Ultrastructural Analysis of Glomerular Endothelium

The kidney specimens were fixed with a 4% paraformaldehyde and then incubated with 1% osmium tetroxide (OsO₄) at 4 °C for 1 h. The fixed specimens were dehydrated with gradient alcohols and embedded in Epon. Next, 70–100 nm ultrathin sections were cut with a Leica EM UC 7 microtome and stained with 1% uranyl acetate and lead citrate. Electron micrographs were taken at 30.000× at 80 kV with a JEM 1400 electron microscope (JEOL, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Microscopic analysis was carried out at the Multiple-access Center for Microscopy of Biological Subjects (Institute of Cytology and Genetics, Novosibirsk, Russia). The number of fenestrae of endotheliocytes of glomerular capillaries and the number of podocyte foot processes were determined for 2 μm of the glomerular basement membrane. Additionally, the thickness of the glomerular membrane and basement membrane of proximal tubular epitheliocytes, as well as the width of podocyte foot processes and slit diaphragm were measured using scale bars.