Dissection of chromosome 18 blood pressure and salt-sensitivity quantitative trait loci in the spontaneously hypertensive rat

Abstract

Hypertension in humans and experimental models has a strong hereditary basis, but identification of causative genes remains challenging. Quantitative trait loci (QTLs) for hypertension and salt sensitivity have been reported on rat chromosome 18. We set out to genetically isolate and prioritize genes within the salt-sensitivity and hypertension QTLs on the spontaneously hypertensive rat (SHR) chromosome 18 by developing and characterizing a series of congenic strains derived from the SHR and normotensive Brown Norway rat strains. The SHR.BN-D18Rat113/D18Rat82 congenic strain exhibits significantly lower blood pressure and is salt resistant compared with the SHR. Transplantation of kidneys from SHR.BN-D18Rat113/D18Rat82 donors into SHR recipients is sufficient to attenuate increased blood pressure but not salt sensitivity. Derivation of congenic sublines allowed for the separation of salt sensitivity from hypertension QTL regions. Renal expression studies with microarray and Solexa-based sequencing in parental and congenic strains identified 4 differentially expressed genes within the hypertension QTL region, one of which is an unannotated transcript encoding a previously undescribed, small, nonprotein coding RNA. Sequencing selected biological candidate genes within the minimal congenic interval revealed a nonsynonymous variant in SHR transcription factor 4. The minimal congenic interval is syntenic to a region of human chromosome 18 where significant linkage to hypertension was observed in family based linkage studies. These congenic lines provide reagents for identifying causative genes that underlie the chromosome 18 SHR QTLs for hypertension and salt sensitivity. Candidate genes identified in these studies merit further investigation as potentially causative hypertension genes in SHR and human hypertension.

Figure 1

Genetic and physical map showing the transferred segment of chromosome 18 in the SHR-18 congenic strain, and in the SHR-18A, SHR-18B and the SHR-18C congenic sublines. Map distances in centiMorgans (cM) and in Megabasepairs (Mbp) are based on the SHRSP x BN genetic map (http://rgd.mcw.edu/) and RGSC3.4, Ensembl release 45 respectively. Black bars represent the chromosomal region transferred from the BN strain, white bars from the SHR background and grey bars represent the recombination zones. The dashed lines represent the limits of the salt sensitivity and blood pressure regulatory regions.

Figure 2

Systolic blood pressure measured by radiotelemetry in (a) 15–17 week old SHR and SHR-18, (b) 19–21 week old SHR and SHR-18A, (c) 15–17 week old SHR and SHR-18B, (d) 15–17 week old SHR and SHR-18C strains. Baseline systolic blood pressure was significantly reduced in SHR-18 (F_1,12=4.84; P=0.048), SHR-18A (F_1,12=10.17; P=0.008) and SHR-18B (F_1,11=12.6; P=0.005). No significant reduction in systolic blood pressure was obtained in SHR-18C (F_1,11=0.27; P=0.61) strain vs SHR. All results according to main effect F values of two way repeated measures ANOVA are reported. Salt-loaded systolic blood pressure was significantly raised in all SHR and congenic strains SHR-18A (F_1,6 =27.7; P= 0.002), SHR-18B (F_1,4 =57.3; P= 0.0016) and SHR-18C (F_1,6 =10.3; P= 0.019) with the exception of SHR-18: SHR-18 (F_1,7 =0.88; P= 0.38). All F values computed for contrast comparison in two-way ANOVA. The baseline period is from −12 to −2 days and salt administration from day 1 as indicated. Values displayed are means ± SEM.

Figure 3

Transplant recipients’ systolic blood pressure during normal and high salt intake (1% NaCl as drinking water) measured by radiotelemetry at the age of 18–20 weeks. Reduction in baseline systolic blood pressure near the significance level was achieved in SHR_SHR18 (F_1,11=4.50; P=0.057) vs SHR_SHR transplanted rats. All results according to main effect F values of two way repeated measures ANOVA. Salt-loaded systolic blood pressure was significantly raised in SHR_SHR-18 (F=101.6; P<0.0001). All F values computed for contrast comparison in two-way ANOVA. The baseline and salt administration period is indicated as in Figure 2.

Figure 4

Northern blot of SHR and BN kidney small RNA, probed with an antisense oligonucleotide derived from the extended RACE product for Affymetrix probe set rc_AA957526_at rat sequence with 5S rRNA as a loading control.