Studies have shown that high levels of serum free fatty acids (FFAs) are associated with lipotoxicity and type 2 diabetes. Palmitic acid (PA) is the predominant circulating saturated FFA, yet its role in the pathogenesis of diabetic nephropathy (DN) is not clear. Recently, one study suggested that mitochondrial superoxide production is related to AMP-activated protein kinase (AMPK) activity in diabetic mice kidneys. To elucidate the link between PA and oxidative stress and AMPK activity in DN, we compared the cultured murine podocytes exposed to PA and oleic acid (OA). Incubation of cells with 250 μM PA or OA induced a translocation of CD36, a fatty acid transport protein, with intracellular lipid accumulation. PA, but not OA, induced mitochondrial superoxide and hydrogen peroxide (H O ) generation in podocytes, as shown by enhanced fluorescence of MitoSOX Red and dichlorofluorescein (DCF), respectively. Costimulation of PA-treated cells with the H O scavenger catalase abolished the PA-induced DCF fluorescence. Only PA induced mitochondrial damage as shown by electron microscopy. The AMPK activity was determined by immunoblotting, measuring the ratio of phosphorylated AMPK (p-AMPK) to total AMPK. Only PA significantly increased the p-AMPK levels compared with controls. Addition of catalase to PA-treated cells did not affect the PA-stimulated p-AMPK levels. Collectively, our results indicate that PA induces mitochondrial superoxide and H O generation in cultured podocytes, which may not be directly linked to AMPK activation. Given that, PA seems to play an important role in the pathogenesis of DN through lipotoxicity initiated by mitochondrial superoxide overproduction.

High levels of serum free fatty acids (FFAs) are associated with lipotoxicity and type 2 diabetes. Palmitic acid (PA) is the predominant circulating saturated FFA. PA induces mitochondrial superoxide and hydrogen peroxide (H O ) generation in cultured podocytes. To elucidate the role of PA in antioxidant defense systems in diabetic nephropathy (DN), cultured podocytes were exposed to 250 μM PA for 1-24 hr, and protein expressions of catalase, peroxiredoxins (Prxs), and glutathione peroxidase (GPx) were examined by western blot analysis. PA induced an early transient increase in the Prx1, Prx2, and GPx1 levels in podocytes, but not catalase. Long-term exposure of PA to podocytes significantly decreased the protein levels of Prx1, Prx2, GPx1, and catalase. Coincubation of PA-treated cells with oleic acid, however, restored the expression of these proteins. In advanced human diabetic glomeruli, H O generation was elevated as shown by increased fluorescence of dichlorofluorescein. Strong immunostaining for Prx1, Prx2, GPx1, and catalase was observed in the podocytes of advanced human DN, wherein transforming growth factor-β1 staining was also positive. These results suggest that podocytes are susceptible to PA-induced oxidative damage with impaired peroxidase activity and that peroxidases have futile antioxidant effects in the podocytes in the late stages of DN. Given this, PA-induced podocyte injury via inadequate peroxidase response to H O appears to play an important role in the pathogenesis of DN.