Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli

Aims/hypothesis: We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-beta 1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. Methods: THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/-THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. Results: In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. Conclusions/interpretation: Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.