JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 14:63-69 (1990)

Rat Kidney Glomerular Basement Membrane Visualized In Situ by Embedment-Free Sectioning and Subsequent Platinum-Carbon Rep1ication HISATAKE KONDO Departrwnt of Anatomy, School of Medicine, Kanazawa University, Kanazawa, 920 Japan

KEY WORDS

Filtration barrier, Renal sections, Resinless ultrastructure

Following the removal of polyethylene glycol (PEG) from thin sections, and viewABSTRACT ing through the endothelial fenestrae and/or the interpedicel spaces, the rat renal glomerular basement membrane in situ was revealed to consist of meshworks and to be electron-transparent when examined a t right angles to the plane of the membrane. By subsequent platinum replication of the embedment-free sections, the lamina densa of the basement membrane appeared as a veil composed of rather closely packed particles. The architecture of the slit diaphragm and the surface morphology of the endothelial cell membrane were also clearly revealed. The present results indicate that the PEG method, with or without replication, can provide valuable information on basement membrane morphology.

INTRODUCTION Through the use of polyethylene glycol (PEG), a highly water-soluble wax, in electron microscopy (Wolosewick, 1980), it is possible to observe biological materials in embedment-free sections by transmission electron microscopy. Resulting ultrastructural images of cells in the embedment-free sections have shown no major alteration compared with conventional epoxy image:$ (Kondo et al., 1982, 1983; Kondo, 1984). In fact, the PEG-processed embedment-free sections have reveahd, within cells, fine strands of varying sizes, termed microtrabeculae. The microtrabeculae are considered to be indistinct or invisible in conventional epoxy sections mainly due to the similarity in electrcn-scattering properties between the microtrabeculae and the epoxy resin (Wolosewick and Porter, 1979; Guatelli et al., 1982). Combining rotary platinum-c srbon replication with the PEG method has made it possible to see the surface structures of biolog cal membranes, the organization of the cytoskeleton, and even some of the extracellular matrix elements with excellent resolution (Cidadao and David Ferreira, 1986; Kondo, 1987). In the present study, the PEG method was utilized to examine the organization of the renal glomerular basement nembrane, which is considered to function as a renal jiltration barrier. This made it possible to reveal the meshwork architecture of the basement membrane in situ. The simultaneous examination of epoxy and embedment-free sectioned images, as well as platinumcarbon replica images from the embedment-free secf same renal tissues, provides additional intions c ~ the formai,ion about basement membrane structure and its spatial relation to the endotherial cells and podocyte pedicels. Portions of this study have appeared previously Ln abstract form (Kondo, 1986).

8 1990 ALAN R. LISS, INC.

MATERIALS AND METHODS Young adult rats of both sexes, weighing 150-200 g, were perfused through the heart with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.5% tannic acid, pH 7.4. The kidneys were removed 10 min after perfusion, minced in the fresh fixative, and immersed in the fixative for a n additional 2 hr. The tissue blocks were postfixed with 1%Os04 in the same buffer for 2 hr and subsequently dehydrated in increasing concentrations ofethanol. The blocks were transferred t o a 50% solution of PEG-4000 in absolute ethanol (viv) and infiltrated for 3 h r at 60°C. The blocks were subsequently placed in pure molten PEG a t 60°C and embedded in PEG contained in well dried gelatin capsules and solidified by immersion in liquid nitrogen. Sections, 200-400 nm thick, were made with dry glass knives and mounted on Formvar-coated grids, with a platinum loop filled with 2.5% sucrose. Immediately after mounting, the sections on grids were transferred to a submerged grid holder in 95% ethanol to remove PEG from the section. After exchange of the 95% ethanol with absolute ethanol several times, the sections on grids were transferred to a critical-point apparatus (Hitachi HCP2 model) and dried with liquid COz. For rotary replication of the embedment-free sections, the dried grids were transferred to a freeze-fracture apparatus (EIKO FD-3, equipped with a n electron gun and a rotary stage for specimens) with the specimen side facing up. The sections on grids were shadowed with platinum a t a n angle of 15" and backed with

Received February 23, 1 9 8 9 accepted in revised form May 9, 1989. Address reprint requests to Hisatake Kondo. Department of Anatomy. School of Medicine. Tohoku University, Sendai, 980 Japan.

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carbon a t an angle of 90" while the specimen stage was rotated a t 60 rpm. Then the grids were floated on household bleach solution with the specimen side facing up for 10 min. Following several rinses with distilled water, replicas on the grids were examined with an electron microscope (Hitachi H-700-H) a t a n accelerating voltage of 200 kV. Additional information on the practical techniques and instruments used with the PEG method have been described in the original paper by Wolosewick (1980) and in other reports by the present author (Kondo, 1984,1987; Kondo and Ushiki, 1985). For comparison of embedment-free section images with the conventional epoxy images, some of the tissue blocks fixed with aldehydes and Os04 were dehydrated and embedded in Epon according to the conventional procedure. Ultrathin sections were obtained and observed with the electron microscope.

RESULTS Conventional epoxy section images of the renal glomerulus are shown in Figures 1, 3, and 4. The basement membrane in a cross-cut view exhibits three distinct layers composed of the electron-dense lamina densa sandwiched by the subendothelial lamina rara interna and the subepithelial lamina rara externa. The total thickness including the three layers is 120-170 nm in a cross view, and the lamina densa is 50-70 nm in thickness. The grazing (en face) image exhibits a homogenously dense, finely fibrillar veil. The endothelial fenestrae in grazing views are not circular but oval in shape, 70-150 nm in size, and randomly distributed. No distinct structures except for some flocculent material are visible within the fenestrae even a t higher magnification (Fig. 4). In embedment-free sections, the cross-cut image of the basement membrane exhibits three layers comparable to those in epoxy sections, but several differences are noted (Fig. 2). A layer comparable to the lamina densa is highly electron-dense and 30-45 nm in thickness. Layers comparable to the laminae rara externa and interna are 40-60 nm in thickness, respectively (vs. 40-50 rim each in Epon sections). The laminae rara externa and interna consist of thin strands, approximately 4 nm in thickness, which are rather loosely arranged and cross link the lamina densa with podocyte pedicels or endothelial cells. The interpedicel space is approximately 60-80 nm in distance (vs. 4050 nm in epoxy sections) where single or sometimes multiple strands, 6 nm in thickness, are clearly seen to cross link. The strands are regarded a s the slit diaphragm. At higher magnification of the embedment-free sections containing grazed renal glomeruli, three structural elements, including fenestrated endothelial cells, podocyte pedicels, and intercalating basement membrane altogether, or two of the three elements, are seen overlapping each other (Fig. 5-7). Through the endothclial fenestrae (80-200 nm in diameter) without

overlapping the interpedicel spaces, membranes are visible with highly heterogenous electron density, which appear as meshworks composed of strands, 1020 nm in thickness, and interlacing polygonal or oval spaces, 15-35 nm in size. When viewed through the interpedicel spaces with or without overlapping fenestrae, the meshwork is superimposed on strands forming the slit diaphragm. Rotary replica images of the PEG-processed renal glomerulus are shown in Figures 8-10, in which the luminal surface morphology of the fenestrated endothelium is clearly seen. At the periphery of the replica, the en face image of the basement membrane is also visible. At higher magnification of the replica, the luminal surface of the endothelial cell is covered by closely packed, fine granules a s reported elsewhere (Kondo, 1987). The lamina densa of the basement membrane appears as a veil composed of rather closely packed particles, 15-20 in diameter. Where the interspace between particles may be slightly widened, the particles appear to be aligned in short lines. The lamina rara externa is composed of strands, 13-20 nm in thickness, which are loosely arranged and cross bridged between the pedicel and the lamina densa as in embedment-free sections. Similar strands are seen in the lamina rara interna. The en face image of the slit diaphragm in the interpedicel space is also obtained in the replica of the embedment-free section (Fig. 10). The transverse st rands, 14-20 nm in thickness, are closely arranged in a zipper form and connect adjacent pedicels of the podocytes.

DISCUSSION Since the contrast of objects prepared by conventional epoxy embedment procedures is based mainly on the relative difference in electron scattering between objects and epoxy resins (Pease and Porter, 1981 1, one great advantage of this embedment-free observation is

Figs. 1,2. Transverse section images of the glomerular basement membrane in conventional epoxy-embedded (Fig. 1)and embedmentfree (Fig. 2 ) specimens. Arrows indicate slit diaphragm. C, capillary endothelium; D, lamina densa; E, lamina rara externa; I, lamina rara interna; P, podocyte pedicel; x 79,200 Figs. 3, 4. Tangential section image of the glomerular capillary in epoxy embedded specimen (Fig. 3) and high-magnification en face view of the endothelial fenestrae located close to the grazing edge of the endothelium in transit to the basement membrane (B) (Fig. 4). Note flocculent material seen within the fenestrae. C, capillary endothelium; P, podocyte pedicel. ~ 2 6 , 4 0 0(Fig. 31, 79,200 (Fig. 4). Fig. 5. Tangential section image of the glomelular capillary in the

PEG embedment-free specimen. A l , area containing endot.helium only; A2, area in which endothelium and basement membrane are superimposed; A3, area in which endothelium, basement membrane, and podocyte pedicels (P)are superimposed; A4, area containing basement membrane only. Arrows indicate grazing edge of endothelium, Note meshwork structures of the basement membrane in A2. 43. and A4. L, capillary lumen. x 52,800.

EXAMINATION OF GLOMERULAR BASEMENT MEMBRANE

Figs. 1-5.

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Figs. 6 and 7.

EXAMINATION OF GLOMERULAR BASEMENT MEMBRANE

the enhancement of contrast. This is illustrated by the fact that all the electron micrographs of the embedment-free sections shown in this report are not treated with the usual heavy metal staining. This advantage is expressed remarkably in areas containing the endothelial fenestrae and the glomerular basement membrane cut tangentially: The meshwork architecture of the in situ basement membrane, mainly the lamina densa, is clearlj. visible for the first time through the endothelial fenesti-ae and/or the interpedicel space. Furthermore, it is noted from the embedment-free observation that the basement membrane is basically electron-transparent in a direction at right angles, and the transparency makes it possible to look through the three superimposed elements including endothelial and epithelial cellular elements and basement membrane. Since it has been suggested that networks of fibrils 3-4 nin in thickness are embedded in the lamina densa, and the fibrils are composed of type IV collagen, laminin, f bronectin, and proteoglycan (Farquhar, 1982; Yurchenco and Rubin, 1987; Reale et al., 19831, it is likely that meshwork strands viewed in the en face image of the lamina densa might correspond to those macromolecules. This PEG procedure has also been shown to be applicable to immunoelectron microscopy (Kondo, 1984), and the chemical compositon of individual strands of the meshwork is currently being examined. In contrast to the en face and look-through images of the lamina densa of the basement membrane in the embedment-free sections, its replica presents an appearance of veils composed of packed granules. This granular image of the lamina densa is basically similar to that observed in the rapid-freezing and deep-etch replica method (Kubosawa and Kondo, 19851, although the veil in the latter method appears more compact than that in the present method, probably because the chemical fixation extracts substances from specimens to some degree. Regarding the difference in appearance of the lamina densa between the embedment-free section a id its replica, it should be noted that the appearance cf the surface morphology of every element is enhanced in the replica, whereas the contour of every element based on differential contrast is the main information obtained in the section. It is also clear that the final size of every bulky object becomes larger and that of interposing empty spaces is smaller than those in the embedment-free sections due to the thickness of the replica covering every object. In addition, any objects having a much lower electron-scattering property than that of surrounding objects may be viewed as empty in the embedment-free sections. With these reasons, It is understandable that the “granular veil” im-

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age instead of the fibrous “mesh” one derived from the same lamina densa is dominant in the replica of the embedment-free section and that no distinct interlacing spaces or pores are clearly visible in the replica. In previous studies by means of negative staining and transmission electron microscopy, meshwork architecture consisting of strands and pores has been shown in unfixed basement membrane isolated from human and some mammalian kidneys (Ota et al., 1980). Ota et al. interpreted the meshwork architecture as representing the size barrier of the glomerular filtration. Although it is likely that the meshwork revealed in the present study corresponds to the negatively stained counterparts, the interlacing space in the embedment-free section is larger in size than the pores in the negative image. In this regard the difference in size of various biological elements should be noted between images of the embedment-free sections and the epoxy sections. In rough estimation, the size of bulky elements such as podocyte pedicels, endothelial cells, and the lamina densa reduces by 30-40%, whereas the size of surrounding empty spaces such as the interpedicel space and the endothelial fenestrae increases correspondingly. This dimensional change in similar ranges is known to occur commonly in specimen preparation for scanning electron microscopy, although the change is isotropic (Boyde et al., 1977). Therefore, the main cause of the dimensional change seems to be the dehydration procedure. The interlacing space or pore is thus regarded as more enlarged than the real counterpart. Furthermore, from careful comparison of published micrographs showing both epoxy images and freezeetched replica images of the same specimens, such as Figures 1 and 2 in a study by Hirokawa and Heuser (1982), we can easily note that similar dimensional changes also occur in the intercellular distance and vesicle sizes in the neuromuscular junction by the freeze-etch procedure, which is another embedmentfree preparation method. Although hydrated specimens are frozen in the normal freeze-etch procedure, this resultant dimensional change, as compared with epoxy images, implies that the etching itself is essentially equivalent to the dehydration, although detailed mechanisms leading to this change by the freeze etching remain to be elucidated. Consequently, the dimensional change under discussion must be unavoidable in the embedment-free preparation procedures regardless of techniques employed. With careful use and critical comparison with other methods as well as its application to normal and pathological specimens, this PEG method should provide us with valuable information on the basement membrane.

Figs. 6, 7. Stereo pairs of higher-magnification electron micrographs of the areas A2 (Fig. 6) and A3 (Fig. 7). Note meshwork structure in stereo of the basement membrane viewed through endothelial fenestrae. Arrows indicate grazing edges of the endothelium. Asterisks indicate basement membrane superimposed on slit diaphragms between podocyte pedicels (P). x 79,200.

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Figs. 8-10

EXAMINATION OF GLOMERULAR BASEMENT MEMBRANE Fig. 8. Replica electron micrograph of the PEG embedment-free section of the renal glomerulus showing en face images of the capillary endothelium (Cj and its fenestrae. B, basement membrane; P, podocytc pedicles; x 26,400. Fig. 9. Higher-magnification electron micrograph of a region enclosed b,y a rectangle in Figure 8. En face images of the lamina densa (D) and capillary endothelial cell (C) are clearly shown. E, lamina rara externa; P, podocyte pedicels. x 79,200. Fig. 10. Replica image of PEG-sectioned podocyte pedicels iP) showing slit diaphragm (*) in a zipper form. Arrows indicate the lamina rart externa. C, capillary endothelium; D, lamina densa of the basement membrane; E, lamina rara externa, I, lamina rara interna. X 79,201).

ACKNOWLEDGMENTS The author thanks Mr. S. Yamazaki and Mrs. Y. Akabori for their photographic assistance and secretarial help. 'This study was supported by Grant-in-Aid for Gener a1 Scientific Research 62480092 from the Ministry of Education, Science and Culture of Japan and a grant from Ueno Hospital Foundation in Hitachi, Japan. REFERENCES Boyde, A,, Bailey, E., Jones, S.J., and Tamarinn A (1977) Dimensional changes during specimen preparation for scanning electron microscopy. Scanning Electron Microsc., 1507-518. Cidadao, A.J., and David-Ferreira, J.F. (1986) A method for TEM visualization of the extracellular matrix three-dimensional organization in tissues. J . Microsc., 14249-62. Farquhar. M.G. (1982) Structure and function in glomerular capillaries: role of the basement membrane in glomerular filtration. In: Biology and Chemistry of Basement Membranes. N.A. Kefalides, ed. Amdemic Press, Inc., New York, pp. 43-88. Guatelli, J.C., Porter, K.R., Anderson, K.L., and Boggs, D.P. (1982)

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Ultrastructure of the cytoplasmic and nuclear matrix of human lymphocytes observed using high voltage electron microscopy or embedment-free sections. Biol. Cell, 43:69-80. Hirokawa, N., and Heuser, J.E. (1982) Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction. J . Neurocytol., 11:487-510. Kondo, H. (1984) Polyethylene glycol (PEG) embedding and subsequent deembedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy. J . Electron Microsc. Tech., 1:227-241. Kondo, H. (1986) Ultrastructure of the basement membrane of the rat kidney as revealed by the embedment-free sections using polyethylene glycol embedding and subsequent deembedding. Acta Anat Nipponica, 61:512 Kondo, H. (1987) Visualization of the inner and outer surfaces of the cell membrane and cytoskeleton by polyethylene glycol embedding, subsequent deembedding, and rotary replication with platinum. J. Electron Microsc. Tech., 7:17-27. Kondo, H., Pappas, G.D., and Wolosewick, J.J. (1983)The cytoskeletal lattice of the neurohypophysial cells. Biol. Cell, 49:99-108. Kondo, H., and Ushiki, T. (1985) Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy, scanning electron microscopy, and transmission electron microscopy. J . Electron Microsc. Tech., 2:457-462. Kondo, H., Wolosewick, J . J . and Pappas G.D. (1982) The microtrabecular lattice of the adrenal medulla revealed by polyethylene glycol embedding and stereo electron microscopy. J. Neurosci., 257-65. Kubosawa, H., and Kondo, Y. (1985) Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method. Cell Tissue Res., 242:33-39. Ota, Z., Makino, H., Takaya, Y., and Ofuji, T. (1980) Molecular sieve in renal golmerular and tubular basement membranes as revealed by electron microscopy. Renal Physiol., 3:317-323. Pease, D.C., and Porter, K.R. (1981) Electron microscopy and ultramicrotomy. J. Cell Biol., 91:2875-2925. Reale, E., Luciano, L., and Kuhn, K.W. (1983) Ultrastructural architecture of proteoglycans in the glomerular basement membrane: A cytochemical approach. J. Histochem., 31:662-668. Wolosewick, J . J . (1980) The application of polyethylene glycol (PEG) to electron microscopy. J. Cell Biol., 86:675-681. Wolosewick, J.J., and Porter, K.R. (1979) Microtrabecular lattice of the cytoplasmic ground substance: artifact or reality. J. Cell Biol., 82:114-139. Yurchenco, P., and Ruben, G.C. (1987) Basement membrane structure in situ: Evidence for lateral associations in the type IV collagen network. J. Cell Biol., 105:2559-2568.

Rat kidney glomerular basement membrane visualized in situ by embedment-free sectioning and subsequent platinum-carbon replication.

Following the removal of polyethylene glycol (PEG) from thin sections, and viewing through the endothelial fenestrae and/or the interpedicel spaces, t...
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