The interaction between IgG and Fc-γ receptors in glomeruli contributes to

The interaction between IgG and Fc-γ 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. 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 = 0.48). Serum levels of IgG2b were generally undetectable in SHR-A3 at both 18 and 30 weeks of age. Figure 2. Serum IgG subclass levels in SHR-A3 and SHR-B2 show persistent differences (IgG1 IgG2b IgG2c) at 18 and 30wks of age. In SHR-A3 no IgG2b can be detected by ELISA. DAPK Substrate Peptide Heritability of IgG subclass levels was assessed using trait variance in F1 and F2 animals (25 weeks of age). Heritability of each subclass (IgG1 IgG2a IgG2b and IgG2c) was estimated at 95.5 59.4 93.4 and 92.7% respectively. DAPK Substrate Peptide Because of evidence of heritable factors influencing serum IgG subclass levels we used genetic mapping to determine whether we could identify a genomic quantitative trait locus (QTL) influencing the level of each serum IgG subclass. For each of the 25-week-old F2 progeny of an SHR-A3 × SHR-B2 intercross we measured serum IgG subclass levels and determined single nucleotide polymorphism (SNP) genotypes at genomic regions where these two closely related lines were not identical by descent. No significant QTL could be mapped for IgG2a (Figure 3). For each of the other three subclasses a major QTL was identified that in every case mapped to the chromosome 6 haplotype block that contains the Ig heavy chain gene (IgH) from which the IgG isotype subclasses are transcribed. Chromosome 6 is 98% genetically identical by descent between SHR-A3 and SHR-B2. There are two haplotype blocks of nonidentical alleles. The one we have mapped is in the distal part of the chromosome and is tagged by four SNPs (Supplemental Table 1). This block extends over ~7 Mb and in addition to the IgH locus contains 20 rat RefSeq genes. This suggests that genome sequence variation in or near the IgH region influences serum IgG subclass levels. Figure 3. Major IgG subclass quantitative trait locus peaks are detected for IgG1 IgG2b and IgG2c each center on the rat IgH locus on chromosome 6 (P<0.00001 for each). The genome-wide LOD scores for IgG subclass levels in the F2 progeny of an SHR-A3 x ... The haplotype block was initially marked by adjacent mapping SNPs that were ~5 Mb from the IgH gene. Because there were no informative SNPs to indicate the presence of sequence difference in the IgH gene segments encoding the Fc region of IgG we performed resequencing of the IgH locus focusing on regions encoding the Fc exons of the IgG subclasses to determine whether DAPK Substrate Peptide sequence variation could be detected. GenBank cDNA sequences were used to identify genomic regions encoding the IgH gene. These regions were then amplified from genomic DNA of each parental line and submitted for sequence analysis. The results show that SHR-A3 contains a large amount of sequence variation in this region that includes a high degree of nonsynonymous (Table 1 and Supplemental Table 2) and other nonprotein sequence altering variations (GenBank accession numbers HQ640950-3 and HQ693704-7). In contrast the genomic sequence in SHR-B2 was much more similar to the rat genome reference sequence (derived from inbred Brown-Norway rats) and to the GenBank IgG subclass sequences obtained largely from PVG rats.5 SHR-A3 appears to have DAPK Substrate Peptide fixed an IgH locus that is highly diverged from that observed in other rat strains (SHR-B2 PVG and Brown-Norway strain [BN]) and that contains extensive alterations GLP-1 (7-37) Acetate in the predicted amino-acid composition of IgG Fc regions. Table 1. Nonsynonymous variation in rat IgG subclass Fc region To assess whether genetic variation at this important immunological locus was associated with hypertensive renal injury a trait by which SHR-A3 and SHR-B2 contrast notably 13 we examined whether urinary albumin excretion a biomarker of renal injury was related to the inheritance of SHR-A3 alleles in the haplotype block containing the IgH locus. There was a statistically significant relationship between inheritance of zero one or two SHR-A3 alleles at the IgH locus and the level of albuminuria (< 0.05 determined by linear regression analysis). This correlation predicted a doubling of urinary albumin-creatinine ratio from 9.4 to 18.7 mg/mg in F2 animals inheriting two SHR-A3 alleles compared with F2 animals inheriting only SHR-B2 alleles. Figure 4 shows the mean albumin excretion in the F2.