Detergent-resistant globotriaosyl ceramide may define verotoxin/glomeruli-restricted hemolytic uremic syndrome pathology
2009; Elsevier BV; Volume: 75; Issue: 11 Linguagem: Inglês
10.1038/ki.2009.7
ISSN1523-1755
AutoresFahima Khan, François Proulx, Clifford A. Lingwood,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoVerotoxin binding to its receptor, globotriaosyl ceramide(Gb3) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb3 is expressed in both tubular and glomerular cells. Gb3 within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against verotoxin glomerular binding. Mouse glomeruli remained verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb3 in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb3 ligand) was detergent sensitive, tubule-restricted, and inhibited by verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb3 organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome. Verotoxin binding to its receptor, globotriaosyl ceramide(Gb3) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb3 is expressed in both tubular and glomerular cells. Gb3 within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against verotoxin glomerular binding. Mouse glomeruli remained verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb3 in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb3 ligand) was detergent sensitive, tubule-restricted, and inhibited by verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb3 organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome. The more rigid membrane domains of the liquid ordered phase are easily demonstrable in model systems1.London E. Insights into lipid raft structure and formation from experiments in model membranes.Curr Opin Struct Biol. 2002; 12: 480Crossref PubMed Scopus (223) Google Scholar but the relevance of such models in terms of normal cellular physiology have frequently been questioned,2.Munro S. Lipid rafts: elusive or illusive?.Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 3.Lagerholm B.C. Weinreb G.E. Jacobson K. et al.Detecting microdomains in intact cell membranes.Annu Rev Phys Chem. 2005; 56: 309-336Crossref PubMed Scopus (181) Google Scholar primarily due to the difficulty of isolation of such microdomains (or 'lipid rafts') from living cells. 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Such infections and lesions spontaneously resolve in most cases, but in a minor subset of individuals, primarily in the very young and old, a second phase of pathology may occur.23.Tarr P.I. Neill M.A. Clausen C.R. et al.Escherichia coli O157:H7 and the hemolytic uremic syndrome: importance of early cultures in establishing the etiology.J Infect Dis. 1990; 162: 553-556Crossref PubMed Scopus (192) Google Scholar This is termed hemolytic uremic syndrome (HUS),24.Andreoli S.P. The pathophysiology of the hemolytic uremic syndrome.Curr Opin Nephrol Hypertens. 1999; 8: 459-464Crossref PubMed Scopus (44) Google Scholar whereby systemic verotoxemia targets the renal microvasculature to induce infarcts within the glomerulus and the ensuing thrombocytopenia and hemolysis can yet be fatal.25.Goldwater P.N. 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Gao F. et al.Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy.Nat Med. 2002; 8: 522-526Crossref PubMed Scopus (246) Google Scholar Gb3 can be found in DRMs in the plasma membrane of cultured cells and mediate transmembrane signals.34.Katagiri Y. Mori T. Nakajima H. et al.Activation of Src family kinase induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains.J Biol Chem. 1999; 274: 35278-35282Crossref PubMed Scopus (148) Google Scholar, 35.Mori T. Kiyokawa N. Katagiri Y.U. et al.Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor-mediated apoptosis by regulating Lyn kinase activity in human B cells.Exp Hematol. 2000; 28: 1260-1268Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 36.Takenouchi H. Kiyokawa N. Taguchi T. et al.Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells.J Cell Sci. 2004; 117: 3911-3922Crossref PubMed Scopus (69) Google Scholar VT cytotoxicity in cell culture is dependent on Gb3 presentation within a DRM format.37.Falguieres T. Mallard F. Baron C. et al.Targeting of Shiga toxin b-subunit to retrograde transport route in association with detergent-resistant membranes.Mol Biol Cell. 2001; 12: 2453-2468Crossref PubMed Scopus (223) Google Scholar, 38.Hoey D.E.E. Currie C. Lingwood C.A. et al.Binding of verotoxin 1 to primary intestinal epithelial cells expressing Gb3 results in trafficking of toxin to lysosomal compartments.Cell Microbiol. 2003; 5: 85-97Crossref PubMed Scopus (81) Google Scholar, 39.Smith D.C. Sillence D.J. Falguieres T. et al.The association of Shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for a cytotoxic effect.Mol Biol Cell. 2006; 17: 1375-1387Crossref PubMed Scopus (80) Google Scholar, 40.Tam P. Mahfoud R. Nutikka A. et al.Differential intracellular trafficking and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.J Cell Physiol. 2008; 216: 750-763Crossref PubMed Scopus (47) Google Scholar Cytotoxicity depends on the retrograde transport of VT1 or VT2 from the cell surface to the Golgi/ER40.Tam P. Mahfoud R. Nutikka A. et al.Differential intracellular trafficking and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.J Cell Physiol. 2008; 216: 750-763Crossref PubMed Scopus (47) Google Scholar for cytosolic transit of the A subunit.41.Tam P. Lingwood C. Membrane-cytosolic translocation of verotoxin A1-subunit in target cells.Microbiol. 2007; 153: 2700-2710Crossref PubMed Scopus (53) Google Scholar Non-DRM plasma membrane Gb3 mediates binding and subsequent traffic to lysosomes for VT degradation.37.Falguieres T. Mallard F. Baron C. et al.Targeting of Shiga toxin b-subunit to retrograde transport route in association with detergent-resistant membranes.Mol Biol Cell. 2001; 12: 2453-2468Crossref PubMed Scopus (223) Google Scholar, 38.Hoey D.E.E. Currie C. Lingwood C.A. et al.Binding of verotoxin 1 to primary intestinal epithelial cells expressing Gb3 results in trafficking of toxin to lysosomal compartments.Cell Microbiol. 2003; 5: 85-97Crossref PubMed Scopus (81) Google Scholar Our present studies indicate that differential DRM expression of Gb3 also plays a major age-related role in VT-induced renal pathology in man. The role played by Gb3 expression on renal glomerular endothelial cells in the susceptibility to HUC following gastrointestinal verocytotoxin-producing E. coli infection remains to be clearly defined. Our early studies using direct labeled VT1 showed selective binding of pediatric as opposed to adult renal glomeruli,42.Lingwood C.A. Verotoxin-binding in human renal sections.Nephron. 1994; 66: 21-28Crossref PubMed Scopus (176) Google Scholar which correlates with the incidence of HUS. However, later studies using indirect immunostaining showed that VT1 stained some, and VT2 all, adult glomeruli and that cholesterol depletion induced VT1 glomerular staining.43.Chark D. Nutikka A. Trusevych N. et al.Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and monoclonal antibody: Role in human renal glomerular binding.Eur J Biochem. 2004; 271: 1-13Crossref PubMed Scopus (42) Google Scholar We now confirm these results but show that the differential glomerular/tubular VT binding is a function of resistance to detergent extraction as a function of age. These studies imply that the membrane organization of Gb3 is different in tubular epithelial and glomerular endothelial cells and that this changes with age such that HUS susceptibility may be lipid raft-based. Double labeling of human pediatric renal frozen tissue with VT1 and VT2 showed strong VT2 glomerular binding with (Figure 1a), or without (Figure 1b and c), coincident VT1 staining, according to sample. Samples with selective VT2 glomerular staining showed pronounced VT1 tubular binding. VT2 blood vessel staining was more prominent (arrows) than VT1. Similar results were obtained by separate VT1 and VT2 staining of serial pediatric renal sections (Figure 2). Although VT1 glomerular was greater than tubular staining (Figure 2A, a), VT2 glomerular staining was more pronounced and VT2 tubular staining was also more significant (cf. Figure 2A, a vs c).Figure 2Effect of tissue section Triton extraction on VT1 and VT2 human renal section staining. (A) Pediatric serial sections (a) VT1 staining, (b) VT1 staining of serial section after detergent extraction, (c) serial section VT2 staining, (d) serial section VT2 staining after detergent extraction. The same two glomeruli are marked with arrows in each section and a small blood vessel marked with an asterisk in each unextracted section. (B) Adult (83 years) frozen renal cortical sections stained with VT1. (a, c) Serial sections were stained without or (b, c) with prior detergent extraction. Boxed glomerulus in a, b is expanded in c, d. Glomerular staining is enhanced but tubular staining was removed after extraction. Image analysis showed glomerular staining was increased by 260±103% (n=9), while tubular staining was reduced to 23.6±5.6% (n=7) by detergent extraction. (C) Renal cortex (60 years). Serial section staining: (a, b) VT1 or (c, d) VT2. (a, c) Untreated or (b, d) after detergent extraction counterstained with hematoxylin and eosin. Arrows indicate the same glomerulus in each panel. In this case VT1 shows stronger glomerular staining than VT2. Tubular, but not glomerular, staining is lost after detergent extraction. Image analysis showed VT1 glomerular staining was increased by 117±21.9% (n=5) and VT2 staining was increased by 179±49% (n=8), while VT1 tubular staining was reduced to 37.7±11.5% (n=5) and VT2 to 18.2±7.7% (n=5) by detergent extraction. (D) Adult (24 years) serial renal sections were stained with (a, b) VT1 or (c, d) VT2. (a, c) Untreated or (b, d) after detergent extraction. The same glomerulus (arrow) is seen in each panel. This glomerulus is more strongly stained by VT2, but after detergent extraction both VT1 and VT2 staining is markedly enhanced (371.6±210% (n=8), 159.6±52% (n=9), respectively).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparison of pediatric renal section staining with VT1 or VT2 before and after Triton extraction at 4°C (cf. Figure 2A, a, c vs b, d), clearly showed that glomerular VT1 and VT2 staining are marginally affected by detergent extraction (Figure 2A, b and d). In the serial sections shown, the same two glomeruli are apparent (arrows) in each section. VT2 staining of both glomeruli is more pronounced than VT1. For VT1 only, the right glomerulus is more extensively stained than the left, consistent with the differential VT2 glomerular staining observed by double labeling in pediatric (Figure 1) and adult43.Chark D. Nutikka A. Trusevych N. et al.Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and monoclonal antibody: Role in human renal glomerular binding.Eur J Biochem. 2004; 271: 1-13Crossref PubMed Scopus (42) Google Scholar renal sections. In untreated sections, VT1 glomerular staining is particularly prevalent as we reported.42.Lingwood C.A. Verotoxin-binding in human renal sections.Nephron. 1994; 66: 21-28Crossref PubMed Scopus (176) Google Scholar VT1 and VT2 blood vessel staining is seen (*). VT2 tubular staining is greater than VT1. Following Triton extraction, VT1 staining of the left glomerulus is enhanced and staining of the right is unaffected (that is, detergent resistant) but blood vessel staining is lost. The left glomerulus, less well stained with VT1, is strongly stained by VT2. VT2 glomerular staining is largely resistant to detergent extraction but, similar to VT1, VT2 blood vessel staining is lost. VT2 tubular binding is detergent sensitive. In adult renal sections (Figure 2B), VT1 glomerular staining is significantly less than that seen for pediatric sections (though some pediatric glomeruli are less VT1-reactive than others). Adult tubular VT1 staining is remarkably increased relative to pediatric sections (Figure 2B, a and c). This strong VT1 tubular staining is virtually eliminated in detergent-treated sections (Figure 2B, b and d) but glomerular staining is increased. In serial sections, it can be seen that the same glomerulus initially VT1/2 reactive can become intensely bound by VT1 and VT2 following detergent extraction (Figure 2B, b and d). Some adult samples contained glomeruli preferentially reactive with VT1 (cf. Figure 2C, a vs c). In this case, detergent extraction enhanced VT2 staining (cf. Figure 2C, c vs d). In contrast, tubular VT1 and VT2 staining was entirely detergent sensitive (Figure 2C, a, c vs b, d). In the more common samples in which VT2 glomerular staining was predominant (Figure 2D), serial sections showed that detergent extraction enhanced both VT1 and VT2 glomerular staining. For all adult sections tested, tubular staining was lost following detergent extraction and glomerular staining was increased (to a variable degree). We had shown that treatment with β-methylcyclodextrin (β-MCD) to extract cholesterol enhanced VT1 glomerular binding.43.Chark D. Nutikka A. Trusevych N. et al.Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and monoclonal antibody: Role in human renal glomerular binding.Eur J Biochem. 2004; 271: 1-13Crossref PubMed Scopus (42) Google Scholar This was therefore compared with detergent extraction (Figure 3). As expected, MCD extraction had no effect on tubular VT1 staining and detergent extraction eliminated tubular, and enhanced glomerular VT1 staining. Surprisingly however, MCD extraction increased VT1 glomerular staining more than detergent extraction (cf. Figure 3c vs b and). In sequential extractions of serial sections (Figure 3g–p), MCD-induced glomerular staining was largely detergent resistant. After detergent extraction, MCD induction of VT1, but not VT2, glomerular binding was still evident. Thus, even after detergent extraction, Gb3-binding sites within the glomerulus can be hidden by cholesterol. As mouse renal glomeruli do not bind VT1 or VT2,26.Rutjes N. Binnington B. Smith C. et al.Differential tissue targeting and pathogenesis of verotoxins 1 and 2 in the mouse animal model.Kidney Int. 2002; 62: 832-845Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 44.Mattocks M. Bagovich M. De Rosa M. et al.Treatment of neutral glycosphingolipid storage disease via inhibition of the ABC drug transporter, MDR1: Cyclosporin A can lower serum and some tissue globotriaosyl ceramide levels in the Fabry's mouse model.FASEB J. 2006; 273: 2064-2075Google Scholarwe assessed the effect of detergent extract on mouse glomerular binding (Figure 4). The intense VT1 and VT2 tubular binding was largely eliminated by detergent extraction, but the unstained glomeruli (Figure 4 a, c and e) remained completely VT1/VT2 unreactive after detergent extraction (Figure 4b, d and f). Study of the renal binding of the HIV adhesin gp120, which also binds Gb345.Mahfoud R. Mylvaganam M. Lingwood C.A. et al.A novel soluble analog of the HIV-1 fusion cofactor, globotriaosylceramide(Gb3), eliminates the cholesterol requirement for high affinity gp120/Gb3 interaction.J Lipid Res. 2002; 43: 1670-1679Crossref PubMed Scopus (58) Google Scholar (among other GSLs46.Bhat S. Mettus R.V. Reddy E.P. et al.The galactosyl ceramide/sulfatide receptor binding region of HIV-1 gp120 maps to amino acids 206–275.AIDS Res Hum Retroviruses. 1993; 9: 175-181Crossref PubMed Scopus (80) Google Scholar) served to further emphasize the complex regulation of Gb3 receptor function within the kidney. Gp120 bound exclusively to the renal tubular epithelium (Figure 5A) and this binding was significantly reduced when the sections were pretreated with VT1 B subunit, suggesting that a large fraction of the gp120 renal binding is Gb3 mediated. Following detergent extraction, the gp120 tubular binding was lost (as for VT1, VT2). However, unlike for VT1/2 staining, there was no induction of gp120 glomerular binding (Figure 5b). Serial section staining confirmed that while detergent extraction increased VT1 staining of a glomerulus (Figure 2B) gp120 staining of the same glomerulus remained negative. Thus, detergent extraction has a selective effect to 'unmask' VT1 (VT2) glomerular Gb3 binding. Aglycone regulation of GSL receptor function, both by the lipid moiety of the GSL itself, and by the membrane environment has been described in many reports.47.Lingwood C.A. Aglycone Modulation of Glycolipid Receptor Function.Glycoconj J. 1996; 13: 495-503Crossref PubMed Scopus (69) Google Scholar Although Gb3 is the only functional receptor for VT1 and VT2,48.Okuda T. Tokuda N. Numata S. et al.Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins.J Biol Chem. 2006; 281: 10230-10235Crossref PubMed Scopus (139) Google Scholar VT1 and VT2 can preferentially bind different Gb3 'lipid assemblies' in vitro, and following internalization, undergo differential membrane retrograde trafficking to the Golgi and ER in cultured cells.40.Tam P. Mahfoud R. Nutikka A. et al.Differential intracellular trafficking and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.J Cell Physiol. 2008; 216: 750-763Crossref PubMed Scopus (47) Google Scholar VT1 and VT2 recognize overlapping epitopes within the Gb3 carbohydrate sequence,43.Chark D. Nutikka A. Trusevych N. et al.Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and monoclonal antibody: Role in human renal glomerular binding.Eur J Biochem. 2004; 271: 1-13Crossref PubMed Scopus (42) Google Scholar and VT1 (but not VT2) binding to Gb3 within the renal glomerulus43.Chark D. Nutikka A. Trusevych N. et al.Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and monoclonal antibody: Role in human renal glomerular binding.Eur J Biochem. 2004; 271: 1-13Crossref PubMed Scopus (42) Google Scholar and to Gb3 vesicle constructs40.Tam P. Mahfoud R. Nutikka A. et al.Differential intracellular trafficking and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.J Cell Physiol. 2008; 216: 750-763Crossref PubMed Scopus (47) Google Scholar can be inhibited by excess cholesterol. The relevance of sucrose gradient separated DRMs in normal cellular physiology remains a matter of debate. Such DRMs, although useful to assess changing parameters of cell membrane physiology, represent an amalgum of potentially separate microdomains, which might be originally present in the cell plasma membrane.10.Lingwood D. Simons K. Detergent resistance as a tool in membrane research.Nat Protoc. 2007; 2: 2159-2165Crossref PubMed Scopus (213) Google Scholar However, detergent extraction/resistance of tissue sections potentially represents a more physiologically relevant probe of cellular/tissue DRMs, as the residual antigens are, by definition, detergent resistant and therefore, more representative of their original cellular presentation and location. The use of this approach to investigate differences in membrane domains in tissue sections has not been previously used, but examination of intracellular membranes of cultured cells according to detergent resistance has been reported.37.Falguieres T. Mallard F. Baron C. et al.Targeting of Shiga toxin b-subunit to retrograde transport route in association with detergent-resistant membranes.Mol Biol Cell. 2001; 12: 2453-2468Crossref PubMed Scopus (223) Google Scholar, 40.Tam P. Mahfoud R. Nutikka A. et al.Differential intracellular trafficking and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.J Cell Physiol. 2008; 216: 750-763Crossref PubMed Scopus (47) Google Scholar Detergent extraction of tissue sections in situ may provide a new tool to examine the role of lipid rafts in cellular physiology, and perhaps more importantly, in disease. In simple model Gb3/cholesterol DRM vesicles, we have found that a significant fraction of the Gb3 is unavailable for ligand binding.49R. Mahfoud, A. Manis,
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