Harrison Latta

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.

Sialoglycoproteins and filtration barriers in the glomerular capillary wall.

The free surface coat on foot processes of rat glomerular epithelium stains particularly heavily with ruthenium red and osmium tetroxide, indicating a polyanionic sialoglycoprotein. It is compressible and expansile, containing densely packed filaments 12–25 wide. The coat covers the filtration slit membrane and under some conditions fills the filtration slit between foot processes. Previous studies have indicated that this free surface coat has the characteristics necessary to act as a fine glomerular filtration barrier for serum albumin. Other epithelial and endothelial surface coats with different characteristics contribute to the inner and outer layers of the basement membrane. The central layer of the basement membrane, which acts as a course filter, seems to contain glycoprotein with very little sialic acid. Most fibrils in this layer are 12–25 wide, but some are up to 50 wide. The fibrils are more loosely packed than those in the free surface coat. The surface coats of human glomerular capillary walls stain like those of the rat.

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 glomerular capillary wall

Observations of the glomerular capillary wall of rats and rabbits in normal and experimental conditions, including hemoglobin excretion, clarify the fine structure of the different components involved in the process of glomerular filtration. During excretion of large amounts of hemoglobin, the protein fills the inner and outer layers of the basement membrane, indicating a fairly loose gel structure. The lesser density of the central layer of the basement membrane and of the surface coat on the foot processes indicate rather compact structures without much space for hemoglobin to accumulate, although it must pass through both layers. The absence of hemoglobin between most foot processes when other parts of Bowmans space are filled with it, suggest that normally in vivo the surface coats between adjacent foot processes are usually in contact for the entire height of the processes. The glomerular filtrate must pass through the two surface coats filling the slit approximately 240 A wide between the foot processes. Several lines of evidence support the following conclusions. Glomerular endothelial fenestrations offer little or no barrier to filtration. The central layer of the basement membrane is a dense structure normally stopping molecules larger than about 100 A. The slit membrane and the polysaccharide surface coat beyond it appear to act as a finer filter, but are permeable to hemoglobin (64 A in largest diameter) and smaller molecules.

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.

Interstitial cells of the renal medulla

Interstitial cells in the medulla of the rat kidney have been studied by electron microscopy after fixation by perfusion with glutaraldehyde. In the outer zone of the medulla the interstitial cells resemble fibroblasts and are associated with collagen and a small amount of ground substance. In the inner zone of the medulla the interstitial cells have some characteristics of fibroblasts, but also show several unusual features. They have remarkably enlarged perinuclear cisternae. Groups of vesicles are found outside cells, and may be associated with apparent gaps in the plasma membrane. The cells and their processes are more closely related to capillaries and thin limbs of the loop of Henle than to collecting tubule cells. The most prominent feature of the interstitial cells of the inner medulla is their content of numerous osmiophilic droplets of unknown function.

Variations in fine structure of renal tubular epithelium under different conditions of fixation.

Variations in fine structure of renal tubular cells under different conditions prior to and during fixation were studied. Various procedures may affect cells before the fixative reaches them in effective concentrations. The resultant cellular alterations indicate reactive mechanisms that could take place in the living animal. The best fixation for electron microscopy is obtained by dripping the fixative on the surface of the kidney in the living animal, without removing the capsule. Good fixation is found, by both light and electron microscopy, from 100 to 200 μ down from the surface. Tubule lumina are open and capillaries distended. The lumina of proximal convoluted tubules are closed in blocks fixed by immersion or in the deep layers of blocks fixed by dripping. Particular attention was given extracellular compartments, which on the basis of previous work seem to indicate physiologically active sites of water resorption. The major non-physiologic procedures producing compartments in normal kidneys with an intact circulation seem to involve mechanical trauma, such as removing the renal capsule, rubbing the surface, or excising tissue. Hypertonic solutions produce large compartments readily in excised tissue, but an intact circulation renders the kidney resistant to the effects of moderately hypertonic or hypotonic solutions, and of exposure to air. Extracellular compartments, therefore, represent sensitive cellular reactions which can appear and disappear in a few minutes. Distal convoluted and collecting tubules are much less affected by the conditions studied in these experiments, and glomeruli are even more resistant.

The thin limbs of the loop of Henle

The renal medulla of male rats, fixed by perfusion with 6% glutaraldehyde and postfixed in osmium tetroxide, was examined at different levels by electron microscopy. The descending thin limb of the loop of Henle, identified at its origin from the proximal tubule as well as by its occurrence in the outer medulla, is composed of a single layer of interdigitating cell processes which have smaller basal, lateral, and luminal projections as well as microvilli on the luminal surface. The ascending thin limb of the loop of Henle, identified at its junction with the distal tubule, is composed of interdigitating cell processes which have very few small basal projections and short microvilli. Both types of thin segments have been found at different levels of the inner medulla, but the descending type becomes less frequent and disappears toward the tip of the pyramid. It seems likely, therefore, that the transition between the two types occurs in the descending limbs in the middle layer of the inner medulla. The basement membranes around thin limbs and medullary capillaries are frequently covered with multilayered segments of ground substance. A close association between thin limbs, vessels, and interstitial cells rich in osmiophilic droplets occurs in the inner medulla. Although physiologic evidence at present indicates that the descending and ascending limbs have different functions in countercurrent concentration, certain problems arise in attempting a detailed correlation.

Collagen in normal rat glomeruli.

Collagen fibers have been found by electron microscopy in the glomeruli of six normal rats. They occur in scattered small groups adjacent to intercapillary cells in centrolobular regions on the capillary side of the glomerular basement membrane. The fibers may lie at considerable distances from the glomerular hilus. The presence of collagen next to normal intercapillary cells is additional evidence that these cells should be regarded as a special cell type and not just as endothelial cells. This supports other evidence that the centrolobular part of the glomerulus is a special region having a unique role in renal function and disease.

Acute reactions with collagen production in renal glomeruli of rats as studied electron microscopically.

Striking glomerular changes are seen electron microscopically in rat kidneys after acute uranyl nitrate poisoning. More interesting than the several glomerular epithelial cell reactions to this injury, perhaps, is the development of the nodular centrolobular lesion containing collagen fibers. The glomerular epithelial cell changes include loss of foot processes , the appearance of dense deposits in foot processes or in the larger portions of cytoplasm which replace lost foot processes, hyaline droplets , other cytoplasmic bodies with varying internal membranes and granules, myelin figures , and cytoplasmic vacuoles . The functional significances of these changes and of the observed proteinuria, polyuria, and oliguria are discussed. The nodular centrolobular lesion was identified with a trichrome stain after embedding in methacrylate. As seen with the electron microscope it consists of several components lying between the central dense layer of the basement membrane and endothelial cells. Separately distinguished components include intercapillary cells and their processes lying in intimate contact with extracellular (a) small dense granules 30 m μ in diameter, (b) large bodies 400 m μ in diameter, vaguely forming rough cords, and (c) collagen fibers , randomly oriented and appearing within four days after injection of uranium. The intercapillary cells, often containing clear vacuoles with very dense and thick osmiophilic walls, may later prove to be endothelial cells but their intimate association with collagen fibers raises other alternatives.