Rajakaruna, Gayathri Kumari; (2019) Investigating the role of connective tissue growth factor as a critical mediator of cryoglobulinaemic glomerulonephritis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The research was based on a serendipitous finding that over-expression of connective tissue growth factor (CTGF) using a particular promoter in mice resulted in cryoglobulinaemic glomerulonephritis. Cryoglobulins are immunoglobulins (Ig) that reversibly precipitate at temperatures less than 37 0 C. To test the hypothesis that CTGF is a critical mediator of cryoglobulinaemia in vivo and in vitro investigations were carried out using a Thymic Stromal Lymphopoetin (TSLP) transgenic (Tg) animals, which develop typical type 3 cryoglobulins and a glomerulonephritis similar to human disease. I found that CTGF was similarly overexpressed in these mice suggesting that it may be a key molecule in cryoglobulin formation, and so I tried to suppress disease using CTGF anti-sense oligonucleotides (ASO). I carried out treatment of TSLP Tg animals with CTGF ASO and control ASO and found significantly lower histological injury and a trend towards reduced proteinuria, clinical lesions and lower serum creatinine in the CTGF ASO-treated animals compared to untreated animals. These findings however, were similar to those with control ASO that also supressed CTGF levels, making it difficult to be certain about the specificity of the effect being mediated through CTGF. Previous data suggested the importance of glycosylation in cryo-precipitation. Serum cryoglobulins and immunoglobulins were isolated from the TSLP Tg animals and compared to immunoglobulins from Wild Type (WT) littermates. I could not confirm a difference in glycosylation patterns between these two animals, suggesting that at least in this model there is no major impact of altered glycosylation in mediating the ability of immunoglobulins to form cryoprecipitates. Since TSLP Tg mice had elevated levels of CTGF, I performed a series of in vitro experiments on human peripheral blood mononuclear cells, murine lymphoid cells and mesangial cells to investigate the relationship between TSLP and CTGF. There was no direct cell proliferation response of any of these cells to CTGF, and there was no increased production of TSLP. However, TSLP did induce a significant dose dependent cell proliferation response, but did not lead to CTGF production by these cells. This study suggested that CTGF may be a key molecule in development of cryoglobulinaemia, and that its suppression, using ASO, showed some promising results with regards disease attenuation in TSLP transgenic animals, however, further investigation is required to identify the link between CTGF and TSLP.

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