Accumulation of Immune Complexes in Glomerular Disease Is Independent of Locally Synthesized C3
Neil S. Sheerin,
Katsushige Abe,
Paul Risley and
Steven H. Sacks
Department of Nephrology and Transplantation, Guy's Hospital, King's College London, London, United Kingdom
Address correspondence to: Dr. Neil Sheerin, Department of Nephrology and Transplantation, 5th Floor, Thomas Guy House, Guy’s Hospital, St. Thomas’ Street, London, SE1 9RT. Phone: +44-207-955-4305; Fax: +44-207-955-4303; neil.sheerin{at}kcl.ac.uk
Received for publication July 1, 2004.
Accepted for publication December 7, 2005.
Although complement activation can make immune complex glomerulonephritisworse, the third complement component also can solubilize immunecomplexes and thus reduce the severity of disease. How C3 thatis produced within the kidney contributes to this balance isunknown. This study therefore investigated the relative rolesof systemic and local C3 production in a model of glomerularimmune complex disease. Injection of sheep anti–glomerularbasement membrane antibody into preimmunized mice resulted inaccumulation of immune complexes and progressive loss of functionover 14 d that was much more marked in C3-deficient (C3–/–)mice. In C3-sufficient mice that received a transplant of aC3–/– mouse kidney and in C3–/– micewith C3-sufficient mouse kidney transplants, the severity andthe pattern of injury went with the complement status of therecipient. That is, mice with deficient circulating C3 developedsevere glomerular immune complex disease, whereas those witha high level of circulating C3 had well-preserved glomerularstructure and function. It is concluded that circulating C3is a critical factor in reducing the glomerular accumulationof immune complexes. Local synthesis of C3 did not have a majorinfluence on this aspect of glomerular disease.
It is widely known that complement plays a role in the pathogenesisof immune complex disease. However, the nature of this roleis controversial, because complement activation can mediateinflammatory injury (1–3), whereas deficiency, especiallyof the early components, can predispose toward immune complexdisease. In particular, C1q deficiency has been associated withreduced clearance of apoptotic cells (4) and the developmentof antibody-mediated glomerulonephritis (5). C3 has an importantrole in the removal of immune complexes both from the circulationand from the glomerulus (6–8), and failure can resultin glomerulonephritis. It is likely that the effect of complementin the pathogenesis of glomerular disease may be dependent onthe stage of the disease (9). Acute glomerular inflammationafter a single injection of anti–glomerular basement antibodyis reduced in the absence of complement (1,10), whereas theslower accumulation of glomerular complexes that are formedby autologous antibody may be increased (9).
A further complexity is that whereas a large amount of complement,produced in the liver (11), is present in the circulation, smaller,although significant, quantities can be synthesized locallyin the extravascular space. Circulating components are instantlyavailable, whereas tissue-specific components are produced ina regulated manner after the induction of disease or after exposureof cultured cells to various inflammatory stimuli. In the kidney,glomerular epithelial (12), mesangial (13,14), and endothelialcells (15) have the capacity to synthesize C3, a pivotal componentof the complement cascade that is able to mediate the clearanceof immune complexes as well as induce inflammation. In addition,renal tubule cells can produce C3 (16). After the inductionof glomerular disease in rats, tissue expression of C3 mRNAis increased, and the degree of protein leak from the injuredkidney correlates positively with the level of C3 gene expression(17). It therefore has been proposed that locally synthesizedC3 may have a role in the pathogenesis of renal injury and thatthis function may differ from that of circulating complement.
To assess the contribution of systemic and local productionof C3, we investigated the development of immune complex glomerulonephritisafter the administration of anti–glomerular basement antibodyto mice. We specifically examined the development of this diseasein mice that lack either circulating C3 or renally producedC3, achieved by transplanting kidneys between mice that areC3 deficient and C3 sufficient.
Mice
The C3-deficient (C3–/–) mice were originally generatedby homologous recombination in the laboratory of Prof. M.C.Carroll (18). The mice have no detectable C3 by ELISA. Althoughoriginally generated on a mixed C57Bl/6 x 129 background, theC3–/– mice that were used in these experiments hadbeen backcrossed six generations onto a C57Bl/6 background.Skin that was engrafted from these mice onto C57Bl/6 mice showedno evidence of rejection (data not shown). C3-sufficient C57Bl/6(C3+/+) mice were bred in our own facility. All animal experimentswere performed in accordance with United Kingdom Home Officeregulations.
Measurement of Anti-Sheep IgG Titers
To determine whether C3–/– and C3+/+ mice had similaranti-sheep IgG antibody titers, we injected 1 mg of sheep IgGin complete Freund’s adjuvant (CFA) into 10 mice fromeach strain. Blood was taken at days 5, 12, and 19. Ninety-six-wellplates were coated with 20 µg/ml sheep IgG in PBS overnightat 4°C. Plates were blocked with 1% BSA, and serial dilutionsof serum in PBS–1% BSA were added for 1 h at 37°C.After washing, the plates were incubated with 1:1000 dilutionof goat anti-mouse IgG horseradish peroxidase (HRP) conjugate(Stratech Scientific Ltd, Soham, UK) or rabbit isotype-specificalkaline phosphate conjugate (Southern Biotechnology AssociatesInc., Birmingham, AL) for 1 h at 37°C, washed, and developedwith O-phenylenediamine or p-Nitrophenyl phosphate.
Induction of Glomerular Injury and the Transplant Model
Anti–glomerular basement membrane (GBM) antibody–mediatedglomerular injury was induced in 6- to 8-wk-old female C3+/+and C3–/– mice (13 mice per group). Five days beforethe injection of anti-GBM antiserum, mice were immunized subcutaneouslywith 1 mg of sheep IgG (Sigma, Dorset, UK) in CFA. Before theinjection of anti-GBM antiserum, mice were housed in metaboliccages to measure baseline albuminuria, and blood was taken tomeasure serum albumin and urea concentrations. Mice receivedan injection of 50 µl of sheep anti-GBM antiserum (Dr.D.J. Salant, Boston, MA) intravenously on 3 consecutive days.Urine and blood were collected at days 7 and 14 after the firstinjection of anti-GBM antiserum. Mice were killed at day 14,and their kidneys were harvested for histologic analysis.
Heterotropic kidney transplantation was performed in two donor–recipientcombinations: Group 1, C3–/– donor mouse to C3+/+mouse recipient (n = 4); and group 2, C3+/+ donor mouse to C3–/–mouse recipient (n = 4). The resulting mice either were deficientin locally produced C3 in the transplanted kidney but with intactsystemic production (group 1) or had intact local C3 productionbut deficient systemic production (group 2). The donor and recipientaorta and vena cava were joined by end-to-side anastomosis.A patch of donor bladder was attached to the recipient bladder(19). Unilateral recipient nephrectomy was performed at thetime of transplantation. After a period of recovery (10 d),a second native nephrectomy was performed so that the only sourceof renal function was from the transplanted kidney. Preliminarystudies demonstrated that the transplanted kidney was histologicallynormal 14 d after transplantation, with donor and graft survivalfor at least 3 mo with no evidence of rejection. Fourteen daysafter transplantation, anti-GBM antibody–mediated glomerularinjury was induced.
Assessment of Renal Functional Injury
Urine albumin concentration was measured by ELISA, and the 24-hurinary albumin excretion was calculated. Ninety-six-well plateswere coated with 5 µg/ml goat anti-mouse albumin (NordicImmunological Laboratories, Tilburg, The Netherlands) in carbonatebuffer (pH 9.6) overnight at 4°C. After blocking with 2%BSA at 37°C for 2 h, samples or control dilutions of mousealbumin (Sigma) in blocking buffer were added to the platesfor 2 h at 37°C. A secondary HRP-conjugated goat anti-mousealbumin (Nordic) in blocking buffer was added for 2 h at 37°C.Absorbance was measured after incubation with O-phenylenediamine.Serum urea and albumin concentrations were measured using Sigmakits according to the manufacturer’s protocols.
Assessment of Histologic Injury
To assess histologic injury we stained formalin-fixed, wax-embeddedkidney sections (2 µm thick) with periodic acid-Schiffreagent (PAS). The severity of injury was scored by a blindedobserver according to the following scheme: 0, normal; 1, mild(small areas of glomerular abnormalities); 2, moderate (<50%of the glomerulus affected by necrosis or crescent formation);and 3, severe (>50% of the glomerulus affected by necrosisor crescent formation) (20). Fifty glomeruli were assessed foreach animal.
Immunohistochemical Staining
Cryostat sections (5 µm) were fixed with acetone at 4°Cfor 10 min. Deposition of mouse IgG, mouse IgM, and sheep IgGwas detected with HRP-conjugated anti-mouse IgG, HRP-conjugatedanti-mouse IgM (both Stratech), and HRP-conjugated anti-sheepIgG (Serotec, Oxford, UK; preabsorbed against sheep and mouseIg, respectively, and lack of cross-reactivity tested by ELISA[data not shown]). For C3 staining, the primary antibody wasrabbit anti-human C3d (Dako Ltd, Cambridgeshire, UK) and a secondaryHRP-conjugated goat anti-rabbit IgG. For C4 staining, the primaryantibody was rat anti-mouse C4 and a secondary HRP-conjugateddonkey anti-rat (Stratech). HRP-antibody was detected with diaminobenzidine.Sections were counterstained with methyl green.
Fifty glomeruli from each animal were examined, and a semiquantitativeassessment of the intensity of the immunochemical staining wasperformed using a scale from 0 to 3 (0, negative; 1, weak; 2,moderate; 3, strong staining) according to the methods describedpreviously (21).
Electron Microscopy
Tissue was prefixed with 2.5% glutaraldehyde and postfixed withosmium tetroxide. Sections were stained with lead citrate andviewed on a transmission electron microscope (Hitachi H7000;Hitachi, Berkshire, UK). A minimum of three glomeruli were assessedin each mouse.
Demonstration of Renal C3 Synthesis
Using standard phenol-chloroform methods, RNA was extractedfrom the renal cortex of transplanted kidney, and cDNA was synthesized.C3 gene expression was detected by reverse transcription–PCRusing the primer sequences 5'-TCACACACCGAAGAAGACTGCC-3' and5'-GTGGCTGATGAACTTGCGTTGC-3' (product size 407 bp). -Actin geneexpression (5'-GAGCAAGAGAGGTATCCTGACC-3' and 5'-GGATGCCACAGGATTCCATACC-3')was used as an internal control. All amplifications were performedin the linear phase of amplification (28 cycles: 94°C for1 min; 60°C for 1 min; 72°C for 2 min). The specificPCR product was not seen in non–reverse-transcribed mRNAor genomic DNA controls with the primers used (data not shown).
In situ hybridization for C3 mRNA was performed using a 30-baseantisense oligonucleotide probe, corresponding to bases 167to 196 of mouse C3 cDNA. The probe was labeled using digoxigenin(DIG) oligonucleotide tailing kit according to the manufacturer’sinstructions (Boehringer Mannheim, Lewes, UK). Frozen sections(4 µm) were fixed with 4% paraformaldehyde in PBS. Thesections were deproteinized by using HCl and proteinase K, prehybridized,and then hybridized with DIG-labeled oligonucleotide probe inprehybridization buffer at 37°C overnight. After washingwith 2x SSC, the DIG-labeled probe was visualized using HRP-conjugatedsheep polyclonal anti-DIG antibody (Boehringer Mannheim) anddiaminobenzidine (22). Control studies with a sense probe andcompetitive binding studies with sense and antisense probe confirmedspecificity.
Statistical Analyses
Data were expressed as mean ± SD. Differences betweendifferent groups were tested for statistical significance usingone-way ANOVA with Scheffe’s F test. P < 0.05 was takenas statistically significant.
In the first phase, anti-GBM antibody–mediated glomerulonephritiswas studied in the C3+/+ and C3–/– back-crossedmice. The second phase used a transplantation strategy to definewhether locally synthesized C3 plays any role in the developmentof injury.
Assessment of Anti-Sheep IgG Titers in C3+/+ and C3–/– Mice
C3+/+ and C3–/– mice developed similar anti-sheepIgG titers 12 (Figure 1A) and 19 (Figure 1B) days after immunizationwith 1 mg of sheep IgG in CFA. Titers were low in both groupsat day 5. In addition, the titers of IgG1 and IgG2b were similarin the two groups of mice (Figure 1, C and D). Very little IgG2aor IgG3 was detected in either group of mice (data not shown).
Figure 1. Anti-sheep antibody titers in C3+/+ () and C3–/– () mice. IgG titers were measured 12 (A) and 19 (B) days after the injection of 1 mg of sheep IgG in complete Freund’s adjuvant (CFA). IgG1 (C) and IgG2b (D) titers were also assessed in the day 19 sample. Serum from nonimmunized mice was also included (). n = 10 per group.
Assessment of Antibody-Mediated Injury in C3+/+ and C3–/– Mice
Before the injection of anti-GBM antiserum, C3+/+ and C3–/–mice had equivalent low levels of albuminuria. Seven days afterthe first injection, C3–/– mice had significantlygreater albuminuria than the C3+/+ mice (57.5 ± 30.1versus 4.3 ± 3.6 mg/24 h, respectively; P < 0.01).This difference persisted to day 14 (62.3 ± 17.4 versus9.9 ± 4.5 mg/24 h; P < 0.01). By day 14 after theinduction of disease, the serum urea was significantly higherin the C3–/– mice than in the C3+/+ mice (31.2 ±8.0 and 9.5 ± 2.1 mmol/L, respectively; P < 0.01).These observations confirm greater disruption to glomerularfiltration function in the mice that are deficient in C3.
Histologic analysis 14 d after the induction of disease demonstratedthat severity of the injury was dependent on the complementstatus of the mice: The C3+/+ mice showed only minor glomerularchanges (Figure 2, A and C). In contrast, the kidneys from C3–/–mice showed significant glomerular injury with glomerular hypercellularityand crescent formation. There was also capillary wall thickeningwith deposition of PAS-positive material (Figure 2, B and C).
Figure 2. Histology of mouse kidney 14 d after the induction of anti–glomerular basement membrane (anti-GBM) antibody (Ab)-mediated glomerulonephritis. Representative histology of C3+/+ mice (A) and C3–/– mice (B) is shown. (C) Semiquantitative analysis of the severity of glomerular damage was performed on a minimum of 50 glomeruli from all mice (n = 13) in each group. Magnification, x400 in A and B, periodic acid-Schiff (PAS) staining.
Immunohistochemical Analysis of Diseased Kidneys
The kidneys from C3+/+ and C3–/– mice 14 d afterdisease induction were examined for the presence of C3, C4,sheep IgG, and mouse IgG and IgM. C3, C4, and IgM can be detectedin the glomeruli of normal mice in a mesangial distribution(data not shown). Increased C3 deposition was detected in theglomeruli of C3+/+ mice after disease induction. C3 was depositedin both a pericapillary and mesangial distribution (Figure 3A).As expected, there was no C3 deposition in the kidneys fromC3–/– mice (Figure 3B). C4 staining was seen inthe glomeruli of both C3+/+ and C3–/– mice predominantlyin a mesangial distribution (Figure 3, C and D, respectively).The intensity of staining was greater, as assessed on a semiquantitativescale, in the C3–/– mice (Table 1).
Figure 3. Immunohistochemistry of mouse kidney after the induction of anti-GBM Ab-mediated glomerulonephritis. Staining for C3 (A and B), C4 (C and D), sheep IgG (E and F), mouse IgG (G and H), and mouse IgM (I and J) was performed on C3+/+ mice (A, C, E, G, and I) and C3–/– mice (B, D, F, H, and J). Magnification, x400.
Table 1. Semiquantitative assessment of the intensity of staining for complement and Ig in the C3+/+ and C3–/– mice 14 d after disease inductiona
Staining for sheep IgG (Figure 3, E and F), mouse IgG (Figure 3, G and H),and mouse IgM (Figure 3, I and J) was more intensein the C3–/– mice than in the C3+/+ mice. Antibodystaining was predominantly in a pericapillary distribution,although some mesangial staining was also seen. Using a semiquantitativescoring system, this reached statistical significance in allthree cases (Table 1). Increased deposition of Ig in the glomeruliof C3–/– mice could explain the increased amountsof C4 detected in these mice. We previously reported that theC3–/– mice have immune complexes within their glomerulimore frequently than do control C3+/+ mice (9). This was trueagain with this group of mice (data not shown) and is consistentwith the increased glomerular deposition of Ig and C4 in C3–/–mice.
Assessment of Functional Injury in Mice with a Transplant
The data above suggest that, after intravenous injection ofanti-GBM antibody, mice that are totally deficient in C3 accumulateimmune-reactive material in their glomeruli and subsequentlydevelop more severe renal injury. To dissect the relative importanceof circulating and locally synthesized C3, we used a renal transplantationmodel.
Fourteen days after the second native nephrectomy, the micethat received a transplant in both groups 1 and 2 seemed healthy.Before the induction of anti-GBM antibody–mediated glomerulonephritis,the serum urea was equivalent in both groups but approximatelytwo-fold higher than in normal unmanipulated mice (Table 2),as a consequence of the animals’ surviving on a singletransplanted kidney. The serum albumin was also higher in themice that received a transplant before the induction of disease.Before disease induction, there was minimal albuminuria in micefrom both transplant groups (Figure 4). After the inductionof disease, albuminuria developed in both groups but was significantlygreater in mice from group 2 (C3+/+ donor: C3–/–recipient) at both days 7 and 14 (P < 0.01 at both time points).Fourteen days after the induction of disease, C3–/–mice that received a kidney from a C3+/+ donor (group 2) hadsevere functional disturbance with a significant rise in serumurea and reduction in serum albumin (P < 0.01; Table 2).In contrast, the renal function of mice in group 1 was not alteredsignificantly after the induction of disease. Therefore, thefunctional disturbance seen after disease induction in the micethat received a transplant was dependent on the complement statusof the recipient rather than that of the donor kidney.
Figure 4. 24-hour urinary albumin excretion (mg/24 h) in the mice that received a transplant. Albuminuria in group 1 mice (C3–/– donor: C3+/+ recipient; dotted line) was significantly less than group 2 mice (C3+/+ donor: C3–/– recipient; solid line); n = 4 per group.
Assessment of Histologic Injury in Kidney Transplants
The glomeruli of the group 1 mice were hypercellular and exhibitedareas of minor sclerosis (Figure 5A). The glomeruli of micein group 2 showed marked histologic damage, including capillaryocclusion with PAS-positive material and areas of focal proliferationand crescent formation (Figure 5B). Scoring of the glomerularhistologic changes confirmed the greater injury in group 2 mice(2.5 ± 0.6) compared with group 1 mice (1.4 ±0.2; P < 0.01). Similarly, the tubulointerstitium in C3+/+mice that received a C3–/– kidney (group 1) waswell preserved in contrast to mice from group 2 that had tubulardilation, epithelial cell flattening expansion of the interstitialcompartment with evidence of a mononuclear cell infiltrate.
Figure 5. Histology of the transplanted kidneys. Representative glomerular histology from group 1 mice (C3–/– donor: C3+/+ recipient; A) and group 2 mice (C3+/+ donor: C3–/– recipient; B) is shown. The glomeruli from group 2 mice showed severe histologic damage including capillary occlusion with PAS-positive material and areas of focal proliferation and crescent formation. Magnification, x100, PAS staining.
Immunohistochemical Analysis of Transplanted Kidneys
Complement C3 was deposited in the glomeruli of mice from bothtransplant groups 14 d after disease induction (Figure 6, A through D).Because mice from group 2 lack systemic C3 production,the C3 that is deposited in the glomeruli must be of donor kidneyorigin, presumably derived from resident renal cells. Stainingfor C3 was present in a capillary wall and mesangial distribution.C4 was also seen in the glomeruli of mice from both groups,as in the mice that did not received a transplant, in a mesangialdistribution, suggesting activation of the classical pathwayof complement (Figure 6, E and F). Sheep IgG and mouse IgG andIgM were deposited in the glomeruli of mice from both groupsin both the capillary walls and the mesangium (Figure 7). Semiquantitativeassessment of the intensity of staining showed greater intensityof C4, sheep IgG, and mouse IgG and IgM in group 2 mice (Table 3).Therefore, as with the functional changes, the pattern ofdeposition of immunoreactive proteins followed the complementstatus of the recipient. We cannot exclude an effect of nonspecificprotein trapping in the glomeruli of the more proteinuric micein group 2, in particular the mesangial IgM.
Figure 6. Immunochemistry of transplanted kidneys. C3 staining was demonstrated in mice from both group 1 (A and C) and group 2 (B and D). Glomerular C4 staining was also demonstrated in both groups (E and F) but with greater intensity in group 2 mice (C3+/+ donor: C3–/– recipient; F). Magnification, x160 in A and B, x400 in C through F.
Figure 7. Immunochemistry of mice that received a transplant. Sheep IgG (A and B), mouse IgG (C and D), and mouse IgM (E and F) all were detected in mice from both groups. However, the staining for all three immunoreactants was greater in mice from group 2 (C3+/+ donor: C3–/– recipient; B, D, and F) compared with group 1 mice (C3–/– donor: C3+/+ recipient; A, C, and E). Magnification, x400.
Table 3. Semiquantitative assessment of the intensity of staining for complement and Ig in the mice that received a transplant
Electron Microscopy on Kidneys from Mice with a Transplant
The kidneys from mice in group 1 (Figure 8A) and group 2 (Figure 8B)were examined by electron microscopy. In the glomeruli ofmice from group 2, there were electron-dense immune complexesin all mice examined (three of three). In addition, there wasepithelial cell foot-process effacement and endothelial cellswelling. Evidence of immune complex deposition was detectedin only one mouse (one of four) from group 1, and the structuralchanges were less severe than those seen in group 2.
Figure 8. Electron microscopy of transplanted kidneys. Representative glomerular changes in group 1 (C3–/– donor: C3+/+ recipient; A) and group 2 (C3+/+ donor: C3–/– recipient; B) mice is shown. Group 2 mice demonstrated accumulation of electron-dense material in the subendothelial space (arrows). Bar = 500 nm. Magnification, x20,000.
C3 Production within Transplanted Kidneys
PCR was used to analyze reverse-transcribed mRNA from the renalcortex of mice that received a transplant. C3 mRNA was detectedin the cortex of both groups (Figure 9A), suggesting that C3gene expression was occurring in both infiltrating cells (group1) and native renal cells (group 2). To localize cortical C3gene expression further, we performed in situ hybridization.In kidney transplants that were derived from a C3–/–donor, C3 message was detected only in infiltrating cells arounddamaged tubules (Figure 9B). When the kidney was derived froma C3+/+ donor, glomerular and tubular C3 gene expression wasevident (Figure 9C). The result with sense probe is shown asa control (Figure 9D). Therefore, during the development ofrenal injury, C3 that is synthesized within the kidney couldbe derived from both native and infiltrating renal cells.
Figure 9. C3 mRNA analysis in transplanted kidneys. Reverse transcription–PCR was used to determine the presence of C3 gene transcription in mice from both group 1 and group 2. In situ hybridization was used to identify the site of C3 gene expression in mice from group 1 (B) and group 2 (C). Staining with sense probe of group 2 tissue is shown as control (D). G, glomerulus; T, tubule. Magnification, x300 in B, x400 in C, x250 in D.
This work supports a protective role for the complement systemin the later, autologous phase of anti-GBM antibody–mediatedglomerulonephritis. The protective effect is mediated by circulatingcomplement rather than complement that is produced from residentglomerular cells.
Patients with circulating antibody directed against the GBMdevelop a severe, rapidly progressive glomerulonephritis. Antibodyis deposited along the basement membrane, activating the complementsystem, components of which can be demonstrated on the GBM byimmunohistochemistry. This led to the hypothesis that complementactivation was important in the generation of injury, eitherby causing direct cell damage or by promoting an inflammatorycell infiltrate. Early studies in animal models using complementdepletion (23,24) supported this view; however, this was nota universal finding, particularly in the mouse (25,26). Morerecent studies using C3–/– mice support a role forcomplement activation. After the injection of anti-GBM antibody,there is rapid complement activation and neutrophil influx.At this stage of the disease, complement activation seems tohave a harmful effect as deficiency (1,27) or inhibition (28)reduces injury. However, as the disease progresses and the animaldevelops autologous antibody against the heterologous serum,the absence of C3 seems to have a detrimental effect, both onrenal function and on histologic injury (9).
The results presented here suggest that the reason for the greaterfunctional injury in the C3–/– mice is the accumulationof immune complexes in the glomerular capillary wall. This couldbe explained by either reduced clearance of circulating immunecomplexes or reduced clearance of complexes from within theglomerulus. C3 is readily incorporated into immune complexesbecause of the high density of Ig Fc regions, thereby disruptingimmune complex structure and increasing solubility (29). Inaddition, in some mammals (not rodents), C3 within immune complexesacts as the ligand for complement receptor 1 on erythrocytes,binding to which facilitates immune complex transport to phagocyticcells of the reticuloendothelial system (30). However, the modelof anti-GBM antibody–mediated glomerulonephritis thatis used in this study relies on affinity of the heterologousserum for the GBM. Therefore, the immune complex disease seenin this model is probably strongly dependent on in situ immunecomplex formation. This does not exclude the possibility thatreduced clearance of circulating complexes plays a role in thepathogenesis of glomerular disease in the C3–/–mice.
Complement also has a function in the solubilization of immunecomplexes from within the glomerulus (31) and the transfer ofimmune complexes across the GBM. Studies in animals have shownthat, in contrast to the normal passage of antigen and antibodythrough the GBM, in animals that are depleted of complementwith cobra venom factor, antigen remains in a subendothelialposition (32,33). The disruption to the structure of the immunecomplex that is caused by complement binding probably facilitatestransfer through the GBM, although the exact mechanism remainsunknown.
In the C3–/– mice described in this study, the absenceof one or more of these mechanisms leads to the accumulationof immunoreactants and immune complexes within the glomerulusand therefore greater functional injury. A similar increasein severity of immune complex glomerular disease was seen whenthe targeted C3 deletion was back-crossed onto an MRL/lpr background.Greater glomerular IgG deposition and albuminuria was observedin the C3–/– mice (34). However, the role of complementin this model is complex, and mice that are specifically deficientin the alternative pathway (35) or in which complement activationis inhibited (36) have reduced disease severity. Overall, thissuggests a predominant role for the classical pathway of complementactivation in protecting from glomerular immune complex disease.
We next addressed the issue of the source of C3 that reducesthe build-up of immune complexes in the glomerulus. Native cellswithin the glomerulus, including mesangial (13,37), epithelial(38), and endothelial cells (15), as well as tubular epithelialcells (16,39) have the capacity to synthesize C3. Although itis well documented that glomerular expression of C3 mRNA isincreased in glomerulonephritis, the data presented here provideno evidence for a functional role for intraglomerular complementsynthesis. Logically, in this context, the local synthesis ofC3 should protect against immune complex accumulation, a clearlydefined physiologic role of the complement system. In our modelof immune complex–mediated disease, however, we were unableto demonstrate this effect of locally synthesized C3, despiteevidence of C3 production in the glomerulus during disease development.
It is possible that because the glomerular capillaries are exposedto high concentrations of circulating complement components,locally synthesized C3 has only minor importance in diseasethat is induced by anti-GBM antibodies. Moreover, in other modelsof glomerulonephritis, the glomerular expression of C3 is timedependent, and maximal expression is achieved after 14 d (17).Therefore, local synthesis of C3 may have a greater contributionto make in more protracted models of renal injury than in themodel described here. In models of very acute injury, such asthe heterologous phase of this model, local synthesis of C3is even less likely to contribute to disease expression.
It should also be noted that the main site of complement geneexpression in the kidney is the renal tubule (40), and expressionat this site is upregulated during renal injury. It is possiblethat locally synthesized complement that is secreted into theextravascular, interstitial compartment has a greater impacton the development of injury than glomerular-produced complement.For example, there is increasing evidence that complement activationmay contribute to the damage to the tubulointerstitial compartmentin proteinuria (41–43). In addition, renal tubular epithelialcell production of C3 is increased by exposure to serum proteins.However, as yet, there is no direct evidence for involvementof locally synthesized complement proteins in this phase ofrenal disease.
Thus, although local expression of C3 clearly is upregulatedby the induction of immune complex glomerulonephritis, thisstudy does not show that local synthesis of C3 plays a contributingrole in the pathogenesis of glomerular dysfunction. Rather,these data suggest that circulating complement plays a vitalrole in prevention of glomerular disease, consistent with theremoval of circulating or planted immune complexes mediatedby C3. It seems likely that locally produced complement mayhave greater impact on diseases of the tubulointerstitium, themain intrarenal site of complement synthesis in such disorders(44).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-Actin gene expression (5'-GAGCAAGAGAGGTATCCTGACC-3' and 5'-GGATGCCACAGGATTCCATACC-3') was used as an internal control. All amplifications were performed in the linear phase of amplification (28 cycles: 94°C for 1 min; 60°C for 1 min; 72°C for 2 min). The specific PCR product was not seen in non–reverse-transcribed mRNA or genomic DNA controls with the primers used (data not shown). 
) and C3–/– (
) mice. IgG titers were measured 12 (A) and 19 (B) days after the injection of 1 mg of sheep IgG in complete Freund’s adjuvant (CFA). IgG1 (C) and IgG2b (D) titers were also assessed in the day 19 sample. Serum from nonimmunized mice was also included (
). n = 10 per group.
