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Ultrastructural localization of advanced glycation end products and beta2-microglobulin in dialysis amyloidosis
D Brancaccio1, M Gallieni, T Niwa
1Renal Unit, San Paolo Hospital, Milan, Italy. gallieni@iol.it
Background:
beta2-microglobulin (beta2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis (DRA, Abeta2M amyloidosis). beta2m modified with advanced glycation end products (AGE) may be an important factor in the pathogenesis of DRA. The presence of AGE in beta2m-positive amyloid deposits and surrounding macrophages has been demonstrated by immunohistochemical techniques in light microscopy.
Methods:
In order to better define the localization of beta2m and AGE in amyloid deposits and in cells, carpal tunnel connective tissues obtained from surgical specimens in six patients with DRA were studied by immunohistochemistry and electron microscopy, using the avidine-biotine complex and immunogold staining procedures, respectively. A polyclonal rabbit anti-human beta2m and two monoclonal mouse anti-AGE antibodies [AG-1 anti-imidazolone and AG-10 anti-N(epsilon)-carboxymethyl-lysine] enabled us to label their respective antigens at the optical and ultrastructural level.
Results:
with both techniques, extracellular amyloid deposits strongly reacted with anti-beta2m and anti-AGE antibodies, although the immunoreactivity of beta2m was more intense. Macrophage-like synovial cells (CD-68 positive) surrounding amyloid deposits were also immunoreactive for beta2m and AGE, which were detected in lysosomes and in intracellular fibrillar material. Anti-AGE reactivity was also evident in collagenous structures in the absence of beta2m or amyloid deposits, supporting the proposal that AGE modification of collagen might have pathogenic relevance in the development of DRA.
Conclusions:
The co-localization of AGE and beta2m, both intra- and extra-cellularly, in amyloid fibrils was confirmed by immunoelectron microscopy; however, the positivity of collagen to anti-AGE antibodies and a different pattern of intracellular localization suggest that molecules other than beta2m may also be modified by AGE and may be involved in the pathogenesis of DRA.
Insights
Advanced glycation end products (AGE) and beta2-microglobulin (beta2m) coexist in dialysis-related amyloidosis (DRA) fibrils. Collagen modification by AGE may also contribute to DRA pathogenesis.
Area of Science:
- Nephrology
- Pathology
- Biochemistry
Background:
- Dialysis-related amyloidosis (DRA) is linked to beta2-microglobulin (beta2m) as the amyloid precursor.
- Advanced glycation end products (AGE) modification of beta2m may play a role in DRA pathogenesis.
- Previous studies showed AGE in beta2m deposits and macrophages.
Purpose of the Study:
- To precisely localize beta2m and AGE in amyloid deposits and cells in DRA.
- To investigate the role of AGE modification in DRA pathogenesis.
Main Methods:
- Immunohistochemistry and immunoelectron microscopy on carpal tunnel tissues from DRA patients.
- Use of anti-beta2m and anti-AGE antibodies (anti-imidazolone, anti-N(epsilon)-carboxymethyl-lysine).
Main Results:
- Extracellular amyloid deposits showed strong immunoreactivity for both beta2m and AGE.
- Beta2m and AGE were detected within macrophage-like cells (CD-68 positive) in lysosomes and intracellular fibrils.
- Collagenous structures exhibited AGE reactivity independently of beta2m or amyloid deposits.
Conclusions:
- Co-localization of AGE and beta2m within amyloid fibrils was confirmed ultrastructurally.
- AGE modification of collagen suggests other molecules besides beta2m may be involved in DRA pathogenesis.
- Intracellular localization patterns indicate potential roles for AGE-modified non-beta2m proteins in DRA.