Collagen in normal rat glomeruli

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Collagen in Normal Rat GlomerulP HARRISON LATTA~ W i t h the technical c o l l a b o r a t i o n of MARGERY L. COOK

Laboratoire de Microscopie Olectronique, Institut de Recherches scientifiques sur le Cancer, Villejuif (Seine), France Received March 27, 1961 Collagen fibers have been found by electron microscopy in the glomeruli of six normal rats. They occur in scattered small groups adjacent to intercapillary cells in centrolobular regions on the capillary side of the glomerular basement membrane, The fibers may lie at considerable distances from the glomerular hilus. The presence of collagen next to normal intercapillary cells is additional evidence that these cells should be regarded as a special cell type and not just as endothelial cells. This supports other evidence that the centrolobular part of the glomerulus is a special region having a unique role in renal function and disease. Collagen has n o t previously been f o u n d b y electron m i c r o s c o p y in the n o r m a l glomeruli of m a m m a l i a n kidneys, in spite of their intensive study in m a n y l a b o r a t o r i e s . I n the course of studying the c e n t r o l o b u l a r region with a variety of staining a n d histochemical procedures, a m e t h o d was f o u n d which stained collagen in m a r k e d c o n t r a s t to adjacent tissue structures. A f t e r collagen fibers were observed in one c e n t r o l o b u l a r area, an intensive search was instituted for collagen in the glomeruli of several n o r m a l rats. MATERIAL AND METHODS The kidneys of six normal white male rats weighing 100-150 g were fixed by dripping buffered osmium tetroxide on the encapsulated surface in the living animal (1, 13). Tissue blocks were dehydrated and embedded in Epon (14). After sections were cut and picked up on a grid, with or without a film, they were floated on 10% phosphotungstic acid (PTA) for 10 minutes and then washed for 5 minutes or more (23). Some sections were examined after this procedure and some were counterstained with 5% uranyl acetate (Ur Ac) for 1 Supported in part by research Grant RG-5762 from the National Institutes of Health, United States Public Health Service, and the Commonwealth Fund. 2 On sabbatical leave from the Department of Pathology, School of Medicine, University of California, Los Angeles 24, California. Fellowship support from the Commonwealth Fund is gratefully acknowledged.

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15-60 minutes. Lead acetate was tried with less satisfactory results. Renal tissue embedded in butyl methacrylate was also stained with PTA. The sections were examined in a Siemens or an RCA EMU 2 Electron Microscope. For light microscopy, hematoxylin and eosin stains were performed on kidney sections of five rats. The same kidney was used from which the blocks were taken for electron microscopy. Some periodic acid-Schiff stains were also prepared. RESULTS Collagen fibers were found by electron microscopy in the centrolobular regions of the renal glomeruli of six rats. The same kidney in each of five of these rats was examined by light microscopy and found to have normal glomeruli and tubules. There was no evidence of scarring or of thickening of the basement membranes. The collagen fibers are not found easily or in large numbers. They are best located by examining the tissue stained only with PTA. With sections of kidney embedded in Epon and stained by PTA, collagen is quite dark and stands out in marked contrast to adjacent tissue structures, which exhibit a relatively low density to electrons (Fig. 1). (The only other structure in the glomerulus staining with PTA under these conditions is the plasma membrane of the visceral epithelium of Bowman's capsule. This will be the subject of a separate report.) Collagen can be found at moderate magnifications in these sections ( x 5000-8000) but it is usually necessary to search through severn glomeruli before a group of fibers is located. After being found by the above procedure, collagen fibers could then be located in glomeruli embedded in methacrylate and stained with PTA. The fibers may occur in centrolobular areas in any part of the glomerulus. (The centrolobular region has also been called mesangial, intercapillary, axial, or interluminal.) The relationships of the fibers to cells and other tissue components are best demonstrated in the sections counterstained with uranyl acetate after PTA, although fibers are more difficult to find in such sections. In several glomeruli, collagen was found on the side opposite the hilus and at a considerable distance from the afferent or efferent arteriole (Figs. 2 and 3). The fibers form small groups of up to 12 or 15 fibers lying in the intercapillary space in any part of the centrolobular area. They may lie beneath basement membranes, but without any definite orientation or relationship to them (Figs. 1 and 4). They seem to be more closely related to intercapillary cells which lie beneath basement membranes and endothelial cells in the central portion of glomerular lobules (Fig. 5). They are frequently associated with the intercellular substance or matrix (12, 13) (Figs. 4 and 5). The fibers in the small groups usually run together, although some bend or curl in random directions. While fibers become thin as they pass out of the plane of the section, the ends do not seem to taper. They are about 300-500 A wide. They show an

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asymmetric or polarized periodicity in which the crossbands vary in width, density, and spacing (Fig. 1). A major period of about 500-700 A (average about 600 A) is found in several places. Five or six subperiods are frequently found with a spacing averaging about 100 A. This subperiod is often observed where the major period is not evident. In places light and dark segments alternate with an apparent period of about 200 A. These segments seem to form one-third of the major period. Occasionally, a very fine subperiod of about 25 A is seen. A recording densitometer was not available to enable a more detailed analysis. However, the periodicities of the glomerular fibers are quite similar to those of collagen fibers found in larger quantities between the renal tubules and elsewhere in the body. DISCUSSION

Previous studies of the centrolobular region The structure of the centrolobular region of mammalian glomeruli has been a subject of controversy for many years. Early investigators using light microscopy described fibrocytes in the "mesangium" (26), and even fibers extending into the lobules of glomeruli (3). Nevertheless, a number of investigators using electron microscopy have studied normal mammalian glomeruli in considerable detail without finding collagen fibers (1, 4-6, 10-13, 17, 22, 24). Collagen has been described in the frog glomerulus (25), but the investigator stated that his study was limited and that the kidneys might have had pathological changes. Other electron microscopic investigations of glomeruli in marsupials, birds, reptiles, amphibians, and fish have revealed no collagen (17). Human glomeruli in a variety of diseases have been carefully studied without the demonstration of collagen (4-6, 11). Sclerosis or hyalinization of glomeruli is apparently due largely to thickening of basement membranes or deposition of basement membrane-like material (5). In one case in which collagen was related to a severely damaged glomerulus (21), it was difficult to distinguish the limits of the glomerulus in the periglomerular fibrosis. In a continuing study of over 300 renal biopsies, one diseased glomerulus was found with a fiber having a period of about 210 ~ (onethird of the collagen period) (15), and more recently another case showed nodular glomerular lesions containing many fibers with a similar period (8).

FIG-. 1. Collagen fibers in the centrolobular region of a glomerulus stained with relative specificity by phosphotungstic acid after embedding in Epon. They stand out in contrast to adjacent tissue structures. The subperiods of about 100 ~_ are most easily seen here. They form an asymmetric or polarized pattern with bands varying in width, density, and spacing. The major period of about 600 ]~ is less frequently seen. In all these characteristics the fibers resemble collagen fibers between renal tubules. The plasma membrane of the glomerular epithelium stains also. Siemens. x 118,000.

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For these reasons it was surprising to find in rats with acute uranium poisoning a centrolobular lesion which contained many newly-formed collagen fibers (1, 2). No collagen was found in electron microscopic studies of the normal control animals used in these experiments. However, attention was focused on the special nature of the centrolobular region and further investigations were conducted (.11-13) which led to the present observations. Several factors probably have combined to hinder previous recognition of collagen in mammalian glomeruli. It is sparse, being encountered infrequently in the rat even in the best of conditions. The recently developed Epon embedding (14) gives better preservation of tissue detail than has been obtained in much of the previous work using methacrylate embedding. Although collagen can be found in glomeruli embedded in methacrylate, the high contrast of other tissue elements fixed with osmium tetroxide makes the discovery of a few fibers quite difficult. Epon obviates this difficulty. We have also the impression that the periodicity of newly-formed collagen is more apparent than that of mature collagen occurring in normal glomeruli. This impression comes from comparing our present studies with earlier studies of the collagen developing with acute uranium poisoning (1).

The specificity of phosphotungstic acid for collagen The relative specificity of PTA for collagen is more apparent after embedding in Epon than in other media. Although PTA has long been used to stain purified collagen fibers (19), and staining has been observed in thin sections (23), the electron density of other tissue components fixed with osmium and embedded in methacrylate has generally tended to obscure the staining of collagen in sections. Epon may enhance the contrast either by virtue of its resistance to sublimation in the electron beam or by chemical alteration of tissue components to decrease their staining with osmium or PTA. Some evidence for chemical alteration of proteins embedded in epoxy resins

FIG. 2. Collagen fibers (arrows) in the center of a glomerular lobule at a considerable distance from the hilus of the glomerulus. The hilus lies outside the picture at the top. This section, as well as the ones in the following figures, has been stained with PTA and uranyl acetate to demonstrate the position and relationships of the collagen. The nucleic acid masses at the periphery of the nuclei seem to be denser in the endothelial cells (End) than in the intercapillary cells (I). RCA. x 5000. FIG. 3. Higher magnification of the centrolobular region shown in Fig. 2. PTA and Ur Ac. RCA. x 28,000. FIG. 4. Collagen (arrow) associated with the intercellular substance or matrix between an intercapillary cell and the basement membrane. Note that the only cell processes associated with a long stretch of the basement membrane (BM) to the right of the intercapillary channel (IC) are epithelial. PTA and Ur Ac. Siemens. x 26,000. Fro. 5. Collagen fibers between intercapillary cell processes and a cell body at some distance from the basement membranes. PTA and Ur Ac. RCA. × 16,000.

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has been presented (20), and we have noted a change in the specificity of the periodic acid-Schiff reaction on tissue embedded in Aquon (7). The basis of the staining of collagen by PTA is now fairly well established (9, 19). The stained bands represent regions of high concentrations of basic groups, particularly arginine. The bands stained by cationic uranyl salts occur at the same levels but differ in intensity. These bands represent concentrations of acid residues such as glutamic and aspartic acid. It may be concluded only that under the present experimental conditions collagen presents a higher concentration of free basic groups than do most other tissue components.

The significance of collagen in the centrolobular region The main significance of these observations at present seems to lie in the light they shed on the nature of the centrolobular region. Collagen fibers are so few in the rat glomerulus that they probably play only a minor role in structural support. Perhaps they are only a remnant of evolution. Other studies indicate that they are rarely encountered in human renal disease. The association of collagen with normal intercapillary cells indicates that the latter may not be simple endothelial cells. Smooth muscle cells may be associated with collagen (18), and intercapillary cells may show an arrangement of cytoplasmic fibers associated with attachment bodies (15) similar to those found in smooth muscle. The intercapillary cells in glomerular lobules may be an extension of the pseudoMeissnerian cells of the "lacis" or cellulo-conjunctive lace-work of the juxtaglomerular apparatus (16). The close relationship of the cells to the intercapillary substance may indicate a role in its formation (12, 13). Although the cells could be fibroblasts, as suggested long ago (26), the development of collagen in association with cells does not necessarily require them to be fibroblasts. A macromolecular precursor collagen, such as tropocollagen (19), could reach the centrolobular region by the bloodstream and polymerize there into collagen fibers. The penetration of relatively large particles into this region (12, 13) demonstrates that protein molecules could also enter easily. The occurrence of collagen in the normal centrolobular region serves to call attention to other evidence that this region is anatomically distinct (12, 13) and functionally significant (1). The rapid penetration of large particles and the great surface area presented by the branching intercapillary cells (12, 13) suggest that this region is strategically located to react to substances in the blood plasma and possibly to control the flow of blood through the glomerulus. Studies, by different investigators, of a variety of types of human renal disease (11) show prominent involvement of the centrolobular region, and the rapid development of large deposits there in uranium poisoning (1, 2) is a demonstration of its potential reactivity.

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REFERENCES 1. BENCOSME, S. A., STONE, R. S., LATTA, H. and MADDEN, S. C., J. Ultrastrueture Research 3, 171 (1959). 2. - A. M. A. Arch. PathoI. 69, 470 (1960). 3. DENSELY,R. R. and DENSELY,R. D., Anat. Record 47, 147 (1930). 4. BERGSTRAND,A. and BUCHT, H., J. Pathol. Bacteriol. 77, 231 (1959). 5. FARQUHAR,M. G., VERNIER,R. L. and GOOD, R. A., J. ExptI. Med. 106, 649 (1957). 6. FARQUHAR,M. G., HOPPER, J. and MOON, H. O., Am. J. Pathol. 35, 727 (1959). 7. GIBBONS,I. R., Nature 184, 375 (1959). 8. HINGLAIS-GUILLAUD,N., personal communication. 9. KUHN, K., GRASSMANN,W. and HOFMANN, U., Z. Naturforseh. 13b, 154 (1958). 10. KURTZ, S. M. and MCMANuS, J. F. A., Am. Heart J. 58, 357 (1959). ll. LATTA,H., Calif. Med. 93, 272 (1960). 12. LATTA, H. and MAUNSBACH, A. B., Intern. Congr. Nephrology, 1st Congr. Excerpta Mediea, Intern. Congr. Ser. 29, 52 (1960). 13. LATTA, H., MAUNSBACH, A. B. and MADDEN, S. C., d. Ultrastrueture Research 4, 455 (1960). 14. Luvz, J. H., quoted by PEASE,D. C., in Histological Techniques for Electron Microscopy, p. 84. Academic Press, New York, 1960. 15. MICHIELSEN,P., personal communication. 16. OBERLING,C. and HART, P. Y., Ann. anat. pathol. 5, 441 (1960). 17. PAK POY, R. K. F., Am. J. Pathol. 5, 885 (1958). 18. PEASE, D. C. and MOLINARI,S., Y. Ultrastructure Research 3, 447 (1960). 19. SCHMITT,F. O. and HODGE, A. J., J. Soe. Leather Trades' Chemists 44, 217 (1960). 20. SPENDLOVE,R. S. and SINOER, S. J., Proc. Natl. Acad. Sei. U.S. 47, 14 (1961). 21. SPmo, D., Am. J. Pathol. 35, 47 (1959). 22. SUZUKT,Y., Keio J. Med. 8, 129 (1959). 23. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). 24. YAMADA, E., J. Biophys. Biochem. Cytol. 1, 551 (1955). 25. - - - - ibid. 7, 407 (1960). 26. ZIMMERMANN,K. W., Z. mikroskop, anat. Forsch. 32, 176 (1932).