American Journal ofPathology, Vol. 136, No. 5, May 1990 Copyright © American Association ofPathologists
Immunogold Quantitation of Immunoglobulin Light Chains in Renal Amyloidosis and Kappa Light Chain Nephropathy
Meredith M. Silver,* Stephen A. Hearnt John C. Walton,t Lois A. Lines,* and Virginia M. Walleyt From the Department ofPathology,* Hospitalfor Sick Children and University of Toronto, Toronto; the Department of Pathology,j St. Joseph 's Hospital, London; and the Department ofPathology,* Ottawa Civic Hospital and University of Ottawa, Ottawa, Ontario, Canada
By quantitative immunoelectron microscopy using protein A-gold, the authors compared the content and distribution of immunoglobulin light chain (LC) antigens in glomeruli from 1I cases of renal amyloidosis with that in two cases of kappa LC glomerulopathy and two cases ofdiabetic glomerulosclerosis. In a supplementary study and using a similar immunogold technique, the authors identified amyloid A in deparaffinized renal tissue from three of the I I cases of renal amyloidosis. Each patient had similar clinical manifestations (chronic renalfailure with proteinuria) and similarglomerular morphology (thickened glomerular basement membranes and nodular expansion of the mesangium). In 12 cases (10 amyloid, 2 kappa LC), immunoelectron microscopy localized LC antigens over the glomerular deposits and allowed indirect tissue quantitation of each LC antigen to the various cellular and interstitial compartments. In 6 of the 1 I cases of renal amyloidosis, the amyloid labeled onlyfor lambda, and in one, onlyfor kappa. In one patient with Waldenstrom 's macroglobulinemia, who had a biclonal gammopathy, both LC were identified in the amyloid. In two cases, both of whom had a history of chronic suppurative lung disease, both LC antigens as well as amyloid A were localized to the amyloidfibrils. In only one case, in which glomerular amyloid labeled for amyloid A, the amyloid did not label for either LC. Whereas lambda LC-derived fibrils often appeared as spicules in the glomerular subepithelial space, other amyloid deposits usually accumulated in the subendothelial zone and did notform spicules. The epimembranous location of spicules suggested that
the amyloid precursor protein transformed into amyloid fibrils after filtration into the urinary space. Presence ofepimembranous spicules may explain the more severeproteinuric renalfailure and the more rapid progression to glomerulosclerosis described in primary amyloidosis. (Am J Pathol 1990, 136:997-1007)
Amyloid fibrils in the renal glomerulus may be composed of immunoglobulin light chains (LC), lambda LC being more common than kappa.1'4 An infiltrate of kappa LC that is noncongophilic and granular rather than fibrillar by electron microscopy (called para-amyloid hereafter) has been mistaken for diabetic glomerulosclerosis (DGS) in the past.58 Secondary renal amyloidosis due to amyloid A (AA) is more common than LC-derived (primary or AL) amyloid in some studies.4 In each of these conditions, the glomeruli show hyaline thickening of the glomerular basement membrane (GBM) associated with expansion of the mesangial region and, usually, normal glomerular cellularity. Para-amyloid deposits are strongly positive in periodic acid-Schiff (PAS) stain, whereas AA and AL deposits are only weakly PAS-positive. Amyloid glomerulopathy is easily distinguished from DGS because the latter is noncongophilic. The congophilia of AA amyloid is usually abolished by potassium permanganate pretreatment, whereas this reduces but does not usually abolish the congophilia of AL amyloid.4 9 Sensitivity to this bleaching treatment is not, however, specific to AA amyloid.'° Although LC antigens have been identified in glomerular deposits by immunofluorescence and immunoperoxidase methods at the optical microscope level,1-8 LC antigens have been demonstrated in renal amyloid deposits by immunoelectron microscopy (IEM) in only a few studies.578 We used an IEM technique with protein A-gold to Supported in part by grant MA 8930 from the Medical Research Council of Canada (MMS). Accepted for publication December 13, 1989. Address reprint requests to Dr. Meredith M. Silver, Department of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Canada M5G 1 X8.
997
998
Silver et al
A/P May 1990, Vol. 136, No. 5
localize LC antigens in 11 cases of renal amyloidosis, in order to identify the exact site of the amyloid within the glomerulus and also to indirectly quantitate LC antigen in the glomerulus by measuring the labeling intensity over various tissue compartments.711 We compared the LC content of renal amyloidosis with that present in other kinds of nodular glomerulosclerosis (para-amyloid and DGS) that may be confused with renal amyloidosis.
Materials and Methods Patient Material Renal biopsy (8) and autopsy-derived (4) renal tissue was available from 11 subjects with renal amyloidosis. Three of the subjects were male and eight were female; clinical data is tabulated (Table 1). In addition, renal tissue (both biopsy and autopsy) was derived from two male subjects with para-amyloidosis, and (renal biopsy) from two cases (male, female) of DGS (Table 1). For light microscopy, renal biopsies were fixed in either neutral buffered formalin (NBF), Helly's fluid, or DuboscqBrasil. Autopsy tissues were fixed in NBF. Light microscopy stains always included hematoxylin and eosin (H & E), trichrome, PAS, silver methenamine, Giemsa, and congo red; the latter was done with and without pretreatment with potassium permanganate.9 For electron microscopy, renal biopsies were fixed in either 2.5% glutaraldehyde in 0.1 mol/l (molar) cacodylate buffer, or in 4% paraformaldehyde-2% glutaraldehyde in phosphate-buffered saline (PBS). Autopsy-derived renal tissue was fixed in NBF, washed overnight, then refixed in 4% paraformaldehyde-2% glutaraldehyde in PBS. All samples were postfixed in 1 % osmium tetroxide and processed into epoxy resin (either Epon, Polybed, or Spurr's); 1 -,g sections stained with toluidine blue were used to locate glomeruli. Ultrathin sections of glomeruli, mounted on copper grids and stained with uranyl and lead, were viewed in a Philip's 410 electron microscope (Eindhoven, The Netherlands).
Immunoelectron Microscopy for Light Chain Antigens Our immunolabeling method localizes tissue antigens on the surface of ultrathin, nickel grid-mounted epoxy sections by the use of a specific antibody after pretreating the section with an oxidizer, either 4% sodium metaperiodate or 10% hydrogen peroxide.7'11'12 Such oxidizers 'unmask' the antigen on the surface of the resin section, making it accessible to a specific antibody.13'14 Antibodyantigen reaction sites were labeled with protein A-gold
(PAG), the gold grain size being 15 nm, made up as previously described.12 Primary antibodies used were anti-human LC antisera (Dako Corp, Santa Barbara, CA); anti-lambda was used at a dilution of 1:2000 and anti-kappa, at 1:3000. To reduce nonspecific labeling, sections were preincubated with 10% bovine serum albumin (BSA). Incubation time for the primary antibody was 16 hours at 40C. Control ultrathin nickel grid-mounted consecutive tissue sections included: 1) substitution of PBS for the primary antibody; 2) substitution of an irrelevant antibody (anti-insulin) for the primary antibody; 3) substitution of antibody that had been preabsorbed with an excess of purified gamma G immunoglobulin (IgG); 4) use of native protein A before PAG, to block antigen-primary antibody reaction sites; 5) use of PAG absorbed with an excess of
purified IgG. Immunolabeled sections were counterstained with uranyl and lead. Electron micrographs of each glomerulus were taken at a magnification (x 30,000) that we find suitable for quantitation of the labeling density.7 To be nonselective, the photographs were taken in the four corners of a grid square randomly positioned over the most central area of a glomerulus, at a magnification of X 5400. From each photographic negative, four prints were prepared at a magnification of X 30,000: a 2400 lines/mm grating was used to calibrate magnification on the negative, with specimen height being adjusted for each using a goniometer stage, and the photographic enlargement was calculated with a millimeter scale in the plane of the photographic negative. For each micrograph (maximum 16 per case), labeling density over the glomerular compartments was calculated by dividing the number of gold particles over each compartment by the area of that compartment. Areas were estimated with a point counting technique according to Weibel,'s using a 168-point multipurpose overlay (distance between points, 15 mm). The mean labeling density for each compartment was calculated from a tally of the counts for each micrograph. Counts from representative cases are tabulated (Table 2). Immunolabeling was assessed as specific if labeling density for kappa or lambda over a compartment exceeded that over the same compartment in the PBS control section by 8x or greater. The latter was chosen arbitrarily because 1) compartments that labeled 8X control or greater were always assessed as showing clearly positive labeling when viewed independently without knowledge of the corresponding morphometric assessment, and 2) the strongest labeling for LC antigens over the GBM-mesangial compartment in DGS was only 4X control (Table 2). In addition to comparing the labeling density over deposits with that over the same deposits in the PBS control
Immunogold EM in Renal Amyloidosis
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Immunogold Quantitation of Immunoglobulin Light Chains in Renal Amyloidosis and Kappa Light Chain Nephropathy
Meredith M. Silver,* Stephen A. Hearnt John C. Walton,t Lois A. Lines,* and Virginia M. Walleyt From the Department ofPathology,* Hospitalfor Sick Children and University of Toronto, Toronto; the Department of Pathology,j St. Joseph 's Hospital, London; and the Department ofPathology,* Ottawa Civic Hospital and University of Ottawa, Ottawa, Ontario, Canada
By quantitative immunoelectron microscopy using protein A-gold, the authors compared the content and distribution of immunoglobulin light chain (LC) antigens in glomeruli from 1I cases of renal amyloidosis with that in two cases of kappa LC glomerulopathy and two cases ofdiabetic glomerulosclerosis. In a supplementary study and using a similar immunogold technique, the authors identified amyloid A in deparaffinized renal tissue from three of the I I cases of renal amyloidosis. Each patient had similar clinical manifestations (chronic renalfailure with proteinuria) and similarglomerular morphology (thickened glomerular basement membranes and nodular expansion of the mesangium). In 12 cases (10 amyloid, 2 kappa LC), immunoelectron microscopy localized LC antigens over the glomerular deposits and allowed indirect tissue quantitation of each LC antigen to the various cellular and interstitial compartments. In 6 of the 1 I cases of renal amyloidosis, the amyloid labeled onlyfor lambda, and in one, onlyfor kappa. In one patient with Waldenstrom 's macroglobulinemia, who had a biclonal gammopathy, both LC were identified in the amyloid. In two cases, both of whom had a history of chronic suppurative lung disease, both LC antigens as well as amyloid A were localized to the amyloidfibrils. In only one case, in which glomerular amyloid labeled for amyloid A, the amyloid did not label for either LC. Whereas lambda LC-derived fibrils often appeared as spicules in the glomerular subepithelial space, other amyloid deposits usually accumulated in the subendothelial zone and did notform spicules. The epimembranous location of spicules suggested that
the amyloid precursor protein transformed into amyloid fibrils after filtration into the urinary space. Presence ofepimembranous spicules may explain the more severeproteinuric renalfailure and the more rapid progression to glomerulosclerosis described in primary amyloidosis. (Am J Pathol 1990, 136:997-1007)
Amyloid fibrils in the renal glomerulus may be composed of immunoglobulin light chains (LC), lambda LC being more common than kappa.1'4 An infiltrate of kappa LC that is noncongophilic and granular rather than fibrillar by electron microscopy (called para-amyloid hereafter) has been mistaken for diabetic glomerulosclerosis (DGS) in the past.58 Secondary renal amyloidosis due to amyloid A (AA) is more common than LC-derived (primary or AL) amyloid in some studies.4 In each of these conditions, the glomeruli show hyaline thickening of the glomerular basement membrane (GBM) associated with expansion of the mesangial region and, usually, normal glomerular cellularity. Para-amyloid deposits are strongly positive in periodic acid-Schiff (PAS) stain, whereas AA and AL deposits are only weakly PAS-positive. Amyloid glomerulopathy is easily distinguished from DGS because the latter is noncongophilic. The congophilia of AA amyloid is usually abolished by potassium permanganate pretreatment, whereas this reduces but does not usually abolish the congophilia of AL amyloid.4 9 Sensitivity to this bleaching treatment is not, however, specific to AA amyloid.'° Although LC antigens have been identified in glomerular deposits by immunofluorescence and immunoperoxidase methods at the optical microscope level,1-8 LC antigens have been demonstrated in renal amyloid deposits by immunoelectron microscopy (IEM) in only a few studies.578 We used an IEM technique with protein A-gold to Supported in part by grant MA 8930 from the Medical Research Council of Canada (MMS). Accepted for publication December 13, 1989. Address reprint requests to Dr. Meredith M. Silver, Department of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Canada M5G 1 X8.
997
998
Silver et al
A/P May 1990, Vol. 136, No. 5
localize LC antigens in 11 cases of renal amyloidosis, in order to identify the exact site of the amyloid within the glomerulus and also to indirectly quantitate LC antigen in the glomerulus by measuring the labeling intensity over various tissue compartments.711 We compared the LC content of renal amyloidosis with that present in other kinds of nodular glomerulosclerosis (para-amyloid and DGS) that may be confused with renal amyloidosis.
Materials and Methods Patient Material Renal biopsy (8) and autopsy-derived (4) renal tissue was available from 11 subjects with renal amyloidosis. Three of the subjects were male and eight were female; clinical data is tabulated (Table 1). In addition, renal tissue (both biopsy and autopsy) was derived from two male subjects with para-amyloidosis, and (renal biopsy) from two cases (male, female) of DGS (Table 1). For light microscopy, renal biopsies were fixed in either neutral buffered formalin (NBF), Helly's fluid, or DuboscqBrasil. Autopsy tissues were fixed in NBF. Light microscopy stains always included hematoxylin and eosin (H & E), trichrome, PAS, silver methenamine, Giemsa, and congo red; the latter was done with and without pretreatment with potassium permanganate.9 For electron microscopy, renal biopsies were fixed in either 2.5% glutaraldehyde in 0.1 mol/l (molar) cacodylate buffer, or in 4% paraformaldehyde-2% glutaraldehyde in phosphate-buffered saline (PBS). Autopsy-derived renal tissue was fixed in NBF, washed overnight, then refixed in 4% paraformaldehyde-2% glutaraldehyde in PBS. All samples were postfixed in 1 % osmium tetroxide and processed into epoxy resin (either Epon, Polybed, or Spurr's); 1 -,g sections stained with toluidine blue were used to locate glomeruli. Ultrathin sections of glomeruli, mounted on copper grids and stained with uranyl and lead, were viewed in a Philip's 410 electron microscope (Eindhoven, The Netherlands).
Immunoelectron Microscopy for Light Chain Antigens Our immunolabeling method localizes tissue antigens on the surface of ultrathin, nickel grid-mounted epoxy sections by the use of a specific antibody after pretreating the section with an oxidizer, either 4% sodium metaperiodate or 10% hydrogen peroxide.7'11'12 Such oxidizers 'unmask' the antigen on the surface of the resin section, making it accessible to a specific antibody.13'14 Antibodyantigen reaction sites were labeled with protein A-gold
(PAG), the gold grain size being 15 nm, made up as previously described.12 Primary antibodies used were anti-human LC antisera (Dako Corp, Santa Barbara, CA); anti-lambda was used at a dilution of 1:2000 and anti-kappa, at 1:3000. To reduce nonspecific labeling, sections were preincubated with 10% bovine serum albumin (BSA). Incubation time for the primary antibody was 16 hours at 40C. Control ultrathin nickel grid-mounted consecutive tissue sections included: 1) substitution of PBS for the primary antibody; 2) substitution of an irrelevant antibody (anti-insulin) for the primary antibody; 3) substitution of antibody that had been preabsorbed with an excess of purified gamma G immunoglobulin (IgG); 4) use of native protein A before PAG, to block antigen-primary antibody reaction sites; 5) use of PAG absorbed with an excess of
purified IgG. Immunolabeled sections were counterstained with uranyl and lead. Electron micrographs of each glomerulus were taken at a magnification (x 30,000) that we find suitable for quantitation of the labeling density.7 To be nonselective, the photographs were taken in the four corners of a grid square randomly positioned over the most central area of a glomerulus, at a magnification of X 5400. From each photographic negative, four prints were prepared at a magnification of X 30,000: a 2400 lines/mm grating was used to calibrate magnification on the negative, with specimen height being adjusted for each using a goniometer stage, and the photographic enlargement was calculated with a millimeter scale in the plane of the photographic negative. For each micrograph (maximum 16 per case), labeling density over the glomerular compartments was calculated by dividing the number of gold particles over each compartment by the area of that compartment. Areas were estimated with a point counting technique according to Weibel,'s using a 168-point multipurpose overlay (distance between points, 15 mm). The mean labeling density for each compartment was calculated from a tally of the counts for each micrograph. Counts from representative cases are tabulated (Table 2). Immunolabeling was assessed as specific if labeling density for kappa or lambda over a compartment exceeded that over the same compartment in the PBS control section by 8x or greater. The latter was chosen arbitrarily because 1) compartments that labeled 8X control or greater were always assessed as showing clearly positive labeling when viewed independently without knowledge of the corresponding morphometric assessment, and 2) the strongest labeling for LC antigens over the GBM-mesangial compartment in DGS was only 4X control (Table 2). In addition to comparing the labeling density over deposits with that over the same deposits in the PBS control
Immunogold EM in Renal Amyloidosis
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AJPMay 1990, Vol. 136, No. 5
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