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Many forms of human glomerulonephritis share the common features of mesangial proliferation and the deposition of extracellular matrix. It is thought that mesangial cells make a significant contribution to the accumulation of extracellular matrix. The factors involved in regulating matrix accumulation are not clearly defined but cytokines particularly TGF-β1 are thought to be involved. One of the matrix components shown to accumulate in such diseases is fibronectin. Fibronectin exists in two forms; 1. a soluble dimer which circulates in the blood stream, and 2. as an insoluble multimer which is incorporated into extracellular matrix as linked dimers. The incorporation of fibronectin into extracellular matrix is mediated by the fibronectin receptors. There are three known receptors for fibronectin, VLA3, VLA4 and VLA5. The differences between the two types of fibronectin arise by alternative splicing of the fibronectin pre-mRNA. Three regions of alternative splicing are known, two additional cell binding sites are found within the IIICS region of alternative splicing, therefore a possible function of alternative splicing is to alter composition of the extracellular matrix. The expression of the fibronectin gene can be altered by cytokines, particularly transforming-growth factor-Beta-1. The effect of TGF-β1 and PDGF-BB upon fibronectin synthesis in human mesangial cells was determined. Fibronectin gene expression and pre-mRNA splicing as well as the synthesis of fibronectin protein were investigated. The localisation of fibronectin synthesised in response to cytokine stimulation was also determined. In order to test the interaction of these two cytokines in human mesangial cells neutralising antibodies were used to inactivate TGF-β when cells were stimulated with PDGF-BB or PDGF was inactivated upon TGF-β1 stimulation. The results show that TGF-β1 stimulation is independent of PDGF, but that PDGF-BB acts, at least partially, by inducing TGF-β. The results reported in this thesis indicate a central role for TGF-β1 in the development of mesangial sclerosis resulting from excess matrix synthesis and deposition. Furthermore they suggest that clinical regimens aimed at preventing the progression of glomerulonephritis should be targeted at blocking the action of this cytokine. |
