Possible continuity of subplasmalemmal cytoplasmic network with basement membrane cord network: ultrastructural study

The ultrastructure of the subplasmalemmal cytoplasm of the cell and the associated basement membrane as well as the area of the cell-basement membrane border were observed with high resolution electron microscopy after preparation of the tissues with cryofixation or glutaraldehyde fixation followed by freeze substitution. The subplasmalemmal cytoplasm of the smooth muscle cells of rat epididymal tubules and the podocyte processes of the mouse glomerular visceral epithelium were found to be composed of a fine network of irregular anastomosing strands. This network closely resembled the previously characterized cord network of the basement membrane. The cords are known to be composed of a 1.5 to 3 nm thick core filament made up of type IV collagen which is surrounded by an irregular ‘sheath’ of other components. The strands in the subplasmalemmal network showed ultrastructural features similar to those of the cord network. Ribbon-like, 4.5 nm wide heparan sulfate proteoglycan ‘double tracks’ were previously reported to be associated with the cord network. Structures similar in size and appearance to the double tracks were also found in the subplasmalemmal network. At the cell-basement membrane border, the lamina densa of the basement membrane was in contact with the cell without the intervening space of a lamina lucida which was recently found to be an artefact caused by conventional tissue processing. Furthermore, the subplasmalemmal network appeared to be continuous through the plasma membrane, with the cord network of the basement membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption and transport of ferritin and exogenous horseradish peroxidase in the opisthonephric kidney of the sea lamprey. I. The renal corpuscle

The single, elongate renal corpuscle in the opisthonephros of the sea lamprey deals with exogenous protein tracers in much the same manner as the renal corpuscles in the kidneys of higher vertebrates. Horseradish peroxidase readily diffuses from the capillaries through the mesangium, basement membrane, and slit diaphragms of the visceral epithelium into the urinary space, where it is transported to the tubule. Ferritin diffuses more slowly but follows a similar pathway. Protein tracers which come in contact with the visceral and parietal epithelium, either through diffusion from the circulation or after direct introduction of these substances into the urinary space, are readily absorbed by both types of epithelium. Permeability of the parietal epithelium is suggested by the presence of the tracers in intercellular spaces. The localization of these tracers in the urinary space and epithelial cells both anterior and posterior to the site of their introduction suggests that the elongate glomus is surrounded by a single tortuous urinary space resulting from the continuity of the capsules.

GLOMERULAR PERMEABILITY

Ferritin was used as a tracer to investigate pathways and mechanisms for transfer across the various layers of the glomerular capillary wall. Kidney tissue, fixed at intervals of 2 minutes to 2 hours following an intravenous injection of ferritin, was examined by electron microscopy. The observations confirmed the existence of three distinct and successive layers in the glomerular capillary wall (the endothelium, the basement membrane, and the visceral epithelium). In addition, they demonstrated a number of new structural features: namely (a) discrete fibrils in the subendothelial spaces; (b) a characteristic, highly elaborate, cytoplasmic organization in the visceral epithelium; and (c) special structures resembling "desmosomes" in the slits between foot processes. In animals sacrificed at short time intervals (2 to 15 minutes) following ferritin administration, ferritin molecules were found at high concentration in the lumen and endothelial fenestrae, at low concentration in the basement membrane, and in very small numbers within the epithelium. Later (1 to 2 hours), the tracer particles were still present in the lumen and within endothelial fenestrae, and, in addition, had accumulated on the luminal side of the basement membrane, especially in the axial regions of the vessels. Larger numbers of ferritin molecules were also found in the epithelium—in invaginations of the cell membrane at the base of the foot processes, and in various membrane-limited bodies (vesicles, multivesicular bodies, vacuoles, and dense bodies) present within the cytoplasm. These observations suggest that the endothelial fenestrae are patent and that the basement membrane is the main filtration barrier. Since the basement membrane has no demonstrable pores, it is probably not a simple sieve but presumably is a gel-like structure with two fine fibrillar components embedded in an amorphous matrix. Both the epithelium and endothelium may be concerned with building and maintaining this structure. Finally, the intracellular accumulation of particles in the epithelium suggests that the latter acts as a monitor that recovers, at least in part, the small amounts of protein which normally leak through the filter.

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.