Heparan sulfate in glomerular inflammation
From NIER
by Dr. Angelique Rops and Dr. Johan van der Vlag
Nephrology Research Laboratory, Nijmegen Medical Centre for Molecular Life Sciences, Division of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Originally published as general introduction to the PhD thesis of Dr. Rops, which she published in June 2007 under supervision of her promotor Prof. dr. Jo H.M. Berden and co-promotores Dr. Johan van der Vlag, Dr. Toin H. van Kuppevelt and Dr. Lambert P.W.J. van den Heuvel at the Nijmegen Medical Centre for Molecular Life Sciences and Radboud University Nijmegen Medical Centre in Nijmegen, The Netherlands.
Contents |
[edit] Abstract
Proliferative forms of glomerulonephritis are characterized by the influx of inflammatory cells (leukocytes), proteinuria, hematuria and decline in renal function. Heparan sulfate proteoglycans (HSPGs) play a prominent role in inflammatory processes. HSPGs consist of a core protein to which linear heparan sulfate (HS) side chains are attached. HS chains consist of up to 150 disaccharide units of α(1-4)-uronic acid-β(1-4)-glucosamine, which can be modified at different positions by numerous enzymes, which include N-deacetylases, N- and O-sulfotransferases, C5-epimerase, sulfatases and hydrolases. The possible HS modifications give rise to an enormous dynamic and structural complexity of the HS chain, which corresponds to the variety of biologic functions mediated by HS, including its role in inflammation. During inflammation, HS in the extracellular matrix (ECM) and at the surfaces of leukocytes and endothelial cells binds chemokines, which directs the movement and activation of leukocytes. Endothelial and leukocyte cell surface HS also interacts directly with adhesion molecules like selectins and integrins. In particular HS on activated endothelium is crucial for the interaction with leukocytes. The common practice for treatment of glomerulonephritis is administration of immunosuppressive drugs like corticosteroids and cytotoxic immunosuppressives. However, these drugs have unwanted adverse effects and it takes several days before they become effective. During this window phase, extensive structural damage of the glomerulus can occur. It remains elusive which structures within HS on activated glomerular endothelium are important for the interaction with leukocytes. Identification of these structures could lead to the rational development of defined glycomimetics (HS-based therapeutics) for the supplementary treatment of glomerulonephritis. In this thesis, the expression of specific HS domains by glomerular endothelial cells and their function in the interaction with leukocytes are evaluated in vitro in leukocyte-endothelium adhesion assays, in vivo in experimental forms of glomerulonephritis and in patients with systemic lupus erythematosus (SLE) with glomerulonephritis.
[edit] The glomerulus
The glomerulus consists of a complex network of capillaries within Bowman’s capsule, which enables the ultrafiltration process. The main barrier in the ultrafiltration process is the glomerular capillary wall, which consists of a layer of fenestrated endothelial cells that are attached to the glomerular basement membrane (GBM), which is covered at the outer, urinary space with visceral epithelial cells, also called podocytes. The mesangial cell is the third glomerular cell type and located between the capillaries and regulates dynamic forces1,2 (Figure 1). The GBM is organized as a dense network of interacting ECM molecules, such as collagens, laminins and HSPGs. The glomerular capillary wall dictates the charge- and size-properties by allowing the passage of small and positively charged molecules into the urinary space, but by restricting the passage of large and negatively charged molecules into the urinary space3. In recent years, it has been shown that proteins like nephrin, podocin and CD2AP in the slit diaphragm between the podocyte foot processes are of prime importance for the shape and function of the podocyte. Patients and mice with mutations in genes encoding components of the slit diaphragm protein complex develop proteinuria, which is associated with effacement of podocyte foot processes4-6.
Figure 1. Schematic representation of the structure of the glomerular capillary wall. The glomerular capillary wall consists of fenestrated endothelium, which lines the lumen of the microcapillaries and covers the glomerular basement membrane (GBM) that is covered at the urinary site with podocytes. Mesangium is situated between capillary loops. Reproduced with permission from W. Kriz (Institute of Anatomy and Cell Biology, University of Heidelberg, Germany).
The charge-dependent permeability of the glomerular capillary wall and in particular the GBM may be determined by the negatively charged HS polysaccharide side chains of HSPGs in the GBM. Furthermore, negatively charged sialoproteins like podocalyxin and α-dystroglycan, and glycosaminoglycans (GAGs) at the cell surface of both glomerular endothelial cells and podocytes may contribute to the charge-selective properties of the capillary filter7-10. Apart from a possible function in filtration, HS in the glycocalyx on glomerular endothelium plays an important role in the interaction with leukocytes, as will be discussed below. Furthermore, it dictates the availability and function of several chemokines, growth factors and coagulation factors. It is also important as a defense mechanism for reactive oxygen species generated during inflammation.
[edit] Proliferative glomerulonephritis
Glomerulonephritis can be classified as a primary renal disease, i.e. when only the glomerulus is affected, or as a secondary renal disease, i.e. when the glomerulus is damaged as a result of a systemic disease like a systemic autoimmune disease11. Proliferative forms of glomerulonephritis are characterized by an acute or chronic inflammation of the glomeruli, which can involve all three glomerular cell types, i.e. glomerular endothelial cells, podocytes and mesangial cells, respectively. Several types of glomerulonephritis are distinguished by light microscopy, immunofluorescence and electron microscopy. By light microscopy the proliferating cell types are identified, while by immunofluorescence and electron microscopy, the localization of immune complexes or immunoglobulin (Ig) deposits is identified. Glomerulonephritis characterized by an endothelial pattern of glomerular injury is manifested by glomerular influx of leukocytes like polymorphonuclear granulocytes (PMNs), endothelial swelling and cellular proliferation associated with proteinuria and hematuria. Glomerulonephritis characterized by an epithelial (podocytic) pattern of glomerular injury is manifested by podocyte degeneration and dedifferentiation, podocyte detachment from the GBM and severe proteinuria without glomerular leukocyte influx or cellular proliferation. Glomerulonephritis characterized by a mesangial pattern of glomerular injury is manifested by mesangial cell proliferation and matrix expansion associated with proteinuria, hematuria and variable glomerular leukocyte infiltration11,12.
Glomerulonephritis with a typical glomerular endothelial pattern of injury includes anti-GBM nephritis, lupus nephritis, postinfectious glomerulonephritis, mesangiocapillary glomerulonephritis, dense deposit disease, systemic vasculitis glomerulonephritis, antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis and cryoglobulinemia-associated glomerulonephritis. Frequently, glomerulonephritis with this typical glomerular endothelial pattern of injury is induced by the binding of antibodies to an antigen in the GBM or the accumulation of antibodies or antigen-antibody complexes in the subendothelial space or in the GBM, which then may lead to complement activation and glomerular PMN influx. The PMNs may cause glomerular endothelial injury by secretion of proteases. At a later stage, after capillary loop necrosis, extracapillary cell proliferation may be observed in the urinary space between Bowman’s capsule and the capillary tuft, which then results in crescent formation and finally to loss of renal function13.
[edit] Heparan sulfate proteoglycans
HSPGs consist of a core protein with covalently attached HS sugar side chains, which are located in basement membranes and at cell surfaces. HSPGs are involved in various biologic processes such as cell adhesion, migration, proliferation and differentiation, which are mediated by binding of chemokines, cytokines, enzymes, growth factors or other bio-active molecules to the HS side chain. HS belongs to the family of strongly negatively charged GAGs that also includes heavily sulfated heparin and unsulfated hyaluran. HS is a linear chain that consists of up to 150 repeating disaccharide units of glucosamine α(1-4)-uronic acid-β(1-4). The HS chain can be modified extensively, which starts with the N-deacetylation and N-sulfation of N-acetyl-glucosamine by different isoforms of N-deacetylase/N-sulfotransferases (NDSTs) followed by C5-epimerization of glucuronate to iduronate by C5-epimerase and O-sulfation at various positions (C-2, C-3, and C-6) by different sulfotransferases14-16. The modifications of the HS chain result in a block-like structure of alternating N-acetylated, mixed N-acetylated/N- and O-sulfated and N- and O-sulfated (heparin-like) domains. The HS chains may also be modified by (extracellular) heparanase (HPSE), a β(1-4)-endoglucuronidase that cleaves at specific sites within HS, while 6-O-sulfate groups may be removed by two extracellular HS 6-O-endosulfatases (Sulf-1 and Sulf-2). Finally, several lysosomal enzymes (hydrolases, N- and O-sulfatases) are responsible for the intracellular degradation of HS. The modification process gives rise to an enormous structural diversity within one HS chain, which is associated with the binding of a myriad of different factors and corresponding functions14,16-18.
[edit] The role of heparan sulfate in (glomerular) inflammation
A central theme in inflammatory events is the attraction and transmigration of leukocytes from the circulation through the endothelium and the ECM into the injured tissue. The leukocyte-endothelium interaction occurs in a sequential four-step process19: (i) tethering of leukocytes to the activated endothelium; (ii) rolling of leukocytes over the endothelium; (iii) firm adhesion of leukocytes to the endothelium; and (iv) transmigration (diapedesis) of leukocytes through the endothelium into the injured tissue. In all these steps, HS plays a prominent role20 (Figure 2). Tethering of leukocytes involves binding of chemokines to HS on activated endothelial cells and in the ECM. For some chemokines it has been established that O-sulfation of HS promotes chemokine binding22,23. It has been reported that activation of microvascular endothelial cells by proinflammatory cytokines leads to an increased expression of sulfated HS domains and binding of RANTES (regulated upon activation, normal T cell expressed and secreted), which was mediated by an increased NDST-1 expression24. Rolling of leukocytes is mediated by L-selectin expressed by leukocytes and E- and P-selectin expressed by endothelial cells and/or platelets. HS can serve as a ligand for both L- and P-selectin, which has also been shown in the kidney25-29. Selectins bind 6-O-sulfated HS domains, whereas N-sulfation seems to be less important30-32. In a recent study it has been shown that a mouse conditionally deficient in NDST-1 and/or NDST-2 in both endothelial cells and leukocytes showed an impaired inflammatory response, which was associated with a 2-3-fold reduction of N-, 2-O- and 6-O-sulfated HS domains on the endothelium and a higher velocity of rolling leukocytes33. Firm adhesion of leukocytes involves interactions between integrins and integrin ligands like intercellular adhesion molecule (ICAM)-1. Endothelial HS is a ligand for the integrin Mac-1 that is expressed by leukocytes and both N- and O-sulfation seem to be important for this process32-35. Transmigration of leukocytes also involves HS-binding factors like HS-bound chemokines that attract/direct leukocytes through the endothelium into the injured tissue, while HS/heparin can influence this process, in which N-sulfation seems not to be essential22,33,34,36.
Figure 2. Multiple roles of heparan sulfate in leukocyte entry into sites of inflammation. HS on activated endothelium binds L-selectin on leukocytes (granulocytes, monocytes and lymphocytes) during the rolling phase of leukocytes over the endothelium. Endothelial HS binds and presents chemokines to chemokine receptors on leukocytes, which leads to activation of leukocytes and movement of leukocytes towards the site of inflammation, while HS is also involved in the transport of chemokines across the endothelial cell barrier. Endothelial HS binds integrins on leukocytes like Mac-1 (not shown in this figure), which results in the firm adhesion of leukocytes to the activated endothelium. Finally, endothelial HS may play a role in the transmigration of leukocytes through the endothelial layer into the tissue that may involve heparin-binding protein (not shown in this figure).
ICAM, intercellular adhesion molecule; PSGL-1, P-selectin glycoprotein ligand-1; TNF, tumor necrosis factor. Reproduced with permission from Macmillan Publishers Ltd: Nature Immunology21.
In addition to the role of HS in the process of leukocyte trafficking, HPSE that is expressed by activated endothelial cells and leukocytes degrades HS in the inflamed area, thus facilitating transmigration of leukocytes37,38. The cleavage of HS chains may also affect the binding of chemokines or other HS ligands important for tethering/chemotaxis, rolling, firm adhesion or transmigration of leukocytes. Interestingly, a HS cleavage product produced by HPSE is able to neutralize the inflammatory cytokine tumor necrosis factor (TNF)-α39. Elastase, a protease that is expressed by activated leukocytes, thereby facilitating their migration, can also be inhibited by HS/heparin40.
There are few data on (structural) changes in the expression of glomerular HS during glomerulonephritis. In various proliferative forms of glomerulonephritis a reduction of HS in the GBM has been observed41, but the expression of HS on glomerular endothelium was not evaluated. In (experimental) lupus nephritis it appeared that the decreased staining of HS in the GBM was negatively correlated with proteinuria and the presence of Ig deposits42. Also in Heymann nephritis, which is a model for membranous glomerulopathy, a decreased expression of HS in the GBM was demonstrated43. Finally, in vitro studies with bovine glomerular endothelial and activated human microvascular cells showed that activation with proinflammatory cytokines led to an increased expression and secretion of sulfated HS24,44. In summary, it has been shown that N-sulfated and 6-O-sulfated HS residues on activated (microvascular) endothelial cells from different vascular beds play a role in the interaction with leukocytes20,31,33,45. However, it remains elusive which HS structures on activated glomerular endothelium are important for the interaction with leukocytes.
[edit] Experimental models used in this study
[edit] In vitro adhesion of leukocytes to cultured glomerular endothelial cells
The functional role of endothelial HS structures in the interaction with leukocytes can be studied by performing adhesion experiments under either static or dynamic flow conditions. Under static conditions only the firm adhesion of leukocytes to endothelium can be determined. Adhesion under conditions of dynamic blood flow can be mimicked by using a flow chamber, which enables the separation of the rolling and firmly adhering phase of leukocytes on endothelium, while also the rolling velocity of the leukocytes can be determined46. The shear forces in a flow chamber, originated by the fluid flow, are indicated by the wall shear rate, while the friction between individual blood elements or between blood elements and the vessel wall can be calculated by multiplying the shear rate with the fluid viscosity, which is called the wall shear stress (τ). In a flow chamber the wall shear stress τ (in dynes/cm2) can be calculated according to the Navier Stokes equation:
τ = (6 x Q x µ)/(w x h2), in which Q is the volumetric flow rate, µ is the viscosity of the fluid, w is the slid width and h is the height of the flow chamber.
[edit] Experimental anti-GBM nephritis
Experimental anti-GBM nephritis is a well-characterized model of glomerulonephritis, which is induced by the administration of heterologous anti-GBM antibodies47. Two phases can be distinguished in experimental anti-GBM nephritis. The first, acute or heterologous phase is the result of a rapid fixation of the injected heterologous anti-GBM antibodies to the glomerular capillary wall and is associated with glomerular leukocyte influx. The second, autologous phase is characterized by the immune response of the host against the heterologous antibodies, which leads to an amplification of the disease.
[edit] Systemic lupus erythematosus (SLE)
SLE is a systemic autoimmune disease that is characterized by the development of autoantibodies against nuclear antigens. SLE is associated with a broad spectrum of clinical manifestations that frequently involves glomerulonephritis. SLE glomerulonephritis is characterized by granular deposits of Ig and complement factors along the glomerular capillary wall and in the mesangium. The extent of Ig deposits in the capillary loops and the albuminuria is correlated with a decreased expression of HS in the GBM42. Several mouse strains, including the MRL/lpr mouse, spontaneously develop an autoimmune disease similar to human SLE48.
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