泰和鸡肾小球旁器的观察

本文用光镜和透射电镜对泰和鸡(乌骨鸡)的肾小球旁器进行了观察。结果表明,泰和鸡的肾小球旁器由球旁细胞、过渡型致密斑、球外间膜细胞和极周细胞所组成。极周细胞在鸟类还属首次报道,它位于肾小囊脏层与壁层上皮移行处,环绕着肾小体的血管极,其结构与哺乳动物的相似。本文还就泰和鸡肾小球旁器的结构与功能的关系作了讨论。     

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.     

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowman's capsule).     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.     

ELECTRON MICROSCOPY OF THE AVIAN RENAL GLOMERULUS

Electron microscopy of sections of chicken glomeruli shows them to possess a large central cell mass, occupying the hilum and the centre of the glomerulus, and continuous with the adventitia of the afferent and efferent arterioles. The glomerular capillaries form a much simpler system than in mammals and are spread over the surface of the central cell mass. Between the capillaries the mass is limited externally by the major component of the glomerular capillary basement membrane, which continues over the surface of the mass from one capillary to the next. Projections of the central cell mass characteristically form the support for glomerular capillaries, and smaller knobs of the central mass may project actually into the lumen of the capillaries, but always carry a layer of endothelial cytoplasm before them. They are never in direct contact with blood. The basement membrane of the glomerular capillary loop has a central dense layer and two lateral less dense layers as in mammals. The central dense layer is continuous with similar appearing dense material in the intercellular spaces of the adventitiae of the arterioles, and also with that of the central cell mass. The two less dense layers can also be traced into direct continuity with the less dense regions of this intercellular substance. The endothelial cytoplasm is spread as a thin sheet over the inner surface of the capillary basement membrane, and shows scattered "pores" resembling those described in mammals. Epithelial cells with interlacing pedicels are at least as prominent as those in mammals. Bowman's capsular membrane also possesses three layers similar to but less wide than those of the capillary basement membrane, and all three layers can be traced into continuity with the dark and light regions of the intercellular material of the adventitial cells of the arterioles, and beyond them with that of the central cell mass. At the hilum Bowman's capsular membrane also fuses with the capillary basement membrane.     

Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

Distribution of actin bundles in Bowman's capsule of rat kidney

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.     

Ultrastructure of the Tegumental Basement Membrane of Fasciola hepatica(Trematoda)

The fine–structural characteristics of the basement membrane of the tegument of F. hepatica were examined following extraction fixations and tannic acid infiltration. The basement membrane was shown to consist of three layers: lamina lucida, lamina densa, and lamina reticularis. The lamina densa appeared amorphous and homogeneous with tannic acid impregnation. The lamina reticularis appeared as a dense network of 10–12 nm fibrils. Anchoring fibrils cross this layer and form loops. Along their length they contact hemidesmosomes of muscles, thus connecting muscle to muscle and to tegument. The tegument/basement membrane contact is enhanced by extensions of the lamina densa into infoldings of the tegumental basal membrane. Where tegumental spines reach the basement membrane, the contact is reinforced by hemidesmosomes that connect to anchoring fibrils reaching toward the underlying muscles. The basement membrane thus seems to be a complex structure integrating the distal tegumental layer with underlying tissues and transducing muscle contractions to the tegument and its spines.     

Three-dimensional network of cords: the main component of basement membranes

Basement membranes were divided into two types: 1) thin basement membranes, such as those of the epidermis, trachea, jejunum, seminiferous tubule, and vas deferens of the rat, the ciliary process of the mouse, and the seminiferous tubule of the monkey, and 2) thick basement membranes, such as the lens capsule of the mouse and Reichert's membrane of the rat. High-magnification electron microscopy was used to examine both types after fixation either in glutaraldehyde followed by postosmication or in potassium permanganate. The basic structure of thin and thick basement membranes was found to be a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the diameter of these cords was variable, but averaged 4 nm in all cases examined. The spaces separating the cords differed, however. In the lamina densa of thin basement membranes, the diameter of these spaces averaged about 14 nm in every case, whereas in the lamina lucida it ranged up to more than 40 nm. Intermediate values were recorded in thick basement membranes. Finally, the third, inconstant layer of thin basement membranes, pars fibroreticularis, was composed of discontinuous elements bound to the lamina densa: i.e., anchoring fibrils, microfibrils, or collagen fibrils. In particular, collagen fibrils were often surrounded by processes continuous with the lamina densa and likewise composed of a typical cord network. Finally, two features were encountered in every basement membrane: 1) a few cords were in continuity with a 1.4- to 3.2-nm thick filament or showed such a filament within them; the filaments became numerous after treatment of the seminiferous tubule basement membrane with the proteolytic enzyme, plasmin, since cords decreased in thickness and could be reduced to a filament, and 2) at the cord surface, it was occasionally possible to see 4.5-nm-wide sets of two parallel lines, referred to as "double tracks." On the basis of evidence that the filaments are type IV collagen molecules and the double tracks are polymerized heparan sulfate proteoglycan, it is proposed that cords are composed of an axial filament of type IV collagen to which are associated glycoprotein components (laminin, entactin, fibronectin) and the double tracks of the proteoglycan.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

THE COLUMNAR CELLS OCCURRING IN THE PARIETAL LAYER OF BOWMAN''S CAPSULE : Cellular Fine Structure and Protein Transport

The renal corpuscles of adult, C3H Swiss, male mice contain testosterone-sensitive, columnar cells in the parietal layer of Bowman's capsule. A study of the normal fine structure of these cells reveals several distinctive characteristics: a microvillous brush border; apical tubular invaginations and apical tubules; an elaborate infolding of the basal surface membrane forming cellular compartments, which contain numerous mitochondria; and a complex group of membrane-limited cytoplasmic inclusions. This appearance is remarkably similar to the fine structure of cells in the proximal convoluted tubule. 1 hr after an in vivo injection of horseradish peroxidase, numerous protein-absorption droplets occur in the columnar cell cytoplasm. The speed and cytomorphology of protein transport by these capsular cells closely resemble the handling of peroxidase by the proximal convoluted tubule. Origins for these testosterone-sensitive cells are discussed briefly. Morphological evidence is presented for the differentiation of squamous cells in Bowman's parietal capsule into columnar cells, which appear structurally and functionally identical with proximal convoluted tubular epithelium.     

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.     

Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains

To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site approximately 15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this mAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina rara and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different sites on the short arms by rotary shadowing and five that blotted to a large pepsin-resistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.     

Heterogeneity of distribution pattern at the electron microscopic level of heparan sulfate in various basement membranes.

Using the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique and enzymatic digestion, we investigated the ultrastructural distribution pattern of heparan sulfate side chains of heparan sulfate proteoglycan (HSPG) in various basement membranes (nerve, capillary, oral epithelial, muscle, and dental basement membranes). Four different distribution patterns of stain deposits were identified as heparan sulfate on the basis of enzymatic degradation by heparitinase. In some basement membranes associated with tooth germs and oral epithelium, HID-TCH-SP stain deposits were regularly located at both sides of the lamina densa, but few were observed in the lamina densa itself. In nerve, muscle, and capillary basement membranes, the stain deposits were localized at the external side of the lamina densa adjacent to the underlying connective tissue, but were not found in the laminae lucida and densa. In the internal basal lamina of junctional epithelium of gingiva, the stain deposits were detected mainly in the lamina lucida region. Finally, in some dental and oral epithelial basement membranes, the stain deposits were randomly distributed throughout both laminae lucida and densa. Thus, the present study demonstrated distinct differences in heparan sulfate distribution pattern among various basement membranes, suggesting their architectural heterogeneity.     

Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney

Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin , heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study. Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida. When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick "cords," which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as " basotubules "; and (3) 3.5-nm elements composed of minute paired rods, referred to as "double pegs." The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern. It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin , heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae . Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

Localization and quantitation of anionic charge sites in fetal and neonatal alveolar basement membranes

A method utilizing biopsy sized samples of lung for anionic charge site localization in alveolar and capillary basement membranes in human tissue is discussed. Tissue fixed in either paraformaldehyde-lysine-periodate or 1% paraformaldehyde with 0.05% glutaraldehyde, cut into 30 mu sections, and incubated with the cationic probe, polyethyleneimine, was processed for electron microscopic analysis using standard techniques. Anionic charge sites were identified and regularly distributed in increments of approximately 40-50 nm in the lamina rara externa of the alveolar basement membrane, with lesser amounts found in the lamina rara interna and lamina densa. Anionic charge sites were also demonstrated in the interstitial portion of the capillary basement membrane and on cell surfaces. These methods can be used to more broadly define the localization of anionic charge sites in human lung tissue in both normal and pathologic states.     

Incomplete fusion of the epithelial and endothelial basement membranes in interspecies hybrid glomeruli

Avascular, undifferentiated mouse kidneys transplanted onto quail chorioallantoic membrane differentiate and become vascularized by quail vessels. The glomeruli which form under these conditions consist of mouse podocytes and quail endothelial cells. Immunohistochemistry has shown that the glomerular basement membrane (GBM) has a dual origin, as integral basement membrane components are produced by both podocytes and endothelial cells. In electron microscopy this GBM is composed of two partially separated layers, an epithelial and an endothelial basal lamina which both have a lamina densa and a lamina rara. These two basal laminas are partially fused, but there are large areas where this fusion does not occur. In some places of incomplete fusion, fibrillar extracellular material is seen between and beneath the GBM. It is concluded that basement membrane components derived from the different species can interact partially, but the fusion is incomplete. The abnormal assembly of the epithelial and the endothelial basal laminas might be due to molecular differences between the components produced by the two cell lineages. In spite of the incomplete fusion, the system used serves as a good model-system to study basement membrane formation, since the cells organize in a histiotypic fashion and form true vascularized glomeruli.