Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice

Abstract

Complement activation has a deep pathogenic influence in immunoglobulin (Ig)A nephropathy (IgAN). C3a and C5a, small cleavage fragments generated by complement activation, are key mediators of inflammation. The fragments exert broad proinflammatory effects by binding to specific receptors (C3aR and C5aR, respectively). However, no studies thus far have investigated the effects of C3a, C5a and their receptors on IgAN. We observed that C3aR and C5aR antagonists repressed IgA-induced cell proliferation and interleukin (IL)-6 and monocyte chemotactic protein 1 (MCP-1) production in cultured human mesangial cells (HMCs). Furthermore, an IgAN mouse model induced by Sendai virus infection was employed to investigate the effects of C3aR and C5aR on IgAN in vivo for the first time. Wild-type (WT) and several knock-out mouse strains (C3aR^-/- or C5aR^-/- ) were immunized intranasally with increasing doses of inactivated virus for 14 weeks and were subjected to two intravenous viral challenges during the time-period indicated. In the Sendai virus-induced IgAN model, C3aR/C5aR-deficient mice had significantly reduced proteinuria, lower renal IgA and C3 deposition, less histological damage and reduced mesangial proliferation compared with WT mice. Both C3aR deficiency and C5aR deficiency, especially C3aR deficiency, inhibited renal tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β, IL-1β, IL-6 and MCP-1 expression significantly. However, C3aR/C5aR-deficient and WT mice with IgAN did not differ with respect to their blood urea nitrogen (BUN) and serum creatinine levels. Our findings provide further support for the idea that C3aR and C5aR are crucially important in IgAN, and suggest that pharmaceutically targeting C3aR/C5aR may hold promise for the treatment of IgAN.

Keywords: C3a receptor; C5a receptor; IgA nephropathy; complement.

Figure 1

Cell proliferation and cytokine mRNA and protein expression in cultured human mesangial cells (HMCs). The HMCs were treated with blank medium or medium containing 100 μg/ml immunoglobulin (Ig)A, 100 μg/ml IgA + 100 μM C3aR antagonist (C3aRA) or 100 μg/ml IgA + 100 μM C5aR antagonist (C5aRA) for 48 h. The data are expressed as the mean ± standard deviation (s.d.). (a) HMC proliferation was measured using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (n = 6 for each group). (b) Interleukin (IL)‐6 (left row) and monocyte chemotactic protein 1 (MCP‐1) (right row) gene expression levels relative to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were determined by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). (c) Western blot analysis of IL‐6, MCP‐1 and GAPDH protein expression. (d) Quantitative analysis for Western blot of IL‐6 and MCP‐1 protein expression. *P < 0·05, **P < 0·01, ***P < 0·001 versus negative control.

Figure 2

Wild‐type (WT), C3aR deficient (C3aR^–/–) and C5aR^–/– mice were immunized with inactivated Sendai virus to induce immunoglobulin (Ig)A nephropathy (IgAN). After 14 weeks of immunization, IgA and C3 deposition in the mesangium and the associated histological changes were analysed. (a,b) Glomerular IgA (upper row) and C3 (low row) deposition was measured by immunofluorescence staining of kidney sections from WT, C3aR^–/– and C5aR^–/– IgAN mice and negative controls. (c) Mesangial matrix expansion and cell proliferation were demonstrated by periodic acid‐Schiff (PAS) staining of renal tissue in the following four groups of mice: negative controls and WT, C3aR^–/– and C5aR^–/– IgAN mice. Magnification ×400. Scale bars represent 100 μM.

Figure 3

Representative images of immunohistochemical staining for interleukin (IL)‐6 and monocyte chemotactic protein 1 (MCP‐1) in the kidney sections of wild‐type (WT), C3aR deficient (C3aR^–/–) and C5aR^–/– immunoglobulin (Ig)A nephropathy (IgAN) mice and negative controls. C3aR and C5aR deficiency reduced renal IL‐6 and MCP‐1 expression compared to WT mice. Scale bars represent 100 μM.

Figure 4

Cytokine and chemokine gene expression in the renal tissues of the following four groups of mice: negative controls and wild‐type (WT), C3aR deficient (C3aR^–/–) and C5aR^–/– immunoglobulin (Ig)A nephropathy (IgAN) mice (n = 7 for each group). Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) was used to quantify the mRNA expression levels of (a) tumour necrosis factor (TNF)‐α, (b) transforming growth factor (TGF)‐β, (c) interleukin (IL)‐1β, (d) interleukin (IL)‐6 and (e) monocyte chemotactic protein 1 (MCP‐1) relative to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Data are expressed as the mean ± standard deviation. *P < 0·05, **P < 0·01, ***P < 0·001 versus negative control.