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J Am Soc Nephrol. Jun 2008; 19(6): 1158–1167.
PMCID: PMC2396931

Complement-Mediated Dysfunction of Glomerular Filtration Barrier Accelerates Progressive Renal Injury


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 interventions to minimize proteinuria would more effectively prevent the progression of renal disease.

Chronic kidney diseases (CKD) are a major global and increasing health epidemic, with expected cumulative costs of dialysis and kidney transplantation exceeding $1 trillion in the next decade.1 Epidemiology shows tight association between CKD and cardiovascular disease, predisposition of the patients to cardiovascular events, and albuminuria as an independent risk factor for complications and premature death.24 Experimental evidence indicates that once diseases of various etiology destroy a critical amount of the renal mass, injury develops in remaining nephrons as a consequence of maladaptive increases in intraglomerular capillary pressure and flow.5,6 In addition to playing a pivotal role in this injury, the dysfunction of the physiologic filtering barrier against the loss of plasma proteins into the urine may act as an independent causative factor.7 Abnormally ultrafiltered proteins alter gene expression in proximal tubular cells, resulting in proinflammatory and fibrogenic phenotypic changes.8 Angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers prevent the progressive impairment of the barrier and delay kidney failure. Drugs' renoprotective effects cannot be dissociated from their action of lowering proteinuria, although they may not be achieved invariably among patients. Clarifying further the pathophysiologic significance of the abnormal passage of proteins to the luminal compartment of the nephron is mandatory in the search of drug targets for renal and cardiovascular protection.

Intrarenal complement activation is a powerful mechanism of tubulointerstitial injury by eliciting cytotoxic and proinflammatory responses.9 C3 and other complement proteins were found in proximal tubules in human renal biopsy material10,11 and in kidneys of rats after extensive renal mass reduction1214 or other proteinuric models.13,1517 Protective effects of limiting complement activation were revealed using C6-deficient animals16,17 or genetically modified mice overexpressing a soluble C3 inhibitor18 and by pharmacologic manipulations.16,17,19,20 Stimulatory factors underlying complement-mediated injury were not yet clarified. This is an important issue because complement inhibitory approaches must be targeted timely and at specific steps. One hypothesis is that the exposure of proximal tubular cells to excess plasma proteins as a consequence of glomerular barrier dysfunction could play a role.13,17,21,22 With proteinuria, a key putative factor is the excess filtration of C3 (molecular weight 180 kD),14 the central molecule for the system to exert proinflammatory potential.14,23 In rats after severe reduction of the renal mass, C3 was localized to proximal tubular cells engaged in high protein uptake.13,14,21 Complement proteins also accumulate in renal tubules of rats with protein overload proteinuria, a model in which plasma protein toxicity is specifically studied.24 ACEi treatment by limiting the transglomerular passage of proteins was an effective maneuver to reduce both high protein and C3 load of tubular cells in remnant kidneys14,21; however, no studies tested the role of ultrafiltered complement in tubulointerstitial injury. Furthermore, proximal tubular cells are able to synthesize C3 and other complement factors23,25 and, in vitro, upregulate C3 in response to serum proteins,8,9 raising the possibility that tubular C3 synthesis may be required to activate pathogenic pathways.

We used C3-deficient mice and transplant experiments to assess whether (1) protein overload proteinuria promotes renal injury through a C3-mediated pathway and (2) the abnormal exposure of proximal tubular cells to ultrafiltered complement could be a trigger of the inflammatory response. We analyzed effects of antiproteinuric treatment with ACEi to validate further the enhanced passage of proteins across the glomerular capillary barrier as a determinant of ultrafiltered protein and complement challenge of tubular cells.


C3 Deficiency Protects Mice against Renal Damage Induced by Protein Overload

To assess the role of C3 in the pathogenesis of proteinuria-induced tubulointerstitial damage, we first characterized the renal disease phenotype of protein overload proteinuria in C3 gene–disrupted (C3−/−) mice. Wild-type (WT) mice given BSA (WT-BSA) for 4 wk had high levels of urinary protein excretion (Figure 1A). WT-BSA mice developed tubular luminal casts and dilation and focal glomerular sclerotic lesions (Figure 1, B and C). C3−/− mice given BSA (C3−/−BSA) had lower degrees of proteinuria (Figure 1A) and tubular damage (Figure 1, B and C). Percentages of glomeruli showing sclerotic changes were lower in C3−/−BSA than in WT-BSA mice (0.3 ± 0.2 versus 5 ± 1; P < 0.01). Control WT or C3−/− mice given saline showed no changes. Structural changes in WT-BSA mice were associated with accumulation of F4/80-positive cells (Figure 2A) and α-smooth muscle actin (α-SMA)-positive cells (Figure 2B) into the interstitium. As compared with WT-BSA mice, the interstitium of C3−/− BSA mice contained significantly lower numbers of F4/80-positive cells and α-SMA–positive cells. Given the protection exerted by C3 deficiency against proteinuria and glomerular sclerosis, we performed studies to assess the effects of C3 deficiency on the structural integrity of the glomerular barrier in this model. Neither circulating anti-BSA antibodies (data not shown) nor electron-dense immune deposits or infiltrating immune-type cells were detectable in WT-BSA mice (Figure 3), thus excluding a typical form of immunologically mediated glomerulonephritis. WT-BSA mice developed podocyte damage consisting of protein droplet accumulation, cytoplasmic vacuolization, effacement of foot processes, and formation of microvilli (Figure 3B). Podocyte damage was markedly attenuated in glomeruli of C3−/− BSA mice (Figure 3C). Confocal microscopy showed absence of C3 staining in the glomeruli of WT mice given saline (Figure 3D) and, in contrast, C3 fixation of WT-BSA glomeruli, with a granular pattern corresponding to intracellular droplets of podocytes in the epithelial cell areas (Figure 3E).

Figure 1.
Effect of C3 deficiency on protein overload nephropathy in mice. (A and B) Urinary protein excretion (A) and tubular damage (B) in WT mice (n = 5) and C3−/− mice (n = 5) receiving saline and in mice that were administered ...
Figure 2.
C3-dependent interstitial inflammatory and fibrogenic response to tubular protein overload. (A and B) Interstitial F4/80+ cells (A) and α-SMA+ cells (B) in renal cortex of WT and C3−/− mice receiving saline or BSA. ...
Figure 3.
Podocyte injury is associated with C3 fixation in the absence of electron-dense immune deposits in WT mice with BSA overload and is attenuated in C3−/− BSA mice. (A through C) Electron micrographs show glomerular capillaries of WT mice ...

To test whether C3 deficiency may be protective against tubulointerstitial changes by abolishing the tubular load of C3 and other plasma proteins, which acts as a trigger of tubular cell activation and proinflammatory phenotypic change, we performed immunohistochemical studies for detection of filtered plasma proteins in tubular epithelial cells. In kidneys of WT mice, C3 had peritubular distribution with no apical or intracellular staining (Figure 4A). In contrast, in WT-BSA mice, C3 was detected on apical cell surface and within proximal tubular cells in addition to the normal peritubular staining (Figure 4A). No IgG was found in tubular cells of WT (Figure 4B) or C3−/− controls (data not shown). Kidneys of C3 −/−BSA mice showed abnormal staining of IgG in proximal tubules, albeit to a lesser degree with respect to WT-BSA mice (Figure 4B). Kidneys of WT-BSA mice killed at an early stage of disease (week 1) showed abnormal staining for C3 and IgG in proximal tubules, before the onset of tubular changes (Figure 5). These findings indicate that C3 deficiency may exert protective effects on the tubulointerstitium in the presence of residual dysfunction of the glomerular barrier, without fully abrogating the exposure of proximal tubular cells to ultrafiltered proteins.

Figure 4.
(A and B) Proximal tubular staining for C3 (A) and IgG (B) in renal cortex of WT mice or C3−/− mice receiving saline and of mice that were administered daily intraperitoneal injections of BSA (WT-BSA and C3−/−BSA). Data ...
Figure 5.
C3 accumulation and protein overload of proximal tubules in early stage of protein overload nephropathy (week 1). Sections of renal cortex of mice that received daily intraperitoneal injections of BSA (A, C, and E) or saline (B, D, and F), stained for ...

Effects of Transplantation of C3−/− Kidneys into WT mice or WT Kidneys into C3−/− Mice on Renal Injury of Protein Overload Proteinuria

We tested the role of plasma-derived C3 by transplantation experiments in which congenic C3−/− kidneys were transplanted into WT recipients before exposure to protein overload proteinuria, and vice versa. WT mice given WT kidneys developed protein overload proteinuria in response to BSA injections (Table 1). Proteinuria (Table 1) and tubular injury (score 1.00 ± 0.00 versus 0.96 ± 0.04) of WT mice that received a transplant of C3-deficient kidneys were comparable to those found in the WT mice that received a transplant of WT kidneys in this model. The percentage of glomeruli showing sclerotic lesion (mean percentage 5.6% [range 3 to 7%] and 6% [range 5 to 7%]) was comparable in both groups, which also showed marked podocyte damage and glomerular C3 fixation (Figure 6). Abnormal staining for C3 was found within the cells and on apical surface in proximal tubules in transplanted kidneys (Figure 7), to similar extent in both groups (Table 1). These changes were associated with protein overload of proximal tubules, as reflected by evidence of IgG accumulation at this site (data not shown). F4/80+ cells into the interstitium were lower but not to a significant extent in transplanted C3−/− kidneys as compared with WT kidneys (Table 1, Figure 7). C3−/− mice that received a transplant of WT kidneys and were exposed to BSA overload had less severe proteinuria (Table 1) and tubular injury (score 0.16 ± 0.16; P < 0.05 versus WT/WT BSA mice and KO/WT BSA mice), a lower percentage of glomeruli showing sclerosis (0.33%; range 0 to 1%; P < 0.05 versus WT/WT BSA mice and KO/WT BSA mice), and reduced interstitial F4/80+ cell accumulation (Table 1, Figure 7). The protective effects of systemic C3 deficiency were associated with less prominent podocyte injury (Figure 6), in the absence of abnormal staining of C3 both in the glomeruli (Figure 6) and at tubular level (Table 1, Figure 7).

Figure 6.
Electron micrographs of podocytes and immunofluorescence staining for C3 in glomeruli of transplanted kidneys in BSA overload. WT mice received a transplant of either a WT kidney (left) or a C3−/− kidney (middle), and the C3−/− ...
Figure 7.
Effects of selectively abrogating intrarenal synthesis of C3 versus circulating C3 on the accumulation of C3 in proximal tubules and interstitial F4/80+ cells of BSA overload nephropathy. WT mice received a transplant of either a WT kidney (left) ...
Table 1.
Effects of C3−/− kidney transplantation in WT mice or WT kidney transplantation in C3−/− mice on proteinuria, accumulation of C3 in proximal tubules, and interstitial F4/80+ cell infiltration of BSA overload nephropathy ...

Lisinopril Limits C3 Accumulation and C3 mRNA Upregulation in Proximal Tubules and Tubulointerstitial Changes in Mice with Protein Overload Proteinuria

ACEi was previously found to limit both proteinuria and C3 deposition in proximal tubules in rats with progressive tubulointerstitial injury.14,21 To verify further excess protein load on proximal tubular cells as a stimulus for the activation of complement-mediated injury, we investigated whether ACEi treatment by reducing the high protein load could lead to less C3 accumulation in proximal tubules and less tubulointerstitial damage. Lisinopril treatment significantly reduced both proteinuria and the proximal tubular cell staining of C3 and IgG as compared with untreated mice with BSA overload (Table 2). Lisinopril reduced tubular damage score and numbers of F4/80+ cells in the renal interstitium (Table 2).

Table 2.
Effects of lisinopril treatment on proteinuria, tubular damage, and C3 and IgG accumulation in proximal tubules and interstitial F4/80+ cells in WT mice receiving BSAa

To establish whether protein overload may increase C3 synthesis by tubular cells, we first evaluated by real-time reverse transcriptase–PCR (RT-PCR) the effects of BSA overload on C3 mRNA in whole kidneys. BSA overload caused a 14-fold upregulation of C3 mRNA (P < 0.05 versus saline-injected mice; Figure 8A). In situ hybridization analysis showed focal C3 mRNA staining in tubular epithelial cells of kidney sections of WT control mice that were administered an injection of saline (Figure 8B). A strong increase in C3 mRNA was detected in tubular epithelial cells of both proximal tubules and distal segments in WT-BSA mice (Figure 8B). No change was found in kidneys of WT-BSA mice killed at early stage (1 wk; Figure 8B). Sense probe produced no or trace nonspecific staining. C3−/− kidneys had no signal (data not shown). Lisinopril caused a significant reduction of whole kidney C3 mRNA levels as compared with BSA mice given vehicle (Figure 8A). In contrast to the strong signal for C3 mRNA that was focally detectable by in situ hybridization in this group, focal perinuclear C3 mRNA staining was found in proximal tubular cells in kidneys of lisinopril-treated BSA mice (Figure 8B).

Figure 8.
Enhanced C3 mRNA expression in kidney and inhibition by antiproteinuric therapy in protein overload nephropathy. (A) Real-time RT-PCR. Data are means ± SEM. °°P < 0.01 versus WT-saline; *P < 0.05 versus ...


The search for mechanisms of progressive renal injury has crucial importance in renal and cardiovascular medicine. Protein overload proteinuria is a unique ad hoc model whereby the role of toxicity of filtered proteins in tubulointerstitial damage can be investigated in mice. Findings that protein-overloaded C3−/− mice were significantly protected from tubular injury and interstitial accumulation of macrophages and myofibroblasts indicate that C3 plays a pivotal role in processes by which the abnormal passage of plasma proteins across the glomerular barrier into the luminal compartment of the nephron damages the kidney. One major novel finding is that despite no evidence of antibody-mediated nephritis, the degrees of proteinuria and glomerular damage were low in C3−/− mice, suggesting that the injury to the glomerular barrier was exacerbated by complement and that the related stimulation of proximal tubular cells was reduced in its absence. Protection by systemic C3 deficiency was exerted even on transplanted kidneys with intact local complement system. Collectively, our data document that complement is recruited locally to augment both podocyte injury and the interstitial inflammatory response and that protein overload is a stimulatory factor. Candidate effector molecules of renal epithelial cell injury have been recognized both in experimental models19,25 and in vitro.26 Alternative pathway complement activation through C3 convertase leads to C5b-9 (MAC) binding on tubular cell surface12 and causes tubular cell dysfunction via reactive oxygen species and cytokines.26 A monocyte-activating amidated form of C3,12 C3a,27,28 and C529 can also be pathogenic.

Glomerular permselective dysfunction in the protein overload model leads to urinary loss of endogenous plasma proteins.30 Consistently, the attenuation of podocyte injury accounted for reduction of proteinuria by C3 deficiency. Previous investigation showed that lisinopril treatment by preserving the integrity of the glomerular barrier31,32 limited the exposure of the tubule to plasma proteins and attenuated both C3 accumulation in proximal tubular cells and renal injury in rat remnant kidneys.14,21 Similar effects were achieved here by lisinopril manipulation, further indicating that protein overload acts as a stimulus for complement accumulation in tubular cells and the associated interstitial inflammatory reaction. In light of less tubulointerstitial damage found in C3−/− mice, reducing the accumulation of complement proteins in proximal tubular cells, or at least the apical exposure of the cells to plasma-derived C3, is in all likelihood a component of the drug's protective action against tubulointerstitial injury. Evidence that complement acts downstream of protein load to enhance tubulointerstitial injury also explains less susceptibility of C6-deficient rats to tubulointerstitial damage despite proteinuria in aminonucleoside of puromycin16 or renal mass reduction.17 Instead, no apical complement deposition in tubules occurred and the lack of C6 was not protective in nonproteinuric models,22 indirectly suggesting the importance of apical exposure to complement to enhance injury in proteinuric nephropathy.

C3 (180 kD) is mostly circulating and of hepatic source; however, a role in renal injury has been suggested for the capacity of kidney cells to synthesize C3, C4, C5, and factor B.25,33 C3 of proximal tubule cell origin is a crucial mediator of injury in experimental acute allograft rejection and after renal ischemia and reperfusion.23,34 Its expression was enhanced in both human and experimental proteinuric nephropathies.35 Moreover, exposing cultured proximal tubular cells to serum proteins at the apical surface enhanced C3 mRNA expression36 and secretion.36,37 Conversely, our transplantation studies done to abrogate C3 synthesis by the kidney show that C3 accumulates in renal tubules to promote tubulointerstitial injury, whereas the latter is less severe in systemically C3-deficient mice with transplanted WT kidneys. These data would not exclude the pathogenic potential of tubular C3 synthesis on more severe glomerular injury and proteinuria as found in WT mice. Here, lisinopril prevented C3 mRNA upregulation in tubular cells of BSA-overloaded mice. In addition, peritubular C3 staining was preserved in WT kidneys transplanted into C3−/− mice in the presence of residual proteinuria; however, the abundance of C3 reaching the ultrafiltrate seems to overwhelm locally synthesized C3. Consistently, C3 overload of proximal tubular cells was present since a stage of disease in which tubular C3 mRNA was not upregulated.

Collectively, our data show that limiting glomerular barrier dysfunction attenuates the excess protein load responsible for abnormal accumulation of C3 in the renal tubule. The finding of significant reduction of proteinuria in C3−/− mice with BSA overload makes it difficult to evaluate to which extent the reduced tubular load with other plasma proteins and the lack of filtered C3 itself, respectively, concurred to protect against tubulointerstitial injury. Both in C3−/− mice and in ACEi-treated mice, residual accumulation of plasma proteins in proximal tubules and tubulointerstitial changes reflects the contribution of complement-independent mechanisms of injury in the presence of excess protein in the ultrafiltrate. They include proximal tubule cell synthesis of mediators (monocyte chemoattractant protein-1, RANTES, fractalkine, endothelin-1, and TGF-β1) and/or other stimulatory effects by protein-bound molecules.7 In humans, data using laser capture microdissection and microarray analysis revealed complex gene regulatory responses of proximal tubular cells in proteinuric nephropathies.38 Cultured proximal tubular cells via the megalin receptor for protein uptake can undergo activation by regulated intramembrane proteolysis39 and cellular damage.40 Whether these responses may be linked to and possibly modify effects of complement pathway(s) is not known. It is noteworthy that, in vitro, excess protein exposure enhanced complement activation via alternative pathway in human proximal tubular cells.41

A key observation of this study is that preventing the accumulation of C3 in podocytes was associated with less severe podocyte damage, both in C3−/− mice with BSA overload and in WT kidneys transplanted in C3−/− mice in this model, resulting in protection against progressive glomerular injury. Thus, whereas locally synthesized C3 seems to be irrelevant to the pathogenesis of proteinuria, the presence of circulating C3 acts as a susceptibility factor that negatively influences proteinuria and its deleterious consequences. Our data are fully consistent with a role of protein overload of glomerular epithelial cells in the development of the sclerotic lesion in proteinuric disease.42 This possibility is strengthened by findings that C3 deficiency attenuated podocyte damage and sclerosis in Adriamycin nephropathy in mice, and, conversely, CD59 deficiency exacerbated the disease.43 Because podocytes normally express complement inhibitory molecules such as complement receptor 1 or its mouse analogue factor H,44 it is reasonable to suggest that perturbed complement regulation may represent an important feature of this process.

Improved success in preventing CKD progression could come from manipulation of intrarenal complement activation, but stimulatory factors and source(s) of complement in proteinuric nephropathy had not been characterized so far. We found that C3 deficiency prevents accumulation of circulating C3 in glomerular cells and attenuates podocyte injury, resulting in protection against proteinuria and glomerular sclerosis. Our data provide the evidence that the abnormal passage of C3 protein across the glomerular filtering barrier is the factor underlying the accumulation of C3 together with other filtered proteins in proximal tubules, and systemic C3 deficiency attenuates interstitial injury. Finally, protective effects of lisinopril treatment are due to its antiproteinuric action, at least partly, via reduction of tubular load by C3 and/or ultrafiltered proteins. C5 antibodies blocked complement activation in phase II clinical trials in patients with paroxysmal nocturnal hemoglobinuria45 or myocardial infarction.46 A soluble form of the C3/C5 convertase inhibitor CR1 (TP10) decreased complement activation and protected vascular function in infants with cardiopulmonary bypass.47 Hopefully, complement inhibitors will be useful to prevent progression of human proteinuric renal diseases, when combined with interventions to minimize the dysfunction of the glomerular barrier to proteins.



Male C3-deficient mice (strain B6.129S4-C3tm1Crr) of C57BL/6 genetic background and age-matched C57BL/6 WT mice were created by heterozygote × heterozygote breeder pairs from Jackson Laboratory (Bar Harbor, ME). Genotypes were determined by PCR of DNA accordingly to the protocol C3tm1Crr from Jackson Laboratory.

Animal care and treatment were in accordance with institutional guidelines in compliance with national (Decreto Legislativo n.116, Gazzetta Ufficiale suppl 40, 18 febbraio 1992, Circolare n.8, Gazzetta Ufficiale 14 luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJL358-1, December 1987; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996). Animals were housed at a constant room temperature, 12-h dark/12-h light cycle, and fed a standard diet.

Murine Model of Protein Overload Proteinuria

BSA overload proteinuria was induced in WT or C3-deficient mice. Uninephrectomy was performed 5 d before starting BSA.48,49 Low endotoxin BSA (Sigma A-9430, St. Louis, MO) was given 5 d weekly intraperitoneally (days 1 through 5) at the dosage of 15 mg/g body wt for 4 wk. Control WT and C3-deficient mice received the same volume of saline. For establishment of whether C3 deposition may ensue in advance of tubular changes, two groups of mice given BSA or saline were killed at 1 wk for immunohistology. In another set of experiments, beginning on day 1 of BSA injection, uninephrectomized mice received daily either lisinopril (Astra Zeneca, Basiglio, Milano, Italy; 60 mg/L in the drinking water) or no treatment up to 4 wk. Proteinuria was determined as described previously.48

Anti-BSA Antibodies

At variance with other protocols,50,51 this model does not cause immune complex disease.24 For further exclusion of immunologic factors, plasma of mice with BSA overload proteinuria (4 wk) versus controls (n = 3 each group) was analyzed for BSA antibodies on serial dilutions using the double gel diffusion method of Ouchterlony.52

Mouse Kidney Transplantation

WT mice received a transplant of either C3−/− kidney or WT kidney, and C3−/− mice received a transplant of WT kidney.53 The donor's left kidney was flushed with ice-cold heparinized saline and removed together with ureter and vessels. Recipient mice underwent left-sided nephrectomy and orthotopic implantation. The ureter was inserted into the bladder and pulled through.54 The right native kidney was removed on day 15 postoperatively, whereupon recipients became dependent on a functioning graft. Animals without graft function died within 36 h of the contralateral nephrectomy. Protein overload proteinuria was induced starting on day 15 after removal of the native kidney.

Renal Histology

Kidneys were processed as described previously.48 Tubular (atrophy, casts, and dilation) and interstitial changes (fibrosis and inflammation) were graded from 0 to 3+ (0, no changes; 1+, changes affecting <25% of the sample; 2+, changes affecting 25 to 50% of the sample; 3+, changes affecting >50% of the sample). At least 100 glomeruli were examined for each animal. Percentages of glomeruli presenting sclerotic lesions were recorded. Biopsies were analyzed by one pathologist, who was unaware of experimental groups.


Cortical samples were embedded in OCT compound and fresh-frozen in liquid nitrogen. Acetone-fixed sections were stained (1 h) with FITC-conjugated sheep anti-mouse IgG (20 μg/ml; Sigma) or goat anti-mouse C3 antibody (10 μg/ml; Cappel, Durham, NC). Proximal tubule staining was scored as follows: 0, no IgG or linear peripheral C3; 1, intracellular and/or brush border staining in few tubular profiles (<5% in each microscopic field); 2, staining affecting 5 to 50% of profiles per field; 3, >50% of profiles per field. Proximal tubules were identified on the basis of morphology, presence of brush border, and topography. At least 20 randomly selected high-power microscopic fields (×400) were analyzed per animal. Glomerular C3 localization was further assessed using an inverted confocal laser microscope (LSM 510 meta; Zeiss, Jena, Germany).

Both F4/80 and α-SMA have been used as end-point markers for inflammation and fibrosis in this model.55,56 F4/80 analysis was performed as described previously.48 The primary antibody (Caltag Laboratories, Burlingame, CA), was incubated overnight (2.5 μg/ml, 4°C), followed by biotinylated goat anti-rat IgG (Vector Laboratories), avidin-biotin peroxidase complex, and DAB. For detection of α-SMA, 3-μm frozen sections were blocked with PBS/1% BSA and incubated (1 h, room temperature) with Cy3-conjugated anti–α-SMA antibody (1A4, 1:200; Sigma). At least 20 randomly selected high-power fields per section were assigned a score: 0, no staining; 1+, scattered α-SMA–positive cells in peritubular areas; 2+, moderate α-SMA peritubular staining; 3+, marked accumulation of cells surrounding >50% of the tubules. The investigator was unaware of experimental groups.

Electron Microscopy

Cortical kidney fragments were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and embedded in Epon resin by standard methods. Glomeruli showing no sclerotic lesion (n = 4 glomeruli from three animals per group) were selected on semithin sections and further processed for analysis.

Quantitative Real-Time RT-PCR

RT-PCR was performed as described previously.57 The following primers were used: Murine C3 (300 nM) forward 5′-AAGCCAAGGACTGCAGACTGA-3′ and reverse 5′-GGCTTGGTGCACTCAAGATCT-3′ and 18S (50 nM) forward 5′-ACGGCTACCACATCCAAGGA-3′ and reverse 5′- CGGGAGTGGGTAATTTGCG-3′.

In Situ Hybridization

A 301-bp fragment of C3 cDNA was cloned into transcription vector pSPT19 between SP6 and T7 promoters. Mouse C3 antisense and sense riboprobes were prepared and labeled by in vitro transcription using digoxigenin RNA labeling kit (Roche Diagnostic, Monza, Italy).

In situ hybridization was performed as described previously.58 Sections were hybridized overnight with RNA probes at final concentration of 0.5 ng/μl, in 2× SSC, 10% dextran sulfate, 1× Denhardt's solution, 20 mM Vanadil Ribonucleoside Complex (New England Biolabs, Frankfurt Am Main, Germany), and 0.1 M sodium phosphate in a moist chamber at 50°C.

Statistical Analyses

Results are expressed as means ± SEM. Statistical analysis was performed using nonparametric Kruskal-Wallis or Mann Whitney tests or ANOVA followed by Tukey test for multiple comparisons. Statistical significance level was defined as P < 0.05.




Part of this work received support by a Genzyme Renal Innovation Program grant. S.B. was a recipient of a fellowship “Fondazione ARMR—The Nando Peretti Foundation.”

Part of this work was presented at the annual meeting of the American Society of Nephrology; October 27 through November 1, 2004; St. Louis, MO.

We thank Drs. Daniela Macconi and Simona Buelli for helpful technical advice and discussion. Manuela Passera helped to prepare the manuscript.


Published online ahead of print. Publication date available at www.jasn.org.


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