Preparation and Histoarchitecture of Ultrastructurally Pure Glomerular Basement Membrane

Abstract

Our recent morphological studies showed that basement membranes isolated from renal tubules tended to collapse and form folded sheets while glomerular basement membranes were more resilient. In an effort to study the shapes of various isolated basement membranes in undissociated tissues, a method was developed to remove all cellular elements and leave the extracellular matrix and associated basement membranes intact. Accordingly, transplant quality human kidneys were harvested, perfused with Collin’s medium and transported to the laboratory on ice. The renal cortex was then peeled away by blunt dissection, further minced to 2 mm³ and placed in 1 mM EDTA (with gentle intermittent stirring) for 72 h at 4°C. Solubilization of cellular materials was carried out by successive washings with 3% Triton X-100, 0.025% DNAse and 1–4% sodium deoxycholate (all with gentle stirring or shaking at 22 °C). Each solution contained 0.1% sodium azide. At the level of fine structure, glomerular, tubular, Bowman’s capsular and peritubular capillary basement membranes all maintained their respective shapes and did not collapse. Glomerular basement membrane was particularly striking in this regard and exhibited an open, lobulated form that closely resembled its in vivo histoarchitecture. Moreover, when the acellular tissue blocks were prepared for scanning electron microscopic observation, the glomerular basement membranes exposed at the surface of the block showed a remarkable structural rigidity. These basement membranes were free-standing, convoluted electron-dense sheets, continuous with highly folded central mesangial regions. It seems significant that glomerular basement membranes maintain their in vivo conformation irrespective of the presence of other extracellular matrix components while removal of these materials by organ subfractionation results in folding and general shapelessness of tubular basement membrane. It is possible that in addition to its unique role in filtration, glomerular basement membrane may also serve to preserve glomerular shape, regardless of changing cell populations or alterations in hydrostatic pressures.

Ultrastructural Analyses of Acellular Glomerular Basement Membranes and Mesangial Matrix in a Spontaneously Diabetic Rhesus Monkey

“…1982;Carl son et al. 1986], By SEM their external surfaces appear smooth, evenly contoured and similar to those observed in normal humans [Carlson and Kenney. 1980: Carl son and Chatterjee.…”

Ultrastructural Analysis of Major Basement Membrane Types in Rhesus Monkey <i>Macaca mulatta</i> Acellular Renal Cortex

“…Of special interest are the GBMs which are not nor mally associated with large numbers of collagenous fib rils, yet invariably maintain their open lobulated shapes despite the complete removal of their cellular compo nents [Carlson and Kenney, 1980;Carlson and Hinds, 1983]. Rhesus monkey GBMs are particularly rigid following detergent extraction and resemble closely their human counterparts [Carlson and Chatterjee, 1983], As in the human, isolated monkey GBMs pre pared for SEM studies show spheroid forms comprised of highly folded BM sheets with smoothly contoured external surfaces.…”

“…Kidneys were excised and transported on ice in Earle's Balanced Salt Solution to the laboratory. Renal cortices were rendered acellular by a modification [Carlson and Kenney, 1980] of the sequential detergent extraction procedure developed by Meezan et al [1975], Briefly, the parenchyma of the kidney was dissected free from the renal capsule and associated perirenal fat. It was sliced (2mm thick) with a sharp razor blade followed by removal of the cortex (outer 3 mm of each slice).…”

“…We have developed a technique that renders renal cortical tissue blocks acellular while maintaining the histoarchitecture of BMs and associated structural compo nents of the ECM [Carlson and Kenney, 1980]. By this procedure all major BM types are available for study by LM, SEM or TEM.…”

Escape of Red Blood Cells through Gaps in Glomerular Basement Membrane in a Patient with Mixed Connective Tissue Disease

“…Frozen blocks used for immunofluorescence studies were treated sequentially in the following solutions according to the modified method of Carlson and Kenney [7] as previously described [8]:…”