Ultrastructural Study of Glomerular Capillary Loops at Different Perf usion Pressures as Revealed by Quick-Freezing, Freeze-Substitution and Conventional Fixation Methods

Abstract

Wistar rat kidneys were perfused with some fixatives at different pressures of 100, 150, 200 or 250 cm H₂0 via the aorta and were studied by the quick-freezing and freeze-substitution (QF-FS) or deep-etching (QF-DE) methods, in addition to the conventional immersion or perfusion fixation method. Different parts of glomerular capillary walls were selected for morphometric analyses. It was demonstrated that the widths of glomerular slit diaphragms and the spaces between foot processes were more widely dilated at higher perfusion pressures (200 and 250 cm H₂0) than those seen at both normal perfusion pressure (150 cm H₂O) and lower perfusion pressure (100 cm H₂O). On the other hand, the glomerular basement membranes were thinner at higher perfusion pressures. By the QF-FS and QF-DE methods, the foot processes showed different shapes from those revealed by the conventional preparation methods, even at the same perfusion pressure. It is concluded that the widths of glomerular slit diaphragms and glomerular basement membranes and the spaces between foot processes may be significantly changed in vivo, depending on the hemodynamics in the glomerular capillary.

Ultrastructural Study of Anionic Sites in Glomerular Basement Membranes at Different Perfusion Pressures by Quick-Freezing and Deep-Etching Method

“…3), typical three-layered structures of GBM were observed under each perfusion pressure condition, as previously reported [19]. Moreover, the widths of the LRE and LRI were thicker than those seen by the QF-DE method (fig.…”

“…The ultrastructures of glomerular capillary walls were gradually changed at different perfusion pressures, as reported before [19]. By the QF-DE method for pure morphology, filamentous structures in the GBM were observed more clearly than those seen on the ultrathin sections (fig.…”

“…The GBM became also thinner when the perfusion pressures were changed from low levels to higher ones, as reported before (fig. 4) [19]. …”

“…Some tissue slices obtained from the right kidneys were quickly plunged into liquid isopentane-propane mixture (around –193°C) cooled in liquid nitrogen, as reported before [19]. Then the frozen tissues were freeze-substituted in absolute acetone containing 2% osmium tetroxide nearly at –80°C for 20 h. After the freeze-substitution fixation, the samples were kept at –20°C for 2 h and then at 4°C for 2 h. Finally, they were raised up to a room temperature and washed twice in absolute acetone, and then embedded in Luveak-812.…”

Nephrin is specifically located at the slit diaphragm of glomerular podocytes

“…The actual slit diaphragm is considered to be a rather rigid structure. However, it has recently been suggested that at least the slit area might increase with increasing perfusion pressures of the glomerulus (34,35). Because the actual molecular structure of the slit diaphragm is, as yet, unresolved, it is premature to speculate on a molecular assembly allowing change in the width of the slit.…”