Refined mapping of a hypertension susceptibility locus on rat chromosome 12

Abstract

Previously, we found that transferring 6.1 Mb of salt-sensitive (SS) chromosome 12 (13.4-19.5 Mb) onto the consomic SS-12(BN) background significantly elevated mean arterial pressure in response to an 8% NaCl diet (178±7 versus 144±2 mm Hg; P<0.001). Using congenic mapping, we have now narrowed the blood pressure locus by 86% from a 6.1-Mb region containing 133 genes to an 830-kb region (chr12:14.36-15.19 Mb) with 14 genes. Compared with the SS-12(BN) consomic, the 830-kb blood pressure locus was associated with a ∆+15 mm Hg (P<0.01) increase in blood pressure, which coincided with elevated albuminuria (∆+32 mg/d; P<0.001), proteinuria (∆+48 mg/d; P<0.01), protein casting (∆+154%; P<0.05), and renal fibrosis (∆+79%; P<0.05). Of the 14 genes residing in the 830-kb locus, 8 were differentially expressed, and among these, Chst12 (carbohydrate chondroitin 4 sulfotransferase 12) was most consistently downregulated by 2.6- to 4.5-fold (P<0.05) in both the renal medulla and cortex under normotensive and hypertensive conditions. Moreover, whole genome sequence analysis of overlapping blood pressure loci revealed an ≈86-kb region (chr12:14 541 567-14 627 442 bp) containing single-nucleotide variants near Chst12 that are unique to the hypertensive SS strain when compared with the normotensive Brown Norway, Dahl salt-resistant, and Wistar-Kyoto strains. Finally, the 830-kb interval is syntenic to a region on human chromosome 7 that has been genetically linked to blood pressure, suggesting that insight gained from our SS-12(BN) congenic strain may be translated to a better understanding of human hypertension.

Keywords: hypertension; rats, inbred BN; rats, inbred Dahl.

Figure 1

Experimental timeline for diet switches, urine collection, blood pressure measurements, and tissue collections (A). D0, day of transmitter implants; D#, number of days post-implant. B, Schematic representation of the overlapping salt-sensitive (SS)-12^BN congenic strains that were generated by introgressing segments of the SS chromosome 12 (black) into the genetic background of the SS-12^BN consomic rat (white) by marker-assisted breeding. Mean arterial pressure (MAP) of the congenic strains while on 1% NaCl diets and stressed for 3 weeks on 8% NaCl diets. Data were analyzed by 1-way ANOVA on ranks followed by Dunn's multiple comparison test. The sample size for each group is shown in the figure. The dashed lines indicate the boundaries (markers D12Hmgc13 to D12Hmgc14) of the narrowed candidate region after excluding the Cb and Cc genetic regions. The thin black bars represent chromosomal regions that could be either BN or SS. There were no statistical differences between the strains on 1% NaCl.*P<0.05 vs. SS-12^BN on 8% salt diet.

Figure 2

Urinary albumin (A) and protein (B) excretion on a 1% NaCl diet (n=26 SS-12^BN, 20 C, 14 Ca, 6 Cb, and 5 Cc animals). H&E-stained (C) SS-12^BN (BN12), Ca, and Cc kidneys for examining protein casting after an 8% salt load. Trichrome-stained SS-12^BN (BN12), Ca, and Cc kidneys for examining tubulointerstitial fibrosis (D) and glomerular sclerosis (E). F, Quantification of percent area of protein casting in the outer medulla (n=5 per group). G, Quantification of percent area of tubulointerstitial fibrosis (n=5 per group). H, Quantification of glomerular sclerosis scores (n=5 per group). Data are presented as mean ± SEM. *P<0.05, †P<0.01, and ‡P<0.001 vs. BN12. §P<0.05, ‖P<0.01, and ¶P<0.001 vs. Cc.

Figure 3

Comparison of Dahl salt-sensitive (SS/JrHsdMcwi), Dahl salt-resistant (SR/JrHsd), Brown Norway (BN/NHsdMcwi), and Wistar-Kyoto (WKY/NHsd) sequences over the 830 kb interval that is uniquely SS in the Ca congenic strain. Data are presented as number of single nucleotide variants per 0.05 Mb bin where SS has a different allele than SR, BN, and WKY. The locations of the 14 genes within this region are also shown.