Macrophage Metalloelastase as a Major Factor for Glomerular Injury in Anti-Glomerular Basement Membrane Nephritis

Northern blot analysis of rat MMP-12 mRNA in the kidneys of anti-GBM nephritis rats. Total RNA (20 μg) purified from kidneys in untreated normal rat, anti-GBM nephritis rat killed on days 1, 3, and 7, or control serum-administered rat killed on days 1, 3, and 7 was hybridized with alkaline phosphatase-labeled rat MMP-12 cDNA probe.

In situ hybridization of rat MMP-12 mRNA in the kidneys of anti-GBM nephritis rats. RNA in the kidneys on paraffin sections from anti-GBM nephritis rats killed on days 7 (A–D, H, and I), 3 (E), and 1 (F), or from untreated normal rat (G) was hybridized with Dig-labeled rat MMP-12 cRNA probe, and the macrophage marker was detected with anti-rat ED-1 mAb. A, mRNA in multinuclear giant cells forming crescent in the glomerulus was hybridized with antisense cRNA probe (blue). B, Sense cRNA probe was used as a control. C, Macrophages and multinuclear giant cells were stained with anti-rat ED-1 mAb (brown) after the hybridization with antisense cRNA probe (blue). D, Serial section was stained with PAS. MMP-12 mRNA was mildly expressed in macrophages infiltrated into some glomeruli on day 3 (E), but not in the kidney on day 1 or normal control (F, G). H, MMP-12 mRNA was detected in most of the glomeruli with crescent (arrow), but not in the glomeruli, with only mild proliferative change (arrowhead). I, MMP-12 mRNA was not detected in the macrophages infiltrated within the interstitium. (Original magnifications: A–G, ×200; H and I, ×100.)

A, Western blot analysis with anti-rat rMMP-12 Ab. Rat rMMP-12 (0.5 μg) was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated with anti-rat rMMP-12 Ab or rabbit control IgG, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG Ab. B, In vitro digestion of natural substrates by rat rMMP-12, and inhibition of its digestive effect by anti-rat rMMP-12 Ab. Ten micrograms of human fibronectin was incubated with 0.2 μg of rat rMMP-12, with or without anti-rat rMMP-12 Ab at room temperature for 16 h, and analyzed by 5% SDS-PAGE. Fibronectin was completely degraded by incubation with rat rMMP-12. Two micrograms of anti-rat rMMP-12 Ab inhibited the digestive effect of rat rMMP-12 almost completely, and the inhibition was dose dependent. Same dose of control IgG did not inhibit the digestion. C, Fifty micrograms of bovine solubilized elastin was incubated with 4 μg of rat rMMP-12, with or without anti-rat rMMP-12 Ab at room temperature for 16 h. After intact elastin or Ab was precipitated with trichloroacetic acid, 100 μl of supernatant containing released peptides was blended with 100 μl of ninhydrin reagent, and was quantitatively measured as an absorbance at 570 nm after dilution with 800 μl of 50% (v/v) ethanol. Released peptides besides background release were reduced by 57% by addition of 10 μg of anti-rat rMMP-12 Ab. Values are mean ± SEM in triplicate samples; ∗, p = < 0.0001 vs released peptides from digested elastin by rat rMMP-12 without blocking.

Immunohistochemical analysis of the kidney in anti-GBM nephritis rats with anti-rat rMMP-12 Ab. Serial paraffin sections from anti-GBM nephritis rat killed on day 7 were stained with anti-rat rMMP-12 Ab (A, D, E), or rabbit control IgG (B), or stained with PAS (C). A, Large granules in the cytoplasm of the crescent-forming cells or multinuclear giant cells were stained with anti-rat rMMP-12 Ab (red). D, Double staining with anti-rat ED-1 mAb (brown) and anti-rat rMMP-12 Ab (red). MMP-12 was detected in ED-1-positive cells in the glomeruli with severe crescent. E, These cells containing MMP-12 were seen in several glomeruli with severe crescentic formation (arrow), but not in the glomeruli almost intact or with mild proliferative change (arrowhead). (Original magnifications: A–D, ×200; E, ×100.)

Photomicrographs of representative kidney sections from rats with anti-GBM nephritis treated with anti-rat rMMP-12 Ab (A, C) or control Ig (B, D) on days 0, 2, 4, and 6 after the injection of anti-GBM antiserum. The kidneys were removed on day 7. Paraffin sections were stained with PAM (A, B) or anti-rat ED-1 mAb (C, D). In these sections, numbers of glomeruli with crescent (arrow) were 1 of 10 (A), and 7 of 9 (B). Only several numbers of ED-1-positive cells (brown) per glomerulus were counted in the kidney of the rat treated with anti-rat rMMP-12 Ab (C), in contrast to 10–40 of ED-1-positive cells per glomerulus in the kidney of the rat treated with control Ig (D). (Original magnifications: ×40.)