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.

Background: Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown.Animals: One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included.Methods: Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels.Results: Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status.Conclusions and Clinical Importance: The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.

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.

Background: Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown. Animals: One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included. Methods: Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels. Results: Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status. Conclusions and Clinical Importance: The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.