IntroductionThe renin-angiotensin system plays a critical role in blood pressure regulation and fluid hemodynamics. Pharmacologic inhibitors of this system are routinely used to treat hypertension and congestive heart failure. One of the most controversial effects of the reninangiotensin system has been the interplay of this system with erythrocyte production. A variety of clinical reports have noted an association between activation of the renin-angiotensin system and increased erythropoiesis (1-3). These studies have come from analyses of patients with a variety of chronic diseases including chronic obstructive pulmonary disease, heart failure, and renal transplantation. Other investigators have suggested a link between angiotensin-converting enzyme (ACE) inhibitors and worsened anemia, particularly in patients with chronic renal failure (4-6). While research has focused on the interplay of the renin-angiotensin system and erythropoietin, no mechanistic explanation for these observations has been generally accepted.Central to the renin-angiotensin system is ACE, a peptidase that converts angiotensin I to angiotensin II (7). In mammals, most ACE is bound to tissues such as endothelium, but enzymatic cleavage results in a circulating form within plasma. In vitro, ACE is capable of cleaving many small peptides besides angiotensin I. However, in vivo, with the exception of bradykinin, the significance of nonangiotensin peptides as ACE substrates is not well understood. ACE is a protein with two independent catalytic domains. While both catalytic sites hydrolyze angiotensin I with roughly equal efficiency, the amino-and carboxy-terminal catalytic domains differ in their rate constants for other peptides.Using targeted homologous recombination in embryonic stem (ES) cells, our laboratory created two lines of mice with modifications of the ACE gene (8, 9). These animals are termed ACE.1 and ACE.2. Mice homozygous for the ACE.1 allele (ACE.1 knockout mice) are null for all ACE production. They have a marked reduction of blood pressure, and a renal lesion characterized by hypoplasia of the renal medulla and papilla. In contrast to this null phenotype, animals homozygous for the ACE.2 allele (ACE.2 knockout mice) have a partial restoration of ACE activity. These animals express a truncated ACE protein containing only the amino-terminal catalytic domain. Since this shortened ACE protein lacks the carboxy-terminal domain that normally anchors ACE to cell membranes, the ACE.2 protein is exported from cells into blood and other extracellular fluids. Thus, while the plasma of ACE.2 mice converts angiotensin I to angiotensin II with about 34% of the activity of wild-type mouse plasma, tissues such as the lung and kidney completely lack ACE protein or activity. The systolic blood pressure of ACE.2 knockout mice averaged 75 mmHg, as low as that of the ACE.1 knockout animals.Here, we investigate an unexpected finding concerning the phenotypes of both the ACE.1 and ACE.2 mice. These animals are anemic. ACE.2 knockout mice are a p...
Angiotensin-converting enzyme (ACE) produces the vasoconstrictor angiotensin II. The ACE protein is composed of two homologous domains, each binding zinc and each independently catalytic. To assess the physiologic significance of the two ACE catalytic domains, we used gene targeting in mice to introduce two point mutations (H395K and H399K) that selectively inactivated the ACE N-terminal catalytic site. This modification does not affect C-terminal enzymatic activity or ACE protein expression. In addition, the testis ACE isozyme is not affected by the mutations. Analysis of homozygous mutant mice (termed ACE 7/7) showed normal plasma levels of angiotensin II but an elevation of plasma and urine N-acetyl-Ser-Asp-Lys-Pro, a peptide suggested to inhibit bone marrow maturation. Despite this, ACE 7/7 mice had blood pressure, renal function, and hematocrit that were indistinguishable from wild-type mice. We also studied compound heterozygous mice in which one ACE allele was null (no ACE expression) and the second allele encoded the mutations selectively inactivating the N-terminal catalytic domain. These mice produced approximately half the normal levels of ACE, with the ACE protein lacking N-terminal catalytic activity. Despite this, the mice have a phenotype indistinguishable from wild-type animals. This study shows that, in vivo, the presence of the C-terminal ACE catalytic domain is sufficient to maintain a functional renin-angiotensin system. It also strongly suggests that the anemia present in ACE null mice is not due to the accumulation of the peptide N-acetyl-Ser-Asp-Lys-Pro.The major role of the renin-angiotensin system (RAS) 1 is the regulation of electrolyte homeostasis and blood pressure in mammals. The major effector peptide of the RAS is the octapeptide angiotensin II, which raises blood pressure through a variety of mechanisms, including vasoconstriction and the control of aldosterone release. Angiotensin II is the product of two successive enzymatic cleavages of angiotensinogen. First, renin releases the intermediate decapeptide angiotensin I. Then the last two C terminus amino acids of angiotensin I are removed by the zinc-metallopeptidase, angiotensin-converting enzyme (ACE) (1). ACE inhibitors markedly reduce the formation of angiotensin II and are widely used in the treatment of hypertension, congestive heart failure, and diabetic nephropathy.
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...
We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of bla KPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the bla KPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum -lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the bla KPC PCR assay was used to confirm the presence of bla KPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected bla KPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of bla KPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two bla KPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with bla KPC-2 and bla KPC-3 , respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.