The aim of this study was to determine whether aminoguanidine (AG), an inhibitor of advanced glycation, prevents expression of the profibrotic cytokine, connective tissue growth factor (CTGF), as well as accumulation of the previously reported CTGF-dependent matrix protein, fibronectin, in a model of experimental diabetic nephropathy. Diabetic animals were randomly allocated into groups receiving 32 wk of AG or vehicle. Diabetic rats showed increases in CTGF mRNA and protein expression as well as in advanced glycation end-product (AGE) and fibronectin immunostaining, compared with nondiabetic rats. In the diabetic kidney, the increase in CTGF gene and protein expression as well as expression of the extracellular matrix protein fibronectin were prevented by AG. To further explore the relationship between AGEs and mesangial CTGF and fibronectin production, cultured human mesangial cells were exposed in vitro to soluble AGE-BSA and carboxymethyl lysine-BSA, and this led to induction of both CTGF and fibronectin. On the basis of our in vitro findings in mesangial cells linking AGEs to CTGF expression, the known prosclerotic effects of CTGF, and the ability of AG to attenuate mesangial expansion, it is postulated that the antifibrotic effects of AG in this animal model may be partially mediated by CTGF.

The accumulation of extracellular matrix (ECM) within the kidney is an ultrastructural hallmark of diabetic nephropathy and is directly linked to a decline in renal function [1]. This increase of ECM can result from either increased synthesis and decreased degradative activity or both. Several studies have shown that high glucose concentration increases synthesis of ECM proteins in both mesangial and tubular epithelial cells [2]. However, more recently it has also Diabetologia (2002) Abstract Aims/hypothesis. Extracellular matrix accumulation is thought to be involved in the pathogenesis of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting, have not been fully explored. Furthermore, the effect of renoprotective therapies on matrix accumulation through these pathways has not been examined. We investigated the degradative pathway of type IV collagen and the effects of ACE inhibition in experimental diabetic nephropathy. Methods. Diabetes was induced in 16 rats by administrating streptozocin; 8 of the diabetic rats were allocated at random to receive the ACE inhibitor perindopril (2 mg/l) in their drinking water and 8 age and weight matched rats served as controls. Gene expression of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) was measured by RT-PCR and type IV collagen content by immunohistochemistry. MMP activities were determined by degradation of a radiolabelled substrate and by zymography.Results. Six months of diabetes was associated with a decrease in mRNA and enzymatic activity of MMP-9 (21 % and 51 % respectively, p < 0.05 vs control) and a 51 % increase in TIMP-1 mRNA (p < 0.05 vs control). By contrast, MMP-2 mRNA was increased but its activity decreased (43 % and 43 % respectively, p < 0.05 vs control). Total degradative capacity of kidney tissue from diabetic rats was also lower (Control: 48 7 %, Diabetic: 33 6 %, p < 0.05). Activation of latent MMPs with amino-phenylmercuric acetate increased matrix degradation by two-fold. However the relative decrease associated with experimental diabetes still remained. All diabetes-associated changes in MMP and TIMP mRNA and activities were attenuated by perindopril treatment in association with reduced type IV collagen accumulation. Conclusions/interpretation. These results indicate that the impairment of matrix degradation contributes to matrix accumulation in diabetic nephropathy and that the beneficial effects of ACE inhibition could in part be mediated by modulation of changes in matrix degradative pathways. [Diabetologia (2002) 45: 268±275]

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