Recent insights into the structure and functions of heparan sulfate proteoglycans in the human glomerular basement membrane

Alexander J. A. Groffen1, Jacques H. Veerkamp2, Leo A. H. Monnens1 and Lambert P. W. J. van den Heuvel1

1 Department of Pediatrics and 2 Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands

Correspondence and offprint requests to: Dr L. P. van den Heuvel, Department of Pediatrics, University Hospital Nijmegen, Box 9101, 6500 HB Nijmegen, The Netherlands.

Abstract

As the first barrier to be crossed on the way to urinary space, the glomerular basement membrane (GBM) plays a key role in renal function. The permeability of the GBM for a given molecule is highly dependent on its size, shape and charge. As early as 1980, the charge-selective permeability was demonstrated to relate to the electrostatic properties of covalently bound heparan sulfates (HS) within the GBM. Since the identification of perlecan as a heparan sulfate proteoglycan (HSPG) of basement membranes, the hypothesis that perlecan could be a crucial determinant of GBM permselectivity received considerable attention. In addition to perlecan, the GBM also contains other HSPG species, one of which was identified as agrin. The high local expression of agrin in the GBM, together with the presence of agrin receptors at the cell–matrix interface, suggests that this HSPG contributes to glomerular function in multiple ways. Here, we review the current knowledge regarding the structure and functions of HSPGs in the GBM, and discuss how these molecules could be involved in various glomerular diseases. Possible directions for future investigation are suggested.

Introduction

The charge-selective permeability of the glomerular basement membrane (GBM) was demonstrated to relate to the electrostatic properties of covalently bound heparan sulfates (HS) in this structure [1,2]. Polyanionic sites in the GBM, visualized by various cationic probes [3], appear in a regular pattern and clearly dissociated from the epithelial and endothelial cell membrane on both sides of the GBM [4]. The polyanionic sites are sensitive to heparitinase treatment [5], which also increases the permeability of the GBM for anionic macromolecules such as native ferritin or albumin [1,6]. These observations form the basis of the hypothesis that extracellular heparan sulfate proteoglycans (HSPGs) are crucial determinants of the charge-selective permeability of the GBM. At present, this hypothesis is still widely accepted. Notwithstanding this, other parts of the glomerular filtration barrier may contribute to charge-selective permeability [7]. This is true in particular for the glycocalyx that covers the outer surface of the podocyte's foot processes and for endothelial cells. Among the constituents of the glycocalyx are podocalyxin [8,9], {alpha}-dystroglycan [10,11], integrins and cell surface proteoglycans. The net negative charge of this layer can be stained by cationic dyes such as colloidal iron [12], and is susceptible to enzymatic degradation by neuraminidase. Neutralization of the anionic charge results in fusion of the foot processes and detachment from the GBM [13,14]. A direct influence of the glycocalyx on the selective passage of macromolecules was not in agreement with experiments using native and modified ferritins. They were retarded primarily in the GBM [12,15]. Given these data, and given its close association with both the cytoskeleton and the GBM, the major function of the glycocalyx may be related to the structural organization of the filtration barrier [16].

Molecular architecture of the glomerular permselectivity barrier

A major step forward in the investigation of the molecular organization of basement membranes was the characterization of the Engelbreth–Holm–Swarm (EHS) mouse tumour that produces large amounts of extracellular matrix [17]. The main molecular components were identified as type IV collagens, laminins, nidogen and proteoglycans [18]. Although the overall organization of the GBM resembles that of the EHS matrix, both matrices contain different isoforms of type IV collagen and laminin, and a different range of proteoglycans.

Type IV collagen

The basic scaffold of the GBM is formed by collagen type IV, a fibrillar protein that aggregates into a polymeric mesh. Each collagen type IV monomer consist of three polypeptide chains folded into a triple-stranded helix [18,19]. The monomers aggregate by three types of homotypic interaction: the covalent association of the C-termini into dimers, the covalent association of N-termini into tetramers and lateral interactions between adjacent collagenous segments [20]. At least six different collagen type IV polypeptide chains, named {alpha}1–{alpha}6, are encoded by separate genes named COL4A1COL4A6. Only two stable collagen IV networks have been characterized; the first composed of the {alpha}1 and {alpha}2 chains, and the second containing {alpha}3, {alpha}4 and {alpha}5 [21]. The latter network is presumably stronger, as illustrated by the progressively disrupted structure of the GBM in Alport syndrome. Here the deficient assembly of the {alpha}3-, {alpha}4- and {alpha}5-containing network is associated with a sustained expression of the {alpha}1 and {alpha}2 chains [19,22]. During maturation of the GBM, the collagen IV composition is changed gradually from an {alpha}1-and {alpha}2-containing to an {alpha}3-, {alpha}4- and {alpha}5-containing network [23,24].

Laminins

Laminins are assembled from three polypeptide chains designated the laminin {alpha}, ß and {gamma} chain. For each individual chain, different isoforms were identified. They were designated {alpha}1–5, ß1–4 and {gamma}1–3. While most variants are encoded by a different gene, truncated chains may also be produced by alternative splicing (e.g. {alpha}3) [25] or proteolytic processing (e.g. {alpha}2, ß3 and {gamma}2) [26]. During nephrogenesis, the laminin composition of the GBM is altered importantly. The {alpha}1 and {alpha}4 chains are replaced gradually by the {alpha}5 chain, and the ß1 chain by ß2 [25], thus yielding a predominating laminin isoform composed of {alpha}5ß2{gamma}1 (also designated laminin-11) in the mature GBM. The laminin ß2 chain seems to have an important function in the GBM, since mutant mice that lack this chain display a nephrotic syndrome in the second week after birth [27]. Interestingly, their massive proteinuria is associated with fusion of the foot processes, resembling the phenotype of minimal change nephrosis. This suggests an important role for laminin-11 in cell–matrix interaction. Such a function is conceivable since laminins interact with multiple components of the GBM, including agrin [28,29], nidogen [26] and perlecan [30]. Laminins also interact with cell surface receptors, such as {alpha}3ß1 integrin [31], {alpha}6ß1 integrin [32] and {alpha}-dystroglycan [33]. Both receptors appear on the cell surface of the developing nephron after the mesenchymal to epithelial transition, which corresponds to the onset of expression of laminin-1 [11,34].

Nidogen

Nidogen, also called entactin, is a 158 kDa large single chain glycoprotein first purified from the extracellular matrix produced by the EHS tumour. It is composed of three globular domains (G1, G2 and G3) separated by rod-like segments [35]. The C-terminal globule (G3) binds non-covalently and in equimolar amounts to domain III of the laminin {gamma}1 chain. Domain G2 associates with collagen IV, thus linking the collagen matrix to the laminin network. This domain also has a perlecan-binding function. Given these cross-linking properties, nidogen is an important component in the assembly of BMs [18,20].

Proteoglycans

The majority (>80%) of proteoglycans in the mature GBM are associated with HS rather than with other glycosaminoglycans (chondroitin sulfate, dermatan sulfate, keratan sulfate or hyaluronic acid) [36,37]. A chondroitin sulfate proteoglycan named bamacan is expressed transiently during nephrogenesis, but disappears from the GBM upon maturation [38,39]. Characterization and molecular identification of the HSPG species involved in the charge-selective ultrafiltration process proved a difficult task: extraction and purification from basement membranes are complicated by the strongly cross-linked characteristics of the proteoglycans and their sensitivity to proteolysis [40]. Additionally, the heterogeneity in size, charge and structure of the HS residues results in a heterogeneous electrophoretic mobility, which impairs molecular mass estimations. Current insights into the identity, structure and functions of HSPGs will be reviewed in the next sections.

Agrin as a glomerular basement membrane component

The structural and functional properties of agrin have been the subject of extensive investigation in the field of neuroscience, especially in the developing chick, rat and mouse. Agrin is expressed in the specialized basement membrane of the neuromuscular junction, where it plays a key role in the development of the synaptic apparatus [41,42]. Agrin proved to be an HSPG [43].

As well as at the neuromuscular junction, agrin is also expressed in other basement membranes, including those of the kidney, lung and microvasculature [4447]. The expression pattern of non-neural agrin isoforms suggests a possible function in the selective exchange of molecules between compartments. In the developing blood—brain barrier, the onset of agrin expression coincides with the time at which the basement membrane of capillary vessels becomes impermeable [46]. A possible function of agrin in regulating the molecular access through basement membranes is especially intriguing with respect to renal function. As shown by immunoelectron microscopy, agrin is expressed in glomeruli where it is localized predominantly within the GBM (Figure 1Go). The large amounts of agrin that accumulate in the GBM, and its homogenous distribution therein, suggest a major role in the maintenance of glomerular permselectivity [48].



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Fig. 1. Ultrastructural localization of agrin in mouse glomeruli analysed by immunoelectron microscopy. Anti-agrin serum strongly stained the GBM in a homogenous linear manner. A less intense staining was observed in the mesangial matrix. The antibodies and procedures used were described previously [48]. Bar = 1 µm.

 
Structure of agrin

The human agrin gene, named AGRN, was assigned to the locus 1p36.1–1pter [49]. This is relatively close to the perlecan gene (1p35–1p36.1). The human agrin mRNA molecule exceeds 7 kb in length and encodes a core protein of 212 kDa [47]. The primary structure shows a multimodular composition (Figure 2AGo) containing a globular laminin-binding domain, nine follistatin-like protease inhibitor domains, two laminin-like epidermal growth factor (EGF) repeats, two serine/threonine-rich domains, an SEA module, four EGF repeats and three domains sharing homology with globules of the laminin {alpha} chains [41,47]. The four globules (the N-terminal laminin-binding domain and the three laminin-like domains) can be distinguished by electron microscopy [30]. The globules are connected by flexible rod-like structures. The agrin protein contains two regions particularly favourable for HS attachment, both located in the central part of the molecule: the first region, that contains the amino acid sequence SGGSGSGED, is located just upstream from the eighth follistatin-like domain, and the second, that contains the sequence SGDQEASGGGSGG, directly precedes the SEA module. Rotary shadowing electron microscopy of full-length recombinant agrin provided experimental support for the attachment of two or three HS chains to the central region of the molecule in chick [30].



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Fig. 2. The domain organization of human agrin (a) and human perlecan (b). EG, EGF-like repeat; FS, follistatin-like repeat; Ig, Ig-like repeat similar to those of neural cell adhesion molecule; LA, LDL receptor class A module; LamB, laminin IV-like domain; LamG, laminin G-like domain; LE, laminin III-like EGF repeat; NtA, N-terminal agrin (laminin-binding domain); SEA, module first found in sperm protein, enterokinase and agrin; SS, signal peptide; S/T, serine/threonine-rich. The arrows indicate putative glycosaminoglycan attachment sites. Redrawn from [47,67,69], with minor modifications.

 
Various isoforms of agrin are expressed through tissue-dependent alternative splicing. The four sites at which splice variants were reported are designated c, x, y and z in mammalian species (C, A and B in chick). As the differences between agrin splice variants are restricted to short oligopeptide sequences, they cannot be distinguished by polyacrylamide gel electrophoresis. Different tissues produce different splice variants of agrin, each with different functional properties.

Functions of agrin

Neuronal agrin triggers the clustering of acetylcholine receptors (AChRs) on the muscle cell membrane [41,42,50]. The cell surface receptor responsible for this activity, referred to as `MASC', has not yet been identified. The clustering activity requires the presence of an insert at the z splice site [51] and involves the activation of a muscle-specific kinase (MuSK) [52]. MuSK becomes immobilized in the synaptic region, where it is finally included in a large multimolecular network that traverses the cell membrane. Other components included in this structure are neuregulins, perlecan, AChRs, rapsyn-associated transmembrane linker, rapsyn, the dystrophin-associated glycoprotein complex and the cytoskeleton [41]. Muscle-derived isoforms of agrin do not have AChR clustering activity. Instead, they induce the differentiation of the nerve terminal, a process which includes the clustering of synaptotagmin in the developing presynaptic density [53].

In the GBM, agrin may have various other functions. The N-terminal region of agrin binds to laminin, and thereby mediates the anchorage of agrin within the basement membrane [30]. The laminin-binding globule is spatially separated from the x, y, and z sites, suggesting that agrin binding to laminin is not influenced by alternative splicing at these sites. The binding was localized to a central region in the long arm of laminin (Figure 3c and dGo) [30]. Agrin binding to laminin may be essential to immobilize the HS chains in a regular distribution into the GBM.



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Fig. 3. Involvement of agrin in cell–matrix and matrix–matrix interactions [29,34,54,56]. Agrin can bind to the podocyte cell surface via {alpha}-dystroglycan, a peripheral membrane glycoprotein linked to the dystrophin-associated glycoprotein complex (a) and via {alpha}vß1 integrin (b). Agrin also binds to the mature GBM via laminin-11, an integral component of the GBM. Laminin may associate with {alpha}-dystroglycan (c) or {alpha}3ß1 integrin (d) on the podocyte cell surface. Note the tight association of extracellular matrix molecules with the contractile cytoskeleton of the podocyte. Integrin-mediated cell binding may also take place on the endothelial and mesangial cell surface, where the expression of isoforms {alpha}vß1 and {alpha}3ß1 has been reported [57,58]. DG, dystroglycan; D/U, dystrophin/utrophin-related protein (not yet identified in the differentiated podocyte); pp44, podocyte 44 kDa protein (or synaptopodin).

 
At the interface between the GBM and the cell surface, agrin may play an important role in cell– matrix adhesion. One cell surface receptor for agrin, {alpha}-dystroglycan (Figure 3aGo), was shown to be expressed in the epithelium of developing and mature kidney [11]. In the mature glomerulus, {alpha}-dystroglycan, like agrin, is expressed prominently in the GBM [11]. Interestingly, {alpha}-dystroglycan localizes to the basal side of the epithelium where the cell surface is in contact with the basement membrane. The localization of agrin and {alpha}-dystroglycan in the GBM thus supports a function in cell–matrix interaction. Co-immunoprecipitation of agrin with {alpha}-dystroglycan [54] provided experimental evidence that this interaction indeed occurs in adult kidney. The function of this binding may be to provide a structural association between the basement membrane and the cytoskeleton of the foot processes. A tight association of the cytoskeleton with the GBM is not only essential to resist the high local blood pressures, it is also required to adapt the structure of the glomerular filtration surface in response to haemodynamic changes. The contractile cytoskeleton of the podocyte is thought to play a key role in this process [55].

A second mechanism for agrin adhesion to the cell surface is mediated by integrins (Figure 3bGo). Inhibition studies with various anti-integrin antibodies indicated that this adhesion is mediated by {alpha}vß1 integrin, although an involvement of other ß1 integrins is not excluded [56]. In the glomerulus, {alpha}vß1 integrin was shown to be present on the surface of podocytes, mesangium and endothelium [57,58]. The interaction of integrins with agrin provides an additional structural link between the cytoskeleton and the extracellular matrix.

Agrin could also be involved in transmembrane signalling. This could occur via {alpha}-dystroglycan as well as via integrins, as suggested by the following arguments. First, {alpha}-dystroglycan is known to be associated with the dystrophin-associated glycoprotein complex (DGC), and hence linked to several enzymes of intracellular signalling pathways (including a nitric oxide synthase) [10]. Although little is known regarding the composition of the DGC as it exists in the podocyte, the relevance of {alpha}-dystroglycan-mediated signal transduction in kidney was supported experimentally by a study where anti-dystroglycan antibodies resulted in aberrant nephrogenesis in vitro [10,11,33]. Not only agrin, but also laminin may be involved in such processes. Second, integrins also have the potential to influence cellular differentiation. In fact, integrins may represent the agrin receptor `MASC' that triggers AChR clustering in the muscle cell [56]. As the cellular response may differ between cell types, it will be interesting to investigate how agrin influences the differentiation of glomerular cells by this mechanism. Besides {alpha}vß1 integrin, mature podocytes predominantly express {alpha}3ß1 integrin [57]. This integrin also mediates adhesion to extracellular matrix components, including laminin (Figure 3dGo). These data suggest that both agrin and laminin may influence cellular differentiation through {alpha}-dystroglycan- and integrin-mediated signalling pathways.

Finally, another component that interacts with agrin is heparin (a useful property for purification procedures). Not all agrin isoforms show this activity; the binding requires the presence of a basic tetrapeptide cluster at the y splice site [59]. It would be attractive to hypothesize that heparin binding to (residual) agrin in the GBM could explain the beneficial effect of heparin administration in diabetes (see below). This is, however, not supported by the sequence of a cDNA clone encoding a renal agrin isoform, which was devoid of an insert at the y site [47]. Heparin binding could be a mechanism to modulate the ability of agrin to bind {alpha}-dystroglycan. In isoforms containing a four amino acid insert at the y site, {alpha}-dystroglycan binding is still possible but it can be inhibited in the presence of heparin [59,60]. Consistently, the {alpha}-dystroglycan-binding activity of isoforms that lack the y insert is not inhibited by heparin [59,60]. Additional functions of agrin may be related to protease inhibition [45] or interactions with various growth factors.

Perlecan as a glomerular basement membrane component

The first identified HSPG of basement membranes was isolated from the EHS mouse tumour [2] and named perlecan with reference to its beads-on-a-string-like appearance in rotary shadowing electron microscopy [61]. For more than a decade, perlecan was the only identified HSPG known to be associated with basement membranes. The hypothesis that perlecan could be a crucial determinant of GBM-associated polyanionic sites therefore received considerable attention. Despite this, no clear evidence was obtained for this idea. Perlecan is widely expressed in basement membranes, including those of skin, lung, colon, liver, heart, connective tissue, thymus, prostate, spleen, pituitary gland, kidney, placenta, skeletal muscle and blood vessels [62]. In the developing kidney, perlecan expression is initiated as early as the vesicle stage [63]. During further nephrogenesis, perlecan expression is accompanied by agrin expression in the GBM. In the mature glomerulus, the majority of perlecan immunoreactivity is observed in Bowman's capsule and the mesangial matrix [48,64]. Along the glomerular capillary loops, perlecan expression is then limited to focal accumulations in the endothelial side of the GBM [48]. The absence of perlecan from parts of the glomerular capillary loop indicates that it cannot be the sole HSPG involved in charge-selective ultrafiltration. This is also suggested by the relatively low concentration of perlecan in isolated glomeruli, compared with that of agrin [48].

Structural properties of perlecan

Perlecan is encoded by the gene HSPG2 that is located on the short arm of chromosome 1 (1p35–p36.1), comprises >120 kb and contains 94 exons [65]. The cDNA sequence of human perlecan (14 kb in length) encodes a core protein of 467 kDa composed of five structural domains (Figure 2BGo, roman numbers) [6668]. The N-terminal domain has a primary structure unique for perlecan. Based on secondary structure prediction, this domain was identified as an SEA module [69]. Domain II is composed of four low-density-lipoprotein receptor class A (LA) modules. The similarity to the low-density lipoprotein receptor includes the ligand-binding domain of the receptor. Immediately downstream lies a single IgG repeat that resembles the 21 further repeats found in domain IV. Domain III shares similarity with the short arms of laminin chains. This domain contains four cysteine-rich LE modules similar to domain III of the laminin short arms, alternated by three cysteine-free (LamB) modules resembling domain IV of the laminin short arms. Domain IV, encoded by 2010 nucleotides, is the largest domain of perlecan. The 21 Ig-like repeats found in this domain are similar to neural cell adhesion molecules (N-CAMs), and presumably fold into a globular structure typical of the immunoglobulin superfamily. Domain V, finally, displays similarity to the large globular G domain located on the long arm of the laminin {alpha}1 chain. The six globules of the perlecan core protein (visible by electron microscopy) are probably constituted as follows: domains I and II together form the first globule, domain III provides the second, third and fourth globule, domain IV forms the fifth globule, and domain V represents the C-terminal globule [66,70].

Three glycosaminoglycan chains are linked to domain I, where each residue is attached to a serine hydroxyl group of an SGD tripeptide [71]. The glycosaminoglycan residues contribute to ~50% of the total molecular mass of the proteoglycan. It is important to note that as well as with HS, perlecan may also be glycosylated with other glycosaminoglycans such as chondroitin and dermatan sulfate, or be expressed as a hybrid proteoglycan containing a mixture of glycosaminoglycans [7274]. The glycosylation of perlecan may be regulated differentially in tissues and developmental stages, and influence the functional activities of the molecule.

Functions of perlecan

As well its contribution to glomerular polyanionic sites, multiple functions were demonstrated for perlecan. First, the HS residues of perlecan were shown to participate in mitogenesis and angiogenesis. This activity is mediated by basic fibroblast growth factor (bFGF). After HS binding, bFGF becomes activated and subsequently presented to its functional receptor located on the cell surface [75,76]. The oligosaccharide structure involved in bFGF binding appears to occur preferentially on perlecan, since the cell surface HSPGs syndecan, fibroglycan and glypican showed no bFGF-binding capacity. The bFGF-mediated mitogenic activity of perlecan explains the strongly mitogenic and angiogenic behaviour of perlecan-expressing tumours. Consistently, antisense perlecan cDNA transcription reduces the metastatic potential of human melanoma cells [77]. Besides bFGF, other growth factors were also shown to bind to HS. These include heparin-binding EGF, acidic FGF, platelet-derived growth factor, granulocyte—macrophage colony-stimulating factor, hepatocyte growth factor/scatter factor, interferon-{gamma} and several interleukins.

Second, perlecan mediates cell attachment as shown for endothelial cells and fibroblasts, and acts as a repulsive component for other cell types [74,7881]. Two independent mechanisms were described that may be responsible for these activities: the binding of domain V to ß1 integrins [81], and the binding of the RGD site (of mouse perlecan) in domain III to ß3 integrins [78]. In human perlecan, the RGD site is not conserved, raising the question of whether an alternative mechanism for ß3-mediated cell adhesion could exist.

Perlecan can bind various other molecules in the basement membrane, including nidogen, laminin (enhanced by nidogen), collagen IV and fibronectin. These interactions anchor the proteoglycan in the matrix and increase its strength. Self-association of perlecan was also reported [82]. Perlecan may also be involved in lipoporotein metabolism, since the primary structure of domain II is very similar to the ligand-binding region of the low-density lipoprotein receptor. In addition to a possible lipoprotein-binding function of the perlecan core protein, lipoprotein lipase and apolipoproteins are known to interact with HS. Experimental evidence to confirm this hypothesis is, however, still lacking. Furthermore, the HS chains of perlecan may bind to and activate antithrombin III, which has an anticoagulant effect. This activity requires the presence of specific sequences within the glycosaminoglycan chain, which also occur in heparin [83].

Additional HSPGs in the glomerular basement membrane

Although agrin and perlecan are the only identified HSPGs in the GBM, several lines of evidence indicate the existence of additional HSPGs in the GBM. Fractionation of proteoglycans from GBM extracts yielded distinct classes of HSPGs, that could be distinguished by density [84]. In addition to the low-density HSPGs (now known to represent perlecan and agrin), high-density HSPGs were described containing four HS chains and a much smaller core protein with an estimated molecular mass of 18–128 kDa [85,86]. The immunological relatedness of different HSPGs extracted from the GBM to core proteins of Mr 250, 150 and 75 kDa suggested that they could be generated by proteolytic processing of perlecan [87]. This finding, however, did not rule out the possibility that additional HSPG species exist in the GBM.

More recently, the presence of a `unique' high-density HSPG with a core protein of 22 kDa in the human GBM was demonstrated convincingly [88]. Tryptic fragments from the core protein of this basement membrane-associated molecule contained amino acid sequences that do not occur in human perlecan or human agrin, indicating that the HSPG is encoded by a different gene. Urinary release of this HSPG during episodes of post-exercise proteinuria suggested that it may contribute to charge-selective ultrafiltration [88].

Heparan sulfate proteoglycans and proteinuria

The essential role of HSPGs in glomerular ultrafiltration suggested that alterations in their structure or quantity could be a cause of proteinuria under pathogenic conditions. Under experimental conditions, this proved indeed possible, as selective degradation of HS-associated polyanionic sites induced the passage of anionic ferritin [1]. Human diseases marked by a defective permselectivity of the renal ultrafiltration barrier may, however, originate from more complex mechanisms of pathogenesis. Numerous investigations were performed to analyse the quantity and structure of GBM HSPGs in various nephropathies. The results suggest that the proposed mechanism may contribute to proteinuria in at least some diseases. Below, we discuss the possible role of HSPGs in diabetic nephropathy, minimal change nephrotic syndrome, Denys–Drash syndrome, congenital nephrotic syndrome of the Finnish type and post-exercise proteinuria

Diabetic glomerulopathy

Diabetic nephropathy is perhaps the most illustrative example of a disease where proteinuria is associated with alterations in HSPG composition of the GBM. Patients with diabetes mellitus are at high risk of developing nephropathy as a secondary effect. About 40% of diabetic patients develop microalbuminuria after 10–15 years, which subsequently can progress into overt proteinuria. Especially in juvenile-onset diabetes, end-stage renal failure develops frequently. In most cases of diabetic nephropathy, structural alterations are observed in the glomerulus (diabetic glomerulopathy). Glomeruli typically show a substantial widening of the GBM and expansion of the mesangial matrix. This is reflected by an increased production of matrix components such as collagens, laminin and fibronectin [89]. The thickened diabetic GBM was also shown to contain the chondroitin sulfate proteoglycan bamacan, which is abnormal for the mature GBM [90]. Numerous independent studies have demonstrated a relative decrease of HSPG in the diabetic GBM and in the matrix produced by cultured glomerular epithelial cells in medium containing high glucose levels [89,9193]. Poorly understood is the finding that heparin administration has a protective effect on the structure and function of diabetic glomeruli. Heparin treatment prevented the thickening of the GBM, the decrease in anionic content and the increase in albumin excretion [94,95]. Perlecan transcription is inhibited by high glucose [96], but a major impact of this on GBM permselectivity is not likely. More importantly, agrin is down-regulated in diabetes as shown by a reduced staining of its core protein [89,91]. Elucidation of the molecular mechanisms that influence agrin expression in diabetic glomerulopathy will be of great interest to gain insight into its mode of pathogenesis.

Minimal change nephrotic syndrome

Amongst children, minimal change nephrotic syndrome (MCNS) accounts for 85% of all cases of nephrotic syndrome. Most cases present before the age of 6 years, typically with oedema and a heavy proteinuria of high selectivity. MCNS is treated successfully by steroids, and its prognosis is favourable, although multiple relapses of the nephrotic syndrome are common. The glomeruli appear normal by light microscopy but, at the electron microscopic level, effacement of the epithelial foot processes becomes visible. The nephrotic syndrome was shown to be associated with a reduction of polyanionic sites in the GBM, especially in the lamina rara interna [97,98]. Immunological studies showed a reduction of HS in the GBM, while an antibody against the agrin core protein produced undiminished staining [99]. The loss of the recognized epitope (that is part of the HS chain) may either reflect: (i) a decreased expression of an HSPG other than agrin; (ii) a reduction in the length or number of glycosaminoglycans attached to a core protein expressed in normal amounts; (iii) an altered fine structure of the chain, including its pattern of N-deacetylation/sulfation, O-sulfation and C5- epimerization; or (iv) depolymerization of the chain by reactive oxygen species [12,100]. The observed changes in polyanionic sites may be part of a complex mechanism of pathogenesis. A nephrotic syndrome with the features of MCNS was also observed in laminin ß2-deficient transgenic mice [28]. Finally, the involvement of a circulating factor was suggested by the finding that T-lymphocytes from patients with MCNS produce agents that induce the same disease in rats [97].

Denys–Drash syndrome

Denys–Drash syndrome (DDS) is a severe congenital disease characterized by renal and gonadal abnormalities, including the appearance of Wilms' tumours and severe nephrotic syndrome [101]. The cause of DDS is a heterozygous mutation that affects the DNA-binding domains of the WT-1 gene product, a transcription factor that is expressed during the mesenchymal to epithelial transition of developing kidney and gonads [101,102]. In the mature nephron, the expression of WT-1 is sustained in the podocytes. To investigate the involvement of HSPG in the development of the nephrotic syndrome, the GBM of a patient with DDS was analysed for glycosaminoglycan content. Although the total glycosaminoglycan content of the GBM was normal, the GBM showed a reduced HS content in DDS, and CS levels appeared to be elevated [103]. The immunoreactivity of the agrin core protein was normal.

Congenital nephrotic syndrome of the Finnish type

The congenital nephrotic syndrome of the Finnish type (CNS-F) is a rare disorder in most populations, with the highest incidence in Finland (1 per 8000 deliveries) [104]. Typical clinical features include a premature delivery with enlarged placenta, proteinuria starting in fetal stages, and oedema observed immediately post-partum or soon after birth. Morphological abnormalities include enlarged glomeruli and increased mesangial cells and matrix. In electron microscopy, the GBM shows a normal width, but the epithelial filtration slits are reduced in number [105107]. Although a role for HSPGs in the pathogenesis of CNS-F was suggested [108], several other investigations have contradicted this hypothesis [109,110]. Linkage analysis in both Finnish and non-Finnish pedigrees mapped the gene responsible to the long arm of chromosome 19 (locus 19q13.1) [104,111]. Recently, this novel gene was cloned and named nephrin [112]. The nephrin gene product is a cell surface protein expressed specifically in glomerular slit diaphragms, suggesting an important role in renal ultrafiltration [112]. The possibility that nephrin could be an HSPG has not yet been excluded.

Post-exercise proteinuria

The induction of proteinuria by maximal exercise is common in healthy subjects [113]. The mechanism that underlies this phenomenon is largely unknown, but data suggest that both increased glomerular permeability and reduced tubular uptake are involved [114]. The proteinuria is associated with a temporary decrease in the electrostatic charge of the GBM [113] and urinary excretion of an unidentified GBM HSPG [88]. Standardized exercise protocols may provide a powerful novel tool to investigate if altered expression of this small HSPG could be involved in various types of nephropathy, without the need for a kidney biopsy [115].

Concluding remarks

The hypothesis that HSPGs play a vital role in determining charge-selectivity is still valid at present. There is convincing evidence that GBM polyanionic charge is reduced in several diseases associated with proteinuria. However, the exact mechanisms that may invoke these changes are largely unknown. As the podocyte is a major source of de novo HSPG synthesis, much progress is to be expected from a more detailed investigation of the signalling processes that influence the metabolism, shape and adhesion of the podocyte [16]. The identification of agrin and perlecan as two members of the HSPG population of the GBM provides novel clues for the functioning of the glomerular permselectivity barrier in renal ultrafiltration. The accumulation of agrin in the GBM points to a possible determining role in charge-selective permeability. Agrin could also be involved in cell adhesion and transmembrane signalling. The ultrastructural localization of a third, unidentified HSPG in the GBM and the elucidation of its primary structure are of great interest.

Acknowledgments

The authors are grateful to H. Dijkman for his expert technical assistance in immunoelectron microscopy. This study was supported by grant C93.1309 from the Dutch Kidney Foundation. The laboratory participated in a concerted action entitled `Alterations in extracellular matrix components in diabetic nephropathy and other glomerular diseases', which is financially supported by the EC and the Biomed I program BMH1-CT92–1766.

References

  1. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the GBM to ferritin after removal of glycosaminoglycans (HS) by enzyme digestion. J Cell Biol 1980; 86: 688–693[Abstract]
  2. Hassell JR, Robey PG, Barrach HJ, Wilczek J, Rennard SI, Martin GR. Isolation of a HS-containing proteoglycan from basement membrane. Proc Natl Acad Sci USA 1980; 77: 4494–4498[Abstract]
  3. van Kuppevelt TH, Veerkamp JH. Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes. Microsc Res Tech 1994; 28: 125–140[ISI][Medline]
  4. Kanwar YS, Farquhar MG. Anionic sites in the GBM. In vivo and in vitro localization to the laminae rarae by cationic probes. J Cell Biol 1979; 81: 137–153[Abstract]
  5. Kanwar YS. Biophysiology of glomerular filtration and proteinuria. Lab Invest 1984; 51: 7–21[ISI][Medline]
  6. Rosenzweig LJ, Kanwar YS. Removal of sulfated (HS) or nonsulfated (hyaluronic acid) glycosaminoglycans results in increased permeability of the GBM to 125I-bovine serum albumin. Lab Invest 1982; 47: 177–184[ISI][Medline]
  7. Goode NP, Shires M, Davison AM. The GBM charge-selectivity barrier: an oversimplified concept? Nephrol Dial Transplant 1996; 11: 1714–1716[ISI][Medline]
  8. Kerjaschki D, Sharky DJ, Farquhar MG. Identification and characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 1984; 98: 1591–1596[Abstract]
  9. Kershaw DB, Thomas PE, Wharram BL et al. Molecular cloning, expression, and characterization of podocalyxin-like protein 1 from rabbit as a transmembrane protein of glomerular podocytes and vascular epithelium. J Biol Chem 1995; 270: 29439–29446[Abstract/Free Full Text]
  10. Matsumura K, Yamada Saito F, Sunada Y, Shimizu T. The role of dystroglycan, a novel receptor of laminin and agrin, in cell differentiation. Histol Histopathol 1997; 12: 195–203[ISI][Medline]
  11. Durbeej M, Henry MD, Ferletta M, Campbell KP, Ekblom P. Distribution of dystroglycan in normal adult mouse tissues. J Histochem Cytochem 1998; 46: 449–457[Abstract/Free Full Text]
  12. Kanwar YS, Liu ZZ, Kashihara N, Wallner EI. Current status of the structural and functional basis of glomerular filtration and proteinuria. Semin Nephrol 1991; 11: 390–413[ISI][Medline]
  13. Seiler MW, Venkatachalam MA, Cotran RS. Glomerular epithelium: structural alterations induced by polycations. Science 1975; 189: 390–393[ISI][Medline]
  14. Kanwar YS, Farquhar MG. Detachment of endothelium and epithelium from the GBM produced by kidney perfusion with neuraminidase. Lab Invest 1980; 42: 375–384[ISI][Medline]
  15. Rennke HG, Cotran RS, Venkatachalam MA. Role of molecular charge in glomerular permeability. Tracer studies with cationized ferritins. J Cell Biol 1975; 67: 638–646[Abstract]
  16. Smoyer WE, Mundel P. Regulation of podocyte structure during the development of nephrotic syndrome. J Mol Med 1998; 76: 172–183[ISI][Medline]
  17. Orkin RW, Gehron P, McGoodwin EB, Martin GR, Valentine T, Swarm R. A murine tumor producing a matrix of basement membrane. J Exp Med 1977; 145: 204–220[Abstract]
  18. Paulsson M. Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Mol Biol 1992; 27: 93–127[Abstract]
  19. Hudson BG, Reeders ST, Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol Chem 1993; 268: 26033–26036[Free Full Text]
  20. Yurchenco PD, Schittny JC. Molecular architecture of basement membranes. FASEB J 1990; 4: 1577–1590[Abstract/Free Full Text]
  21. Lohi J, Korhonen M, Leivo I et al. Expression of type IV collagen {alpha}1(IV)–{alpha}6(IV) polypeptides in normal and developing human kidney and in renal cell carcinomas and oncocytomas. Int J Cancer 1997; 72: 43–49[ISI][Medline]
  22. Lemmink HH, Schroder CH, Monnens LA, Smeets HJ. The clinical spectrum of type IV collagen mutations. Hum Mutat 1997; 9: 477–499[ISI][Medline]
  23. Miner JH, Sanes JR. Collagen IV {alpha}3, {alpha}4, and {alpha}5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches. J Cell Biol 1994; 127: 879–891[Abstract]
  24. Kalluri R, Shield CF, Todd P, Hudson BG, Neilson EG. Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis. J Clin Invest 1997; 99: 2470–2478[Abstract/Free Full Text]
  25. Miner JH, Patton BL, Lentz SI et al. The laminin {alpha} chains: expression, developmental transitions, and chromosomal locations of {alpha}1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel {alpha}3 isoform. J Cell Biol 1997; 137: 685–701[Abstract/Free Full Text]
  26. Timpl R, Brown JC. The laminins. Matrix Biol 1994; 14: 275–281[ISI][Medline]
  27. Noakes PG, Miner JH, Gautam M, Cunningham JM, Sanes JR, Merlie JP. The renal glomerulus of mice lacking s-laminin/laminin ß2: nephrosis despite molecular compensation by laminin ß1. Nature Genet 1995; 10: 400–406[ISI][Medline]
  28. Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA. Agrin binds to the nerve–muscle basal lamina via laminin. J Cell Biol 1997; 137: 671–683[Abstract/Free Full Text]
  29. Denzer AJ, Schulthess T, Fauser C et al. Electron microscopic structure of agrin and mapping of its binding site in laminin-1. EMBO J 1998; 17: 335–343[Abstract/Free Full Text]
  30. Battaglia C, Mayer U, Aumailley M, Timpl R. Basement-membrane HSPG binds to laminin by its HS chains and to nidogen by sites in the protein core. Eur J Biochem 1992; 208: 359–366[Abstract]
  31. Reiser J, Kriz W, Wang Z, Kreidberg JA, Mundel P. Establishment and characterization of {alpha}3 integrin deficient mouse podocytes. J Am Soc Nephrol1998;526A (abstract)
  32. Falk M, Salmivirta K, Durbeej M et al. Integrin {alpha}6B ß1 is involved in kidney tubulogenesis in vitro. J Cell Sci 1996; 109: 2801–2810[Abstract/Free Full Text]
  33. Durbeej M, Ekblom P. Dystroglycan and laminins: glycoconjugates involved in branching epithelial morphogenesis. Exp Lung Res 1997; 23: 109–118[ISI][Medline]
  34. Ekblom P. Receptors for laminins during epithelial morphogenesis. Curr Opin Cell Biol 1996; 8: 700–706[ISI][Medline]
  35. Olsen DR, Nagayoshi T, Fazio MJ et al. Human nidogen: cDNA cloning, cellular expression, and mapping of the gene to chromosome 1q43. Am J Hum Genet 1998; 44: 876
  36. Kanwar YS, Hascall VC, Farquhar MG. Partial characterization of newly synthesized proteoglycans isolated from the GBM. J Cell Biol 1981; 90: 527–532[Abstract]
  37. van den Heuvel LP, Veerkamp JH, Monnens LA, Schroder CH. HSPG from human and equine glomeruli and tubules. Int J Biochem 1988; 20: 1391–1400[ISI][Medline]
  38. McCarthy KJ, Bynum K, St.John PL, Abrahamson DR, Couchman JR. Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development. J Histochem Cytochem 1993; 41: 401–414[Abstract/Free Full Text]
  39. Wu RR, Couchman JR. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs. J Cell Biol 1997; 136: 433–444[Abstract/Free Full Text]
  40. Van den Heuvel LP, van den Born J, van de Velden TJ et al. Isolation and partial characterization of HSPG from the human GBM. Biochem J 1989; 264: 457–465[ISI][Medline]
  41. Ruegg MA, Bixby JL. Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction. Trends Neurosci 1998; 21: 22–27[ISI][Medline]
  42. Denzer AJ, Gesemann M, Ruegg MA. Diverse functions of the extracellular matrix molecule agrin. Semin Neurosci 1996; 8: 357–366[ISI]
  43. Tsen G, Halfter W, Kroger S, Cole GJ. Agrin is a HSPG. J Biol Chem 1995; 270: 3392–3399[Abstract/Free Full Text]
  44. Godfrey EW. Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein. Exp Cell Res 1991; 195: 99–109[ISI][Medline]
  45. Biroc SL, Payan DG, Fisher JM. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors. Brain Res Dev Brain Res 1993; 75: 119–129[ISI][Medline]
  46. Barber AJ, Lieth E. Agrin accumulates in the brain microvascular basal lamina during development of the blood–brain barrier. Dev Dynam 1997; 208: 62–74[ISI][Medline]
  47. Groffen AJ, Buskens CA, van Kuppevelt TH, Veerkamp JH, Monnens LA, van den Heuvel LP. Primary structure and high expression of human agrin in basement membranes of adult lung and kidney. Eur J Biochem 1998; 254: 123–128[Abstract]
  48. Groffen AJ, Ruegg MA, Dijkman H et al. Agrin is a major HSPG in the human GBM. J Histochem Cytochem 1998; 46: 19–27[Abstract/Free Full Text]
  49. Rupp F, Ozcelik T, Linial M, Peterson K, Francke U, Scheller R. Structure and chromosomal localization of the mammalian agrin gene. J Neurosci 1992; 12: 3535–3544[Abstract]
  50. Cole GJ, Halfter W. Agrin: an extracellular matrix HSPG involved in cell interactions and synaptogenesis. Perspect Dev Neurobiol 1996; 3: 359–371[ISI][Medline]
  51. Gautam M, Noakes PG, Moscoso L et al. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 1996; 85: 525–535[ISI][Medline]
  52. Hopf C, Hoch W. Tyrosine phosphorylation of the muscle-specific kinase is exclusively induced by AChR-aggregating agrin fragments. Eur J Biochem 1998; 253: 382–389[Abstract]
  53. Meier T, Masciulli F, Moore C et al. Agrin can mediate AChR gene expression in muscle by aggregation of muscle-derived neuregulins. J Cell Biol 1998; 141: 715–726[Abstract/Free Full Text]
  54. Campagna JA, Ruegg MA, Bixby JL. Agrin is a differentiation-inducing `stop signal' for motoneurons in vitro. Neuron 1995; 15: 1365–1374[ISI][Medline]
  55. Gesemann M, Brancaccio A, Schumacher B, Ruegg MA. Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue. J Biol Chem 1998; 273: 600–605[Abstract/Free Full Text]
  56. Kriz W, Hackenthal E, Nobling R, Sakai T, Elger M, Hahnel B. A role for podocytes to counteract capillary wall distension. Kidney Int 1994; 45: 369–376[ISI][Medline]
  57. Martin PT, Sanes JR. Integrins mediate adhesion to agrin and modulate agrin signaling. Development 1997; 124: 3909–3917[Abstract/Free Full Text]
  58. Sterk LM, de Melker AA, Kramer DC et al. Glomerular extracellular matrix components and integrins. Cell Adhesion Commun 1998; 5: 177–192[ISI][Medline]
  59. Adler S, Eng B. Integrin receptors and function on cultured glomerular endothelial cells. Kidney Int 1993; 44: 278–284[ISI][Medline]
  60. O'Toole JJ, Deyst KA, Bowe MA, Nastuk MA, McKechnie BA, Fallon JR. Alternative splicing of agrin regulates its binding to heparin {alpha}-dystroglycan, and the cell surface. Proc Natl Acad Sci USA 1996; 93: 7369–7374[Abstract/Free Full Text]
  61. Gesemann M, Cavalli V, Denzer AJ, Brancaccio A, Schumacher B, Ruegg MA. Alternative splicing of agrin alters its binding to heparin, dystroglycan, and the putative agrin receptor. Neuron 1996; 16: 755–767[ISI][Medline]
  62. Paulsson M, Yurchenco PD, Ruben GC, Engel J, Timpl R. Structure of low density HSPG isolated from a mouse tumor basement membrane. J Mol Biol 1987; 197: 297–313[ISI][Medline]
  63. Murdoch AD, Liu B, Schwarting R, Tuan RS, Iozzo RV. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem 1994; 42: 239–249[Abstract/Free Full Text]
  64. Handler M, Yurchenco PD, Iozzo RV. Developmental expression of perlecan during murine embryogenesis. Dev Dynam 1997; 210: 130–145[ISI][Medline]
  65. Groffen AJ, Hop FW, Tryggvason K et al. Evidence for the existence of multiple HSPGs in the human GBM and mesangial matrix. Eur J Biochem 1997; 247: 175–182[Abstract]
  66. Cohen IR, Grassel S, Murdoch AD, Iozzo RV. Structural characterization of the complete human perlecan gene and its promoter. Proc Natl Acad Sci USA 1993; 90: 10404–10408[Abstract]
  67. Noonan DM, Fulle A, Valente P et al. The complete sequence of perlecan, a basement membrane HSPG, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. J Biol Chem 1991; 266: 22939–22947[Abstract/Free Full Text]
  68. Kallunki P, Tryggvason K. Human basement membrane HSPG core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Cell Biol 1992; 116: 559–571[Abstract]
  69. Murdoch AD, Dodge GR, Cohen I, Tuan RS, Iozzo RV. Primary structure of the human HSPG from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Biol Chem 1992; 267: 8544–8557[Abstract/Free Full Text]
  70. Bork P, Patthy L. The SEA module: a new extracellular domain associated with O-glycosylation. Protein Sci 1995; 4: 1421–1425[Abstract/Free Full Text]
  71. Schulze B, Sasaki T, Costell M, Mann K, Timpl R. Structural and cell-adhesive properties of three recombinant fragments derived from perlecan domain III. Matrix Biol 1996; 15: 349–357[ISI][Medline]
  72. Costell M, Mann K, Yamada Y, Timpl R. Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides. Eur J Biochem 1997; 243: 115–121[Abstract]
  73. Couchman JR, Kapoor R, Sthanam M, Wu RR. Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Engelbreth–Holm–Swarm tumor matrix. J Biol Chem 1996; 271: 9595–9602[Abstract/Free Full Text]
  74. Kokenyesi R, Silbert JE. Formation of heparan sulfate or chondroitin/dermatan sulfate on recombinant domain I of mouse perlecan expressed in Chinese hamster ovary cells. Biochem Biophys Res Commun 1995; 211: 262–267[ISI][Medline]
  75. SundarRaj N, Fite D, Ledbetter S, Chakravarti S, Hassell JR. Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J Cell Sci 1995; 108: 2663–2672[Abstract/Free Full Text]
  76. Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 1994; 79: 1005–1013[ISI][Medline]
  77. Guillonneau X, Tassin J, Berrou E, Bryckaert M, Courtois Y, Mascarelli F. In vitro changes in plasma membrane HSPGs and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity. J Cell Physiol 1996; 166: 170–187[ISI][Medline]
  78. Adatia R, Albini A, Carlone S et al. Suppression of invasive behavior of melanoma cells by stable expression of anti-sense perlecan cDNA. Ann Oncol 1997; 8: 1257–1261[Abstract]
  79. Hayashi K, Madri JA, Yurchenco PD. Endothelial cells interact with the core protein of basement membrane perlecan through ß1 and ß3 integrins: an adhesion modulated by glycosaminoglycan. J Cell Biol 1992; 119: 945–959[Abstract]
  80. Klein G, Conzelmann S, Beck S, Timpl R, Muller CA. Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells. Matrix Biol 1995; 14: 457–465[ISI][Medline]
  81. Chakravarti S, Horchar T, Jefferson B, Laurie GW, Hassell JR. Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence. J Biol Chem 1995; 270: 404–409[Abstract/Free Full Text]
  82. Brown JC, Sasaki T, Gohring W, Yamada Y, Timpl R. The C-terminal domain V of perlecan promotes ß1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem 1997; 250: 39–46[Abstract]
  83. Yurchenco PD, Cheng YS, Ruben GC. Self-assembly of a high molecular weight basement membrane HSPG into dimers and oligomers. J Biol Chem 1987; 262: 17668–17676[Abstract/Free Full Text]
  84. Pejler G, Backstrom G, Lindahl U et al. Structure and affinity for antithrombin of HS chains derived from basement membrane proteoglycans. J Biol Chem 1987; 262: 5036–5043[Abstract/Free Full Text]
  85. Hassell JR, Leyshon WC, Ledbetter SR et al. Isolation of two forms of basement membrane proteoglycans. J Biol Chem 1985; 260: 8098–8105[Abstract/Free Full Text]
  86. Kanwar YS, Veis A, Kimura JH, Jakubowski ML. Characterization of HSPG of glomerular basement membranes. Proc Natl Acad Sci USA 1984; 81: 762–766[Abstract]
  87. Edge AS, Spiro RG. Selective deglycosylation of the HSPG of bovine GBM and identification of the core protein. J Biol Chem 1987; 262: 6893–6898[Abstract/Free Full Text]
  88. Klein DJ, Brown DM, Oegema TR et al. GBM proteoglycans are derived from a large precursor. J Cell Biol 1988; 106: 963–970[Abstract]
  89. Heintz B, Stöcker G, Mrowka C et al. Decreased GBM HSPG in essential hypertension. Hypertension 1995; 25: 399–407[Abstract/Free Full Text]
  90. van den Born J, van Kraats AA, Bakker MA et al. Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in GBM heparan sulphate. Diabetologia 1995; 38: 161–172[ISI][Medline]
  91. McCarthy KJ, Abrahamson DR, Bynum KR, St. John PL, Couchman JR. Basement membrane-specific chondroitin sulfate proteoglycan is abnormally associated with the glomerular capillary basement membrane of diabetic rats. J Histochem Cytochem 1994; 42: 473–484[Abstract/Free Full Text]
  92. Tamsma JT, van den Born J, Bruijn JA et al. Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the GBM. Diabetologia 1994; 37: 313–320[ISI][Medline]
  93. Goode NP, Shires M, Crellin DM, Aparicio SR, Davison AM. Alterations of GBM charge and structure in diabetic nephropathy. Diabetologia 1995; 38: 1455–1465[ISI][Medline]
  94. van Det NF, van den Born J, Tamsma JT et al. Effects of high glucose on the production of HSPG by mesangial and epithelial cells. Kidney Int 1996; 49: 1079–1089[ISI][Medline]
  95. Ceol M, Nerlich A, Baggio B et al. Increased glomerular {alpha}1 (IV) collagen expression and deposition in long-term diabetic rats is prevented by chronic glycosaminoglycan treatment. Lab Invest 1996; 74: 484–495[ISI][Medline]
  96. Myrup B, Hansen PM, Jensen T et al. Effect of low-dose heparin on urinary albumin excretion in insulin-dependent diabetes mellitus. Lancet 1995; 345: 421–422[ISI][Medline]
  97. Kasinath BS, Grellier P, Choudhury GG, Abboud SL. Regulation of basement membrane HSPG, perlecan, gene expression in glomerular epithelial cells by high glucose medium. J Cell Physiol 1996; 167: 131–136[ISI][Medline]
  98. Maruyama K, Tomizawa S, Shimabukuro N, Fukuda T, Johshita T, Kuroume T. Effect of supernatants derived from T lymphocyte culture in minimal change nephrotic syndrome on rat kidney capillaries. Nephron 1989; 51: 73–76[ISI][Medline]
  99. Cheung PK, Baller JF, Bakker WW. Impairment of endothelial and subendothelial sites by a circulating plasma factor associated with minimal change disease. Nephrol Dial Transplant 1996; 11: 2185–2191[Abstract]
  100. van den Born J, van den Heuvel LP, Bakker MA et al. Distribution of GBM HSPG core protein and side chains in human glomerular diseases. Kidney Int 1993; 43: 454–463[ISI][Medline]
  101. Raats CJ, Bakker MA, van den Born J, Berden JH. Hydroxyl radicals depolymerize glomerular HS in vitro and in experimental nephrotic syndrome. J Biol Chem 1997; 272: 26734–26741[Abstract/Free Full Text]
  102. Little M, Holmes G, Bickmore W, van Heyningen V, Hastie N, Wainwright B. DNA binding capacity of the WT1 protein is abolished by Denys–Drash syndrome WT1 point mutations. Hum Mol Genet 1995; 4: 351–358[Abstract]
  103. Rauscher FJ, III. The WT1 Wilms tumor gene product: a developmentally regulated transcription factor in the kidney that functions as a tumor suppressor. FASEB J 1993; 7: 896–903[Abstract/Free Full Text]
  104. van den Heuvel LP, Westenend PJ, van den Born J, Assmann KJ, Knoers N, Monnens LA. Aberrant proteoglycan composition of the GBM in a patient with Denys–Drash syndrome. Nephrol Dial Transplant 1995; 10: 2205–2211[Abstract]
  105. Mannikko M, Lenkkeri U, Kashtan CE, Kestila M, Holmberg C, Tryggvason K. Haplotype analysis of congenital nephrotic syndrome of the Finnish type in non-Finnish families. J Am Soc Nephrol 1996; 7: 2700–2703[Abstract]
  106. Autio-Harmainen H, Vaananen R, Rapola J. Scanning electron microscopic study of normal human glomerulogenesis and of fetal glomeruli in congenital nephrotic syndrome of the Finnish type. Kidney Int 1981; 20: 747–752[ISI][Medline]
  107. Autio-Harmainen H, Rapola J. The thickness of the GBM in congenital nephrotic syndrome of the Finnish type. Nephron 1983; 34: 48–50[ISI][Medline]
  108. Haltia A, Solin ML, Holmberg C, Reivinen J, Miettinen A, Holthofer H. Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome. Pediatr Res 1998; 43: 410–414[Abstract]
  109. Vernier RL, Klein DJ, Sisson SP, Mahan JD, Oegema TR, Brown DM. HS-rich anionic sites in the human GBM. Decreased concentration in congenital nephrotic syndrome. N Engl J Med 1983; 309: 1001–1009[Abstract]
  110. van den Heuvel LP, van den Born J, Jalanko H et al. The glycosaminoglycan content of renal basement membranes in the congenital nephrotic syndrome of the Finnish type. Pediatr Nephrol 1992; 6: 10–15[ISI][Medline]
  111. Ljungberg P, Rapola J, Holmberg C, Holthofer H, Jalanko H. Glomerular anionic charge in congenital nephrotic syndrome of the Finnish type. Histochem J 1995; 27: 536–546[ISI][Medline]
  112. Mannikko M, Kestaila M, Holmberg C et al. Fine mapping and haplotype analysis of the locus for congenital nephrotic syndrome on chromosome 19q13.1. Am J Hum Genet 1995; 57: 1377–1383[ISI][Medline]
  113. Kestila M, Lenkkeri U, Mannikko M et al. Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome. Mol Cell 1998; 1: 575–582[ISI][Medline]
  114. Poortmans JR, Vanderstraeten J. Kidney function during exercise in healthy and diseased humans. An update. Sports Med 1994; 18: 419–437[ISI][Medline]
  115. Poortmans JR, Brauman H, Staroukine M, Verniory A, Decaestecker C, Leclercq R. Indirect evidence of glomerular/tubular mixed-type postexercise proteinuria in healthy humans. Am J Physiol 1988; 254: F277–F283[Abstract/Free Full Text]
  116. Stocker G, Stickeler E, Switalla S, Fischer DC, Greiling H, Haubeck HD. Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney. Eur J Clin Chem Clin Biochem 1997; 35: 95–99[ISI][Medline]