C3a receptor activation promotes uric acid or LPS-induced CCL2 production in proximal tubular epithelial cells

Background To identify the role of the interaction of complement fragment 3a (C3a) and its receptor C3aR in uric acid (UA) or lipopolysaccharide (LPS)-induced CCL2 expression in human renal proximal tubular epithelial cell line HK-2. Methods HK-2 cells were cultured in vitro and treated with UA or LPS to induce the production of CCL2. To determine the role of C3a-C3aR interaction in CCL2 production, HK-2 cells exposed to UA or LPS were pretreated with the small molecule sb290157 (1μmol/l) or recombinant C3a protein (100nmol/l) to block or amplify C3a-C3aR interaction. The expression of CCL2, C3 and C3aR were detected by real-time quantitative PCR (Q-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blotting. Results 12 hours of UA (150μmol/L) stimulation or 8 hours of LPS (5μg/ml) treatment significantly induced CCL2 and C3 mRNA transcription in HK-2 cells. The expression of CCL2 induced by UA or LPS could be abrogated by C3aR blockade. C3a stimulation alone has little effect on inducing CCL2 expression in HK-2 cells. However, when it stimulated the HK-2 cells together with UA or LPS, it could remarkably potentiate UA or LPS-induced CCL2 expression.

It is related to the activation of Nuclear Factor-κB (NF-κB) in the renal tubular epithelial cells [3,4] . Complement system, which includes over 30 kinds of soluble and membrane contained proteins, is an important part of the innate immune system [5,6] .
Once the complement system is activated, Membrane Attack Complex (MAC) is produced and the system will play a role in immune regulation. Similarly, the system will also produce two important proinflammatory factors C3a and C5a. When some cells such as skin cutin cells or umbilical vein endothelial cells were induced to highly express the C3aR, exogenous C3a intervention will significantly induce CCL2 expression [7,8] . Previous studies provide evidence that UA and LPS could induce the expression of CCL2 in renal tubular epithelial cells and also could activate the complement system [9][10][11][12] . In the current study UA and LPS were used as an inducer to upregulate the expression of CCL2 in HK-2 cells. C3aR was blocked or activated to observe how it affected the expression of CCL2, after being induced by UA or LPS. Expert was adopted to calculate sample concentration. Sample concentration multiplied by the dilution multiple was the final concentration.

Western Blotting
The proximal tubular epithelial cells were lysed with RIPA lysis buffer and the protein concentration was measured by BCA protein assay kit. The proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Primary and secondary antibodies were added for Western blotting analysis. GAPDH was used as an internal control. The proteins were visualized by enhanced chemiluminescencewith FluorChem E imaging system. The ratios of interest proteins and internal control were analysed by ImagJ.

Statistical analysis
The data were presented as mean with SEM. All data were analyzed using SPSS version 11.5. Independent sample T test was used to evaluate differences between each experimental group and control group. The differences among every experimental group were analyzed by one-way analysis of variance. Q-PCR showed that 150μmol/L UA significantly upregulated C3, C3aR, CCL2mRNA transcription of HK-2 cells in 12 hours. Pretreatment of 1μmol/L C3aR blocker sb290157 significantly suppressed CCL2mRNA transcription induced by UA (Figure 1).

C3aR blocker sb290157 suppressed CCL2 expression of HK-2 cells in concentratedependent manner.
CCL2 is one kind of secreted protein which can be detected from HK-2 cells supernatant after intervention. ELISA and Western Blotting showed that CCL2 protein significantly increased in HK-2 cells supernatant after 12 hours of 150μmol/L UA intervention.
sb290157 significantly suppressed the expression of CCL2 protein induced by UA in concentrate-dependent manner (Figure 2, Figure 6).

Blocking C3a-C3aR interaction inhibited CCL2mRNA transcription induced by LPS in HK-2 cells.
LPS, another strong inducer of CCL2, was also used to interfer with the cells. C3 and CCL2 transcription significantly increased in HK-2 cells after 8 hours of intervention with 5μg/ml LPS. Pre-treatment of 1μmol/L sb290157 significantly suppressed CCL2mRNA transcription and upregulated C3aRmRNA transcription induced by LPS. However, it had no effect on the C3mRNA transcription ( Figure 3).

C3aR activation promoted the CCL2mRNA transcription, induced by UA or LPS
In order to verify how C3aR activation affect CCL2 expression in HK-2 cells, the current study used 100nmol/L C3a to pre-treat the cells in different intervention, then CCL2 transcription and protein expression were detected by Q-PCR and ELISA. C3a intervention for 8 hours slightly upregulated CCL2mRNA transcription. Whereas C3a together with 150μmol/L UA or 5μg/ml LPS intervention for the same time significantly upregulated CCL2mRNA transcription (Figure 4).

C3aR activation promoted CCL2 protein secretion induced by UA or LPS
ELISA and Western Blotting showed CCL2 protein significantly increased in HK-2 cells supernatant after UA or LPS intervention. C3a further up-reguated CCL2 protein expression induced by UA or LPS ( Figure 5, Figure 7).

Discussion
Mononuclear macrophages infiltration in kidney have been regarded to be related closely to renal interstitial fibrosis and chronic renal failure in a lot of renal diseases [13][14][15] . The expression of CCL2 in kidney has an obvious correlation with the infiltration of macrophages. The expression of CCL2 in urine has a correlation with the activity of kidney diseases and it can predict early renal diseases [16][17][18][19] . CCL2 is a member of CC chemotactic factor family. It is lowly expressed in normal human proximal convoluted tubule epithelial cells. It is highly expressed when the cells are stimulated by certain pathological factor such as urine protein or inflammatory mediator [20] . CCL2 is a chemoattractant for monocytes such as mononuclear macrophages, lymphocytes, eosinophilic granulocytes and basophilic granulocytes. It promotes leukocyte infiltration by adjusting the expression of integrin and matrix-degrading enzymes on the surface of macrophages [21,22] . As mentioned in the prior studies, by inhibiting the expression of CCL2 or its receptor CCR2 would significantly improve inflammatory cell infiltration in renal interstitial, tubular atrophy and renal interstitial fibrosis in UUO model mice [23,24] . DNA vaccination with naked DNA encoding CCL2 by inducing autoantibodies against CCL2 could alleviate the progress of kidney damage in adriamycin nephropathy rats [25] .
The activation of complement system had been proven to be involved in the tubulointerstitial injury in chronic kidney disease (CKD). When glomerular filtration membrane was injured, complement in the blood would go through the membrane. Then the complement would be presented in the renal tubules. Brush border of lumen surface of the renal tubular epithelial cells had the activity of endogenous C3 invertase, which could activate the complement deposited in the renal tubules [26] . Literature provide evidence that C3a could aggravate tubulointerstitial injury by inducing generation of TGF-β1 and collagen I and by inducing epithelial-mesenchymal transition of the renal tubular epithelial cells [27] . C3aR is a protein with 55kDa, which belongs to the G-Protein Coupled Receptor (GPCR) family. In the human kidney C3aR is mainly expressed in the epithelium of renal tubule and renal capsule [28] . C3a-C3aR was likely to promote renal fibrosis in diabetic nephropathy model by TGF-β/Smad3 signal path [29] . Previous study has shown that deficiency of C3aR and C5aR mitigates Ang II-induced regulatory T cell in kidneys.
Numbers of both CD4 + T cells and CD8 + T cells in the kidneys were substantially increased by Ang II infusion, and the increase of CD4 + T cells was notably reduced in DKO (C3aR and C5aR double knockout) mice. T cells infiltrate the kidney and release inflammatory cytokines to alter renal function and promote end-kidney damage. [30] In the Unilateral Ureteral Obstruction (UUO) model knocking out C3 gene could significantly inhibit the expression of renin and angiotensin II, while the over expression of angiotensin II had been proven to be related to the activation of NF-κB and the mass induction of CCL2 [31] .
Thurman et al found that knocking out Factor B, the important ingredient of complement alternative pathway, would significantly inhibit the transcription of CCL2 mRNA in mice kidney damaged by ischemia reperfusion. This means that the expression of CCL2 may be related to the activation of complement alternative pathway [32] .
UA has a close relationship with CKD. The epidemiological studies have proven serum UA level is a key factor to predict the renal disease progression [33] . LPS had also been verified to be a strong inducer for CCL2 expression in renal tubular epithelial cells [12,34] .
In this current study it was found that UA and LPS both upregulate the expression of CCL2

Conclusion
The activation of C3aR provides an important co-stimulating signal for CCL2 production in HK-2 cells and blocking the interaction of C3a-C3aR will significantly inhibit UA or LPS induced CCL2 production.        Western Blotting showed sb290157 significantly suppressed the expression of CCL2 protein induced by UA in concentrate-dependent manner.