Abstract-To test the hypothesis that local vascular production of angiotensin II is necessary for the normal regulation of blood pressure, we engineered a new line of genetically altered mice that lack endothelial angiotensin-converting enzyme (ACE). This was accomplished using a novel strategy of targeted homologous recombination to separate the transcriptional control of somatic ACE from its endogenous promoter and to substitute control to the albumin promoter. These new mice, termed ACE.3, do not produce ACE within the lung, the aorta, or any vascular structure. ACE activity within the kidney is only about 14% that of wild-type mice and is limited to tubular epithelium. Key Words: knockout mice Ⅲ angiotensin-converting enzyme Ⅲ angiotensin II Ⅲ endothelium Ⅲ blood pressure T he renin-angiotensin system plays a central role in controlling mammalian blood pressure: mice with a genetic block of this system have systolic blood pressures 35 mm Hg lower than control animals. 1-3 Less clear is whether this system functions as a systemic endocrine network or whether it functions more as a series of local autocrine/paracrine networks within various organs and segments of the vasculature. 4 Indeed, some (including us) have hypothesized that the local production of angiotensin II by endothelial-bound angiotensin-converting enzyme (ACE) is a critical feature of normal vascular function. 3,5 To test the hypothesis that local vascular production of angiotensin II is necessary for the normal regulation of blood pressure, we genetically altered mice to lack endothelial ACE. This was accomplished using a novel strategy of targeted homologous recombination to separate the transcriptional control of somatic ACE from its endogenous promoter and to substitute control to the albumin promoter. These new mice, termed ACE.3, produce ACE within hepatocytes but do not make ACE within the lung, the aorta, or any vascular structure. Surprisingly, the expression of ACE by the liver is capable of substituting for endothelial ACE expression in that ACE.3 Ϫ/Ϫ mice have a normal blood pressure and normal renal function. Materials and Methods Creation of Homozygous Mutant MiceA 10.7-kb fragment of mouse genomic DNA was cloned from a mouse CC1.2 embryonic stem (ES) cell library derived from mouse strain 129 DNA. This contained 2.4 kb of the somatic ACE promoter, the somatic ACE transcription start site, and 8.3 kb of genomic sequence encompassing somatic ACE exons 1 through 12. Using a unique BssHII restriction site, a neomycin cassette was inserted 3Ј to the somatic ACE transcriptional start site but 5Ј to somatic ACE exon 1. Next, a 2.3-kb albumin promoter/enhancer was placed immediately 3Ј to the neomycin cassette. The albumin promoter/enhancer, termed NB, was a gift of Dr Richard Palmiter (University of Washington). Animal care was supervised by the Emory University Division of Animal Research and followed accepted practices of the American Veterinary Medical Association.The targeting construct was linearized and electroporated into R1 ES c...

mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 Ϯ 5, 6 Ϯ 6, and 39 Ϯ 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, NaϪ cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 Ϯ 11 and 29 Ϯ 6%, respectively, whereas Na ϩ -K ϩ -ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 Ϯ 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins.