BASIC RESEARCH www.jasn.org Complement-Mediated Dysfunction of Glomerular Filtration Barrier Accelerates Progressive Renal Injury Mauro Abbate,* Carla Zoja,* Daniela Corna,* Daniela Rottoli,* Cristina Zanchi,* Nadia Azzollini,* Susanna Tomasoni,* Silvia Berlingeri,* Marina Noris,* Marina Morigi,* and Giuseppe Remuzzi*† *Mario Negri Institute for Pharmacological Research and †Unit of Nephrology and Dialysis, Azienda Ospedaliera Ospedali Riuniti di Bergamo, Bergamo, Italy ABSTRACT Intrarenal complement activation leads to chronic tubulointerstitial injury in animal models of proteinuric nephropathies, making this process a potential target for therapy. This study investigated whether a C3-mediated pathway promotes renal injury in the protein overload model and whether the abnormal exposure of proximal tubular cells to filtered complement could trigger the resulting inflammatory response. Mice with C3 deficiency were protected to a significant degree against the protein overload–induced interstitial inflammatory response and tissue damage, and they had less severe podocyte injury and less proteinuria. When the same injury was induced in wild-type (WT) mice, antiproteinuric treatment with the angiotensin-converting enzyme inhibitor lisinopril reduced the amount of plasma protein filtered, decreased the accumulation of C3 by proximal tubular cells, and protected against interstitial inflammation and damage. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell–derived C3, C3-deficient kidneys were transplanted into WT mice. Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when WT kidneys were transplanted into C3-deficient mice, protein overload led to a more mild disease and abnormal C3 deposition was not observed. These data suggest that the presence of C3 increases the glomerular filtration barrier’s susceptibility to injury, ultrafiltered C3 contributes more to tubulointerstitial damage induced by protein overload than locally synthesized C3, and local C3 synthesis is irrelevant to the development of proteinuria. It is speculated that therapies targeting complement combined with interven- tions to minimize proteinuria would more effectively prevent the progression of renal disease. J Am Soc Nephrol 19: 1158–1167, 2008. doi: 10.1681/ASN.2007060686 Chronic kidney diseases (CKD) are a major global this injury, the dysfunction of the physiologic fil- and increasing health epidemic, with expected cu- tering barrier against the loss of plasma proteins mulative costs of dialysis and kidney transplanta- into the urine may act as an independent caus- tion exceeding $1 trillion in the next decade.1 Epi- ative factor.7 Abnormally ultrafiltered proteins demiology shows tight association between CKD alter gene expression in proximal tubular cells, and cardiovascular disease, predisposition of the patients to cardiovascular events, and albuminuria Received June 20, 2007. Accepted January 11, 2008. as an independent risk factor for complications and 2–4 Published online ahead of print. Publication date available at premature death. Experimental evidence indi- www.jasn.org. cates that once diseases of various etiology destroy a Correspondence: Dr. Mauro Abbate, Mario Negri Institute for critical amount of the renal mass, injury develops in Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, remaining nephrons as a consequence of maladap- Italy. Phone: ϩ39-035-319888; Fax: ϩ39-035-319331; E-mail: tive increases in intraglomerular capillary pressure [email protected] and flow.5,6 In addition to playing a pivotal role in Copyright ᮊ 2008 by the American Society of Nephrology 1158 ISSN : 1046-6673/1906-1158 J Am Soc Nephrol 19: 1158–1167, 2008 www.jasn.org BASIC RESEARCH Figure 1. Effect of C3 deficiency on protein overload nephrop- athy in mice. (A and B) Urinary protein excretion (A) and tubular Ϫ Ϫ damage (B) in WT mice (n ϭ 5) and C3 / mice (n ϭ 5) receiving saline and in mice that were administered daily intraperitoneal Ϫ Ϫ injections of BSA (15 mg/g) for 4 wk (WT-BSA, n ϭ 9; C3 / BSA, n ϭ 7). ЊЊP Ͻ 0.01 versus WT-saline; **P Ͻ 0.01 versus WT-BSA. (C) Periodic acid-Schiff–stained sections of renal cortex of WT and Ϫ Ϫ C3 / mice given saline or BSA. Magnification, ϫ150. Figure 2. C3-dependent interstitial inflammatory and fibrogenic ϩ response to tubular protein overload. (A and B) Interstitial F4/80 ϩ Ϫ Ϫ resulting in proinflammatory and fibrogenic phenotypic cells (A) and ␣-SMA cells (B) in renal cortex of WT and C3 / changes.8 Angiotensin-converting enzyme inhibitors mice receiving saline or BSA. Data are means Ϯ SEM. ЊP Ͻ 0.05, ЊЊP Ͻ 0.01 versus WT-saline; *P Ͻ 0.05,**P Ͻ 0.01 versus (ACEi) and angiotensin receptor blockers prevent the pro- Ϫ Ϫ WT-BSA; #P Ͻ 0.01 versus C3 / saline. HPF, high-power field gressive impairment of the barrier and delay kidney failure. ϩ (ϫ400). The photomicrographs show F4/80 cells (A) and Drugs’ renoprotective effects cannot be dissociated from ϩ ␣-SMA cells (B), as revealed by immunoperoxidase and im- their action of lowering proteinuria, although they may not munofluorescence analysis, in kidney sections of WT mice Ϫ Ϫ be achieved invariably among patients. Clarifying further given saline or BSA and of a C3 / mouse given BSA. Magni- the pathophysiologic significance of the abnormal passage of fication, ϫ250. proteins to the luminal compartment of the nephron is man- datory in the search of drug targets for renal and cardiovascular protection. Complement proteins also accumulate in renal tubules of Intrarenal complement activation is a powerful mechanism of rats with protein overload proteinuria, a model in which tubulointerstitial injury by eliciting cytotoxic and proinflamma- plasma protein toxicity is specifically studied.24 ACEi treat- tory responses.9 C3 and other complement proteins were found in ment by limiting the transglomerular passage of proteins was proximal tubules in human renal biopsy material10,11 and in an effective maneuver to reduce both high protein and C3 load kidneys of rats after extensive renal mass reduction12–14 or of tubular cells in remnant kidneys14,21; however, no studies other proteinuric models.13,15–17 Protective effects of limiting tested the role of ultrafiltered complement in tubulointerstitial complement activation were revealed using C6-deficient ani- injury. Furthermore, proximal tubular cells are able to synthe- mals16,17 or genetically modified mice overexpressing a soluble size C3 and other complement factors23,25 and, in vitro, up- C3 inhibitor18 and by pharmacologic manipulations.16,17,19,20 regulate C3 in response to serum proteins,8,9 raising the possi- Stimulatory factors underlying complement-mediated injury bility that tubular C3 synthesis may be required to activate were not yet clarified. This is an important issue because com- pathogenic pathways. plement inhibitory approaches must be targeted timely and at We used C3-deficient mice and transplant experiments to specific steps. One hypothesis is that the exposure of proximal assess whether (1) protein overload proteinuria promotes re- tubular cells to excess plasma proteins as a consequence of nal injury through a C3-mediated pathway and (2) the abnor- glomerular barrier dysfunction could play a role.13,17,21,22 With mal exposure of proximal tubular cells to ultrafiltered comple- proteinuria, a key putative factor is the excess filtration of C3 ment could be a trigger of the inflammatory response. We (molecular weight 180 kD),14 the central molecule for the analyzed effects of antiproteinuric treatment with ACEi to vali- system to exert proinflammatory potential.14,23 In rats after date further the enhanced passage of proteins across the glomer- severe reduction of the renal mass, C3 was localized to prox- ular capillary barrier as a determinant of ultrafiltered protein and imal tubular cells engaged in high protein uptake.13,14,21 complement challenge of tubular cells. J Am Soc Nephrol 19: 1158–1167, 2008 Filtered Protein Toxicity via C3 1159 BASIC RESEARCH www.jasn.org droplet accumulation, cytoplasmic vacu- olization, effacement of foot processes, and formation of microvilli (Figure 3B). Podocyte damage was markedly attenu- ated in glomeruli of C3Ϫ/Ϫ BSA mice (Fig- ure 3C). Confocal microscopy showed ab- sence of C3 staining in the glomeruli of WT mice given saline (Figure 3D) and, in con- trast, C3 fixation of WT-BSA glomeruli, with a granular pattern corresponding to intracellular droplets of podocytes in the epithelial cell areas (Figure 3E). To test whether C3 deficiency may Figure 3. Podocyte injury is associated with C3 fixation in the absence of electron-dense be protective against tubulointerstitial Ϫ Ϫ immune deposits in WT mice with BSA overload and is attenuated in C3 / BSA mice. (A changes by abolishing the tubular load of through C) Electron micrographs show glomerular capillaries of WT mice given saline (A) C3 and other plasma proteins, which acts Ϫ/Ϫ or BSA (B) and of a C3 BSA mouse (C). The WT-BSA mouse glomerulus (B) shows as a trigger of tubular cell activation and podocyte swelling and protein droplets (arrows) and foot process fusion in the absence Ϫ Ϫ proinflammatory phenotypic change, we of electron-dense deposits. Podocyte changes are less marked in C3 / BSA (C). (D and performed immunohistochemical stud- E) In contrast to WT mouse given saline (D) showing only interrupted staining of Bow- man’s capsule (BC), the detection of C3 by confocal microscopy in a WT-BSA glomerulus ies for detection of filtered plasma pro- reveals C3 fixation of WT-BSA glomeruli, with a granular pattern corresponding to teins in tubular epithelial cells. In kid- intracellular droplets of podocytes in glomerular epithelial cell areas (E, arrows). Magni- neys of WT mice, C3 had peritubular fications: ϫ5600 in A through C; ϫ1000 in D and E. distribution with no apical or intracellu- lar staining (Figure 4A). In contrast, in RESULTS WT-BSA mice, C3 was detected on apical cell surface and within proximal tubular cells in addition to the normal peri- C3 Deficiency Protects Mice against Renal Damage tubular staining (Figure 4A).