Virchows Archiv B Cell Pathol (1992) 61:351-358
WwchowsArch&B CellPathology 9 Springer-Verlag1992
Ultrastructural study of the glomerular slit diaphragm in fresh unfixed kidneys by a quick-freezing method Shinichi Ohno 1, Kumiko Hora 2, Takeshi Furukawa 2, and Hisao Oguehi 2 1 Departments of Anatomy and 2 Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan Received June 25 / Accepted September 20, 1991
Summary. In normal kidneys fixed by perfusion with tannic acid and glutaraldehyde, glomerular slit diaphragms have been reported to consist of highly ordered and isoporous substructures with a zipper-like configuration. We have re-evaluated the ultrastructure of fixed or unfixed glomeruli using quick-freezing and deep-etching (QF-DE) and freeze-substitution (QF-FS) methods. In the fixed slit diaphragms, zipper-like substructures were often observed by the QF-DE method. In contrast, in fresh unfixed glomeruli the slit diaphragms mainly consisted of non-porous substructures. The slit diaphragms were more widely opened in the fixed glomeruli, as examined by the QF-FS method. These results suggest that the foot processes shrink during tissue preparation by conventional methods with chemical fixatives, and that the broadening of slit diaphragms and zipperlike substructures are formed by the pulling apart of adjacent foot processes due to shrinkage. Key words: Glomerular slit diaphragm - Quick-freezing - Ultrastructure
Introduction The renal glomerulus serves both as a charge-selective and size-selective sieve capable of excluding most plasma proteins from the urine (Brenner et al. 1976, 1977, 1978; Kanwar 1984). Proteins larger than albumin are largely restricted from passing into the urinary space (Venkatachalam et al. 1970; Caulfield and Farquhar 1974; Ryan and Karnovsky 1976; Rennke and Venkatachalam 1977; Bariety et al. 1978; Olivetti et al. 1981). To gain access to the urinary space, proteins in capillary lumina must cross the glomerular wall, passing sequentially through the fenestrated endothelium, the basement membrane and the narrow epithelial slits. In the past, the slit diaphragm, a structure bridging the epithelial slits near the Offprint requests to: S. Ohno
basement membrane, appeared to have a filtering function for proteins in addition to basement membrane (Graham and Karnovsky 1966; Venkatachalam et al. 1970). However, a fully detailed description of the slit diaphragm was not reported before 1974, because of the limited electron density of its structure after conventional fixation and staining techniques. In 1974 and 1975, the glomerular slit diaphragms in the normal rat, mouse and human kidney were reported to consist of regular patterns of zipper-like substructures with periodic cross-bridges extending from the foot processes to a central filament (Rodewald and Karnovsky 1974; Karnovsky and Ryan 1975; Ryan et al. 1975; Schneeberger et al. 1975). The preparations studied were fixed with a solution of tannic acid, glutaraldehyde and osmium tetroxide (TGO) and ultrathin sections were cut for electron microscopy. The use of tannic acid fixation increased the electron density of extracellular structures in the glomeruli and revealed the isoporous substructures of the slit diaphragm. The glomerular slit diaphragms were also reported to show zipper-like or web-like substructures by a conventional freeze-fracture replica method (Karnovsky and Ryan 1975), and Kubosawa and Kondo (1985) demonstrated that they consisted of irregular zipper-like substructures by a quick-freezing and deep-etching method. Despite these reports, it could be still argued that these substructures simply reflect the artifactual appearances of the glycocalyx within epithelial slits as adjacent foot processes shrink and pull apart during tissue preparation. Recently, we reported that the slit diaphragms in isolated glomeruli were easily modified to form zipperlike, ladder-like and sheet-like substructures by various fixation methods (Hora et al. 1990). This suggested that their rearrangement might easily occur during the specimen preparation. We also demonstrated that anionic sites on the glomerular basement membrane were dramatically changed by conventional fixation with osmium tetroxide, as revealed by the QF-DE method (Yoshimura et al. 1991). On the other hand, basement membrane images obtained by freeze-substitution (FS) fixation are
352 more closely representative of the in vivo ultrastructure than those prepared by conventional procedures with chemical fixatives (Goldberg 1986; F u r u k a w a etal. 1989, 1991). We have previously reported the three-dimensional ultrastructure of the glomerular basement m e m b r a n e and mesangial matrix in normal and diseased kidneys by the quick-freezing and deep-etching method (Takami et al. 1990, 1991; N a r a m o t o et al. 1991a, 1991 b). In the present study, we used the quick-freezing and deep-etching (QF-DE) and the freeze-substitution (QF-FS) methods to re-evaluate the ultrastructure of slit diaphragms in fresh unfixed glomeruli and have demonstrated that they appear to be composed of substructures with a sheet-like configuration.
Materials and methods A total of 15 normal male Wistar rats, weighing 100 g to 130 g, were used for this study. Under pentobarbital anesthesia, small pieces of cortex were excised from the kidneys with a razor blade. They were processed for the following quick-freezing steps after dicing into small pieces as reported previously (Takami et al. 1990, 1991). Fresh unfixed tissue fragments were plunged into a liquid isopentane-propane mixture (around -193 ~ C) cooled in liquid nitrogen (Ohno and Fujii 1990, 1991; Naramoto etal. 1991a, 1991b). The frozen tissues were freeze-substituted in acetone containing 2% or 0.1% osmium tetroxide as follows. Freeze-substitution was performed by placing the samples in an acetone solution kept at about - 8 0 ~ for 20 h (Furukawa et al. 1989, 1991). The temperature of the samples was then raised, firstly to - 2 0 ~ C for 2 h, then to 4~ C for 2 h and finally to a room temperature. They were washed twice in absolute acetone and embedded in Luveak812 (Nacalai Tesque, Inc., Kyoto, Japan) (Shinagawa et al. 1980). Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined in a Hitachi HS-9 electron microscope. Preparations of replica membranes from fresh kidneys were made as follows. The previously described frozen tissues were carefully fractured with a scalpel in liquid nitrogen (Ohno and Fujii 1990, 1991). The frozen specimens were deeply etched under vacuum conditions of 2-6 • 10 -~ Torr for 20-30 min at a temperature of - 9 5 ~ (Naramoto et al. 1991a, 1991b). After deep-etching, the specimens on a rotary stage were first shadowed with platinum at an angle of 37~ for several seconds and then rotary shadowed up to the total thickness of about 2 nm. They were additionally coated with carbon at an angle of 90~ A drop of 2 0 collodion in amylacetate was put onto the replicas as soon as the specimens were taken out from the machine to prevent their breaking into pieces during the following digestion procedure. The replicas coated with dried collodion were floated on household bleach for 1530 rain to dissolve the tissue components. The large replica membranes were washed in distilled water and cut into small pieces with scissors. They were picked up on Formvar-filmed copper grids and immersed in amylacetate solution to dissolve the collodion film. They were then observed in a Hitachi HS-9 electron microscope at an accelerating voltage of 75 kV and electron micrographs were printed from the inverted negative films. Preparations of TGO fixed kidneys were made as follows. Under pentobarbital anesthesia, the kidneys were first perfused with 0.1 M phosphate buffer (PB), pH 7.4, for 1-2 min and then fixed with 1% tannic acid and 1% glutaraldehyde in phosphate buffer for 15 min (Rodewald and Karnovsky 1974; Furukawa et al. 1989, 1991). They were cut into small pieces and postfixed with 1% osmium tetroxide for 60 min. The fixed samples were rinsed in 10% methanol diluted with distilled water. Excess fluid was removed with filter paper, and the specimens were then rapidly frozen in an isopentane-propane mixture (around - 193~ C) cooled
in liquid nitrogen. They were transferred into liquid nitrogen, shaken briskly to remove adherent isopentane-propane mixture and fractured with a scalpel in liquid nitrogen. They were processed as described above. Some fixed tissues were routinely dehydrated and embedded for ultrathin sections.
Results In all the specimens prepared by FS fixation, tissue layers of acceptable preservation were located within 10 pm f r o m the frozen surface areas. The ultrastructure of the glomeruli was well preserved in the FS fixatives containing 2% or 0.1% osmium tetroxide in acetone (Fig. 1 a and b). The cell surface coat of the foot processes showed electron-dense layers along the cell m e m branes. Where ultrathin sections cut foot processes vertically, some slit diaphragms could be distinguished as single lines between the foot processes. Their width, i.e. the distance from cell m e m b r a n e to cell membrane, had a mean value near 34 nm (n = 50). A detailed description of the m o r p h o m e t r i c data to determine the widths of slit diaphragms will be published in a separate paper (Furukawa et al. 1991). As shrinkage and pulling apart of foot processes were minimized, the epithelial clefts ran relatively straight and narrow courses. This was due to the close apposition o f the glycocalyx on adjacent foot processes in vivo. Freeze-fractured capillary loops from the same samples were observed on replica m e m branes by the Q F - D E method (Fig. 1 c). The width of slit diaphragms, as measured from the outer surface of one foot process to the next, averaged 25 nm (n = 50). The replica m e m b r a n e s freeze-fractured parallel to the basement m e m b r a n e revealed the ultrastructure of the slit diaphragms (Fig. 2a). Interdigitating foot processes were in close apposition, and sheet-like substructures were detected between the foot processes. Ultrathin sections by the Q F - F S method show the glomerular ultrastructure (Fig. 2b). Where the plane of section passed almost parallel to the basement membrane, the slit diaphragms were observed as consistently heterogeneous structures connecting the cell m e m b r a n e s of adjacent foot processes. Figure 2c illustrates that sheet-like substructures were identified in some parts of obliquely or horizontally freeze-fractured capillary loops. Figure 3 illustrates electron micrographs of the glomeruli fixed with T G O . The trilaminar structure o f the basement m e m b r a n e was m o r e clearly apparent (compare Fig. 3 a with Fig. 1 a and b). The electron-dense lamina densa was sandwiched between thin electron-lucent layers, the laminae rarae interna and externa. In the lamina rara externa, fibrils appeared to radiate from the lower surfaces o f foot processes into the lamina densa. Foot processes were contracted during fixation with T G O , except for their bases attached to the basement membrane. Unlike the foot processes fixed by the Q F - F S method, those of TGO-fixed glomeruli showed shrunken cell surface contours. Widely opened clefts were formed between the glycocalyx layers of adjacent foot processes; in addition, the slit diaphragms were pulled apart, their width averaging 47 nm ( n = 50). In the replica m e m branes, the slit diaphragms were observed between the
353
Fig. I a--c. Electron micrographs of glomerular capillary loops prepared by the QF-FS method with 2% osmium tetroxide (a) or 0.1% osmium tetroxide (b) in acetone, and the QF-DE method (e). Bar; 0.5 ~tm. In (a) and (b) the unit membranes of the foot processes (F) are clearly seen and their external surfaces are coated by fibrillar glycocalyx. A large amount of flocculent material is localized within the basement membrane (BM), urinary space (US) and capillary lumen (CL) in (a) and (b). The epithelial slits run relatively straight and narrow courses (arrows). Parts of the slits are closed by a thin slit diaphragm which is seen spanning the filtration clefts (large arrowheads). Central layers of the basement membrane contain networks of fine fibrils, as shown in (a) and
(b). However, the lamina densa and lamina rara externa or interna are less discerned in these specimens than in Fig. 3 a. Some crossbridging filaments appear to be inserted into the base of foot processes in the lamina rara externa (small arrowheads). E; endothelium. a; • 50,300. b; • 52,400. In (c) a replica electron micrograph of a capillary loop freeze-fractured transversely to the basement membrane in an unfixed sample is shown. The slit diaphragms are observed between foot processes (large arrowheads). Extracellular granular material is seen and fibrillar structures are identified on the surfaces of foot processes (small arrowhead). The epithelial slits run relatively straight courses (arrows). BM; basement membrane. F; foot process. E; endothelium. • 90,000
s h r u n k e n f o o t processes a n d c o n s i s t e d o f i r r e g u l a r zipp e r - l i k e s u b s t r u c t u r e s (Fig. 3b). I n s o m e sites, zigzag s u b s t r u c t u r e s c o u l d be seen r u n n i n g c e n t r a l l y a l o n g the space. Slit d i a p h r a g m s were also f o u n d b e t w e e n s h r u n k e n f o o t processes, w h i c h were f r e e z e - f r a c t u r e d a l m o s t
h o r i z o n t a l l y to the b a s e m e n t m e m b r a n e (Fig. 3c). T h e c r o s s - b r i d g e s a p p e a r e d to e x t e n d b e t w e e n the t w o sides o f a c e n t r a l f i l a m e n t , t h u s giving the slit d i a p h r a g m a zipper-like appearance.
354
355 Discussion
It has been argued that the surface coats between adjacent foot processes are normally in close contact over the entire extent of the epithelial slits (Latta 1970). However, the actual existence of slit diaphragms has been questioned, because they varied in appearance, depending on the techniques used for fixing and staining (Karnovsky and Ainsworth 1972). Thus the slit diaphragms were suggested to be artifacts resulting from shrinkage of adjacent foot processes during conventional tissue preparation. However, some experiments with small molecular tracers suggested that a filtration barrier occurred in the region of epithelial slits as well as at the glomerular basement membrane (Graham and Karnovsky 1966; Venkatachalam et al. 1970). In our morphologic study, the slit diaphragms in fresh unfixed glomeruli actually showed sheet-like substructures, although they were composed of regular zipper-like substructures in glomeruli fixed with TGO. These findings indicate that slit diaphragms really do exist in the glomeruli in vivo. It has been reported that the slit diaphragms have a regular pattern when observed by transmission electron microscopy after TGO fixation and the conventional freeze-fracture replica method (Rodewald and Karnovsky 1974; Karnovsky and Ryan 1975; Ryan et al. 1975; Schneeberger etal. 1975). They have periodic cross-bridges extending from the foot processes to a central filament, to form homogeneous pores (about 4 x 14 nm) and are continuous between all foot processes. On the other hand, morphologic and physiological data indicated that the glomerular filter functions as an isoporous membrane which excluded proteins larger than serum albumin (Caulfield and Farquhar 1974; Purtell et al. 1979). In our study, however, the slit diaphragms rarely had an isoporous substructure consisting of regularly spaced cross-bridges. These were changed to zipper-like substructures, in the preparations fixed
WwchowsArch&B CellPathology 9 Springer-Verlag1992
Ultrastructural study of the glomerular slit diaphragm in fresh unfixed kidneys by a quick-freezing method Shinichi Ohno 1, Kumiko Hora 2, Takeshi Furukawa 2, and Hisao Oguehi 2 1 Departments of Anatomy and 2 Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan Received June 25 / Accepted September 20, 1991
Summary. In normal kidneys fixed by perfusion with tannic acid and glutaraldehyde, glomerular slit diaphragms have been reported to consist of highly ordered and isoporous substructures with a zipper-like configuration. We have re-evaluated the ultrastructure of fixed or unfixed glomeruli using quick-freezing and deep-etching (QF-DE) and freeze-substitution (QF-FS) methods. In the fixed slit diaphragms, zipper-like substructures were often observed by the QF-DE method. In contrast, in fresh unfixed glomeruli the slit diaphragms mainly consisted of non-porous substructures. The slit diaphragms were more widely opened in the fixed glomeruli, as examined by the QF-FS method. These results suggest that the foot processes shrink during tissue preparation by conventional methods with chemical fixatives, and that the broadening of slit diaphragms and zipperlike substructures are formed by the pulling apart of adjacent foot processes due to shrinkage. Key words: Glomerular slit diaphragm - Quick-freezing - Ultrastructure
Introduction The renal glomerulus serves both as a charge-selective and size-selective sieve capable of excluding most plasma proteins from the urine (Brenner et al. 1976, 1977, 1978; Kanwar 1984). Proteins larger than albumin are largely restricted from passing into the urinary space (Venkatachalam et al. 1970; Caulfield and Farquhar 1974; Ryan and Karnovsky 1976; Rennke and Venkatachalam 1977; Bariety et al. 1978; Olivetti et al. 1981). To gain access to the urinary space, proteins in capillary lumina must cross the glomerular wall, passing sequentially through the fenestrated endothelium, the basement membrane and the narrow epithelial slits. In the past, the slit diaphragm, a structure bridging the epithelial slits near the Offprint requests to: S. Ohno
basement membrane, appeared to have a filtering function for proteins in addition to basement membrane (Graham and Karnovsky 1966; Venkatachalam et al. 1970). However, a fully detailed description of the slit diaphragm was not reported before 1974, because of the limited electron density of its structure after conventional fixation and staining techniques. In 1974 and 1975, the glomerular slit diaphragms in the normal rat, mouse and human kidney were reported to consist of regular patterns of zipper-like substructures with periodic cross-bridges extending from the foot processes to a central filament (Rodewald and Karnovsky 1974; Karnovsky and Ryan 1975; Ryan et al. 1975; Schneeberger et al. 1975). The preparations studied were fixed with a solution of tannic acid, glutaraldehyde and osmium tetroxide (TGO) and ultrathin sections were cut for electron microscopy. The use of tannic acid fixation increased the electron density of extracellular structures in the glomeruli and revealed the isoporous substructures of the slit diaphragm. The glomerular slit diaphragms were also reported to show zipper-like or web-like substructures by a conventional freeze-fracture replica method (Karnovsky and Ryan 1975), and Kubosawa and Kondo (1985) demonstrated that they consisted of irregular zipper-like substructures by a quick-freezing and deep-etching method. Despite these reports, it could be still argued that these substructures simply reflect the artifactual appearances of the glycocalyx within epithelial slits as adjacent foot processes shrink and pull apart during tissue preparation. Recently, we reported that the slit diaphragms in isolated glomeruli were easily modified to form zipperlike, ladder-like and sheet-like substructures by various fixation methods (Hora et al. 1990). This suggested that their rearrangement might easily occur during the specimen preparation. We also demonstrated that anionic sites on the glomerular basement membrane were dramatically changed by conventional fixation with osmium tetroxide, as revealed by the QF-DE method (Yoshimura et al. 1991). On the other hand, basement membrane images obtained by freeze-substitution (FS) fixation are
352 more closely representative of the in vivo ultrastructure than those prepared by conventional procedures with chemical fixatives (Goldberg 1986; F u r u k a w a etal. 1989, 1991). We have previously reported the three-dimensional ultrastructure of the glomerular basement m e m b r a n e and mesangial matrix in normal and diseased kidneys by the quick-freezing and deep-etching method (Takami et al. 1990, 1991; N a r a m o t o et al. 1991a, 1991 b). In the present study, we used the quick-freezing and deep-etching (QF-DE) and the freeze-substitution (QF-FS) methods to re-evaluate the ultrastructure of slit diaphragms in fresh unfixed glomeruli and have demonstrated that they appear to be composed of substructures with a sheet-like configuration.
Materials and methods A total of 15 normal male Wistar rats, weighing 100 g to 130 g, were used for this study. Under pentobarbital anesthesia, small pieces of cortex were excised from the kidneys with a razor blade. They were processed for the following quick-freezing steps after dicing into small pieces as reported previously (Takami et al. 1990, 1991). Fresh unfixed tissue fragments were plunged into a liquid isopentane-propane mixture (around -193 ~ C) cooled in liquid nitrogen (Ohno and Fujii 1990, 1991; Naramoto etal. 1991a, 1991b). The frozen tissues were freeze-substituted in acetone containing 2% or 0.1% osmium tetroxide as follows. Freeze-substitution was performed by placing the samples in an acetone solution kept at about - 8 0 ~ for 20 h (Furukawa et al. 1989, 1991). The temperature of the samples was then raised, firstly to - 2 0 ~ C for 2 h, then to 4~ C for 2 h and finally to a room temperature. They were washed twice in absolute acetone and embedded in Luveak812 (Nacalai Tesque, Inc., Kyoto, Japan) (Shinagawa et al. 1980). Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined in a Hitachi HS-9 electron microscope. Preparations of replica membranes from fresh kidneys were made as follows. The previously described frozen tissues were carefully fractured with a scalpel in liquid nitrogen (Ohno and Fujii 1990, 1991). The frozen specimens were deeply etched under vacuum conditions of 2-6 • 10 -~ Torr for 20-30 min at a temperature of - 9 5 ~ (Naramoto et al. 1991a, 1991b). After deep-etching, the specimens on a rotary stage were first shadowed with platinum at an angle of 37~ for several seconds and then rotary shadowed up to the total thickness of about 2 nm. They were additionally coated with carbon at an angle of 90~ A drop of 2 0 collodion in amylacetate was put onto the replicas as soon as the specimens were taken out from the machine to prevent their breaking into pieces during the following digestion procedure. The replicas coated with dried collodion were floated on household bleach for 1530 rain to dissolve the tissue components. The large replica membranes were washed in distilled water and cut into small pieces with scissors. They were picked up on Formvar-filmed copper grids and immersed in amylacetate solution to dissolve the collodion film. They were then observed in a Hitachi HS-9 electron microscope at an accelerating voltage of 75 kV and electron micrographs were printed from the inverted negative films. Preparations of TGO fixed kidneys were made as follows. Under pentobarbital anesthesia, the kidneys were first perfused with 0.1 M phosphate buffer (PB), pH 7.4, for 1-2 min and then fixed with 1% tannic acid and 1% glutaraldehyde in phosphate buffer for 15 min (Rodewald and Karnovsky 1974; Furukawa et al. 1989, 1991). They were cut into small pieces and postfixed with 1% osmium tetroxide for 60 min. The fixed samples were rinsed in 10% methanol diluted with distilled water. Excess fluid was removed with filter paper, and the specimens were then rapidly frozen in an isopentane-propane mixture (around - 193~ C) cooled
in liquid nitrogen. They were transferred into liquid nitrogen, shaken briskly to remove adherent isopentane-propane mixture and fractured with a scalpel in liquid nitrogen. They were processed as described above. Some fixed tissues were routinely dehydrated and embedded for ultrathin sections.
Results In all the specimens prepared by FS fixation, tissue layers of acceptable preservation were located within 10 pm f r o m the frozen surface areas. The ultrastructure of the glomeruli was well preserved in the FS fixatives containing 2% or 0.1% osmium tetroxide in acetone (Fig. 1 a and b). The cell surface coat of the foot processes showed electron-dense layers along the cell m e m branes. Where ultrathin sections cut foot processes vertically, some slit diaphragms could be distinguished as single lines between the foot processes. Their width, i.e. the distance from cell m e m b r a n e to cell membrane, had a mean value near 34 nm (n = 50). A detailed description of the m o r p h o m e t r i c data to determine the widths of slit diaphragms will be published in a separate paper (Furukawa et al. 1991). As shrinkage and pulling apart of foot processes were minimized, the epithelial clefts ran relatively straight and narrow courses. This was due to the close apposition o f the glycocalyx on adjacent foot processes in vivo. Freeze-fractured capillary loops from the same samples were observed on replica m e m branes by the Q F - D E method (Fig. 1 c). The width of slit diaphragms, as measured from the outer surface of one foot process to the next, averaged 25 nm (n = 50). The replica m e m b r a n e s freeze-fractured parallel to the basement m e m b r a n e revealed the ultrastructure of the slit diaphragms (Fig. 2a). Interdigitating foot processes were in close apposition, and sheet-like substructures were detected between the foot processes. Ultrathin sections by the Q F - F S method show the glomerular ultrastructure (Fig. 2b). Where the plane of section passed almost parallel to the basement membrane, the slit diaphragms were observed as consistently heterogeneous structures connecting the cell m e m b r a n e s of adjacent foot processes. Figure 2c illustrates that sheet-like substructures were identified in some parts of obliquely or horizontally freeze-fractured capillary loops. Figure 3 illustrates electron micrographs of the glomeruli fixed with T G O . The trilaminar structure o f the basement m e m b r a n e was m o r e clearly apparent (compare Fig. 3 a with Fig. 1 a and b). The electron-dense lamina densa was sandwiched between thin electron-lucent layers, the laminae rarae interna and externa. In the lamina rara externa, fibrils appeared to radiate from the lower surfaces o f foot processes into the lamina densa. Foot processes were contracted during fixation with T G O , except for their bases attached to the basement membrane. Unlike the foot processes fixed by the Q F - F S method, those of TGO-fixed glomeruli showed shrunken cell surface contours. Widely opened clefts were formed between the glycocalyx layers of adjacent foot processes; in addition, the slit diaphragms were pulled apart, their width averaging 47 nm ( n = 50). In the replica m e m branes, the slit diaphragms were observed between the
353
Fig. I a--c. Electron micrographs of glomerular capillary loops prepared by the QF-FS method with 2% osmium tetroxide (a) or 0.1% osmium tetroxide (b) in acetone, and the QF-DE method (e). Bar; 0.5 ~tm. In (a) and (b) the unit membranes of the foot processes (F) are clearly seen and their external surfaces are coated by fibrillar glycocalyx. A large amount of flocculent material is localized within the basement membrane (BM), urinary space (US) and capillary lumen (CL) in (a) and (b). The epithelial slits run relatively straight and narrow courses (arrows). Parts of the slits are closed by a thin slit diaphragm which is seen spanning the filtration clefts (large arrowheads). Central layers of the basement membrane contain networks of fine fibrils, as shown in (a) and
(b). However, the lamina densa and lamina rara externa or interna are less discerned in these specimens than in Fig. 3 a. Some crossbridging filaments appear to be inserted into the base of foot processes in the lamina rara externa (small arrowheads). E; endothelium. a; • 50,300. b; • 52,400. In (c) a replica electron micrograph of a capillary loop freeze-fractured transversely to the basement membrane in an unfixed sample is shown. The slit diaphragms are observed between foot processes (large arrowheads). Extracellular granular material is seen and fibrillar structures are identified on the surfaces of foot processes (small arrowhead). The epithelial slits run relatively straight courses (arrows). BM; basement membrane. F; foot process. E; endothelium. • 90,000
s h r u n k e n f o o t processes a n d c o n s i s t e d o f i r r e g u l a r zipp e r - l i k e s u b s t r u c t u r e s (Fig. 3b). I n s o m e sites, zigzag s u b s t r u c t u r e s c o u l d be seen r u n n i n g c e n t r a l l y a l o n g the space. Slit d i a p h r a g m s were also f o u n d b e t w e e n s h r u n k e n f o o t processes, w h i c h were f r e e z e - f r a c t u r e d a l m o s t
h o r i z o n t a l l y to the b a s e m e n t m e m b r a n e (Fig. 3c). T h e c r o s s - b r i d g e s a p p e a r e d to e x t e n d b e t w e e n the t w o sides o f a c e n t r a l f i l a m e n t , t h u s giving the slit d i a p h r a g m a zipper-like appearance.
354
355 Discussion
It has been argued that the surface coats between adjacent foot processes are normally in close contact over the entire extent of the epithelial slits (Latta 1970). However, the actual existence of slit diaphragms has been questioned, because they varied in appearance, depending on the techniques used for fixing and staining (Karnovsky and Ainsworth 1972). Thus the slit diaphragms were suggested to be artifacts resulting from shrinkage of adjacent foot processes during conventional tissue preparation. However, some experiments with small molecular tracers suggested that a filtration barrier occurred in the region of epithelial slits as well as at the glomerular basement membrane (Graham and Karnovsky 1966; Venkatachalam et al. 1970). In our morphologic study, the slit diaphragms in fresh unfixed glomeruli actually showed sheet-like substructures, although they were composed of regular zipper-like substructures in glomeruli fixed with TGO. These findings indicate that slit diaphragms really do exist in the glomeruli in vivo. It has been reported that the slit diaphragms have a regular pattern when observed by transmission electron microscopy after TGO fixation and the conventional freeze-fracture replica method (Rodewald and Karnovsky 1974; Karnovsky and Ryan 1975; Ryan et al. 1975; Schneeberger etal. 1975). They have periodic cross-bridges extending from the foot processes to a central filament, to form homogeneous pores (about 4 x 14 nm) and are continuous between all foot processes. On the other hand, morphologic and physiological data indicated that the glomerular filter functions as an isoporous membrane which excluded proteins larger than serum albumin (Caulfield and Farquhar 1974; Purtell et al. 1979). In our study, however, the slit diaphragms rarely had an isoporous substructure consisting of regularly spaced cross-bridges. These were changed to zipper-like substructures, in the preparations fixed
