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. 2006 May;144(2):326–334. doi: 10.1111/j.1365-2249.2006.03069.x

Sublytic complement C5b-9 complexes induce thrombospondin-1 production in rat glomerular mesangial cells via PI3-k/Akt: association with activation of latent transforming growth factor-β1

L Gao 1, W Qiu 1, Y Wang 1, W Xu 1, J Xu 1, J Tong 1
PMCID: PMC1809655  PMID: 16634807

Abstract

Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury. Complement C5b-9 is a prime candidate to mediate mesangial cell proliferation, especially sublytic C5b-9, which can induce the production of multiple inflammatory factors and cytokines. Transforming growth factor (TGF)-β1 plays a major role in the accumulation of extracellular matrix (ECM), while thrombospondin (TSP)-1 has been identified as an activator of latent TGF-β1 in an in vitro system. Using rat glomerular mesangial cells (GMCs) as a model system, we assessed the effect of sublytic C5b-9 on the expression of TSP-1 and TGF-β1 and explored the relevant pathway of signal transduction. First, we ensured the concentrations of anti-Thy1 antibody and complement, which were regarded as a sublytic C5b-9 dose, and examined whether the sublytic C5b-9 induced expression of TSP-1 in rat GMCs which, in turn, activated latent TGF-β1 by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, we investigated the role of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 production in rat GMCs by Western blot analysis. The addition of sublytic C5b-9 (5% anti-Thy1 antibody and 4% normal serum) to rat GMCs induced activation of latent TGF-β1 via TSP-1. The addition of sublytic C5b-9 apparently increased the protein of Akt phosphorylation, whereas PI3-k inhibitor LY294002 could clearly reduce the increase of TSP-1 induced by sublytic C5b-9. These results indicate that TSP-1 is an activator of latent TGF-β1 in sublytic C5b-9-induced rat GMCs; furthermore, the PI3-k/Akt signal transduction pathway may play a key role in sublytic C5b-9-induced TSP-1 production.

Keywords: anti-Thy1 antibody, glomerular mesangial cells (GMCs), mesangioproliferative glomerulonephritis, PI3-k/Akt, sublytic C5b-9, TGF-β1, TSP-1

Introduction

Anti-Thy1 nephritis is an acknowledged model of human mesangioproliferative glomerulonephritis (MPGN). Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury in animal models [1,2]. Early studies of anti-Thy1 nephritis demonstrate that glomerular injury, including mesangial cell lysis, proliferation and proteinuria, are complement-dependent and neutrophil-independent. The complement system mediates inflammation and cytolysis, etc. In mesangial cells, C5b-9 can induce release of oxidants, cytokines such as interleukin (IL)-1, tumour necrosis factor (TNF) and extracellular matrix (ECM) components, including collagen IV and laminin. Subsequent studies have confirmed and extended these observations to show that systemic complement depletion reduces mesangiolysis, mesangial cell proliferation and expansion [3].

Complement is a critical mediator of antibody-induced renal disease [4,5]. Sublytic C5b-9 can result in a variety of responses in glomerular mesangial cells (GMCs). Previous reports have revealed that proteinuria is mediated primarily by sublytic C5b-9 through similar mechanisms that also involve the glomerular endothelial cell as a target and production of oxidants, proteases and transforming growth factor (TGF)-β1 in response to sublytic C5b-9 attack. TGF-β1 is thought to be the key mediator of matrix accumulation in rat GMC proliferation [6,7]. ECM protein is synthesized in rat GMCs through the induction of TGF-β1. Mesangial cells stimulated by sublytic C5b-9 results in an increase of both total TGF-β1 production and levels of activated TGF-β1. However, the mechanism of the sublytic C5b-9 effect on TGF-β1 has not yet been defined.

Thrombospondin (TSP)-1, the matricellular protein, is a 450-kDa trimeric extracellular matrix glycoprotein found in platelet β-granules and other cell types, including GMCs. Many studies indicate that TSP-1 has diverse effects on cell behaviour [8,9]; for example, TSP-1 is a major physiological regulator of TGF-β1 activation [10], suggesting that TSP-1 may exert an important role in TGF-β1 driving the accumulation of ECM. However, the relationship between sublytic C5b-9-induced TSP-1 and TGF-β1 in rat GMCs remains unclear.

PI3-k/Akt is involved in modulation, chemotaxis and cell proliferation [11–15]. PI3-k catalyses the addition of a phosphate molecule to the inositol ring of phosphoinositides generating phosphoinositol-3,4,5-P3 which, after binding to the pleckstrin homology domain of Akt, permits association of the phosphatidylinositol-dependent kinases PDK1 and PDK2 and activation of Akt through phosphorylation of Thr308 and Ser473. JNK activities were also induced by sublytic C5b-9 [16], but regulation of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 in rat GMCs has not been demonstrated fully in vitro or in vivo.

Materials and methods

Reagents

Specific PI3-k inhibitors LY294002, mouse monoclonal antibody against phospho-Akt phototope-horseradish peroxidase (HRP) Western blot detection system, including anti-mouse IgG, HRP-linked antibody, biotinylated protein ladder, 20× LumiGLO reagent and 20× peroxide were purchased from Cell Signalling Technology (Beverly, MA, USA). A rat TGF-β1 immunoassay kit was purchased from Bio-source (Camarillo, CA, USA). Human complement C6-deficient serum (C6DS) was obtained from Sigma (St Louis, MO, USA). Mouse monoclonal IgG antibody against Akt and goat polyclonal antibodies against TSP-1 and TGF-β1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The TSP-1-blocking peptides (GGWSHW) were synthesized by Shanghai Sangon Co. Ltd (Shanghai, China). M-MLV reverse transcriptase was obtained from Promega (Madison, WI, USA); Trizol reagent was purchased from Life Technology (Gaitherburg, MD, USA). Normal human serum (NS) obtained from several healthy adult donors was pooled and used as a source of serum complement. Heat-inactivated serum (HIS) was obtained by incubating the NS at 56°C for 30 min. Rabbit polyclonal antibody (anti-Thy1 antibody, titre 1 : 320) against Thy1 antigen of rat thymocyte was prepared according to Yamamoto and Wilson [17].

Rat mesangial cell culture

The rat GMCs strain was provided by the China Centre for Type Culture Collection (Wuhan, China). The rat GMCs were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin and 10% non-essential amino acid at 37°C in a 5% CO2 incubator. Five to seven passages of rat GMCs were used in the experiments. After an initial incubation in medium + 10% FCS to approximately 80% confluence, the rat GMCs (108 cells/l) were divided into seven treatments (three wells per treatment, 3 ml/well); namely, 5% anti-Thy1 antibody + 4% NS (sublytic C5b-9), 5% anti-Thy1 antibody + 4% C6DS, LY294002 (30 µM) + sublytic C5b-9, 5% anti-Thy1 antibody + 4% HIS, only 5% anti-Thy1 antibody, TSP-1-blocking peptide + sublytic C5b-9 and 3 ml culture medium alone (control).

Measurement of complement-dependent cytotoxicity

To induce sublytic C5b-9 attack [5], the selection of the anti-Thy1 antibody and complement concentration used in the study was based on checkerboard titration studies to define the maximal amounts of anti-Thy1 antibody and complement that could be used without producing rat GMC injury or lysis. Anti-Thy1 antibody was diluted to 2%, 5%, 6%, 7%, 10% and 33% (v/v), respectively; meanwhile, the NS was diluted to 4%, 5%, 6%, 8%, 16% and 33% (v/v) in serum free Kerbs–Henseleit (K–H) buffer [18] contain-ing 145 mM NaCl, 5 mM KCl, 0·5 mM MgSO4, 1 mM Na2HPO4, 0·5 mMCaCl2, 5 mM glucose and 20 mM HEPES, pH 7·4, and applied to the anti-Thy1 antibody-sensitized rat GMCs for 40 min at 37C to allow C5b-9 formation and insertion [19]. Rat GMCs were then washed with serum free K–H media to remove any unbound complement. The concentration of complement proteins used in this study are sublytic for rat GMCs, as determined by staining cells with vital dye trypan blue (less than 5% death cells was regarded as sublytic) [5].

Western blot analysis

The rat GMCs were treated with different media, such as sublytic C5b-9, anti-Thy1 antibody + C6DS, LY294002 + sublytic C5b-9, 5% anti-Thy1 antibody + 4% HIS, 5% anti-Thy1 antibody, TSP-1-blocking peptide + sublytic C5b-9 and cultured medium for the desired time and were then aspirated. The rat GMCs lysates were lysed by adding 1 × sodium dodecyl sulphate (SDS) sample buffer (500 µl per plate of 10 cm plates) and heated for 5 min at 95°C. Equal amounts of protein were subjected to 11% and 6% SDS-polyacrylamide gel electrophoresis (PAGE) gel, respectively. After electrophoresis, protein was transferred to polyvinyl difluoride (PVDF) membranes and probed with monocloned antibodies against serine 473-phosphospecific Akt, total-Akt and TSP-1, respectively. The primary antibodies (diluted 1 : 1000) and a second antibody consisting of HRP-conjugated anti-mouse IgG diluted 1 : 2000) were used for the detection of phosphospecific Akt, total-Akt and TSP-1. The bands were visualized by the electrochemiluminescence (ECL) detection system with 5–10 min exposure after extensive washing of the membranes. Controls for protein loading were identified using β-actin as the internal standard [20].

Immunoassays

Rat GMCs were seeded on 24-well plates and grown to 80% confluence. Cells then were incubated in the sublytic C5b-9, the TSP-1 blocking peptide + sublytic C5b-9, the TSP-1 blocking peptide and cultured medium, respectively, for the specified time. The culture medium was then collected and centrifuged, and the supernatant was stored at −20°C until assayed. TGF-β1 in culture media was measured by enzyme-linked immunosorbent assay (ELISA). The total amount of TGF-β1 was acidified before assay, whereas activated TGF-β1 was determined without acidification according to the manufacturer's instructions. The activated TGF-β1 was normalized by the total amount of TGF-β1.

Real-time quantitative polymerase chain reaction (PCR)

The rat GMCs were treated with the above-mentioned media, including 5% sublytic C5b-9, 5% anti-Thy1 antibody + 4% C6DS, LY294002 (30 µM) + sublytic C5b-9, 5% anti-Thy1 antibody + 4% HIS, only 5% anti-Thy1 antibody and 3 ml culture medium alone (control). cDNA templates for PCR amplification were prepared from the rat GMCs using random hexamer and M-MLV-RT. Specific probes and primers (TaqMan probe, FAM-labelled) were obtained from Applied Biosystems. Each reaction contained a cDNA template from 0·1 µg total RNA, 1 × TaqMan Universal PCR Master Mix with AmpErase UNG, 900 nM of forward and reverse primers and 200 nM of TaqMan probes (1 × TaqMan Target Mix). The real-time PCR was performed using ABI PRISM 7000 sequence detection system (Applied Biosystems) with the following thermal cycling conditions: 2 min at 50°C, 10 min at 95°C followed by a total of 40 cycles of 15 s at 95°C and 1 min at 60°C. Each reaction was made three times. Rat β-actin (FAM labelled) was co-amplified as an internal control. The relative quantification of gene expression was calculated using the manufacturer's recommendations, with Ct representing the threshold cycle number as defined by the software when the PCR reaction reaches the start of its exponential stage. This value was related logarithmically and conversely to the input copies of the transcript of interest.

Quantification of TSP-1 and TGF-β1 genes by real-time PCR

To investigate overexpression of the TSP-1 [21] and TGF-β1 genes in treated rat GMCs, quantification of gene expression was performed by real-time PCR using cDNA templates prepared from the rat GMCs. PCR reactions were performed under the same conditions as described above (β-actin was used as an endogenous control). The relative levels of gene expression were obtained by calculating the ratio of cycle numbers of the initial exponential amplification phase, as determined by the sequence detection system for specific target genes and rat β-actin, using the following formula:

Ct value ofβ-actin/Ct value of target gene

Statistical analysis

Data are expresssed as the mean ± s.e. Statistical evaluation of the data was performed using Student's t-test, with a P-value of < 0·05 considered significant.

Results

C5b-9-induced rat GMCs lysis is concentration-dependent

Rat GMCs used in the experiment were from five to seven passages. After an initial incubation in medium plus 10% FCS until approximately 80% confluence, the anti-Thy1 antibody-sensitized rat GMCs were treated with C6DS, HIS or different concentrations of NS.

The morphology of anti-Thy1 antibody-sensitized rat GMCs had apparently changed after treatment with different concentrations of complement for 40 min. When anti-Thy1 antibody-sensitized rat GMCs were incubated with serially increasing doses of complement, rat GMCs showed increasing cytolysis. Rat GMC morphology changed from intact polygon to cytolysis with 4%, 6%, 8% and 16% NS, respectively, but the morphology of rat GMCs was similar to normal cells when incubated with HIS. Rat GMCs occurred as a mass of lysis when the concentration of anti-Thy1 antibody and complement were all 33%, which was stained by trypan blue. When the concentration of anti-Thy1 antibody was 5% and complement was 4%, respectively, the number of dead rat GMCs was 9%; meanwhile, in rat GMCs treated by cultured medium (controls), the number of dead rat GMCs was 5% (Fig. 1). Therefore, the number of dead rat GMCs owing to complement-mediated cytolysis or death was 4% (Stuart et al. reported that less than 5% death rat GMCs was regarded as sublytic) [5]. We concluded preliminarily that the current concentration (5% anti-Thy1 antibody and 4% NS) could be regarded as sublytic.

Fig. 1.

Fig. 1

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Rat glomerular mesangial cells (GMCs) were incubated with anti-Thy1 antibody (30 min) and different concentrations of normal human serum (NS) (to form C5b-9) or heat-inactivated serum (HIS) in controls (40 min). The concentration of anti-Thy1 antibody was maintained at 5% with the increase of NS concentration, C5b-9 induced increasing rat GMC lysis, but the number of dead rat GMCs did not increase following 5% anti-Thy1 antibody and 4% NS. When the concentration of anti-Thy1 antibody and NS were 5% and 16%, respectively, a mass of rat GMC death and lysis was found. The number of dead rat GMCs increased along with the concentration of anti-Thy1 antibody and increased complement.

Sublytic C5b-9-induced expression of TSP-1 in rat GMCs

To determine whether sublytic C5b-9 is involved in up-regulation of TSP-1 gene expression, rat GMCs were treated with sublytic C5b-9, anti-Thy1 antibody, anti-Thy1 antibody + C6DS, anti-Thy1 antibody + HIS and cultured medium for 18 h. Levels of TSP-1 mRNA and protein in rat GMCs were measured using real-time PCR and Western blotting, respectively. The expression of TSP-1 increased significantly after exposure to sublytic C5b-9 in the defined time. Sublytic C5b-9 up-regulated TSP-1 mRNA and protein were 1·6-fold and 2·5-fold, respectively, compared with controls (Fig. 2), and other groups such as anti-Thy1 antibody, anti-Thy1 antibody + C6DS and anti-Thy1 antibody + HIS showed no apparent increase of TSP-1 mRNA and protein (Fig. 2). The level of TSP-1 was also maintained at the basal level, which was equivalent to those in controls. These findings, together, suggest that sublytic C5b-9 can induce expression of TSP-1 in rat GMCs.

Fig. 2.

Fig. 2

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(a) Relative gene expression levels at 18 h after administration of sublytic C5b-9, anti-Thy1 antibody + C6DS, culture medium alone, anti-Thy1 antibody and anti-Thy1 antibody + heat-inactivated serum (HIS). The amplification plot of thrombospondin (TSP)-1 and β-actin are shown in this figure. Differential expression of TSP-1 by real-time polymerase chain reaction. The TSP-1 gene in the sublytic C5b-9 group at 18 h was overexpressed, **P< 0·01 versus control group. (b) TSP-1 protein expression in rat glomerular mesangial cells using Western blotting. The graph showing the relative TSP-1 protein levels normalized to β-actin relative to controls. The results are expressed as the mean ± s.e. of three separate experiments. **P< 0·05 versus control group.

To ensure that the increase of TSP-1 expression was indeed caused by the C5b-9 components, we performed contrasting experiments between anti-Thy1 antibody + NS (which could form C5b-9) and anti-Thy1 antibody + C6DS, anti-Thy1 antibody + HIS. Figure 2 shows that the expression of TSP-1 in rat GMCs exposed to anti-Thy1 antibody + C6DS, anti-Thy1 antibody + HIS was clearly lower than that exposed to anti-Thy1 antibody + NS. The results indicated that the production of TSP-1 was due to the assembly of C5b-9 on the membrane of rat GMCs.

TSP-1 is the activator of latent TGF-β1 in rat GMCs

Rat GMCs were stimulated with sublytic C5b-9, anti-Thy1 antibody, anti-Thy1 antibody + C6DS, anti-Thy1 antibody + HIS and cultured medium for 18 h. Rat GMCs incubated with sublytic C5b-9 induced a significant increase of TGF-β1 mRNA and protein (Fig. 3a). With the decreasing contents of TSP-1, the expression of TGF-β1 also decreased (Fig. 3b). To determine whether TSP-1 can activate latent TGF-β1, the amount of total and activated TGF-β1 were measured by ELISA using acidified or non-acidified samples, respectively. When rat GMCs were incubated with sublytic C5b-9, the levels of activated TGF-β1 showed a significant increase. However, when the rat GMCs were incubated with the TSP-1 blocking peptide + sublytic C5b-9 for the time specified, the ELISA results showed that the levels of activated TGF-β1 were not apparently increased (Fig. 3c). The above-mentioned results indicate that TSP-1 induced the activation of latent TGF-β1 in rat GMCs operated by the sublytic C5b-9.

Fig. 3.

Fig. 3

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Rat glomerular mesangial cells (GMCs) were treated with different stimulation in a defined time. (a) Relative gene expression levels at 18 h after treated of sublytic C5b-9, anti-Thy1 antibody + C6DS, culture medium alone, anti-Thy1 antibody and anti-Thy1 antibody + heat-inactivated serum (HIS). The amplification plot of transforming growth factor (TGF)-β1 and β-actin by real-time polymerase chain reaction (PCR). The Ct values of TGF-β1/β-actin are shown in this figure. Differential expression of TGF-β1 by real-time PCR. The TGF-β1 gene in the sublytic C5b-9 group at 18 h was overexpressed, *P< 0·05 versus control group. (b) The relationship between thrombospondin (TSP)-1 and TGF-β1 expression under different stimulation. With the decrease of TSP-1, the contents of TGF-β1 were also decreasing. (c) TGF-β1 protein expression was analysed by enzyme-linked immunosorbent assay. The graph showing the relative activated TGF-β1 protein levels normalized to total TGF-β1 relative to control. Data are shown as mean ± s.e., *P< 0·05 versus control group.

Effect of different concentrations of complement on phosphorylated Akt in rat GMCs

Incubation with serially increasing doses of anti-Thy1 antibody and NS can activate diverse cell signal transduction. In the experiment, when the correct concentration of anti-Thy1 antibody and complement were selected, the results showed that when the rat GMCs were incubated with 5% anti-Thy1 antibody and 4% NS the expression of phosphorylated Akt reached 1·8-fold compared with control, whereas when incubated with the low dose (2% anti-Thy1 antibody and 4% NS) or the high dose (7% anti-Thy1 antibody and 8% NS), expression of phosphorylated Akt was not increased significantly compared with control or anti-Thy1 antibody + HIS. Taken together, these results revealed that a concentration of 5% anti-Thy1 antibody and 4% NS could induce the maximal expression of phosphorylated Akt (Fig. 4).

Fig. 4.

Fig. 4

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(a) Expression of phosphorylated Akt protein in rat glomerular mesangial cells (GMCs) by Western blotting. Rat GMCs were incubated with anti-Thy1 antibody and normal human serum (NS) or heat-inactivated serum (HIS) for 40 min. When the concentration of anti-Thy1 antibody and NS were 2% and 8%, 7% and 4%, respectively, the expression of phosphorylated Akt increased 1·3-fold, whereas with incubation of rat GMCs with 5% anti-Thy1 antibody and 4% NS, the expression of phosphorylated Akt increased 1·8-fold, *P< 0·05 versus control group. (b) Incubation of rat GMCs with 5% anti-Thy1 antibody and 4% NS; the expression of phosphorylated Akt maximal increased twofold (n = 3, *P< 0·05, compared with control).

Activity of PI3-k/Akt induced by sublytic C5b-9

In this experiment, we also focused upon whether the activation of Akt in rat GMCs needed sublytic C5b-9 stimulation, or the effects of sublytic C5b-9 on rat GMCs required the PI3-k/Akt pathway; we pretreated cells with specific PI3-K inhibitors. Rat GMCs were cultured for 40 min in dissimilar medium, LY294002 + sublytic C5b-9, sublytic C5b-9, anti-Thy1 antibody + C6DS and cultured medium. Akt activation by sublytic C5b-9 was determined by in vitro kinase assay and by assessing the phosphorylated Akt at Ser473, as shown in Fig. 5. Akt phosphorylation at Ser473 reached a maximum which was activated by sublytic C5b-9, compared with LY294002 + sublytic C5b-9 and anti-Thy1 antibody + C6DS. Moreover, inhibition of PI3-k with 30 µM LY294002 abolished the stimulatory effect of sublytic C5b-9 on Akt. These results suggest that the sublytic C5b-9 could activate the phosphorylated Akt and LY294002 could abolish this activation.

Fig. 5.

Fig. 5

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Activation of PI3-k/Akt by sublytic C5b-9 in cultured rat glomerular mesangial cells (GMCs). Rat GMCs were treated with sublytic C5b-9, anti-Thy1 antibody + C6DS or only anti-Thy1 antibody for 30 min. Rat GMCs were lysed and examined for phosphorylation of Akt by Western blotting. The different activity of PI3-k was determined as described. The significant increase of phosphorylation of Akt was seen in rat GMCs with sublytic C5b-9 attacking. The graph shows the relative phosphorylation of Akt protein levels normalized to total Akt relative to control. Results were expressed as mean ± s.e. of three separate experiments, *P< 0·05 versus control group.

To verify that complement-induced Akt phosphorylation was due to C5b-9 assembly, anti-Thy1 antibody-sensitized rat GMCs were incubated with C6DS or NS. Akt phosphorylation was increased markedly with NS treatment, whereas phosphorylation Akt was not changed in anti-Thy1 antibody + C6DS compared with controls. The results implied that phosphorylation of Akt needed C5b-9 formation and insertion into the membrane of rat GMCs.

Production of TSP-1 induced by sublytic C5b-9 via PI3-k/Akt

Sublytic C5b-9-induced production of TSP-1 may be involved in parallel in various signalling pathways, but it is unclear as to which one plays the main role. In this experiment, we utilized the inhibitor LY294002 (a selective inhibitor of PI3-k) to treat rat GMCs, and the results revealed that 30 µM LY294002 could substantially reduce the sublytic C5b-9-stimulated synthesis of TSP-1 in rat GMCs. The sublytic C5b-9-induced release of TSP-1 was amplified, compared with LY294002 + sublytic C5b-9 and controls (Fig. 6). Therefore, these results indicate that the expression of TSP-1 mediated by the sublytic C5b-9 complex in rat GMCs might be associated primarily with the PI3-k/Akt signalling pathway.

Fig. 6.

Fig. 6

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Sublytic C5b-9 induced thrombospondin (TSP)-1 production in rat glomerular mesangial cells (GMCs) via PI3-k/Akt. Rat GMCs were stimulated with sublytic C5b-9 or 30 µM LY294002 inhibitor + sublytic C5b-9 for 18 h. (a) Relative gene expression levels at 18 h after administration of LY294002 + sublytic C5b-9, sublytic C5b-9 and culture medium alone. The amplification plot of TSP-1 and β-actin are shown in this figure. Differential expression of TSP-1 by real-time polymerase chain reaction. The TSP-1 gene in the sublytic C5b-9 group at 18 h was overexpressed, **P< 0·01 versus control group. (b) Graph shows the relative TSP-1 protein levels normalized to β-actin relative to control. Results were expressed as mean ± s.e. of three separate experiments, **P< 0·01 versus control group.

Discussion

Mesangial cell proliferation is a characteristic feature of many forms of glomerular diseases, including IgA nephropathy [18]. Considerable evidence suggests that excessive proliferation of GMCs is associated usually with matrix expansion, leading to the development of glomerulosclerosis [2]. The most extensively studied animal model of mesangioproliferative glomerulonephritis is anti-Thy1 nephritis, induced by injection of anti-Thy1 antibody to the Thy1 antigen expressed on the plasma membrane of the mesangial cell.

In anti-Thy1 nephritis, an increase in mesangial cell proliferation has been shown to occur very early. The factors that mediate mesangial cell proliferation in anti-Thy1 nephritis are multiple. Previous studies have shown a possibility that stimulation or activation of mesangial cells could lead to the increased synthesis of mesangial cell-derived growth factor, including growth factors such as bFGF and PDGF, from intact cells that could then mediate a mitogenic effect in an autocrine manner [22,23].

It has been reported that several inflammatory cellular processes in the anti-Thy1 nephritis model are complement-dependent and neutrophils are not present. Activation of the complement cascade near the cell surface leads to the assembly of terminal components in the plasma membrane. Assembly of C5b-9 can result in the formation of transmembrane channels or the rearrangement of membrane lipids, with a loss of membrane integrity. However, the injury by C5b-9 to nucleated cells is almost non-lysis (sublytic) because the surfaces of the cells have many homologous restriction factors such as crry protein, CD59 and MCP [5]. In anti-Thy1 nephritis, anti-Thy1 antibody binds to glomerular mesangial cell antigen and leads to the formation of immune complexes (IC), and activates the complement system resulting in C5b-9 assembly in the membrane of rat GMCs. Previous studies have demonstrated that sublytic C5b-9 can induce sublethal injury and activate various metabolic processes to rat GMCs, which can produce multiple cytokine and growth factors, bFGF, PDGF, and so on [23]. In our experiments, when the concentrations of anti-Thy1 antibody and complement are at higher doses, the formation of C5b-9 can disrupt the integrity of the rat GMCs membrane. As a consequence, increasing rat GMCs occurred by lysis or death; but the concentrations of anti-Thy1 antibody and complement were 5% and 4%, respectively, which could activate the maximal expression of TSP-1 and TGF-β1 in rat GMCs.

It has become increasingly evident that cell-secreted and extracellular matrix-bound proteins such as TSP-1 [24,25], which affects nearly all phases of repair, promotes proliferation. Moreover, it has been reported that the function of TGF-β1 can promote the accumulation of ECM [26]. However, the relationship between sublytic C5b-9 and TSP-1, TGF-β1 has not been defined.

TSP-1 is a multi-domain protein that is synthesized by many cell types and is exported to the extracellular matrix [27–29]. TGF-β1 is considered the major cytokine that causes tissue fibrosis in many inflammatory diseases [30]. TGF-β1 is secreted by many cells as a latent procytokine complex that requires extracellular activation before it can interact with its receptors. Excessive matrix protein secretion by rat GMCs has been associated with autocrine overexpression of TGF-β1. TGF-β1 is secreted as a inactive complex. The complexes require cleavage to an active form, which is then able to bind to the TGF-β1 receptors on the cell surface and exert a biological effect. Many studies indicate that TSP-1 is an important endogenous activator of TGF-β1 in inflammatory kidney disease [30,31].

The major finding in this study was that TSP-1 is an important endogenous activator of TGF-β1 in anti-Thy1 nephritis. In our experiment comparing the degree of the TSP-1-blocking peptide (GGWSHW) inhibited the expression of TGF-β1. The data presented here suggest that TSP-1 was the major activator of TGF-β1. Nevertheless, participation of other activators of TGF-β1 or direct secretion of the active cytokine by glomerular cells cannot be excluded completely. This study provides evidence that the activation of latent TGF-β1 production in rat GMCs was, in part, dependent on TSP-1; because the expression of TSP-1 in rat GMCs was up-regulated using sublytic C5b-9 stimulation for 18 h, the production of TGF-β1 synthesis was related to TSP-1. Moreover, the production of TSP-1 along with TGF-β1 synthesis in the rat GMCs were at the same time, which is consistent with previous reports [9].

Previous studies have demonstrated that sublytic C5b-9 assembly induces transactivation of receptor tyrosine kinases and an increase in the PI3-k/Akt signal pathway [20]. Sublytic C5b-9 is known to induce a variety of responses via the PI3-k/Akt pathway. For example, activation of PI3-k/Akt by sublytic C5b-9 protects oligodendrocytes from death. PI3-k is a heterodimeric cytoplasmic enzyme that associates physically with tyrosine-phosphorylated membrane-bound cellular proteins via the SH2 domain of it (85 kD regulatory subunit). Akt is one of the PI3-k effectors that plays an important role in mediating the transformation and anti-apoptotic effects of PI3-k [32,33]. Akt itself is a serine/threonine kinase that phosphorylates and regulates the activities of the cell cycle regulatory proteins glycogen synthase kinase 3 and cyclin D [34–36]. These studies led to the hypothesis that translocation and membrane localization of PI-3-k is necessary for its activation in vivo. C5b-9 can activate heterotrimeric G proteins of the Gi–Go subfamily [37] and transcription factors, including c-jun, jun-D and c-fos with increased AP-1 binding activity as well as increased JNK activity [38,39]. Our data show that sublytic C5b-9 could activate the PI3-k/Akt signal transduction pathway, and the expression of TSP-1 could be decreased by using LY294002 to inhibit PI3-k activity. Thus, activated PI3-k/Akt is an essential enzymatic pathway for sublytic C5b-9-induced production of TSP-1 in rat GMCs.

The role of PI3-k/Akt signal transduction in sublytic C5b-9-induced TGF-β1 activation is more complex than being required simply for the up-regulation of TSP-1. Incubation of rat GMCs with the PI3-k inhibitor prevented a marked increase in the total amount of TGF-β1 in response to sublytic C5b-9 stimulation. Therefore, sublytic C5b-9-induced PI3-k/Akt signalling is necessary for the production of TGF-β1. Our experiments indicate that sublytic C5b-9 induced the expression of TGF-β1 via TSP-1 in rat GMCs. Sublytic C5b-9 stimulation can increase TSP-1 mRNA and protein through the PI3-k/Akt signalling pathway.

Acknowledgments

We thank Professors Xiao Han and Yuqing Wu for their helpful collaboration in the reviewing the manuscript. We also thank Jun Guo for technical assistance. The study was supported in part by the National Natural Science Foundation of China (No.30471615) and the Provincial Natural Science Foundation (No.03KJA310074).

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