Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy

We characterized a rabbit polyclonal antibody raised against human recombin ant connective tissue growth factor (CTGF). The antibody recognised a highe r molecular mass form (approx. 56 kDa) of CTGF in mesangial cell lysates as well as the monomeric (36-38 kDa) and lower molecular mass forms (<30 kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeru li of kidneys of non-obese diabetic mice 14 days after the onset of diabete s, and this was prominent by 70 days, CTGF protein is also present in glome ruli of human patients with diabetic nephropathy. No CTGF was detected in e ither normal murine or human glomeruli. Transient transfection of a transfo rmed human mesangial cell line with a CTGF-V5 epitope fusion protein marked ly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4 mM) conditions a CTGF-antisense c onstruct reduced the elevated synthesis of these proteins in high glucose ( 30 mM) cultures. Culture of primary human mesangial cells for 14 days in hi gh glucose. or in low glucose supplemented with recombinant CTGF or transfo rming growth factor <beta>1, markedly increased CTGF mRNA levels and fibron ectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucl eotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it o nly partially reduced fibronectin mRNA levels and synthesis. A chick anti-C TGF neutralizing antibody had a similar effect on fibronectin synthesis. Th us both CTGF and CTGF-independent pathways mediate increased fibronectin sy nthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys i s likely to be a key event in the development of glomerulosclerosis by affe cting both matrix synthesis and, potentially through plasminogen activator inhibitor-1. its turnover.