Genetic analysis of intracapillary glomerular lipoprotein deposits in aging mice

Abstract

Background: Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes.

Methods: Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0-4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping.

Results: Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3.

Conclusions: By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponses.

Figure 1. Intracapillary glomerular deposits.

Representative example of an unaffected NZW 6-month-old, an affected NZW 20-month-old and an unaffected C57BLKS 20-month-old male mouse using PAS (A–C), apoE (D–F), apoB (G–I), and apoA-IV (J–L) stainings. Deposits are seen in the glomerular capillary lumina (arrows). Immunohistochemistry staining shows that these deposits are strongly positive for apoE (E, arrows) compared to the younger NZW mouse (D) and a mouse of a strain negative for glomerular deposits (C57BLKS) (I). The deposits are weakly positive for apoB (H, arrows) and apoA-IV (K, arrows) compared to the 6-month old NZW mouse (G, J) and C57BLKS control (I, L).

Figure 2. Severity of the deposits.

Scoring of the severity of the deposits in 20-month-old male mice of the six affected strains: B10 (n = 14), C57BR (n = 15), NOD.B10 (n = 6), NZW (n = 10), C3 H (n = 10) and NON (n = 12). The score represents the mean percentage of the total of 50 glomeruli affected (grade 0–4).

Figure 3. Characterization of the glomerular deposits.

Elektron microscopy of an affected glomerulus of a 20-month-old NOD.B10 mouse shows filling of the intracapillary lumina with optically lucent areas, suggestive of lipid vacuoles. Intracapillary lumina (closed arrow), nucleus (*) and podocyte (dashed arrow).

Figure 4. Glomerular deposits express a lipid droplet surface protein.

Glomeruli with intracapillary deposits show positive staining for a protein that binds to the surface of lipid droplets, perilipin-2 (B, D) in contrast to glomeruli without deposits, which are negative (A, C).

Figure 5. Genome wide HAM results.

Genome-wide scan for intracapillary glomerular deposits. A peak reaching the significant threshold of P<10⁻⁶ is seen on Chr 1 (A). The haplotype block of six SNPs between rs48848476 and rs46020199 is associated with glomerular deposits. The region spanning 1 Mb on each site from this area includes the genes Spata17, Gpatch2, Esrrg, Ush2a, and Kctd3 (B).

Figure 6. Expression levels of the five associated genes.

Rt-PCR for Spata17, Gpatch2, Esrrg, Ush2a, and Kctd3 (n = 3–5). The results represent the mean ± SE. Comparing all animals (regardless of strain) with and without deposits using a t-test showed no significant difference for any of the genes.