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Sublytic C5b-9 Induces CCL3/4 Production and Macrophage Accumulation in Thy-1N Rats via PKC-α/p65/IRF-8 Axis
Wenbo Wang1, Baomei Qian1, Chenhui Zhao2
1Department of Immunology, and Key Laboratory of Immunological Environment and Disease, Nanjing Medical University, Nanjing, Jiangsu 211166, China.
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 (Mφ) accumulation in rat renal tissues. In this study, we found that the expression or phosphorylation of chemokine CCL3/4, CD68 (Mφ marker), IRF-8, PKC-α and NF-κB-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-α (p-PKC-α) 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 Mφ 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-α, p65, IRF-8 and CCL3/4 genes could inhibit CCL3/4 production, Mφ accumulation, GMC proliferation and proteinuria of Thy-1N rats. Furthermore, p-PKC-α, p-p65, IRF-8, CCL3/4 expression and Mφ accumulation were also increased in the renal tissues of MsPGN patients. Collectively, these findings indicate that sublytic C5b-9 induces CCL3/4 production and Mφ accumulation via PKC-α/p65/IRF-8 axis, and finally aggravates the pathological changes of MsPGN.
Insights
Sublytic C5b-9 triggers kidney inflammation and macrophage accumulation in mesangioproliferative glomerulonephritis (MsPGN) by activating the PKC-α/p65/IRF-8 pathway, leading to increased CCL3/4 production and disease progression.
Area of Science:
- Nephrology
- Immunology
- Molecular Biology
Background:
- Mesangioproliferative glomerulonephritis (MsPGN) is a common kidney disease.
- Rat Thy-1 nephritis (Thy-1N) is a key animal model for studying MsPGN, involving complement component C5b-9, inflammation, and macrophage infiltration.
Purpose of the Study:
- To elucidate the molecular mechanisms by which sublytic C5b-9 induces inflammation and macrophage accumulation in MsPGN.
- To investigate the role of the PKC-α/p65/IRF-8 signaling axis in regulating chemokine production and macrophage recruitment in MsPGN.
Main Methods:
- Utilized both *in vivo* (Thy-1N rat model) and *in vitro* (glomerular mesangial cells) approaches.
- Assessed the expression and phosphorylation of key proteins (PKC-α, NF-κB-p65, IRF-8) and chemokines (CCL3/4).
- Employed gene knockdown techniques to determine the functional significance of identified molecules in disease pathogenesis.
Main Results:
- Sublytic C5b-9 stimulation upregulated PKC-α, p65, IRF-8, and CCL3/4 expression in renal tissues and glomerular mesangial cells.
- PKC-α activation promoted p65 phosphorylation, which in turn enhanced IRF-8 expression.
- IRF-8 directly regulated CCL3/4 transcription, driving macrophage chemotaxis and accumulation; gene knockdown ameliorated disease markers.
Conclusions:
- Sublytic C5b-9 activates the PKC-α/p65/IRF-8 signaling pathway, leading to CCL3/4 production and macrophage accumulation in MsPGN.
- This pathway is a critical driver of MsPGN pathogenesis, contributing to glomerular mesangial cell proliferation and proteinuria.
- Targeting this axis represents a potential therapeutic strategy for managing MsPGN.
