Morphometric study of glomerular slit diaphragms fixed by rapid-freezing and freeze-substitution

Abstract

The widths of slit diaphragms in rat glomeruli were quantitatively measured on electron micrographs obtained by the rapid-freezing and freeze-substitution (FS) fixation method and compared to those prepared by conventional chemical fixation procedures, such as tannic acid, glutaraldehyde and osmium tetroxide (TGO) or glutaraldehyde and osmium tetroxide (GO) fixation. When fixed by FS using 2% osmium tetroxide, glomerular slit diaphragms had widths of 33.8 +/- 3.6 nm, but when fixed by TGO or GO they had widths of 47.0 +/- 4.3 and 48.7 +/- 5.5 nm, respectively. The width of slit diaphragms was significantly narrower after cryofixation than after conventional TGO or GO fixation (P less than 0.001). Similarly, the distance between neighboring foot processes at 50 nm from slit diaphragms was significantly narrower after cryofixation (P less than 0.001). The widths were 98.6 +/- 17.1 nm after TGO fixation, 122.0 +/- 29.3 nm after GO fixation and only 54.8 +/- 18.7 nm after FS fixation. These foot processes were significantly broader after FS fixation. The foot processes were 179.2 +/- 59.4 nm in breadth after TGO fixation (P less than 0.001), 188.8 +/- 78.2 nm after GO fixation (P less than 0.01) and 235.1 +/- 87.2 nm after FS fixation. These results suggest that the cytoplasm of foot processes shrinks when the tissue is prepared by conventional chemical fixation methods, and that the broadening of slit diaphragms is an artifact separation of the margins of adjacent foot processes due to shrinkage. We propose that TGO fixation alters the structure of slit diaphragms by causing them to shrink.

Nephrin is specifically located at the slit diaphragm of glomerular podocytes

“…Because these studies used the conventional thin-sectioning method requiring harsh chemical treatments in sample preparation, it has later been suggested that the zipper-looking structure might be due to an artifact. Since then, the podocytepodocyte junction has also been studied by several other electron microscopic methods (32,33). These studies have indicated that the width of this junction varies between 20 and 50 nm.…”

Glomerular epithelial foot processes and filtration slits in IDDM patients

“…The classification of true widths provides an UNBiased estimate of widths, since the probability of perpendicular sectioning is independent of the slit width. The absolute measures of the slit width vary with the tissue procurement [25]. In this investigation all of the biopsies were treated in the same way except that in the non-diabetic control group a different buffer was used and the biopsies were taken under anaesthesia after a period of ischaemia during the transplantation of the kidney.…”

Cell Biology of the Glomerular Podocyte

“…The filtration slits have a constant width of ϳ30 -40 nm (133,374) and are bridged by the slit diaphragm. Based on transmission electron microscopy findings in chemically fixed, tannic acid-stained material, Rodewald and Karnovsky (374) have published a model of the substructure of the slit membrane.…”