Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat.
We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximately 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.
Kotlo
Kumar
K
Department of Medicine, University of Illinois at Chicago, 840 S Wood St, Chicago, IL 60612. RDanziger@aol.com.
Hughes
Douglas E
DE
Herrera
Victoria L M
VL
Ruiz-Opazo
Nelson
N
Costa
Robert H
RH
Robey
R Brooks
RB
Danziger
Robert S
RS
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics; D018826:CD13 Antigens / Q000235:genetics*; D004195:Disease Models, Animal; D006973:Hypertension / Q000235:genetics*; D008297:Male; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D020303:Rats, Inbred Dahl; D011917:Rats, Inbred Lew
D022763:CCAAT-Enhancer-Binding Protein-alpha; D018826:CD13 Antigens