AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys

Diabetic nephropathy is characterized by diffuse mesangial matrix expansion and is largely dependent on the TGF-beta/Smad signaling pathway. Smad4 is required for TGF-beta signaling; however, its regulation has not been well characterized in diabetic kidney disease. Here, we report that high glucose is sufficient to stimulate nuclear translocation of Smad4 in mesangial cells and that stimulation of the major energy sensor AMP-activated protein kinase (AMPK) has a potent effect to block Smad4 nuclear translocation. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) inhibited high glucose-induced and TGF-beta stimulation of nuclear Smad4. To identify which of the catalytic alpha-subunits may be involved, small interfering (si) RNA-based inhibition of AMPK alpha 1- or alpha 2-subunit was employed. Inhibition of either subunit reduced overall AMPK activity and contributed to Smad4 nuclear accumulation. In an animal model of early diabetic kidney disease, induction of diabetes was found to markedly stimulate Smad4 protein levels and enhance nuclear accumulation. AMPK activation with AICAR completely prevented the upregulation of Smad4 and reduced mesangial matrix accumulation. We conclude that stimulation of Smad4 in cell culture and in in vivo models of early diabetic kidney disease is dependent on AMPK.