JOURNAL OF PATHOLOGY, VOL.
161: 137-143 (1990)
ULTRASTRUCTURAL IMMUNOGOLD STUDIES OF HEPARAN SULPHATE PROTEOGLYCAN IN NORMAL HUMAN GLOMERULI AND GLOMERULONEPHRITIS JILL MOSS*, DAVID F. WOODROW*, IAN SHORE*, PETER GOWERt, MALCOLM PHILLIPST AND ROBERT G . SPIRO;
Department of Histopathology* and Medicine?, Charing Cross and Westminster Medical School. London, U.K.; SEIIiott P . Joslin Research Laboratory, Harvard Medical School, Boston, Massachusetts, U.S.A. Received 8 December I989 Accepted 23 January 1990
SUMMARY The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis,mesangial IgA disease, and anti-glomerularbasement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on parafoxmaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis,no HSPG was found in the electron-dense deposits.These findings contrast with our previous studiesusing the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM. KEY
woms-Immunogold, heparan sulphate proteoglycans, ultrastructure, glomerular basement membranes, glomerulonephri tis.
INTRODUCTION The proteoglycan heparan sulphate (HSPG) is a normal component of the glomerular basement membrane (GBM) and it contributes principally to the charge of the glomerulus and so prevents the loss of many constituents of the blood through the GBM filter (for reviews see refs 1 and 2). It has been found to be decreased in amount in the glomeruli of rats with nephrotic syndrome3 and also in the glomeruli of patients with diabetes m e l l i t ~ sThe . ~ distribution of HSPG has not been demonstrated ultrastructurally in human GBM, although the region of the lamina rara externa is thought to be the main site of deposition. The evidence for this is that following preincubation with heparatinase there is a reduction in the number of anionic sites in this area of the Addressee for correspondence: Dr J. Moss, Department of Histopathology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, U.K.
0022-341 7/90/060137-07 $05.00 0 1990by John Wiley & Sons, Ltd.
basement membrane, as demonstrated using the cationic probe polyethyleneimine (PEI).576 The GBM also contains other structural components including ty IV collagen, laminin, entactin, and fibronectin,’.cnd we have previously shown that there is a zonation in the distribution of components in normal human glomeruli and in the immune complex glomerulonephritis, mesangial IgA disease. Type IV collagen and fibronectin are found predominantly on the endothelial aspects of the GBM and throughout the mesangial matrix, being less evident on the epithelial aspect.’ This zone beneath the epithelial cells is the region which previous work has suggested contains much of the HSPG.’T~ We have now used the same immunogold ultrastructural technique to study the distribution of HSPG in normal human glomeruli and in the GBM of patients with mesangial IgA disease, membranous glomerulonephritis, and anti-glomerular basement membrane (anti-GBM) disease.
I38
J. MOSS ET A L .
Table I-Clinical
details of patients at presentation
~
.Mc.rotl~yiol/,?A
C.D.*t
F
16 years
Microscopic haernaturia 1.96 g proteinQ4 h
R.B.t
M
40 years
Microscopic haematuria 0.46 g pr0tein~24h
.M~~nlh~attou.s Gj\' F 76 years
N.F.t M.L.t C.W.t E.L.*:
F M F
80years 73years 56years
5.6 g proteinl24 h 4.0 g protein24 h 12.0 g proteini24 h I .4 g protein24 h
. ~ ~ z I I - G~B~ M SCUSC (Rapidly progressive glornerulonephritis with 95 per cent crcscen ts) S.D.: F 76years Acute renal failure Conirol pcirietir J.H.*t
F
45years
*Biopsy processed for routine electron microscopy. tFresh biopsy. :Previously frozen biopsy processed for immunoelectron microscopy.
MATERIALS A N D M E T H O D S Needle biopsies of kidneys were obtained from two cases of mesangial IgA disease, four cases of membranous giomerulonephritis. and one case of anti-GBM disease. A surgical biopsy ofmorphologically normal kidney was obtained from a 45-year-old female patient who had a nephrectomy for a renal adenocarcinoma. The clinical findings are shown in Table I. Immunological reagents Rabbit anti-bovine heparan sulphate proteoglycan was prepared at the Elliott P. Joslin Research Laboratory. Boston. The specificity of the antiserum was verified by solid-phase radioimmunoassaysand by immunobiotting ofelectrophoretically separated antigens.' By indirect immunofluorescence using FI TC-conj ugated goat an ti-rabbit I g G (ICN Biomedicals Ltd., C.K.) in the normal kidney, the anti-HSPG antiserum was localized exclusively to basement membranes. In theglomeruli, the capillary loops stained in a linear pattern and the mesangial regions were less intensely stained. All tubule basement membranes were outlined. as were the capillary membranes and the matrix around the smooth muscle cells of the arteries (Fig. I ) . This
Fig. I-lrnmunofluorescence micrograph to show the localization of HSPG around glomerular capillary loops from the control patient (J.H.). Less HSPG IS present in the rnesangiurn. the Bowman's capsule. and tubule basement membranes
distribution confirms that previously reported in humans'and rats." For immunoelectron microscopy goat anti-rabbit IgG conjugated to 10nm gold was obtained from Janssen Life Sciences Products. For diagnostic immunofluorescence FITCconjugated rabbit anti-human IgA. IgG, IgM. and C 3 were obtained from Dakopatts. Light microscop). The renal biopsies were fixed in Dubosq-Brazil solution and were routinely processed and stained as previously described." Immunojuorescence microscop!, Fresh renal tissue was embedded in OCT compound and snap-frozen in isopentane immersed in
IMMUNOGOLD STUDY OF HEPARAN SULPHATE IN GBM
139
Fig. 2-Electron micrograph of a glomerulus from the control patient (J.H.) to show the immunogold localization of HSPG predominantly on the epithelial aspect of (a) the mesangium and (b) the GBM. (a) x 24 000; (b) x 30 0oO
liquid nitrogen. Cryostat sections (6 pm) were incubated with the antisera as in previous studies.’’ Routine electron microscop), Specimens were fixed in 3 per cent glutaraldehyde in 0.1 M phosphate buffer, post-fixed in 2 per cent osmium tetroxide, block-stained in uranyl acetate, dehydrated through ascending grades of alcohol. and embedded in Spurr’s resin. Where insufficient tissue was available, the material embedded in Lowicryl K4M for immunoelectron microscopy proved adequate for rountine examination. Ultrathin sections were stained with aqueous uranyl acetate and Reynold’s lead citrate. lmmunoelectron microscopy Fresh tissue or the material previously frozen for immunofluorescence was placed in 4 per cent paraformaldehyde in 0. I M phosphate buffer (pH 7.2). dissected into I mm3, left for 3 h at 4°C. and stored in I per cent paraformaldehyde at 4°C for I week.
The samples were dehydrated through ascending grades of ethanol, infiltrated with Lowicryl K4M resin (Taab Laboratories Ltd., U.K.) and embedded under ultraviolet light at low temperature.’ The immunogold technique was carried out at room temperature as previously described.’ Briefly. ultrathin sections were preincubated in an ovalbumin block, followed by rabbit anti-bovine HSPG. and then gold-conjugated goat anti-rabbit IgG. Sections were stained in aqueous uranyl acetate and Reynold’s lead citrate prior to examination with a Philips EM 300. Controls consisted of omitting the primary antiserum and replacing the primary antiserum with non-immune rabbit serum. RESULTS Normal kidney (one case) The gold particles indicating the presence of HSPG were found throughout the GBM but were more numerous on the epithelial aspect. Similarly, in the mesangium the HSPG was most abundant in
140
J . MOSS ET A L .
Fig. >Electron micrograph of a glomerulus from a patient (C.D.)with mesangial IgA disease to show electrondense deposits and the immunogold localization of HSPG. x 23 OOO
peripheral regions with fewer gold particles seen within the central regions of the matrix (Fig. 2). Mesangial I g A disease ( t w o cases) The distribution of HSPG was similar to that seen in the control patient. being found throughout the GBM and present predominantly on the epithelial aspect. In the mesangium, the HSPG was found mainly at the periphery of the matrix and only infrequently within the central regions. Gold particles were not seen over the mesangial electron-dense deposits (Fig. 3). Membranous glomerulonephritis (four cases) In the lesions in which the subepithelial electrondense deposits were separated by spike-like projections of GBM. the HSPG was around and beneath the electron-dense deposits and was also present in the spikes (Fig. 4). Less HSPG was seen on the endothelial aspect of the GBM and it was not localized in the electron-dense deposits. In older lesions,' where the GBM covered the electron-dense deposits on the epithelial aspects, the HSPG was present in this new basement membrane. As in the control patient, gold
particles were more numerous in peripheral as compared with central regions of the mesangial matrix. Anti-glomerular basement membrane disease (one case) The distribution of HSPG was similar to that found in the normal glomerulus. being predominantly on the epithelial aspects of the GBM and the mesangium (Fig. 5).
DISCUSSION These results show that HSPG is present in normal glomeruli and in those of patients with mesangial IgA disease, membranous glomerulonephritis, and anti-GBM disease. In all cases, there is a zonation in distribution of the HSPG since it is found predominantly on the epithelial aspect of the GBM and the mesangium with little in the central regions of the mesangial matrix. This finding contrasts with our previous results in normal glomeruli and in mesangial IgA disease. where type IV collagen and fibronectin were found predominantly along the
Fig. +Electron micrograph of a glomerulus from a patient (M.L.) with membranous GN to show the distribution of HSPG (*: capillary lumen). x 32 000
Fig. 5-Electron micrograph o f a glomerulus from a patient (S.D.)with anti-GBM disease to show the localization of HSPG predominanrly on the epithelial aspect of the GBM (*: capillary lumen). x 37 000
142
J. MOSS ETAL.
endothelial aspect of the GBM and throughout the the lamina densa,” while in humans the type IV mesangial matrix, being less abundant on the epi- collagenwaslocalizedmoreon theendothelialaspect thelial aspect.’ Thus, the human GBM appears to of the GBM.’ It is also possible that any differences have a layered structure with the HSPG mainly may be explained in part by the source of antibody, present in the sub-epithelial region where it is ideally methods of fixation, and techniques for localization situated to play a major filtration role in relation to of the antibody.” Human glomerular epithelial and mesangial cells electrostatic charge of the filtered molecules. The localization of the HSPG on the epithelial aspect have been shown to produce proteoglycans in tissue may also reflect a diffusion gradient within the culture,’’ and there has been a previous report of the GBM, as observations in rat glomeruli suggest that detection of HSPG in the rough endoplasmic reticuthe epithelial cells play a predominant role in the lum and Golgi cisternae of visceral and parietal epithelial cells of rat glomeruli using horseradish synthesis of basement membrane HSPG.” In the six cases of immune complex disease peroxidase-labelled antibodies. I’ In the present studied, HSPG was not localized within the electron- study, no HSPG was localized within epithelial, dense deposits. Indeed, in membranous glomerulo- endothelial, or mesangial cells. The present qualitative studies on immune comnephritis the deposits appear to displace the HSPG deeper into the GBM as the complex develops in size plex disease have not revealed any major loss of beneath the epithelial cells. HSPG was also present HSPG in patients with proteinuria. While this may throughout the spikes of new basement membrane reflect the sensitivity of the ultrastructural method, it which form following the accumulation of the com- is in agreementwith one earlier study of GBM charge plexes. Thus, in these particular glomerulopathies using the PEI technique where no diminution in there is no evidence to suggest a binding of anti- anionic sites was demonstrated in human glomeruli bodies with the sequestered HSPG within the GBM. of patients with heavy proteinuria,6 although in This observation is of interest in relation to the human congenital nephrotic syndrome a reduction reports of anti-DNA antibodies cross-reacting in the number of anionic sites has been observed.’ This study has shown that HSPG is present both with HSPG in some patients with systemic lupus erythemato~us,’~ and also to the finding of anti- in the GBM of normal human glomeruli and in the bodies to HSPG in patients with post-streptococcal GBM in some types of glomerulonephritis. It appears to be confined predominantly to the outer gl~merulonephritis.’~ Since the HSPG is predominantly on the epi- aspect of the GBM and mesangium and is not thelial aspect of the GBM, this may explain the lack localized in the immune complexes. of disruption of HSPG in the anti-GBM disease as the autoimmune reaction with the Goodpasture antigen present on type IV collagen would occur REFERENCES mainly on the endothelial aspect of the GBM.” While these are the first ultrastructural immuno- 1. Martinez-Hernandez A, Amenta PS. The basement membrane in pathology. Lab Invest 1983;48:656471. gold studies of HSPG distribution in human DR. Recent studies on the structure and pathology of glomeruli, other ultrastructural antibody studies on 2. Abrahamson basement membranes. J Pathol1986; 149 257-278. the distribution of this component within glomeruli 3. Myndersc LA, Hassell JR, Kleinman HK, Martin GR, MartinezHernandez A. Loss of heparan sulfate proteoglycan from glomerular have been undertaken in rats. In one study, the antibasement membrane of nephrotic rats. Lab invest 1983; 48:292-302. rat HSPG was concentrated in the lamina rara 4. Shimomura H. Spiro RG. Studies on macromolecular components of interna and externa of the GBM,” while in others, human glomerular basement membrane and alterations in diabetes. Decreased levels of heparan sulfate proteoglycan and laminin. antibodies directed against HSPG purified from the 1987; M:3 7 4 3 8 1. Engelbreth-Holm Swarm sarcoma bound equally to 5 . Diabetes Vernier RL. Klein DJ, Sisson SP, Mahan JD, Oegema TR, Brown the lamina densa and laminae rarae of the GBM.3q’6 DM. Heparan sulphate-rich anionic sites in the human glomerular basement membrane. N Engl J Med 1983; 309:1001-1M)9. Since from the results reported here HSPG is pre- 6 . Furness PN, Turner DR. Cotton RE. Basement membrane charge in dominantly localized on the epithelial aspect of the human glomerular disease. J Parhol1986; 150: 267-278. human GBM, these findings may reflect species dif- 7 . Woodrow DF, Shore 1, Moss J, Gower P, Phillips M. lmmunoelectron microscopic studies of immune complex deposits and basement ferences, as our previous results showed a different membrane components in IgA nephropathy. J Pathol 1989; 157: distribution in type IV collagen and fibronectin in 47-57. humans from that reported in rat GBM. Type IV 8. Mohan PS. Spiro RG. Macromolecular organization of basement membranes. Characterization and comparison of glomerular basecollagen in the rat glomeruli was found to be ment membrane and lens capsule components by immunochemical distributed across the GBM but predominantly in and lectin affinity procedures. J Biol Chem 1986 261: 43284336.
IMMUNOGOLD STUDY O F HEPARAN SULPHATE IN GBM
9. Schleicher ED. Wagner E-M. Olgemoller B. Nerlich AG. Gerbitz KD. Characterization and localization of basement membrane-associated heparan sulfate proteoglycan human tissues. Lab Invesr 1989; 61: 323-332. 10. Couchman JR. Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues. J Cell Biol 1987: 105 190I - 1916. I I. kdinford D. Woodrow DF. Sloper JC. de Wardener HE. Griffiths I. Post-meningococcal acute glomerular nephritis. Clin Nephrol1978:9: 249-253. I?. Stow JL. Sdwada H, Farquhar MG. Basement membrane heparan sulphate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus. Pror Narl Acad Sci USA 1985: 8 2 32963300. 13. Faaber P, Rijke TP, van de Putte LB. Capel PJ. Burden JH. Crossreactivity of human and murine anti-DNA antibodies with heparan sulphate. The major glycosaminoglycan in glomerular basement membrane. JClin Invesr 1986:77: 1824-1830. 14. Fillit H.Damle SP. Gregory JD. Volin C. Poon-King T, Zabriskie J. Sera from patients with poststreptococcal glomerulonephntiscontain antibodies to glomerular heparan sulfate proteoglycan. J €xp Med 1985:161:277-289.
143
15. Butkowski RJ. Weislander J. Wisdom BJ. er a / . Properties of
the globular domain of type IV collagen and its relationship to the Goodpasture antigen. J Biol Chem 1985;260: 3739-3747. 16. Laurie GW, Leblond CP. Inoue S. Martin GR. Chung AE. Structure of the basement membrane and immunolocalization of five basement membrane components to the lamina densa and its extensions in both glomeruli and tubules of the rat kidney. Am J Anar 1984: 169: 463-48 I. 17. Bendayan M. Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach. Diaberologia 1985: 28: 373-378. 18. Kejaxhki D. Sawada H. Farquhar MG. Immunoelectron microscopy in kidney research: some contributions and limitations. Kidney h r 1986: 30:229-245. 19. Davies M. Thomas GJ. Jenner L, Mason RM. Human glomerular epithelial and mesangial cells produce different populations of proteoglycans. In: Gubler MC, Sternberg M. eds. Progress in Basement Membrane Research. Renal and Related Aspects in Health and Disease. London: John Libbey Eurotext Ltd.. 1988: 35-41.
161: 137-143 (1990)
ULTRASTRUCTURAL IMMUNOGOLD STUDIES OF HEPARAN SULPHATE PROTEOGLYCAN IN NORMAL HUMAN GLOMERULI AND GLOMERULONEPHRITIS JILL MOSS*, DAVID F. WOODROW*, IAN SHORE*, PETER GOWERt, MALCOLM PHILLIPST AND ROBERT G . SPIRO;
Department of Histopathology* and Medicine?, Charing Cross and Westminster Medical School. London, U.K.; SEIIiott P . Joslin Research Laboratory, Harvard Medical School, Boston, Massachusetts, U.S.A. Received 8 December I989 Accepted 23 January 1990
SUMMARY The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis,mesangial IgA disease, and anti-glomerularbasement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on parafoxmaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis,no HSPG was found in the electron-dense deposits.These findings contrast with our previous studiesusing the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM. KEY
woms-Immunogold, heparan sulphate proteoglycans, ultrastructure, glomerular basement membranes, glomerulonephri tis.
INTRODUCTION The proteoglycan heparan sulphate (HSPG) is a normal component of the glomerular basement membrane (GBM) and it contributes principally to the charge of the glomerulus and so prevents the loss of many constituents of the blood through the GBM filter (for reviews see refs 1 and 2). It has been found to be decreased in amount in the glomeruli of rats with nephrotic syndrome3 and also in the glomeruli of patients with diabetes m e l l i t ~ sThe . ~ distribution of HSPG has not been demonstrated ultrastructurally in human GBM, although the region of the lamina rara externa is thought to be the main site of deposition. The evidence for this is that following preincubation with heparatinase there is a reduction in the number of anionic sites in this area of the Addressee for correspondence: Dr J. Moss, Department of Histopathology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, U.K.
0022-341 7/90/060137-07 $05.00 0 1990by John Wiley & Sons, Ltd.
basement membrane, as demonstrated using the cationic probe polyethyleneimine (PEI).576 The GBM also contains other structural components including ty IV collagen, laminin, entactin, and fibronectin,’.cnd we have previously shown that there is a zonation in the distribution of components in normal human glomeruli and in the immune complex glomerulonephritis, mesangial IgA disease. Type IV collagen and fibronectin are found predominantly on the endothelial aspects of the GBM and throughout the mesangial matrix, being less evident on the epithelial aspect.’ This zone beneath the epithelial cells is the region which previous work has suggested contains much of the HSPG.’T~ We have now used the same immunogold ultrastructural technique to study the distribution of HSPG in normal human glomeruli and in the GBM of patients with mesangial IgA disease, membranous glomerulonephritis, and anti-glomerular basement membrane (anti-GBM) disease.
I38
J. MOSS ET A L .
Table I-Clinical
details of patients at presentation
~
.Mc.rotl~yiol/,?A
C.D.*t
F
16 years
Microscopic haernaturia 1.96 g proteinQ4 h
R.B.t
M
40 years
Microscopic haematuria 0.46 g pr0tein~24h
.M~~nlh~attou.s Gj\' F 76 years
N.F.t M.L.t C.W.t E.L.*:
F M F
80years 73years 56years
5.6 g proteinl24 h 4.0 g protein24 h 12.0 g proteini24 h I .4 g protein24 h
. ~ ~ z I I - G~B~ M SCUSC (Rapidly progressive glornerulonephritis with 95 per cent crcscen ts) S.D.: F 76years Acute renal failure Conirol pcirietir J.H.*t
F
45years
*Biopsy processed for routine electron microscopy. tFresh biopsy. :Previously frozen biopsy processed for immunoelectron microscopy.
MATERIALS A N D M E T H O D S Needle biopsies of kidneys were obtained from two cases of mesangial IgA disease, four cases of membranous giomerulonephritis. and one case of anti-GBM disease. A surgical biopsy ofmorphologically normal kidney was obtained from a 45-year-old female patient who had a nephrectomy for a renal adenocarcinoma. The clinical findings are shown in Table I. Immunological reagents Rabbit anti-bovine heparan sulphate proteoglycan was prepared at the Elliott P. Joslin Research Laboratory. Boston. The specificity of the antiserum was verified by solid-phase radioimmunoassaysand by immunobiotting ofelectrophoretically separated antigens.' By indirect immunofluorescence using FI TC-conj ugated goat an ti-rabbit I g G (ICN Biomedicals Ltd., C.K.) in the normal kidney, the anti-HSPG antiserum was localized exclusively to basement membranes. In theglomeruli, the capillary loops stained in a linear pattern and the mesangial regions were less intensely stained. All tubule basement membranes were outlined. as were the capillary membranes and the matrix around the smooth muscle cells of the arteries (Fig. I ) . This
Fig. I-lrnmunofluorescence micrograph to show the localization of HSPG around glomerular capillary loops from the control patient (J.H.). Less HSPG IS present in the rnesangiurn. the Bowman's capsule. and tubule basement membranes
distribution confirms that previously reported in humans'and rats." For immunoelectron microscopy goat anti-rabbit IgG conjugated to 10nm gold was obtained from Janssen Life Sciences Products. For diagnostic immunofluorescence FITCconjugated rabbit anti-human IgA. IgG, IgM. and C 3 were obtained from Dakopatts. Light microscop). The renal biopsies were fixed in Dubosq-Brazil solution and were routinely processed and stained as previously described." Immunojuorescence microscop!, Fresh renal tissue was embedded in OCT compound and snap-frozen in isopentane immersed in
IMMUNOGOLD STUDY OF HEPARAN SULPHATE IN GBM
139
Fig. 2-Electron micrograph of a glomerulus from the control patient (J.H.) to show the immunogold localization of HSPG predominantly on the epithelial aspect of (a) the mesangium and (b) the GBM. (a) x 24 000; (b) x 30 0oO
liquid nitrogen. Cryostat sections (6 pm) were incubated with the antisera as in previous studies.’’ Routine electron microscop), Specimens were fixed in 3 per cent glutaraldehyde in 0.1 M phosphate buffer, post-fixed in 2 per cent osmium tetroxide, block-stained in uranyl acetate, dehydrated through ascending grades of alcohol. and embedded in Spurr’s resin. Where insufficient tissue was available, the material embedded in Lowicryl K4M for immunoelectron microscopy proved adequate for rountine examination. Ultrathin sections were stained with aqueous uranyl acetate and Reynold’s lead citrate. lmmunoelectron microscopy Fresh tissue or the material previously frozen for immunofluorescence was placed in 4 per cent paraformaldehyde in 0. I M phosphate buffer (pH 7.2). dissected into I mm3, left for 3 h at 4°C. and stored in I per cent paraformaldehyde at 4°C for I week.
The samples were dehydrated through ascending grades of ethanol, infiltrated with Lowicryl K4M resin (Taab Laboratories Ltd., U.K.) and embedded under ultraviolet light at low temperature.’ The immunogold technique was carried out at room temperature as previously described.’ Briefly. ultrathin sections were preincubated in an ovalbumin block, followed by rabbit anti-bovine HSPG. and then gold-conjugated goat anti-rabbit IgG. Sections were stained in aqueous uranyl acetate and Reynold’s lead citrate prior to examination with a Philips EM 300. Controls consisted of omitting the primary antiserum and replacing the primary antiserum with non-immune rabbit serum. RESULTS Normal kidney (one case) The gold particles indicating the presence of HSPG were found throughout the GBM but were more numerous on the epithelial aspect. Similarly, in the mesangium the HSPG was most abundant in
140
J . MOSS ET A L .
Fig. >Electron micrograph of a glomerulus from a patient (C.D.)with mesangial IgA disease to show electrondense deposits and the immunogold localization of HSPG. x 23 OOO
peripheral regions with fewer gold particles seen within the central regions of the matrix (Fig. 2). Mesangial I g A disease ( t w o cases) The distribution of HSPG was similar to that seen in the control patient. being found throughout the GBM and present predominantly on the epithelial aspect. In the mesangium, the HSPG was found mainly at the periphery of the matrix and only infrequently within the central regions. Gold particles were not seen over the mesangial electron-dense deposits (Fig. 3). Membranous glomerulonephritis (four cases) In the lesions in which the subepithelial electrondense deposits were separated by spike-like projections of GBM. the HSPG was around and beneath the electron-dense deposits and was also present in the spikes (Fig. 4). Less HSPG was seen on the endothelial aspect of the GBM and it was not localized in the electron-dense deposits. In older lesions,' where the GBM covered the electron-dense deposits on the epithelial aspects, the HSPG was present in this new basement membrane. As in the control patient, gold
particles were more numerous in peripheral as compared with central regions of the mesangial matrix. Anti-glomerular basement membrane disease (one case) The distribution of HSPG was similar to that found in the normal glomerulus. being predominantly on the epithelial aspects of the GBM and the mesangium (Fig. 5).
DISCUSSION These results show that HSPG is present in normal glomeruli and in those of patients with mesangial IgA disease, membranous glomerulonephritis, and anti-GBM disease. In all cases, there is a zonation in distribution of the HSPG since it is found predominantly on the epithelial aspect of the GBM and the mesangium with little in the central regions of the mesangial matrix. This finding contrasts with our previous results in normal glomeruli and in mesangial IgA disease. where type IV collagen and fibronectin were found predominantly along the
Fig. +Electron micrograph of a glomerulus from a patient (M.L.) with membranous GN to show the distribution of HSPG (*: capillary lumen). x 32 000
Fig. 5-Electron micrograph o f a glomerulus from a patient (S.D.)with anti-GBM disease to show the localization of HSPG predominanrly on the epithelial aspect of the GBM (*: capillary lumen). x 37 000
142
J. MOSS ETAL.
endothelial aspect of the GBM and throughout the the lamina densa,” while in humans the type IV mesangial matrix, being less abundant on the epi- collagenwaslocalizedmoreon theendothelialaspect thelial aspect.’ Thus, the human GBM appears to of the GBM.’ It is also possible that any differences have a layered structure with the HSPG mainly may be explained in part by the source of antibody, present in the sub-epithelial region where it is ideally methods of fixation, and techniques for localization situated to play a major filtration role in relation to of the antibody.” Human glomerular epithelial and mesangial cells electrostatic charge of the filtered molecules. The localization of the HSPG on the epithelial aspect have been shown to produce proteoglycans in tissue may also reflect a diffusion gradient within the culture,’’ and there has been a previous report of the GBM, as observations in rat glomeruli suggest that detection of HSPG in the rough endoplasmic reticuthe epithelial cells play a predominant role in the lum and Golgi cisternae of visceral and parietal epithelial cells of rat glomeruli using horseradish synthesis of basement membrane HSPG.” In the six cases of immune complex disease peroxidase-labelled antibodies. I’ In the present studied, HSPG was not localized within the electron- study, no HSPG was localized within epithelial, dense deposits. Indeed, in membranous glomerulo- endothelial, or mesangial cells. The present qualitative studies on immune comnephritis the deposits appear to displace the HSPG deeper into the GBM as the complex develops in size plex disease have not revealed any major loss of beneath the epithelial cells. HSPG was also present HSPG in patients with proteinuria. While this may throughout the spikes of new basement membrane reflect the sensitivity of the ultrastructural method, it which form following the accumulation of the com- is in agreementwith one earlier study of GBM charge plexes. Thus, in these particular glomerulopathies using the PEI technique where no diminution in there is no evidence to suggest a binding of anti- anionic sites was demonstrated in human glomeruli bodies with the sequestered HSPG within the GBM. of patients with heavy proteinuria,6 although in This observation is of interest in relation to the human congenital nephrotic syndrome a reduction reports of anti-DNA antibodies cross-reacting in the number of anionic sites has been observed.’ This study has shown that HSPG is present both with HSPG in some patients with systemic lupus erythemato~us,’~ and also to the finding of anti- in the GBM of normal human glomeruli and in the bodies to HSPG in patients with post-streptococcal GBM in some types of glomerulonephritis. It appears to be confined predominantly to the outer gl~merulonephritis.’~ Since the HSPG is predominantly on the epi- aspect of the GBM and mesangium and is not thelial aspect of the GBM, this may explain the lack localized in the immune complexes. of disruption of HSPG in the anti-GBM disease as the autoimmune reaction with the Goodpasture antigen present on type IV collagen would occur REFERENCES mainly on the endothelial aspect of the GBM.” While these are the first ultrastructural immuno- 1. Martinez-Hernandez A, Amenta PS. The basement membrane in pathology. Lab Invest 1983;48:656471. gold studies of HSPG distribution in human DR. Recent studies on the structure and pathology of glomeruli, other ultrastructural antibody studies on 2. Abrahamson basement membranes. J Pathol1986; 149 257-278. the distribution of this component within glomeruli 3. Myndersc LA, Hassell JR, Kleinman HK, Martin GR, MartinezHernandez A. 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