Genetic analysis of mesangial matrix expansion in aging mice and identification of Far2 as a candidate gene

Abstract

Aging of the kidney is associated with renal damage, in particular mesangial matrix expansion (MME). Identifying the genes involved in this process will help to unravel the mechanisms of aging and aid in the design of novel therapeutic modalities aimed at prevention and regression. In this study, structural changes in glomeruli of 24 inbred mouse strains were characterized in male mice at 6, 12, and 20 months of age. Haplotype association mapping was used to determine genetic loci associated with the presence of MME at 20 months. This analysis identified a significant association with a 200-kb haplotype block on chromosome 6 containing Far2. Sequencing revealed that mouse strains with MME contain a 9-bp sequence in the 5' untranslated region of Far2 that is absent in most of the strains without MME. Real-time PCR showed a two-fold increase in the expression of Far2 in the kidneys of strains with the insert, and subsequent experiments performed in vitro with luciferase reporter vectors showed that this sequence difference causes differential expression of Far2. Overexpression of Far2 in a mouse mesangial cell line induced upregulation of platelet activating factor and the fibrotic marker TGF-β. This upregulation of MME-promoting factors may result, in part, from the FAR2-catalyzed reduction of fatty acyl-coenzyme A to fatty alcohols, which are possible precursors of platelet activating factor. Overall, these data suggest the identification of a novel pathway involved in renal aging that may yield therapeutic targets for reducing MME.

Figure 1.

Periodic acid-Schiff staining reveals MME in NZW/LacJ, KK, and SM mice but not in LP/J, SWR, and WSB mice at 20 months of age. (A–C) Glomerulus of a 20-month-old male mouse without MME, LP/J (A), SWR (B), and WSB (C). (D-F) Glomerulus of a male NZW/LacJ (D), KK (E), and SM (F) mouse with MME. Broadened mesangium (arrow). Original magnification, ×200.

Figure 2.

Strain and age-dependent increase in matrix accumulation. For each strain the mean percentage and SD of affected glomeruli are shown at 20 (gray bar), 12 (white bar), and 6 (black bar) months of age. Scoring at a younger time point was performed only if the threshold of 10% was reached at the 20-month time point. At each time point, n=6, except for P at 12 months (n=3). The results represent the mean ± SD.

Figure 3.

Genome-wide scan for MME. The significant threshold was set to P<10⁻⁶.

Figure 4.

Association and expression of Far2. (A) The haplotype block between rs32310749 and rs30560187 is associated with MME. The only gene found in this region is Far2. The 9-bp sequence CTCGAGTGC is absent in 7 of 11 strains without MME. (B) Far2 expression levels correlate with MME. The results represent the mean ± SEM. Comparing all animals (regardless of strain) with and without MME using a t test showed a significant difference (P<0.01).

Figure 5.

The 9-bp indel in the promoter region of Far2 influences gene expression. (A) Both alleles of the 5′UTR of Far2 were cloned in front of the luciferase gene of pGL4.10 and co-transfected with 4.73 into MES 13 mesangial cells and NMu3Li liver cells. (B) Relative luciferase expression in the allele with the 9-bp insert is significantly higher compared with the allele without the insert. The results represent the mean ± SEM.

Figure 6.

Far2 overexpression causes upregulation of PAF and TGF-β. PAF protein levels are significantly upregulated in the media of MES-13 cells transfected with pCMV6-Far2 compared with the media of cells transfected with pCMV6 control vector (P<0.05). The expression of TGF-β is also significantly increased in the cells overexpressing Far2 compared with the control (P<0.02). The results represent the mean ± SEM.