Background The recent development of a large panel of genome-wide single nucleotide polymorphisms (SNPs) provides the opportunity to examine genetic relationships between distinct SHR lines that share hypertension, but differ in their susceptibility to hypertensive end-organ disease. Methods and Results We compared genotypes at nearly 10,000 SNPs obtained for the hypertension end-organ injury-susceptible SHR-A3 (SHRSP, SHR-stroke prone) line and the injury-resistant SHR-B2 line. This revealed that that the two lines were genetically identical by descent (IBD) across 86.6% of the genome. Areas of the genome that were not IBD were distributed across 19 of the 20 autosomes and the X chromosome. A block structure of non-IBD comprising a total of 121 haplotype blocks was formed by clustering of SNPs inherited from different ancestors. To test the null hypothesis that distinct SHR lines share a common set of hypertension susceptibility alleles we compared blood pressure in adult SHR animals from both lines and their F1 and F2 progeny using telemetry. In 16–18wk old animals fed a normal diet, systolic blood pressure (SBP, mm Hg) in SHR-A3 was 205.7 ± 3.86 (mean ± SEM, n = 26), while in similar SHR-B2 animals SBP was 186.7 ± 2.53 (n = 20). In F1 and F2 animals, SBP was 188.2 ± 4.23, (n = 19) and 185.6 ± 1.1 (n = 211) respectively (p<10−6, ANOVA). In order to identify non-IBD haplotype blocks contributing to blood pressure differences between these SHR lines we developed a high throughput SNP genotyping system to genotype SNPs marking non-IBD blocks. We mapped a single non-IBD block on chromosome 17 extending over less than 10Mb at which SHR-A3 alleles significantly elevate blood pressure compared with SHR-B2. Conclusions Thus hypertension in SHR-A3 and -B2 appears to arise from an overlapping set of susceptibility alleles, with SHR-A3 possessing an additional hypertension locus that contributes to further increase blood pressure.

Background The spontaneously hypertensive rat (SHR) strain exists in lines that contrast strongly in susceptibility to renal injury in hypertension. These inbred lines share common ancestry and only 13% of their genomes arise from different ancestors. Methods and Results We used next gen sequencing to detect natural allelic variation in 5 genes of the immunoreceptor signaling pathway (IgH, Dok3, Src, Syk and JunD) that arise from different ancestors in the injury-prone SHR-A3 and the resistant SHR-B2 lines. We created an intercross between these lines and in the F2 progeny we observed that the inheritance of haplotype blocks containing the SHR-A3 alleles of these 5 genes correlated with increased albuminuria and histological measures of renal injury. To test whether accumulated genetic variation in this pathway may create a therapeutic target in hypertensive renal injury, rats of both lines were treated with the immunosuppressant mycophenolate mofetil (MMF). MMF reduced proteinuria (albumin to creatinine ratio, uACR) from 6.6 to 1.2 mg/mg (p<0.001) in SHR-A3. Glomerular injury scores were reduced in MMF treated SHR-A3 from 1.6 to 1.4 (p<0.002). Tubulo-interstitial injury was reduced in MMF-treated SHR-A3 from 2.62 to 2.0 (p=0.001). MMF treatment also reduced renal fibrosis in SHR-A3, (3.9 vs. 2.0, p<0.001). Conclusions Polygenic susceptibility to renal injury in hypertension arises in association with genetic variation in genes that participate in immune responses and is dramatically improved by reduction of immune system activity.

The interaction between IgG and Fc-␥ receptors in glomeruli contributes to the development of several types of proteinuric glomerular disease, but the involvement of immunological mechanisms in hypertensive renal injury is incompletely understood. Here, we investigated serum IgG levels in SHR-A3 rats, which develop hypertensive injury, and compared them with the injury-resistant SHR-B2 line. At 18 weeks old, SHR-A3 rats had serum total IgG levels nearly twice those of SHR-B2 rats, although subclass IgG2b was undetectable in SHR-A3 rats compared with mean levels (Ϯ SEM) of 80.7 Ϯ 12.8 mg/dl (18 weeks) and 116.6 Ϯ 19.0 mg/dl (30 weeks) in SHR-B2 rats. In addition, these two strains had significantly different serum levels of IgG1, IgG2a, and IgG2c; differences persisted at 30 weeks for all subclasses except IgG2a. Genetic mapping revealed that a locus on chromosome 6 linked to IgG subclass levels that affected IgG1, IgG2b, and IgG2c but not IgG2a. The mapped haplotype block contains IgH, suggesting regulation of three of four serum IgG subclass levels in cis. Resequencing revealed variation in the sequence of the Fc portion of the IgG heavy chain, which predicts important functional changes. To examine whether there is any relationship between this haplotype block and susceptibility to renal injury, we examined the effect of SHR-A3 and SHR-B2 alleles at this block on albumin excretion in an F2 intercross. Albuminuria doubled with inheritance of SHR-A3 alleles. In summary, allelic variation in IgH or nearby genes may modulate the susceptibility to hypertensive renal injury in SHR-A3 rats.

SHR lines differ in their susceptibility to hypertensive end-organ disease and may provide an informative model of genetic risk of disease. Lines derived from the original SHR-B and SHR-C clades are highly resistant to hypertensive end-organ disease, while lines derived from the SHR-A clade were selected for stroke susceptibility and experience hypertensive renal disease. Here we characterize the temporal development of progressive renal injury in SHR-A3 animals consuming 0.3% sodium in the diet and drinking water. SHR-A3 rats demonstrate albuminuria, glomerular damage, tubulo-interstitial injury and renal fibrosis that emerge at 18 weeks of age and progress. Mortality of SHR-A3 animals was 50% at 40 weeks of age, and animals surviving to this age had reduced renal function. In contrast SHR-B2, which are 87% genetically identical to SHR-A3, are substantially protected from renal injury and demonstrate only moderate changes in albuminuria and renal histological injury over this time period. At 40 weeks of age, electron microscopy of the renal glomerulus revealed severe podocyte effacement in SHR-A3, but slit diaphragm architecture in SHR-B2 at this age was well preserved. Renal injury traits in the F1 and F2 progeny of an intercross between SHR-A3 and SHR-B2 were measured to determine heritability of renal injury in this model. Heritability of albuminuria, glomerular injury and tubulo-interstitial injury were estimated at 48.9, 66.5 and 58.6% respectively. We assessed the relationship between blood pressure and renal injury measures in the F2 animals and found some correlation between these variables that explain up to 26% of the trait variation. Quantitative trait locus (QTL) mapping was performed using over 200 SNP markers distributed across the 13% of the genome that differs between these two closely related lines. Mapping of albuminuria, tubulo-interstitial injury and renal fibrosis failed to identify loci linked with disease susceptibility, suggesting a complex inheritance of disease risk. We detected a single QTL conferring susceptibility to glomerular injury that was confined to a small haplotype block at chromosome 14:70–76Mb.

Xpc-null (Xpc-/-) mice, deficient in the global genome repair subpathway of nucleotide excision repair (NER-GGR), were exposed by intraperitoneal (i.p.) injection to a 300 mg/kg mutagenic dose of 3,4-epoxy-1-butene (EB), to investigate NER's potential role in repairing butadiene (BD) epoxide DNA lesions. Mutagenic sensitivity was assessed using the Hprt assay. Xpc-/- mice were significantly more sensitive to EB exposure, exhibiting an average 2.8-fold increase in Hprt mutant frequency (MF) relative to those of exposed Xpc+/+ (wild-type) mice. As a positive control for NER-GGR, additional mice were exposed by i.p. injection to a 150 mg/kg mutagenic dose of benzo[a]pyrene (B[a]P). The Xpc-/- mice had MFs 2.9-fold higher than those of exposed Xpc+/+ mice. These results suggest that NER-GGR plays a role in recognizing and repairing some of the DNA adducts formed following in vivo exposure to EB. Additional research is needed to examine the response of Xpc-/- mice, as well as other NER-deficient strains, to inhaled BD. Furthermore, it is likely that alternative DNA repair pathways also are involved in restoring genomic integrity compromised by BD-epoxide DNA damage. Collaborative studies are currently underway to address these critical issues.