Table 1.
Demographics of island foxes with systemic amyloidosis between 1987 and 2010.
Figure 1.
Histochemical and immunohistochemical staining of island fox amyloid.
A: Amyloid markedly thickens the capillary loops of the glomerular tuft, labels with anti-AA antibody and shows birefringence when stained by Congo red. B: The renal medullary interstitium is expanded and tubules compressed by congophilic AA amyloid. C: Glomeruli and interstitium without amyloid show no immunoreactivity to canine AA antibody and are not congophilic. D: In the spleen, amyloid deposits in extensive, expansile nodules, replacing the white pulp and red pulp, and compressing smooth muscle trabeculae. E: Glossal papillae show hyperplasia and hyperkeratosis and a diffuse, thick submucosal band of amyloid that extends along the basement membrane. Scale bars = 50 µm [A, B, C, D (middle and right), E (middle)]; 200 µm [E (left and right)]; and 500 µm [D (right)].
Table 2.
Organs affected in cases of systemic amyloidosis.
Figure 2.
Congo red staining and electron microscopy of amyloid.
A: Amyloid markedly expands colonic submucosal arteries (arrow) and accumulates diffusely throughout the muscularis mucosae (black star). B: The epiglottis mucosa is undulating and hyperplastic and is expanded by submucosal amyloid (black star). C: Amyloid deposits thicken the capillary walls in the alveolar septae of the lung (arrow). D: Amyloid widens the Space of Disse in the liver (arrows) disrupting hepatic plates and isolating hepatocytes. E: The renal medullary interstitium from an amyloid positive fox shows long, thin, non-branching fibrils approximately 10 nm diameter (white arrowheads) arranged haphazardly amongst thicker bundles of collagen (black arrowheads). F: The renal medulla from an amyloid negative fox contains basement membrane (white star) and collagen (black arrowheads), but no fibrils. Scale bars = 200 µm (A, B); 50 µm (C, D); 200 nm (E); and 50 nm (F).
Figure 3.
SDS-PAGE and western blot of the dominant insoluble protein in amyloid-laden kidneys.
Coomassie blue stain of insoluble proteins from the renal cortex and medulla show a dominant band at 12 kDa and one or two lower molecular weight bands at 10 and 8 kDa, consistent with full length and fragments of SAA. An immunoblot shows that 12 kDa and lower molecular weight bands react with anti-canine AA antibody.
Table 3.
Percent of total insoluble protein composed of amyloidogenic proteins in renal cortex and medulla.
Figure 4.
Island fox SAA protein sequence and relative abundance of N- and C-terminal peptides.
A: The island fox SAA protein, reassembled from tryptic fragments (black arrows), is composed of 111 amino acids. A, B: Island fox SAA protein has 8 sites of amino acid variation at positions 6, 7, 55, 59, 60, 61, 70, and 72 (*). Some isoforms are shared with domestic dog and arctic fox; however five amino acid differences are unique to island fox amongst carnivores (_). C: The contribution of N-terminal and C-terminal peptides to insoluble SAA varies in renal cortex and medulla. Peptides that are within 2 amino acids of the C-terminus contribute to insoluble amyloid in 5 of 9 samples (+). Animal identifiers correspond to animals in Figure 4. D. Prediction of amyloidogenic regions of island fox SAA polymorphisms using the algorithm Tango [15].
Figure 5.
Protein abundance in amyloid and non-amyloid cases.
Mass spectrometry analysis reveals SAA as the dominant insoluble protein, only in cases with histologic amyloid. Amyloid-associated proteins apolipoprotein E and A-IV were significantly increased by 1.6 and 1.3-fold, respectively, in cases of amyloidosis, as were complement C3 and C4, fibrinogen, and inter-α-trypsin inhibitor heavy chain.