Experiment
The goal of the current study was to investigate the underlying genetics that determine albuminuria in KK/H1J (KK) mice. Elevated albumin-to-creatinine ratio (ACR), a condition also called albuminuria, is a phenotypic marker of chronic kidney disease in both humans and mice. In KK mice hyperglycemia is often accompanied by a strong tendency to develop type 2 diabetes (TD2) and diabetic nephropathy characterized by increased kidney weight, albuminuria, and proteinuria.
In the current study KK/H1J mice, the albuminuria-sensitive strain, were crossed with C57BL/6J (B6) mice, an albuminuria-resistant strain, to perform quantitative trait analysis (QTL) to identify the genetic basis for chronic kidney failure.
309 ((KK x B6) x (KK x B6))F2 mice were generated. Only male progeny were used due to the low or undetectable ACR in female mice. At 10 days of age tail tissue was collected for subsequent genotyping analysis and at 21 days of age mice were weaned. Mice were raised and maintained on a 6% fat containing chow diet. At 8 weeks of age, urine was collected in the parental strains, F1 and F2 mice over a three day period. Albumin and creatinine levels were measured and individual albumin-creatinine-ratios (ACRs) were calculated. DNA was extracted from tail tissues for genotyping using the Illumina platform and 385 unique polymorphic markers were analyzed. Because lipid levels were also examined in the cross, liver was collected and used as a surrogate for kidney tissue gene expression in the study.
QTL analysis was performed using R/qtl. Significant (p<0.05) and suggestive (p<0.63) thresholds were based on 1,000 permutations for the autosomes and 16,404 permutations for the X chromosome.
Results, Table 2:
The results identified a significant QTL, Albq21, (albuminuria QTL 21) on Chromosome X peaking at 27.5 cM nearest marker rs13483751 with a LOD score of 3.8 in a 95% confidence interval mapping between 20.5 and 89.0 Mb. At Albq21 B6 conferred the high ACR allele. To identify candidate genes within the confidence interval a variety of bioinformatics tools were applied and integrated. Gm6880, 4930595M18Rik, Rhox4g, Gpc4, Mospd1, BC023829, Idh3g, Pls3, Avpr2, Cul4b, Irak1, Fln1 and Xpnpep were identified as potential candidate genes for QTL Albq21.
Suggestive loci mapped to Chromosome 6 (23.7 cM, LOD=3.0); Chromosome 7 (79.2 cM, LOD=2.5); Chromosome 12 (20.7 cM, LOD=2.5) and Chromosome 13 (16.9 cM, LOD=3.1).
The scantwo function of R/qtl identified an interaction between the suggestive locus on Chromosome 6 at 23.7 cM and Chromosome 8 at 27.4 cM. Homozygous KK mice had higher ACR compared to homozygous B6 mice if they were also heterozygous at the Chr 6 QTL.
Analysis Tools