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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&lt;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&lt;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.

Functional Polymorphism of the Anpep Gene Increases Promoter Activity in the Dahl Salt-Resistant Rat