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• W2020114266 abstract "Angiotensin-dependent gene expression in the developing rat kidney. We aimed to identify genes involved in the growth effects of angiotensin II (Ang II) during kidney development. In rats treated from birth with the Ang II type-1 receptor blocker losartan, expression of transforming growth factor β1 (TGF-β1), platelet-derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), as measured by Northern blot, did not change significantly (N = 4 to 6 per group each). Differential display methods, used to identify genes with Ang II-dependent expression, produced mostly false positives. We identified one novel rat partial cDNA, termed AD.5, that is related to a human orphan receptor. AD.5 was expressed in a developmentally regulated pattern and may be involved in kidney development and/or the trophic actions of Ang II. Angiotensin-dependent gene expression in the developing rat kidney. We aimed to identify genes involved in the growth effects of angiotensin II (Ang II) during kidney development. In rats treated from birth with the Ang II type-1 receptor blocker losartan, expression of transforming growth factor β1 (TGF-β1), platelet-derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), as measured by Northern blot, did not change significantly (N = 4 to 6 per group each). Differential display methods, used to identify genes with Ang II-dependent expression, produced mostly false positives. We identified one novel rat partial cDNA, termed AD.5, that is related to a human orphan receptor. AD.5 was expressed in a developmentally regulated pattern and may be involved in kidney development and/or the trophic actions of Ang II. angiotensin II differential display epidermal growth factor polymerase chain reaction platelet-derived growth factor B transforming growth factor β1 vascular endothelial growth factor The vasoconstrictor peptide angiotensin II (Ang II) is necessary for the normal development of the kidney, in particular for renal vessels and tubules1.Tufro-Mc Reddie A. Romano L.M. Harris J.M. Ferder L. Gomez R.A. Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar,4.Hilgers K.F. Norwood V.F. Gomez R.A. Angiotensin's role in renal development.Semin Nephrol. 1997; 17: 492-501PubMed Google Scholar. To identify genes exhibiting Ang II-dependent expression during kidney development, we studied the expression of growth factors [transforming growth factor β1 (TGF-β1), platelet-derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF)] after blockade of the Ang II type-1 receptor in neonatal rats using polymerase chain reaction (PCR)-based differential display (DD)5.Liang P. Averboukh L. Pardee A.B. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization.Nucleic Acid Res. 1993; 21: 3269-3275Crossref PubMed Scopus (875) Google Scholar and RNA fingerprinting6.Welsh J. Chada K. Dalal S.S. Cheng R. Ralph D. Mc Clelland M. Arbitrarily primed PCR fingerprinting of RNA.Nucleic Acid Res. 1992; 20: 4965-4970Crossref PubMed Scopus (482) Google Scholar. Neonatal Sprague-Dawley rats (Hilltop Farms, Scottsdale, PA, USA) were treated from the day of birth with daily subcantaneous injections of the Ang II type-1 receptor antagonist losartan (25 mg/kg) or saline (N = 20 each) as described1.Tufro-Mc Reddie A. Romano L.M. Harris J.M. Ferder L. Gomez R.A. Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar. Rats were sacrificed on day 13, and the kidney RNA was extracted. Northern blots3.Hilgers K.F. Reddi V. Krege J.H. Smithies O. Gomez R.A. Aberrant renal vascular morphology and renin expression in mutant mice lacking angiotensin converting enzyme.Hypertension. 1997; 29: 216-221Crossref PubMed Google Scholar loaded with 10 to 20 μg total RNA or 2 μg poly(A+) RNA were hybridized with a 206-bp probe for VEGF, with full-length cDNAs for renin, PDGF-B, TGF-β1, and EGF, as well as with DD cDNA fragments. Probes were labeled with α[32]P-CTP (DuPont NEN, Boston, MA, USA) by random prime or PCR labeling3.Hilgers K.F. Reddi V. Krege J.H. Smithies O. Gomez R.A. Aberrant renal vascular morphology and renin expression in mutant mice lacking angiotensin converting enzyme.Hypertension. 1997; 29: 216-221Crossref PubMed Google Scholar. Signals were quantified by a PhosphoImage screen (Molecular Dynamics, Sunnyvale, CA, USA). Data were subjected to analysis of variance and are expressed as mean ± sem. DD-PCR was performed using the polyA+-anchored primer 5′-TTTTTTTTTTTTMA-3′ for reverse transcription and PCR; cycling conditions and arbitrary 10mer primers were selected as described by Liang et al5.Liang P. Averboukh L. Pardee A.B. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization.Nucleic Acid Res. 1993; 21: 3269-3275Crossref PubMed Scopus (875) Google Scholar. DD-PCR products were displayed on a 5% acrylamide-urea gel; differentially expressed bands were eluted and reamplified5.Liang P. Averboukh L. Pardee A.B. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization.Nucleic Acid Res. 1993; 21: 3269-3275Crossref PubMed Scopus (875) Google Scholar. The reamplified PCR products were cloned into the pCNTR vector (5 Prime-3 Prime Inc., Boulder, CO, USA). In addition, we also used a modification of the RNA fingerprinting method6.Welsh J. Chada K. Dalal S.S. Cheng R. Ralph D. Mc Clelland M. Arbitrarily primed PCR fingerprinting of RNA.Nucleic Acid Res. 1992; 20: 4965-4970Crossref PubMed Scopus (482) Google Scholar. Poly(A+)-RNA was used for reverse transcription; primers, reverse transcription, and PCR conditions were adapted from a modification of the method7.Kojima R. Randall J. Brenner B.M. Gullans S.R. Osmotic stress protein 94: A new member of the HSP110/SSE gene subfamily.J Biol Chem. 1996; 271: 12327-12332Crossref PubMed Scopus (91) Google Scholar. Sequencing was performed using the Sequenase 2.0 kit (Amersham, Arlington Heights, IL, USA). Neonatal losartan treatment reduced the kidney weight/body weight ratio (5.13 ± 0.09 vs. 5.52 ± 0.11 mg/g in controls, N = 20 each, P < 0.05). Kidney renin expression was increased 35-fold by losartan (321 ± 40 vs. 9 ± 1 arbitrary units in controls, N = 6 each, P < 0.05). However, the expression of TGF-β1, PDGF-BB, VEGF, and EGF (N = 4 to 6 per group) was unchanged by losartan. Five differential display PCR fragments exhibiting apparent down-regulation by losartan in a reproducible manner were tested by Northern blotting. For one fragment, no expression was detected, whereas three further fragments showed unchanged expression. Only one fragment, termed AD.5, exhibited a slightly lower expression in losartan-treated kidneys than in controls Figure 1. Using the RNA fingerprinting method, approximately 80% of apparently differential bands were not reproducible. Five fragments with reproducible apparent differential expression were tested by Northern blotting. All but one labeled multiple transcripts, and none were differentially expressed. With the exception of AD.5, all cloned DD-PCR or RNA fingerprinting bands contained more than one (3–7) cDNA sequences of the same length, all containing the expected primer sequences at both ends. Expression of the 2.0-kb transcript of AD.5 was highest in lung, kidney, and heart and was weakest in brain Figure 1. Age-dependent expression of AD.5 in the kidney was found in 2-week-old rats>adult>newborn Figure 1. Of the 307 nucleotide sequence of AD.5, the 5′ 133 bases exhibited 86% identity to a human seven-transmembrane orphan receptor (GenBank accession #AF000546). A hypothetical translation yielded 88% identity and 93% similarity of the amino acid sequences of AD.5 and the orphan receptor Figure 1. The function and the ligand of this receptor are unknown. A chicken homologue (termed 6H1) of this orphan receptor was initially considered to be a P2Y purinoceptor8.Webb T.E. Kaplan M.G. Barnard E.A. Identification of 6H1 as a P2Y purinoceptor: P2Y5.Biochem Biophys Res Commun. 1996; 219: 105-110Crossref PubMed Scopus (95) Google Scholar, but recent reports refute this9.Li Q. Schachter J.B. Harden T.K. Nicholas R.A. The 6H1 orphan receptor, claimed to be the P2Y5 receptor, does not mediate nucleotide-promoted second messenger responses.Biochem Biophys Res Commun. 1997; 236: 455-460Crossref PubMed Scopus (38) Google Scholar. In this study, we screened the expression of various growth factors in kidneys with an abnormal structure due to neonatal Ang II receptor blockade1.Tufro-Mc Reddie A. Romano L.M. Harris J.M. Ferder L. Gomez R.A. Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar. Although some of these factors are thought to be involved in the trophic actions of Ang II10.Itoh H. Mukoyama M. Pratt R.E. Gibbons G.H. Dzau V.J. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II.J Clin Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (453) Google Scholar, we found no altered expression. Our findings do not exclude more subtle or localized changes in the expression of these growth factors, as described by some authors in similar models2.Niimura F. Labosky P.A. Kakuchi J. Okubo S. Yoshida H. Oikawa T. Ichiki T. Naftilan A.J. Fogo A. Inagami T. Hogan B.L.M. Ichikawa I. Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (309) Google Scholar. However, massive structural abnormalities are present in rats treated neonatally with losartan1.Tufro-Mc Reddie A. Romano L.M. Harris J.M. Ferder L. Gomez R.A. Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar, and subtle or equivocal changes in the expression of growth factors are not likely to explain these striking effects of Ang II receptor blockade2.Niimura F. Labosky P.A. Kakuchi J. Okubo S. Yoshida H. Oikawa T. Ichiki T. Naftilan A.J. Fogo A. Inagami T. Hogan B.L.M. Ichikawa I. Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (309) Google Scholar,4.Hilgers K.F. Norwood V.F. Gomez R.A. Angiotensin's role in renal development.Semin Nephrol. 1997; 17: 492-501PubMed Google Scholar. We turned to PCR-based methods to identify genes that are down-regulated by neonatal Ang II receptor blockade. Both DD-PCR5.Liang P. Averboukh L. Pardee A.B. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization.Nucleic Acid Res. 1993; 21: 3269-3275Crossref PubMed Scopus (875) Google Scholar and a modification7.Kojima R. Randall J. Brenner B.M. Gullans S.R. Osmotic stress protein 94: A new member of the HSP110/SSE gene subfamily.J Biol Chem. 1996; 271: 12327-12332Crossref PubMed Scopus (91) Google Scholar of RNA fingerprinting6.Welsh J. Chada K. Dalal S.S. Cheng R. Ralph D. Mc Clelland M. Arbitrarily primed PCR fingerprinting of RNA.Nucleic Acid Res. 1992; 20: 4965-4970Crossref PubMed Scopus (482) Google Scholar produced mostly false positives. Moreover, more than one sequence was almost invariably found after cloning cDNA fragments. In our view, these pitfalls limit the use of these methods for systematic comparison of gene expression in complex animal tissues such as the kidney. These PCR-based methods are very useful for identifying differentially expressed genes in cell culture systems1.Tufro-Mc Reddie A. Romano L.M. Harris J.M. Ferder L. Gomez R.A. Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar. DD-PCR of cultured kidney cells has previously identified novel genes with cell-specific expression11.Hilgers K.F. Nagaraji S.N. Karginiova E.A. Kazakova I.G. Pentz E.S. Gomez R.A. Cloning of PTSA, a novel gene expressed exclusively in mature proximal tubular cells.J Am Soc Nephrol. 1997; 8 (abstract): 360AGoogle Scholar,12.Karginova E.A. Pentz E.S. Kazakova I.G. Carey R.M. Gomez R.A. Zis, a developmentally regulated gene expressed in juxtaglomerular cells.Am J Physiol. 1997; 273: F731-F738PubMed Google Scholar. Despite the high rate of false positives produced by DD-PCR, we did identify novel rat cDNAs that were down-regulated by losartan. One novel gene, termed WOT.113.Hilgers K.F. Karginova E.A. Tufro-Mc Reddie A. Pentz E.S. Gomez R.A. WOT.1, a novel gene involved in angiotensin-dependent renal development.Hypertension. 1995; 26 (abstract): 541Google Scholar, will be described in detail elsewhere. Here, we describe the partial sequence of AD.5, which appears to be the rat homologue of a previously cloned human orphan receptor. Because the function of this orphan receptor is unknown, we cannot draw conclusions on the possible function of the AD.5 product. However, the AD.5 transcript exhibits organ-specific and developmentally regulated expression, and its expression is affected by neonatal losartan treatment. Thus, the AD.5 product may be involved in the development of structure and function of the kidney and/or in the trophic actions of Ang II. This study was supported by National Institutes of Health grants DK-44756, DK-45179, and HD-22910. K.F.H. was the recipient of a DFG habilitation scholarship (Hi 510/5-1)." @default.
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• W2020114266 title "Angiotensin-dependent gene expression in the developing rat kidney" @default.
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