Protein Load Impairs Factor H Binding Promoting Complement-Dependent Dysfunction of Proximal Tubular Cells

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector original article http://www.kidney-international.org & 2009 International Society of Nephrology Protein load impairs factor H binding promoting complement-dependent dysfunction of proximal tubular cells Simona Buelli1,6, Mauro Abbate1,6, Marina Morigi1, Daniela Moioli1, Cristina Zanchi1, Marina Noris1, Carla Zoja1, Charles D. Pusey2, Peter F. Zipfel3,4 and Giuseppe Remuzzi1,5 1‘Mario Negri’ Institute for Pharmacological Research, Bergamo, Italy; 2Renal Section, Imperial College London, Hammersmith Hospital, London, UK; 3Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany; 4Friedrich Schiller University, Jena, Germany and 5Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy Intrarenal complement activation plays an important role in Progressive nephropathies with severe defects of the glomer- the progression of chronic kidney disease. A key target of the ular filtration barrier to proteins are accompanied by activated complement cascade is the proximal tubule, a site tubulointerstitial damage eventually contributing to loss of where abnormally filtered plasma proteins and complement renal function.1 The clinical problem has grown enormously1,2 factors combine to promote injury. This study determined and understanding the basic mechanisms is mandatory. whether protein overloading of human proximal tubular cells Experimental observations suggested that ultrafiltered plasma (HK-2) in culture enhances complement activation by proteins exert renal toxicity, which is partly mediated by impairing complement regulation. Addition of albumin or activation of inflammatory and fibrogenic pathways by transferrin to the cells incubated with diluted human serum proximal tubular cells.3–9 Among processes underlying injury, asasourceofcomplementcausedincreasedapicalC3 the activation of the complement cascade in the renal tubule deposition. Soluble complement receptor-1 (an inhibitor of has major effects.10 Complement proteins can be abnormally all 3 activation pathways) blocked complement deposition filtered across the altered glomerular barrier leading to while the classical and lectin pathway inhibitor, magnesium intratubular deposition of C3 and formation of membrane chloride–EGTA, was, ineffective. Media containing albumin as attack complex (MAC).11–13 Proximal tubular epithelial cells well as complement had additive proinflammatory effects as also synthesize most components of the activation cascade.14,15 shown by increased fractalkine and transforming growth Investigation using proteinuric models of glomerular injury factor-b mRNA expression. This paralleled active C3 and showed less tubulointerstitial damage in complement-depleted C5b-9 generations, effects not shared by transferrin. Factor H, rats12,13 and C6-deficient rats,16 or upon treatment with one of the main natural inhibitors of the alternative pathway, complement inhibitory molecules.13,17 Inflammation and a binds to heparan sulfate proteoglycans. Both the density of fibrogenic reaction ensue upon proximal tubule overloading heparan sulfate and factor H binding were reduced with by C3 and ultrafiltered proteins in rats with remnant protein loading, thereby enhancing the albumin- and kidneys.18,19 We have recently found that ultrafiltered serum-dependent complement activation potential. Thus, complement factors are a key component of proteinuria- protein overload reduces the ability of the tubule cell to bind associated renal injury. Thus, plasma-derived C3 is recruited factor H and counteract complement activation, effects with other ultrafiltered proteins in the tubular compartment to instrumental to renal disease progression. mediate interstitial macrophage accumulation and renal Kidney International (2009) 75, 1050–1059; doi:10.1038/ki.2009.8; structural damage in the absence of C3 synthesis by the published online 25 February 2009 kidney, in a mouse model of protein-overload proteinuria.20 KEYWORDS: albumin; chemokines; complement; cytokines; factor H; Overall, proteinuria is required for complement to fully exert transferrin harmful effects on the kidney,21 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 Correspondence: Mauro Abbate, ‘Mario Negri’ Institute for Pharmacological proteins to induce tubular cell dysfunction. However, Research, Via Gavazzeni 11, 24125 Bergamo, Italy. approaches using cultured proximal tubular epithelial cells E-mail: [email protected] are most appropriate to address this issue. 6These authors contributed equally to this paper. In human proximal tubular cells exposed to serum, Received 23 April 2008; revised 19 November 2008; accepted 23 complement activation through the alternative pathway leads December 2008; published online 25 February 2009 to surface deposition of C3 and MAC and upregulation of 1050 Kidney International (2009) 75, 1050–1059 S Buelli et al.: Complement-activating environment by protein load original article cytokines.22,23 Complement activation might be dependent to 25% HS in comparison with control cells (10% HS: 91±5, on relative lack of complement inhibitors. Cell-associated 25% HS: 71±4 versus control: 100% viable cells). The regulators at C3 level, decay accelerating factor and concentration of 10% HS was therefore established to be membrane cofactor protein, are hardly detectable on apical sublytic. HK-2 cells upon exposure to 10% HS activated and membranes of proximal tubular cells. Conversely, human fixed C3 and MAC on the apical side of the cell surface as proximal tubular cells produce factor H,24 a 150-kDa plasma showed by appearance of granular staining by immunofluor- glycoprotein acting as the major soluble inhibitor of the escence analysis (Figure 1) on cell surface. In contrast, formation of alternative pathway C3 convertase on the cell unstimulated cells showed no fluorescent signal (Figure 1). surface25,26 and possibly representing a unique defense Addition of sCR-1, a soluble inhibitor of all the three mechanism against complement activation. No studies have pathways of complement activation, completely blocked C3/ yet documented actual binding of factor H by tubular MAC deposition on cell surface, indicating specificity of C3 epithelial cells. Relevant binding sites on the cell surface, as and MAC signals (Figure 1). Similarly, no complement shown in endothelial cells, may include heparan sulfate protein deposition was detected using heat-inactivated HS proteoglycans.27 (data not shown). The aim of this study was to investigate whether (1) To study whether protein overload could enhance the protein overload may influence complement activation and ability of proximal tubular epithelial cells to fix complement deposition on the surface of proximal tubular cells exposed to molecules, immunofluorescence analysis of C3 and MAC serum complement, a condition that mimics the exposure of deposits was performed on HK-2 cells, first challenged with tubular cells to ultrafiltered complement in proteinuric albumin or transferrin and then incubated with 10% disease; (2) this effect was related to reduced binding of HS as described above. Both plasma proteins caused factor H on the cell surface accounting for reduced capability significant increases in C3 staining as detected at the end of of the cell to counteract complement activation; and (3) loss the experiment, in comparison with cells exposed to HS of heparan sulfate sites contributed to reduced factor H alone (Figure 2a). Exposure of HK-2 cells to plasma proteins binding to the cell. in the absence of HS resulted in no significant changes in C3 staining as compared with unstimulated cells, thus RESULTS excluding that the abnormal accumulation of C3 on the cell Protein overload enhances the activation of serum-derived surface in the presence of HS could be attributed to the complement by human proximal tubular cells induction of C3 synthesis in response to albumin and With the aim of studying the effect of plasma protein transferrin. To test whether complement activation may challenge on complement deposition, preliminary experi- occur through the alternative pathway, cells were incubated ments were carried out to identify the sublytic concentration with HS containing sCR-1 or with MgCl2–EGTA that blocks of human serum (HS), used as source of complement. Cell the activation of the classic and the lectin pathways. The viability, as assessed by trypan blue dye exclusion at 90 min, addition of sCR-1 resulted in complete inhibition of C3 was not affected by exposure to 10% HS, whereas a deposition. In contrast, MgCl2–EGTA had no significant significant (Po0.01) reduction was observed upon exposure effect (Table 1). Control HS 10% HS 10% + sCR-1 C3 Inset Control HS 10% HS 10% + sCR-1 MAC Inset Figure 1 | C3 and MAC (membrane attack complex) deposition on tubular epithelial cell surface in response to human serum (HS). Immunofluorescence staining of C3 and MAC (green) on HK-2 cells exposed for 90 min to medium or 10% HS in the presence or absence of soluble complement receptor-1 (sCR-1, 100 mg/ml). No C3 or MAC fixation was observed on unstimulated cells. HK-2 cells incubated with 10% HS exhibited C3 and MAC staining with granular distribution on cell surface (inset: two-fold enlargement of selected areas). sCR-1 treatment completely

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