Oxidized LDL immune complexes stimulate collagen IV production in mesangial cells via Fc gamma receptors I and III

Abstract

Diabetic nephropathy is characterized by progressive mesangial expansion. Although we have reported that circulating oxidized LDL-containing immune complexes (oxLDL-IC) are associated with abnormal levels of albuminuria, the underlying mechanisms have not been investigated. In this study, we have studied the effect of oxLDL-IC on collagen IV expression by mesangial cells. We found that oxLDL-IC markedly stimulated collagen IV expression in a concentration- and time-dependent fashion while oxLDL only had moderate effect. We also found that oxLDL-IC stimulated collagen IV expression by engaging Fc gamma receptor (FcγR) I and III, but not FcγRII, and that p38 MAPK, JNK and PKC pathways were involved in collagen IV expression. Furthermore, we found that oxLDL-IC stimulated FcγRI expression, suggesting a positive feedback mechanism involved in oxLDL-IC-stimulated collagen IV expression. Taken together, this study showed that oxLDL-IC stimulated collagen IV in mesangial cells via FcγRI and FcγRIII, and the expression of FcγRI was increased by oxLDL-IC.

Figure 1

The effect of oxLDL-ICs, anti-oxLDL antibodies and oxLDL on collagen IV production by mesangial cells. Human mesangial cells were treated with 50 µg/ml of oxLDL-ICs, 30 µg/ml of anti-oxLDL antibody or 20 µg/ml of oxLDL for 24 h. After the treatment, cellular proteins were extracted and subjected to immunoblotting to detect collagen IV and β-actin. The blot is representative of at least 3 separate experiments showing similar results.

Figure 2

Concentration- and time-dependent stimulation of collagen IV production by oxLDL-ICs. Human mesangial cells were treated with different concentrations of oxLDL-ICs (A) for 24 h or with 50 µg/ml of oxLDL-ICs for different time periods (B). After treatment, cellular proteins were extracted and subjected to immunoblotting to detect collagen IV. The image from the time course study was analyzed using densitometry after normalization of collagen IV to β-actin (C). The blot is representative of at least 3 separate experiments showing the similar results.

Figure 3

The effects of monomeric IgG1 and blocking antibodies to FcγRII (CD32), III (CD16) or CD36 on collagen IV production. Human mesangial cells were treated with 50 µg/ml of oxLDL-ICs in the absence or presence of 50 µg/ml of IgG1, 10 µg/ml of CD32, CD36, or CD16 blocking antibodies for 24 h. After the treatment, cellular proteins were extracted and subjected to immunoblotting to detect collagen IV. The blot is representative of at least 3 separate experiments showing similar results.

Figure 4

Time course of FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and CD36 surface expression in mesangial cells treated with IFNγ. Human mesangial cells were treated with 100 ng/ml of IFNγ for different time periods (2–72 h) and the surface expression of FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and CD36 was determined at each time point using flow cytometry as described in Methods. A. Plots from flow cytometry analysis for FcγRI, FcγRII and FcγRIII surface expression recorded before and after IFNγ treatments. B. Quantification of the data from flow cytometry analysis. The data presented is representative of at least 3 separate experiments showing similar results.

Figure 5

Stimulation of FcγRI gene expression in mesangial cells by oxLDL-ICs. Human mesangial cells were treated with 50 µg/ml of oxLDL-ICs for different time periods (1–12 h). After treatment, FcγRI mRNA was quantified using real-time PCR and normalized to GAPDH mRNA. The data presented is representative of at least 3 separate experiments showing similar results.

Figure 6

The effect of signaling pathway inhibitors on oxLDL-IC-stimulated collagen IV production by mesangial cells. A and B: Human mesangial cells were treated with 50 µg/ml of oxLDL-ICs in absence or presence of different concentrations of SB203580 (SB), SP600125 (SP), calphostin C (Calph), staurosporine (Staur), AG490 (AG), PD98059 (PD) or U0126 for 24h. After the treatment, cellular proteins were extracted and subjected to immunoblotting to detect collagen IV (A), and collagen IV was quantified using densitometric scanning (B). C: Mesangial cells were treated with different concentrations of oxLDL-ICs for 5 min and cellular protein was extracted after the treatment. The phosphorylated ERK, JNK, p38 MAPK and PKCβ2 were detected using specific anti-ERK, JNK, p38 MAPK and PKCβ2 antibodies. For control, GAPDH was detected using anti-GAPDH antibody. The blot was representative of at least 3 separate experiments showing similar results.