J. Rostgaard

Sieve Plugs in Fenestrae of Glomerular Capillaries – Site of the Filtration Barrier?

The exact location of the filtration barrier of the glomerular capillary wall, which consists of an endothelium, a basement membrane and a visceral epithelium, has not yet been determined. Apparent discrepancies between different investigators in the past could be explained if postmortem artifactual tissue changes, due to subnormal blood pressure or anoxia, have taken place in the endothelium before the tissue and tracers have been sufficiently fixed and immobilized by the fixative. To test this supposition, a new method of fixation, which includes a technique to maintain a physiological perfusion pressure and a fixative composed of an oxygen-carrying blood substitute fluid containing glutaraldehyde, was employed combined with contrast enhancement. New observations of the glomerular capillary wall revealed that filamentous plugs (about 90 nm in height) filled the capillary fenestrae and a filamentous surface coat about 60 nm thick covered the interfenestral domains of the endothelial cell. Based on these purely morphological data, we dare to suggest that the fenestral plugs are the primary site of the glomerular filtration barrier – albeit highly speculative, nevertheless a logical location – and consequently that the glomerular filtration process is a ‘tangential-flow’ as opposed to a ‘dead-end’ filtration process. A tangential-flow filtration would minimize ‘clogging’ and ‘concentration polarization’ in the ‘filter’.

Similar Endothelial Glycocalyx Structures in Microvessels from a Range of Mammalian Tissues: Evidence for a Common Filtering Mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.

Electron microscopy of filaments in the basal part of rat kidney tubule cells, and their in situ interaction with heavy meromyosin

SummaryBy electron microscopy, the prominent bundles of filaments occurring in the basal part of proximal and distal tubule cells and in interstitial cells of rat kidney cortex were studied in cells fixed by vascular perfusion, in glycerol-extracted cells and in glycerol-extracted cells treated with heavy meromyosin (HMM).The studies of perfusion-fixed tissue showed that the proximal tubule cells contained in their most basal part filamentous bundles oriented transversely around the tubule. The bundles consisted of trightly packed thin filaments (50–80 Å in diameter). Similar but less prominent bundles were found in distal tubule cells and in interstitial cells. The dimension of these filaments was similar to that of actin filaments and their insertion in the basal cell membrane of the tubule epithelial cells resembled the insertion of actin filaments in the cell membrane of smooth muscle cells.The studies on glycerol-extracted cells revealed that some tubule cells contained two types of filaments (60–80 Å and 130–170 Å in diameter) located side by side in the basal filamentous bundles. The dimension of the thick filaments corresponds well to the values for myosin filaments in glycerinated smooth and skeletal muscle.The studies on HMM-reacted renal tissue revealed that the thin filaments (60–80 Å) described in tubule and interstitial cells are probably actin filaments, as they formed characteristic arrowhead complexes morphologically indistinguishable from the complexes of HMM with actin filaments in smooth and striated muscle cells.Our results provide strong evidence that a two-filament contractile system, based on interaction of actin and myosin filaments, exists in renal tubule and interstitial cells. As a hypothesis it is proposed that it is changes in tonus of the basal filamentous system in the proximal tubule cells which stabilize the intratubular pressure, possibly via angiotensin.

3D Reconstruction of the Glycocalyx Structure in Mammalian Capillaries using Electron Tomography

Please cite this paper as: Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 19: 343–351, 2012.

Three-dimensional organization of a transcellular tubulocisternal endoplasmic reticulum in epithelial cells of Reissner's membrane in the guinea-pig.

SummaryThe ultrastructure of the epithelial cells of Reissners membrane (membrana vestibularis) in the guinea-pig is described following vascular perfusion with glutaraldehyde of live, anaesthetised and artificially respirated animals. Postfixation in a solution containing OsO4 and potassium ferricyanide revealed a well-developed tubulocisternal endoplasmic reticulum, not previously described, the continuity of which has been mapped by serial sectioning and reconstruction. Large disc-shaped subsurface cisternae lining the cell membrane, but separated from it by a space approximately 10 nm wide, are in continuity with the smooth endoplasmic reticulum, forming an elaborated transcellular canalicular pathway. This structure is compared to that found in solute-transporting epithelia, e.g., renal proximal tubule, gall bladder, small intestine and choroid plexus. The fixation method used in the present study is compared to other techniques used for preservation of Reissners membrane. Each epithelial cell of Reissners membrane is endowed with one kinocilium, one to four multivesicular bodies, and a number of intercalated bodies. The functional significance of the canalicular pathway is discussed.

Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney

SummaryThe ultrastructure of the plasma membrane and the core of microvilli of proximal tubule cells has been investigated by electron microscopy using sectioned and negatively stained material. By the technique of negative staining, a particulated coat is disclosed on the outside of the plasma membrane of microvilli of brush borders isolated from rat, rabbit and ox. This coat is composed of 30 to 60 Å particles and is 150 to 300 Å thick and appears to be a distinguishing feature for the luminal plasma membrane (brush border) of proximal tubule cells. The plasma membrane of the basal part of tubule cells is found to be smooth. By thin sectioning, an axial bundle of 50 to 70 Å diameter filaments regularly arranged in an “1+6 configuration”, one axially located filament being surrounded by a ring of six, is disclosed. The distance from the ring of filaments to the inner surface of the plasma membrane is 250–300 Å, the diameter of the ring 300 Å and the center-to-center distance between filaments 120 Å. Negative staining also discloses 60 Å filaments in microvilli of isolated brush borders. Broken off, single microvilli (fingerstalls) are observed with thin filaments projecting from their broken ends. Filaments up to 1 μ in length are seen. At high magnification, the filaments appear beaded and show strong resemblance with actin filaments isolated from skeletal muscle. Based on present evidence, it is postulated that microvilli constituting renal brush borders possess contractile properties, which may play a role in the absorption process operating at the luminal part of the cells.

A two-filament system and interaction of heavy meromyosin (HMM) with thin filaments in smooth muscle

SummaryThe structure of glycerinated smooth muscle from small intestine of adult rat was investigated by electron microscopy. In the central parts of the tissue blocks a two-filament system was found, consisting of parallel thick and thin filaments with regularly spaced interconnections, closely resembling that of striated muscle. In the peripheral parts of the blocks only thin filaments were found. The thin filaments were identified as actin by the formation of arrowhead complexes after incubation with heavy meromyosin.

Surface morphology of the endolymphatic duct in the rat: a scanning electron microscopy study

Following intracardiac vascular perfusion fixation of 8 rats with glutaraldehyde in a buffered and oxygenated blood substitute, the vestibular aqueduct and endolymphatic duct were opened by microsurgery of the resulting 16 temporal bones. Optimum preservation of the epithelium for scanning electron microscopy was attained by coating of the specimens with osmium tetroxide and thiocarbohy drazide followed by a continuous dehydration procedure. This technique permitted, for the first time, an investigation of the surface morphology of the epithelial cells in the endolymphatic duct. Three types of cells were identified with the scanning electron microscope. A polygonal and oblong epithelial cell was observed in the largest number throughout the duct, and in the juxtasaccular half of the duct, two additional types of epithelial cells were observed. The scanning electron microscopic observations are compared and discussed with reference to previous transmission electron microscopic studies of the endolymphatic duct.

Variations in myoneme birefringence in relation to length changes in Stentor coeruleus.

Abstract The stalk segment of the heterotrich ciliate, Stentor coeruleus , appears nearly isotropic in the contracted state and develops a characteristic birefringence during extension. The birefringence occurs in stripes and is associated with the myonemes, one of the two longitudinally running subpellicular fiber systems. Electron microscopical investigations reveal changes in the ultrastructure of the myonemes from the extended to the contracted state. The relaxed myonemes consist mainly of 3 nm filaments running in the longitudinal direction, while the contracted myonemes show 10 nm tubular-like filaments, more randomly oriented. It is suggested that during elongation the randomly oriented tubular filaments undergo a conformational change to more regularly arranged thin filaments, thus causing development of birefringence.

Effect of cyclosporine during initiation of transplant arteriosclerosis. An ultrastructural study in the aorta-transplanted rabbit

The immunosuppressant cyclosporine protects against the development of experimental transplant arteriosclerosis. To investigate the mechanism underlying this effect, aorta-allografted rabbits were randomly assigned to cyclosporine (n = 6) in the human therapeutic range or to its vehicle (n = 5). Perfusion fixation was performed 2 weeks after the transplantation, followed by light, scanning and transmission electron microscopy examination. Intimal proliferation was absent in native aortas, present in all grafts from vehicle-treated animals, and either absent or sparse in grafts from cyclosporine-treated rabbits. The endothelium of native aortas from both vehicle- and cyclosporine-treated rabbits was normal. Aortic allografts from cyclosporine-treated rabbits exhibited a normal endothelium with only a few adhering mononuclear cells, whereas aortic allografts from vehicle-treated rabbits exhibited an endothelial surface ranging from near-normal with only few adhering platelets and mononuclear cells, to an almost destroyed endothelium, lined with lymphocyte-like cells, monocytes/macrophages, platelets, erythrocytes and fibrin; in the subendothelial layer, mononuclear cells and smooth muscle cells were abundant. These results suggest that damage to the endothelial cells as well as invasion of lymphocytes, monocytes/macrophages and smooth muscle cells into the subendothelial space are important events during initiation of transplant arteriosclerosis, and that cyclosporine largely attenuates these early pathological changes.