Yifeng Lin

OBJECTIVE: Diabetic nephropathy is one of the major causes of renal failure, which is accompanied by the production of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls the antioxidant response essential for maintaining cellular redox homeostasis. Here, we report our findings demonstrating a protective role of Nrf2 against diabetic nephropathy. RESEARCH DESIGN AND METHODS: We explore the protective role of Nrf2 against diabetic nephropathy using human kidney biopsy tissues from diabetic nephropathy patients, a streptozotocin-induced diabetic nephropathy model in Nrf2(-/-) mice, and cultured human mesangial cells. RESULTS: The glomeruli of human diabetic nephropathy patients were under oxidative stress and had elevated Nrf2 levels. In the animal study, Nrf2 was demonstrated to be crucial in ameliorating streptozotocin-induced renal damage. This is evident by Nrf2(-/-) mice having higher ROS production and suffering from greater oxidative DNA damage and renal injury compared with Nrf2(+/+) mice. Mechanistic studies in both in vivo and in vitro systems showed that the Nrf2-mediated protection against diabetic nephropathy is, at least, partially through inhibition of transforming growth factor-beta1 (TGF-beta1) and reduction of extracellular matrix production. In human renal mesangial cells, high glucose induced ROS production and activated expression of Nrf2 and its downstream genes. Furthermore, activation or overexpression of Nrf2 inhibited the promoter activity of TGF-beta1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-beta1 transcription and fibronectin production. CONCLUSIONS: This work clearly indicates a protective role of Nrf2 in diabetic nephropathy, suggesting that dietary or therapeutic activation of Nrf2 could be used as a strategy to prevent or slow down the progression of diabetic nephropathy.

BACKGROUND: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines. RESULTS: We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter. CONCLUSION: In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

AIM: To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC). METHODS: The vector, pcDNA3-AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC. CONCLUSION: AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1.