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Ultrastructural and immunohistochemical similarities between glomerular arteriolar and mesangial lesions in experimental diabetes
Diabetes Research and Clinical Practice, 16 (1992) 0
1992 Elsevier Science Publishers
27-35
27
B.V. All rights reserved 0168-8227/92/$05.00
DIABET 00613
Ultrastructural and immunohistochemical similarities between glomerular arteriolar and mesangial lesions in experimental diabetes K. Ina’,
H. Kitamura’,
M. Nakamura’,
J. 0n02
and R. Takaki2
‘Departmerit of Anatomy and ‘First Department of Medicine, Medical College of Oita. Oita, Japan
(Received 12 August 1991) (Revision accepted 10 November 1991)
Summary
It has thus far been speculated that plasma proteins have leaked and accumulated in diabetic expanded mesangium. We have previously shown that glomerular arteriolar hyalinosis has frequently occurred and has consisted of basement membrane-like extracellular matrix in alloxan diabetic rats. In the present study, the two diabetic glomerular lesions, arteriolar hyaline nodules and increased mesangial matrix, were comparatively evaluated by means of electron microscopy and immunohistochemistry. It was revealed that both of these lesions contained, in common, finely particulate extracellular matrix mingled with cytoplasmic fragments, and exhibited intense immunoreactions for IgG and IgM. It is indicated that both types of diabetic glomerular lesions are essentially of the same nature.
Key words: Glomerular
arteriolar hyalinosis; IgM ; Alloxan
Mesangial expansion;
Introduction
Glomerular basement membrane thickening [l] and mesangial expansion [2], both of which appear at early stage of diabetes, have thus far been extensively studied by means of light and electron microscopy and immunohistochemistry [3-71. In particular, it has been reported by Mauer and associates that such plasma proteins as IgG, IgM and C3 have accumulated in diabetic
Correspondence to: K. Ina, Department College of Oita, Oita, Japan.
of Anatomy, Medical
Immunohistochemistry
for IgG and
mesangium [4]. As compared with basement membranous and mesangial lesions, a fewer number of studies have been made on glomerular arteriolar hyalinosis [&lo], which has been regarded as an exudative diabetic lesion. In our foregoing study using conventional electron microscopy, we have revealed that prominent hyalinosis of glomerular arterioles has frequently occurred in the rats alloxan-diabetic for 10 months and that the lesion has consisted of basement membrane-like extracellular matrix [ 111. In terms of such results, we have held a view that this arteriolar lesion is essentially similar to mesangial expansion.
28 In the mesangial by means and light
present study, these arteriolar and lesions were comparatively evaluated of conventional electron microscopy and electron immunohistochemistry.
Materials and Methods Animals A total of ten male Wistar rats were employed. They were reared on free access to rat chow (CE-2, Japan CREA, Japan) and tap water. Induction of’ experimental diabetes Rats weighing 180 to 200 g were given 40 mg/kg alloxan (Wako Pure Chemical, Japan) in I”(, aqueous solution via tail vein. Hyperglycemia was confirmed by determining the non-fasting plasma glucose levels by means of glucose-oxidase method at 1 month intervals, and only those individuals which persistently exhibited plasma glucose values higher than 400 mg/dl were used as diabetic animals. None of the diabetic animals were maintained on insulin therapy. Five diabetic and five age- and sex-matched intact control rats were offered for study at 10 months after induction of diabetes. At the corresponding time, albuminuria has been confirmed to be significantly increased in diabetic as compared with control rats (DM, 301 k 159,ug/day versus C, 33 2 25 pg/ day; P < 0.05) [ 111. Preparation of tissue specimens Animals were deeply anesthetized by Nembutal injection. Left kidneys were excised after ligation of supplying vessels, to be subjected to conventional electron-microscopic examination (see below). Animals were then perfusion-fixed, and right kidneys were immunohistochemically examined (see below). Conventional electron microscopy Renal cortical tissues were obtained and immersed in our modified Dalton’s fixative [ 121 for 2 h at 4 “C under constant stirring. Tissues were then dehydrated in a graded ethanol series,
immersed in QY-1 and embedded in epon-epoxy resin. Ultrathin sections were made and mounted on neoprene-coated copper grids. They were stained with methanolic uranyl acetate [ 131 and aqueous lead citrate [ 141. The sections were then observed and photographed under a transmission electron microscope at 80 kV, at the magnihcations of 3300 to 20000. Photographic negatives were enlarge-printed as desired. Immunohistochemical procedures After animals were perfused with two-fold diluted Karnovsky’s fixative [ 151 for about 10 min (see above), right kidneys were removed, and their cortical tissues were sliced and immersion-fixed in non-diluted solution of the same fixative for another two hours. For examination at the lightmicroscopic level, fixed tissues were dehydrated in a graded ethanol series, cleared in xylene and vacuum-embedded in parafhn. Four micra-thick sections were made, mounted on glass slides and were processed as briefed below. Sections were then deparaffinized, hydrated and were incubated for 12 h at 4 ‘C with either rabbit antirat IgG antibody (Seikagaku Kogyo, Japan) or rabbit antirat IgM antibody (Zymed Laboratories Inc., U.S.A.). After being rinsed in phosphate buffer (pH 7.4) the sections were further incubated for 1 h at room temperature with gold-labelled goat antirabbit IgG antibody (Amersham, U.K.). These sections were then physically developed for 40 min at 20 “C in our physical developer [ 161 which contains, 0.8”,; citric acid, 0.3”,, bromohydroquinone, 15 ‘,, gum Arabic and 0.2”,, silver nitrate. Finally, sections were counterstained with Kernechtrot. For examination at the electron microscopic level, the tissues fixed in Karnovsky’s fixative were dehydrated in a graded ethanol series and embedded in Lowicryl K4M resin (Chemische Werke Lowi, Waldkraiburg, Germany) at - 20 ‘C with ultraviolet lighting (Toshiba SHL1OOUVQ mercury lamp equipped with Toshiba UV-D35 filter, Toshiba, Japan) for 10 h. Ultrathin sections were mounted on uncoated nickel grids and immunostained according to the sche-
29 dule briefed below. Sections were firstly reacted with either rabbit antirat IgG antibody or rabbit
antirat IgM antibody for 36 h at 4 “C. They were then rinsed in phosphate buffer (pH 7.4) and
Fig 1. Electron micrographs of glomerular arterioles from control (a and b) and diabetic (c and d) rats. (a) Medial extracellular matrix (MEM) shows age-related slight increase ( x 5750). (b) A high magnification electron micrograph of the area encircled by a rectangle in panel a. MEM exhibits finely particulate substructure, in contrast to subepithelial basement membrane (BM) which is rather homogeneous and less electron dense ( x 34400). (c) MEM of a diabetic glomerular arteriole exhibits marked increase ( x 5750). (d) A high magnification electron micrograph of the area encircled by a rectangle in panel c. Ultrastructures of MEM and BM are essentially the same as in panel b. A cytoplasmic fragment (CF) is seen ( x 34400). AL, arteriolar lumen; JG, juxtaglomerular cell; SM, smooth muscle cell; P, podocyte; BC, Bowman’s capsule.
30 further incubated for 1.5 h at room temperature with gold-labelled goat antirabbit IgG antibody. As in light-microscopic survey, sections were finally physically developed at 20 “C, for shorter
periods of time (8 min). In addition, somewhat different formula of physical developer was adopted. Thus the developer contained 0.5”~ citric acid, 0.17 9,0bromohydroquinone, 17 9; gum
Fig. 2. Electron micrographs of mesangium from control (a and b) and diabetic (c and d) rats. (a) Mesangial matrix (MM) exhibits age-related slight increase ( x 5750). (b) A high-magnification electron micrograph of the area encircled by a rectangle in panel a ( x 34400). (c) MM of a diabetic rat exhibits marked increase ( x 5750). (d) A high-magnification electron micrograph of the area encircled by a rectangle in panel c ( x 34400). CL, capillary lumen; P, podocyte; BM, basement membrane.
31
Arabic and 0.17 P; silver nitrate. Immunostained sections were examined and photographed electron microscopically at 60 kV. In order to check the specificity of immunostainings, some tissue sections were incubated in preabsorbed antibodies, but were otherwise processed as mentioned above.
Results Conventional electron microscopy
In control and diabetic rats arteriolar medial extracellular
examined here, matrix located
around smooth muscle cells exhibited finely clustered substructures (Fig. la and c), being in marked contrast to subepithelial and subendothelial basement membrane, where rather homogeneous, less electron dense substructure prevailed (Fig. lb and d). In diabetic rats, arteriolar medial extracellular matrix often underwent massive enlargement and contained cytoplasmic fragments (Fig. lc). Apparently the lesion corresponds to light-microscopic hyaline nodule. Interestingly, the mesa&al matrix of control and diabetic rats was of essentially the same substructure and similarly increased in amount in diabetes (Fig. 2).
Fig. 3. Light micrographs ofglomeruli from control and diabetic rats immunostained for IgG and IgM, followed by Kernechtrot counterstaining. Appreciable stainings for IgG (a) and IgM (c) are not seen over the glomeruli from control rats. Distinct stainings for IgG (b) and IgM (d) are observed in association with the arteriole and mesangium from diabetic rats ( x 330).
32
Immunohistochemical light microscopy Appreciable stainings for IgG and IgM were not observed over the glomeruli from control rats (Fig. 3a and c). In marked contrast, most glomeruli from diabetic rats exhibited distinct stainings for these plasma proteins in association with arterioles and the mesangium (Fig. 3b and d). Positive immunostainings for IgG and IgM were localized in the mesangium in 45 out of 50 and 48 out of 50 diabetic glomeruli, respectively. On the other hand, positive immunostainings for the same proteins were noted in association with
the arteriole in 21 out of 25 and 24 out of 25 diabetic glomeruli where one or two arterioles were seen, respectively. Immunoelectron microscopy In coincidence with the light microscopic data, only sporadic labellings for IgG and IgM were observed over the entire elements of the glomeruli from control rats (data not shown). The glomeruli of diabetic rats exhibited intense immunolabellings for IgG in arteriolar hyaline nodules (Fig. 4a) and mesangial matrix and cells (Fig. 4b). Sub-
Fig. 4. Electron micrographs of the glomerular arteriole (a) and mesangium (b) of a diabetic rat immunostained for IgG. (a) Labellings for IgG are intense in association with arteriolar medial extracellular matrix (MEM) exhibiting increase and podocyte (P), and are feeble over basement membrane (BM) and endothelial cell ( x 12300). (b) Labellings for IgG are intense in association with mesangial matrix (MM) and podocyte, and are feeble over BM and endothelial cell ( x 12 300). AL, arteriolar lumen; CL. capillary lumen.
33 stantial intensity of labellings was also recognized in association with the cytosol and lysosomes of the glomerular podocyte of diabetic rats (Fig. 4). In addition, feeble labellings were found to be localized in the glomerular basement membrane (Fig. 4). On the other hand, labellings for IgM were intense in arteriolar hyaline nodules (Fig. 5a) as well as the mesangial matrix (Fig. 5b), but were trace in extent in the podocyte and almost completely missing from the glomerular basement membrane (Fig. 5).
Control immunostainings In those light and electron microscopic sections which were subjected to control immunostainings, reactions for IgG and IgM were virtually absent (data not shown).
Discussion In terms of the present conventional electron microscopic findings, the two diabetic glomerular
;
.
.
Fig. 5. Electron micrographs of the glomerular arteriole (a) and mesangium (b) of a diabetic rat immunostained for IgM. (a) Labellings for IgM are intense in association with arteriolar medial extracellular matrix (MEM) exhibiting increase, but are trace in extent over other structures observed ( x 12 300). (b) Labellings for IgM are intense in association with mesangial matrix (MM), but are trace in extent over other structures observed (x 12300). AL, arteriolar lumen; CL, capillary lumen; P, podocyte.
34
lesions, arteriolar hyaline nodules and increased mesangial matrix, closely resembled each other, both being composed of extracellular matrix and cytoplasmic fragments. In addition, large amounts of IgG and IgM were detected in both type lesions. Taken together, we are prone to speculate that these lesions are essentially of the same nature. In this context, Mauer and associates have indicated abundance of plasma proteins including IgG, IgM and C3 in diabetic mesangium, and ascribed this to functional incompetence of such mesangium [4]. These authors have used the immunofluorescence technique, and their observations have been necessarily made only at the light microscopic level. In the present study, we successfully identified electron microscopic localization of IgG and IgM within the rat glomerulus with high discrimination by virtue of a modern immunostaining technique. This led us to a new concept on the nature of diabetic glomerular lesions. Two mechanisms can be presumed to explain accumulation of plasma proteins in the arteriolar wall. Thus, plasma proteins already present in the mesangium could be transported to the arteriolar wall through the ‘route of drainage of material from mesangium’ previously proposed by Leiper et al. on the basis of tracer experiment [ 171. Alternatively, it is also possible that these proteins exude through the arteriolar endothelium and accumulate in the arteriolar media because of raised vascular permeability [ 18-201. As a circumferential evidence for the latter possibility, the following present finding is to be added. As apparent from Fig. 1 the segment of the glomerular arteriole susceptible to diabetic change is lined with endothelial cells which contain appreciable numbers of pinocytotic vesicles and exhibit localized attenuation and fenestration. The podocytes and basement membrane of diabetic rats were immunolabelled for IgG but not for IgM. Unequivocal explanation for such differential labelling is not available at present. Permselectivity of tested proteins related to the size, shape and charge of molecules appears to be involved.
References 1 Osterby, R. (1965) A quantitative estimate of the peripheral glomerular basement membrane in recent juvenile diabetes. Diabetologia 1, 97-100. 2 Osterby, R. (1973) A quantitative electron microscopic study ofmesangial regions in glomeruli from patients with short term juvenile diabetes mellitus. Lab. Invest. 29, 99-l 10. 3 Miller, K. and Michael, A.F. (1976) Immunopathology of renal extracellular membranes in diabetes mellitus. Specificity of tubular basement-membrane immunofluorescence. Diabetes 25, 701-708. 4 Mauer, S.M., Steffes, M.W., Sutherland, D.E.R., Najarian, J.S., Michael, A.F. and Brown, D.M. (1975) Studies of the rate of regression of the glomerular lesions in diabetic rats treated with pancreatic islet transplantation. Diabetes 24, 280-285. 5 Bower, G., Brown, D.M., Steffes, M.W., Vernier, R.L. and Mauer, S.M. (1980) Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse. Lab. Invest. 43, 333-341. 6 Miller, L.L., Izzo, M.J., Wemett, D.. Panner, B.J. and Schenk, E.A. (1988) Increased plasma IgA, sIgA, and C3and IgA-containing immune complexes with renal glomerular deposits in diabetic rats. Diabetes 37, 185-193. 7 Bendayan, M., Gingras, D. and Charest, P. (1986) Distribution of endogenous albumin in the glomerular wall of streptozotocin induced diabetic rats as revealed by highresolution immunocytochemistry. Diabetologia 29, 868-875. 8 Mauer, S.M., Barbosa, J., Vernier, R.L., Kjellstrand, C.M., Buselmeier,T.J., Simmons, R.L., Najarian, J.S. and Goetz, F.C. (1976) Development of diabetic vascular lesions in normal kidneys transplanted into patients with diabetes mellitus. N. Engl. J. Med. 295, 916-920. 9 Bader, H. and Meyer, D.S. (1977) The size of the juxtaglomerular apparatus in diabetic glomerulosclerosis and its correlation with arteriolosclerosis and arterial hypertension: a morphometric light microscopic study on human renal biopsies. Clin. Nephrol. 8, 308-311. 10 Nakamura, R., Saruta, T., Yamagami, K., Saito, I., Kondo. K. and Matsuki, S. (1978) Renin and juxtaglomerular apparatus in diabetic nephropathy. J. Am. Geriatr. Sot. 26, 17-21. 11 lna, K., Kitamura, H., Ono, J.,Takaki, R. and Nakamura, M. (1990) Afferent arteriolar hyalinosis in experimentally induced diabetes. J. Japan Diab. Sot. 33, 979-981. 12 Nakamura, M., Shimada, T. and Fujimori, 0. (1980) Ultrastructural studies on the pancreatic polypeptide cell of the rat with special reference to pancreatic regional differences and changes induced by alloxan diabetes. Acta Anat. 108. 193-201. 13 Stempak, J.H. and Ward. R.T. (1964) An improved
35
14
15
16
17
staining method for electron microscopy. J. Ceil. Biol. 22, 697-701. Reynolds, E.S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol. 17. 208-212. Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol. 27, 137A. Takita. K.. Shimada. T., Kitamura, H., Fujii, S. and Nakamura, M. (1990) Studies on the physical developer for use in immunohistochemistry. Acta Histochem. Cytothem. 23, 647-662. Leiper. J.M.,Thomson, D. and MacDonald, M.K. (1977) Uptake and transport of Imposil by the glomerular mesangium in the mouse. Lab. Invest. 37. 526-533.
18 Alpert, J.S., Coffman, J.D., Balodimos, M.C., Koncz, L. and Soeldner, J.S. (1972) Capillary permeability and blood flow in skeletal muscle of patients with diabetes mellitus and genetic prediabetes. N. Engl. J. Med. 286, 454-460. 19 Parving, H-H. and Rasmussen, S.M. (1973) Transcapillary escape rate of albumin and plasma volume in short- and long-term juvenile diabetes. Stand. J. Clin. Lab. Invest. 32, 81-87. 20 Colantuoni, A., Berardi, P.G. and Orefice, G. (1979) Increased permeability of the microcirculation to high molecular weight dextran in experimental diabetes, shown by intravital microfluorimetry. J. Nucl. Med. Allied. Sci. 23, 49-54.
1992 Elsevier Science Publishers
27-35
27
B.V. All rights reserved 0168-8227/92/$05.00
DIABET 00613
Ultrastructural and immunohistochemical similarities between glomerular arteriolar and mesangial lesions in experimental diabetes K. Ina’,
H. Kitamura’,
M. Nakamura’,
J. 0n02
and R. Takaki2
‘Departmerit of Anatomy and ‘First Department of Medicine, Medical College of Oita. Oita, Japan
(Received 12 August 1991) (Revision accepted 10 November 1991)
Summary
It has thus far been speculated that plasma proteins have leaked and accumulated in diabetic expanded mesangium. We have previously shown that glomerular arteriolar hyalinosis has frequently occurred and has consisted of basement membrane-like extracellular matrix in alloxan diabetic rats. In the present study, the two diabetic glomerular lesions, arteriolar hyaline nodules and increased mesangial matrix, were comparatively evaluated by means of electron microscopy and immunohistochemistry. It was revealed that both of these lesions contained, in common, finely particulate extracellular matrix mingled with cytoplasmic fragments, and exhibited intense immunoreactions for IgG and IgM. It is indicated that both types of diabetic glomerular lesions are essentially of the same nature.
Key words: Glomerular
arteriolar hyalinosis; IgM ; Alloxan
Mesangial expansion;
Introduction
Glomerular basement membrane thickening [l] and mesangial expansion [2], both of which appear at early stage of diabetes, have thus far been extensively studied by means of light and electron microscopy and immunohistochemistry [3-71. In particular, it has been reported by Mauer and associates that such plasma proteins as IgG, IgM and C3 have accumulated in diabetic
Correspondence to: K. Ina, Department College of Oita, Oita, Japan.
of Anatomy, Medical
Immunohistochemistry
for IgG and
mesangium [4]. As compared with basement membranous and mesangial lesions, a fewer number of studies have been made on glomerular arteriolar hyalinosis [&lo], which has been regarded as an exudative diabetic lesion. In our foregoing study using conventional electron microscopy, we have revealed that prominent hyalinosis of glomerular arterioles has frequently occurred in the rats alloxan-diabetic for 10 months and that the lesion has consisted of basement membrane-like extracellular matrix [ 111. In terms of such results, we have held a view that this arteriolar lesion is essentially similar to mesangial expansion.
28 In the mesangial by means and light
present study, these arteriolar and lesions were comparatively evaluated of conventional electron microscopy and electron immunohistochemistry.
Materials and Methods Animals A total of ten male Wistar rats were employed. They were reared on free access to rat chow (CE-2, Japan CREA, Japan) and tap water. Induction of’ experimental diabetes Rats weighing 180 to 200 g were given 40 mg/kg alloxan (Wako Pure Chemical, Japan) in I”(, aqueous solution via tail vein. Hyperglycemia was confirmed by determining the non-fasting plasma glucose levels by means of glucose-oxidase method at 1 month intervals, and only those individuals which persistently exhibited plasma glucose values higher than 400 mg/dl were used as diabetic animals. None of the diabetic animals were maintained on insulin therapy. Five diabetic and five age- and sex-matched intact control rats were offered for study at 10 months after induction of diabetes. At the corresponding time, albuminuria has been confirmed to be significantly increased in diabetic as compared with control rats (DM, 301 k 159,ug/day versus C, 33 2 25 pg/ day; P < 0.05) [ 111. Preparation of tissue specimens Animals were deeply anesthetized by Nembutal injection. Left kidneys were excised after ligation of supplying vessels, to be subjected to conventional electron-microscopic examination (see below). Animals were then perfusion-fixed, and right kidneys were immunohistochemically examined (see below). Conventional electron microscopy Renal cortical tissues were obtained and immersed in our modified Dalton’s fixative [ 121 for 2 h at 4 “C under constant stirring. Tissues were then dehydrated in a graded ethanol series,
immersed in QY-1 and embedded in epon-epoxy resin. Ultrathin sections were made and mounted on neoprene-coated copper grids. They were stained with methanolic uranyl acetate [ 131 and aqueous lead citrate [ 141. The sections were then observed and photographed under a transmission electron microscope at 80 kV, at the magnihcations of 3300 to 20000. Photographic negatives were enlarge-printed as desired. Immunohistochemical procedures After animals were perfused with two-fold diluted Karnovsky’s fixative [ 151 for about 10 min (see above), right kidneys were removed, and their cortical tissues were sliced and immersion-fixed in non-diluted solution of the same fixative for another two hours. For examination at the lightmicroscopic level, fixed tissues were dehydrated in a graded ethanol series, cleared in xylene and vacuum-embedded in parafhn. Four micra-thick sections were made, mounted on glass slides and were processed as briefed below. Sections were then deparaffinized, hydrated and were incubated for 12 h at 4 ‘C with either rabbit antirat IgG antibody (Seikagaku Kogyo, Japan) or rabbit antirat IgM antibody (Zymed Laboratories Inc., U.S.A.). After being rinsed in phosphate buffer (pH 7.4) the sections were further incubated for 1 h at room temperature with gold-labelled goat antirabbit IgG antibody (Amersham, U.K.). These sections were then physically developed for 40 min at 20 “C in our physical developer [ 161 which contains, 0.8”,; citric acid, 0.3”,, bromohydroquinone, 15 ‘,, gum Arabic and 0.2”,, silver nitrate. Finally, sections were counterstained with Kernechtrot. For examination at the electron microscopic level, the tissues fixed in Karnovsky’s fixative were dehydrated in a graded ethanol series and embedded in Lowicryl K4M resin (Chemische Werke Lowi, Waldkraiburg, Germany) at - 20 ‘C with ultraviolet lighting (Toshiba SHL1OOUVQ mercury lamp equipped with Toshiba UV-D35 filter, Toshiba, Japan) for 10 h. Ultrathin sections were mounted on uncoated nickel grids and immunostained according to the sche-
29 dule briefed below. Sections were firstly reacted with either rabbit antirat IgG antibody or rabbit
antirat IgM antibody for 36 h at 4 “C. They were then rinsed in phosphate buffer (pH 7.4) and
Fig 1. Electron micrographs of glomerular arterioles from control (a and b) and diabetic (c and d) rats. (a) Medial extracellular matrix (MEM) shows age-related slight increase ( x 5750). (b) A high magnification electron micrograph of the area encircled by a rectangle in panel a. MEM exhibits finely particulate substructure, in contrast to subepithelial basement membrane (BM) which is rather homogeneous and less electron dense ( x 34400). (c) MEM of a diabetic glomerular arteriole exhibits marked increase ( x 5750). (d) A high magnification electron micrograph of the area encircled by a rectangle in panel c. Ultrastructures of MEM and BM are essentially the same as in panel b. A cytoplasmic fragment (CF) is seen ( x 34400). AL, arteriolar lumen; JG, juxtaglomerular cell; SM, smooth muscle cell; P, podocyte; BC, Bowman’s capsule.
30 further incubated for 1.5 h at room temperature with gold-labelled goat antirabbit IgG antibody. As in light-microscopic survey, sections were finally physically developed at 20 “C, for shorter
periods of time (8 min). In addition, somewhat different formula of physical developer was adopted. Thus the developer contained 0.5”~ citric acid, 0.17 9,0bromohydroquinone, 17 9; gum
Fig. 2. Electron micrographs of mesangium from control (a and b) and diabetic (c and d) rats. (a) Mesangial matrix (MM) exhibits age-related slight increase ( x 5750). (b) A high-magnification electron micrograph of the area encircled by a rectangle in panel a ( x 34400). (c) MM of a diabetic rat exhibits marked increase ( x 5750). (d) A high-magnification electron micrograph of the area encircled by a rectangle in panel c ( x 34400). CL, capillary lumen; P, podocyte; BM, basement membrane.
31
Arabic and 0.17 P; silver nitrate. Immunostained sections were examined and photographed electron microscopically at 60 kV. In order to check the specificity of immunostainings, some tissue sections were incubated in preabsorbed antibodies, but were otherwise processed as mentioned above.
Results Conventional electron microscopy
In control and diabetic rats arteriolar medial extracellular
examined here, matrix located
around smooth muscle cells exhibited finely clustered substructures (Fig. la and c), being in marked contrast to subepithelial and subendothelial basement membrane, where rather homogeneous, less electron dense substructure prevailed (Fig. lb and d). In diabetic rats, arteriolar medial extracellular matrix often underwent massive enlargement and contained cytoplasmic fragments (Fig. lc). Apparently the lesion corresponds to light-microscopic hyaline nodule. Interestingly, the mesa&al matrix of control and diabetic rats was of essentially the same substructure and similarly increased in amount in diabetes (Fig. 2).
Fig. 3. Light micrographs ofglomeruli from control and diabetic rats immunostained for IgG and IgM, followed by Kernechtrot counterstaining. Appreciable stainings for IgG (a) and IgM (c) are not seen over the glomeruli from control rats. Distinct stainings for IgG (b) and IgM (d) are observed in association with the arteriole and mesangium from diabetic rats ( x 330).
32
Immunohistochemical light microscopy Appreciable stainings for IgG and IgM were not observed over the glomeruli from control rats (Fig. 3a and c). In marked contrast, most glomeruli from diabetic rats exhibited distinct stainings for these plasma proteins in association with arterioles and the mesangium (Fig. 3b and d). Positive immunostainings for IgG and IgM were localized in the mesangium in 45 out of 50 and 48 out of 50 diabetic glomeruli, respectively. On the other hand, positive immunostainings for the same proteins were noted in association with
the arteriole in 21 out of 25 and 24 out of 25 diabetic glomeruli where one or two arterioles were seen, respectively. Immunoelectron microscopy In coincidence with the light microscopic data, only sporadic labellings for IgG and IgM were observed over the entire elements of the glomeruli from control rats (data not shown). The glomeruli of diabetic rats exhibited intense immunolabellings for IgG in arteriolar hyaline nodules (Fig. 4a) and mesangial matrix and cells (Fig. 4b). Sub-
Fig. 4. Electron micrographs of the glomerular arteriole (a) and mesangium (b) of a diabetic rat immunostained for IgG. (a) Labellings for IgG are intense in association with arteriolar medial extracellular matrix (MEM) exhibiting increase and podocyte (P), and are feeble over basement membrane (BM) and endothelial cell ( x 12300). (b) Labellings for IgG are intense in association with mesangial matrix (MM) and podocyte, and are feeble over BM and endothelial cell ( x 12 300). AL, arteriolar lumen; CL. capillary lumen.
33 stantial intensity of labellings was also recognized in association with the cytosol and lysosomes of the glomerular podocyte of diabetic rats (Fig. 4). In addition, feeble labellings were found to be localized in the glomerular basement membrane (Fig. 4). On the other hand, labellings for IgM were intense in arteriolar hyaline nodules (Fig. 5a) as well as the mesangial matrix (Fig. 5b), but were trace in extent in the podocyte and almost completely missing from the glomerular basement membrane (Fig. 5).
Control immunostainings In those light and electron microscopic sections which were subjected to control immunostainings, reactions for IgG and IgM were virtually absent (data not shown).
Discussion In terms of the present conventional electron microscopic findings, the two diabetic glomerular
;
.
.
Fig. 5. Electron micrographs of the glomerular arteriole (a) and mesangium (b) of a diabetic rat immunostained for IgM. (a) Labellings for IgM are intense in association with arteriolar medial extracellular matrix (MEM) exhibiting increase, but are trace in extent over other structures observed ( x 12 300). (b) Labellings for IgM are intense in association with mesangial matrix (MM), but are trace in extent over other structures observed (x 12300). AL, arteriolar lumen; CL, capillary lumen; P, podocyte.
34
lesions, arteriolar hyaline nodules and increased mesangial matrix, closely resembled each other, both being composed of extracellular matrix and cytoplasmic fragments. In addition, large amounts of IgG and IgM were detected in both type lesions. Taken together, we are prone to speculate that these lesions are essentially of the same nature. In this context, Mauer and associates have indicated abundance of plasma proteins including IgG, IgM and C3 in diabetic mesangium, and ascribed this to functional incompetence of such mesangium [4]. These authors have used the immunofluorescence technique, and their observations have been necessarily made only at the light microscopic level. In the present study, we successfully identified electron microscopic localization of IgG and IgM within the rat glomerulus with high discrimination by virtue of a modern immunostaining technique. This led us to a new concept on the nature of diabetic glomerular lesions. Two mechanisms can be presumed to explain accumulation of plasma proteins in the arteriolar wall. Thus, plasma proteins already present in the mesangium could be transported to the arteriolar wall through the ‘route of drainage of material from mesangium’ previously proposed by Leiper et al. on the basis of tracer experiment [ 171. Alternatively, it is also possible that these proteins exude through the arteriolar endothelium and accumulate in the arteriolar media because of raised vascular permeability [ 18-201. As a circumferential evidence for the latter possibility, the following present finding is to be added. As apparent from Fig. 1 the segment of the glomerular arteriole susceptible to diabetic change is lined with endothelial cells which contain appreciable numbers of pinocytotic vesicles and exhibit localized attenuation and fenestration. The podocytes and basement membrane of diabetic rats were immunolabelled for IgG but not for IgM. Unequivocal explanation for such differential labelling is not available at present. Permselectivity of tested proteins related to the size, shape and charge of molecules appears to be involved.
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