Sublytic C5b-9 Induces CCL3/4 Production and Macrophage Accumulation in Thy-1N Rats via PKC-α/p65/IRF-8 Axis

Abstract

Mesangioproliferative glomerulonephritis (MsPGN) is a common human kidney disease. Rat Thy-1 nephritis (Thy-1N) is an animal model widely used for the study of MsPGN. Thy-1N is not only sublytic C5b-9-dependent, but also related to pro-inflammatory cytokine production and macrophage (M) accumulation in rat renal tissues. In this study, we found that the expression or phosphorylation of chemokine CCL3/4, CD68 (M marker), IRF-8, PKC- and NF-B-p65 (p65) were all up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the glomerular mesangial cells (GMCs) upon sublytic C5b-9 stimulation (in vitro). Further experiments in vitro revealed that the phosphorylated PKC- (p-PKC-) could promote p65 phosphorylation, and then p-p65 enhanced IRF-8 expression through binding to IRF-8 promotor (-591 ~ -582 nt and -299 ~ -290 nt). Additionally, up-regulation or silencing of IRF-8 gene promoted or reduced CCL3/4 production, and then regulated M chemotaxis. The underlying mechanism involved in IRF-8 binding to CCL3 promoter (-249 ~ -236 nt), which resulted in CCL3 gene transcription. The experiments in vivo showed that knockdown of renal PKC-, p65, IRF-8 and CCL3/4 genes could inhibit CCL3/4 production, M accumulation, GMC proliferation and proteinuria of Thy-1N rats. Furthermore, p-PKC-, p-p65, IRF-8, CCL3/4 expression and M accumulation were also increased in the renal tissues of MsPGN patients. Collectively, these findings indicate that sublytic C5b-9 induces CCL3/4 production and M accumulation via PKC-/p65/IRF-8 axis, and finally aggravates the pathological changes of MsPGN.