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The glycoprotein inner layer of glomerular capillary basement membrane as a filtration barrier
J O U R N A L OF U L T R A S T R U C T U R E R E S E A R C H
57, 65-67 (1976)
The Glycoprotein Inner Layer of Glomerular Capillary Basement Membrane as a Filtration Barrier 1 HARRISON LATTA AND WILLIAM H. JOHNSTON
Department of Pathology, University of California School of Medicine, Los Angeles, California 90024 Received March 4, 1976, and in revised form, August 4, 1976 The inner layer of the glomerular capillary basement membrane extends across the inner portion of endothelial fenestrations and is continuous with the surface coat material beneath endothelial cytoplasm. It is in a position to account for the filtration barrier to plasma albumin observed by Ryan and Karnovsky [Fed. Proc. 34, 877 (1975); Kidney Int. 9, 36 (1976)] with normal blood flow. Different parts of the glomerular capillary wall can act as barriers to other tracers studied under different conditions by various investigators.
Various investigators have found that the inner layer of the glomerular basement membrane is only focally penetrated by particles of 100-140 /~ diameter, and that the central dense layer appears to act as a coarse filter for most particles greater than 100 /~ in diameter [see review by Latta (3)]. Other evidence points to the slit membrane or to the outer surface coat of the foot processes as a fine filter for proteins larger than hemoglobin, which would include plasma albumin (3). The slit membrane or diaphragm has a zipperlike structure of rectangular pores after fixation by perfusion with tannic acid (5). The surface coat which covers the slit membrane and may also fill the filtration slits in vivo appears as a dense mass of sialoglycoprotein filaments after staining with ruthenium red and osmium tetroxide
in contrast to protein molecules (2). The dextrans do not stain in the dense layer and they are stained only infrequently in the outer layer of the basement membrane. Farquhar (2) has discussed problems of interpretation that arise when enzymatic proteins are used as tracers for glomerular permeability. Recent studies by Ryan and Karnovsky (6, 7) using an electron microscopic immunoperoxidase technique with labeled antibody Fab fragments found that during norreal blood flow in rats, serum albumin was confined to the glomerular capillary lumen and endothelial fenestrations. Interruption of blood flow for 5 min allowed serum albumin to penetrate throughout the basement membrane to the urinary space. Because the above study pointed to a barrier at the level of endothelial fenestrations in normally functioning kidneys, we have examined this region further, using ruthenium red and osmium tetroxide stains as in our earlier studies of acidic polysaccharides or glycoproteins in glomerular capillaries (4). Figure 1 demonstrates that endothelial fenestrations open on the inner layer of the basement membrahe and that the inner layer contains fibrillar material staining with ruthenium red and osmium which is similar to and continuous with the portions of inner layer
(4). Caulfield and Farquhar (1) believe that fractionated dextran molecules of larger size (with Einstein-Stokes radii of 55/~ or more) are blocked by the dense layer of the basement membrane. However, interpretation of these experiments is complicated because dextran molecules are not charged and do not have a fixed shape in solution, I Supported by Research Grant No. AM 06074 from the National Institute of Arthritis, Metabolism and Digestive Diseases, U.S. Public Health Service. 65 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
LATTA AND JOHNSTON
'! iii!':!!i:i !
Fro. 1. Glomerular capillary wall st ained with ruthenium red and osmium tetroxide to demonstrate mainly glycoproteins. The inner layer (IL) of the basement membrane (lamina rara interna) is a continuous layer on the inner surface of the central dense layer (CL) (lamina densa). This staining reverses the contrast of the layers customarily seen with metal stains. The inner layer is continuous with the surface coat of the endothelium and lies beneath the fenestrations. The more homogeneous appearance beneath the fenestration on the left (arrow) approximates what would be expected for a finer filtration barrier. The more irregular or granular appearance beneath the fenestration on the right may be artifactual. Counterstained with uranyl acetate and lead citrate, x 140 000. b e n e a t h the endothelial cytoplasm. Similar r e s u l t s are seen less clearly in the illust r a t i o n s of our earlier p a p e r (4), b u t Fig. 1 h e r e shows a m o r e h o m o g e n e o u s l a y e r ext e n d i n g across the i n n e r p a r t of fenestrations a n d b e n e a t h the e n d o t h e l i u m . This i n n e r l a y e r a p p e a r s to be continuous w i t h the surface coat of the e n d o t h e l i u m , a n d it s e e m s likely t h a t it is derived f r o m it. As a lightly stained glycoprotein it would h a v e a lower n e g a t i v e c h a r g e a n d sialic acid content t h a n the free surface coat of glom e r u l a r foot processes. The glycoproteins in the g l o m e r u l a r c a p i l l a r y wall are diag r a m m e d on the r i g h t side of Fig. 2. Because this i n n e r l a y e r is in the position of the filtration b a r r i e r to p l a s m a alb u m i n d e m o n s t r a t e d by R y a n a n d K a r n o v s k y (7) it would s e e m to be responsible in p a r t for the r e s u l t s t h e y observed d u r i n g n o r m a l blood flow. This suggests t h a t the l a y e r is r e l a t i v e l y h o m o g e n e o u s in vivo a t such t i m e s a n d t h a t the g r e a t e r i r r e g u l a r -
ity seen in our earlier p a p e r (4) is a n artifact which occurs w h e n n o r m a l blood flow ceases a n d the tissue is fixed. The irregul a r i t y could r e s u l t f r o m c l u m p i n g or loss of m a t e r i a l , or both. Indeed, one possible exp l a n a t i o n of the rapid loss of i m p e r m e a b i l ity offered by R y a n a n d K a r n o v s k y (7) is t h a t the n o r m a l i m p e r m e a b i l i t y m a y depend on l a r g e r p l a s m a proteins b e i n g conc e n t r a t e d in or on the i n n e r surface of t h e filter a n d stearically blocking the p a s s a g e of a l b u m i n molecules. S t o p p i n g filtration for a n y r e a s o n would allow the p l a s m a proteins to diffuse a w a y a n d a l b u m i n to p e n e t r a t e the previously existing b a r r i e r . Accepting the i n n e r l a y e r of the basem e n t m e m b r a n e as t h e b a r r i e r to a l b u m i n filtration u n d e r n o r m a l conditions does not m e a n t h a t it is n e c e s s a r y to discard the evidence obtained f r o m previous studies. O t h e r b a r r i e r s in the g l o m e r u l a r c a p i l l a r y wall m a y operate in different c i r c u m s t a n ces a n d m a y be quite helpful u n d e r less
GLOMERULAR BASEMENT MEMBRANE
Ep
67
Mf
5 ' obo
(9 Fro. 2. Glyeoproteins in the glomerular capillary wall as stained with r u t h e n i u m red and osmium tetroxide are indicated with heavy lines on the r i g h t side. This diagram combines the present observations on the inner layer with earlier observations on the glomerular capillary wall. The inner layer of the b a s e m e n t m e m b r a n e (lamina r a r a interna) seems to consist largely of'glyeoproteins continuous with the surface coat of the endothelial ceils (En). It lies b e n e a t h the endothelial fenestrations (F) and would be the first filtration barrier encountered by the glomerular filtrate. Some glycoprotein fibrils r u n in the plane of the dense central layer (CL) (lamina densa) and some r u n across it. The portion of the foot processes (FP) embedded in the outer layer (OL) of the basement m e m b r a n e (lamina r a r a externa) has a surface coat t h a t seems to contribute considerable material to the outer layer. Dense surface coats cover the plasma m e m b r a n e (PM) of the free surface of foot processes and extend across the slit m e m b r a n e (SM) or diaphragm. These surface coats partially or completely fill the filtration slits between the foot processes. On the left side of the diagram the fine lines show the effect of metal stains and probably represent mainly protein components of the capillary wall. Epithelial cell (Ep), myofilaments (Mf), Bowman's space (BS). Modified from LATTA, H., J. Ultrastruct. Res. 32, 526 (1970). than ideal conditions in preventing massive losses of plasma proteins. The ease with which the inner layer loses its impermeability would make the other barriers quite important. REFERENCES 1. CAULFIELD,J. P., AND FARQUHAR, M. G., J. Cell Biol. 63, 883 (1974). 2. FARQUHAR,M. G., Kidney Int. 8, 197 (1975).
3. LATTA,H., in ORLOFF,J., BERLINER,R. W., AND GEIGER, S. R. (Eds.), Handbook of Physiology, Sect. 8, p. 1. Amer. Physiol. Soc., Washington, 1973. 4. LATTA, H., JOHNSTON, W. H., AND STANLEY,T. M., J. Ultrastruct. Res. 51, 354 (1975). 5. RODEWALD,R., AND KAaNOVSKY, M. J., J. Cell Biol. 60, 423 (1974). 6. RYAN,G. B., ANDKARNOVSKY,M. J., Fed. Proc. 34, 877 (1975) (Abstr. 3722). 7. RYAN,G. B., and KARNOVSKY,M. J., Kidney Int. 9, 36 (1976).
57, 65-67 (1976)
The Glycoprotein Inner Layer of Glomerular Capillary Basement Membrane as a Filtration Barrier 1 HARRISON LATTA AND WILLIAM H. JOHNSTON
Department of Pathology, University of California School of Medicine, Los Angeles, California 90024 Received March 4, 1976, and in revised form, August 4, 1976 The inner layer of the glomerular capillary basement membrane extends across the inner portion of endothelial fenestrations and is continuous with the surface coat material beneath endothelial cytoplasm. It is in a position to account for the filtration barrier to plasma albumin observed by Ryan and Karnovsky [Fed. Proc. 34, 877 (1975); Kidney Int. 9, 36 (1976)] with normal blood flow. Different parts of the glomerular capillary wall can act as barriers to other tracers studied under different conditions by various investigators.
Various investigators have found that the inner layer of the glomerular basement membrane is only focally penetrated by particles of 100-140 /~ diameter, and that the central dense layer appears to act as a coarse filter for most particles greater than 100 /~ in diameter [see review by Latta (3)]. Other evidence points to the slit membrane or to the outer surface coat of the foot processes as a fine filter for proteins larger than hemoglobin, which would include plasma albumin (3). The slit membrane or diaphragm has a zipperlike structure of rectangular pores after fixation by perfusion with tannic acid (5). The surface coat which covers the slit membrane and may also fill the filtration slits in vivo appears as a dense mass of sialoglycoprotein filaments after staining with ruthenium red and osmium tetroxide
in contrast to protein molecules (2). The dextrans do not stain in the dense layer and they are stained only infrequently in the outer layer of the basement membrane. Farquhar (2) has discussed problems of interpretation that arise when enzymatic proteins are used as tracers for glomerular permeability. Recent studies by Ryan and Karnovsky (6, 7) using an electron microscopic immunoperoxidase technique with labeled antibody Fab fragments found that during norreal blood flow in rats, serum albumin was confined to the glomerular capillary lumen and endothelial fenestrations. Interruption of blood flow for 5 min allowed serum albumin to penetrate throughout the basement membrane to the urinary space. Because the above study pointed to a barrier at the level of endothelial fenestrations in normally functioning kidneys, we have examined this region further, using ruthenium red and osmium tetroxide stains as in our earlier studies of acidic polysaccharides or glycoproteins in glomerular capillaries (4). Figure 1 demonstrates that endothelial fenestrations open on the inner layer of the basement membrahe and that the inner layer contains fibrillar material staining with ruthenium red and osmium which is similar to and continuous with the portions of inner layer
(4). Caulfield and Farquhar (1) believe that fractionated dextran molecules of larger size (with Einstein-Stokes radii of 55/~ or more) are blocked by the dense layer of the basement membrane. However, interpretation of these experiments is complicated because dextran molecules are not charged and do not have a fixed shape in solution, I Supported by Research Grant No. AM 06074 from the National Institute of Arthritis, Metabolism and Digestive Diseases, U.S. Public Health Service. 65 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
LATTA AND JOHNSTON
'! iii!':!!i:i !
Fro. 1. Glomerular capillary wall st ained with ruthenium red and osmium tetroxide to demonstrate mainly glycoproteins. The inner layer (IL) of the basement membrane (lamina rara interna) is a continuous layer on the inner surface of the central dense layer (CL) (lamina densa). This staining reverses the contrast of the layers customarily seen with metal stains. The inner layer is continuous with the surface coat of the endothelium and lies beneath the fenestrations. The more homogeneous appearance beneath the fenestration on the left (arrow) approximates what would be expected for a finer filtration barrier. The more irregular or granular appearance beneath the fenestration on the right may be artifactual. Counterstained with uranyl acetate and lead citrate, x 140 000. b e n e a t h the endothelial cytoplasm. Similar r e s u l t s are seen less clearly in the illust r a t i o n s of our earlier p a p e r (4), b u t Fig. 1 h e r e shows a m o r e h o m o g e n e o u s l a y e r ext e n d i n g across the i n n e r p a r t of fenestrations a n d b e n e a t h the e n d o t h e l i u m . This i n n e r l a y e r a p p e a r s to be continuous w i t h the surface coat of the e n d o t h e l i u m , a n d it s e e m s likely t h a t it is derived f r o m it. As a lightly stained glycoprotein it would h a v e a lower n e g a t i v e c h a r g e a n d sialic acid content t h a n the free surface coat of glom e r u l a r foot processes. The glycoproteins in the g l o m e r u l a r c a p i l l a r y wall are diag r a m m e d on the r i g h t side of Fig. 2. Because this i n n e r l a y e r is in the position of the filtration b a r r i e r to p l a s m a alb u m i n d e m o n s t r a t e d by R y a n a n d K a r n o v s k y (7) it would s e e m to be responsible in p a r t for the r e s u l t s t h e y observed d u r i n g n o r m a l blood flow. This suggests t h a t the l a y e r is r e l a t i v e l y h o m o g e n e o u s in vivo a t such t i m e s a n d t h a t the g r e a t e r i r r e g u l a r -
ity seen in our earlier p a p e r (4) is a n artifact which occurs w h e n n o r m a l blood flow ceases a n d the tissue is fixed. The irregul a r i t y could r e s u l t f r o m c l u m p i n g or loss of m a t e r i a l , or both. Indeed, one possible exp l a n a t i o n of the rapid loss of i m p e r m e a b i l ity offered by R y a n a n d K a r n o v s k y (7) is t h a t the n o r m a l i m p e r m e a b i l i t y m a y depend on l a r g e r p l a s m a proteins b e i n g conc e n t r a t e d in or on the i n n e r surface of t h e filter a n d stearically blocking the p a s s a g e of a l b u m i n molecules. S t o p p i n g filtration for a n y r e a s o n would allow the p l a s m a proteins to diffuse a w a y a n d a l b u m i n to p e n e t r a t e the previously existing b a r r i e r . Accepting the i n n e r l a y e r of the basem e n t m e m b r a n e as t h e b a r r i e r to a l b u m i n filtration u n d e r n o r m a l conditions does not m e a n t h a t it is n e c e s s a r y to discard the evidence obtained f r o m previous studies. O t h e r b a r r i e r s in the g l o m e r u l a r c a p i l l a r y wall m a y operate in different c i r c u m s t a n ces a n d m a y be quite helpful u n d e r less
GLOMERULAR BASEMENT MEMBRANE
Ep
67
Mf
5 ' obo
(9 Fro. 2. Glyeoproteins in the glomerular capillary wall as stained with r u t h e n i u m red and osmium tetroxide are indicated with heavy lines on the r i g h t side. This diagram combines the present observations on the inner layer with earlier observations on the glomerular capillary wall. The inner layer of the b a s e m e n t m e m b r a n e (lamina r a r a interna) seems to consist largely of'glyeoproteins continuous with the surface coat of the endothelial ceils (En). It lies b e n e a t h the endothelial fenestrations (F) and would be the first filtration barrier encountered by the glomerular filtrate. Some glycoprotein fibrils r u n in the plane of the dense central layer (CL) (lamina densa) and some r u n across it. The portion of the foot processes (FP) embedded in the outer layer (OL) of the basement m e m b r a n e (lamina r a r a externa) has a surface coat t h a t seems to contribute considerable material to the outer layer. Dense surface coats cover the plasma m e m b r a n e (PM) of the free surface of foot processes and extend across the slit m e m b r a n e (SM) or diaphragm. These surface coats partially or completely fill the filtration slits between the foot processes. On the left side of the diagram the fine lines show the effect of metal stains and probably represent mainly protein components of the capillary wall. Epithelial cell (Ep), myofilaments (Mf), Bowman's space (BS). Modified from LATTA, H., J. Ultrastruct. Res. 32, 526 (1970). than ideal conditions in preventing massive losses of plasma proteins. The ease with which the inner layer loses its impermeability would make the other barriers quite important. REFERENCES 1. CAULFIELD,J. P., AND FARQUHAR, M. G., J. Cell Biol. 63, 883 (1974). 2. FARQUHAR,M. G., Kidney Int. 8, 197 (1975).
3. LATTA,H., in ORLOFF,J., BERLINER,R. W., AND GEIGER, S. R. (Eds.), Handbook of Physiology, Sect. 8, p. 1. Amer. Physiol. Soc., Washington, 1973. 4. LATTA, H., JOHNSTON, W. H., AND STANLEY,T. M., J. Ultrastruct. Res. 51, 354 (1975). 5. RODEWALD,R., AND KAaNOVSKY, M. J., J. Cell Biol. 60, 423 (1974). 6. RYAN,G. B., ANDKARNOVSKY,M. J., Fed. Proc. 34, 877 (1975) (Abstr. 3722). 7. RYAN,G. B., and KARNOVSKY,M. J., Kidney Int. 9, 36 (1976).