Characterization of the genomic structure and function of regions influencing renin and angiogenesis in the SS rat

Abstract

Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.

Congenic Mapping and Sequence Analysis of the Renin Locus

“…11 They reported that the SR Renin region increased BP (Δ+30 mmHg; P <0.05) in the SS-13 SR congenic (referred to as S/ren rr ) compared with SS (Figure 2A). 11 Also similar to line 9 13, 14 , the S/ren rr congenic restores plasma renin activity (PRA), 19 angiogenesis, 19 and vasoreactivity, 20 indicating that line 9 and S/ren rr share some functional allele(s), but not all. Most notably, the protective region around Renin (46.1–47.2 Mb) did not offset the hypertensive BP allele(s) in S/ren rr , 11 as was detected in line 9 (Figure 2).…”

“…We compared the SS/Jr (used by Jiang et al 11 ), the SS/JrMcwi (used for our SS-13 BN congenics 13 ), the SR, and the BN genomic sequences across the entire 3.7 Mb line 9 congenic interval to identify common and unique variants among the strains (Supplementary Table 2). Across the line 9 interval, only 2 variants (<0.0001%) were polymorphic between SS/Jr and SS/JrMcwi, and therefore, we hereafter use SS to denote both strains.…”

“…Combined, the SS had 267 variants that differed from both SR and BN, of which 1 nonsynonymous variant was predicted to be damaging. These common variants likely include shared SR and BN allele(s) that regulate renin expression, 13, 19 angiogenesis, 13, 19 and vasoreactivity 14, 20 that were restored in both line 9 and S/ren rr .…”

“…Genomic boundaries for congenic lines 9, 9A, 9B, and 9C were described previously, 13 but have been further refined here (Figure 1). …”

“…13–15 This revealed a protective region (chr13:46.1–47.2 Mb) promoting lower blood pressure (referred to as line9 BP1 ) that is offset by an adjacent region promoting higher blood pressure (chr13:47.2–49.0 Mb; referred to as line9 BP2 ), both derived from the same strain. We also reanalyzed archived DNA from the hypertensive S/ren rr congenic strain (SR Renin on a SS background) used in a 1996 study published in Hypertension by Jiang et al 11 This reduced by 85% the undefined congenic interval of S/ren rr , enabling us to compare overlapping sequence between SS, SR, and Brown Norway (BN) in our congenic region.…”