Motohide Isono

Increased Glomerular and Tubular Expression of Transforming Growth Factor-β1, Its Type II Receptor, and Activation of the Smad Signaling Pathway in the db/db Mouse

Activation of the renal transforming growth factor-beta (TGF-beta) system likely mediates the excess production of extracellular matrix in the diabetic kidney. To establish the role of the TGF-beta system in type 2 diabetic nephropathy, we examined the intrarenal localization and expression of the TGF-beta1 isoform, the TGF-beta type II receptor, and the Smad signaling pathway in the 16-week-old db/db mouse, a genetic model of type 2 diabetes that exhibits mesangial matrix expansion, glomerular basement membrane thickening, and renal insufficiency that closely resemble the human disease. Compared with its nondiabetic db/m littermate, the db/db mouse showed significantly increased TGF-beta1 mRNA expression by in situ hybridization in both glomerular and tubular compartments. Likewise, TGF-beta1 protein, by immunohistochemical staining, was increased in both renal compartments, but the fractional expression of TGF-beta1 protein was less than that of the mRNA in the glomerulus. In situ hybridization and immunohistochemical staining for the TGF-beta type II receptor revealed concordant and significant increases of both mRNA and protein in the glomerular and tubular compartments of diabetic animals. Finally, immunohistochemistry showed preferential accumulation of Smad3 in the nuclei of glomerular and tubular cells in diabetes. The complementary technique of Southwestern histochemistry using a labeled Smad-binding element demonstrated increased binding of nuclear proteins to Smad-binding element, indicating active signaling downstream of the TGF-beta stimulus. We therefore propose that the TGF-beta system is up-regulated at the ligand, receptor, and signaling levels throughout the renal cortex in this animal model of type 2 diabetes. Our findings suggest that the profibrotic effects of TGF-beta may underlie the progression to glomerulosclerosis and tubulointerstitial fibrosis that characterize diabetic nephropathy.

Smad pathway is activated in the diabetic mouse kidney and smad3 mediates TGF-β-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.

Reversibility of established diabetic glomerulopathy by anti-TGF-β antibodies in db/db mice

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.

THE KEY ROLE OF THE TRANSFORMING GROWTH FACTOR-β SYSTEM IN THE PATHOGENESIS OF DIABETIC NEPHROPATHY

Progressive renal injury in diabetes mellitus leads to majormorbidity and mortality. The manifestations of diabetic nephropathy may bea consequence of the actions of certain cytokines and growth factors. Prominentamong these is transforming growth factor-beta (TGF-β) because it promotesrenal cell hypertrophy and stimulates extracellular matrix accumulation, thetwo hallmarks of diabetic renal disease. In cell culture, high ambient glucoseincreases TGF-β m RNA and protein in proximal tubular, glomerular epithelial,and mesangial cells. Neutralizing anti-TGF-β antibodies prevent the hypertrophicand matrix stimulatory effects of high glucose in these cells. In experimentaland human diabetes mellitus, several reports describe overexpression of TGF-βin the glomeruli and tubulointerstitium. We demonstrate that short-term treatmentof diabetic mice with neutralizing monoclonal antibodies against TGF-βsignificantly reduces kidney weight and glomerular hypertrophy and attenuatesthe increase in extracellular matrix mRNAs. Long-term treatment of diabeticmice further improves the renal pathology and also ameliorates the functionalabnormalities of diabetic nephropathy. Finally, we provide evidence that therenal TGF-β system is significantly up-regulated in human diabetes. Thekidney of a diabetic patient actually elaborates TGF-β1 protein intothe circulation whereas the kidney of a non-diabetic subject extracts TGF-β1from the circulation. The data we review here strongly support the hypothesisthat elevated production or activity of the TGF-β system mediates diabeticrenal hypertrophy and extracellular matrix expansion.

Involvement of the transforming growth factor-β system in the pathogenesis of diabetic nephropathy

The manifestation of diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Prominent among them is transforming growth factor-β (TGF-β), which promotes renal cell hypertrophy and stimulates extracellular matrix accumulation, the two hallmarks of diabetic renal disease. In experimental and human diabetes mellitus, several reports describe overexpression of TGF-β in the glomeruli and tubulointerstitium. In renal cell cultures, hypertrophy and matrix production are stimulated by high glucose concentrations in the culture media. High glucose, in turn, appears to act through the TGF-β system; high glucose increases TGF-β expression, and the hypertrophic and matrix stimulatory effects of high glucose are prevented by anti-TGF-β therapy. Short-term treatment with the same neutralizing monoclonal antibodies against TGF-β in type 1 diabetic mice significantly reduces kidney weight and glomerular hypertrophy and attenuates the increase in extracellular matrix mRNA. Similar treatment of type 2 diabetic mice in the long term further diminishes the renal pathology and ameliorates the functional abnormalities of diabetic nephropathy. Finally, the intrarenal TGF-β system is significantly up-regulated in human diabetes. Whereas the kidney of a nondiabetic subject extracts TGF-β1 from the circulation, the kidney of a diabetic patient elaborates TGF-β1 protein into the circulation. The data we review here strongly support the hypothesis that elevated production or activity of the TGF-β system mediates diabetic renal hypertrophy and extracellular matrix expansion.