Date of Award

12-10-1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

The purpose of this study is to utilize high resolution SEM to examine topographical features of acellular renal cortical basement membranes (BMs) in the rat. Alterations in ultrastructure of protease-digested samples are evaluated in terms of the concept of BM heterogeneity, and an attempt is made to correlate observed changes with known components of BMs.Renal cortical blocks were rendered acellular by sequential detergent extraction, and digested either with 150 $\mu$g/ml Streptomyces griseus protease (SGP) or with 5 mg/ml pepsin and conventionally prepared for SEM and correlative TEM.With both proteases, Bowman's capsular BM (BCBM), tubular BM (TBM) and glomerular BM (GBM) showed time-dependent digestion, with BCBM least and GBM most resistant. At low magnifications, all acellular surfaces initially appeared smooth. With SGP, within 2 hrs BCBM showed ragged openings which exposed subjacent 50-60 nm fibrils. Between 2-4 hrs, BCBM was completely digested, and holes and other disruptions appeared in TBMs. At 6 hrs, most TBMs appeared only as isolated strips lying across extracellular matrix (ECM) fibrils. Unlike SGP, even after 184 hrs pepsin digestion was only complete with BCBM, while TBM and GBM appeared unaffected.At high magnification all control BM surfaces appeared continuous and consisted of 15-60 nm granules. Following protease digestion, however, surfaces appeared rougher and distinctly "pebbled". With SGP digestion, all BMs (most prominently TBMs) showed linear arrays of granules, which resembled "beads-on-a-string." The most notable effect of pepsin digestion occurred after 44 hrs digestion when 50-70 nm granules were aligned in "linear arrays" on GBM surfaces. With continued digestion this "fibrillar" component became more extensive, forming an interconnected meshwork which appeared to extend throughout the granular component of the GBM.The current study demonstrates the utility of high resolution SEM in assessing normal and altered BM morphology, thus extending the concept of BM heterogeneity to surface topography at the level of ultrastructure. Identification of observed ultrastructure with known components of BM was not possible, but correlation of BM granules with proteoglycans, and the extensive linear arrays visible on the GBM external surface following pepsin digestion with proposed models of Type IV collagen meshwork, is discussed.

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