Activation mechanism of NF-kB and development of therapeutic strategy through its regulation.

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NF-kB plays an important role in cellular response to injury, such as mesangial cell (MC) proliferation or infiltration of inflammatory cells, in glomerulonephritis through activation of a number of cytokines, growth factors and adhesion molecules. We attempted to modulate NF-kB activity by using several reagents, such as glucocorticoid, anti-oxidant (PDTC). We also verified feasibility of oligonucleotide (ODN) which contains the consensus NF-kB binding site (decoy DNA) in an attempt to inhibit NF-kB activation. We evaluated inhibitory effect of these reagents on mesangial cell proliferation in vitro and therapeutic effects in the rat anti-Thyl.l nephritis model as well. First, the effect of these reagents on MC proliferation was assessed in vitro. The nuclear extracts were prepared from the cells treated with these reagents. Electrophoretic mobility-shift assay (EMSA) was performed to confirm the effect on NF-kB DNA binding activity. Secondly, these three reagents were used in vivo. T … More he rat anti-Thyl.l model was induced by injection of monoclonal anti-Thyl.l antibody (oX-7). Renal biopsy was performed at day 8.The total cell number and the number of proliferating nuclear cell antigen (PCNA)-positive cells per glomeralar cross section were determined. Glomerular mRNA was extracted and the mRNA expressions of IL-1, TNF-a, MCP-1 and ICAM-1 of which gene expression was regulated by NF-kB, were measured by 'reverse transcriptase polymerase chain reaction (RT-PCR) method. NF-kB decoy inhibited the MC growth in a dose dependent manner in vitro. Ten mM of decoy inhibited the MC growth by 75%. EMSA revealed that this inhibitory effect was mediated by decreased NF-kB binding activity. Glucocorticoid, PDTC and NF-kB decoy suppressed MC proliferation in the Thy1.1 glomerulonephritis model. The number of glomeralar cell decreased by 25%. Decreased mRNA expressions of IL-1, TNF-a, MCP-1 and ICAM-1 were recognized in the experimental group. Thus, inhibition of mesangial cell proliferation was possibly resulted from suppressed expression of these cytokines at least in part.These results indicate that the reagents which modulate NF-kB function would be a novel therapeutic agent for inflammatory glomeralar diseases. Less