Deficiency of a transcriptional regulator, inhibitor of differentiation 3, induces glomerulonephritis in apolipoprotein E-deficient mice: a model linking hyperlipidemia and renal disease

Abstract

The clinical association between hyperlipidemia and renal disease is well established, yet hyperlipidemia as a cause for renal disease is rare. Apolipoprotein E-deficient (ApoE(-/-)) mice develop hyperlipidemia and are a model for atherosclerosis. Introducing deficiency of inhibitor of differentiation 3 (Id3) in ApoE(-/-) mice further exacerbates atherosclerosis. ID3 is a transcription regulator expressed in multiple cell types. Id3(-/-) mice develop antibodies to self-antigens and salivary gland autoimmunity. This study was undertaken to investigate a link between hyperlipidemia, autoimmunity, and renal disease. ApoE(-/-), Id3(-/-), and ApoE(-/-)Id3(-/-) double-knockout (DKO) mice were studied at different ages for renal pathological features and function. Serum samples were analyzed for the presence of autoantibodies. At 16 weeks, DKO mice developed mesangioproliferative glomerulonephritis (GN), leading to severe proteinuria. GN was associated with glomerular deposition of lipids and immune complexes and with macrophage infiltration. DKO mice had high levels of circulating autoantibodies. Although ApoE(-/-) mice had glomerular lipid deposits and Id3(-/-) mice had circulating autoantibodies, neither group of age-matched single-knockout mice developed GN. These data provide support for the hypothesis that induction of renal disease in hyperlipidemia is dictated by additional factors. Our study shows that some of these factors are regulated by ID3. Thus, ID3 is a novel risk factor linking cardiovascular and renal disease.

Figure 1

Renal pathological features in B6 WT, Id3^−/−, ApoE^−/−, and DKO female mice at the age of 24 weeks. Representative photomicrographs of renal histological features showing PAS-stained kidney at low (A–D) and high (E–H) magnification. There were enlarged and hypercellular glomeruli in DKO mice (H). Arrows show areas of PAS-positive material. Immunohistochemical staining for Ki-67 antigen (I–L) in glomeruli. Arrows (L) show positive staining in nuclei. Detection of fibronectin (M–P) by indirect immunofluorescence, showing renal cortex with glomeruli (g). Arrowheads show glomerular fibronectin (P). Nuclei were stained with DAPI. Scale bar = 20 μm (A–L).

Figure 2

Severity of GN in WT, Id3^−/−, ApoE^−/−, and DKO female mice at the ages of 16 weeks (A) and 24 weeks (B). H&E-stained kidney sections were evaluated for renal pathological features, as described in Materials and Methods. Each data point represents one mouse. *P = 0.022; ***P < 0.0001 compared with WT. nd, not determined.

Figure 3

DKO female mice (n = 10) develop significant proteinuria at the age of 38 to 42 weeks, compared with ApoE^−/− females (n = 6) at the age of 43 weeks. Mice were housed individually in metabolic cages, and a 24-hour urine sample was assayed for albumin/creatinine ratio. Data are given as the mean ± SEM of urinary albumin/creatinine ratios.

Figure 4

Quantitation of Ki-67⁺ cells in B6 WT, Id3^−/−, ApoE^−/−, and DKO female mice at the age of 24 weeks. Glomeruli in one cross section of each kidney were evaluated for frequency of glomeruli with Ki-67⁺ cells (A) and number of Ki-67⁺ cells per glomerulus (B). Data are represented as the mean ± SEM in each group (n = 4 per group, except for Id3^−/− mice, for which n = 2). ***P < 0.0001, **P = 0.002 compared with WT.

Figure 5

Representative electron micrographs of glomeruli from ApoE^−/− and DKO female mice at the age of 24 weeks. ApoE^−/− glomerulus (A) showing a glomerular capillary (c) with normal fenestrated endothelium, podocyte foot processes (p), and mesangium (m). B: A DKO glomerulus shows significant mesangial expansion. Scale bar = 5 μm. C: Another example of a DKO glomerulus at a lower power, showing extensive mesangial involvement. Scale bar = 10 μm.

Figure 6

Localization of CD68⁺ (A–D) and F4/80⁺ (E–H) macrophages in the renal cortex of WT, Id3^−/−, ApoE^−/−, and DKO female mice at the age of 24 weeks. Glomeruli (g) showing CD68⁺ and F4/80⁺ cells labeled with red fluorescence in the cortical interstitium (arrows). CD68⁺ cells infiltrating glomerular cells (arrowheads) are seen in DKO mice (D). Colocalization of major histocompatibility complex (MHC) II staining (green) with CD68 or F4/80 markers is indicative of macrophage activation. Nuclei are stained with DAPI. Scale bar = 20 μm.

Figure 7

Glomerular deposits of neutral lipids (A–D) by Oil-red O staining, IgG (E–H), and C3 complement (I–L) by direct immunofluorescence in B6 WT, Id3^−/−, ApoE^−/−, and DKO female mice at the age of 24 weeks. Arrows indicate lipid deposits (C and D).

Figure 8

Western blot analysis showing reactivity of serum samples from WT, Id3^−/−, ApoE^−/−, and DKO female mice at the age of 24 weeks. WEHI 7.1 cell lysates were separated on a 12.5% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with serum samples at 1:100 dilution, followed by goat anti-mouse IgG horseradish peroxidase conjugate. Bound antibody was detected with chemiluminescence, as previously described. Each lane represents serum from one mouse (m). −, negative control (not incubated with serum); +, pooled serum samples from MRL/lpr mice with lupus at 1:1000 dilution.