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• W2105755732 abstract "Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression ofHAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice. Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression ofHAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice. The renin-angiotensin system (RAS) 1The abbreviations used are: RAS, renin-angiotensin system; AGT, angiotensinogen; Ang-I or II, angiotensin I or II; PCT, proximal convoluted tubule; KAP, kidney androgen-regulated protein; bp, base pair(s); PCR, polymerase chain reaction; kb, kilobase pair(s); HAGT, human AGT. is a classical endocrine system activated by the release of renin from the kidney and angiotensinogen (AGT) from the liver. In blood, renin proteolytically cleaves AGT to form angiotensin I (Ang-I) which is further processed by angiotensin converting enzyme to form Ang-II, a potent vasoconstrictor and antinatriuretic peptide. The RAS has been implicated in the genetic basis of hypertension and pre-eclampsia (1Jeunemaitre X. Soubrier F. Kotelevtsev Y.V. Lifton R.P. Williams C.S. Charru A. Hunt S.C. Hopkins P.N. Williams R.R. Lalouel J. Corvol P. Cell. 1992; 71: 169-180Abstract Full Text PDF PubMed Scopus (1715) Google Scholar, 2Ward K. Hata A. Jeunemaitre X. Helin C. Nelson L. Namikawa C. Farrington P.F. Ogasawara M. Suzumori K. Tomoda S. Berrebi S. Sasaki M. Corvol P. Lifton R.P. Lalouel J. Nat. Genet. 1993; 4: 59-61Crossref PubMed Scopus (449) Google Scholar, 3Rapp J.P. Wang S.M. Dene H. Science. 1989; 243: 542-544Crossref PubMed Scopus (283) Google Scholar, 4Kurtz T.W. Simonet L. Kabra P.M. Wolfe S. Chan L. Hjelle B.L. J. Clin. Invest. 1990; 85: 1328-1332Crossref PubMed Scopus (160) Google Scholar). Our understanding of the RAS in normal and pathophysiological regulation of blood pressure has been complicated by the fact that in addition to its actions as an endocrine system, certain individual tissues, such as the kidney (5Johnston C.I. Fabris B. Jandeleit K. Kidney Int. 1993; 42: S59-S63Google Scholar, 6Hall J.E. Compr. Ther. 1991; 17: 8-17PubMed Google Scholar, 7Levens N.R. Peach M.J. Carey R.M. Circ. Res. 1981; 48: 157-167Crossref PubMed Scopus (162) Google Scholar), heart (8Lindpaintner K. Ganten D. Cardiology. 1991; 79: 32-44Crossref PubMed Scopus (18) Google Scholar, 9Lindpaintner K. Ganten D. Acta Cardiol. 1991; XLVI: 385-397Google Scholar), brain (10Bunnemann B. Fuxe K. Ganten D. J. Cardiovasc. Pharmacol. 1992; : S51-S62Crossref PubMed Scopus (79) Google Scholar), and vasculature (11Dzau V.J. Hypertension. 1986; 8: 553-559Crossref PubMed Scopus (270) Google Scholar), contain all the components of the RAS cascade and therefore have the potential for local synthesis and action of Ang-II. In the kidney, for example, renin, AGT and ACE mRNAs, and proteins are synthesized in juxtaglomerular cells, proximal convoluted tubule (PCT) cells, and endothelial and tubular cells, respectively, and Ang-II type-1 (AT-1) and type-2 (AT-2) receptors are localized in glomeruli, collecting ducts, tubules, and vasa recta (12Ingelfinger J.R. Zuo W.M. Fon E.A. Ellison K.E. Dzau V.J. J. Clin. Invest. 1990; 85: 417-423Crossref PubMed Scopus (305) Google Scholar, 13Kakinuma Y. Fogo A. Inagami T. Ichikawa I. Kidney Int. 1993; 43: 1229-1235Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 14Taugner R. Hackenthal E. Helmchen U. Ganten D. Kugler P. Marin-Grez Nobiling R. Unger T. Lockwald I. Keilbach R. Klin. Wochenschr. 1982; 60: 1218-1222Crossref PubMed Scopus (73) Google Scholar, 15Gomez R.A. Lynch K.R. Chevalier R.L. Wilfong N. Everett A. Carey R.M. Peach M.J. Am. J. Physiol. 1988; 254: F582-F587Crossref PubMed Google Scholar, 16Tufro-McReddie A. Harrison J.K. Everett A.D. Gomez R.A. J. Clin. Invest. 1993; 91: 530-537Crossref PubMed Scopus (127) Google Scholar, 17Jones C.A. Sigmund C.D. McGowan R. Kane-Haas C. Gross K.W. Mol. Endocrinol. 1990; 4: 375-383Crossref PubMed Scopus (105) Google Scholar, 18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). The intrarenal RAS has been postulated to regulate various aspects of renal function including blood flow, natriuresis, and tubular-glomerular feedback, and may therefore participate in the pathogenesis of hypertension (19Hall J.E. Guyton A.C. Mizelle H.L. Acta Physiol. Scand. Suppl. 1990; 591: 48-62PubMed Google Scholar, 20Hall J.E. Guyton A.C. Tripodo N.C. Lohmeier T.E. McCaa R.E. Cowley A.W.J. Am. J. Physiol. 1977; 232: F538-F544PubMed Google Scholar, 21Mattson D.L. Raff H. Roman R.J. Am. J. Physiol. 1991; 260: R1200-R1209PubMed Google Scholar). Our current understanding of the relative importance of the intrarenalversus systemic RAS comes largely from pharmacological studies (22Peng Y. Knox F.G. Am. J. Physiol. 1995; 269: F40-F46PubMed Google Scholar) which have been limited by the specificity of inhibitors, the ability to deliver agents to specific regions of the kidney, and their differential actions in the kidney and systemic circulation. Indeed, progress in our understanding of the role of the intrarenal RAS has been hampered by the lack of tools that separate the effects of the endocrine (blood borne) RAS from individual tissue RAS. Normally, circulating AGT is derived from the liver where it is expressed at a high level and constitutively released. Transgenic mice containing a human AGT (HAGT) gene construct containing its own endogenous promoter express the transgene in hepatocytes of the liver, but also in PCT cells of the kidney, and in a number of other tissues (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). Double transgenic mice containing both the HAGT gene and also the human renin gene, encoding the species-specific processing protease for HAGT, are chronically hypertensive (23Merrill D.C. Thompson M.W. Carney C.L. Granwehr B.P. Schlager G. Robillard J.E. Sigmund C.D. J. Clin. Invest. 1996; 97: 1047-1055Crossref PubMed Scopus (159) Google Scholar, 24Fukamizu A. Sugimura K. Takimoto E. Sugiyama F. Seo M.-S. Takahashi S. Hatae T. Kajiwara N. Yagami K. Murakami K. J. Biol. Chem. 1993; 268: 11617-11621Abstract Full Text PDF PubMed Google Scholar) due to co-activation of both endocrine and tissue RAS. Clearly, the identification and characterization of a promoter capable of specifically targeting renal PCT cells would lead to the development of a novel mechanism to specifically activate the intrarenal RAS independently of the systemic RAS. The kidney androgen-regulated protein (KAP) was originally identified as an abundant 20,000-Da polypeptide product derived from in vitro translation of mouse kidney RNA (25Toole J.J. Hastie N.D. Held W.A. Cell. 1979; 17: 441-448Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Although the function of the KAP protein remains unknown, it is encoded by a highly abundant 850-bp mRNA which accumulates to 4–5% of the total poly(A) mRNA in kidney of androgen-stimulated mice, suggesting the use of a highly active and efficient promoter (26Catterall J.F. Kontula K.K. Watson C.S. Seppanen P.J. Funkenstein B. Melanitou E. Hickok N.J. Bardin C.W. Janne O.A. Recent Prog. Horm. Res. 1986; 42: 71-103PubMed Google Scholar). Androgen treatment causes a 3–4-fold stimulation of KAP mRNA (26Catterall J.F. Kontula K.K. Watson C.S. Seppanen P.J. Funkenstein B. Melanitou E. Hickok N.J. Bardin C.W. Janne O.A. Recent Prog. Horm. Res. 1986; 42: 71-103PubMed Google Scholar) and a similar accumulation of angiotensinogen mRNA (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar) in the kidney. In situhybridization studies showed that KAP mRNA is localized in PCT cells of the renal cortex in normal male and testosterone-induced female mice (27Meseguer A. Catterall J.F. Mol. Endocrinol. 1987; 1: 535-541Crossref PubMed Scopus (48) Google Scholar); and its expression is under complex hormonal control involving androgens, estrogens, and pituitary hormones (28Meseguer A. Catterall J.F. Mol. Endocrinol. 1990; 4: 1240-1248Crossref PubMed Scopus (39) Google Scholar, 29Meseguer A. Catterall J.F. Mol. Cell Endocrinol. 1992; 89: 153-162Crossref PubMed Scopus (21) Google Scholar). Based on the cellular localization of KAP mRNA in the kidney and its induction by androgen, we hypothesized that the KAP promoter and regulatory sequences would specifically target expression of theHAGT gene to the renal PCT cells. We show herein that under the control of 1542 bp of the KAP promoter, HAGT gene expression is restricted to renal PCT cells and is strongly induced in female mice in response to androgen treatment. The transgene used herein consists of 1542 bp of the KAP promoter fused to the coding region of the HAGT gene. In the first step of the construction, aSacI to XbaI fragment encoding a portion of the 5′-flanking region of the KAP gene (−1542 to −466) was excised from a λ phage genomic clone (30Niu E.M. Meseguer A. Catterall J.F. DNA Cell Biol. 1991; 10: 41-48Crossref PubMed Scopus (13) Google Scholar) and joined with a PCR amplified fragment encoding DNA from the same XbaI site (−466) to coordinate −1 relative to the start site of transcription. This PCR fragment was engineered to contain a BglII site downstream of the PCR product. The first A in the BglII recognition sequence (AGATCT) became the transcription start site (+1) of the transgene. The resulting SacI to BglII fragment extending from −1542 to +6 was then cloned first into pGL2-Basic (Promega, Madison, WI) to form the plasmid 1542-KAP-Luc, and then subcloned as a KpnI to HindIII fragment into pBluescript II SK− to form the plasmid 1542-KAP-SK. TheHAGT coding region was cloned from a previously described genomic clone (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar) as a BglII to NheI fragment into the unique BglII and SpeI sites in 1542-KAP-SK to form the plasmid 1542-KAP-HAGT.NheI and SpeI have compatible ends. TheBglII site in HAGT resides within intron I, 70 bp upstream of exon II, and in this construct forms the 5′-untranslated region of the gene. Translation initiation of HAGT starts 4 bp into exon II. All cloning junctions were confirmed by sequence analysis. The transgene segment of the final plasmid was excised by digestion with KpnI and NotI and the transgene segment was purified away from the remainder of the plasmid backbone by agarose gel electrophoresis. The transgene DNA was recovered using the Qia-Quick purification kit (Qiagen) and was microinjected at a concentration of 2 ng/μl in 10 mm Tris-Cl, pH 7.5, 0.1 mm EDTA made with embryo culture-certified water into one-cell fertilized mouse embryos obtained from superovulated C57BL/6J X SJL/J (B6SJL F2) mice using standard procedures (31Hogan E. Costantini F. Lacy E. Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar, 32Sigmund C.D. Hypertension. 1993; 22: 599-607Crossref PubMed Scopus (45) Google Scholar). All transgenic mice were heterozygous for the KAP-HAGT construct. Female mice were treated with testosterone by the administration of a 5-mg testosterone pellet designed for continuous release for 21 days (catalog number A-151, Innovative Research of America, Sarasota, FL). Mice were anesthetized with the inhalation anesthetic metofane and the pellet was implanted subcutaneously in the back and tunneled to the nape of the neck using a 10-gauge trocar. The incision was closed with a 6-0 silk suture and the mice were allowed to recover on a heating pad. The duration of the procedure was less than 5 min. The testosterone treatment was allowed to proceed for 5 days at which time experimental and sham operated control mice were killed. All mice were fed standard mouse chow and water ad libitum. Care of the mice used in the experiments met or exceeded the standards set forth by the National Institutes of Health in their guidelines for the care and use of experimental animals. All procedures were approved by the University Animal Care and Use Committee at the University of Iowa. Genomic DNA was purified from tail biopsies and subjected to Southern blot analysis for the identification of founder animals or to PCR analysis for the identification of transgenic offspring (33Sigmund C.D. Jones C.A. Kane C.M. Wu C. Lang J.A. Gross K.W. Circ. Res. 1992; 70: 1070-1079Crossref PubMed Scopus (95) Google Scholar). To identify transgenic founders, 10 μg of tail genomic DNA was digested withBglII and BamHI and probed with a genomic segment encompassing exon 2 and intron 2 of HAGT (see Fig. 1). A 2.9-kb band was diagnostic of the presence of the transgene. There was no significant cross-reactivity of this probe with mouse genomic DNA. PCR analysis was performed on approximately 10–50 ng of tail DNA using primers specific for HAGT and amplified a 539-bp segment internal to exon 2 as described previously (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). Tissue-specific expression studies were performed by Northern blot analysis using: 1) a HAGT cDNA probe derived from exon 2 at nucleotide coordinates 302–840 relative to the start site of transcription (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar), and 2) a KAP cDNA probe derived by cloning a reverse transcriptase-PCR product encompassing coordinates 93–521. The oligonucleotides used to clone the KAP cDNA were 5′-ACTGTGGCTTTCCCCCTGTC-3′ and 5′-CTTCCTCGTTCTTTCTTCTTTG-3′. Both probes were cloned using an AT cloning kit (Invitrogen); and the orientation of each probe in the vector was confirmed by sequence analysis. Single-stranded antisense RNA was generated by SP6 transcription of the HAGT vector and by T7 of the KAP vector as described previously (33Sigmund C.D. Jones C.A. Kane C.M. Wu C. Lang J.A. Gross K.W. Circ. Res. 1992; 70: 1070-1079Crossref PubMed Scopus (95) Google Scholar). Total tissue RNAs were isolated by homogenization in guanidine isothiocyanate followed by phenol emulsion extraction at pH 4.0 using a modification of the method previously described (34Burson J.M. Aguilera G. Gross K.W. Sigmund C.D. Am. J. Physiol. 1994; 267: E260-E267PubMed Google Scholar, 35Chomczynski P. Sacchi N. Anal. Biochem. 1987; 161: 156-159Crossref Scopus (63232) Google Scholar). Homogenization was scaled up to 2.5 ml to increase RNA yield and quality. To ensure the specific detection ofHAGT and KAP transcripts, Northern blots were treated with 1.0 μg/ml RNase A in 2 × standard saline citrate for 15 min at room temperature. We have previously demonstrated that this procedure removes nonspecific hybridization of single-stranded RNA probes (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar,33Sigmund C.D. Jones C.A. Kane C.M. Wu C. Lang J.A. Gross K.W. Circ. Res. 1992; 70: 1070-1079Crossref PubMed Scopus (95) Google Scholar). Primer extension was performed using a modification of the procedure previously reported (36Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, New York1989Google Scholar). A 30-base oligonucleotide with the sequence 5′-AAATATCATTTTGCAAAGGGTGAAAGGTGG-3′ was end labeled using [γ-32P]ATP and T4 polynucleotide kinase. This oligonucleotide was designed antisense to KAP-HAGT mRNA in the 5′-untranslated region so that a 37-base extension would result if transcription was initiated at the major KAP transcriptional start site. The labeled oligonucleotide was purified from a denaturing 8% polyacrylamide, 7 m urea gel, eluted into 0.5 mammonium acetate and 10 mm magnesium acetate, and ethanol precipitated. 50,000 dpm of labeled oligonucleotide was hybridized with 40 μg of total kidney RNA in a total volume of 20 μl containing Sequenase reaction buffer (U. S. Biochemical Corp.) in a thermal cycler (MJ Research). We used a thermal profile in which the samples were first incubated at 95 °C for 2 min, followed by a gradual ramped cooling cycle to 65 °C over 6 min, incubation at 65 °C for 90 min, followed by a gradual cooling to room temperature at a rate of 1.5 °C/min. The annealing mixture was diluted into avian myeloblastosis virus reverse transcriptase buffer containing potassium chloride (provided by the manufacturer) in a volume of 40 μl containing 0.25 mm dATP, dCTP, dGTP, TTP, and 2.5 units of avian myeloblastosis virus reverse transcriptase (Bethesda Research Laboratories) and incubated at 42 °C for 90 min. The primer extension products were purified by ethanol precipitation and run on 6% polyacrylamide, 7 m urea sequencing gels. The same end-labeled oligonucleotide was also used to sequence the 1542-KAP-HAGT plasmid DNA using the Sequenase kit using the protocol recommended by the manufacturer (U. S. Biochemical Corp.) for using prelabeled primers. The primer extension products and sequencing reactions were run in adjacent lanes on the same gel. In situ hybridization was used to examine the cellular localization of KAP and KAP-HAGT mRNA in the kidney of transgenic mice. Tissues were removed and immediately frozen in liquid nitrogen. Frozen sections were cut 8 μm on a Reichert-Jung cryostat and were hybridized to antisense and sense HAGT or KAP RNA probes labeled as described previously, except that digoxigenin-labeled uridine triphosphate (UTP) (studies performed at the Population Council) or3H-labeled UTP (studies performed in Iowa) was used as a label (17Jones C.A. Sigmund C.D. McGowan R. Kane-Haas C. Gross K.W. Mol. Endocrinol. 1990; 4: 375-383Crossref PubMed Scopus (105) Google Scholar, 28Meseguer A. Catterall J.F. Mol. Endocrinol. 1990; 4: 1240-1248Crossref PubMed Scopus (39) Google Scholar, 37Sigmund C.D. Jones C.A. Jacob H. Ingelfinger J. Kim U. Gamble D. Dzau V.J. Gross K.W. Am. J. Physiol. 1991; 260: F249-F257Crossref PubMed Google Scholar). The partial cDNA clones described above were used for in situ hybridization. No HAGT signal was detected in kidney tissue from non-transgenic mice. The3H-labeled sections were stained with hematoxylin and eosin. Detection of digoxigenin-labeled probes was by enzyme immunoassay and enzyme-catalyzed color reaction using the protocol provided by the manufacturer (Boehringer Mannheim). Sense and antisense probes were used on adjacent sections from the same tissue block. Mouse and human angiotensinogen protein were determined in plasma as described previously (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). The assay differentiates mouse angiotensinogen from human angiotensinogen on the basis of the species-specificity of the biochemical reaction between renin and angiotensinogen (38Hatae T. Takimoto E. Murakami K. Fukamizu A. Mol. Cell. Biochem. 1994; 131: 43-47Crossref PubMed Scopus (64) Google Scholar). Briefly, transgenic KAP-HAGT and negative control littermates were sacrificed by CO2 asphyxiation. Approximately 0.5 ml of fresh blood was collected from the aorta and placed in chilled tubes and EDTA was added to a final concentration of 2.5 mmol/liter. Plasma was obtained by centrifugation at 14,000 rpm in a microcentrifuge for 5 min and 150 μl of plasma was immediately frozen at −80 C. Radioimmunoassays were performed using the RIANEN Angiotensin I 125I-labeled RIA kit (DuPont Co., Billerica, MA) using the directions and reagents supplied by the manufacturer. Plasma samples were thawed in an ice bath and 3 μl of dimercaprol, 3 μl of 8-hydroxyquinoline, and 300 μl of maleate buffer were added to the plasma (all reagents were obtained with the radioimmunoassay kit). Samples were then split into 2 tubes. Tube A was incubated without any further additives and used endogenous mouse renin. Tube B was incubated with 17 μg (3.33 units/mg specific activity) of purified human renin in normal saline (Scripps Laboratories, San Diego, CA). Both samples were incubated at 37 °C. Samples were removed from incubation at 0, 15, 30, 45, 60, 120, and 240 min. Samples were appropriately diluted with reagent blank so that the radioimmunoassay results were on the linear portion of a standard curve. The amount of angiotensin-I generated at each time point was then obtained by comparison to a standard curve generated each time the assay was performed. The angiotensin-I produced was then plottedversus time and saturation kinetics (due to conversion of all angiotensinogen to angiotensin-I) was observed by 60 min (data not shown). Plasma levels of angiotensinogen were then extrapolated using the 1:1 molar relationship between angiotensin-I and its precursor, angiotensinogen, using the 120-min time point. The amount of human angiotensinogen was calculated by subtracting the total angiotensinogen in tube A (mouse angiotensinogen) from tube B (human plus mouse angiotensinogen). To detect urinary angiotensinogen, the above assay was used except 150 μl of urine replaced the plasma. No other changes were made in the assay protocol. Transgenic mice containing a KAP-HAGT transgene were developed to accomplish two specific goals. The first was to determine if 1542 bp of the KAP promoter would target the expression of a heterologous gene specifically to PCT cells of the kidney. The second and long term goal of these studies is to develop a model to examine the functional importance of the intrarenal RAS by expressingHAGT within the kidney, but not in any other extra-renal tissues. We chose the KAP promoter for these studies by virtue of its kidney-specific expression and because, like angiotensinogen, KAP is expressed in PCT cells and its expression is responsive to androgens. Therefore, expression of HAGT under KAP promoter control would not change the overall cellular expression normally exhibited byAGT in the kidney. Transgenic mice were generated with a construct containing 1542 bp of the KAP promoter fused to a 10.3-kb HAGT genomic clone encompassing exons II, III, IV, and V, the intervening introns, a 70-bp segment derived from the 3′-end of intron I, and the native 3′-end of the HAGT gene containing the poly(A) sites (Fig.1 A). This fusion results in the utilization of DNA normally present in intron I as a 5′-untranslated region. Of 58 live born offspring, 4 transgenic founders containing this construct were identified by both PCR (data not shown) and Southern blot (Fig. 1 B). Three of these founders were successfully bred to establish transgenic lines, two of which, derived from founders 1827/1 (Fig. 1, +1) and 1848/3 (Fig. 1 B, +3), transmitted the transgene to approximately 50% of both males and females, indicating insertion in autosomes. However, the transgene in the third line, derived from founder 1841/1, was transmitted to 100% of males (of 26 tested) but 0% of females (of 15 tested), indicating an insertion into the Y chromosome. This line was discontinued as there was no evidence of transgene expression (data not shown). Fig. 2 shows the expression ofHAGT in male and female transgenic mice containing a genomic construct controlled by its own endogenous promoter (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). Under the control of 1.2 kb of its own promoter, HAGT mRNA is expressed at the highest level in liver, at a moderate level in kidney, white adipose tissue, diaphragm, and aorta, and at low levels in heart, brown adipose tissue, skeletal muscle, and reproductive tissues (Fig.2). In contrast, under control of the KAP promoter, expression ofHAGT mRNA is evident in kidney, but not in liver, heart, lung, brain, adipose tissue, submandibular gland, spleen, muscle, adrenal gland, or vascular tissue (Fig.3, A and B). This kidney-specific pattern of expression was observed in two independent lines of KAP-HAGT mice, although the level of expression in line 1827/1 (Fig. 3 A) was reproducibly higher than in line 1848/3 (Fig. 3 B). As with the expression of many transgenes, there was no correlation between transgene copy number, which was higher in 1848/3 (Fig. 1), and level of transgene expression, which was higher in 1827/1 (Fig. 3). Importantly, the expression ofHAGT in these mice paralleled the tissue-specific expression of the endogenous KAP mRNA (Fig. 3 C).Figure 3Expression of the KAP-HAGTtransgene in male transgenic mice. Northern blots of total tissue RNA from a representative male KAP-HAGT line 1827/1 (A and C) and KAP-HAGT line 1848/3 (B) probed with antisense RNA probes for HAGT(A and B) and endogenous KAP (Panel C). K L, left kidney;K R, right kidney; L, liver;H, heart; Lg, lung; B, brain;Wa, white adipose tissue; Ba, brown adipose tissue; Sg, submandibular gland; Sp, spleen;Sk, skeletal muscle; D, diaphragm; Ag, adrenal gland; Ao, aorta; T, testes. Exposure time was 1 day.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The level of HAGT mRNA in kidney when driven by its endogenous promoter is approximately 4-fold higher in male mice than in female mice (Fig. 2, compare panel A to B), consistent with the androgen responsiveness reported previously by us (18Yang G. Merrill D.C. Thompson M.W. Robillard J.E. Sigmund C.D. J. Biol. Chem. 1994; 269: 32497-32502Abstract Full Text PDF PubMed Google Scholar). Similarly, expression of endogenous KAP mRNA in the kidney is about 4-fold higher in male than in untreated-female mice (Fig.4 B). It is therefore interesting to note that there was essentially no detectable expression of the KAP-HAGT transgene in the kidney of untreated-female mice (Fig. 4 A). This suggests the possibility that the 1542-bp segment of the KAP promoter employed in this construct may have a strong dependence on androgen for expression. To test this directly, female transgenic littermates were either left untreated or were administered a testosterone (T) pellet (9.2 mg/kg/day) for a period of 5 days. The level of endogenous KAP and transgene mRNA was then examined in kidney RNA samples from control male, T-treated female, and control female mice. Representative samples from line 1827/1 are shown in Fig. 4. As expected, T-treatment caused a 3–4-fold induction in endogenous KAP mRNA (Fig. 4 B), consistent with the responsiveness of the KAP promoter to androgens as previously reported (26Catterall J.F. Kontula K.K. Watson C.S. Seppanen P.J. Funkenstein B. Melanitou E. Hickok N.J. Bardin C.W. Janne O.A. Recent Prog. Horm. Res. 1986; 42: 71-103PubMed Google Scholar). This induction resulted in an approximately equal level of KAP mRNA in males and T-treated females. This is in stark contrast to the marked induction (estimated to be at least 100-fold) of KAP-HAGT transgene mRNA in the" @default.
• W2105755732 created "2016-06-24" @default.
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• W2105755732 creator A5046657367 @default.
• W2105755732 creator A5057041329 @default.
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• W2105755732 date "1997-10-01" @default.
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• W2105755732 title "The Kidney Androgen-regulated Protein Promoter Confers Renal Proximal Tubule Cell-specific and Highly Androgen-responsive Expression on the Human Angiotensinogen Gene in Transgenic Mice" @default.
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