Morphofunctional Studies of the Glomerular Wall in Mice Lacking Entactin-1
Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec, Canada (S-PL,DG,MB), and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania (YC,AEC)
Correspondence to: M. Bendayan, Dept. of Pathology and Cell Biology, Université de Montréal, CP6128 Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7. E-mail: moise.bendayan{at}umontreal.ca
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: entactin-1 immunocytochemistry glomerular basement membrane collagen type IV laminin anionic charges permselectivity
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identified in 1977 (Chung et al. 1977; Carlin et al. 1981
), entactin is also known as nidogen (Timpl et al. 1983
). Molecular biology analysis by cDNA cloning has established that both terms refer to the same protein (Durkin et al. 1988
; Mann et al. 1989
). Entactin has recently been pointed out as an important component of the basement membranes (Chung and Durkin 1990
). It has high affinity for laminin, being responsible for the molecular assembly of basement membranes (Chung and Durkin 1990
; Aumailley et al. 1993
; Chung et al. 1993
; Mayer et al. 1998
). It also connects the networks formed by type IV collagen and laminin to create the tridimensional network architecture particular to basement membranes (Chung and Durkin 1990
; Yurchenco and Schittny 1990
; Chung et al. 1993
; Miosge et al. 1999
). Entactin is also known for cell adhesion support (Chakravarti et al. 1990
). In fact, two distinct molecular cell attachment sites have been identified (Dong et al. 1995
). The one located in the second globular G2 domain of entactin binds a member of the ß1 family of integrins receptors, probably the
3ß1 (Dedhar et al. 1992
; Dong et al. 1995
; Wu et al. 1995
), while the second, on the rigid stalk E domain within the RGD sequence, is recognized by the
vß3-integrin (Dong et al. 1995
; Yi et al. 1998
). Moreover, this RGD sequence can also bind the leukocyte response integrin that stimulates neutrophil chemotaxis to sites of injury (Senior et al. 1992
; Gresham et al. 1996
). Recent findings have shown that entactin, through its G2 domain, constitutes a signal for stimulation of the Fc receptor-mediated phagocytosis via ligation of
3ß1 (Gresham et al. 1996
), therefore participating in acute inflammation responses. Furthermore, this protein binds fibrinogen (Wu and Chung 1991
) and human trophoblasts (Yang et al. 1996
), indicating potential roles in hemostasis and embryo implantation.
In this study we evaluated the importance of entactin-1 in the overall structural and functional properties of the glomerular basement membrane (GBM). Using renal tissues from entactin-1-null transgenic mice and applying cytochemical approaches, we evaluated the impact of the absence of entactin-1 on the molecular organization of other basement membrane components, on the distribution of anionic charges of the GBM, and on the expression by glomerular cells of extracellular matrix membrane receptors, the integrins. In view of the major roles played by the GBM in glomerular filtration, we also assessed the functional properties of the glomerular wall in renal tissues from entactin-1-null mice.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Processing
After anesthesia, the kidneys were removed and cut into small samples for immunocytochemistry protocols. They were fixed by immersion with a periodatelysineparaformaldehyde 4% solution for 2 hr at room temperature (RT). Tissues were dehydrated in ethanol and embedded in Lowicryl K4M at -30C (Bendayan 1995). Thin sections were cut and mounted on Parlodion- and carbon-coated nickel grids and processed for immunocytochemistry.
Immunocytochemistry
Some grids were stained with uranyl acetate and lead citrate for electron microscopic examination, and others were subjected to immunocytochemistry. Antigenic sites for entactin-1, type IV collagen, laminin, integrins, and endogenous albumin were revealed using corresponding specific polyclonal and monoclonal antibodies [rabbit anti-entactin-1 (Chung and Durkin 1990); mouse anti-type IV collagen (MAB1430), Chemicon, Temecula, CA; mouse anti-laminin (clone Lam-1), ICN ImmunoBiologicals, Costa Mesa, CA; rabbit anti-integrins for
3 (AB1920) and
n (AB1930) subunits, both from Chemicon; rabbit anti-mouse serum albumin (Cappel, ICN Biomedical); and protein Agold complexes (Bendayan 1995
)]. The postembedding immunocytochemical labeling protocol was carried out as previously described (Bendayan 1995
). Briefly, for the different antigens, the tissue thin sections were first incubated on a drop of 0.15 mol/liter glycine for 15 min and then on a drop of 1% ovalbumin in PBS, pH 7.3, for 20 min. The grids were then placed on a drop of 1% gelatin for an additional 15 min and transferred to a drop of one of the antibodies [anti-entactin-1 (1:100); anti-type IV collagen (1:10); anti-laminin (1:50); anti-integrins (1:100 for
3 and 1:50 for
n)] for an overnight incubation at 4C. After rinsing with PBS, the sections were incubated with 1% ovalbumin for 20 min and moved directly to a drop of the protein Agold complex for 30 min. Protein Agold was prepared with 10-nm gold particles according to protocols described previously (Bendayan 1995
). The grids were then washed with PBS and distilled water before drying. Staining was performed with uranyl acetate before examination with a Philips 410 electron microscope. For endogenous albumin, the grids carrying the tissue thin sections were transferred directly from the PBS to a drop of the anti-mouse albumin polyclonal antibody (1:50) for a 90-min incubation at RT. The incubation step with 1% ovalbumin was omitted. After washing with PBS, they were incubated with the protein Agold complex for 30 min. Specificity of each immunolabeling was demonstrated by control experiments: (a) incubation of the tissue sections with each antibody solution adsorbed with its corresponding antigen, followed by the protein Agold complex, and (b) incubation with the protein Agold complex alone, omitting the antibody step.
In addition, the distribution of the anionic charges through the GBM was revealed using the poly-L-lysinegold (PLG) complex. Previous experiments have demonstrated the specificity of the PLG complex for heparan sulfate proteoglycans (Russo et al. 1993; Londoño et al. in press
). The PLG complex was prepared according to Skutelsky and Roth (1986)
as adapted by Russo et al. (1993)
and applied on the tissue sections as described previously (Russo et al. 1993
; Londoño et al. in press
).
Data and Statistical Analysis
Morphometrical analysis of the labelings was performed using an image processing system (Videoplan 2; Carl Zeiss, Toronto, ONT, Canada). Labeling density for entactin-1 (gold particles/µm2) was evaluated for the glomerular and mesangial basement membranes. For the 3 and
n integrins, the density of labeling present over the cells' plasma membrane was evaluated in reference to the length of the membranes (gold particles/µm2). These measurements were made on 18 recorded fields at a final magnification of x21,000 for each animal in each group. For type IV collagen, laminin, and albumin, as well as for the anionic sites, the exact location of the gold particles over the GBM was analyzed. The distribution of the labeling was carried out as reported in detail previously (Bendayan et al. 1986
; Bendayan 1995
). Briefly, the distance between each gold particle and the endothelial abluminal plasma membrane was first measured. Next, the thickness of the GBM at the same site and the distance between the endothelial abluminal plasma membrane and the podocyte basal plasma membrane was measured. Then, the ratio R = [distance (endotheliumgold particle)/distance (endotheliumepithelium)] was calculated and reported in histograms reflecting the distribution of the labeling over the GBM. These evaluations were carried out individually for each animal in each group. Over 900 measurements were recorded for each animal and for each protocol. Finally, the thickness of the GBM was measured according to methods previously described (Bendayan et al. 1986
; Doucet et al. 2000
). The distance between the endothelial abluminal plasma membrane and that of podocytes was directly measured by planimetry. Care was taken to evaluate fields cut at right angles. This was reflected by the clear presence of slit diaphragms between podocyte foot processes. Again, over 900 measurements were recorded for each animal. The quantitative data were statistically evaluated using the MannWhitney U-test or the KruskalWallis test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transgenic entactin-null animals have been recently generated to assess the role of this component in the formation of basement membranes (Murshed et al. 2000; Dong et al. 2002
). Murshed and colleagues (2000)
did not find modifications in basement membranes formation for the transgenic animals, while Dong and colleagues (2002)
found rather subtle changes in particular basement membranes, i.e., those from the brain capillaries and the lens capsule. These two studies raised the possibility of a compensatory role for entactin-2, a protein related to entactin-1. Entactin-2 is present in most basement membranes and exhibits the same domains as entactin-1, but having different binding affinities with the major constituents of the basement membrane. However, a recent work has reported that entactin-2 is not essential for basement membrane formation or maintenance (Schymeinsky et al. 2002
), although some findings revealed complementary actions of both entactins (Salmivirta et al. 2002
).
The importance of entactin-1 in the overall architecture of the basement membrane prompted us to investigate the consequence of its absence on the conformation of the GBM and on the permselectivity properties of the glomerular wall. The approach consisted of the removal of this component from the tissues by genetic engineering followed by morphocytochemical studies of the glomerular wall (Dong et al. 2002). Immunoblotting together with immunocytochemistry at the light microscopic level and in situ hybridization have demonstrated that basement membranes in entactin-1-null mice effectively lack this protein (Dong et al. 2002
). We confirmed by quantitative immunoelectron microscopy the absence of this protein in the GBM. Despite full agreement with Dong et al. (2002)
of no major morphological alteration of basement membranes in the entactin-1-null mice, we decided to investigate the consequence of the removal of entactin-1 on the functional properties of the GBM. Our analysis made use of different and more sensitive cytochemical techniques. Furthermore, we assessed functional properties of the GBM. We found that entactin-1-null mice exhibit alterations in glomerular filtration. Absence of entactin-1 appears to be sufficient to modify the permselectivity of the glomerular wall. We know that entactin-1 bridges the two independent networks of laminin and type IV collagen, creating a stabilized basement membrane. We found that these two components retained their normal distribution in the GBM despite the absence of entactin-1. Alterations were also detected in the glomerular wall and concerned the thickness of the GBM, the distribution of the anionic charges, and the functional properties. Indeed, the GBM in tissues of entactin-1-null mice showed small but significant thickening and loss of anionic charges on the subendothelial side. The main alteration was the functional one, the glomerular wall being unable to retain albumin. However, despite this leakage of albumin across the glomerular wall, the entactin-1-null mice did not demonstrate any significant proteinuria. This indicates that the epithelial cells along the nephron are efficient enough to reabsorb the filtered albumin. In fact, as illustrated in Figure 8, the glomerular and the tubule epithelial cells of the entactin-1-null animals did exhibit increased number of lysosomes, which contain large amounts of albumin. Such results reflect important endocytotic reabsorptive activities along the nephron.
Integrins are a family of transmembrane receptors composed of - and ß-subunits that interact with components of the extracellular matrix. In the glomerular wall, the
3 and ß1 isoforms are the most abundant and represent the main extracellular matrix receptors along the GBM (Kerjaschki et al. 1989
; Regoli and Bendayan 1997
), suggesting adhesion roles between endothelial as well as epithelial cells and the underly GBM (Adler 1992
). This prompted us to investigate the effect of the lack of a major extracellular matrix component, entactin-1, known to be a ligand of the integrins (Dedhar et al. 1992
; Dong et al. 1995
; Wu et al. 1995
; Yi et al. 1998
), on the expression of two different integrin subunits by the endothelial and epithelial cells of the glomerular wall. Indeed, the
v-integrin increased along the plasma membrane of both the endothelial and epithelial cells in entactin-1-null mice. Knowing that interactions among components of the extracellular matrix and cell surface integrins mediate a variety of cellular responses, including cell adhesion, cell movement, and signal transduction, we can assume that lack of one ligand, i.e., entactin-1, in the extracellular matrix must affect cellular activities in neighboring cells through integrin relays. For the glomerular wall we can assume that changes in endothelial and epithelial cells might also contribute to the loss of filtration properties.
![]() |
Acknowledgments |
---|
We thank Dr Irène Londoño for her appreciated assistance. This article represents part of the work required for the fulfillment of the M.Sc. program for S.P.L.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abrahamson DR (1986) Recent studies on the structure and pathology of basement membranes. J Pathol 149:257278[Medline]
Adal Y, Smith MF, Osicka TM, Comper WD (1995) Albumin interaction with the glomerular capillary wall in vitro. Kidney Int 47:10311038[Medline]
Adler S (1992) Characterization of glomerular epithelial cell matrix receptors. Am J Pathol 141:571578[Abstract]
Arshi K, Bendayan M, Ghitescu LD (2000) Alterations of the rat mesentery vasculature in experimental diabetes. Lab Invest 80:11711184[Medline]
Aumailley M, Battaglia C, Mayer U, Reinhardt D, Nischt R, Timpl R, Fox JW (1993) Nidogen mediates the formation of ternary complexes of basement membrane components. Kidney Int 43: 712[Medline]
Aumailley M, Wiedemann H, Mann K, Timpl R (1989) Binding of nidogen and the laminin-nidogen complex to basement membrane collagen IV. Eur J Biochem 184:241248[Abstract]
Bendayan M (1995) Colloidal gold-post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1159[Medline]
Bendayan M, Gingras D, Charest P (1986) Distribution of endogenous albumin in the glomerular wall of streptozotocin-induced diabetic rats as revealed by high-resolution immunocytochemistry. Diabetologia 29:868875[Medline]
Bendayan M, Rasio E (1981) Hyperglycemia and microangiopathy in the eel. Diabetes 30:317325[Abstract]
Bouchard P, Ghitescu LD, Bendayan M (2002) Morpho-functional studies of the blood-brain-barrier in streptozotocin-induced diabetic rats. Diabetologia 45:10171025[Medline]
Brenner BM, Hostetter TH, Humes HD (1978) Glomerular permselectivity: barrier function based on discrimination of molecular size and charge. Am J Physiol Renal Physiol 234:455460
Carlin B, Jaffe R, Bender B, Chung AE (1981) Entactin a novel basal lamina-associated sulfated glycoprotein. J Biol Chem 256:52095214[Abstract]
Chakravarti S, Tam MF, Chung AE (1990) The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions. J Biol Chem 265:1059710603
Chittenden SJ, Shami SK (1991) Microangiopathy in the diabetes mellitus. Diabetes Res 17:105114[Medline]
Chung AE, Dong LJ, Wu C, Durkin ME (1993) Biological functions of entactin. Kidney Int 43:1319[Medline]
Chung AE, Durkin ME (1990) Entactin: structure and function. Am J Respir Cell Mol Biol 3:275282[Medline]
Chung AE, Freeman IL, Braginski JE (1977) A novel extracellular membrane elaborated by a mouse embryonal carcinoma-derived cell line. Biochem Biophys Res Commun 79:859868[Medline]
Daniels BS, Hauser EB, Deen WM, Hostetter TH (1992) Glomerular basement membrane: in vitro studies of water and protein permeability. Am J Physiol 262:R919926
Dedhar S, Jewell K, Rojiani M, Gray V (1992) The receptor for the basement membrane glycoprotein entactin is the integrin 3/ß1. J Biol Chem 267:1890818914
Deen WD, Bridges CR, Brenner BM (1983) Biophysical basis of glomerular permselectivity. J Membr Biol 71:110[Medline]
Desjardins M, Bendayan M (1989) Heterogeneous distribution of type IV collagen, entactin, heparan sulfate proteoglycans, and laminin among renal basement membranes as demonstrated by quantitative immunocytochemistry. J Histochem Cytochem 37:885897[Abstract]
Dong LJ, Chen Y, Lewis M, Hsieh JC, Reing J, Chaillet R, Howell K, et al. (2002) Neurologic defects and selective disruption of basement membranes in mice lacking entactin-1. Lab Invest 82:16171630[Medline]
Dong LJ, Hsieh JC, Chung AE (1995) Two distinct cell attachment sites in entactin are revealed by amino acid substitutions and deletion of the RGD sequence in the cystein-rich epidermal growth factor repeat 2. J Biol Chem 270:1583815843
Doucet M, Londoño I, GomezPascual A, Bendayan M (2000) Glomerular basement membrane selective permeability in short-term streptozotocin-induced diabetic rats. Int J Exp Diabetes Res 1:1930[Medline]
Durkin ME, Chakravarti S, Bartos BB, Liu SH, Friedman RL, Chung AE (1988) Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor. J Cell Biol 107:27492756[Abstract]
Ghitescu L, Desjardins M, Bendayan M (1992) Immunocytochemical study of glomerular permeability to anionic, neutral and cationic albumins. Kidney Int 42:2532[Medline]
Goode NP, Shires M, Crellin DM, Aparicio SR, Davison AM (1995) Alterations of glomerular basement membrane charge and structure in diabetic nephropathy. Diabetologia 38:14551465[Medline]
Gresham HD, Graham IL, Griffin GL, Hsieh JC, Dong L-J, Chung AE, Senior RM (1996) Domain-specific interactions between entactin and neutrophil integrins. J Biol Chem 48:3058730594
Katz A, Fish AJ, Kleppel MM, Hagen SG, Michael AF, Butkowski RJ (1991) Renal entactin (nidogen): isolation, characterization and tissue distribution. Kidney Int 40:643652[Medline]
Kerjaschki D, Ojha PP, Susani M, Horvat R, Binder S, Hovorka A, Hillemanns P, et al. (1989) A beta 1-integrin receptor for fibronectin in human kidney glomeruli. Am J Pathol 134:481489[Abstract]
Londoño I, Gingras D, Bendayan M (in press) Circulating glycated albumin and glomerular anionic charges. Exp Diabetes Res
Mann K, Deutzmann R, Aumailley M (1989) Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells. EMBO J 8:6572[Abstract]
Mayer U, Kohfeldt E, Timpl R (1998) Structural and genetic analysis of laminin-nidogen interaction. Ann NY Acad Sci 857:130142
Miosge N, Heinemann S, Leissling A, Klenczar C, Herken R (1999) Ultrastructural triple localization of laminin-1, nidogen-1, and collagen type IV helps elucidate basement membrane structure in vivo. Anat Rec 254:382388[Medline]
Miosge N, Sasaki T, Timpl R (2002) Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice. Matrix Biol 21:611621[Medline]
Murshed M, Smyth N, Miosge N, Karolat J, Krieg T, Paulsson M, Nischt R (2000) The absence of nidogen 1 does not affect murine basement membrane formation. Mol Cell Biol 20:70077012
Paulsson M (1988) The role of Ca2+ binding in the self-aggregation of laminin-nidogen complexes. J Biol Chem 263:54255430
Paulsson M (1992) Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Mol Biol 27:93127[Abstract]
Regoli M, Bendayan M (1997) Alterations in the expression of the 3/ß1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus. Diabetologia 40:1522[Medline]
Ruoslahti E (1991) Integrins. J Clin Invest 87:15[Medline]
Russo P, Gingras D, Bendayan M (1993) Poly-L-lysine-gold probe for the detection of anionic sites in normal glomeruli and in idiopathic and experimentally induced nephrosis. A comparative ultrastructural study. Am J Pathol 142:261271[Abstract]
Salmivirta K, Talts JF, Olsson M, Sasaki T, Timpl R, Ekblom P (2002) Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp Cell Res 279:188201[Medline]
Schymeinsky J, Nedbal S, Miosge N, Pöschl E, Rao C, Beier DR, Skarnes WC, et al. (2002) Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement formation in mice. Mol Cell Biol 22:68206830
Senior RM, Gresham HD, Griffin GL, Brown EJ, Chung AE (1992) Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. J Clin Invest 90:22512257[Medline]
Simpson LO, Shand BO (1986) Glomerular permeability. Clin Sci 71:221223[Medline]
Skutelsky E, Roth J (1986) Cationic colloidal golda new probe for the detection of anionic cell surface sites by electron microscopy. J Histochem Cytochem 34:693696[Abstract]
Timpl R, Brown JC (1996) Supramolecular assembly of basement membranes. BioEssays 18:123132[Medline]
Timpl R, Dziadek M (1986) Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol 29:1111[Medline]
Timpl R, Dziadek M, Fujiwara S, Nowack H, Wick G (1983) Nidogen: a new self-aggregating basement membrane protein. Eur J Biochem 137:455465[Abstract]
Weber M (1992) Basement membrane proteins. Kidney Int 41:620628[Medline]
Wu C, Chung AE (1991) Potential role of entactin in hemostasis. J Biol Chem 266:1880218807
Wu C, Chung AE, McDonald JA (1995) A novel role for 3/ß1 integrins in extracellular matrix assembly. J Cell Sci 108:25112523
Yamaji T, Fukuhara T, Kinoshita M (1993) Increased capillary permeability to albumin in diabetic rat myocardium. Circ Res 72:947957[Abstract]
Yang Y, Todt JC, Svinarich DM, Qureshi F, Jacques SM, Graham CH, Chung AE, et al. (1996) Human trophoblast cell adhesion to extracellular matrix protein, entactin. Am J Reprod Immunol 36:2532[Medline]
Yi XY, Wayner EA, Kim Y, Fish AJ (1998) Adhesion of cultured human kidney mesangial cells to native entactin: role of integrin receptors. Cell Adhes Commun 5:237248[Medline]
Yurchenco PD, Schittny JC (1990) Molecular architecture of basement membranes. FASEB J 4:15771590