace-/- we demonstrated P. C. Sen G.C. J. Clin. Invest. 1998; PubMed Scopus Google that expression in male of Ace-/- However, for gACE, that the two are for functions T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In expression of germinal ACE in the normal and renal functions were of blood pressure J.B. T.M. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we have generated one mouse that in vascular endothelial cells and a mouse that in renal proximal cells. In to in the both groups also have in the serum. In to ACE mice, both mice of Ace-/- were as healthy as wild-type Both strains normal renal structure and though all male mice remained Despite wild-type levels of and levels of Ang II in the serum of both only the mice expressing in the vascular endothelial cells had normal blood pressure. mouse kinase J. I. L. J. D. K. 1995; PubMed Google by the and the the The mouse γ-glutamyl J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google by the and the the Both were by and for by to the gene in the activity, and kidney as T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and the were by the phosphoglycerate in the T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the or The and the were from and by and The were from a system to by the as T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). or of the by the to the mouse gene in the The is by the of a wild-type or the 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, T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ACE ACE enzyme ACE cleavage of the Ang I by of from and and mouse kidney or serum. All were in ACE as I. Samanta H. Livingston W.D. Sen G.C. J. Biol. Chem. 1991; 266: 21985-21990Abstract Full Text PDF PubMed Google Scholar, T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). All from II and I each the blood from mice of the and were to a plasma plasma from each were for the of Ang II and Ang I levels as A. H. D. 1998; PubMed Scopus Google Scholar). The results are the of and were and and by the by in the phosphate-buffered were in for and to The were for in serum The Sen G.C. Sen I. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus (63) Google mouse ACE in in to the in a for C. in to each for in the in in All were a and of and mice and mice were male mice to and mice were generated by the in mice 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 for mice T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). male and mice to the male and mice to of all mice by as the of from each of all males by Ace-/- Ace-/- and males a of wild-type for the equivalent to two of two were observed for were within from male the were males for The of a the male mice, express their were wild-type mice, and the of also and mice of the and were in a and and urine for for each for each mouse the A to the were the urine from mice of each to Blood blood pressure system for mice used to blood pressure J.B. T.M. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mice were and a mouse for to to the and the mice, to and in a were two and of All and and each were for A of blood pressure mouse were used to the blood pressure for each The blood pressure for each mouse by the blood pressure of each mouse the to of blood pressure for each a of two mice and a of mice of mice were generated by mice the All mice were of the to the had been by for from the Ace-/- mice from the Smithies 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 of a and splicing from the bovine growth hormone and either the or the were T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. I. L. J. D. K. 1995; PubMed Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google to expression to the vascular endothelial cells or renal proximal Both were by The and the were as in and in for expression by and by kidney cells. ACE of from cells of the from both The of the and were of and in the of of and by a as the as in the and the mice were mice, and the were by All of the the to their and were and and were Because the to be expressed in vascular endothelial we its expression in the a tissue and the of natural of of the that only I, and expressed of sACE, though expressed a expression also in and serum In the to be expressed in of kidney that only and mice expressed in the kidney and express the in kidney or in any tissue. Because I, and expressed the in the mice for both the and the or in these were to produce the or mice that were for the of be either or Ace-/- one of the in the mice sufficient to produce In the a the gene a and the ACE The both the and the mouse gene Expression of the the mice, ACE enzymatic were in the kidney and serum. the of ACE in the kidney and that observed in the all groups had renal ACE wild-type mice the the serum ACE levels of the and mice were that observed in the mice had of wild-type levels of in the serum To the ACE in the serum of the mice produce Ang II, the of both Ang I and Ang II in Ace-/- and The in A and are the of the from The Ang II for the and Ace-/- mice were for strains of mice J. D. H. R.L. E. Capecchi Corvol P. Bernstein K.E. J. Clin. Invest. PubMed Scopus Google Scholar). Although the Ang II levels in all strains of mice were that of Ace-/- mice, the Ang II the observed in The mice Ang II levels to wild-type levels were the group The Ang I levels were to the Ang II the mice Ang II had a Ang I in Ang I or Ang II levels of the the cell type in a Because also mouse ACE, in all were from Ace-/- in the expressed in and In all tissues, expression to the vascular endothelial cell the of blood Expression of in the mice in the renal proximal to the expression of in the in the of the proximal is the natural of expression The proximal tubules and blood of the Ace-/- mice were of ACE to the expression of the in the blood as in Expression of the in the mice to the vascular endothelial cells expression in the vascular cell types or in cells of the proximal tubules in the The of mice were of any However, the mice expressed in the and of the blood A and However, in the vascular endothelial cells of the These results demonstrated the cell expression of in the and In the of Ts, and the observed renal ACE in each of the mice have the of of are and they are in the of their by the of of and all by the of In all Ace-/- mice, the were and were from of the wild-type A of Ace-/- mice is male 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, P. C. Sen G.C. J. Clin. Invest. 1998; PubMed Scopus Google Scholar). in the male The of of mice were by the Ace-/- male mice Although expression of in the vasculature of or male mice it Ace-/- male In as T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google expression of in the of Ace-/- mice its of Ace-/- mice have the of mice of fluid and are the and a in the is by a of the arterial The were in all mouse were to of mice, and their arterial were and of normal kidney structure by expression in the Ace-/- mice is by of fluid and urine in Ace-/- mice as the In the mice, both were to normal A and is decreased by in the Ace-/- mice, to normal levels in the and mice These results demonstrated that expression of sACE, only in the proximal of the mice, also in the vasculature of the mice, a expression in the proximal tubules, is sufficient for maintaining normal kidney structure and The physiological role of ACE is in maintaining normal blood pressure. To the of expression we used the J.H. Hodgin J.B. Hagaman J.R. Smithies O. Hypertension. 1995; PubMed Scopus Google for blood of and in male and mice of the have blood pressure of ACE expression in the Ace-/- mice a in the blood pressure in both the in blood to normal levels in the male and the for the or male and mice, a in the of the and the are in the physiological for ACE expression in of the a is the or the of ACE is to regulate blood pressure by Ang II in the However, the that Ace-/- mice have many in to blood pressure, that of ACE in many be for normal 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, C.R. Marino E.M. Howard T.E. A. Corvol P. Capecchi M.R. Bernstein K.E. J. Clin. Invest. 1997; PubMed Scopus Google Scholar). because of these such as and male are in mice of the RAS, ACE must physiological functions by Ang I (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, J. D. H. R.L. E. Capecchi Corvol P. Bernstein K.E. J. Clin. Invest. PubMed Scopus Google Scholar). The the for functions of of ACE, namely, the conservation of the structurally related enzymatic activity, their physiological (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). To these we have been different strains of mice, to express only one of ACE in only one tissue. a mouse is in a the expression of the ACE in only one tissue. mouse is mice to produce the Because many properties of the such as blood pressure, and renal different strains Biol. 20: PubMed Scopus Google it that all of are to one of the of we the is and the are is and it is to produce mice of To we to the Ace-/- to as by of a mouse the we have demonstrated P. C. Sen G.C. J. Clin. Invest. 1998; PubMed Scopus Google that expression of in is sufficient for male of Ace-/- mice, from kidney and blood pressure because of the lack of ACE expression in somatic In we expressed sACE, of gACE, in sperm. these male mice were that is in the of male expressed in the cell type T.M. J.B. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In we generated mice that levels of germinal ACE in the serum in to the sperm. These mice were as healthy as wild-type mice, normal renal structure and of normal blood pressure J.B. T.M. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we have two different strains of mice, to express in the vascular endothelial cells or the renal proximal cells The used for from the gene and the gene of the expression of is to the and blood and in has been to expression of to vascular endothelial cells J. I. L. J. D. K. 1995; PubMed Google Scholar). In a the because it J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google to a gene in renal proximal cells of a In mice, expressed in many tissues, the expression to the vascular and the expression of the in the the expression of the ACE in both the vascular endothelial and proximal cells of expression of the in the mice to the blood endothelial cells The group expressed as ACE expression to the of renal proximal tubules and in the vasculature However, the expression in the different from expressed in renal proximal the to the is proximal to the that is in mice group also expression in the and of blood expression observed in the endothelial cell group also had levels of in their serum though for physiological of ACE were in the and male kidney and and blood pressure. the and male mice were expression in serum and vasculature demonstrated the of the somatic functions of from the functions of In the of ACE expression the blood pressure of the the the blood of both of the mice were in the normal the of in the vascular sufficient for the blood pressure be that ACE in the serum of the mice, as ACE in the serum from ACE by natural cleavage secretion Sen I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the mouse also had serum ACE levels the to the functions of ACE and serum ACE in The blood pressure of the mice remained as as Ace-/- mice their serum ACE that of the and the in mice in ACE and ACE mouse strains expressing germinal ACE, both of had blood pressure serum activity, and normal renal structure and J.B. T.M. Sen G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is also that expression in the renal proximal tubules of mice or in the serum sufficient to renal structure and and of the from is the of blood pressure and renal The of the Ace-/- including the of the arterial were by the of in the serum ACE expression in the proximal tubules to be for any of these physiological the fluid homeostasis of the both the and the urine of the Ace-/- mice were to normal levels in and The urine by in the of the of normal urine it that ACE expression in the kidney proximal tubules is for maintaining normal kidney functions as as ACE is in the serum. In to the kidney functions, maintenance of normal blood pressure in mice the of ACE blood pressure only in the mice, the mice, the that mice had serum ACE levels to of mice the of a J. L. K. Corvol P. Capecchi M.R. Bernstein K.E. Res. PubMed Scopus Google that serum ACE levels blood pressure. The for the are However, the strains of the mice used in the two were that genetic a the mice in the expressed of ACE in the as the that blood pressure in their mice, because Ang II levels were to The in that the of Ang II in the plasma blood pressure. The had Ang II levels the the mice, their blood pressure that the production of Ang II or the vascular endothelial cells is for blood pressure Ang II results also that endothelial ACE is for maintaining blood pressure. Ang II by is sufficient for The results that expression of ACE in the endothelial cells of the vasculature is for blood pressure be in the mice, though ACE expressed in cell types of their vasculature in endothelial cells. and John for and and for also Sen and for the and ACE enzyme