Contr. Nephrol., vol. 1, pp. 80-85 (Karger, Basel 1975)
The Fine Structure of the Glomeruli as Revealed by Freeze-Fracturing K. Kuhn 1 and E. Reale Department of Anatomy, Laboratory of Electron Microscopy, Hannover Medical School, Hannover
In troduc tion
Although there are many studies dealing with the fme structure of the glomeruli in thin sections (5, 9, 12) the morphological basis for the passage of proteins in the glomerulus has not yet been presented. To investigate this problem, glomeruli in replicas of freeze-fractured specimens were examined. Until now, only tubular parts (1 , 6 , 8) and interstitial capillaries (2 , 3,7) of the kidney have been studied by this method .
Materials and Methods Kidneys of Wi star rats were fix ed by the dripping method using an aldehyde solution. The glomeruli were separated from the cortex, glycerinated and deep-frozen. Fracturing and shadowing with platinum/carbon was done in a Balzers BA 360 M unit. Some glomeruli were post-fixed in an OsO, -solution and embedded in Epon. Electron microscopes: Siemens Elmiskop I a and 10 I.
Results Endothelium. In figure 1, illustrating the capillary wall, the cleavage plane runs across the pedicles and the basement membrane, then within the plasma membrane of two adjacent endothelial cells. In this figure, endothelial pores with a diameter ranging from 500)\ to 1,000 )\ are clearly visible. A diaphragm across the pores is not revealed in the replicas. Only exceptionally, strands of a 2
Permanent address: Department of Internal Medicine, Hannover Medical School,
3 Hannover (FRG)
Kuhn/Reale
81
zonula occludens (not illustrated) are found on the split membrane of endothelial cells, although intercellular spaces (suture lines, fig. 1) are seen frequently and extensively.
Fig. 1. Glomerular capillary wall. The cleavage plane runs through pedicles (P) and basement membrane (BM) and splits the plasma membrane of two endothelial cells (E, and E,) along their external and sometimes also along their internal faces. Ery = erythrocyte in the lumen of the vessel; S = suture line between the endothelial cells. 24,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing.
Kuhn /Reale
82
Basement membrane. In comparison to thin sections (fig. 2) the lamina densa of the basement membrane appears as a coarse granular structure in the replicas (fig. 3). The granularity is probably due to the fibrillar component of the membrane. Epithelium. In thin sections, the lamina rara externa of the basement membrane is separated from the urinary space by the slit membrane (fig. 2). In the replicas the slit membrane is not demonstrable as a membranous structure. However, the replica of the interpedicular space is smooth and that of the lamina rara externa is coarse, thus giving indirect evidence of the presence of a slit membrane. If the cleavage plane runs within the plasma membrane of the epithelium facing the basement membrane of the capillary wall, en face views of the pedi-
2
3 Fig. 2, 3. Comparison between a thin section and a replica of the freeze-fractured capillary wall. 50,000 X. The arrowhead in the right lower corner in figure 3 indicates the direction of platinum-carbon shadowing. Fig. 4. Fracture through a glomerular capillary wall. The cleavage plane runs within the plasma membrane of pedicles facing the basement membrane of the capillary wall. SP = interpedicular space; MP = micropinocytotic vesicles. 48,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing. Fig. 5. Interdigitations of an endothelial cell (E) with a mesangial cell (M). A basement membrane is missing between these two cells. On the split membrane of the mesangial cell, gap junctions are clearly visible (small arrows). 60,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing.
Freeze-Fracture of Renal Glomeruli
4,5
83
Kuhn/Reale
84
cles are seen (fig. 4). The space between the pedicles has a width up to 800 A. Also in this case the slit membrane with its peculiar structure, recently described in thin sections (10), cannot be identified. This is most propably due to different cleavage levels. Micropinocytotic vesicles are frequently found. Mesangium. On the split membrane of the mesangial cells numerous 'gap junctions' are present (fig. 5). In general, they form patchy and/or linear aggregates of particles. In the replicas the mesangial cells are readily recognized because of their direct contact with the endothelial cells.
Discussion The findings in glomeruli examined by freeze-fracture technique were consistent with those made on thin sections. Further, variably extended faces of the split cell membrane were exposed exhibiting the structures belonging to intercellular junctions (zonula occludens, gap junction). A discon tinuous zonula occludens, which can easily be detected on replicas of fenestrated capillaries of other organs (11), was seen only very rarely between the endothelial cells of the glomerular capillaries. From this observation one can conclude that junctions of the endothelial cells of glomerular capillaries are of a 'leaky' type. In analogy to observations on muscle capillaries (4) with continuous endothelium, this may be important for the permeation of molecules through the intercellular clefts. On replicas of cleaved plasma membranes of mesangial cells, gap junctions are numerous, indicating a coupling with other glomerular cells. Such an intercellular linkage can be established either with endothelial cells or with processes of neighbouring mesangial cells.
Summary Isolated glomeruli of the rat kidney were investigated using the freeze-fracture method. The observations were consistent with those made on thin sections. Only exceptionally components of zonulae occludentes were seen between the capillary endothelial cells. The split membrane of mesangial cells revealed aspects of gap junctions.
References 2 2
Claude, P. and Goodenough, D.A.: Fracture faces of zonulae occludentes from 'tight' and 'leaky' epithelia. 1. Cell BioI. 53: 390 (1973). Friederici, H.H.R.: The tridimensional ultrastructure of fenestrated capillaries. 1. Ultrastruct. Res. 23: 444 (1968).
Freeze-Fracture of Renal Glomeruli 3 4
5
6 7 8
9 10 11 12
85
Friederici, H.H.R.: On the diaphragm across fenestrae of capillary endothelium. 1. Ultrastruct. Res. 27: 373 (1969). Karnovsky, MJ.: Morphology of capillaries with special reference to muscle capillaries: in Crone and Lassen Capillary permeability, the transfer of molecules and ions between capillary blood and tissue. Proc. Alfred Benzon Symp. II, Copenhagen, 1970, Munksgaard, p. 341. Latta, H.: The glomerular capillary wall. 1. Ultrastruct. Res. 32: 526 (1970). Leak, L. V.: Ultrastructure of proximal tubule cells in mouse kidney as revealed by freeze etching. 1. Ultrastruct. Res. 25: 253 (1968). Maul, G. G.: Structure and formation of pores in fenestrated capillaries. 1. Ultrastruct. Res. 36: 768 (1971). Pricam, c.; Humbert, F. ; Perreiet, A ., and Orci, L.: A freeze-etch study of the tight junctions of the rat kidney tubules. Lab. Invest. 30: 286 (1974). Rhodin, J.A. G.: Structure of the kidney; in Strauss and Welt Diseases of the kidney; 2nd ed., p. 1. Copyright 1971 (Little, Brown, Boston 1971). Rodewald, R. and Kamovsky, M.J.: Porous substructure of the glomerular slit diaphragm in the rat and mouse. 1. Cell BioI. 60: 423 (1974). Spitznas, M. and Reale, E.: Fracture faces of fenestrations and junctions of endothelial cells in human choroidal vessels. Investve Ophthal. (in press, 1975). Trump, B.F. and Bulger, R.E.: Morphology of the kidney; in Becker Structural basis of renal disease, p. 1. Copyright 1968 by Hoeber Medical Division, 1.
Dr. K. Kiihn, Anatomisches Institut, Abteilung Elektronenmikroskopie, Medizinische Hochschule, Kari-Wiechert-Allee 9, D-3 Hannover (FRG)
The Fine Structure of the Glomeruli as Revealed by Freeze-Fracturing K. Kuhn 1 and E. Reale Department of Anatomy, Laboratory of Electron Microscopy, Hannover Medical School, Hannover
In troduc tion
Although there are many studies dealing with the fme structure of the glomeruli in thin sections (5, 9, 12) the morphological basis for the passage of proteins in the glomerulus has not yet been presented. To investigate this problem, glomeruli in replicas of freeze-fractured specimens were examined. Until now, only tubular parts (1 , 6 , 8) and interstitial capillaries (2 , 3,7) of the kidney have been studied by this method .
Materials and Methods Kidneys of Wi star rats were fix ed by the dripping method using an aldehyde solution. The glomeruli were separated from the cortex, glycerinated and deep-frozen. Fracturing and shadowing with platinum/carbon was done in a Balzers BA 360 M unit. Some glomeruli were post-fixed in an OsO, -solution and embedded in Epon. Electron microscopes: Siemens Elmiskop I a and 10 I.
Results Endothelium. In figure 1, illustrating the capillary wall, the cleavage plane runs across the pedicles and the basement membrane, then within the plasma membrane of two adjacent endothelial cells. In this figure, endothelial pores with a diameter ranging from 500)\ to 1,000 )\ are clearly visible. A diaphragm across the pores is not revealed in the replicas. Only exceptionally, strands of a 2
Permanent address: Department of Internal Medicine, Hannover Medical School,
3 Hannover (FRG)
Kuhn/Reale
81
zonula occludens (not illustrated) are found on the split membrane of endothelial cells, although intercellular spaces (suture lines, fig. 1) are seen frequently and extensively.
Fig. 1. Glomerular capillary wall. The cleavage plane runs through pedicles (P) and basement membrane (BM) and splits the plasma membrane of two endothelial cells (E, and E,) along their external and sometimes also along their internal faces. Ery = erythrocyte in the lumen of the vessel; S = suture line between the endothelial cells. 24,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing.
Kuhn /Reale
82
Basement membrane. In comparison to thin sections (fig. 2) the lamina densa of the basement membrane appears as a coarse granular structure in the replicas (fig. 3). The granularity is probably due to the fibrillar component of the membrane. Epithelium. In thin sections, the lamina rara externa of the basement membrane is separated from the urinary space by the slit membrane (fig. 2). In the replicas the slit membrane is not demonstrable as a membranous structure. However, the replica of the interpedicular space is smooth and that of the lamina rara externa is coarse, thus giving indirect evidence of the presence of a slit membrane. If the cleavage plane runs within the plasma membrane of the epithelium facing the basement membrane of the capillary wall, en face views of the pedi-
2
3 Fig. 2, 3. Comparison between a thin section and a replica of the freeze-fractured capillary wall. 50,000 X. The arrowhead in the right lower corner in figure 3 indicates the direction of platinum-carbon shadowing. Fig. 4. Fracture through a glomerular capillary wall. The cleavage plane runs within the plasma membrane of pedicles facing the basement membrane of the capillary wall. SP = interpedicular space; MP = micropinocytotic vesicles. 48,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing. Fig. 5. Interdigitations of an endothelial cell (E) with a mesangial cell (M). A basement membrane is missing between these two cells. On the split membrane of the mesangial cell, gap junctions are clearly visible (small arrows). 60,000 X. The arrowhead in the right lower corner indicates the direction of platinum-carbon shadowing.
Freeze-Fracture of Renal Glomeruli
4,5
83
Kuhn/Reale
84
cles are seen (fig. 4). The space between the pedicles has a width up to 800 A. Also in this case the slit membrane with its peculiar structure, recently described in thin sections (10), cannot be identified. This is most propably due to different cleavage levels. Micropinocytotic vesicles are frequently found. Mesangium. On the split membrane of the mesangial cells numerous 'gap junctions' are present (fig. 5). In general, they form patchy and/or linear aggregates of particles. In the replicas the mesangial cells are readily recognized because of their direct contact with the endothelial cells.
Discussion The findings in glomeruli examined by freeze-fracture technique were consistent with those made on thin sections. Further, variably extended faces of the split cell membrane were exposed exhibiting the structures belonging to intercellular junctions (zonula occludens, gap junction). A discon tinuous zonula occludens, which can easily be detected on replicas of fenestrated capillaries of other organs (11), was seen only very rarely between the endothelial cells of the glomerular capillaries. From this observation one can conclude that junctions of the endothelial cells of glomerular capillaries are of a 'leaky' type. In analogy to observations on muscle capillaries (4) with continuous endothelium, this may be important for the permeation of molecules through the intercellular clefts. On replicas of cleaved plasma membranes of mesangial cells, gap junctions are numerous, indicating a coupling with other glomerular cells. Such an intercellular linkage can be established either with endothelial cells or with processes of neighbouring mesangial cells.
Summary Isolated glomeruli of the rat kidney were investigated using the freeze-fracture method. The observations were consistent with those made on thin sections. Only exceptionally components of zonulae occludentes were seen between the capillary endothelial cells. The split membrane of mesangial cells revealed aspects of gap junctions.
References 2 2
Claude, P. and Goodenough, D.A.: Fracture faces of zonulae occludentes from 'tight' and 'leaky' epithelia. 1. Cell BioI. 53: 390 (1973). Friederici, H.H.R.: The tridimensional ultrastructure of fenestrated capillaries. 1. Ultrastruct. Res. 23: 444 (1968).
Freeze-Fracture of Renal Glomeruli 3 4
5
6 7 8
9 10 11 12
85
Friederici, H.H.R.: On the diaphragm across fenestrae of capillary endothelium. 1. Ultrastruct. Res. 27: 373 (1969). Karnovsky, MJ.: Morphology of capillaries with special reference to muscle capillaries: in Crone and Lassen Capillary permeability, the transfer of molecules and ions between capillary blood and tissue. Proc. Alfred Benzon Symp. II, Copenhagen, 1970, Munksgaard, p. 341. Latta, H.: The glomerular capillary wall. 1. Ultrastruct. Res. 32: 526 (1970). Leak, L. V.: Ultrastructure of proximal tubule cells in mouse kidney as revealed by freeze etching. 1. Ultrastruct. Res. 25: 253 (1968). Maul, G. G.: Structure and formation of pores in fenestrated capillaries. 1. Ultrastruct. Res. 36: 768 (1971). Pricam, c.; Humbert, F. ; Perreiet, A ., and Orci, L.: A freeze-etch study of the tight junctions of the rat kidney tubules. Lab. Invest. 30: 286 (1974). Rhodin, J.A. G.: Structure of the kidney; in Strauss and Welt Diseases of the kidney; 2nd ed., p. 1. Copyright 1971 (Little, Brown, Boston 1971). Rodewald, R. and Kamovsky, M.J.: Porous substructure of the glomerular slit diaphragm in the rat and mouse. 1. Cell BioI. 60: 423 (1974). Spitznas, M. and Reale, E.: Fracture faces of fenestrations and junctions of endothelial cells in human choroidal vessels. Investve Ophthal. (in press, 1975). Trump, B.F. and Bulger, R.E.: Morphology of the kidney; in Becker Structural basis of renal disease, p. 1. Copyright 1968 by Hoeber Medical Division, 1.
Dr. K. Kiihn, Anatomisches Institut, Abteilung Elektronenmikroskopie, Medizinische Hochschule, Kari-Wiechert-Allee 9, D-3 Hannover (FRG)
