Protein load impairs factor H binding promoting complement-dependent dysfunction of proximal tubular cells
2009; Elsevier BV; Volume: 75; Issue: 10 Linguagem: Inglês
10.1038/ki.2009.8
ISSN1523-1755
AutoresSimona Buelli, Mauro Abbate, Marina Morigi, Daniela Moioli, Cristina Zanchi, Marina Noris, Carla Zoja, Charles D. Pusey, Peter F. Zipfel, Giuseppe Remuzzi,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoIntrarenal complement activation plays an important role in the progression of chronic kidney disease. A key target of the activated complement cascade is the proximal tubule, a site where abnormally filtered plasma proteins and complement factors combine to promote injury. This study determined whether protein overloading of human proximal tubular cells (HK-2) in culture enhances complement activation by impairing complement regulation. Addition of albumin or transferrin to the cells incubated with diluted human serum as a source of complement caused increased apical C3 deposition. Soluble complement receptor-1 (an inhibitor of all 3 activation pathways) blocked complement deposition while the classical and lectin pathway inhibitor, magnesium chloride–EGTA, was, ineffective. Media containing albumin as well as complement had additive proinflammatory effects as shown by increased fractalkine and transforming growth factor-β mRNA expression. This paralleled active C3 and C5b-9 generations, effects not shared by transferrin. Factor H, one of the main natural inhibitors of the alternative pathway, binds to heparan sulfate proteoglycans. Both the density of heparan sulfate and factor H binding were reduced with protein loading, thereby enhancing the albumin- and serum-dependent complement activation potential. Thus, protein overload reduces the ability of the tubule cell to bind factor H and counteract complement activation, effects instrumental to renal disease progression. Intrarenal complement activation plays an important role in the progression of chronic kidney disease. A key target of the activated complement cascade is the proximal tubule, a site where abnormally filtered plasma proteins and complement factors combine to promote injury. This study determined whether protein overloading of human proximal tubular cells (HK-2) in culture enhances complement activation by impairing complement regulation. Addition of albumin or transferrin to the cells incubated with diluted human serum as a source of complement caused increased apical C3 deposition. Soluble complement receptor-1 (an inhibitor of all 3 activation pathways) blocked complement deposition while the classical and lectin pathway inhibitor, magnesium chloride–EGTA, was, ineffective. Media containing albumin as well as complement had additive proinflammatory effects as shown by increased fractalkine and transforming growth factor-β mRNA expression. This paralleled active C3 and C5b-9 generations, effects not shared by transferrin. Factor H, one of the main natural inhibitors of the alternative pathway, binds to heparan sulfate proteoglycans. Both the density of heparan sulfate and factor H binding were reduced with protein loading, thereby enhancing the albumin- and serum-dependent complement activation potential. Thus, protein overload reduces the ability of the tubule cell to bind factor H and counteract complement activation, effects instrumental to renal disease progression. Progressive nephropathies with severe defects of the glomerular filtration barrier to proteins are accompanied by tubulointerstitial damage eventually contributing to loss of renal function.1.Remuzzi G. Bertani T. Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1115) Google Scholar The clinical problem has grown enormously1.Remuzzi G. Bertani T. Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1115) Google Scholar, 2.Perico N. Codreanu I. Schieppati A. et al.The future of renoprotection.Kidney Int Suppl. 2005; 97: S95-S101Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar and understanding the basic mechanisms is mandatory. Experimental observations suggested that ultrafiltered plasma proteins exert renal toxicity, which is partly mediated by activation of inflammatory and fibrogenic pathways by proximal tubular cells.3.Zoja C. Morigi M. Figliuzzi M. et al.Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins.Am J Kidney Dis. 1995; 26: 934-941Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 4.Morigi M. Macconi D. Zoja C. et al.Protein overload-induced NF-kB activation in proximal tubular cells requires H2O2 through a PKC-dependent pathway.J Am Soc Nephrol. 2002; 13: 1179-1189PubMed Google Scholar, 5.Wang Y. Chen J. Chen L. et al.Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein.J Am Soc Nephrol. 1997; 8: 1537-1545PubMed Google Scholar, 6.Zoja C. Donadelli R. 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Morigi M. et al.Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways.J Am Soc Nephrol. 2003; 14: 2436-2446Crossref PubMed Scopus (105) Google Scholar Among processes underlying injury, the activation of the complement cascade in the renal tubule has major effects.10.Matsuo S. Morita Y. Mizuno M. et al.Proteinuria and damage to tubular cells—is complement a culprit?.Nephrol Dial Transplant. 1998; 13: 2723-2726Crossref PubMed Scopus (26) Google Scholar Complement proteins can be abnormally filtered across the altered glomerular barrier leading to intratubular deposition of C3 and formation of membrane attack complex (MAC).11.Eddy A.A. Interstitial nephritis induced by protein-overload proteinuria.Am J Pathol. 1989; 135: 719-733PubMed Google Scholar, 12.Morita Y. Nomura A. Yuzawa Y. et al.The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis.J Am Soc Nephrol. 1997; 8: 1363-1372Crossref PubMed Google Scholar, 13.Nomura A. Morita Y. Maruyama S. et al.Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.Am J Pathol. 1997; 151: 539-547PubMed Google Scholar Proximal tubular epithelial cells also synthesize most components of the activation cascade.14.Zhou W. Marsh J.E. Sacks S.H. Intrarenal synthesis of complement.Kidney Int. 2001; 59: 1227-1235Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15.Tang S. Lai K.N. Chan T.M. et al.Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.Am J Kidney Dis. 2001; 37: 94-103Abstract Full Text PDF PubMed Scopus (40) Google Scholar Investigation using proteinuric models of glomerular injury showed less tubulointerstitial damage in complement-depleted rats12.Morita Y. Nomura A. Yuzawa Y. et al.The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis.J Am Soc Nephrol. 1997; 8: 1363-1372Crossref PubMed Google Scholar, 13.Nomura A. Morita Y. Maruyama S. et al.Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.Am J Pathol. 1997; 151: 539-547PubMed Google Scholar and C6-deficient rats,16.Nangaku M. Pippin J. Couser W.G. Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome.J Am Soc Nephrol. 1999; 10: 2323-2331PubMed Google Scholar or upon treatment with complement inhibitory molecules.13.Nomura A. Morita Y. Maruyama S. et al.Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.Am J Pathol. 1997; 151: 539-547PubMed Google Scholar, 17.He C. Imai M. Song H. et al.Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9.J Immunol. 2005; 174: 5750-5757Crossref PubMed Scopus (59) Google Scholar Inflammation and a fibrogenic reaction ensue upon proximal tubule overloading by C3 and ultrafiltered proteins in rats with remnant kidneys.18.Abbate M. Zoja C. Rottoli D. et al.Antiproteinuric therapy while preventing the abnormal protein traffic in proximal tubule abrogates protein and complement-dependent interstitial inflammation in experimental renal disease.J Am Soc Nephrol. 1999; 10: 804-813PubMed Google Scholar, 19.Abbate M. Zoja C. Rottoli D. et al.Proximal tubular cells promote fibrogenesis by TGF-β1-mediated induction of peritubular myofibroblasts.Kidney Int. 2002; 61: 2066-2077Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar We have recently found that ultrafiltered complement factors are a key component of proteinuria-associated renal injury. Thus, plasma-derived C3 is recruited with other ultrafiltered proteins in the tubular compartment to mediate interstitial macrophage accumulation and renal structural damage in the absence of C3 synthesis by the kidney, in a mouse model of protein-overload proteinuria.20.Abbate M. Zoja C. Corna D. et al.Complement-mediated dysfunction of glomerular filtration barrier accelerates progressive renal injury.J Am Soc Nephrol. 2008; 19: 1158-1167Crossref PubMed Scopus (49) Google Scholar Overall, proteinuria is required for complement to fully exert harmful effects on the kidney,21.Rangan G.K. Pippin J.W. Coombes J.D. et al.C5b-9 does not mediate chronic tubulointerstitial disease in the absence of proteinuria.Kidney Int. 2005; 67: 492-503Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar possibly by enhancing the vulnerability of proximal tubular cells to luminal attack by complement. This makes it difficult to dissect mechanisms by which complement could be engaged with other ultrafiltered proteins to induce tubular cell dysfunction. However, approaches using cultured proximal tubular epithelial cells are most appropriate to address this issue. In human proximal tubular cells exposed to serum, complement activation through the alternative pathway leads to surface deposition of C3 and MAC and upregulation of cytokines.22.Biancone L. David S. Della Pietra V. et al.Alternative pathway activation of complement by cultured human proximal tubular epithelial cells.Kidney Int. 1994; 45: 451-460Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 23.David S. Biancone L. Caserta C. et al.Alternative pathway complement activation induces proinflammatory activity in human proximal tubular epithelial cells.Nephrol Dial Transplant. 1997; 12: 51-56Crossref PubMed Scopus (4) Google Scholar Complement activation might be dependent on relative lack of complement inhibitors. Cell-associated regulators at C3 level, decay accelerating factor and membrane cofactor protein, are hardly detectable on apical membranes of proximal tubular cells. Conversely, human proximal tubular cells produce factor H,24.Gerritsma J.S. Gerritsen A.F. De Ley M. et al.Interferon-gamma induces biosynthesis of complement components C2, C4 and factor H by human proximal tubular epithelial cells.Cytokine. 1997; 9: 276-283Crossref PubMed Scopus (55) Google Scholar a 150-kDa plasma glycoprotein acting as the major soluble inhibitor of the formation of alternative pathway C3 convertase on the cell surface25.Manuelian T. Hellwage J. Meri S. et al.Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome.J Clin Invest. 2003; 111: 1181-1190Crossref PubMed Scopus (289) Google Scholar, 26.Jozsi M. Manuelian T. Heinen S. et al.Attachment of the soluble complement regulator factor H to cell and tissue surfaces: relevance for pathology.Histol Histopathol. 2004; 19: 251-258PubMed Google Scholar and possibly representing a unique defense mechanism against complement activation. No studies have yet documented actual binding of factor H by tubular epithelial cells. Relevant binding sites on the cell surface, as shown in endothelial cells, may include heparan sulfate proteoglycans.27.Jokiranta T.S. Cheng Z.Z. Seeberger H. et al.Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.Am J Pathol. 2005; 167: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar The aim of this study was to investigate whether (1) protein overload may influence complement activation and deposition on the surface of proximal tubular cells exposed to serum complement, a condition that mimics the exposure of tubular cells to ultrafiltered complement in proteinuric disease; (2) this effect was related to reduced binding of factor H on the cell surface accounting for reduced capability of the cell to counteract complement activation; and (3) loss of heparan sulfate sites contributed to reduced factor H binding to the cell. With the aim of studying the effect of plasma protein challenge on complement deposition, preliminary experiments were carried out to identify the sublytic concentration of human serum (HS), used as source of complement. Cell viability, as assessed by trypan blue dye exclusion at 90 min, was not affected by exposure to 10% HS, whereas a significant (P<0.01) reduction was observed upon exposure to 25% HS in comparison with control cells (10% HS: 91±5, 25% HS: 71±4 versus control: 100% viable cells). The concentration of 10% HS was therefore established to be sublytic. HK-2 cells upon exposure to 10% HS activated and fixed C3 and MAC on the apical side of the cell surface as showed by appearance of granular staining by immunofluorescence analysis (Figure 1) on cell surface. In contrast, unstimulated cells showed no fluorescent signal (Figure 1). Addition of sCR-1, a soluble inhibitor of all the three pathways of complement activation, completely blocked C3/MAC deposition on cell surface, indicating specificity of C3 and MAC signals (Figure 1). Similarly, no complement protein deposition was detected using heat-inactivated HS (data not shown). To study whether protein overload could enhance the ability of proximal tubular epithelial cells to fix complement molecules, immunofluorescence analysis of C3 and MAC deposits was performed on HK-2 cells, first challenged with albumin or transferrin and then incubated with 10% HS as described above. Both plasma proteins caused significant increases in C3 staining as detected at the end of the experiment, in comparison with cells exposed to HS alone (Figure 2a). Exposure of HK-2 cells to plasma proteins in the absence of HS resulted in no significant changes in C3 staining as compared with unstimulated cells, thus excluding that the abnormal accumulation of C3 on the cell surface in the presence of HS could be attributed to the induction of C3 synthesis in response to albumin and transferrin. To test whether complement activation may occur through the alternative pathway, cells were incubated with HS containing sCR-1 or with MgCl2–EGTA that blocks the activation of the classic and the lectin pathways. The addition of sCR-1 resulted in complete inhibition of C3 deposition. In contrast, MgCl2–EGTA had no significant effect (Table 1).Table 1Effect of MgCl2–EGTA and sCR-1 on C3 deposition in HK-2 cellsC3 deposit area (pixel2)-+MgCl2-EGTA+sCR-110% HS4458±2314770±25126±1°Albumin +10% HS8045±483°8033±63323±4#Transferrin +10% HS9716±1436°9939±145625±2§Data are expressed as mean±s.e. of 15 fields for each sample (n=4).°P<0.01 versus 10% HS, #P<0.01 versus albumin+10% HS, §P<0.01 versus transferrin+10% HS.HK-2 cells were exposed for 24 h to medium alone, albumin, or transferrin (10 mg/ml) before incubation with 10% human serum (HS) for 90 min in the presence or absence of MgCl2–EGTA (5 mM) or sCR-1 (100 μg/ml). Open table in a new tab Data are expressed as mean±s.e. of 15 fields for each sample (n=4). °P<0.01 versus 10% HS, #P<0.01 versus albumin+10% HS, §P 1. When cells were exposed to HS alone, the C3c/iC3b ratio was 2.15±0.05 (Figure 2b). The addition of albumin significantly increased active C3 deposits (3.60±0.09), whereas transferrin had no significant effect as compared with HS alone (2.41±0.06) (Figure 2b). Then we investigated whether complement activation on tubular cells by albumin and transferrin proceeded to the formation of MAC. MAC fixation also further increased (P<0.01) in cells exposed to albumin followed by the addition of HS, as compared with exposure to HS alone (Figure 2c), an effect that was not observed with transferrin (Figure 2c). The viability of HK-2 cells was not altered by 24-h exposure to albumin or transferrin alone (albumin: 96.8±13.8, transferrin: 93.2±2.9 versus control: 100% viable cells). No additional effect on cell viability was observed when either proteins were given as pretreatment before 10% HS (albumin+HS: 118.3±10.1, transferrin+HS: 83.9±4.4 versus control: 100% viable cells). To investigate the functional consequences of protein overload-induced activation of complement on HK-2 cells, we studied the expression of fractalkine and transforming growth factor-β (TGF-β), taken as markers of cell activation and known mediators of tubulointerstitial inflammation and fibrosis.28.Abbate M. Zoja C. Remuzzi G. How does proteinuria cause progressive renal damage?.J Am Soc Nephrol. 2006; 17: 2974-2984Crossref PubMed Scopus (544) Google Scholar Serum complement enhanced the expression of both cytokines as reflected by combined findings of increased fractalkine and TGF-β mRNA by 10% HS, and abrogation in the presence of complement inhibitor sCR-1 (Figure 3). The combination of HS and albumin challenge had additive effects as shown by further significant (P<0.05) increases in mRNA levels of both mediators as compared with each stimulus alone. The exposure of cells to transferrin alone caused fractalkine and TGF-β mRNA upregulation (P<0.05), and no further increases were observed when combined with HS (Figure 3). To identify the mechanisms underlying the increased serum C3 and MAC deposition on HK-2 cell surface in response to protein overload, we focused on factor H, one of the main natural inhibitors of the alternative pathway of complement, which could act on proximal tubular epithelial cells. For this purpose, we studied first whether resting HK-2 cells bind factor H. Unstimulated HK-2 cells expressed low levels of this complement regulatory protein (Figure 4). Exogenous purified factor H added to HK-2 cells markedly bound to the cell surface (Figure 4), as shown by the increased percentage of fluorescent cells detected by flow cytometry (Table 2). The binding of factor H on the cell surface was decreased by pre-exposure of HK-2 cells to albumin or transferrin (Figure 4 and Table 2).Table 2Plasma proteins decrease exogenous factor H binding on HK-2 cellsFluorescent cells (%)-+Factor HControl23.35±1.4437.87±0.93°Albumin21.89±0.7825.86±1.81**Transferrin27.19±1.1929.28±3.90*FACS analysis of factor H binding on cell surface. Results (mean±s.e.) are expressed as % of fluorescent cells (n=8).°P<0.01 versus control; *P<0.05, **P<0.01 versus control+factor H.HK-2 cells were exposed for 24 h to medium (control), albumin, or transferrin. Then, human factor H (10 μg) was added for 3 h. Open table in a new tab FACS analysis of factor H binding on cell surface. Results (mean±s.e.) are expressed as % of fluorescent cells (n=8). °P<0.01 versus control; *P<0.05, **P<0.01 versus control+factor H. HK-2 cells were exposed for 24 h to medium (control), albumin, or transferrin. Then, human factor H (10 μg) was added for 3 h. To understand whether plasma proteins could affect endogenous factor H synthesis, western blot analysis was performed for detection of factor H production by HK-2 cells exposed to medium alone, albumin, or transferrin for 24 h. Unstimulated cells constitutively produced factor H, as showed by detection in the cell supernatants of a band of approximately 150 kDa, corresponding to factor H (see Figure S1). This band was not detectable in supernatants derived from HK-2 cells incubated with albumin or transferrin (Figure S1), thereby indicating an inhibitory effect of protein overload on factor H production by proximal tubular cells. Download .doc (.47 MB) Help with doc files Supplementary Information To define in our experimental setting the relation between factor H binding and complement deposition, we first assessed the level of active C3 deposits on tubular cells after exposure to HS, a source of factor H in addition to complement. Active C3 deposition was evaluated by flow cytometry as the ratio C3c/iC3b. When HK-2 cells were exposed to HS, the mean fluorescence intensity (MFI) of C3c was higher than iC3b MFI (Figure 5a) and the ratio C3c/iC3b was 2.3±0.4 (Figure 5b). Active C3 level in this condition results from a balance between complement activation spontaneously occurring on cell surface and its inactivation by factor H. Serum factor H was capable of binding to HK-2 cells, as showed by immunofluorescence staining of factor H on tubular cell surface after 90-min HS exposure (data not shown). The blockade of the factor H catalytic site by pretreating HS with N22 Ab29.Oppermann M. Manuelian T. Jozsi M. et al.The C-terminus of complement regulator factor H mediates target recognition: evidence for a compact conformation of the native protein.Clin Exp Immunol. 2006; 144: 342-352Crossref PubMed Scopus (127) Google Scholar significantly impaired factor H-dependent C3b inactivation leading to an increase of total C3 (Figure 5a) and active C3 deposits as indicated by the ratio C3c/iC3b=5.0±0.3 (Figure 5b). Similarly, L20 Ab, which blocks the factor H cell membrane-binding site,29.Oppermann M. Manuelian T. Jozsi M. et al.The C-terminus of complement regulator factor H mediates target recognition: evidence for a compact conformation of the native protein.Clin Exp Immunol. 2006; 144: 342-352Crossref PubMed Scopus (127) Google Scholar increased both total C3 (Figure 5a) and the C3c/iC3b ratio (5.9±1.4) (Figure 5b), indicating more deposition of active C3 on the tubular cell surface. In contrast, addition of irrelevant control anti-CD13 Ab had no effects as compared with HK-2 cells exposed to HS alone (Figure 5a and b). The influence of factor H activity on the presence of MAC deposits on HK-2 cells was also evaluated. The inhibition of either the catalytic site or the cell-binding site of factor H by using N22 and L20 Abs, respectively, significantly increased the amount of MAC deposits on cell surface as compared with cells incubated with HS either alone or plus control irrelevant Ab (Figure 5c). Immunofluorescence experiments showed no inhibitory effects of N22 Ab on exogenous factor H binding to the cell surface, which instead was almost completely prevented by L20 Ab (Figure 5d). Both Abs showed no binding to the cell surface in the absence of factor H (data not shown). Polyanionic molecules and specifically cell-associated proteoglycans are major binding sites for factor H, as shown by studies in cell-free systems30.Meri S. Pangburn M.K. Regulation of alternative pathway complement activation by glycosaminoglycans: specificity of the polyanion binding site on factor H.Biochem Biophys Res Commun. 1994; 198: 52-59Crossref PubMed Scopus (105) Google Scholar and endothelial cells.26.Jozsi M. Manuelian T. Heinen S. et al.Attachment of the soluble complement regulator factor H to cell and tissue surfaces: relevance for pathology.Histol Histopathol. 2004; 19: 251-258PubMed Google Scholar, 27.Jokiranta T.S. Cheng Z.Z. Seeberger H. et al.Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.Am J Pathol. 2005; 167: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar To assess whether heparan sulfate residues could mediate factor H binding to tubular cell surface, HK-2 cells were exposed to an Ab against N-sulfated glucosamine residues of heparan sulfate before the addition of factor H. In comparison with cells exposed to factor H alone (Figure 6), a marked reduction of factor H binding was observed in cells treated with anti-heparan sulfate Ab (Figure 6). No decrease in factor H binding was found in the presence of an irrelevant control Ab (anti-CD26) that binds to a tubular epithelial surface target (Figure 6). To further characterize the effect of protein load on factor H binding capability, we investigated by flow cytometry whether plasma protein challenge could modify the surface expression of heparan sulfate on HK-2 cells. Incubation with albumin or transferrin for 24 h caused a significant reduction of heparan sulfate on HK-2 cell surface as reflected by lower mean fluorescence intensity in comparison with unstimulated cells (Figure 7). To identify the possible mechanism(s) by which plasma proteins lead to a reduction of heparan sulfate residues on the HK-2 cell surface, we focused on heparanase 1 (HPSE-1), an enzyme involved in the heparan sulfate cleavage.31.van den Hoven M.J. Rops A.L. Vlodavsky I. et al.Heparanase in glomerular diseases.Kidney Int. 2007; 72: 543-548Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Western blot analysis of HPSE-1 protein expression was performed in protein cell lysates obtained from HK-2 cells incubated for 15 h with medium (control), albumin, or transferrin. HPSE-1 was constitutively expressed in untreated cells (see Figure S2). In response to plasma protein exposure, an increase in the level of the 65-kDa form of HPSE-1 was observed and confirmed by densitometric analysis (Figure S2). Finally, we assessed whether heparan sulfate, by binding factor H, may be implicated in its inhibitory activity on complement activation on HK-2 cells. Treatment of cells with a specific Ab blocking the heparan sulfate residues before HS addition significantly increased active C3 (Figure 8a) and MAC deposition (Figure 8b) as compared with cells exposed to HS alone or to HS plus the irrelevant control Ab (Figure 8a and b). We established a model to evaluate the effects of sublethal challenge by complement on proximal tubular epithelial cells under conditions that mimic the renal tubule in proteinuric nephropathy. This allowed us to investigate whether excess plasma proteins may lead to proximal tubular cell dysfunction induced by the abnormal presence of complement as found in the ultrafiltrate, and by which mechanisms. Findings that albumin overload enhances the deposition of serum-derived C3 on the cell surface through the alternative pathway leading to enhanced assembly of MAC suggest a role of excess proteins within the ultrafiltrate in facilitating the activation of the complement cascade. In particular, these data explain the high vulnerability of proximal tubular cells to abnormal exposure to complement in chronic proteinuric diseases. Along this line, C6 deficiency failed to significantly impact on tubulointerstitial damage in nonproteinuric models of renal disease, suggesting that in the absence of proteinuria, C5-b9 was not sufficient to enhance injury.21.Rangan G.K. Pippin J.W. Coombes J.D. et al.C5b-9 does not mediate chronic tubulointerstitial disease in the absence of proteinuria.Kidney Int. 2005; 67: 492-503Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar If translated in vivo, increased complement deposition and activation by protein load on tubular cell surface may play a pivotal role in exacerbating tubulointerstitial injury. We previously documented that antiproteinuric treatment by ameliorating the damage of the glomerular barrier to proteins limits tubular and interstitial injury in chronic proteinuric nephropathy.18.Abbate M. Zoja C. Rottoli D. et al.Antiproteinuric therapy while preventing the abnormal protein traffic in proximal tubule abrogates protein and complement-dependent interstitial inflammation in experimental renal disease.J Am Soc Nephrol. 1999; 10: 804-813PubMed Google Scholar, 19.Abbate M. Zoja C. Rottoli D. et al.Proximal tubular cells promote fibrogenesis by TGF-β1-mediated induction of peritubular myofibroblasts.Kidney Int. 2002; 61: 2066-2077Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 20.Abbate M. Zoja C. Corna D. et al.Complement-mediated dysfunction of glomerular filtration barrier accelerates progressive renal injury.J Am Soc Nephrol. 2008; 19: 1158-1167Crossref PubMed Scopus (49) Google Scholar The reduced tubular overload with filtered proteins and C3 was associated with reduced proinflammatory activation of tubular cells, which could account for renopro
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