Figure 6.

HG regulates mRNA of fibronectin expression. (A) Tubular epithelial cells pretreated with actinomycin D (50 μM) before exposure of the cells to HG (HG+A). Total RNA from tubular cells grown in normal or HG and pretreated with and without actinomycin D was extracted and subjected to RT-PCR. Products were separated on agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light. (B) Intensity of each band was quantified by densitometry. Histograms represent mean ± SEM (n=3). ^§P<0.01, significant difference from cells treated with NG or HG. Blocking mTOR activity significantly reduced the promoter activity of fibronectin in primary cells. (C) Rapamycin prevents HG-induced increase in fibronectin promoter transcriptional activity in TSC2^+/+ and TSC2^+/− PPTECs. A reporter plasmid containing the fibronectin promoter driving expression of the luciferase and a control Renilla reporter gene were co-transfected into the cells using Lipofectamine Plus Reagent. After treatment with rapamycin and HG for 24 hours, the cells were washed with PBS and cell lysates were prepared. Luciferase activity was determined using the Luciferase Reporter Assay System by a luminometer and normalized by Renilla reporter activity. Histograms represent mean ± SEM (n=3). ^§P<0.01, significant differences between NG and HG; ^¶P<0.01, differences in rapamycin-treated cells and nontreated cells; ^ϑP<0.01, differences in HG+R and HG; and ^ϕP>0.01, differences in TSC2^+/− cells and TSC2^+/+ cells.