Arvid B. Maunsbach

The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells: I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives

The ultrastructure of rat kidney proximal tubule cells was compared following fixation by different methods (microperfusion of single tubules, different forms of vascular perfusion, dripping of the fixative on the kidney surface). A comparison was also made between glutaraldehyde, formaldehyde and osmium tetroxide fixatives. The rat kidney cortex could be uniformely fixed by vascular perfusion with both aldehyde and osmium tetroxide fixatives. Characteristic ultrastructural differences were observed between cells fixed with the different fixation methods and with the different fixatives. The observations demonstrate a great sensitivity of the proximal tubule cells to different conditions of fixation and illustrate the necessity of considering this when analyzing the ultrastructural organization of these cells.

Absorption of I125-labeled homologous albumin by rat kidney proximal tubule cells: A study of microperfused single proximal tubules by electron microscopic autoradiography and histochemistry

Small amounts of I 125 -labeled rat albumin were injected through micropipettes into single proximal tubules of the rat kidney. After different time-intervals the injected tubules were microperfusion-fixed with glutaraldehyde and the tissue analyzed by electron microscopic autoradiography. No ultrastructural alterations were observed in the proximal tubule cells during the absorption of the labeled protein. Six to 10 minutes after start of I 125 -albumin perfusion most of the absorbed protein was located in the large apical vacuoles in the proximal tubule cells but some label was also associated with small apical vacuoles. The observations suggest that the labeled albumin was transferred to the large apical vacuoles via small apical vacuoles which most likely represented pinched-off apical cell membrane invaginations. After 30 minutes most of the label was present in cytoplasmic bodies, which were limited by a triple-layered membrane about 90 A in thickness and had an electron-dense content, and which were located in the middle or apical regions of the cells. After 60 minutes the label was confined to similar cytoplasmic bodies located in all regions of the cells. Combined electron microscopic autoradiography and histochemistry demonstrated that the labeled cytoplasmic bodies were acid phosphatase positive. The latter observation indicates that the cytoplasmic bodies, in which the I 125 -albumin was located, were lysosomes. It is suggested that at least some of the absorbed albumin was degraded in these lysosomes. There was no autoradiographic or morphologic evidence that the labeled albumin crossed the wall of the proximal tubule by passing between the cells or that vacuoles or cytoplasmic bodies containing absorbed albumin emptied into the peritubular space.

Observations on the segmentation of the proximal tubule in the rat kidney. Comparison of results from phase contrast, fluorescence and electron microscopy.

The segmentation of the proximal tubule in the normal rat kidney was studied by phase contrast, fluorescence and electron microscopy. Three different segments were observed with all methods. The first segment comprised the beginning of the convoluted part of the proximal tubule, the second segment included the end of the convoluted and the beginning of the descending part of the proximal tubule, and the remaining portion of the descending part formed the third segment. Pronounced differences were present between cells in the three segments with respect to size and frequency of occurrence of acid phosphatase containing cytoplasmic bodies, length of brush border projections, size and arrangement of mitochondria, and extent to which the proximal tubule cells interdigitated. Characteristic differences were also observed between proximal tubules from male and female rats.

The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. II. Effects of varying osmolality, ionic strength, buffer system and fixative concentration of glutaraldehyde solutions.

The influence of different glutaraldehyde fixatives on the ultrastructure of proximal tubule cells in the rat kidney was studied. The fixative solutions were systematically varied with respect to sodium chloride concentration, buffer system and glutaraldehyde concentration. The ultrastructure of the proximal tubule cells differed markedly when the sodium chloride concentration of the fixative vehicle was varied. Smaller, yet significant, differences were observed when the concentration of glutaraldehyde was changed. Almost no differences were found between differently buffered glutaraldehyde fixatives when the buffers had approximately the same osmolality.

Observations on the ultrastructure and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubule cells: With a comment on their classification

The ultrastructure and acid phosphatase acitivity of the cytoplasmic bodies in normal proximal tubule cells of the rat kidney was investigated. The cytoplasmic bodies were divided into four different types on the basis of ultrastructural differences between their limiting membranes or boundaries. Cytoplasmic bodies type I (microbodies) were limited by a single triple-layered membrane about 65 A in thickness. Cytoplasmic bodies type II were limited by a single triple-layered membrane about 90 A in thickness. Cytoplasmic bodies type III were not limited by distinct membranes. Cytoplasmic bodies type IV were limited by two triple-layered membranes each 65 A in thickness. Acid phosphatase activity was demonstrable by electron microscopic histochemistry in cytoplasmic bodies type II and type IV which indicates that these bodies are lysosomes.

The centrolobular region of the renal glomerulus studied by electron microscopy.

Centrolobular regions of rat kidney glomeruli were studied electron microscopically after fixation in the living animal and embedding in Araldite or Epon. Intercapillary cells within these regions are separated from adjacent capillary lumens by endothelial cells. The endothelial cell may be represented by a portion containing the nucleus, but more often by a thin layer of cytoplasm having occasional gaps or holes. In contrast to endothelial cells, intercapillary cells have many branching processes which have not been found to extend to the capillary lumen. An amorphous intercellular substance fills much but not all of the centrolobular space between intercapillary cells and their processes. The central dense layer of the basement membrane follows the epithelium over the centrolobular region without splitting and entering it or sending a layer beneath the central portions of endothelial cells. After intravenous injection of thorotrast, particles enter the centrolobular region in high concentrations within 5 to 10 minutes, penetrating intercapillary channels through the intercellular substance between intercapillary cells. These observations suggest that centrolobular regions and intercapillary cells may play special roles in the control of capillary flow and in reactions to pathologic agents.

Absorption of ferritin by rat kidney proximal tubule cells: Electron microscopic observations of the initial uptake phase in cells of microperfused single proximal tubules

Ferritin was injected through micropipettes into single proximal tubules of the rat kidney. After different time-intervals the injected tubules were microperfusion-fixed with glutaraldehyde and analyzed by electron microscopy. Five minutes after start of ferritin perfusion most of the absorbed protein was located in small and large apical vacuoles. No ferritin was observed in the cytoplasm outside the vacuoles. In the tubule lumen ferritin molecules were associated with the outer electron dense coating of the apical cell membrane invaginations. Sixteen to 20 minutes after start of perfusion most small apical vacuoles were devoid of ferritin. The observations indicated that ferritin was transported to the large apical vacuoles via small apical vacuoles, which represented pinched off plasma membrane invaginations. There was no indication that ferritin crossed the wall of the proximal tubule by passing between the cells or that the ferritin-containing vacuoles emptied into the peritubular space.

Effects on tissue fine structure of variations in colloid osmotic pressure of glutaraldehyde fixatives

The effects on tissue fine structure of variations in the colloid osmotic pressure of fixative solutions used for perfusion fixation were studied. Groups of rats were perfusion-fixed with glutaraldehyde solutions which varied only with respect to their concentration of dextran or polyvinylpyrrolidone (PVP). Tissues from several organs, including kidney, pancreas, and intestine, were analyzed by light and electron microscopy. In all organs there were distinct differences in structure between tissues fixed with fixatives containing dextran or PVP and those fixed with solutions devoid of macromolecules. Following fixation without macromolecules in the fixative, the extravascular spaces were greatly enlarged in comparison to similar spaces in tissues fixed in an identical way but with dextran or PVP in the fixative. It is proposed that these and other observed differences are due to differences in the colloid osmotic pressure of the fixations. The results indicate that oncotically active substances should be included in aldehyde fixatives used for perfusion, in particular when structures or cellular relationships in labile extravascular spaces are studied.

Relations of the centrolobular region of the glomerulus to the juxtaglomerular apparatus

Intercapillary or mesangial cells in the centrolobular region of renal glomeruli of rats are similar to and continuous with “lacis” (pseudo-meissnerian) cells of the juxtaglomerular apparatus. They have electron microscopic features which also relate them to the granular epithelioid cells of the juxtaglomerular apparatus and to smooth muscle cells. Their structural features and functions help to differentiate them from adjacent endothelial cells. Intercapillary cells are bathed by a rapid flow of blood plasma through the centrolobular region. They are actively phagocytic and associated with the PAS-positive intercellular substance in normal glomeruli and its increase in disease, with occasional bundles of collagen in the normal glomerulus, and with the formation of collagen in some glomerular diseases in animals and human beings. Intercapillary cells seem to have various mechanisms with which they could supplement the juxtaglomerular apparatus in altering the amount or composition of the urine.

The juxtaglomerular apparatus as studied electron microscopically.

The juxtaglomerular apparatus of the rat kidney has been studied electron microscopically. Three parts and their intimate relationships are described in some detail: (1) the macula densa of the distal convoluted tubule, (2) the granular epithelioid cells, commonly found in the wall of the afferent arteriole, and (3) the cells of the “lacis” of Oberling and Hatt, or the pseudo-meissnerian cells of Goormaghtigh. The cells of the macula densa vary in fine structure from other cells of the distal convoluted tubule, and lying close under them are processes of both granular and lacis cells. Granular cells may contain, besides two or more types of granules, fibrillar bundles resembling those of smooth muscle. Lacis cells may lie beneath the endothelium of the afferent arteriole. Their cytoplasm usually contains prominent membrane systems, and may have granules and fibrillar bundles like those of granular cells. The evidence suggests that lacis cells are related to granular cells and that both may be derived from smooth muscle. Functional implications of these observations are discussed.