In the current study, EM imaging was performed on slices of peripheral kidney tissue from healthy C57BL/6 J wild-type mice. All experiments were approved by the local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz NRW, Germany). Ketamine/xylazine anesthesia was applied before perfusion with 5 ml freshly prepared fixative solution (pH 7.3, sodium cacodylate 0.1 M, 2% glutaraldehyde, 2% lanthanum, 2% sucrose) via the left ventricle for 2 min. The left kidney was explanted immediately after perfusion, stored in fixative overnight at 4 °C and transferred to a glutaraldehyde-free solution the next day for further preparation. Samples were treated with osmium tetroxide, counterstained with uranyl acetate in 70% ethanol, dehydrated, and embedded in Durcupan resin. Resin blocks were prepared, and ultrathin sections were cut with a Leica Ultracut S (Mannheim, Germany). Sections were adsorbed onto glow-discharged Formvar carbon-coated copper grids. Images were taken using a Zeiss LEO 910 electron microscope (Zeiss, Oberkochen, Germany) equipped with a TRS sharpeye CCD camera and manufacturer’s software (Troendle, Moorenweis, Germany).

This approach to visualize the GCX in murine renal vessels (Fig. 2) produced images that, to our knowledge, are the first to be convincingly consistent with the concept of GCX described above. In larger vessels, we first observed dense, but undoubtedly hairy and at the same time delicate glycocalyx bundles with an average thickness of about 250 nm (Fig. 2A, B). Such bundles have been described in numerous publications, but it is still not clear whether they represent the entire GCX or only the lower core of this layer bound to the plasma membrane. When we looked at peritubular capillaries, we found an even more compact glycocalyx-compatible layer that continuously covered the endothelium and filled almost the entire lumen. The surprisingly high thickness of about 800 nm exceeded the height of the individual tufts in the large vessels by a factor of 3 (Fig. 2C, D).

Electron microscopy images of GCX in murine kidney vessels. EM imaging shows GCX bundles in larger vessels of murine kidneys with an average thickness of about 250 nm (A + B). In capillary vessels a dense GCX-compatible layer continuously covers the endothelial surface and fills nearly the whole lumen, reaching a thickness of up to 800 nm. Passing blood cells cause compression of GCX which appears (re-)expanded in-between again (C + D). Scale bar in A and D is 500 nm and 100 nm B and C

Some of the indirect methods for measuring GCX take advantage of the more or less pronounced compression of the glycocalyx by circulating blood cells and estimate the thickness of the GCX from the resulting variability in the lateral deflection of these cells [10]. However, the dynamics in the ultrastructure of the GCX cannot yet be visualized methodically. Fortunately, we were able to capture exactly this (compressive) passage of circulating blood cells through the dense capillary GCX in a rare EM snapshot (Fig. 2D). Coincidentally, a passing nucleus-carrying blood cell (e.g. leukocyte) and an erythrocyte can be seen in the capillary lumen. However, the GCX between the two passing blood cells is not compressed at all. Assuming that one of the two cells must have already passed through the capillary, this indicates a very rapid and complete re-expansion of the GCX.