Quantitating glomerular endothelial fenestration: an unbiased stereological approach

Abstract

Background/aims: Glomerular endothelial cells are fenestrated, allowing for especially high transcellular hydraulic conductivity. Current knowledge about endothelial fenestration structural changes in disease conditions is limited, partly due to the absence of robust methodologies to quantitate these structures. Herein, we propose a novel method for estimating the percentage of endothelial fenestration.

Methods: An unbiased stereological method based on contiguity of two phases and surface area density estimation using isotropic uniform random line probes was developed. A line grid for intercept counting and classifying endothelial coverage of fenestrated versus non-fenestrated areas was designed. The method was applied to renal biopsies from 15 patients with Fabry disease and 9 normal living kidney donor controls.

Results: The percentage of glomerular capillary endothelial coverage which was fenestrated was lower in Fabry patients (43 ± 12%) versus controls (53 ± 9%; p = 0.047). The fraction of endothelial surface which was fenestrated was greater on the peripheral versus mesangial zones of the capillary walls in both Fabry patients (p = 0.00002) and controls (p = 0.0005).

Conclusion: The proposed method provides an unbiased tool to quantitate endothelial fenestration changes in glomeruli. The practical example introduced showed reduced glomerular endothelial fenestration in Fabry nephropathy.

Copyright © 2011 S. Karger AG, Basel.

Figures

Fig. 1

The grid used to estimate %EF is composed of an unbiased counting frame with inclusion (dashed) and exclusion (continuous) borders, and parallel lines 4 mm apart. The shape of the counting frame was chosen based upon the use of the same grid for slit diaphragm length density (not required for endothelial fenestration studies). There is 1 coarse line (where the intercepts with endothelial coverage are counted) per 7 fine lines. The short lines on the coarse lines are 4 mm apart and, similar to fine lines, are used to define fenestrated versusnon-fenestrated coverage. The endothelial coverage was arbitrarily called non-fenestrated if the distance between the two fenestrae on either side of the coarse line was more than 3 fine and coarse lines, otherwise it was called fenestrated.

Fig. 2

A The grid in figure 1 is superimposed on a high magnification (about ×35,000) of a glomerular capillary loop. The arrow marks a myelin figure inclusion (characteristic of Fabry disease) in a podocyte. The arrowhead points to capillary endothelial lining. The asterisk denotes the glomerular basement membrane. The white square shows the area magnified on the right. B Magnified view of a small portion of glomerular basement membrane with endothelial coverage on the bottom and podocyte foot processes on the top. The endothelial lining is traced with bold lines and shaded solid grey for easier visualization. Fenestrae are traced with white lines. Only intercepts of coarse lines (thicker with horizontal short lines) with endothelial lining are counted. The count of fine lines between closest fenestrae on either side of a coarse line (a–a’ and b–b’) arbitrarily classifies the endothelial coverage as fenestrated versus non-fenestrated.

Fig. 3

Schema of systematic uniform random sampling of a glomerular profile simulated on a montage image (×3,000) of a glomerulus from a Fabry patient. Asterisk marks a random starting point on the top left corner of the glomerulus. Squares simulate high-magnification images (×35,000) obtained. Arrows show the pattern of moving the thin section grid using control wheels in a transmission electron microscope to uniformly sample the entire glomerular profile.