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Rescue of Angiotensinogen-Knockout Mice
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
252, 610 – 616 (1998)
RC989707
Rescue of Angiotensinogen-Knockout Mice Junji Ishida,*,†,1 Fumihiro Sugiyama,†,‡ Keiji Tanimoto,* Keiko Taniguchi,*,† Mikio Syouji,† Eriko Takimoto,* Hisashi Horiguchi,§ Kazuo Murakami,*,‡ Ken-ichi Yagami,† and Akiyoshi Fukamizu*,‡,¶,2 *Institute of Applied Biochemistry, †Laboratory Research Animal Center, Institute of Basic Medical Sciences, and ‡Center for the Tsukuba Advanced Research Alliance (TARA), University of Tsukuba; §Molecular Pathology Center, Ibaraki Prefectural University of Health Sciences; and ¶National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba, Ibaraki 305-8572, Japan
Received October 15, 1998
Angiotensinogen, the precursor of angiotensins I and II, is a critical component of the renin-angiotensin system that plays an important role in regulating blood pressure and electrolyte homeostasis. Genetically altered mice lacking angiotensinogen (Agt-KO) showed an expected phenotype, such as marked hypotension, but unexpected ones including abnormal kidney morphology, reduced survival rates of newborns, and impaired blood–brain barrier function after cold injury. To examine whether disruption of the angiotensinogen gene is responsible for the observed phenotypes, we generated a line of mice heterozygous for the mouse angiotensinogen gene under the control of a mouse metallothionein-I promoter (MT-Agt) and crossmated transgenic mice with Agt-KO mice. The resulting mice (MT-Agt(1/2)/ Agt(2/2):MT-Agt/KO) produced the plasma level of angiotensin I comparable to that of wild-type mice, and the mutant phenotypes were rescued. These results indicated that the resultant phenotypes due to the genetic deficiency of mouse angiotensinogen can be corrected by restoring angiotensinogen and angiotensin I in the circulation. © 1998 Academic Press Key Words: angiotensinogen; angiotensin II receptors; genetic rescue; knockout mouse; renin-angiotensin system; transgenic mouse.
Angiotensinogen is constitutively secreted into the circulation mainly from the liver. Circulating renin, 1
Research Fellow of the Japan Society for the Promotion of Science. 2 To whom correspondence should be addressed at Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305, Japan. Fax: 81-298-53-6599. E-mail: akif @sakura.cc.tsukuba.ac.jp. Abbreviations used: Agt, angiotensinogen; angiotensin II type 1 receptor, AT1; angiotensin II type 2 receptor, AT2; bp, base pair; kb, kilobase; KO, knockout; MT, metallothionein. 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
synthesized predominantly in the kidney, cleaves angiotensinogen, the only known substrate for renin, to release a decapepide angiotensin I. Further processing of a physiologically inactive angiotensin I by angiotensin converting enzyme (ACE) generates an octapeptide angiotensin II, which plays an important role in blood pressure, fluid, and sodium homeostasis (1, 2). To study the role of angiotensinogen in vivo, we and other investigators generated angiotensinogen-knockout (Agt-KO) mice, which showed phenotypes with increased infant death and histopathological renal abnormalities in addition to marked hypotension and overexpression of the renin gene (3–5). More recently, we found remarkably attenuated expression of glial fibrially acidic protein and decrease laminin production in the astrocytes of Agt-KO mice in response to cold injury, and eventually incomplete reconstitution of impaired blood-brain barrier function. Furthermore, we demonstrated that these abnormalities were rescued by administration of angiotensin II or angiotensin IV (6). Introduction of desired mutations into embryonic stem cells and generation of knockout mice have now become a routine practice. The extent to which the resultant phenotypes are affected due to such mutations depends on the fact that no other alterations have occurred elsewhere in the manipulated mouse genome. To confirm that the cardiovascular defects found in Agt-KO mice are only due to an engineered mutation in the mouse angiotensinogen locus, we have genetically rescued the Agt-KO mice by introducing a fusion gene composed of a mouse metallothionein (MT) promoter and the mouse angiotensinogen gene lacking its own promoter with the first intron (MT-Agt). This clearly shows that angiotensinogen plays a critical role in maintaining blood pressure, regulating renal renin expression, histological renal preservation, and newborn development.
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MATERIALS AND METHODS Animals. Agt-KO mice were generated as previously described (3). C57BL/6J control and CD-1 mice were obtained from CLEA Japan Co. Ltd., Tokyo and Charles River Japan Inc., Yokohama, respectively. Animals were used according to the regulation of “Standards for Human Care and Use of Laboratory Animals, University of Tsukuba”. Construction of transgene. To replace the promoter of mouse angiotensinogen gene, the 1.8-kb DNA fragment (HindIII/XhoI) consisting of the 1.7-kb 59-flanking region and the 70-bp 59-untranslated region of the mouse metallothionein-1 gene was prepared from pMGH (7). The 11.8-kb structural region (XhoI/HindIII) from the second exon to poly(A) signals of the mouse angiotensinogen gene was prepared from pB6A-16B, pB6A-50 and pB6A-113 (8). Both fragments were ligated into HindIII site of pUC119 to construct pMT-Agt. Generation of transgenic mice. Eggs of CD-1 mice superovulated with PMSG-hCG treatment were collected and fertilized in vitro (9) to C57BL/6J mice sperm. Fertilized oocytes were cultured to appearance of pronucleus at 37°C in 5% CO2. The 13.6-kb linearized MTAgt fusion gene digested by HindIII from pMT-Agt (Fig. 1A) was dissolved in 0.9% NaCl solution at concentration of 2 ng/ml. The method of micromanipulation for transgenic procedure was essentially as described (10). The microinjected oocytes were developed to the 2-cell stage in modified Whitten medium. The survived 2-cell embryos were implanted into oviducts of pseudopregnant mice. DNA preparation and Southern blot analysis. Genomic DNA was prepared from mouse tail as described previously (3), digested with BamHI, electrophoresed on a 0.7% agarose gel, and subjected to Southern blot analysis using GeneScreen Plus membrane (DuponNew England Nuclear, Boston, MA). The filter was hybridized to the 32 P-labelled 0.9-kb BglII/BamHI DNA fragment containing the angiotensinogen gene, at 65°C for 16 h and washed twice with 2 3 SSC (SSC 5 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at room temperature for 5 min, twice with 2 3 SSC/1% SDS at 65°C for 30 min, and twice with 0.1 3 SSC at room temperature for 30 min. RNA preparation and Northern blot analysis. Total RNA was prepared from homogenized tissues using ISOGEN (NipponGene) based on the acid guanidium thiocyanate–phenol– chloroform extraction method (11). 15 micrograms of total RNA were denatured with glyoxal, electrophoresed on a 1.2% agarose gel, and transferred to GeneScreen Plus membrane. The filter was probed 32P-labelled DNA fragments as described below, at 60°C for 16 h and washed twice with 2 3 SSC at room temperature for 5 min, twice with 2 3 SSC/1% SDS at 60°C for 30 min, and twice with 0.1 3 SSC at room temperature for 30 min. The used DNA probe are 0.9-kb BglII/BamHI DNA fragment for angiotensinogen from pA-16B and 0.82-kb KpnI/NcoI DNA fragment for renin from pMRn226 (12), respectively. Quantitative analysis of angiotensinogen and angiotensin I. Blood was collected from inferior vena cava of mice anesthetized with pentobarbital (30 mg/kg). Plasma containing anticoagulant, EDTA z 2Na, was immediately separated at 4°C and stored at 270°C until used. Angiotensin I concentration was measured by radioimmunoassay as described (13). The concentration of angiotensinogen was determined by measurement of the amount of angiotensin I after addition of an excess of purified mouse submandibular gland renin (14). Measurement of blood pressure. Blood pressure was measured in the afternoon from 1 to 5 pm. Unanesthetized mice wrapped with a cotton holder were introduced into the warming tube thermostatically controlled at 37°C. The systolic blood pressure was measured with a programmable sphygmomanometer (BP-98A; Softron, Tokyo, Japan) by the tail-cuff method.
Histological analysis. After blood collection, phosphate buffered saline was introduced into the left ventricle via a 26 gauge needle and allowed to flow out of a cut point at the right atrium. When the perfusate became clear, perfusion was continued with 10% formaldehyde in 0.1 M phosphate buffer (PB; pH7.4). For histopathological examinations, the formalin-fixed kidney were embedded in paraffin. The sections cut from the paraffin block and then were stained with hematoxylin and eosin.
RESULTS The linearized 13.6-kb HindIII fragment (MT-Agt fusion gene) (Fig. 1A) was microinjected into a pronucleus of fertilized mouse oocytes. Forty four newborns were obtained from the pseudopregnat recipients to which the 2-cell embryos were transferred. One transgenic founder animal was identified by Southern blot analysis (data not shown) using MT-Agt probe and the line of mice heterozygous for the transgene was outwardly normal and normotensive (105 6 4.1 mmHg vs 103 6 1.5 mmHg of wild type mice (WT)). To test for rescue of the phenotypes of homozygous angiotensinogen-knockout (Agt-KO) mice, the transgene was introduced into mutant background by breeding. Agt-KO mice were first crossbred with the transgenic mice heterozygous for the MT-Agt fusion gene to give F1 mice that were WT, heterozygous for the MTAgt, heterozygous for the mutant angiotensinogen allele and also carried the transgene. The fourth F1 mice were then crossed with Agt-KO mice to give F2 progeny that were Agt-KO mice, heterozygous Agt-KO mice, heterozygous Agt-KO mice heterozygous for the MT-Agt fusion gene, and Agt-KO mice heterozygous for the MT-Agt gene (MT-Agt/KO). MT-Agt/KO mice were identified by Southern blot analysis of tail DNA digested with BglII. The 0.9-kb DNA probe detected the endogenous angiotensinogen gene as a 2-kb band in WT mice, but not in Agt-KO mice, because the corresponding region was replaced to neor cassette (Fig. 1A). On the other hand, in MT-Agt/KO mice, the MT-Agt fusion gene can be detected as a 6-kb band, because the transgene almost lacked the first intron including the BglII sites (Fig. 1A). For further analyses, we used MT-Agt/KO mice in comparison with WT and Agt-KO mice. To examine expression of the angiotensinogen gene of several tissues in MT-Agt/KO mice, we performed Northern blot analysis using the 0.9-kb DNA fragment (Fig 3). Total RNAs were isolated from the brain, heart, liver, and kidney of WT, Agt-KO, and MT-Agt/KO mice. Angiotensinogen mRNA was found in all tissues examined in WT and the highest intensity was detected in the liver, whereas its transcript in Agt-KO mice was not accumulated in any tissues used. In contrast, angiotensinogen transcripts were identified in the four tissues of MT-Agt/KO mice, although its expression level was different from that of corresponding tissues in WT mice. Ohkubo et al. (15) reported that the MT-I promoter fused to the rat angiotensinogen instead of its own promoter at the 59-flanking region can be acti-
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FIG. 1. Schematic representation of the angiotensinogen genes and Southern blot analysis of the three lines of mice. (A) C57BL/6J and Agt-KO mice are indicated as control mice and mice homozygous for the targeted angiotensinogen gene, respectively. MT-Agt/KO mice have the MT-Agt fusion genes on the Agt-KO genetic background. The coding and noncoding exons are shown as closed and open boxes, respectively. BglII, G; HindIII, H; Neomycin resistance gene, Neor; mouse metallothionein promoter, MT. (B) Southern blot analysis. Ten micrograms of genomic DNA were digested with BglII and subjected Southern hybridization with a DNA probe (closed bar in A).
vated, when induced by ZnCl2 in drinking water. No inducible expression of the MT-Agt transgene was found, even in the presence of the heavy metal in drinking water (data not shown), probably due to the integration site effect or differential methylation pattern of the transgene, although there is no direct evidence at the present time. These results indicated that angiotensinogen mRNA in MT-Agt/KO mice is transcribed from the exogenous transgene. To ensure the production of angiotensinogen and angiotensin I in the plasma of MT-Agt/KO mice, we measured their contents by radioimmunoassay. No angiotensinogen was detectable in Agt-KO mice, but its concentration in MT-Agt/KO mice was restored to the level comparable to that of WT mice (Table 1) despite the lower expression of exogenous angiotensinogen in the liver of MT-Agt/KO mice than that of WT mice (Fig. 2). This provided the possibility that circulating angio-
tensinogen may be supplied from other tissues in addition to the liver. Although the plasma level of angiotensin I in Agt-KO mice decreased below to detectable one, there was no significant difference in angiotensin I content between MT-Agt/KO and WT mice. To further test whether the genetical and biochemical restoration of angiotensinogen have physiologically beneficial effects in mice, we measured the systolic blood pressure in the three different strains. As shown in Table 1, a significant decrease in blood pressure was confirmed in Agt-KO mice as compared with WT mice. In contrast, the blood pressure of MT-Agt/KO mice returned to the normal level comparable to that of WT mice. Previously, we reported that the level of renal renin mRNA increased 6- to 8-fold in Agt-KO mice in comparison with that found in WT mice (3). To investigate the level of renal renin mRNA accumulation, we performed Northern blot hybridization using total RNAs from the
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Angiotensinogen, Angiotensin I Contents in Plasma, and Systolic Blood Pressure of the Three Lines of Mice Strains
mAG (mg/ml)
Al (ng/ml)
SBP (mmHg)
C57BL/6J mice Agt-KO mice MT-Agt/KO mice
4.8 6 0.4 (n 5 4) N.D. (n 5 4) 5.2 6 2.9 (n 5 6)
1.9 6 0.4 (n 5 4) N.D. (n 5 4) 1.7 6 0.8 (n 5 6)
103 6 1.5 (n 5 4) 70 6 3.6 (n 5 4)* 109 6 5.0 (n 5 15)
Note. mAG, mouse angiotensinogen; AI, angiotensin I; SBP, systolic blood pressure. Values are means 6 SD with an average of each group; n indicates total number of mice in each group. SBP was measured by a programmable sphygmomanometer using the tail-cuff method. mAG and AI were measured by radioimmunoassay on plasma samples. * P , 0.01 compared with wild-type mice.
kidney of MT-Agt/KO, Agt-KO, and WT mice. Interestingly, expression of the renal renin gene in MT-Agt/KO mice was suppressed to the level found in WT mice (Fig. 3), despite the significant differences of renal angiotensinogen mRNA accumulation between both the normotensive strains (Fig. 2). We next histopathologically examined the kidney in WT (Fig. 4; A and D), Agt-KO (B and E), and MT-Agt/KO (C and F) mice at the age of 8 weeks. The cross-sections stained with hematoxylin and eosin demonstrated inner medulla- and papillaryatrophy, and pelvic dilation in the unilateral or bilateral kidney in Agt-KO mice. Furthermore, the renal cortex thinned and the arterial wall thickened (arrow) comparable with those of WT mice were observed in Agt-KO mice. However, normotensive MT-Agt/KO mice showed normal histological features of the kidney. Early death of infants was observed in Agt-KO mice (3–5, 16, 17). Thus, we investigated survival rates of the pups obtained from Agt-KO mice that were crossed with MT-Agt/KO mice. In this cross experiment, newborns to be obtained were Agt-KO and MT-Agt/KO and
FIG. 2. Tissue distribution of angiotensinogen mRNA in the three lines of mice. Total RNAs were isolated from the brain (a), heart (b), liver (c), and kidney (d) of C57BL/6J, Agt-KO, and MTAgt/KO and subjected to Northern blot analysis (15 mg/lane) using a probe shown in Fig. 1A.
the Mendelian ratio was expected to be 1:1. Of 42 newborns, only 28 infants grew to a weaning stage. In order to examine the genotypes of survived mice, we performed Southern blot analysis using the 0.9-kb DNA as a probe and summarized the results in Table 2. Twenty four and 4 were identified as MT-Agt/KO and Agt-KO mice, respectively. These results suggested the improved survival rates of Agt-KO mice after birth by genetically introducing angiotensinogen. DISCUSSION The gene targeting system is an indispensable means for analysing in vivo function of cloned genes of interest. However, it is possible that the homologous recombination by deletion and replacement with neor cassette unexpectedly introduces a mutation into other gene(s) in addition to the targeted gene. For examples, Ohno et al. produced a gene-targeted mouse lacking CD3h chain to analyze its role in T cell development and function (18). Most of CD3h-deficient mice died within a few days after birth despite of normal development and function in the T cells. Genetic analysis has revealed that CD3h locus encodes a nuclear transcription factor, Oct-1, on the opposite strand (19), suggesting an important possibility of a complicated interpretation in the gene targeting experiment in-
FIG. 3. Northern blot analysis of renal renin mRNA in the three lines of mice. Total RNAs (15 mg) from the kidney of C57BL/6J, Agt-KO, and MT-Agt/KO mice were subjected to Northern blot analysis using the Ren-2 and b-actin DNA probe.
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FIG. 4. Histopathological analysis of kidney section in the three lines of mice. Magnifications of upper and bottom panels are 340 and 3400, respectively. Cross-sections stained with hematoxylin and eosin. Agt-KO mice (B, E) show renal cortex thinned, atrophy of papilla and inner medulla partially, and arterial wall thickened by hyperplasia. The kidney morphology of MT-Agt/KO (C and F) mice is histopathologically consist with that of C57BL/6J (A and D).
duced by unexpected mutation of gene(s) on the opposite strand (20, 21). Therefore, chemical or protein rescue experiments on gene-targeting mice should be necessary to characterize its real functions of the targeted gene, but it is often difficult to assess the functional contribution of administered substances in fetal, neonatal, and infant stages. To avoid such frustrate
situations, we reconstituted the mouse angiotensinogen transgene composed of the MT-I promoter with the angiotensinogen genomic DNA fragment including the exon 2 to 5, resulting in the enforced unidirectional transcription unit (MT-Agt), generated mice heterozygous for the MT-Agt, and introduced it into Agt-KO genetic background by crossbreeding.
TABLE 2
Survival Analysis of MT-Agt/KO and Agt-KO Mice Breeding form
No. of newborns
MT-Agt/KO 3 Agt-KO
42
a
No. of weanings surviveda
No. of Agt-KO mice
No. of MT-Agt/KO mice
Ratio
42 28
21 4
21 24
1:1 0.17:1
Expected Observed
The angiotensinogen genotypes of weanings at 4 weeks of age were detected by Southern blotting. 614
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It has been shown that chronic administrations of ACE inhibitors in mouse or rat enhance intrarenal renin synthesis (22, 23). Like ACE inhibition experiments, we observed the increased accumulation of renin mRNA in the kidney of Agt-KO mice associated with the complete loss of angiotensin I (3). In contrast to Agt-KO mice, the repressed levels of renal renin mRNA comparable to that of WT mice were seen in MT-Agt mice, suggesting that the restoration of circulating angiotensinogen and angiotensin I upon MTAgt/KO mice stimulates a negative regulation of the renin gene expression in the kidney. A recent histological study by Kakuchi et al. (24), using in situ hybridization, indicated that renal AT1 expression in mouse is corresponded temporally and spatially with differentiation and proliferation of glomerular mesangial and tubular cells. Furthermore, it has been shown that neonatal treatments with either an ACE inhibitor or an AT1 blocker in rats produce persistent, irreversible histopathological renal abnormalities including palliary atrophy and pelvic dilation (25). Such histopathological renal abnormalities coincided with those observed in Agt-KO mice (4, 5, 17), but the restoration of circulating angiotensinogen reversed renal abnormalities in MT-Agt/KO mice. After birth, pups, with the genotype of MT-Agt/KO, obtained from crossmating between Agt-KO and MT-Agt/KO mice developed over 4 weeks, but neonates with the genotype of Agt-KO almost died within a week, suggesting that MT-Agt/KO mice could escape early death in an infant stage. The developmental expression of hepatic angiotensinogen on rat is shown to be detected at the higher level in a newborn stage, especially 24 h after birth, than in the adult stage (26). In the case of mice, the angiotensinogen transcripts in the fetal liver became detectable in embryonic day 12.5 (27). In this sense, the MT-I promoter we used for the transgene construction should make it possible to express the exogenous angiotensinogen at those critical stages, because this promoter could be activated during fetal and postnatal (28 –32) stages. These findings clearly indicated that angiotensinogen is an essential factor for kidney and infant developments. Recently, Davisson et al. (33) also demonstrated complementation of reduced survival, hypotension, and renal abnormalities in Agt-KO mice by means of introducing the human renin and human angiotensinogen genes. Their resultant rescued mice were associated with the overproduction of human renin and human angiotensinogen, leading to the significantly elevated blood pressure in comparison with control mice. In contrast, our rescued mice, MT-Agt/KO, were normotensive and the levels of angiotensinogen and angiotensin I in the plasma of the rescued mice was comparable to those of WT. In our hybrid and congenic lines of Agt-KO mice (3, 16), heterozygous Agt-KO mice were also normotensive and pathologically normal in
the kidney, and they survived normally after birth and had the sustained levels of angiotensin I in the plasma despite the reduced angiotensinogen contents. The biological effects of the renin-angiotensin system on cardiovascular functions, are well known to be mediated by specific receptors, such as AT1 and AT2, in a variety of tissues (34). It has been reported that ACE-KO mice and AT1a/AT1b double-KO mice have marked hypotension, and the renal damages consisting of cortical thinning, focal area of atrophy, and renal vascular wall thickening (35, 36). Interestingly, the early death found in Agt-KO mice was also seen in AT1a/AT1b double-KO mice (36). Therefore, we conclude that the continuous expression of angiotensinogen before and after birth can supply angiotensin signals via AT1 receptors, which is required for newborn development, structural formation of kidney, and blood pressure maintenance. ACKNOWLEDGMENTS This work was supported by grants from “Research for the Future” Program (The Japan Society for the Promotion of Science: JSPS— RFTF 97L00804); the Ministry of Education, Science, Sports, and Culture; Uehara Memorial Foundation; Kanae Foundation of Research for New Medicine; The Inamori Foundation; The Asahi Glass Foundation; The Naito Foundation; The Mochida Memorial Foundation for Medical and Pharmaceutical Research; and The Nissan Science Foundation. We especially thank Professors Hiroaki Ohkubo and Ron Evans for their helpful discussion and gift of the plasmid, pMGH. We also thank the expert technical staffs (Laboratory Animal Research Center, University of Tsukuba) for maintenance of angiotensinogen-deficient mice and acknowledge our laboratory members for their helpful discussion and encouragement.
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Rescue of Angiotensinogen-Knockout Mice Junji Ishida,*,†,1 Fumihiro Sugiyama,†,‡ Keiji Tanimoto,* Keiko Taniguchi,*,† Mikio Syouji,† Eriko Takimoto,* Hisashi Horiguchi,§ Kazuo Murakami,*,‡ Ken-ichi Yagami,† and Akiyoshi Fukamizu*,‡,¶,2 *Institute of Applied Biochemistry, †Laboratory Research Animal Center, Institute of Basic Medical Sciences, and ‡Center for the Tsukuba Advanced Research Alliance (TARA), University of Tsukuba; §Molecular Pathology Center, Ibaraki Prefectural University of Health Sciences; and ¶National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba, Ibaraki 305-8572, Japan
Received October 15, 1998
Angiotensinogen, the precursor of angiotensins I and II, is a critical component of the renin-angiotensin system that plays an important role in regulating blood pressure and electrolyte homeostasis. Genetically altered mice lacking angiotensinogen (Agt-KO) showed an expected phenotype, such as marked hypotension, but unexpected ones including abnormal kidney morphology, reduced survival rates of newborns, and impaired blood–brain barrier function after cold injury. To examine whether disruption of the angiotensinogen gene is responsible for the observed phenotypes, we generated a line of mice heterozygous for the mouse angiotensinogen gene under the control of a mouse metallothionein-I promoter (MT-Agt) and crossmated transgenic mice with Agt-KO mice. The resulting mice (MT-Agt(1/2)/ Agt(2/2):MT-Agt/KO) produced the plasma level of angiotensin I comparable to that of wild-type mice, and the mutant phenotypes were rescued. These results indicated that the resultant phenotypes due to the genetic deficiency of mouse angiotensinogen can be corrected by restoring angiotensinogen and angiotensin I in the circulation. © 1998 Academic Press Key Words: angiotensinogen; angiotensin II receptors; genetic rescue; knockout mouse; renin-angiotensin system; transgenic mouse.
Angiotensinogen is constitutively secreted into the circulation mainly from the liver. Circulating renin, 1
Research Fellow of the Japan Society for the Promotion of Science. 2 To whom correspondence should be addressed at Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305, Japan. Fax: 81-298-53-6599. E-mail: akif @sakura.cc.tsukuba.ac.jp. Abbreviations used: Agt, angiotensinogen; angiotensin II type 1 receptor, AT1; angiotensin II type 2 receptor, AT2; bp, base pair; kb, kilobase; KO, knockout; MT, metallothionein. 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
synthesized predominantly in the kidney, cleaves angiotensinogen, the only known substrate for renin, to release a decapepide angiotensin I. Further processing of a physiologically inactive angiotensin I by angiotensin converting enzyme (ACE) generates an octapeptide angiotensin II, which plays an important role in blood pressure, fluid, and sodium homeostasis (1, 2). To study the role of angiotensinogen in vivo, we and other investigators generated angiotensinogen-knockout (Agt-KO) mice, which showed phenotypes with increased infant death and histopathological renal abnormalities in addition to marked hypotension and overexpression of the renin gene (3–5). More recently, we found remarkably attenuated expression of glial fibrially acidic protein and decrease laminin production in the astrocytes of Agt-KO mice in response to cold injury, and eventually incomplete reconstitution of impaired blood-brain barrier function. Furthermore, we demonstrated that these abnormalities were rescued by administration of angiotensin II or angiotensin IV (6). Introduction of desired mutations into embryonic stem cells and generation of knockout mice have now become a routine practice. The extent to which the resultant phenotypes are affected due to such mutations depends on the fact that no other alterations have occurred elsewhere in the manipulated mouse genome. To confirm that the cardiovascular defects found in Agt-KO mice are only due to an engineered mutation in the mouse angiotensinogen locus, we have genetically rescued the Agt-KO mice by introducing a fusion gene composed of a mouse metallothionein (MT) promoter and the mouse angiotensinogen gene lacking its own promoter with the first intron (MT-Agt). This clearly shows that angiotensinogen plays a critical role in maintaining blood pressure, regulating renal renin expression, histological renal preservation, and newborn development.
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MATERIALS AND METHODS Animals. Agt-KO mice were generated as previously described (3). C57BL/6J control and CD-1 mice were obtained from CLEA Japan Co. Ltd., Tokyo and Charles River Japan Inc., Yokohama, respectively. Animals were used according to the regulation of “Standards for Human Care and Use of Laboratory Animals, University of Tsukuba”. Construction of transgene. To replace the promoter of mouse angiotensinogen gene, the 1.8-kb DNA fragment (HindIII/XhoI) consisting of the 1.7-kb 59-flanking region and the 70-bp 59-untranslated region of the mouse metallothionein-1 gene was prepared from pMGH (7). The 11.8-kb structural region (XhoI/HindIII) from the second exon to poly(A) signals of the mouse angiotensinogen gene was prepared from pB6A-16B, pB6A-50 and pB6A-113 (8). Both fragments were ligated into HindIII site of pUC119 to construct pMT-Agt. Generation of transgenic mice. Eggs of CD-1 mice superovulated with PMSG-hCG treatment were collected and fertilized in vitro (9) to C57BL/6J mice sperm. Fertilized oocytes were cultured to appearance of pronucleus at 37°C in 5% CO2. The 13.6-kb linearized MTAgt fusion gene digested by HindIII from pMT-Agt (Fig. 1A) was dissolved in 0.9% NaCl solution at concentration of 2 ng/ml. The method of micromanipulation for transgenic procedure was essentially as described (10). The microinjected oocytes were developed to the 2-cell stage in modified Whitten medium. The survived 2-cell embryos were implanted into oviducts of pseudopregnant mice. DNA preparation and Southern blot analysis. Genomic DNA was prepared from mouse tail as described previously (3), digested with BamHI, electrophoresed on a 0.7% agarose gel, and subjected to Southern blot analysis using GeneScreen Plus membrane (DuponNew England Nuclear, Boston, MA). The filter was hybridized to the 32 P-labelled 0.9-kb BglII/BamHI DNA fragment containing the angiotensinogen gene, at 65°C for 16 h and washed twice with 2 3 SSC (SSC 5 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at room temperature for 5 min, twice with 2 3 SSC/1% SDS at 65°C for 30 min, and twice with 0.1 3 SSC at room temperature for 30 min. RNA preparation and Northern blot analysis. Total RNA was prepared from homogenized tissues using ISOGEN (NipponGene) based on the acid guanidium thiocyanate–phenol– chloroform extraction method (11). 15 micrograms of total RNA were denatured with glyoxal, electrophoresed on a 1.2% agarose gel, and transferred to GeneScreen Plus membrane. The filter was probed 32P-labelled DNA fragments as described below, at 60°C for 16 h and washed twice with 2 3 SSC at room temperature for 5 min, twice with 2 3 SSC/1% SDS at 60°C for 30 min, and twice with 0.1 3 SSC at room temperature for 30 min. The used DNA probe are 0.9-kb BglII/BamHI DNA fragment for angiotensinogen from pA-16B and 0.82-kb KpnI/NcoI DNA fragment for renin from pMRn226 (12), respectively. Quantitative analysis of angiotensinogen and angiotensin I. Blood was collected from inferior vena cava of mice anesthetized with pentobarbital (30 mg/kg). Plasma containing anticoagulant, EDTA z 2Na, was immediately separated at 4°C and stored at 270°C until used. Angiotensin I concentration was measured by radioimmunoassay as described (13). The concentration of angiotensinogen was determined by measurement of the amount of angiotensin I after addition of an excess of purified mouse submandibular gland renin (14). Measurement of blood pressure. Blood pressure was measured in the afternoon from 1 to 5 pm. Unanesthetized mice wrapped with a cotton holder were introduced into the warming tube thermostatically controlled at 37°C. The systolic blood pressure was measured with a programmable sphygmomanometer (BP-98A; Softron, Tokyo, Japan) by the tail-cuff method.
Histological analysis. After blood collection, phosphate buffered saline was introduced into the left ventricle via a 26 gauge needle and allowed to flow out of a cut point at the right atrium. When the perfusate became clear, perfusion was continued with 10% formaldehyde in 0.1 M phosphate buffer (PB; pH7.4). For histopathological examinations, the formalin-fixed kidney were embedded in paraffin. The sections cut from the paraffin block and then were stained with hematoxylin and eosin.
RESULTS The linearized 13.6-kb HindIII fragment (MT-Agt fusion gene) (Fig. 1A) was microinjected into a pronucleus of fertilized mouse oocytes. Forty four newborns were obtained from the pseudopregnat recipients to which the 2-cell embryos were transferred. One transgenic founder animal was identified by Southern blot analysis (data not shown) using MT-Agt probe and the line of mice heterozygous for the transgene was outwardly normal and normotensive (105 6 4.1 mmHg vs 103 6 1.5 mmHg of wild type mice (WT)). To test for rescue of the phenotypes of homozygous angiotensinogen-knockout (Agt-KO) mice, the transgene was introduced into mutant background by breeding. Agt-KO mice were first crossbred with the transgenic mice heterozygous for the MT-Agt fusion gene to give F1 mice that were WT, heterozygous for the MTAgt, heterozygous for the mutant angiotensinogen allele and also carried the transgene. The fourth F1 mice were then crossed with Agt-KO mice to give F2 progeny that were Agt-KO mice, heterozygous Agt-KO mice, heterozygous Agt-KO mice heterozygous for the MT-Agt fusion gene, and Agt-KO mice heterozygous for the MT-Agt gene (MT-Agt/KO). MT-Agt/KO mice were identified by Southern blot analysis of tail DNA digested with BglII. The 0.9-kb DNA probe detected the endogenous angiotensinogen gene as a 2-kb band in WT mice, but not in Agt-KO mice, because the corresponding region was replaced to neor cassette (Fig. 1A). On the other hand, in MT-Agt/KO mice, the MT-Agt fusion gene can be detected as a 6-kb band, because the transgene almost lacked the first intron including the BglII sites (Fig. 1A). For further analyses, we used MT-Agt/KO mice in comparison with WT and Agt-KO mice. To examine expression of the angiotensinogen gene of several tissues in MT-Agt/KO mice, we performed Northern blot analysis using the 0.9-kb DNA fragment (Fig 3). Total RNAs were isolated from the brain, heart, liver, and kidney of WT, Agt-KO, and MT-Agt/KO mice. Angiotensinogen mRNA was found in all tissues examined in WT and the highest intensity was detected in the liver, whereas its transcript in Agt-KO mice was not accumulated in any tissues used. In contrast, angiotensinogen transcripts were identified in the four tissues of MT-Agt/KO mice, although its expression level was different from that of corresponding tissues in WT mice. Ohkubo et al. (15) reported that the MT-I promoter fused to the rat angiotensinogen instead of its own promoter at the 59-flanking region can be acti-
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FIG. 1. Schematic representation of the angiotensinogen genes and Southern blot analysis of the three lines of mice. (A) C57BL/6J and Agt-KO mice are indicated as control mice and mice homozygous for the targeted angiotensinogen gene, respectively. MT-Agt/KO mice have the MT-Agt fusion genes on the Agt-KO genetic background. The coding and noncoding exons are shown as closed and open boxes, respectively. BglII, G; HindIII, H; Neomycin resistance gene, Neor; mouse metallothionein promoter, MT. (B) Southern blot analysis. Ten micrograms of genomic DNA were digested with BglII and subjected Southern hybridization with a DNA probe (closed bar in A).
vated, when induced by ZnCl2 in drinking water. No inducible expression of the MT-Agt transgene was found, even in the presence of the heavy metal in drinking water (data not shown), probably due to the integration site effect or differential methylation pattern of the transgene, although there is no direct evidence at the present time. These results indicated that angiotensinogen mRNA in MT-Agt/KO mice is transcribed from the exogenous transgene. To ensure the production of angiotensinogen and angiotensin I in the plasma of MT-Agt/KO mice, we measured their contents by radioimmunoassay. No angiotensinogen was detectable in Agt-KO mice, but its concentration in MT-Agt/KO mice was restored to the level comparable to that of WT mice (Table 1) despite the lower expression of exogenous angiotensinogen in the liver of MT-Agt/KO mice than that of WT mice (Fig. 2). This provided the possibility that circulating angio-
tensinogen may be supplied from other tissues in addition to the liver. Although the plasma level of angiotensin I in Agt-KO mice decreased below to detectable one, there was no significant difference in angiotensin I content between MT-Agt/KO and WT mice. To further test whether the genetical and biochemical restoration of angiotensinogen have physiologically beneficial effects in mice, we measured the systolic blood pressure in the three different strains. As shown in Table 1, a significant decrease in blood pressure was confirmed in Agt-KO mice as compared with WT mice. In contrast, the blood pressure of MT-Agt/KO mice returned to the normal level comparable to that of WT mice. Previously, we reported that the level of renal renin mRNA increased 6- to 8-fold in Agt-KO mice in comparison with that found in WT mice (3). To investigate the level of renal renin mRNA accumulation, we performed Northern blot hybridization using total RNAs from the
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Angiotensinogen, Angiotensin I Contents in Plasma, and Systolic Blood Pressure of the Three Lines of Mice Strains
mAG (mg/ml)
Al (ng/ml)
SBP (mmHg)
C57BL/6J mice Agt-KO mice MT-Agt/KO mice
4.8 6 0.4 (n 5 4) N.D. (n 5 4) 5.2 6 2.9 (n 5 6)
1.9 6 0.4 (n 5 4) N.D. (n 5 4) 1.7 6 0.8 (n 5 6)
103 6 1.5 (n 5 4) 70 6 3.6 (n 5 4)* 109 6 5.0 (n 5 15)
Note. mAG, mouse angiotensinogen; AI, angiotensin I; SBP, systolic blood pressure. Values are means 6 SD with an average of each group; n indicates total number of mice in each group. SBP was measured by a programmable sphygmomanometer using the tail-cuff method. mAG and AI were measured by radioimmunoassay on plasma samples. * P , 0.01 compared with wild-type mice.
kidney of MT-Agt/KO, Agt-KO, and WT mice. Interestingly, expression of the renal renin gene in MT-Agt/KO mice was suppressed to the level found in WT mice (Fig. 3), despite the significant differences of renal angiotensinogen mRNA accumulation between both the normotensive strains (Fig. 2). We next histopathologically examined the kidney in WT (Fig. 4; A and D), Agt-KO (B and E), and MT-Agt/KO (C and F) mice at the age of 8 weeks. The cross-sections stained with hematoxylin and eosin demonstrated inner medulla- and papillaryatrophy, and pelvic dilation in the unilateral or bilateral kidney in Agt-KO mice. Furthermore, the renal cortex thinned and the arterial wall thickened (arrow) comparable with those of WT mice were observed in Agt-KO mice. However, normotensive MT-Agt/KO mice showed normal histological features of the kidney. Early death of infants was observed in Agt-KO mice (3–5, 16, 17). Thus, we investigated survival rates of the pups obtained from Agt-KO mice that were crossed with MT-Agt/KO mice. In this cross experiment, newborns to be obtained were Agt-KO and MT-Agt/KO and
FIG. 2. Tissue distribution of angiotensinogen mRNA in the three lines of mice. Total RNAs were isolated from the brain (a), heart (b), liver (c), and kidney (d) of C57BL/6J, Agt-KO, and MTAgt/KO and subjected to Northern blot analysis (15 mg/lane) using a probe shown in Fig. 1A.
the Mendelian ratio was expected to be 1:1. Of 42 newborns, only 28 infants grew to a weaning stage. In order to examine the genotypes of survived mice, we performed Southern blot analysis using the 0.9-kb DNA as a probe and summarized the results in Table 2. Twenty four and 4 were identified as MT-Agt/KO and Agt-KO mice, respectively. These results suggested the improved survival rates of Agt-KO mice after birth by genetically introducing angiotensinogen. DISCUSSION The gene targeting system is an indispensable means for analysing in vivo function of cloned genes of interest. However, it is possible that the homologous recombination by deletion and replacement with neor cassette unexpectedly introduces a mutation into other gene(s) in addition to the targeted gene. For examples, Ohno et al. produced a gene-targeted mouse lacking CD3h chain to analyze its role in T cell development and function (18). Most of CD3h-deficient mice died within a few days after birth despite of normal development and function in the T cells. Genetic analysis has revealed that CD3h locus encodes a nuclear transcription factor, Oct-1, on the opposite strand (19), suggesting an important possibility of a complicated interpretation in the gene targeting experiment in-
FIG. 3. Northern blot analysis of renal renin mRNA in the three lines of mice. Total RNAs (15 mg) from the kidney of C57BL/6J, Agt-KO, and MT-Agt/KO mice were subjected to Northern blot analysis using the Ren-2 and b-actin DNA probe.
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FIG. 4. Histopathological analysis of kidney section in the three lines of mice. Magnifications of upper and bottom panels are 340 and 3400, respectively. Cross-sections stained with hematoxylin and eosin. Agt-KO mice (B, E) show renal cortex thinned, atrophy of papilla and inner medulla partially, and arterial wall thickened by hyperplasia. The kidney morphology of MT-Agt/KO (C and F) mice is histopathologically consist with that of C57BL/6J (A and D).
duced by unexpected mutation of gene(s) on the opposite strand (20, 21). Therefore, chemical or protein rescue experiments on gene-targeting mice should be necessary to characterize its real functions of the targeted gene, but it is often difficult to assess the functional contribution of administered substances in fetal, neonatal, and infant stages. To avoid such frustrate
situations, we reconstituted the mouse angiotensinogen transgene composed of the MT-I promoter with the angiotensinogen genomic DNA fragment including the exon 2 to 5, resulting in the enforced unidirectional transcription unit (MT-Agt), generated mice heterozygous for the MT-Agt, and introduced it into Agt-KO genetic background by crossbreeding.
TABLE 2
Survival Analysis of MT-Agt/KO and Agt-KO Mice Breeding form
No. of newborns
MT-Agt/KO 3 Agt-KO
42
a
No. of weanings surviveda
No. of Agt-KO mice
No. of MT-Agt/KO mice
Ratio
42 28
21 4
21 24
1:1 0.17:1
Expected Observed
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It has been shown that chronic administrations of ACE inhibitors in mouse or rat enhance intrarenal renin synthesis (22, 23). Like ACE inhibition experiments, we observed the increased accumulation of renin mRNA in the kidney of Agt-KO mice associated with the complete loss of angiotensin I (3). In contrast to Agt-KO mice, the repressed levels of renal renin mRNA comparable to that of WT mice were seen in MT-Agt mice, suggesting that the restoration of circulating angiotensinogen and angiotensin I upon MTAgt/KO mice stimulates a negative regulation of the renin gene expression in the kidney. A recent histological study by Kakuchi et al. (24), using in situ hybridization, indicated that renal AT1 expression in mouse is corresponded temporally and spatially with differentiation and proliferation of glomerular mesangial and tubular cells. Furthermore, it has been shown that neonatal treatments with either an ACE inhibitor or an AT1 blocker in rats produce persistent, irreversible histopathological renal abnormalities including palliary atrophy and pelvic dilation (25). Such histopathological renal abnormalities coincided with those observed in Agt-KO mice (4, 5, 17), but the restoration of circulating angiotensinogen reversed renal abnormalities in MT-Agt/KO mice. After birth, pups, with the genotype of MT-Agt/KO, obtained from crossmating between Agt-KO and MT-Agt/KO mice developed over 4 weeks, but neonates with the genotype of Agt-KO almost died within a week, suggesting that MT-Agt/KO mice could escape early death in an infant stage. The developmental expression of hepatic angiotensinogen on rat is shown to be detected at the higher level in a newborn stage, especially 24 h after birth, than in the adult stage (26). In the case of mice, the angiotensinogen transcripts in the fetal liver became detectable in embryonic day 12.5 (27). In this sense, the MT-I promoter we used for the transgene construction should make it possible to express the exogenous angiotensinogen at those critical stages, because this promoter could be activated during fetal and postnatal (28 –32) stages. These findings clearly indicated that angiotensinogen is an essential factor for kidney and infant developments. Recently, Davisson et al. (33) also demonstrated complementation of reduced survival, hypotension, and renal abnormalities in Agt-KO mice by means of introducing the human renin and human angiotensinogen genes. Their resultant rescued mice were associated with the overproduction of human renin and human angiotensinogen, leading to the significantly elevated blood pressure in comparison with control mice. In contrast, our rescued mice, MT-Agt/KO, were normotensive and the levels of angiotensinogen and angiotensin I in the plasma of the rescued mice was comparable to those of WT. In our hybrid and congenic lines of Agt-KO mice (3, 16), heterozygous Agt-KO mice were also normotensive and pathologically normal in
the kidney, and they survived normally after birth and had the sustained levels of angiotensin I in the plasma despite the reduced angiotensinogen contents. The biological effects of the renin-angiotensin system on cardiovascular functions, are well known to be mediated by specific receptors, such as AT1 and AT2, in a variety of tissues (34). It has been reported that ACE-KO mice and AT1a/AT1b double-KO mice have marked hypotension, and the renal damages consisting of cortical thinning, focal area of atrophy, and renal vascular wall thickening (35, 36). Interestingly, the early death found in Agt-KO mice was also seen in AT1a/AT1b double-KO mice (36). Therefore, we conclude that the continuous expression of angiotensinogen before and after birth can supply angiotensin signals via AT1 receptors, which is required for newborn development, structural formation of kidney, and blood pressure maintenance. ACKNOWLEDGMENTS This work was supported by grants from “Research for the Future” Program (The Japan Society for the Promotion of Science: JSPS— RFTF 97L00804); the Ministry of Education, Science, Sports, and Culture; Uehara Memorial Foundation; Kanae Foundation of Research for New Medicine; The Inamori Foundation; The Asahi Glass Foundation; The Naito Foundation; The Mochida Memorial Foundation for Medical and Pharmaceutical Research; and The Nissan Science Foundation. We especially thank Professors Hiroaki Ohkubo and Ron Evans for their helpful discussion and gift of the plasmid, pMGH. We also thank the expert technical staffs (Laboratory Animal Research Center, University of Tsukuba) for maintenance of angiotensinogen-deficient mice and acknowledge our laboratory members for their helpful discussion and encouragement.
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