October 29, 2004
American Society of Nephrology (ASN) Renal Week 2004, St. Louis, Missouri
Abstract F-PO839
Poster: Epithelial to Mesenchymal Transition and other Mechanisms for Fibrosis

Connective Tissue Growth Factor Differentially Mediates Transforming Growth Factorbeta1 Induced Fibrogenesis and Immunomodulation. Weier Qi, Xin-Ming Chen, Stephen Twigg, Richard Gilbert, Philip Poronnik, Carol Pollock. Medicine, Kolling Institute; Medicine, Royal Prince Alfred Hospital; Medicine, St. Vincent s Hospital; Biology, University of Queensland, Australia.

Transforming growth factor-beta1 (TGF-beta1) functions as a pro-fibrogenic and immunomodulatory cytokine in the kidney. Emerging evidence suggests that CTGF is a downstream mediator of the profibrotic effects of TGF-beta1. However, the role of CTGF in the TGF-beta1 induced immunomodulation is still unknown. The present study was undertaken to determine whether CTGF is differentially involved in mediating TGF-beta1 induced fibrogenesis and immunomodulation in human kidney-2 (HK-2) cells. Fibronectin was used as a marker of fibrogenesis, and interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were measured as immunomodulatory cytokines. CTGF gene was effectively silenced using small interfering RNA (siRNA) in HK-2 cells. RT-PCR confirmed a 95% reduction in CTGF mRNA levels. Wild-type, CTGF gene silenced and wild-type transfected with non-specific siRNA cells were then exposed to 2 ng/ml TGF-beta1 for 48 hrs to assess the fibrogenic response. Results were expressed relative to control values (wild-type cells in the absence of TGF-beta1). TGF-beta1 induced a 589+/- 215% increase in fibronectin secretion in wild-type cells (P<0.05). In CTGF gene silenced cells, TGF-beta1 induced a 244+/-63% increase in fibronectin secretion, significantly less than that observed in wild-type cells treated with TGF-beta1 (P<0.05). Exposure of wild type cells to TGF-beta1 induced IL-8 and MCP-1 to 356±88% (P<0.05) and 138±9% (P<0.05) respectively compared to control values. This stimulatory response was similar in CTGF gene silenced cells. Additional studies were performed to assess the direct effect of CTGF (20, 200 and 400 ng/ml) on IL-8 and MCP-1 at different time points (24, 48 and 72 hr). No stimulation was observed in wild type cells. These data suggest that TGF-beta1 mediates both fibrosis and immunomodulatory pathways, at least in part through a CTGF-dependent mechanism in HK-2 cells. However, TGF-beta1 induced IL-8 and MCP-1 is independent of CTGF.

CTGF protein was generously supplied by FibroGen, Inc