Immunology and Pathology

Excessive Matrix Accumulation in the Kidneys of MRL/lpr Lupus Mice Is Dependent on Complement Activation

Lihua Bao, Jian Zhou, V. Michael Holers and Richard J. Quigg
JASN October 2003, 14 (10) 2516-2525; DOI: https://doi.org/10.1097/01.ASN.0000089831.96794.0B

Abstract

ABSTRACT. Complement receptor 1–related gene/protein y (Crry) in rodents is a potent membrane complement regulator that inhibits complement C3 activation by both classical and alternative pathways. Complement inhibition with Crry as the recombinant protein Crry-Ig has been demonstrated to prevent MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice from developing proteinuria and renal failure. Crry-Ig–treated mice also showed less glomerulosclerosis compared with control MRL/lpr mice. To clarify how complement inhibition with Crry might affect renal scarring in lupus nephritis, gene transcript profiling was performed comparing Crry-Ig–treated MRL/lpr mice to control-treated MRL/lpr mice as well as to the MRL/+ strain control. Altered gene expression was confirmed by quantitative PCR, and protein quantity with either immunoblotting or immunofluorescence microscopy. Collagens I, III, IV, and VI were overexpressed in control MRL/lpr mice, whereas complement inhibition with Crry reduced the overexpression of these extracellular matrix components toward normal. Plasminogen activator inhibitor 1, connective tissue growth factor, and TGF-β1 were upregulated in MRL/lpr mice compared with MRL/+ mice and were normalized by Crry-Ig treatment, suggesting that the product of these genes may contribute to the progressive glomerulosclerosis in MRL/lpr mice in a complement-dependent fashion. Thus, complement inhibition with Crry has a prominent effect on matrix-related genes and proteins, which translates into improvement in functional renal disease. E-mail: rquigg@medicine.uchicago.edu

The MRL/MpJ-Tnfrsf6lpr (MRL/lpr) strain is a murine model of human systemic lupus erythematosus. These mice develop many features of human systemic lupus erythematosus, including autoantibodies, hypocomplementemia, and proliferative glomerulonephritis (GN) (1,2⇓). MRL/lpr mice differ from the lupus-prone congenic MRL/+ strain by the nearly complete absence of the apoptosis-promoting membrane Fas protein, which is due to a retroviral insertion in the Tnfrsf6 (Fas) gene (3,4⇓). In contrast to MRL/lpr mice, MRL/+ mice develop lupus very late in life, including only mild renal disease. Renal disease in MRL/lpr mice has similar features to the human disease it models, with mesangial proliferative GN early in disease and diffuse proliferative and crescentic GN later in the course. Ultimately, glomerulosclerosis and renal failure occur in the terminal phase of disease and are believed to cause death in these mice (5). Decreased serum C3 levels and deposition of C3 activation fragments and other complement components in kidney suggest that complement is involved in the pathogenesis of murine as well as human lupus nephritis (2).

Complement activation can proceed via the classical, alternative, or mannose-binding lectin pathways (6). Activation through each of the three pathways leads to cleavage of C3 with generation of the proinflammatory and regulatory fragments C3a and C3b. C3b attaches covalently to immune complexes, which is followed by C5 binding and its cleavage to C5a and C5b. The former is a potent inflammatory molecule that can recruit and activate inflammatory cells, and apparently renal mesangial and proximal tubular cells as well, whereas the generation of C5b begins the nonenzymatic assembly of the C5b-9 membrane attack complex, which can result in cellular death or activation after membrane insertion (7–10⇓⇓⇓).

We have previously developed a recombinant soluble form of the complement inhibitor complement receptor 1–related gene/protein y (Crry) fused to the hinge CH2 and CH3 domains of mouse IgG1 (Crry-Ig) (11). In previous studies, we found that complement inhibition with Crry reduced the incidence of renal failure and severe proteinuria in MRL/lpr mice, in conjunction with a significant reduction of glomerulosclerosis (12,13⇓). These results suggest that complement inhibition with Crry acts to reduce the production and/or promote the degradation of extracellular matrix (ECM) in kidney, preventing severe proteinuria and renal failure in these mice.

This study was designed to investigate the expression of ECM components in MRL/lpr mice with or without Crry-Ig treatment by using the potentially powerful technique of transcript profiling with microarrays. These data illustrate possible sites where complement activation can lead to matrix accumulation in MRL/lpr mice.

Materials and Methods

Experimental Protocol

Crry-Ig was produced and purified as described previously (11). To compare with the natural history in MRL/lpr mice, normal saline was used as a control for Crry-Ig. Animal experiments were performed as detailed in our previous study with these mice (13). Briefly, MRL/lpr and MRL/+ mice were purchased from Jackson Laboratories (Bar Harbor, ME). Thirty-eight MRL/lpr male mice were used in this study and were randomly divided into two groups to receive Crry-Ig (n = 20) or saline (n = 18). Starting at 12 wk of age, mice received intraperitoneal injections of 3 mg of Crry-Ig or an equal volume of normal saline every other day. In this study, ten Crry-Ig–treated and eight saline-treated MRL/lpr mice survived to 24 wk of age, at which time phenotypic measures of disease were obtained, including urinary albumin excretion, blood urea nitrogen (BUN) levels, and assessment of histologic disease, with scoring for GN, glomerular crescents, glomerulosclerosis, interstitial nephritis, and arteritis as described previously (13). Three 24-wk-old MRL/+ male mice were used as additional controls.

Microarray Experiments

MRL/lpr mice representative of respective Crry-Ig–treated (n = 3) and saline-treated (n = 6) groups were included in microarray studies. MRL/+ mice (n = 3) served as controls. Given the greater degree of phenotypic variation in the saline-treated MRL/lpr group, we included six of the eight mice in this group whose disease expression spanned the spectrum seen in all animals. Thus, a total of nine MRL/lpr mice (seven Crry-Ig–treated and two saline-treated animals) were excluded in these experiments, which is a practical reality of such studies with microarrays. By ANOVA and Tukey’s pairwise comparisons, we found no significant differences in any phenotypic measure between included and excluded animals, except for albuminuria in the Crry-Ig–treated group, in which case, it was less in those two not included in microarrays (P = 0.05). To address the potential limitations of not studying the entire group of animals in these studies, all follow-up experiments (described below) included the entire group of animals.

Renal cortical RNA was immediately processed with TRIzol reagent (Life Technologies, Manassas, VA) according to the manufacturer’s instruction. Microarrays were performed with the murine genome U74v2 set (Affymetrix, Santa Clara, CA). In brief, 10 μg of total RNA from each sample was used. Quality of all RNA samples was assured by evaluation on an Agilent 2100 Bioanalyzer. Double-stranded cDNA and biotin-labeled antisense cRNA was produced consequently, followed by cRNA fragmentation. Hybridization to the Affymetrix probe array was performed in a hybridization oven at 45°C and 60 rpm for a minimum of 16 h. After washing and staining, the array chips were scanned with a GeneArray scanner (Affymetrix). Absolute expression and comparison analyses were performed with Affymetrix Microarray Suite (version 5.0). In absolute expression analyses, hybridization intensity data from a single array for a single gene was examined to calculate the signal intensity, and to determine a present, marginal, or absent “call” for each transcript as well as to calculate a P value for that detection call. In a comparison expression analysis, the hybridization intensity data from two probe arrays from different groups were examined to determine a change call for each transcript (increased, marginally increased, decreased, marginally decreased, or no change) and to calculate the magnitude of that change expressed as a log2 signal log ratio. Genes were then sorted and graphed by Microsoft Excel and/or GeneSpring software (Silicon Genetics, Redwood City, CA). In this study, we were interested in genes that were significantly changed in either direction in the control MRL/lpr group compared with MRL/+ mice and that were brought back toward normal by complement inhibition with Crry-Ig. Genes were considered to be relevant when the following two conditions were both met: first, genes were “present” in all of the 6 samples in the saline-treated control MRL/lpr group; second, there were at least 10 “increase” calls in the 18 comparisons between the 6 samples in the control MRL/lpr group with the 3 samples in MRL/+ group and 8 “decrease” calls in the 18 comparisons between the 3 samples in the Crry-Ig–treated group with the 6 samples in the control MRL/lpr group. Signal log ratios between the two groups were calculated by the replicate comparisons.

Quantitative Kinetic PCR

cDNA was produced from kidney cortex RNA by a reverse transcription for PCR kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. Quantitative reverse transcriptase–PCR (qRT-PCR) was performed by QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA). Expression data were normalized to 18S RNA. The primers are as follows: collagen VI (α1) chain left: 5′-tgctcaacatgaagcagacc-3′, right: 5′-gccactaaggaccagaccaa-3′; collagen VI (α3) chain left: 5′-gtctccaggcagttctaccg-3′, right: 5′-agctgtgacgtccaggctat-3′; connective tissue growth factor (CTGF) left: 5′-tgctgtgcaggtgataaagc-3′, right: 5′-ccaccccaaaccagtcataa-3′; TGF-β1 left: 5′-ttgcttcagctccacagaga-3′, right: 5′-tggttgtagagggcaaggac-3′.

Immunofluorescence Microscopy

Kidney cortex tissue was snap-frozen in 2-methylbutane in a container on dry ice. Four-micron cryostat sections were processed for immunofluorescence (IF). Sections were fixed in ether-ethanol and stained with biotin-conjugated polyclonal antibodies to human collagens I, III, IV, and VI (SouthernBiotech, Birmingham, AL), cross-reactive and specific for the respective mouse collagens, followed by FITC-conjugated streptavidin (ICN Biomedical, Aurora, OH). A semiquantitative score of staining intensity and distribution from 0 to 4 was provided in a blinded manner as described previously (14).

Immunoblotting

Fresh renal cortical tissue was homogenized in ice-cold lysis buffer containing 200 mM NaCl, 10 mM Tris HCl pH 7.0, 5 mM EDTA, 1 mM PMSF, 10% glycerol, plus a protease inhibitor cocktail (Roche, Indianapolis, IN), followed by a 30-min incubation on ice. Insoluble material was spun down and the concentration of protein in the supernatant was determined by the bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL) with BSA as a standard. Equal amounts of samples (40 μg) were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene diflouride membrane (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with sheep anti-mouse plasminogen activator inhibitor 1 (PAI-1) antibody (American Diagnostica, Greenwich, CT) at a 1:250 dilution for 2 h, or with rabbit anti-mouse CTGF (Cell Sciences, Norwood, MA) at a 1:5000 dilution for 2 h. Membranes were then incubated with peroxidase-conjugated anti-sheep IgG (ICN Biomedical, Aurora, Oh) for PAI-1 detection or with anti-rabbit IgG (Sigma Chemical, St. Louis, MO) for CTGF detection. Membranes were also probed with anti-actin antibody (Sigma) to confirm equal loading of samples.

Statistical Analyses

Statistical analyses were performed by Minitab software (College Park, MD). Data are expressed as mean ± SEM. Statistical significance was determined by ANOVA followed by Tukey’s pairwise comparisons or by t test when only two groups were compared. Potential relationships between transcriptional changes and phenotypic variables were examined by Pearson product moment correlation coefficient.

Results

ECM Components

In this study, we used the Affymetrix MG U74A version 2 arrays with comprehensive coverage of known mouse genes to examine mRNA expression levels in 3 groups of mice: saline-treated MRL/lpr mice, Crry-Ig–treated MRL/lpr mice, and unmanipulated MRL/+ mice. Among the 119 genes classified as ECM components, 18 genes met the conditions described in Materials and Methods (Table 1); therefore, these genes were regarded as being upregulated in saline-treated MRL/lpr mice compared with MRL/+ mice, and the overexpression was brought down by complement inhibition with Crry-Ig.

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Table 1. ECM component genes altered in MRL/lpr mouse kidneys in a complement-dependent manner

To confirm the microarray data, which were only based on a portion of the animals in this study, qRT-PCR was used on all of the samples. Collagen VI (α1) and (α3) chains were chosen as representative of the 18 genes. Similar to microarray analyses, by qRT-PCR, collagen VI (α1) and (α3) mRNA expressions were also significantly increased in the saline control group compared with the MRL/+ strain control (P < 0.01, respectively, ANOVA), and complement inhibition with Crry-Ig decreased their expression (P < 0.05, respectively) (Figure 1). Collagen VI (α1) and (α3) gene expressions in Crry-Ig–treated mice were not significantly different from normal levels in MRL/+ mice (P > 0.05, respectively). Thus, collagen VI (α1) and (α3) chain mRNA expressions were upregulated in MRL/lpr mice compared with MRL/+ control mice, and complement inhibition with Crry-Ig brought them down to normal levels.

Figure1

Figure 1. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces collagen VI (α1) and (α3) chain mRNA overexpression in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse kidneys. Shown are quantitative reverse transcriptase–PCR data in which relative mRNA expression levels were derived by normalizing individual levels to 18S RNA expression. Data are presented as mean ± SEM. The numbers of animals studied were three for MRL/+, ten for MRL/lpr + Crry-Ig, and eight for MRL/lpr + saline. #P < 0.01 compared with MRL/+ normal control; *P < 0.05 compared with MRL/lpr saline control. Expression levels from Crry-Ig–treated MRL/lpr mice were not significantly different from those of MRL/+ mice.

Of the 18 genes included in Table 1, 13 were for collagen genes, and the changes in collagen VI were validated by qRT-PCR. We were next interested in whether the mRNA changes were also translated at the protein level as determined by IF microscopy. In MRL/+ control mice, there was no staining apparent for collagens I, III, and VI, whereas collagen IV was finely distributed along tubular and glomerular basement membranes (data not shown). All four types of collagen were found in saline-treated MRL/lpr mice (Figure 2). With complement inhibition by Crry-Ig, collagens I, IV, and VI in glomeruli were significantly reduced (P < 0.05 for collagens IV and VI, P < 0.01 for collagen I) compared with the saline control group. Collagen III staining was also decreased with Crry-Ig treatment but did not attain statistical significance.

Figure2

Figure 2. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces collagens I III, IV, and VI accumulations in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse glomeruli. Shown are semiquantitative immunofluorescent staining data presented as mean ± SEM. The numbers of animals studied were ten for MRL/lpr + Crry-Ig and eight for MRL/lpr + saline. #P < 0.005 compared with MRL/lpr saline control; *P < 0.05 compared with MRL/lpr saline control.

In addition to the presence of collagens I, III, IV, and VI in the kidneys of MRL/lpr mice and significant reductions with Crry-Ig treatment, the distribution and pattern of these collagens was also different in the different groups of mice (Figure 3). Collagen I was distributed mainly in sclerotic mesangial areas in MRL/lpr control mice, whereas in Crry-Ig–treated animals, there was much less staining in these areas. Collagens III, IV, and VI were distributed not only in glomeruli but also in the tubulointerstitium. In MRL/lpr control mice, collagens III and VI were found in crescents and sclerotic mesangial areas. Collagen IV had a widespread distribution in tubular and glomerular basement membranes, and it was thicker in glomerular crescents or sclerosis. In mice treated with Crry-Ig, there was only trace staining for collagens III and VI in mesangial areas and along tubular basement membranes. In addition, collagen IV had a largely normal appearance in complement-inhibited mice.

Figure3

Figure 3. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces the accumulations of collagens I, III, IV, and VI in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse glomeruli. Shown are representative immunofluorescence micrographs from 24-wk-old MRL/lpr mice treated with Crry-Ig (A, C, E, G) or saline (B, D, F, H), and stained for collagen I (A and B), collagen III (C and D), collagen IV (E and F), and collagen VI (G and H). In Panel B, there is collagen I staining in a sclerotic mesangial area (arrow). In Panel D, there is collagen III staining in thickened tubular basement membranes (arrow). In Panel F, there is collagen IV staining in a fibrous glomerular crescent (asterisk) and the thickened glomerular basement membrane (arrow). In Panel H, there is collagen VI staining in a fibrous glomerular crescent (asterisk) and the expanded mesangial area (arrow).

ECM Regulatory Proteins and Enzymes

With microarray analyses, we also found altered gene expression levels of profibrotic cytokines and the enzymes that regulate matrix turnover in the three groups. Among the two sets of genes, CTGF and PAI-1 mRNA levels were significantly increased in saline-treated MRL/lpr animals compared with MRL/+ mice and decreased with Crry-Ig treatment compared with saline controls (Table 2). The increases in CTGF and PAI-1 were essentially prevented by complement inhibition: there were six and seven “no change” calls in nine comparisons between Crry-Ig and MRL/+ groups, respectively. To further confirm microarray data with all of the samples in this study, qRT-PCR was performed with CTGF primers. As shown in Figure 4, CTGF mRNA was increased in renal cortices of 24-wk-old MRL/lpr animals compared with MRL/+ mice, and this was significantly reduced by complement inhibition.

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Table 2. ECM regulatory genes altered in MRL/lpr mouse kidneys in a complement-dependent manner

Figure4

Figure 4. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces CTGF mRNA overexpression in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse kidneys by quantitative reverse transcriptase–PCR. Shown are relative connective tissue growth factor (CTGF) mRNA expression levels normalized to 18S RNA expression. Data are presented as mean ± SEM. The numbers of animals studied were three for MRL/+, ten for MRL/lpr + Crry-Ig, and eight for MRL/lpr + saline. #P < 0.01 compared with MRL/+ normal control. *P < 0.05 compared with MRL/lpr saline control. Expression levels from Crry-Ig–treated MRL/lpr mice were not significantly different from those of MRL/+ mice.

In view of the potential link between TGF-β1 and CTGF (15), we examined the former more closely. Although in our array studies, there was marked upregulation of TGF-β1 mRNA comparing control MRL/lpr to MRL/+ (7.13-fold change, with 14 of 18 increase calls), there were only 5 of 18 decrease calls comparing Crry-Ig–treated MRL/lpr mice to control MRL/lpr mice, which did not make the cutoff we imposed (which was at least 8 of 18 decrease calls). Nonetheless, TGF-β1 mRNA was examined in the entire cohort of mice. As shown in Figure 5, TGF-β1 mRNA was increased in renal cortices of 24-wk-old MRL/lpr animals compared with MRL/+ mice, and this was significantly reduced by complement inhibition. Immunoblotting was used to further correlate mRNA with protein expression levels for PAI-1 and CTGF. Corresponding to the microarray data, complement inhibition with Crry-Ig also significantly reduced PAI-1 and CTGF expression at the protein level compared with the saline control group (Figure 6).

Figure5

Figure 5. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces TGF-β1 mRNA overexpression in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse kidneys by quantitative reverse transcriptase–PCR. Shown are relative TGF-β1 mRNA expression levels normalized to 18S RNA expression. Data are presented as mean ± SEM. The numbers of animals studied were three for MRL/+, ten for MRL/lpr + Crry-Ig, and eight for MRL/lpr + saline. #P < 0.01 compared with MRL/+ normal control; *P < 0.05 compared with MRL/lpr saline control.

Figure6

Figure 6. Complement inhibition with complement receptor 1–related gene/protein y (Crry)–Ig reduces connective tissue growth factor (CTGF) and PAI-1 protein overexpression in 24-wk-old MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mouse kidneys. (A) Representative immunoblotting from the nine MRL/lpr animals from which microarray data were obtained. Graphically shown are the relative connective tissue growth factor (CTGF) (B) and PAI-1 (C) protein expression levels from all animals (ten in MRL/lpr + Crry-Ig and eight in MRL/lpr + saline) presented as mean ± SEM. *P < 0.05 compared with MRL/lpr saline control.

Correlation Analysis

From our previous study, we measured BUN and albuminuria levels and pathology scores for these MRL/lpr mice with or without Crry-Ig treatment (13). We used the data derived here to analyze possible relationship between ECM and its regulation genes with renal functional and pathologic changes in these mice. At the mRNA level by qRT-PCR, collagen VI (α1) and (α3) chains, CTGF, and TGF-β1 significantly correlated with glomerulosclerosis and interstitial nephritis, and also with BUN and albuminuria levels (Table 3). At the protein level by IF staining, collagens I, III, IV, and VI also significantly correlated with glomerulosclerosis, interstitial nephritis, BUN, and albuminuria levels (Table 4).

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Table 3. Correlation analyses between mRNA levels and renal disease measurement dataa

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Table 4. Correlation analyses between protein levels and renal disease measurement dataa

Discussion

To investigate the role of chronic complement inhibition in lupus nephritis, we previously used two strategies to provide Crry as a C3 inhibitor in the MRL/lpr lupus mouse model: we used a transgenic mouse line that overexpresses soluble Crry systemically and locally in kidney (12); and we used a recombinant soluble form of Crry by fusing Crry to the CH2 and CH3 domains of mouse IgG1, which has optimal pharmacokinetic properties and is noncomplement activating (13). With both strategies, we found that complement inhibition with Crry can prevent MRL/lpr mice from developing renal failure and severe proteinuria, the two major complications that also occur in human lupus nephritis. More interestingly, Crry-Ig treatment also significantly reduced glomerulosclerosis in 24-wk-old MRL/lpr mice. This reduction also occurred in Crry transgenic mice, although it did not reach statistical significance. To clarify the mechanism of this effect, this study was designed to investigate the role of chronic complement inhibition at the level of the C3 convertase on the components of ECM in MRL/lpr lupus mouse kidneys.

Fibrosis is characterized by an excessive accumulation of ECM caused by increased production and/or insufficient degradation (16). The abnormal ECM in glomerulosclerosis is due to an excessive accumulation of normal components of ECM such as collagen IV, fibronectin, and laminin, and also of ECM components that are not found in normal basement membranes such as collagens I and III (16,17⇓). Complement activation may be linked with such renal fibrogenesis. It has been shown that C6-deficient rats had significant improvement of morphology and function in tubulointerstitial injury and fibrosis in the five-sixths nephrectomy model (18,19⇓), indicating that interstitial fibrosis is mediated predominantly by C5b-9. Reducing C5b-9 attack in tubular cells may slow progression and facilitate recovery.

Interestingly, we identified alterations in genes of eight individual collagen types: collagens I, III, IV, V, VI, VIII, XV, and XVIII. In the MRL/+ strain control, one or more chains of collagens III, IV, VI, and XVIII were scored as present in renal cortex, whereas the others were absent. All eight collagen types were upregulated in MRL/lpr kidneys to a significant extent; those that were present in MRL/+ kidney had increased expression levels in MRL/lpr kidneys, whereas those collagen mRNAs absent in MRL/+ kidneys became detectable in MRL/lpr kidneys. Among these, only collagen IV is known to be a constituent of basement membranes in the kidney. The other seven collagen types are not considered to be normal components in the kidney. In spite of the presence of mRNA for α 1 chains of collagens III and VI in our microarray studies, we were unable to identify intact collagen III and VI protein by IF microscopy in MRL/+ kidneys. Of the eight collagen types we identified here, accumulation of collagens I, III, and IV has been implicated in matrix accumulation in murine lupus nephritis (20). As for a complement dependence, this is the first evidence that the increased transcription and translation of these various collagens are linked to complement activation.

To provide an explanation why these various collagen genes were upregulated, we turned to gene products upstream of collagen transcription. Relevant in this case were genes for TGF-β and CTGF. TGF-β is widely regarded as the key fibrosis-promoting molecule in many organs, including the kidney. In vitro, TGF-β stimulates mouse (21), rat (22) and human (23,24⇓) mesangial cell ECM production. In vivo studies also showed that TGF-β is associated with several human glomerular diseases (25,26⇓). Further evidence supporting a role for TGF-β in renal fibrosis include: TGF-β transgenic mice develop renal fibrosis (27); in vivo transfection of TGF-β into rats induced glomerulosclerosis (28); and antiserum against TGF-β1 suppressed the increase production of ECM and dramatically attenuated histologic manifestations in experimental mesangial proliferative GN (29). TGF-β1 is one of the three mammalian isoforms that has been most extensively investigated in kidney fibrosis, which can be produced by both resident kidney cells and infiltrating leukocytes. Our microarray and qRT-PCR data clearly showed that TGF-β1 mRNA was upregulated in MRL/lpr mice compared with MRL/+ mice and the increased expression was reduced by Crry-Ig treatment. This expression pattern suggests that TGF-β1 is involved in the fibrotic process occurring in MRL/lpr mouse kidney, which is stimulated through complement activation.

CTGF is a cytokine that acts downstream of TGF-β1 to regulate matrix metabolism (30) and can induce fibroblast proliferation and matrix protein synthesis in vitro (31–33⇓⇓). In vivo, CTGF mRNA was reported to be elevated in experimental diabetic glomerulosclerosis (32), and serum CTGF levels were also increased in patients with systemic sclerosis (34) and other human renal diseases characterized by matrix deposition (35). In our microarray study, we found that CTGF mRNA was upregulated in MRL/lpr lupus mouse kidneys, and Crry-Ig treatment reduced this overexpression. We also confirmed this result at a protein level with immunoblotting. To the best of our knowledge, we are the first group to report that CTGF mRNA and protein are elevated in experimental lupus glomerulosclerosis and complement inhibition can reduce this elevation, which potentially can further reduce ECM accumulation.

There are two proteolytic systems involved in matrix degradation: the plasminogen/plasmin system and the matrix metalloproteinase system. PAI-1 is a major inhibitor of plasminogen activators, thereby regulating fibrinolysis and the plasmin-mediated matrix metalloproteinase system (36). PAI-1 mRNA or protein was not found in normal mouse (37) or human (38) kidney but was found to be strongly upregulated in many experimental and human renal diseases, including MRL/lpr mouse kidneys (26,37,39,40⇓⇓⇓). Our study confirmed this at both the mRNA and protein level. Furthermore, complement inhibition with Crry-Ig reduced this overexpression, which may contribute to the reduction of ECM accumulation by this treatment.

That the transcript changes we identified are relevant comes from the strong correlation between the expression of matrix and regulatory genes and functional and histologic measures of renal integrity. Taken together, our data suggest that these ECM components and regulatory proteins are responsible, at least in part, for the glomerulosclerosis and interstitial fibrosis that track with the severe proteinuria and renal failure in these mice. Complement inhibition with Crry-Ig prevented these events by inhibiting their overexpression.

Our data do not allow us to be definitive about which complement component or components acting through which renal cell type or types are responsible for the effects we have observed in this study. This is because Crry acts to prevent generation of C3a, C3b, iC3b, C3d, C5a, and C5b-9, each of which can play a role in renal disease. Relevant cell types in lupus nephritis include intrinsic glomerular, tubular, vascular, and interstitial cells as well as infiltrating inflammatory cells, such as neutrophils, monocytes, and lymphocytes. There is, however, a growing body of data that can provide clues to what might be happening in our experimental system.

Monocytes/macrophages bear receptors for C3a, C3b (CR3), and C5a and are clearly involved in matrix deposition in a variety of experimental conditions (41), including lupus nephritis (42). Hence, prevention of their accumulation and activation in the lupus kidney could reduce matrix accumulation. A recent study in a model of glomerular disease and progressive tubulointerstitial damage with features comparable to lupus nephritis is illuminating (43). In this study, C5a receptor deficiency did not affect the glomerular phenotype at all, whereas it ameliorated the tubulointerstitial disease. Their conclusions were that C5a, generated at least in part through local tubular complement synthesis, was chemoattractant to inflammatory cells bearing C5a receptors and also signaled tubular cells to result in their apoptotic death (43). Finally, C5b-9 can signal a variety of renal cells to result in a number of alterations besides cell death, including involvement in changes relevant to what we have studied here in lupus nephritis (18,44,45⇓⇓). The availability of mice with specific complement protein and receptor deficiencies, as well as the use of renal and bone marrow transplantation techniques, should allow these issues to be sorted out in future studies.

In conclusion, our observations in this study identified the relevant ECM components and regulatory proteins that are involved in renal fibrosis in MRL/lpr mice. Complement inhibition with Crry-Ig inhibited TGF-β1, CTGF and PAI-1 overexpression in the MRL/lpr mouse kidney, and reduced production of some key ECM components including collagens I, III, IV, and VI, as well as potentially enhanced their degradation. The overall effect of complement inhibition was reduction of ECM accumulation in MRL/lpr mouse kidneys, and improved glomerular function as measured by BUN and albuminuria. Our findings provide evidence for the possible mechanisms by which complement inhibition favorably affects lupus nephritis. These data support the strategy of the use of complement inhibitors in human lupus nephritis.

Acknowledgments

This work was supported by NIH grants R01DK55357 and U24DK58820 and by a Biomedical Sciences Grant from the Arthritis Foundation. Dr. Bao was supported by NIH training grant T32DK07510.

References

  1. ↵
    Cameron J: Systemic lupus erythematosus. In: Immunologic Renal Diseases, 2nd Ed., edited by Neilson E, Couser W, Philadelphia, Lippincott Williams & Wilkins, 1997, pp 1055–1098
  2. ↵
    Hahn B: Animal models of systemic lupus erythematosus. In: Dubois’ Lupus Erythematosus, 6th Ed., edited by Wallace DJ, Hahn B, Dubois EL, Philadelphia, Lippincott Williams & Wilkins, 2002, pp 339–388
  3. ↵
    Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S: Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356: 314–317, 1992
    OpenUrlCrossRefPubMed
  4. ↵
    Adachi M, Watanabe-Fukunaga R, Nagata S: Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice. Proc Natl Acad Sci U S A 90: 1756–1760, 1993
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Andrews BS, Eisenberg RA, Theofilopoulos AN, Izui S, Wilson CB, McConahey PJ, Murphy ED, Roths JB, Dixon FJ: Spontaneous murine lupus-like syndromes: Clinical and immunopathological manifestations in several strains. J Exp Med 148: 1198–1215, 1978
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Walport MJ: Complement. First of two parts. N Engl J Med 344: 1058–1066, 2001
    OpenUrlCrossRefPubMed
  7. ↵
    Braun M, Davis AE3rd: Cultured human glomerular mesangial cells express the C5a receptor. Kidney Int 54: 1542–1549, 1998
    OpenUrlCrossRefPubMed
  8. ↵
    Zahedi R, Braun M, Wetsel RA, Ault BH, Khan A, Welch TR, Frenzke M, Davis AE: The C5a receptor is expressed by human renal proximal tubular epithelial cells. Clin Exp Immunol 121: 226–233, 2000
    OpenUrlCrossRefPubMed
  9. ↵
    Wilmer WA, Kaumaya PT, Ember JA, Cosio FG: Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells. J Immunol 160: 5646–5652, 1998
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Taratuska A, Rynkiewicz M, Nicholson-Weller A: Membrane signaling by complement C5b-9, the membrane attack complex. Blood 81: 200–205, 1993
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Quigg RJ, Kozono Y, Berthiaume D, Lim A, Salant DJ, Weinfeld A, Griffin P, Kremmer E, Holers VM: Blockade of antibody-induced glomerulonephritis with Crry-Ig, a soluble murine complement inhibitor. J Immunol 160: 4553–4560, 1998
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Bao L, Haas M, Boackle SA, Kraus DM, Cunningham PN, Park P, Alexander JJ, Anderson RK, Culhane K, Holers VM, Quigg RJ: Transgenic expression of a soluble complement inhibitor protects against renal disease and promotes survival in MRL/lpr mice. J Immunol 168: 3601–3607, 2002
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Bao L, Haas M, Kraus DM, Hack BK, Rakstang JK, Holers VM, Quigg RJ: Administration of a soluble recombinant complement C3 Inhibitor protects against renal disease in MRL/lpr mice. J Am Soc Nephrol 14: 670–679, 2003
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Quigg RJ, Lim A, Haas M, Alexander JJ, He C, Carroll MC: Immune complex glomerulonephritis in C4- and C3-deficient mice. Kidney Int 53: 320–330, 1998
    OpenUrlCrossRefPubMed
  15. ↵
    Gupta S, Clarkson MR, Duggan J, Brady HR: Connective tissue growth factor: Potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 58: 1389–1399, 2000
    OpenUrlCrossRefPubMed
  16. ↵
    Eddy AA: Molecular basis of renal fibrosis. Pediatr Nephrol 15: 290–301, 2000
    OpenUrlCrossRefPubMed
  17. ↵
    Fogo AB: Current concepts in glomerulosclerosis. Am J Kidney Dis 34:liv–lvi, 1999
  18. ↵
    Nangaku M, Pippin J, Couser WG: Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome. J Am Soc Nephrol 10: 2323–2331, 1999
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Nangaku M, Pippin J, Couser WG: C6 mediates chronic progression of tubulointerstitial damage in rats with remnant kidneys. J Am Soc Nephrol 13: 928–936, 2002
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Nakamura T, Ebihara I, Tomino Y, Koide H: Glucocorticoid ameliorates altered gene expression of extracellular matrix components in kidneys of New Zealand black/white F1 mice. Clin Sci (Lond) 83: 701–709, 1992
    OpenUrlPubMed
  21. ↵
    MacKay K, Striker LJ, Stauffer JW, Doi T, Agodoa LY, Striker GE: Transforming growth factor-beta: Murine glomerular receptors and responses of isolated glomerular cells. J Clin Invest 83: 1160–1167, 1989
  22. ↵
    Suzuki S, Ebihara I, Tomino Y, Koide H: Transcriptional activation of matrix genes by transforming growth factor beta 1 in mesangial cells. Exp Nephrol 1: 229–237, 1993
    OpenUrlPubMed
  23. ↵
    Hänsch GM, Wagner C, Bürger A, Dong W, Staehler G, Stoeck M: Matrix protein synthesis by glomerular mesangial cells in culture: Effects of transforming growth factor beta (TGF beta) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA. J Cell Physiol 163: 451–457, 1995
    OpenUrlCrossRefPubMed
  24. ↵
    McKay NG, Khong TF, Haites NE, Power DA: The effect of transforming growth factor beta 1 on mesangial cell fibronectin synthesis: Increased incorporation into the extracellular matrix and reduced pI but no effect on alternative splicing. Exp Mol Pathol 59: 211–224, 1993
    OpenUrlCrossRefPubMed
  25. ↵
    Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H, Maki S: Transforming growth factor-beta protein and mRNA in glomeruli in normal and diseased human kidneys. Lab Invest 68: 154–163, 1993
    OpenUrlPubMed
  26. ↵
    Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border WA: Expression of transforming growth factor-beta isoforms in human glomerular diseases. Kidney Int 49: 461–469, 1996
    OpenUrlCrossRefPubMed
  27. ↵
    Mozes MM, Böttinger EP, Jacot TA, Kopp JB: Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice. J Am Soc Nephrol 10: 271–280, 1999
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: Glomerulosclerosis induced by in vivo transfection of transforming growth factor-beta or platelet-derived growth factor gene into the rat kidney. J Clin Invest 92: 2597–2601, 1993
  29. ↵
    Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1. Nature 346: 371–374, 1990
    OpenUrlCrossRefPubMed
  30. ↵
    Igarashi A, Okochi H, Bradham DM, Grotendorst GR: Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4: 637–645, 1993
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR: Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107: 404–411, 1996
    OpenUrlCrossRefPubMed
  32. ↵
    Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins RG: Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 11: 25–38, 2000
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Murphy M, Godson C, Cannon S, Kato S, Mackenzie HS, Martin F, Brady HR: Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 274: 5830–5834, 1999
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Grotendorst GR, Takehara K: Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. J Invest Dermatol 105: 280–284, 1995
    OpenUrlCrossRefPubMed
  35. ↵
    Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, Goldschmeding R: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 53: 853–861, 1998
    OpenUrlCrossRefPubMed
  36. ↵
    Rondeau E, Sraer J, Schleuning W: The renal plasminogen activating system. In: Molecular Nephrology : Kidney Function in Health and Disease,edited by Schlondorff D, Bonventre JV, New York, Marcel Dekker, 1995, pp 699–715
  37. ↵
    Moll S, Menoud PA, Fulpius T, Pastore Y, Takahashi S, Fossati L, Vassalli JD, Sappino AP, Schifferli JA, Izui S: Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis. Kidney Int 48: 1459–1468, 1995
    OpenUrlCrossRefPubMed
  38. ↵
    Xu Y, Hagege J, Mougenot B, Sraer JD, Rønne E, Rondeau E: Different expression of the plasminogen activation system in renal thrombotic microangiopathy and the normal human kidney. Kidney Int 50: 2011–2019, 1996
    OpenUrlPubMed
  39. ↵
    Rondeau E, Mougenot B, Lacave R, Peraldi MN, Kruithof EK, Sraer JD: Plasminogen activator inhibitor 1 in renal fibrin deposits of human nephropathies. Clin Nephrol 33: 55–60, 1990
    OpenUrlPubMed
  40. ↵
    Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, Border WA: Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci U S A 90: 1814–1818, 1993
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Eddy AA: Interstitial macrophages as mediators of renal fibrosis. Exp Nephrol 3: 76–79, 1995
    OpenUrlPubMed
  42. ↵
    Tesch GH, Maifert S, Schwarting A, Rollins BJ, Kelley VR: Monocyte chemoattractant protein 1–dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice. J Exp Med 190: 1813–1824, 1999
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Welch TR, Frenzke M, Witte D, Davis AE: C5a is important in the tubulointerstitial component of experimental immune complex glomerulonephritis. Clin Exp Immunol 130: 43–48, 2002
    OpenUrlCrossRefPubMed
  44. ↵
    Wagner C, Braunger M, Beer M, Rother K, Hänsch GM: Induction of matrix protein synthesis in human glomerular mesangial cells by the terminal complement complex. Exp Nephrol 2: 51–56, 1994
    OpenUrlPubMed
  45. ↵
    Shankland SJ, Pippin J, Pichler RH, Gordon KL, Friedman S, Gold LI, Johnson RJ, Couser WG: Differential expression of transforming growth factor-beta isoforms and receptors in experimental membranous nephropathy. Kidney Int 50: 116–124, 1996
    OpenUrlPubMed
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Excessive Matrix Accumulation in the Kidneys of MRL/lpr Lupus Mice Is Dependent on Complement Activation
Lihua Bao, Jian Zhou, V. Michael Holers, Richard J. Quigg
JASN Oct 2003, 14 (10) 2516-2525; DOI: 10.1097/01.ASN.0000089831.96794.0B
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