Imaging of the porous ultrastructure of the glomerular epithelial filtration slit

Abstract

The ultrastructure of the glomerular filtration slit is still controversial. In the last 30 years, observations from transmission electron microscopy (TEM) and theoretical analysis of solute clearance produced conflicting results. Here, we used scanning EM with a high-sensitivity detector to image the deepest regions of the filtration slits and report a previously undescribed organization of the slits' ultrastructure. In contrast to previous TEM imaging, we observed circular and ellipsoidal pores in the podocyte junctions mainly located in the central region of the slit diaphragm. The normal mean pore radius estimated by digital morphometric analysis had a log-normal distribution, with an average value of 12.1 nm. In proteinuric pathologic conditions, the mean pore radius values were also log-normally distributed with the presence of some very large pores, exceeding the sizes observed in normal conditions. Our morphologic analysis suggests that the filtration slit is a heteroporous structure instead of the previously proposed zipper-like structure. Selective changes in the ultrastructural organization of the pores may be responsible for the increased filtration of plasma proteins in glomerular disease.

Figure 1.

Visualization of epithelial filtration pores using an in-lens detector with SEM. Representative scanning electron photomicrographs from Wistar rat, taken at low and high magnifications (A: 12,000×; B: 36,000×; C and D: 60,000×; E: 80,000×; F: 100,000×) with conventional detector (A–C) and in-lens detector (D–F) for secondary electrons. In-lens photomicrographs of samples dehydrated with a critical point dryer (A–E) or HMDS (F).

Figure 2.

Schematic representation of the procedure used to evaluate pore dimension. (Top) A representative image obtained at scanning electron microscopy (original magnification, 200,000×) with an enlargement of the filtration slit pores. (Bottom) Representation of the surface plot obtained using the software Image J (v. 1.43 NIH).

Figure 3.

Distribution of slit pore size is unchanged under proteinuria. Distribution of slit pore sizes of Wistar (A) and MWF (B) rats, as measured by digital morphometric analysis and the best fit lognormal probability distribution of pore radii.

Figure 4.

Proteinuric rats contain larger pores. Distribution of fractional pore area (the fraction of total pore area for given pore area interval) calculated for slit pores of Wistar (A) and MWF (B) rats.

Figure 5.

The ultrastructure of the preserved area of glomerular capillary tuft is similar in normal and proteinuric rats. Representative glomerular sections stained by periodic-acid Schiff and transmission electron micrographs of the capillary wall in Wistar (A and C) and MWF (B and D) rats. Original magnifications: 400× (A and B) and 36,000× (C and D).

Figure 6.

The ultrastructure of the filtration slits is similar in different rat strains, both in normal and proteinuric conditions. Representative scanning electron photomicrographs of filtration slit ultrastructure between neighboring podocytes in Wistar (A and B), MWF (C and D), and ZDF (E and F) rats, taken with an in-lens detector. Original magnifications: A and C, 120,000×; B, 85,000×; D, 150,000×; E, 100,000×; F, 140,000×.