JOURNAL OF PATHOLOGY, VOL.

162: 14 1-1 48 (1990)

DISTRIBUTION OF TYPE I COLLAGEN IN HUMAN KIDNEY DISEASES IN COMPARISON WITH TYPE I11 COLLAGEN KAZUO YOSHIOKA, MACH1 TOHDA, TSUKASA TAKEMURA, NORIHISA AKANO, KAZUNORI MATSUBARA, AKIRA OOSHIMA* AND SUNAO MAKI

Department of Pediatrics, Kinki University School ofhledicine, Osaka-sayama 589, Japan; *Department of Pathology, Wakayama Medical College, Wakayama 640, Japan Received 13 February 1990 Accepted 18 May 1990

SUMMARY The distribution of type I collagen in normal and diseased renal tissues was studied using immunofluorescence and immunoelectron microscopy, and was compared with that of type I11 collagen. In normal human kidneys, a monoclonal antibody against type I or type 111collagen reacted with the renal interstitium, but not with the intra-glomerular structures. In various types of glomerulonephritis, immunofluorescent staining for type I collagen was positive in the fibrocellular and fibrous crescents, sclerosed glomeruli, and infrequently within the glomerular mesangium. In the crescents and sclerosed glomeruli, type I collagen was co-localized with type 111collagen. The staining intensity of type I collagen in those areas was generally stronger than that in the interstitium. Mesangial staining for type I collagen was present within the glomeruli, particularly with a marked mesangial matrix increase, but was less in amount and frequency compared with type 111 collagen staining. These findings indicate that the fibrosclerotic process in damaged glomeruli is accompanied by the appearance of interstitial collagens, and that participation of type I collagen is prominent in crescent organization and global glomerular sclerosis, but is less frequent in mesangial expansion, compared with type 111collagen. KEY

WORDS-Type

I collagen, type 111collagen, glomerulonephritis.

INTRODUCTION There are a number of diseases in which quantitative and qualitative changes in the collagen content of tissues are of importance in the destruction of organ architecture and function. Collagen types I and I11 are the major collagenous components which are normally present in the renal interstitium and blood vessels.'-3The ratio of these two collagens in tissues is not fixed, but undergoes changes during develo mental processes and various disease statesJ5 The immunohistochemical properties of collagen and procollagens in the kidney have been studied intensively during the past decade. Type IV collagen is well known to be a major constituent of Addressee for correspondence:Dr K. Yoshioka, Department of Pediatrics, Kinki University School of Medicine, 377-2, Ohno-higashi, Osaka-sayama 589, Japan. 0022-3417/90/ 1001414l8 $05.00 0 1990 by John Wiley & Sons, Ltd

renal basement membranes and an important factor contributing to basement membrane thickening and mesangial Relatively little is known about the role of type I and type I11 collagens in glomerular injury. The development of monoclonal antibodies to these collagens has led us to study the distribution of frequently co-existing, but typedifferent interstitial collagens in tissues."-'3 We previously reported the glomerular localization of type I11 collagen in various types of human kidney disease.I2The data indicated that type TI1 collagen participated in mesangial expansion, crescent organization, and glomerular sclerosis. To extend our previous studies and to further clarify the role of interstitial collagen in human glomerulonephritis, we studied the distribution of type I collagen in normal and diseased kidney tissues using a monoclonal antibody directed against type I collagen.

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MATERIALS AND METHODS

Kidney tissues The tissue sample consisted ofroutine renal biopsy specimens taken from 68 patients (between 3 and 18 years old) with renal disease. The patients were diagnosed according to the histological criteria of WHO:', 26 patients had primary IgA nephritis (Berger's disease), 20 had mesangial proliferative (non-IgA) nephritis, 14 had minimal change nephrotic syndrome, 2 had focal segmental glomerular sclerosis, 2 had reflux nephropathy, 2 had membranoproliferative glomerulonephritis, 1 had haemolytic uraemic syndrome, and 1 had rapidly progressive glomerulonephritis. The mesangial matrix increase in IgA nephritis and mesangial proliferative glomerulonephritis was graded semiquantitatively (mild, moderate, and marked) as described previously. l 2 An tibodies Monoclonal antibodies (mouse IgG) to types I and 111 collagen were produced from hybrids of mouse myeloma cells and spleen cells of mice immunized with the respective antigens isolated from human placenta.' '-I3 The reactivity of the monoclonal antibody to type I collagen was determined by inhibition enzyme-linked immunoassay (ELISA). The specificity of the monoclonal antibody to type I11 collagen was determined by ELISA and immuno-blotting in our previous paper. l 2 Indirect immunojluorescence Indirect immunofluorescent staining was performed as described p r e v i o ~ s l y . ~Cr ~ ,yostat ' ~ ~ ' ~sections ( 3 pm) of kidney specimens were air-dried and then overlaid with a monoclonal antibody for 45 min at room temperature. After washing in phosphate-buffered saline (PBS), the sections were incubated with a secondary antibody, fluoresceinisothiocyanate (F1TC)-labelled goat anti-mouse IgG (Cappel, Malvern, PA, U.S.A.) preabsorbed with normal human plasma for 45 min at room temperature. Inhibition of the binding of antibodies to the tissue sections was carried out by preincubation of the monoclonal antibody with the respective antigen, type I or type 111collagen isolated from human placenta (Cosmo Bio Co., Tokyo, Japan), overnight at 4"C, followed by indirect immunofluorescent staining. As a control experiment, tissue sections were incubated with non-immune mouse sera or

unrelated IgG mouse monoclonal antibody, followed by FITC-goat anti-mouse IgG, or secondary antibody alone. These controls were negative.

Conventional transmission electron microscopy As reported previously, I 2 , l 6 the kidney specimens were prefixed with 2 per cent glutaraldehyde in PBS for 1 h at 4°C. After washing, the specimens were post-fixed with 4 per cent OsO, for 2 h at room temperature. After dehydration in an ethanol series, they were embedded in Epon 812. Ultra-thin sections were cut, stained with uranyl acetate, phosphotungsticacid,andleadcitrate,and thenobservedunderan electron microscope (Hitachi 12A, Tokyo, Japan). Immunoelectron microscopy Immuno old staining was performed as described previously. Small pieces of kidney tissue fixed in paraformaldehyde-lysine periodate (PLP) were embedded in LR-white acrylic resin (The London Resin Co., Sunbury, U.K.) or Lowicryl K4M (Chemisohe Werke Lowi GmbH, Waldkraiburg, F.R.G.). The ultra-thin sections mounted on 200mesh nickel grids were first floated on a drop of PBS for 10 min and then on a drop of 0.1 per cent bovine serum albumin PBS for 10 min. Next the sections were placed on a drop of a monoclonal antibody for 1 h at room temperature. The washed grids were then transferred onto a drop of goat anti-mouse IgG conjugated with gold particles (size: 10 or 20 nm) (Janssen, Olen, Belgium) for 1 h. After incubation, the sections were thoroughly washed with distilled water and then post-fixed with 2 per cent OsO, for 15 min. Finally, the sections were stained with uranyl acetate and examined under an electron microscope.

B

RESULTS

Immunojluorescence In the normal human kidney sections, monoclonal antibodies to types I and I11 collagen bound exclusively to the renal interstitium and blood vessels, but did not react with the structures within the glomeruli (Fig. 1). In various types of glomerulonephritis, type I collagen was present within the glomeruli. Type I collagen, together with type I11 collagen, was found in fibrocellular and fibrous crescents (Figs 2a-2d).

TYPE I COLLAGEN IN GLOMERULONEPHRITIS

143

Immunoelectron microscopy Ultrastructural localization of type I collagen was studied by immunogold staining in kidney tissues from three patients (one with rapidly progressive glomerulonephritis, one with IgA nephritis, and one with membranoproliferative glomerulonephritis type I) and one normal human kidney tissue. Gold particles bound to the collagen fibrils were observed in the interstitium, crescents, and sclerosed glomeruli of the patients. Colloidal gold staining for types I and 111collagen was positive in the mesangial matrix of the patient with membranoproliferative glomerulonephritis, but fibrillar structures of the collagen were not seen in that area (Fig. 5).

Fig. I-Immunofluorescent staining for type I collagen in a normal human kidney. Positive staining of the renal interstitium is seen. Intra-glomerular structures are negative. G = Glomerulus

Within the nearly or totally sclerosed glomeruli, both types of collagen were coarsely distributed (Figs 3a and 3b). Staining for type I collagen in the crescents and sclerosed glomeruli was frequently continuous with the renal interstitium, but more intense than that in the interstitium, while type 111 collagen in these areas had a staining intensity similar to that in the interstitium. In the mesangial area, 12 of 68 patients were positive for type I collagen, and 23 of the 68 patients were positive for type I11 collagen (Table I). Both collagens showed a focal and segmental distribution in most of the cases (Figs 4a-4d). The amount of mesangial type I collagen was small, and it was detected only in glomeruli showing a marked increase in mesangial matrix, while type 111collagen was also present in glomeruli with moderate matrix increases. There were no cases in which type I collagen was present without type 111 collagen. In cases showing positive mesangial types I and 111collagen, serial sections were cut and examined by immunofluorescence. A connection between mesangial staining and interstitial staining was rarely found. Type 111 collagen, without type I collagen, was stained in the vascular pole of glomeruli in six patients.

Conventional electron microscopy Kidney tissues from four patients, one with membranoproliferative glomerulonephritis type I, one with IgA nephritis, one with mesangial proliferative (non-IgA) nephritis, and one with rapidly progressive glomerulonephritis, were examined utilizing conventional electron microscopy with phosphotungstic acid staining. In the patient with membranoproliferative glomerulonephritis type I, cross-banded fibres, characteristic of collagen, were present in the periglomerular regions and in a sclerosed glomerulus. In the mesangial matrix, fibrillar structures of the collagen were rarely seen in any of the cases.

DISCUSSION We studied the distribution of type I collagen in renal biopsy specimens taken from patients with various kidney diseases and compared it with that of another type of interstitial collagen, type 111. By indirect immunofluorescence with monoclonal antibodies, type I and type 111 collagen antigens were observed within the glomeruli. Of the 68 biopsy specimens, type I collagen was detected in 13 cases and type 111 collagen in 23. Both collagens were localized in the crescents, sclerosed glomeruli, and mesangial area. Mesangial localization of type I collagen was also found by Foidart et al.’ in the kidneys of some pregnant patients with chronic or recurrent hypertension. The precise mechanisms for the appearance of interstitial collagens within the glomerulus in kidney diseases are currently unknown. Studies by Striker et al.3have shown type 111 collagen in the synechiae of nephritic kidneys, suggesting extension of this type of collagen from the

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K. YOSHIOKA E T A L .

Fig. 2-Immunofluorescent staining for type I collagen (a, c) and type I11 collagen (b, d) in the glomerular crescents (arrows) of a patient with IgA nephritis (a, b), and of a patient with rapidly progressive glomerulonephritis (c, d). Serial sections, a and b, and c and d, were reacted with monoclonal antibody against types I and Ill collagen. Note the intense staining for type I and type 111 collagen, particularly the former, in the crescents

Fig. 3--Immunofluorescent staining for type 1 (a) and type 111 (b) collagen in a glomerulus showing global sclerosis in a patient with reflux nephropathy. G = Glomerulus

interstitium through disrupted Bowman's capsules. Their view is quite likely to be correct, particularly since intra-glomerular staining for collagen, as seen in crescents and globally sclerosed glomeruli, was continuous to the interstitium. Another possibility

is intra-glomerular synthesis of interstitial type collagens. This is suggested by our findings and the observations of Oomura et d.," showing that types I and I11 collagen were present in the mesangium of the glomeruli, but the collagen staining associated

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Localization

*The number of patients showing positive immunofluorescence for type I, I( +), and type 111, III( +), collagens are shown. ?The increase in mesangial matrix in IgA nephritis and mesangial proliferative GN was graded as mild (+), moderate (+ +), and marked (+ + +). $The number of patients demonstratingpositive immunofluorescence/thenumber of patients showing glomerular crescents or sclerosed glomeruli. GN = glomerulonephritis;NS = nephrotic syndrome; HUS = haemolytic uraemic syndrome.

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60 nm) are labelled only with antibody to type I c ~ l l a g e nFactors . ~ ~ ~ ~other than collagen type are also known to affect the size of collagen fibrils. For example, differences in the types ofglycosamino-

TYPE I COLLAGEN IN GLOMERULONEPHRITIS

147

Fig. 5-Indirect immunogold stainings for type I (a) and type 111(b)collagens in themesangial area of a glomerulus in a patient withmembrano-proliferativeglomerulonephritis typeI. Severalgoldparticles(size:(a) 10 nm; (b)20 nm) bound to the mesangial matrix are seen (arrow-heads). Ep =epithelial cell; MM = mesangial matrix

glycans in the extracellular matrix, differences in the extent of hydroxylation and glycosylation, or the presence of an aminopropeptide could affect the Abnormalities in these factors may explain why fibrillar structures were rarely seen in the mesangial area of nephritic kidneys by conventional and immunoelectron microscopy, although collagen antigens were detected by immunofluorescence in this area. In summary, this study has provided further evidence for the contribution of interstitial collagens to glomerular sclerosis and crescent organization. It seems that participation of type I collagen leads to glomerularinjury due to irreversible fibrosclerotic lesions.

ACKNOWLEDGEMENTS

This work was supported by a Grant for ‘Progressive Kidney Disease’ from the Ministry of Health and Welfare in Japan, and a Grant for ‘Research on a System for Long-term Management of Pediatric Renal Diseases’ from the Ministry of Health and

Welfare. We are grateful to Ms K. Uetani for tissue staining and Ms Y. Shima for preparation of the manuscript. REFERENCES 1 Scheinman JI, Foidart J-M, Michael AF. The immunopathology of

glomerular antigens. V. The collagenous antigens of the glomerulus. Lab Invest 1980; 4 3 373-381. 2 Killen P, Melcion C, Bonadio JF, Morel-Maroger L, Striker GE. Glomerular response to immunologic injury. Studies of progression. Springer Semin Immunopathol1982; 5: 297-320. 3. Striker LM-M, Killen PD, Chi E, Striker GE. The composition of glomerulosclerosis. 1. Studies in focal sclerosis, crescentic glomerulonephritis, and membranoproliferativeglomerulonephritis. Lab Invest 1984;51: 181-192. 4 . Smith LT, Hohrook KA, Madri JA. Collagen types I, 111, and V in human embryonic and fetal skin. Am J Anal 1986; 175 507-521. 5. Amenta PS, Gay S , Vaheri A, Martinez-Hernandez A. The extracellular matrix is an integrated unit: ultrastructural localization of collagen types I, 111, IV, V, VI, fibronectin, and laminin in human term placenta. Cdlagen Res Re1 1986; 6 125-152. 6 . Falk RJ, Scheinman JL, Mauer SM, Michael AF. Polyantigenicexpansion of basement membrane constituents in diabetic nephropathy. Diabetes 1983; 32 (Suppl2): 34-39. 7 . Foidart JM, Nochy D, Nusgens B, el a/. Accumulation of several basement membrane proteins in glomeruli ofpatients with preeclampsia and other hypertensive syndrome of pregnancy. Possible role of renal prostaglandins and fibronectin. Lab Invest 1983; 4 9 250-259. 8. Michael AF, Falk RJ, Platt JL, Melvin T, Yang J-Y. Antigens of the human glomerulus. Adv Nephrol1984; 1 3 203-218.

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9. Yoshioka K, Takemura T, Matsubara K, Miyamoto H, Akano N, Maki S. Immunohistochemical studies of reflux nephropathy: the role ofextracellular matrix, membrane attack complex and immune cells in glomerular sclerosis. Am J Pathol1987;129 223-231. 10. Oomura A, Nakamura T, Arakawa M, Ooshima A, lsemura M. Alterations in the extracellularmatrix components in human glomerular disease. Virchows Arch A [Pathol Anat] 1989;4 1 5 151-1 59. I I . Sakakibara K, Ooshima A, Igarasbi S, Sakakibara J. Immunolocalization of type I11 collagen and procollagen in cirrhotic human liver using monoclonal antibodies. Virchows Arch A ]Path01 Anat] 1986; 409:37-46. 12. Yoshioka K,Takemura T, Tohda M, el al. Glomerular localization of type 111 collagen in human kidney disease. Kidney In[ 1989;3 5 1203-1 21 1 . 13. Ooshima A, Wakasaki H, Muragaki Y. Production of monoclonal antibody to human type I collagen; immunohistochemical localization in human tissue. Virchows Arch A [Parhol Anat] (in press). 14. Churg J, Sobin LH. Renal Disease. Classification and Atlas of Glomerular Diseases. Tokyo: Igaku-Shoin, 1982. 15. Yoshioka K, Michael AF, Verosa J, Fish AJ. Detection of hidden nephritogenic antigen determinants in human renal and nonrenal basement membrane. Am JParhol1985; 121: 156-165. 16. Takemura T, Yoshioka K, Akano N, Miyamoto H, Matsumoto K, Maki S. Glomerular deposition ofcross-linked fibrin in human kidney diseases. Kidney Int 1987;3 2 102-1 11. 17. Foidart JM, Foidart JB, Mahieu PR. Synthesis of collagen and fibronectin by glomerular cells in culture. Renal Physiol 1980;3: 183-192. 18. Scheinman JI, Brown DM, Michael AF. Collagen synthesis by human glomerular cells in culture. Eiochim Eiophys Acru 1987;542: 128-136.

19. Harakon MA, Jacobson HR, Hoover RL. Collagen polymorphism in cultured rat kidney mesangial cells. Lab Invest 1987;57: 5 13-523. 20. Holund B, Clemmensen 1, Junker P, Lyon H. Fibronectin in experimental granulation tissue. Acta Pathol Mirrobiol Immunol ScandSect A 1982;90:159-165. 21. Williams IF, McCullagh KG. Silver IA. The distribution oftype I and I11 collagen and fibronectin in the healing equine tendon. Connect Tissue Res 1984;1 2 21 1-227. 22. Birk DE, Silver FH. Collagen fibrillogenesis in vitro. Comparison of type I, 11 and 111. Arch Biochem Eiophys 1984;235 178-185. 23. Seyer JM, Hutchinson ET, Kang AH. Collagen polymorphism in normal and cirrhotic human liver. J C h Invest 1977;5 9 241-248. 24. Rojkind M, Giambrone MA, Biempica L. Collagen type in normal and cirrhotic liver. Gasrroenrerology 1979;76710-719. 25. Seyer JM, Hutchinson ET, Kang AH. Collagen polymorphism in idiopathic chronic pulmonary fibrosis. J CIin Invest 1976; 57: 1498-1507. 26. Shoemaker CT,Reiser KM, Goetzman BW, Last JA. Elevated ratios of type I / I I I collagen in the lungs of chronically ventilated neonates with respiratory distress. Pedialr Res 1984;18 1176-1 186. 27. Andujar MB, Hartmann DJ, Emonard H, Magloire H. Distribution and synthesis of type I and type 111 collagen in developing mouse molar tooth root. Histochemisrry 1988;8 8 131-140. 28. Olsen BR. Collagen biosynthesis. In: Hay ED, ed. Cell Biology of Extracellular Matrix. N ~ York: N Plenum Press, 1981;139-177. 29. Linsenmayer TF. Collagen. In: Hay ED, ed. Cell Biology of Extracellular Matrix. New York: Plenum Press, 1981; 5-37. 30. Fleischmajer R, Steffen G, Perlish JS, Cesarini JP. Immunoelectron microscopy of type 111 collagen in normal and scleroderma skin. J Invest Dermatol 1980;7 5 189-191.

Distribution of type I collagen in human kidney diseases in comparison with type III collagen.

The distribution of type I collagen in normal and diseased renal tissues was studied using immunofluorescence and immunoelectron microscopy, and was c...
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