Translational Regulation of Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-β1 Generation by Insulin

We have previously demonstrated that the proximal tubular cell may contribute to the pathogenesis of renal interstitial fibrosis in diabetes. Transforming growth factor (TGF)-β1 is one of a group of pro-fibrotic cytokines and growth factors, which have been associated with the development of interstitial fibrosis. The aim of the current study was to examine the effect of insulin on the generation of TGF-β1 by proximal tubular cells. HK-2 cells were grown to confluence in the absence of insulin, and serum deprived for 48 hours before all experimental manipulations. Addition of insulin (5 μg/ml) to the culture medium led to a time-dependent increase in TGF-β1 concentration in the cell culture supernatant, and increased incorporation of radiolabeled amino acids into TGF-β1 suggestive of de novo TGF-β1 protein synthesis. Addition of insulin did not alter TGF-β1 mRNA expression as assessed by reverse transcriptase-polymerase chain reaction or Northern analysis. Insulin-induced increase in TGF-β1 concentration was not abrogated by actinomycin D, however, stimulation by insulin, in the presence of cycloheximide led to a dose-dependent decrease in TGF-β1 production. Addition of insulin had no effect on TGF-β1 mRNA stability as assessed by actinomycin D chase, but led to increased binding of a cytoplasmic protein to a putative stem loop structure in the 5′-UTR of TGF-β1 mRNA, previously implicated in the posttranscriptional control of TGF-β1 synthesis. To address the functional significance of insulin-induced alteration in TGF-β1 synthesis, we examined its effect on matrix turnover. Insulin stimulated type IV collagen gene expression and an increase in the concentrations of the type IV collagen laid down in the extracellular matrix. This increase in type IV collagen was abrogated when cells were stimulated by insulin in the presence of an anti-TGF-β1-blocking antibody. In conclusion the data demonstrate that insulin may directly alter the production of TGF-β1 by renal proximal tubular cells by a posttranscriptional mechanism, and that this may have implications for the increase in extracellular matrix that accompanies diabetic nephropathy.