High resolution SEM analysis of acellular glomerular basement membrane following pepsin digestion: intrinsic...

Microdissection of acellular rat renal cortex with pepsin was carried out to investigate the morphological substructure of glomerular basement membrane (GBM) by high resolution SEM. Renal cortical blocks (less than 5 mm3) from adult male Sprague Dawley rats were rendered acellular by sequential detergent extraction and digested up to 184 hrs with 5 mg/ml pepsin (185 U/mg) in 0.5 M acetic acid (pH 2) at 10-15 degrees C. Samples were conventionally prepared for SEM, and observed at original magnifications of 500-100,000 diameters. At low magnifications (500-5,000x), acellular GBM surfaces appeared smooth at all digestion times. At higher magnifications (50,000-100,000x), control GBM surfaces were finely granular. Granule diameter ranged from 20-80 nm, with most between 30-40 nm. Pepsin digestion did not affect average granule size. Beginning at 44 hrs of digestion, intrinsic fibrillar structures comprised of linear arrays of 20-40 nm granules were observed on/in GBM surfaces. At later incubation times, this component of GBM became more extensive. At 160 hrs, the fibrillar arrays frequently bifurcated and showed distinctive "forked" termini, some of which comprised two sides of a triangle (120-150 nm on a side). Fork "handles" (310-350 nm in length) radiated from each angle of the triangle. These sometimes terminated in large granules (approximately 100 nm in diameter), two of which appeared to connect fibrillar arrays end-to-end. Together with other arrays, the interconnected triangles appeared to comprise a three-dimensional meshwork extending into the GBM and possibly providing support for, its granular components.