Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are s till being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are expo sed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibr onectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has n ot been reported. Prominent among the latter were genes encoding cytoskelet on-associated proteins and connective tissue growth factor (CTGF), a modula tor of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-ind uced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expre ssion in vitro than high glucose, excluding hyperosmolality as the key stim ulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expre ssion of transforming growth factor beta 1 (TGF-beta 1), and addition of TG F-beta 1 to mesangial cells triggered CTGF expression. CTGF expression indu ced by high glucose was partially suppressed by anti-TGF-pl antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest th at 1) high glucose stimulates mesangial CTGF expression by TGF beta 1-depen dent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGF beta 1-driven matrix production within a diabetic milieu.