Complement C3a and C3a Receptor Activation Mediates Podocyte Injuries in the Mechanism of Primary Membranous Nephropathy

Abstract

Background: The complement system is highly activated in primary membranous nephropathy (MN). Identifying the complement components that damage podocytes has important therapeutic implications. This study investigated the role of C3a and the C3a receptor (C3aR) in the pathogenesis of MN.

Methods: C3aR expression in kidneys and circulating levels of C3a of MN patients were examined. Human podocyte damage was assessed after exposure to MN plasma +/- C3aR blockade (SB290157, JR14a). C3aR antagonists were administered to rats with Heymann nephritis on day 0 or after proteinuria. Clinical and pathologic parameters, specific IgG and complement activation, and podocyte injuries were then assessed.

Results: In the glomeruli, C3aR staining merged well with podocin. Overexpression of C3aR correlated positively with proteinuria, serum creatinine, and no response to treatments. Human podocytes exposed to MN plasma showed increased expression of PLA2R, C3aR, and Wnt3/β-catenin, reduced expression of synaptopodin and migration function, downregulated Bcl-2, and decreased cell viability. C3aR antagonists could block these effects. In Heymann nephritis rats, C3aR blockade attenuated proteinuria, electron-dense deposition, foot process width, and glomerular basement membrane thickening in glomeruli. The increased plasma C3a levels and overexpression of C3aR were also alleviated. Specific, but not total, IgG levels decreased, with less deposition of rat IgG in glomeruli and subsequent reduction of C1q, factor B, and C5b-9.

Conclusion: C3a anaphylatoxin is a crucial effector of complement-mediated podocyte damage in MN. The C3aR antagonist may be a potentially viable treatment for this disease.

Keywords: C3a receptor; Heymann nephritis; complement; membrane nephropathy; podocyte.

Graphical abstract

Figure 1.

The expression of C3aR in the kidneys of patients with primary MN and its correlations with clinical features. The granular staining of C3aR along glomerular capillary walls was strongly shown in patients with primary MN, weaker in patients with diabetes nephropathy (DN) and FSGS, and was negative in patients with minimal change disease (MCD) and healthy controls (A, B). The staining intensity was measured by integral OD (IOD). The expression of C3aR merged well with podocin on the podocytes (red: C3aR; green: podocin) (C). In patients with primary MN, the level of C3aR expression was positively correlated with proteinuria (D) and serum creatinine (E). It was significantly lower in the patients who achieved clinical remission after treatments compared with that in the patients of no remission (F).

Figure 2.

The effects on podocytes after exposure to the plasma from MN patients and C3aR antagonists. Human podocytes were exposed to medium containing 10% MN plasma (with C3a 1966.7±259.0 ng/ml), 10% MN plasma + C3aR antagonist SB290157, 10% MN plasma + C3aR antagonist JR14a, 10% MN plasma + human C3a (2 μg/ml), 10% healthy plasma, C3a, or blank control. The expression of PLA2R (A) and C3aR (B) was significantly elevated after exposure to MN plasma, and blocked by C3aR antagonists. Synaptopodin (C) was downregulated in podocytes exposed to MN plasma and recovered by the addition of C3aR antagonists. The migration distance of podocytes (D) was shortened after exposure to MN plasma. C3aR antagonists could attenuate it. The expression of Wnt3 (E) and β-catenin (F) in podocytes was upregulated by exposure of MN plasma and blocked by C3aR antagonists. The apoptosis inhibition gene Bcl-2 (G) was downregulated and the viability of podocytes (H) was decreased in MN plasma, which was attenuated by C3aR antagonists.

Figure 3.

The treatments of C3aR antagonist reduced proteinuria of Heymann nephritis. Sprague-Dawley rats were injected with anti-Fx1A antibody serum 0.8 ml/100 g via the tail vein. The rats developed proteinuria and aggravated gradually (A). At week 7, the rats presented with severe proteinuria (B) and the kidneys (C–E) showed electron-dense deposit in subepithelial area, effusive podocyte foot process fusion, and basement membrane thickening. In the early-treatment groups, the C3aR antagonist, SB290157 (30 mg/kg intraperitoneally, n=6) or JR14a (10 mg/kg intragastrically, n=8), was administrated daily from day 0. In the late-treatment groups, both C3aR antagonists (SB290157, n=8; JR14a, n=6) were administrated from week 3 after the proteinuria was detected. Compared with the disease controls treated with normal saline, the level of proteinuria was significantly reduced in both the early-treatment and late-treatment groups of two kinds of C3aR antagonists (A, B). It was even lower than that at the time point when the treatments were initiated. The amount of electron-dense deposits, the foot process width, and the thickening of GBM were alleviated (C–E). *P<0.05, **P<0.01, ***P<0.001.

Figure 4.

The treatments of C3aR antagonist alleviated plasma level of C3a and glomerular expression of C3aR on Heymann nephritis rats. Sprague-Dawley rats were injected with anti-Fx1A antibody serum via the tail vein. The plasma C3a was significantly elevated in disease controls (A, C). After C3aR antagonist treatments, it was decreased remarkably, both in the early-treatment groups and in the late-treatment groups: SB290157 (B), JR14a (D). In the kidneys, the staining of C3aR was strong in the rats with Heymann nephritis (E), and was attenuated in both treatment groups (F, G). *P<0.05, **P<0.01, ***P<0.001.

Figure 5.

The treatments of C3aR antagonist inhibited specific IgG and complement activation on Heymann nephritis rats. Sprague-Dawley rats were injected with anti-Fx1A antibody serum via the tail vein. In the early-treatment groups, C3aR antagonists (SB290157 or JR14a) was administrated daily from day 0. In the late-treatment groups, both C3aR antagonists were administrated from week 3 after proteinuria was detected. Compared with the disease controls treated with normal saline, the plasma level of specific anti-sheep IgG was decreased in treatment groups of both C3aR antagonists, SB290157 (A) and JR14a (B), whereas the level of total IgG was comparable among the groups. In the glomeruli, the deposit of sheep IgG (C) was comparable among the disease controls and treatment groups. However, the deposit of rat IgG (D) was alleviated in both treatment groups. Along with that, the staining of C1q (E), factor B (F), and C5b-9 (G) was significantly decreased in the treatment groups. *P<0.05, **P<0.01, ***P<0.001.

Figure 6.

The rat podocyte injuries after exposure to the plasma from rats with Heymann nephritis and those with C3aR antagonists. On primary rat podocytes, the expression of C3aR (A) and β-catenin (B) was upregulated, and the expression of synaptopodin (C) and Bcl-2 (D) was downregulated, after exposure to the plasma from disease rats. These effects were attenuated in the treatment groups of both C3aR antagonists. The migration distance (E) and cell viability (F) were reduced after the podocytes were exposed to the disease plasma, which was recovered in treatment groups.