Acta histochemo (lena) 97,343-351 (1995) Gustav Fischer Verlag lena Stuttgart New York 0
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Distribution of extracellular matrix glycoproteins in the human mesonephros Gaetano Magro!, Sebastiano Grasso!, Alfonso ColombattF, Loredana Villari! and Carmela Emmanuele l 1 Institute of Pathological Anatomy and Histology, University of Catania and Biomedical and Technological Sciences, University of Udine, CoR.a. Aviano, Italy
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Accepted 29 April 1995
Summary We analyzed the expression and distribution of collagen types IV and VI, laminin and fibronectin during the development and regression of the mesonephros in human embryos and fetuses ranging from 6 to 12 weeks of gestation by indirect immunoperoxidase methods. Type IV collagen, laminin and fibronectin were detected along the glomerular, tubular and capsular basement membranes of developing and mature nephrons. Only type IV collagen and fibronectin were found in the mesangium. Type VI collagen formed a delicate interstitial fibrillar network and a continuous basement membrane-like structure along the mesonephric nephrons. Basement membranes (GBM) of developing and mature glomeruli showed a distinct continuous staining for this collagen. The mesangial matrix was rich in type VI collagen. Mesonephric involution started during the 8th week of gestation and coincided with a moderate expansion of mesangial matrix and progressive collapse of the capillary walls, while the tubules became thinner and shorter. Staining for all extracellular matrix glycoproteins studied showed GBM wrinkling, gradual disintegration of some capillary loops and glomerulosclerosis. The sclerotic glomeruli were strongly positive for type IV collagen and less positive for type VI collagen and fibronectin. Laminin was absent. Our results indicate that collagen types IV, VI, laminin and fibronectin may be involved in the development and regression of the human mesonephros.
Key words: extracellular matrix - mesonephros - immunohystochemistry - man Introduction During its maturation the human kidney passes through three stages of development: pronephros, mesonephros and metanephros or permanent kidney (Allen, 1951). The mesonephros is the intermediate kidney which differentiates during the fourth week of intrauterine life, achieves full development around the second month and becomes nonfunctional at the end of the fourth month (De Martino and zamboni, 1966; De Martino Correspondence to: S. Grasso
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et al. 1977). Some mesonephric tubules persist as efferent ductules connecting the rete testis to epididymus in the male, while they remain vestigial in the female (Wilson, 1926). Although morphologic differences have been observed between the human mesonephric and metanephric nephron, there are impressive similarities between glomerular structure of these two organs (De Martino and Zamboni, 1966). Electron microscopy studies have shown that, in addition to podocytes with discrete foot processes and endothelial cells with rare fenestrations, the mesonephric glomerular capillaries have mesangial cells surrounded by extracellular matrix (ECM) (De Martino and zamboni, 1966; De Martino et al., 1977). In addition, the mesonephric proximal tubules morphologically are similar to the proximal convoluted tubule of the kidney, while the distal segment shows close similarity to the renal collecting tubule. Although previous immunohistochemical studies have shown that human fetal metanephric nephrons have several ECM glycoproteins including type IV collagen, laminin and fibronectin (Kumar et al., 1986; Mounier et al., 1986), to our knowledge there are no studies on the extracellular matrix (ECM) of the human mesonephros. The purpose of the present study was therefore to analyze immunohistochemically the expression and distribution of collagen types IV, VI, laminin and fibronectin in the human mesonephros. These ECM components were selected because of their possible detection in formaldehyde-fixed and paraffin-embedded tissues.
Materials and Methods Tissue samples. Seven embryos ranging from gestational week (GW) 6 to 7 and 11 male fetuses ranging from GW 8 to 12 were obtained from legal abortions. Stages of embryonic development and weeks of GA were determined according to established morphologic criteria (Gasser, 1975; O'Rahilly and Muller, 1987). Determination of fetal developmental age was based on size, including crown-rump and heel-toe measurements (Singer et al., 1991), menstrual history and ultrasound dating pregnancy. The male sex of all fetuses was histologically seen by the presence of testicular cord differentiation and developing tunica albuginea in gonads (Schlegel, 1969; Gasser, 1975). Tissues were fixed in 10070 buffered formaldehyde for 12 h and embedded in paraffin. Sections were cut and stained with Haematoxylin-Eosin. Ethical permission was obtained for this study. Antibodies. Monoclonal antibodies against type IV collagen (DAKO, dilution 1: 500), laminin (DAKO, dilution 1 : 300) as well as polyclonal antibodies against fibronectin (DAKO, dilution 1 : 300) and type VI collagen (1 :600) were used. Type VI collagen was isolated from pepsin-digested human placenta and purified as described by Treub et al. (1987). Antibodies against the pepsin-digests of human type VI collagen were raised in rabbits by subcutaneous injections of 200 IJ.g of protein in complete Freund's adjuvant. The animals were boosted several times before collecting serum samples. The antiserum was assayed by ELISA assay against several collagens, and no reactivity was found against type I, II, III and V. After treatment of the antiserum with an affinity column of human type IV collagen, the low contaminating reactivity against this collagen was removed as revealed by lack of any reactivity against purified human type IV collagen after an immunoblotting assay carried out according to Towbin et al. (1979) (Fig. 1). Immunohistochemistry. We used 4IJ.m sections deparaffined in xylene, treated with 0.3070 H20 2 in 40070 (VlV) methanol in phosphate-buffered saline (PBS) for 20 min at room temperature to block endogenous peroxidase activity. Enzymatic predigestion was performed with 0.01070 protease (type XVII, Sigma Chemical Co., St. Louis, MO, USA) in 0.01 M PBS (pH 7.4) for 15 min at 37°C to visualize all ECM glycoproteins studied since formaldehyde fixation abolishes their immunoreactivity. Incubation with primary antibodies was performed at 4°C overnight followed by incubation with biotinylated antimouse or anti-rabbit immunoglobulins and then with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature for each step. After each step, sections were washed with 0.01 M PBS (pH 7.4). Bound peroxidase was visualized using 0.05070 3.3' -diaminobenzidine tetrahydrochloride (Sigma) as chromogenic substrate for 10 min at room temperature. Controls included replacement of the primary antibodies by non-immune serum from the same host species or omission of the primary and secondary antibodies.
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Fig. 1. Immunoblotting analysis with anti-collagen IV and anti-collagen VI antibodies. 7S collagen IV tetramers (left panel) were, reparated on reduced 6% SDS·PAGE and pepsin-digested collagen VI tetramers (right panel) on reduced 10070 SDS-PAGE. The identities of the recognized polypeptides and the specifity of the collagen VI antibodies were verified by transfer onto nitrocellulose and immunoblotting with polycIonal antisera. Normal rabbit serum (lanes 1), antiserum to collagen IV (lanes 2), and antiserum to collagen VI (lanes 3).
Results Mesonephrogenesis. In each of the three embryos of GW 6 the cranial nephrons attained
full development, while the more caudal ones appeared as S-shaped tubules (Fig. 2a). The mesonephric nephrons, began as S-shaped tubules whose lateral end blended with, and opened into, the mesonephric or Wolffian duct, while the elongated distal free end was initially in contact with, and then invaginated by, blood capillaries, constituting the mesonephric corpuscle (Fig.2a). These changes involve nephrons in a cranio-caudal direction, and different stages of development are present simultaneously. The mature mesonephric nephron consisted of a glomerulus, a coiled thick walled secretory segment and a thin collecting tubule. Immunohistochemistry showed that type IV collagen, laminin and fibronectin were strongly stained in the basement membranes of the S-shaped tubule and those of the invading capillaries. In subsequent stages and in the mature nephrons, glomerular basement membrane (GBM) and basement membrane of the entire tubule from Bowman's capsule to Wolffian duct were lined by the antibodies against these ECM. Mesangial staining for type IV collagen and fibronectin increased progressively with glomerular maturation (Fig. 2a, 2b), while laminin was absent in the mesangium (data not shown). All these ECM components were not found in the surrounding mesenchyme. Staining for type VI collagen appeared as a fine fibrillar network in the interstitium, and as a continuous line around the mesonephric tubules resulting in a basement membrane-like demarcation. Moreover, GBM of the developing and mature glomeruli showed a distinct and continuous immunoreactivity for this collagen (Fig. 2c). Also the mesangium contained type VI collagen with a similar staining intensity pattern to that observed in GBM. All antibodies used lined all mesenchymal and peritubular capillaries as well as the glomerular afferent and efferent arterioles. At this stage both arterioles were devoid of a tunica media, and only few pericytes formed a discontinuous adventitial layer. Mesonephros regression. Early degenerative changes were observed in the
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Fig. 2. Mesonephros of human embryo of OW 6. (a) A longitudinal section shows the rostro-caudal gradient of the developing nephrons. (b) A transverse section of the upper pole of mesonephros shows three mature glomeruli while a longitudinal section (c) shows three developing glomeruli, the most caudal being on the left. Antibodies against fibronectin (a) and type IV collagen (b) stain the glomerular and extraglomerular basement membranes and
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mesonephros of fetuses of OW 8. They began in the glomeruli of the cranial pole, while the glomeruli and tubules of the caudal portion mantained normal structure. The involution consisted of moderate expansion of the mesangial matrix and progressive reduction followed by complete obliteration of capillary lumina. Initially these changes affected some portions of individual glomeruli more than others, but larger areas were involved as the involution progresses. The normally winding glomerular loops were decreased in number, and the entire glomerulus appeared wilted. Bowman's capsule was lined by a columnar type epithelium and its basement membrane appeared normal. The tubules were thinner and much shorter, and the tubular epithelium showed degenerative changes. The increased mesangial matrix was reactive for all antibodies (Fig. 3) with the exception of laminin (data not shown). Staining for collagen types IV, VI, laminin and fibronectin demonstrated OBM wrinkling (Fig. 4a). Type VI collagen and basement membrane components were still detectable in the more wilted glomerular loops (Fig. 4 b). In the most advanced stages the glomeruli appeared shrunken and transformed into sclerotic masses strongly positive for type IV collagen (Fig.4c, 4d) and less for type VI collagen and fibronectin. No or faint staining for laminin was detected in these areas. The glomeruli disintegrated gradually resulting in round cavities lined by a single layer of cuboidal or columnar epithelium containing cellular debris and fragments of glomerular loops (Fig. 4 b, 5).
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Fig. 4. Mesonephros of a human fetus of OW 12. (a) 'TYpe IV collagen staining demonstrates the OBM wrinkling and decreased capillary loop width. (b, c, d) As the regression progresses the glomerular tuft collapses. Some Bowman spaces are enlarged because of the shrunken glomerular tuft and some Bowman capsules are empty. The tubules are atrophic. 'TYpe VI collagen (b) and type IV collagen (c) delineate the OBM of collapsed glomerular tufts; (d) In this sclerotic glomerulus, type IV collagen is present in the lateral portion but is undetectable in the center. An open arteriole tranverses the rest of the I/;Iomerulus.
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Fig. 5. Human fetus of GW 12. Glomerular loops are decreased in number. Fibronectin staining shows focal disappearence of the GBM (arrows). Unstained cellular debris are found in the glomerulus cavity (arrow head).
Discussion This is the first study describing the immunohistochemical distribution pattern of collagen types IV, VI, laminin and fibronectin in human mesonephros from the S-shaped tubule stage to regression. All these ECM components were expressed in the developing and mature mesonephric nephrons suggesting that the glomerular epithelial, endothelial, mesangial cells, and the tubular epithelial cells are able to synthesize these compounds. It is also coinceivable that the mesonephric GBM is elaborated by both the epithelial and endothelial cells as occurs in the metanephros (Huang, 1979; Ekblom 1981; Mounier et al., 1986), and that it may possess the structural and physical properties required for this organ's filtering mechanism. lYpe IV collagen, fibronectin and laminin, expressed by the mesonephric ECM, appeared to be antigenically similar to those of the fetal metanephros (Kumar et al., 1986; Mounier et al. 1986). However, the antibodies used did not discrimate possible biochemical differences of the equivalent ECM components in the metanephros. Previous studies have shown that during embryonic development, ontogeny of normal tissues is associated with programmed changes in matrix composition and organization. In the human kidney, monoclonal antibodies have shown that basement membranes undergo changes in the expression of some ECM antigens during fetal life and that different isoforms exist for single ECM glycoproteins (Michael et al. 1983; Johansson et al., 1991). The fibronectin polyclonal antibody used in this study (Kumar et al., 1986) did not discriminate the cellular form from that derived from the plasma. However, in studies on human fetal metanephros, Laitinen et al. (1991) showed that a polyclonal antiserum gave results similar to those obtained with a monoclonal antibody
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against a cellular form of fibronectin suggesting that this compound, present in embryonic and fetal tissues, is produced locally and is not a plasm deposit as in adult tissues. In the present investigation type VI collagen yielded a fibrillar staining pattern in the mesonephric mesenchyme and a basement membrane-like staining around the developing epithelial mesonephric structures. These findings suggest that this collagen may act as an anchor component linking the epithelial basement membranes with the ECM. Our results are supported by light and immunomicroscopical studies showing that type VI collagen is associated with basement membranes, anchoring them with other ECM components into a functional unit (Amenta et aI., 1986, 1988; Keene et aI., 1988; Bashey et aI., 1992). We were able to detect type VI collagen as a component of GBM in the developing and mature mesonephric glomeruli and throughout the mesangial matrix. This differs from previous reports showing that in adult kidney glomerular type VI collagen is localized only in the mesangium (Hessle et aI., 1984; von Der Mark et aI., 1984; Mohan et aI., 1990). However, our results agree with recent immunohistochemical studies which clearly demonstrate that type VI collagen is a component of the normal human GBM (Nerlich et ai. 1994; Zhu et ai. 1994). As previously reported (De Martino et aI., 1977; Wartenberg, 1978), we found that alterations of GBM and mesangial matrix were associated with mesonephric glomerular regression. Staining for all ECM components showed GBM wrinkling, moderate expansion of mesangial matrix, collapse of the capillary walls and glomerulosclerosis. Our findings demonstrate that the expanded mesangial matrix and sclerotic lesions contain collagen types IV and VI as well as fibronectin. No information is available on the mechanism involved in mesonephric regression. The involution of the human mesonephros may be initiated by ischemia resulting from the shift of the local circulation from the indirect portal type to the adult type (Bodemer, 1968). It is conceivable that decrease in blood supply may be responsible for the whole sequence which begins with wrinkling and cringling of the capillary basement membrane and terminates as a sclerotic knot. It is noteworthy that the glomerular changes detected in this study, were similar to those occurring in the definitive kidney as a result of ischemic changes in the glomeruli (McManus and Lupton, 1960).
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