Artigo Acesso aberto Revisado por pares

Tubular epithelial syndecan-1 maintains renal function in murine ischemia/reperfusion and human transplantation

2012; Elsevier BV; Volume: 81; Issue: 7 Linguagem: Inglês

10.1038/ki.2011.425

ISSN

1523-1755

Autores

Johanna W.A.M. Celie, K. Katta, Saritha Adepu, Wynand B.W.H. Melenhorst, Rogier M. Reijmers, Edith Slot, Robert H.J. Beelen, Marcel Spaargaren, Rutger J. Ploeg, Gerjan Navis, Jaap J. Homan van der Heide, Marcory C. R. F. van Dijk, Harry van Goor, Jacob van den Born,

Tópico(s)

Hemostasis and retained surgical items

Resumo

Syndecan-1, a heparan sulfate proteoglycan, has an important role in wound healing by binding several growth factors and cytokines. As these processes are also crucial in damage and repair after renal transplantation, we examined syndecan-1 expression in human control kidney tissue, renal allograft protocol biopsies, renal allograft biopsies taken at indication, and non-transplant interstitial fibrosis. Syndecan-1 expression was increased in tubular epithelial cells in renal allograft biopsies compared with control. Increased epithelial syndecan-1 in allografts correlated with low proteinuria and serum creatinine, less interstitial inflammation, less tubular atrophy, and prolonged allograft survival. Knockdown of syndecan-1 in human tubular epithelial cells in vitro reduced cell proliferation. Selective binding of growth factors suggests that syndecan-1 may promote epithelial restoration. Bilateral renal ischemia/reperfusion in syndecan-1–deficient mice resulted in increased initial renal failure and tubular injury compared with wild-type mice. Macrophage and myofibroblast numbers, tubular damage, and plasma urea levels were increased, and tubular proliferation reduced in the kidneys of syndecan-1 deficient compared with wild-type mice 14 days following injury. Hence syndecan-1 promotes tubular survival and repair in murine ischemia/reperfusion injury and correlates with functional improvement in human renal allograft transplantation. Syndecan-1, a heparan sulfate proteoglycan, has an important role in wound healing by binding several growth factors and cytokines. As these processes are also crucial in damage and repair after renal transplantation, we examined syndecan-1 expression in human control kidney tissue, renal allograft protocol biopsies, renal allograft biopsies taken at indication, and non-transplant interstitial fibrosis. Syndecan-1 expression was increased in tubular epithelial cells in renal allograft biopsies compared with control. Increased epithelial syndecan-1 in allografts correlated with low proteinuria and serum creatinine, less interstitial inflammation, less tubular atrophy, and prolonged allograft survival. Knockdown of syndecan-1 in human tubular epithelial cells in vitro reduced cell proliferation. Selective binding of growth factors suggests that syndecan-1 may promote epithelial restoration. Bilateral renal ischemia/reperfusion in syndecan-1–deficient mice resulted in increased initial renal failure and tubular injury compared with wild-type mice. Macrophage and myofibroblast numbers, tubular damage, and plasma urea levels were increased, and tubular proliferation reduced in the kidneys of syndecan-1 deficient compared with wild-type mice 14 days following injury. Hence syndecan-1 promotes tubular survival and repair in murine ischemia/reperfusion injury and correlates with functional improvement in human renal allograft transplantation. Renal allograft transplantation is the treatment of choice for chronic kidney failure. Although significant advances have been made in the limiting (hyper) acute renal allograft rejection, chronic transplant dysfunction poses a serious risk of renal allograft loss at months to years after transplantation. Many factors are known to affect graft function and survival. For example, perioperative ischemia/reperfusion injury (IRI) causes severe damage to the tubular epithelial cells (TECs) of the donor organ, and tubular injury due to ischemia can persist into the chronic phase as a result of vascular changes.1.Grinyo J.M. Role of ischemia-reperfusion injury in the development of chronic renal allograft damage.Transplant Proc. 2001; 33: 3741-3742Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 2.Lien Y.H. Lai L.W. Silva A.L. Pathogenesis of renal ischemia/reperfusion injury: lessons from knockout mice.Life Sci. 2003; 74: 543-552Crossref PubMed Scopus (127) Google Scholar, 3.Hoffmann S.C. Kampen R.L. Amur S. et al.Molecular and immunohistochemical characterization of the onset and resolution of human renal allograft ischemia-reperfusion injury.Transplantation. 2002; 74: 916-923Crossref PubMed Scopus (96) Google Scholar, 4.Nankivell B.J. Chapman J.R. Chronic allograft nephropathy: current concepts and future directions.Transplantation. 2006; 81: 643-654Crossref PubMed Scopus (303) Google Scholar The number and severity of immune-mediated acute allograft rejection episodes, and especially chronic transplant dysfunction, characterized by tubular atrophy, interstitial fibrosis, and transplant glomerulopathy, can result in graft loss. Clearly, repair of tubular damage is a crucial step in restoration of renal function upon transplantation, and the balance between functional repair of tubular epithelium vs. chronic inflammation and nonfunctional scarring, i.e., fibrosis, is a dominant factor determining renal allograft function in the long run.5.Bunnag S. Einecke G. Reeve J. et al.Molecular correlates of renal function in kidney transplant biopsies.J Am Soc Nephrol. 2009; 20: 1149-1160Crossref PubMed Scopus (63) Google Scholar In previous studies, we showed that renal inflammatory responses can be affected by changes in heparan sulfate proteoglycans (HSPGs), as observed in experimental renal IRI, as well as in primary human kidney diseases.6.Celie J.W. Rutjes N. Keuning E.D. et al.Subendothelial heparan sulfate proteoglycans become major L-selectin and MCP-1 ligands upon renal ischemia/reperfusion.Am J Pathol. 2007; 170: 1865-1878Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,7.Celie J.W. Reijmers R.M. Slot E.M. et al.Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria.Am J Physiol Renal Physiol. 2008; 294: F253-F263Crossref PubMed Scopus (36) Google Scholar HSPGs are glycoconjugates that consist of a core protein to which a number of linear carbohydrate side chains (glycosaminoglycans; GAGs) are linked.8.Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu Rev Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1240) Google Scholar,9.Prydz K. Dalen K.T. Synthesis and sorting of proteoglycans.J Cell Science. 2000; 113: 193-205Crossref PubMed Google Scholar HS-GAG chains are typically very heterogeneous because of the coordinated action of various enzymes, which modify the alternating glucuronic acid and N-acetylglucosamine residues, resulting in varying degrees of sulfation and epimerization. HSPGs are involved in diverse biologically processes, including angiogenesis, wound healing, development, and inflammation.10.Alexopoulou A.N. Multhaupt H.A. Couchman J.R. Syndecans in wound healing, inflammation and vascular biology.Int J Biochem Cell Biol. 2007; 39: 505-528Crossref PubMed Scopus (245) Google Scholar, 11.Parish C.R. The role of heparan sulphate in inflammation.Nat Rev Immunol. 2006; 6: 633-643Crossref PubMed Scopus (385) Google Scholar, 12.Whitelock J.M. Iozzo R.V. Heparan sulfate: a complex polymer charged with biological activity.Chem Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (345) Google Scholar HSPGs exert their functions predominantly by binding and presenting growth factors, chemokines, and cytokines, and facilitating adhesion of inflammatory cells by binding to the leukocyte adhesion molecule L-selectin.6.Celie J.W. Rutjes N. Keuning E.D. et al.Subendothelial heparan sulfate proteoglycans become major L-selectin and MCP-1 ligands upon renal ischemia/reperfusion.Am J Pathol. 2007; 170: 1865-1878Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8.Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu Rev Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1240) Google Scholar, 13.Wang L. Fuster M. Sriramarao P. et al.Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses.Nat Immunol. 2005; 6: 902-910Crossref PubMed Scopus (387) Google Scholar Binding of these factors to HSPGs is mediated by the HS-GAG chains, and is dependent on the chemical fine structure of the GAGs.8.Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu Rev Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1240) Google Scholar,14.Kreuger J. Spillmann D. Li J.P. et al.Interactions between heparan sulfate and proteins: the concept of specificity.J Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (399) Google Scholar We previously showed that within 24h after renal IRI, HSPGs expressed at the abluminal side of peritubular capillaries are induced to bind L-selectin and the monocyte chemoattractant protein-1, and that these HSPGs facilitate monocyte extravasation.6.Celie J.W. Rutjes N. Keuning E.D. et al.Subendothelial heparan sulfate proteoglycans become major L-selectin and MCP-1 ligands upon renal ischemia/reperfusion.Am J Pathol. 2007; 170: 1865-1878Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Similar changes were observed in human primary kidney diseases associated with interstitial inflammation.7.Celie J.W. Reijmers R.M. Slot E.M. et al.Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria.Am J Physiol Renal Physiol. 2008; 294: F253-F263Crossref PubMed Scopus (36) Google Scholar In addition, we noted an increased expression of the cell-surface HSPG syndecan-1 on TECs in biopsy specimens of proteinuric patients.7.Celie J.W. Reijmers R.M. Slot E.M. et al.Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria.Am J Physiol Renal Physiol. 2008; 294: F253-F263Crossref PubMed Scopus (36) Google Scholar Syndecan-1 is a transmembrane HSPG involved in re-epithelialization during wound healing and inflammation.10.Alexopoulou A.N. Multhaupt H.A. Couchman J.R. Syndecans in wound healing, inflammation and vascular biology.Int J Biochem Cell Biol. 2007; 39: 505-528Crossref PubMed Scopus (245) Google Scholar, 15.Elenius K. Vainio S. Laato M. et al.Induced expression of syndecan in healing wounds.J Cell Biol. 1991; 114: 585-595Crossref PubMed Scopus (193) Google Scholar, 16.Stepp M.A. Gibson H.E. Gala P.H. et al.Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse.J Cell Sci. 2002; 115: 4517-4531Crossref PubMed Scopus (193) Google Scholar Earlier studies have implicated syndecan-1 in epithelial differentiation.17.Kato M. Saunders S. Nguyen H. et al.Loss of cell surface syndecan-1 causes epithelia to transform into anchorage-independent mesenchyme-like cells.Mol Biol Cell. 1995; 6: 559-576Crossref PubMed Scopus (193) Google Scholar,18.Vainio S. Lehtonen E. Jalkanen M. et al.Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney.Dev Biol. 1989; 134: 382-391Crossref PubMed Scopus (119) Google Scholar These processes are also very relevant in the context of renal allograft transplantation, i.e., inflammation as a result of IRI and donor–acceptor antigenic mismatching, and tubular re-epithelialization and differentiation to restore the damaged tubular epithelium after IRI. Indeed, a recent review by Venkatachalam et al.19.Venkatachalam M.A. Griffin K.A. Lan R. et al.Acute kidney injury: a springboard for progression in chronic kidney disease.Am J Physiol Renal Physiol. 2010; 298: F1078-F1094Crossref PubMed Scopus (391) Google Scholar posed the hypothesis that failure of tubular epithelial differentiation and therefore impaired tubular repair upon larger or smaller acute kidney injury episodes may have a significant role in the progression of kidney disease. On the basis of this background, we hypothesized that syndecan-1 is involved in tubular survival and repair upon renal transplantation. We examined expression of syndecan-1 in human renal allograft biopsies, including protocol biopsies and biopsies taken at indication, and correlated syndecan-1 expression to allograft function and binding of relevant growth factors. To directly study the functional role of syndecan-1, we studied the effect of syndecan-1 knockdown (in vitro) on TEC proliferation. In addition, we performed bilateral renal IRI in syndecan-1 vs. wild-type (WT) mice and studied functional and histological outcome. On the basis of our data, we propose that syndecan-1 is involved in determining the balance between nonfunctional scarring and functional repair, and could be a novel marker indicating prolonged renal allograft survival. In 13 out of 14 control kidneys, weak staining for syndecan-1 protein was detected in a limited percentage of TECs (Figure 1a and b). In contrast, very prominent TEC syndecan-1 staining was observed in all Tx-protocol biopsies (taken according to protocol at 1 year after transplantation), and a large subset of Tx-MHA (minor histological alterations, borderline interstitial inflammation and/or borderline tubular atrophy, and/or borderline intersitital fibrosis, no tubulitis or vasculopathy), acute allograft rejection biopsies (Tx-AR), and Tx-IFTA (interstitial inflammation/tubular atrophy biopsies; Figure 1b). This increased syndecan-1 staining showed distinct localization to the basolateral side of TECs (Figure 1a). Syndecan-1 staining was not observed in glomeruli or interstitial vasculature in any of the biopsies examined. Within the Tx-AR and Tx-IFTA group, the percentage of syndecan-1–positive TECs was heterogeneous throughout the different pathological types/grades (Figure 1b–d), and there was a trend towards a lower percentage of syndecan-1–positive TECs in the more severe Tx-IFTA grades, which was, however, not statistically different. To further characterize the pathological status of transplant biopsies included in this study, we scored for interstitial inflammation, tubulitis, tubular atrophy, C4d status, glomerulosclerosis, vasculopathy, acute tubular necrosis, epithelial vacuolization, and interstitial fibrosis according to the BANFF criteria (as indicated in Supplementary Table S1 online). Correlation analysis across the whole panel of biopsies revealed that increased TEC syndecan-1 is associated with lower scores for interstitial fibrosis (Spearman's ρ=-0.356, P<0.001), interstitial inflammation (Spearman's ρ=-0.305, P<0.005), and tubular atrophy (Spearman's ρ=-0.262, P<0.01). Download .pdf (.01 MB) Help with pdf files Supplementary Table 1 We next examined whether the percentage of syndecan-1–positive TECs was associated with clinical parameters, including time between transplantation and biopsy (time Tx to biopsy), markers for renal function (proteinuria, serum creatinine), and graft survival. A negative correlation was found between the percentage of syndecan-1–positive TECs and time Tx to biopsy (Spearman's ρ=-0.254, P=0.016; analyzed over Tx-protocol, Tx-MHA, Tx-AR, and Tx-IFTA groups together), indicating that a longer time between transplantation and biopsy is associated with lower percentage of syndecan-1–positive TECs. However, in subgroups of Tx-MHA and Tx-IFTA, which were matched according to similar time Tx to biopsy (Figure 2a; time Tx to biopsy between 32 and 288 weeks; Tx-MHA n=6, median 216 weeks, range 32–288 vs. Tx-IFTA n=12, median 188 weeks, range 40–265), the percentage of syndecan-1–positive TECs was significantly higher in the Tx-MHA subgroup compared with the Tx-IFTA subgroup (Figure 2b; P=0.032). Within the Tx-MHA or Tx-IFTA groups, no statistical correlation was observed between time Tx to biopsy and the percentage of syndecan-1–positive TECs (Spearman's ρ=0.150, P=0.495 for Tx-MHA; Spearman's ρ=0.193, P=0.315 for Tx-IFTA). These data suggest that TEC syndecan-1 expression is initially increased upon transplantation and decreases over time, although expression remains higher over time in biopsies with relatively mild histological abnormalities (e.g., Tx-MHA). We next investigated whether TEC syndecan-1 expression in the allografts was related to clinical markers indicative of allograft function (determined at time of biopsy). A statistically significant negative correlation was found between the percentage of syndecan-1–positive TECs and proteinuria (as indicated in Figure 2c; Spearman's ρ=-0.315, P<0.005) and serum creatinine (as indicated in Figure 2d; Spearman's ρ=-0.334, P<0.001; both analyzed across Tx-protocol, Tx-MHA, Tx-AR, and Tx-IFTA groups). These data demonstrate that increased TEC syndecan-1 expression is associated with improved renal allograft function. In control kidneys, 13 out of 14 biopsies showed syndecan-1 expression in 30% or less TECs, which we therefore regarded as a relevant cutoff for survival analysis. There was a statistically significant correlation between an increased percentage of syndecan-1–positive TECs and renal allograft survival (Figure 2e; 30% TEC syndecan-1 mean survival=862 weeks; log-rank test P<0.001). As expected, increased proteinuria was associated with decreased graft survival (log-rank test P=0.039). Together, these data show that TEC syndecan-1 is differentially expressed in renal allografts, and increased TEC syndecan-1 is associated with better renal allograft function and prolonged graft survival. We next examined the effect of short hairpin RNA (shRNA)-mediated silencing of syndecan-1 in the human TEC cell line HK-2 on short-term in vitro re-epithelialization and more long-term proliferation. As shown in Figure 3a, shRNA-mediated silencing of syndecan-1 (synd1-shRNA) reduced cell-surface syndecan-1 expression to background levels, whereas transfection with a control vector did not affect syndecan-1 expression. In the standardized scratch assay, no difference was observed between control vector and synd1-shRNA TECs in short-term re-epithelialization (Figure 3b and c). However, the more long-term proliferation of synd1-shRNA TECs was significantly reduced compared with control vector TECs (Figure 3d). Together, these data show that, although knockdown of syndecan-1 in TECs does not affect short-term (likely predominantly migration-induced) in vitro re-epithelialization, syndecan-1 expression seems to be a prerequisite for proper TEC proliferation. Knowing that HSPGs, including syndecan-1, can be modified to present growth factors depending on cellular activation status, we explored whether the association between syndecan-1 and TEC proliferation could be attributed to growth factor binding. We chose to use FGF-2 (basic FGF) and HB-epidermal growth factor (EGF), which are both known to bind HSPGs,20.Guerrini M. Hricovini M. Torri G. Interaction of heparins with fibroblast growth factors: conformational aspects.Curr Pharm Des. 2007; 13: 2045-2056Crossref PubMed Scopus (32) Google Scholar, 21.Chu C.L. Goerges A.L. Nugent M.A. Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans.Biochemistry. 2005; 44: 12203-12213Crossref PubMed Scopus (35) Google Scholar, 22.Raab G. Klagsbrun M. Heparin-binding EGF-like growth factor.Biochim Biophys Acta. 1997; 1333: F179-F199PubMed Google Scholar although possibly with differences in GAG-chain requirements.20.Guerrini M. Hricovini M. Torri G. Interaction of heparins with fibroblast growth factors: conformational aspects.Curr Pharm Des. 2007; 13: 2045-2056Crossref PubMed Scopus (32) Google Scholar, 21.Chu C.L. Goerges A.L. Nugent M.A. Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans.Biochemistry. 2005; 44: 12203-12213Crossref PubMed Scopus (35) Google Scholar, 22.Raab G. Klagsbrun M. Heparin-binding EGF-like growth factor.Biochim Biophys Acta. 1997; 1333: F179-F199PubMed Google Scholar, 23.Clayton A. Thomas J. Thomas G.J. et al.Cell surface heparan sulfate proteoglycans control the response of renal interstitial fibroblasts to fibroblast growth factor-2.Kidney Int. 2001; 59: 2084-2094Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar In an enzyme-linked immunosorbent assay–type binding assay, we were able to detect dose-dependent binding of FGF-2 and HB-EGF to a heparin-albumin coating, which is used as a model molecule for HSPGs (heparin is a highly sulfated HS-GAG chain; Figure 4a). As shown in Figure 4b and Table 1, no basolateral FGF-2 binding was observed in Tx-MHA biopsies despite high syndecan-1 expression. Limited basolateral TEC binding of FGF-2 was observed in one Tx-IFTA biopsy (Table 1), and binding of FGF-2 to interstitially infiltrated cells was observed in a number of biopsies (predominantly Tx-IFTA and nonTx-IF, likely representing binding to heparin-containing mast cells; not shown). When incubation of FGF-2 was omitted no staining was observed, indicating that binding of the exogenously added FGF-2 was detected rather than endogenously present FGF-2 (not shown). Although FGF-2 is a prototypic HSPG-binding growth factor, it is also a pro-fibrotic growth factor, and therefore increased binding and expression in the renal allograft could be harmful/disadvantageous. We therefore also examined binding of HB-EGF, an epithelial survival factor, which is associated with renal epithelial repair and is expressed in the regeneration phase upon renal injury.24.Zhuang S. Kinsey G.R. Rasbach K. et al.Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells.Am J Physiol Renal Physiol. 2008; 294: F459-F468Crossref PubMed Scopus (28) Google Scholar,25.Sakai M. Zhang M. Homma T. et al.Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules.J Clin Invest. 1997; 99: 2128-2138Crossref PubMed Scopus (90) Google Scholar As shown in Figure 4b and Table 1, prominent binding of HB-EGF to the basolateral side of TECs was observed most dominantly in the Tx-MHA group, although it was also observed to different extents in control tissue, Tx-IFTA, and nonTx-IF. Pretreatment of Tx-MHA tissue sections with heparitinase I, an enzyme that specifically degrades HS-GAG chains, completely diminished HB-EGF binding to TECs, showing that HB-EGF binds to basolateral TEC HSPGs (Figure 4c).Table 1Scoring for TEC syndecan-1 protein expression and binding of FGF-2, HB-EGF, and anti-HS mAb 10E4 in a subset of renal biopsiesCaseDiagnosisSyndecan-1aPercentage of TECs positive for expression/binding.FGF-2 bindingaPercentage of TECs positive for expression/binding.HB-EGF bindingaPercentage of TECs positive for expression/binding.Anti-HS mAb 10E4aPercentage of TECs positive for expression/binding.1Control505nd2Control20030nd3Control1000nd4Control0nd085Control20ndnd466Control5ndnd107Control30ndnd38Tx-MHA80070nd9Tx-MHA600707210Tx-MHA90080nd11Tx-MHA800nd7712Tx-MHA800407313Tx-MHA90ndnd8214Tx-IFTA600305315Tx-IFTA3000116Tx-IFTA2010101317Tx-IFTA000nd18NonTx-IFTA1000219NonTx-IFTA300302220NonTx-IFTA6000nd21NonTx-IFTA60ndnd36Abbreviations: FGF, fibroblast growth factor; HB-EGF, heparin-binding EGF-like growth factor; HS, heparin sulfate; mAb, monoclonal antibody; nd, not determined; TEC, tubular epithelial cell; Tx-IFTA, interstitial inflammation/tubular atrophy; Tx-MHA, minor histological alteration.a Percentage of TECs positive for expression/binding. Open table in a new tab Abbreviations: FGF, fibroblast growth factor; HB-EGF, heparin-binding EGF-like growth factor; HS, heparin sulfate; mAb, monoclonal antibody; nd, not determined; TEC, tubular epithelial cell; Tx-IFTA, interstitial inflammation/tubular atrophy; Tx-MHA, minor histological alteration. To explore the structural characteristics of tubular epithelial HS in more detail, we also evaluated four different anti-HS mAbs, namely JM-403, JM-13, 10E4, and HK249. The various epitope requirements of these anti-HS mAbs have been detailed elsewhere (see Materials and Methods section). All four anti-HS mAbs stained subsets of renal basement membranes; however, 10E4, in addition, stained TECs in a basolateral manner in a number of biopsies. Subgroup analysis revealed a close colocalization of anti-HS mAb 10E4 and syndecan-1 (Table 1 and Figure 5). Together, our data show increased binding of HB-EGF, but not FGF-2, to basolateral TEC HSPGs, colocalizing with syndecan-1 and 10E4 HS expression, in a subset of renal allograft biopsies. Syndecan-1–positive tubules, however, failed to bind with FGF-2, and anti-HS mAbs JM-403, JM-13, and HK249. These data highlight two levels of regulation by which the cells modulate growth factor affinity, namely syndecan-1 protein expression and the formation of specific sulfation motifs in the HS polysaccharide side chains. To determine the in vivo importance of syndecan-1 in renal IRI, we next performed bilateral warm IRI in syndecan-1–deficient mice.16.Stepp M.A. Gibson H.E. Gala P.H. et al.Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse.J Cell Sci. 2002; 115: 4517-4531Crossref PubMed Scopus (193) Google Scholar,26.Alexander C.M. Reichsman F. Hinkes M.T. et al.Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice.Nat Genet. 2000; 25: 329-332Crossref PubMed Scopus (308) Google Scholar Anticipating a more severe phenotype in syndecan-1-/-, we used a relatively mild IRI model (25min bilateral warm ischemia, followed by bilateral reperfusion). Induction of syndecan-1 in the WT mice was evidenced from day 1 onward, which by definition was not observed in the syndecan-1 KO mice (Figure 6a–e). Compared with WT mice, syndecan-1-/- proved to be more susceptible to IRI-induced renal damage (Figures 6 and 7). At day 1 after IRI, plasma creatinine and ureum levels were significantly increased in syndecan-1-/- compared with WT mice (Figure 6f and g). Histologically, clearly more tubular damage was observed in syndecan-1-/- kidneys at day 1 after IRI (Figure 7a and c). Interestingly, tubular damage appeared to remain constant over time in syndecan-1-/- kidneys, whereas damage was largely restored in WT kidneys at day 14 after IRI (Figure 7a and c). By Ki67 staining, we evaluated the proliferative response of the tubular epithelium both in WT and syndecan-1 KO mice. Although there is quite some proliferation at day 3 after I/R (not different between both groups), at day 7 and 14 of the regeneration phase the syndecan-1 KO mice revealed lower proliferation rates compared with the WT (day 14: P=0.0411; Figure 7b). Both macrophages and myofibroblasts are known to have an important role in renal IRI damage/repair responses. We therefore quantified the amounts of these cells by staining for F4/80 (monocytes/macrophages) and α-smooth muscle actin (myofibroblasts). As shown in Figure 7d, monocytes/macrophages steadily accumulated in syndecan-1-/- compared with WT kidneys. Interestingly, also the amount of myofibroblasts was increased in syndecan-1-/- compared with WT kidneys at day 14 after IRI (Figure 7e).Figure 7Increased tubular damage, reduced proliferation, and increased amounts of macrophages and myofibroblasts in syndecan-1-/- kidneys upon bilateral ischemia/reperfusion injury (IRI). Renal IRI was performed by bilateral clamping of renal pedicles for 25min (warm ischemia), followed by bilateral reperfusion. (a) Increased tubular damage was observed at days 1 and 14 after IRI, whereas (b) reduced tubular proliferation (Ki67-positive tubular cells) was observed at day 14 in the syndecan-1 knockout (KO) mice. (c) Representative photographs of periodic acid–Schiff's-stained kidney sections of wild-type (WT) and syndecan-1-/- at day 1 and 14 after IRI. (d) Monocytes/macrophages (F4/80 staining) appear to accumulate over time upon IRI in syndecan-1-/- compared with WT, with significantly more monocyte/macrophages in syndecan-1-/- kidneys at day 14. (e) Myofibroblasts (α-smooth muscle actin staining) are increased in syndecan-1-/- kidneys compared with WT at day 14 after IRI. *P<0.05 (Mann–Whitney U-test).View Large Image Figure ViewerDownload (PPT) Together, these data show that syndecan-1-/- mice have impaired renal function and increased tubular damage upon renal IRI compared with WT mice. In addition, our data indicate that tubular damage is restored less efficiently in syndecan-1-/- mice, and repair mechanisms may be skewed toward fibrosis rather than restoration of tubular epithelium. In this study, we show that syndecan-1 protein expression is increased on the basolateral side of TECs after renal transplantation and that the percentage of syndecan-1–positive TECs is significantly associated with better allograft function and survival. We provide evidence that syndecan-1 is involved in TEC proliferation, and that binding of the growth factor HB-EGF to TEC HSPGs is increased on the basolateral side of TECs that also express syndecan-1. Finally, we show that syndecan-1–deficient mice show more impaired renal function and increased tubular damage associated with reduced tubular repair in an experimental IRI model. On the basis of our data, the role of syndecan-1 in the renal epithelium may be similar to its role in dermal wound healing.10.Alexopoulou A.N. Multhaupt H.A. Couchman J.R. Syndecans in wound healing, inflammation and vascular biology.Int J Biochem Cell Biol. 2007; 39: 505-528Crossref PubMed Scopus (245) Google Scholar,16.Stepp M.A. Gibson H.E. Gala P.H. et al.Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse.J Cell Sci. 2002; 1

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