#5526 COMPLEMENT C3 EXERTS DIRECT PRO-FIBROTIC EFFECTS ON KIDNEY TUBULES

Abstract

Abstract Background and Aims Renal complement expression has been observed in both experimental and human kidney diseases. We have previously reported massive tubular C3 expression in fibrotic TGF-beta1 transgenic mice (Nephrol Dial Transpl 2015, 30; S3:FP301). Still, little is known whether this intra-renal complement is only an inflammatory marker of renal fibrosis or also exerts direct pro-fibrotic effects in the pathogenesis. In the present study we aimed to investigate the effect of C3a receptor activation in murine primary tubular epithelial cells. Method Primary tubular epithelial cells (TEC) were isolated from kidneys of four weeks-old male C57Bl6 mouse. The cells were grown in DMEM/12 medium supplemented with 2% FBS and recombinant 5 ng/ml EGF, and characterized for epithelial and mesenchymal markers. Verified TEC between P4 and P10 passages were used for the experiments. TEC cells were seeded on 6-well plates and treated for 24 hours with PBS (CTL, n=3), 100 nM C3a receptor agonist peptide (C3aR, n=3) or 10 ng/ml recombinant TGF-beta1 (TGFb, n=3). Then, mRNA and protein expressions were assessed and evaluated using Kruskal-Wallis test (p<0.05). Results TGF-beta induced a 2-fold Col1a1 and 1,6-fold Tgfb1 mRNA expression, accompanied by 4-fold Egr2 but did not affect Ccl2 or C3 mRNA expression. In contrast, C3aR group depicted 1,6-fold C3 and 1,8-fold Ccl2 mRNA expression, an only 1,6-fold Egr2 but a 2-fold Col1a1 and 1,5-fold Tgfb1 mRNA expression. Despite the similar Tgfb1 mRNA overexpression in TGFb and C3aR groups, marked TGFB1 protein overexpression (15-fold) was only observed in TGF-beta treated cells but not in the C3aR group. Conclusion Our study data indicate that renal complement C3 production can exert autocrine / paracrine direct pro-fibrotic effects on tubular epithelial cells unrelated to TGF-beta protein levels. Therefore, local C3 overexpression might play a significant pathogenetic role in kidney fibrosis.