title: [Site-directed mutagenesis of atrial natriuretic peptide gene and effect of the mutations on its diuretic activity in nephrotic rats].
abstract: OBJECTIVE
  To prolong the half-life and enhance the biological activity of the human atrial natriuretic peptide (hANP), a peptide hormone, which is synthesized and released mainly by cardiac atrial myocytes and possesses potent natriuretic, diretic, and vasorelaxant properties.
  
  
  METHODS
  The site-directed mutation technique based on polymerase chain reaction was performed to get the mutant of the human ANP gene (mhANP), and the retroviral expression vector, pLHY24, in which mhANP gene is under the transcriptional control of the human cytomegalovirus promoter, was constructed. The naked plasmid DNA of pLHY24 and positive control vector, pLHY19, in which the wild-type hNAP gene is in the same conditions as mhANP gene in pLHY24, and negative control vector, pLNCX without purpose gene, at a dose of 5 mg/kg body weight was injected intramuscularly into the rats with experimental renal disorder induced with adriamycin (ADR), respectively.
  
  
  RESULTS
  DNA sequencing result proved that the respected mhANP gene with the point mutations of TTC(131)/Phe-->TCC/Ser and ATG(135)/Met-->ATA/Ile has been obtained. In comparison with negative control group (87 +/- 7.1 pg/ml), a single intramuscular injection of expression vector harboring mhANP or hANP gene resulted in an obvious increase in plasma level of mhANP (107 +/- 7.8 pg/ml, t = 4.65, P < 0.01) or hANP (113 +/- 8.6 pg/ml, t = 5.71, P < 0.01) 5 days after injection. A significant elevation in the ratio of urine volume to body weight was occurred after both of mhANP gene and hANP gene delivery as compared with negative control and the effect lasted for more than 15 days. The diuretic activity of mhANP gene delivery was 1.6-, 2.0-, and 1.9-fold higher than that of hANP gene 5, 10, and 15 days after gene transfer, respectively. However, there were no statistical differences in the concentrations of K(+) and Na(+) in urine.
  
  
  CONCLUSIONS
  Both of mhANP and hANP gene delivery into the rats with experimental nephropathy could improve their directic function obviously and the diuretic activity of the former is stronger than that of the latter significantly.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Hongyan
authors.initials: H
authors.lastname: Liang
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.forename: Shengdong
authors.initials: S
authors.lastname: Lu
mesh_terms: D000818:Animals; D009320:Atrial Natriuretic Factor / Q000097:blood / Q000235:genetics*; D004231:Diuresis; D015316:Genetic Therapy; D007674:Kidney Diseases / Q000503:physiopathology / Q000628:therapy*; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011507:Proteinuria / Q000628:therapy; D051381:Rats; D017208:Rats, Wistar
chemical_list: D009320:Atrial Natriuretic Factor