Quantitative trait loci influence renal disease progression in a mouse model of Alport syndrome

Abstract

Alport syndrome is a human hereditary glomerulonephritis which results in end-stage renal failure (ESRF) in most cases. It is caused by mutations in any one of the collagen alpha3(IV), alpha4(IV), or alpha5(IV) chain genes (COL4A3-COL4A5). Patients carrying identical mutations can exhibit very different disease courses, suggesting that other genes or the environment influence disease progression. We previously generated a knockout mouse model of Alport syndrome by mutating Col4a3. Here, we show that genetic background strongly influences the timing of onset of disease and rate of progression to ESRF in these mice. On the 129X1/SvJ background, Col4a3 -/- mice reached ESRF at approximately 66 days of age, while on the C57BL/6J background, the mean age at ESRF was 194 days of age. This suggests the existence of modifier genes that influence disease progression. A detailed histopathological analysis revealed that glomerular basement membrane lesions typical of Alport syndrome were significantly more frequent in homozygotes on the 129X1/SvJ background than on the C57BL/6J background as early as two weeks of age, suggesting that modifier genes act by influencing glomerular basement membrane structure. Additional data indicated that differential physiological responses to basement membrane splitting also underlie the differences in disease progression. We attempted to map the modifier genes as quantitative trait loci (QTLs) using age at ESRF as the quantitative trait. Genome scans were performed on mice at the two extremes in a cohort of mutant F1 x C57BL/6J backcross mice. Analysis with Map Manager QT revealed QTLs linked to markers on chromosomes 9 and 16. A more detailed understanding of how these QTLs act could lead to new approaches for therapy in diverse renal diseases.

Figure 1.

Assays for renal function in 129 and B6 Col4a3 +/− and −/− mice. Levels of blood urea nitrogen (BUN) and serum creatinine and the urinary protein:creatinine ratios were determined at various ages for mice of all four cohorts. Renal function declined much faster in 129 vs. B6 Col4a3 −/− mice, as denoted by the rapid rise in all three indicators.

Figure 2.

Comparative histology of 129 and B6 Col4a3 −/− kidneys. By PAS staining, no pathology was evident at 22 days of age on either background. Significant pathologies, including glomerular crescents (asterisks), cellular infiltrates (white arrows), tubular casts (tc), and interstitial fibrosis (black arrows), were evident in the 129 Col4a3 −/− kidney at 47 days of age and later, but only limited pathology was observed in the B6 Col4a3 −/− kidney as late as 82 days of age. g, glomerulus. Scale bar, 50 μm.

Figure 3.

Comparative ultrastructural analysis of 129 and B6 Col4a3 −/− glomerular basement membranes (GBMs). At 14 days of age, widespread splitting was observed in the 129 Col4a3 −/− GBM, while less splitting was observed in the B6 Col4a3 −/− and in the Col4a3 +/− GBMs. At 22 and 44 days of age, significant splitting was observed in both the 129 and B6 Col4a3 −/− GBMs, but not in the Col4a3 +/− GBM. Arrows indicate regions of GBM splitting. Scale bar, 2.3 μm.

Figure 4.

Quantitation of GBM splitting in 129 and B6 Col4a3 −/− and +/− mice. At 14 days of age, the 129 Col4a3 −/− GBMs exhibited significantly more splitting than either the B6 Col4a3 −/− or the heterozygous control GBMs. At 22 days of age, however, both the 129 and B6 Col4a3 −/− GBMs exhibited splitting, but were not significantly different from each other. Error bars indicate S.E.M.

Figure 5.

Immunohistochemical analysis of laminin α2 deposition in 129 and B6 Col4a3 −/− and +/− glomeruli. Laminin α2 was detected in the GBM at both 14 and 22 days in the 129 Col4a3 −/− kidney (arrows in A and D), but was only observed in the mesangium in the B6 Col4a3 −/− and control kidneys. This early deposition of α2 in the GBM may be related to the rapid onset and progression of disease. Scale bar, 25 μm.

Figure 6.

Mean age at ESRF for Col4a3 −/− mice on different genetic backgrounds. The actual values (± SD) were: 129, 66 ± 6 days (n = 16); F1, 113 ± 16 days (n = 26); N2, 136 ± 27 days (n = 51); B6, 194 ± 24 days, (n = 28). The F1 and N2 cohorts exhibited a normal distribution.

Figure 7.

LOD scores for linkage of the markers tested on chromosomes 9 and 16 to QTLs influencing age at ESRF. The analysis incorporated data from 49 backcross mice. Schematic diagrams of the two chromosomes show the regions of linkage. As determined by the permutation test, critical threshold values for highly significant linkage were LOD = 3.26 for chromosome 9 and 3.1 for chromosome 16.