Studies on the interaction between two loci, RF-1 and RF-4, in hypertension-associated renal disease

摘要

End stage renal failure (ESRD) is a major health problem in the United States, with the incidence continuing to increase. The majority of the ESRD cases are associated with hypertension or diabetes with recent studies in both humans and animal models suggesting there are renal disease susceptibility genes independent of the primary diagnosis.;Five quantitative trait loci (QTL) for renal disease, characterized by increased proteinuria, were previously identified in a cross between the Fawn-Hooded Hypertensive (FHH/Eur) rat (a genetic model of hypertension-induced ESRD) and the low-proteinuria ACI strain1,2. Importantly the Rf-1 and Rf-4 loci strongly interact with each other 2,3. In this dissertation we hypothesized that a gene(s) within the Rf-4 locus is interacting with Rf-1 and contributing to the renal disease observed in the FHH model. Based on this hypothesis, the goals were to narrow the region of the Rf-4 locus containing renal disease susceptibility gene(s) using congenic strains and to test 10 candidate genes within the smallest interval for their role in renal disease.;The first step in accomplishing this goal was to generate a physical map for Rf-4 using comparative genomics. Since the rat genome assembly had not yet been released, this physical map was necessary to identify candidate genes within the locus. After the genomic sequence was available, the physical map was compared to the assembly for the Rf-4 region. Using comparative mapping, the regions homologous to Rf-4 were found to be on mouse chromosome 5 and human chromosomes 1 and 4. 111 known genes and 2938 human-mouse conserved elements were identified in these homologous regions. The physical map covered 98% of the QTL and matched the rat genome sequence, providing confidence for gene hunting in the region. In addition, by using the homology data, a more complete list of genes was generated and the importance of the region in human renal disease could be investigated.;The second step was to narrow the region of Rf-4 involved in renal disease. To accomplish this goal, a panel of subcongenic lines (named Rf-1a+4_1 through Rf-1a+4_7) that contained the FHH Rf-1 locus and different portions of the FHH Rf-4 locus introgressed onto the ACI background was generated. The Rf-1a+4_2 subcongenic strain showed a reduction in both albuminuria (UAV) and glomerular permeability (Palb) compared to Rf-1a+4_1 . These phenotypic differences indicated that the 1.7 megabase region (with 24 known genes) that was different between Rf-1a+4_1 and Rf-1a+4_2 contains a causative variant(s) interacting with Rf-1. This interaction contributes to renal disease, specifically by increasing the permeability of the glomerulus.;The third step was to identify and investigate ten candidate genes within the narrowed interval. Expression differences were observed, but there were no sequence variants within the exonic or promoter regions of the genes. From these data we conclude that the coding and promoter regions of these 10 genes do not contain the polymorphism(s) altering renal disease susceptibility.;In summary, we have narrowed the Rf-4 locus to 1.7 megabases. This region interacts with Rf-1 to cause an increase in glomerular permeability, and thereby increasing albumin excretion. Although none of the genes tested can be completely excluded, it appears that the sequence variant within Rf-4 contributing to renal disease is located either in a gene that was not investigated or else in a conserved noncoding sequence that regulates one or more of the genes in the region.