A single nucleotide mutation in the mouse renin promoter disrupts blood pressure regulation
Keiji Tanimoto, … , Kazuyuki Yanai, Akiyoshi Fukamizu
Keiji Tanimoto, … , Kazuyuki Yanai, Akiyoshi Fukamizu
Published February 7, 2008
Citation Information: J Clin Invest. 2008;118(3):1006-1016. https://doi.org/10.1172/JCI33824.
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Research Article Nephrology

A single nucleotide mutation in the mouse renin promoter disrupts blood pressure regulation

Abstract

Renin, a major regulatory component of the renin-angiotensin system, plays a pivotal role in regulating blood pressure and electrolyte homeostasis and is predominantly expressed in the kidney. Several cAMP-responsive elements have been identified within renin gene promoters. Here, we study how 2 such elements, renin proximal promoter element-2 (RP-2) and overlapping cAMP and negative regulatory elements (CNRE), affect the transcriptional regulation of renin. We generated Tg mice (TgM) bearing BACs containing either WT or mutant RP-2 or CNRE, integrated at single chromosomal loci. Analysis of the TgM revealed that RP-2 was essential to basal promoter activity in the kidney, while renin mRNA levels did not significantly change in any tissues tested in the CNRE mutant TgM. To evaluate the physiological significance of these mutations, we used the BAC Tg to rescue hypotensive Renin-null mutant mice. As predicted, no renin expression was observed in the kidneys of RP-2 mutant/Renin-null compound mice, whereas renin expression in CNRE mutant compound mice was indistinguishable from that in control mice. Consistent with this, RP-2 mutant animals were hypotensive, while CNRE mutants had normal blood pressure. Thus, transcriptional regulation of renin expression via RP-2 but not CNRE is critical for blood pressure regulation by this gene.

Authors

Keiji Tanimoto, Akiko Sugiura, Sumiyo Kanafusa, Tomoko Saito, Naoto Masui, Kazuyuki Yanai, Akiyoshi Fukamizu

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Figure 1

Tg coplacement strategy.

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Tg coplacement strategy. (A) Mouse Ren-1C BAC. The 155-kbp BstI restrict...
(A) Mouse Ren-1C BAC. The 155-kbp BstI restriction enzyme fragments with 66 kbp and 80 kbp of 5′ and 3′ flanking sequences, respectively, were used for microinjections. Shown are cis-regulatory elements and 9 exons of the gene. (B, upper left) Map of the Ren-1C promoter region and structure of the targeting vector (pTmRn/mutCNRE), in which WT (open box) and mut CNRE (mut; filled box) sequences as well as homologous sequences to the target locus (thick lines) are included. Restriction enzyme sites with their positions relative to the transcriptional start site (+1) are shown. The loxP2272 and loxP5171 sequences are shown as open and filled triangles, respectively. The targeted locus was generated in E. coli and used to establish TgM lines. Following intercross with Cre-expressing TgM, selective excision in utero of the DNA segment between a pair of loxP5171 or loxP2272 sites generated either WT or mut loci, respectively. (B, upper right) Introduction of the FRT (shaded ovals) and SfiI sites into 3′-UTR of the gene. The targeting and flippase/FRT (FLP/FRT) recombination was carried out in the E. coli strain EL250 (26). The PCR primer set that simultaneously amplifies Tg and endogenous renin transcripts is shown by arrows. (B, bottom) Comparison of WT and mut CNRE promoter sequences. (C, top) Targeting vector (pTmRn/mutRP-2) for modification of the RP-2. Both WT (open circle) and mut (filled circle) RP-2 sequences as well as homologous sequences to the target (thick lines) are included. Tg coplacement was performed as described in B. (C, bottom) Comparison of the WT and mut RP-2 sequences. The HOX/PBX motif is boxed.