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Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.
N P Goode1, M Shires, D M Crellin
1Renal Research Unit St. James's University Hospital, Leeds, United Kingdom.
Summary
This study quantifies glomerular anionic sites using cationic colloidal gold, revealing distinct staining patterns based on pH. Hyaluronic acid appears to be a key component of these sites in the glomerular basement membrane.
Area of Science:
- Nephrology
- Biochemistry
- Cell Biology
Background:
- Glomerular anionic sites are crucial for kidney filtration barrier function.
- Understanding the composition and distribution of these sites is essential for diagnosing kidney diseases.
Purpose of the Study:
- To detect and quantify glomerular anionic sites in kidney tissue sections.
- To investigate the influence of pH and glycosaminoglycan-degrading enzymes on anionic site expression.
- To determine the specific glycosaminoglycans associated with glomerular anionic sites.
Main Methods:
- Utilized cationic colloidal gold for detecting anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections.
- Employed both manual and computer-assisted quantitation methods for site analysis.
- Examined the effects of varying pH (2.5-7.0) and enzymatic digestion (chondroitinase ABC, hyaluronidase, heparitinase I) on staining patterns.
Main Results:
- Both quantitation methods yielded comparable results.
- pH significantly altered staining patterns: pH 2.5 highlighted epithelial/endothelial glycocalyx and lamina rara externa sites, while pH 3.0+ showed intracellular, nuclear, GBM, and mesangial matrix staining.
- Enzymatic digestion indicated that chondroitin sulfate or hyaluronic acid constitute GBM anionic sites, with chondroitinase activity suggesting hyaluronic acid involvement.
Conclusions:
- Glomerular anionic sites exhibit distinct characteristics based on their location and the experimental conditions (pH).
- Hyaluronic acid is suggested as a major component of glomerular anionic sites, particularly within the glomerular basement membrane.
- Lamina rara externa anionic sites are structurally distinct from other glomerular basement membrane anionic sites, as indicated by pH-dependent staining.