Figure 5.

Blockade of 8-OHdG induction (A), reactive oxygen species (ROS) production (B) and AQP-1 induction (C) and elevation of SOD2 induction (C) by tangeretin in glucose-exposed podocytes and in diabetic kidneys. Podocytes were incubated in media containing 5.5 mM glucose, 5.5 mM glucose plus 27.5 mM mannitol as osmotic controls or 33 mM glucose in the absence and presence of 1–20 μM tangeretin for 4 days. For the immunocytochemical analysis of 8-OHdG (A), a FITC-conjugated secondary antibody was used to visualize the induction of 8-OHdG. The nuclear staining was done with DAPI (blue). Each photograph is representative of stained cells. Magnification: 400-fold. The db/db mice were orally supplemented with 10 mg/kg tangeretin for 10 weeks. For the measurement of ROS generation in kidney glomeruli, the dihydroethidium (DHE) staining was performed (B). Each photograph is representative of four mice. Scale bar = 50 μm or 200 μm. Cell lysates and tissue extracts were electrophoresed on 12–15% SDS-PAGE and subject to Western blot analysis with a primary antibody against AQP-1 and SOD2 (C). β-Actin antibody was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative results of blot bands obtained from a densitometer. Values in bar graphs not sharing a common letter are significantly different at p < 0.05.