TGF-β-induced pathogenic changes in mouse podocytes

Abstract

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated TGF-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. Proapoptotic mechanism of TGF-β in immortalized mouse podocytes was investigated. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation which was related to elevated reactive oxygen species (ROS) levels generated by selective upregulation of Nox4. In mouse podocytes, Nox4 was predominantly localized to mitochondria and Nox4 upregulation by TGF-β1 markedly depolarized the mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation was mitigated by an antioxidant, Nox inhibitor DPI, or siRNA for Nox4. A TGF-β receptor I blocker, SB431542, completely reversed the changes triggered by TGF-β1. Knock-down of either Smad2 or Smad3 attenuated increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential and caspase-3 activation by TGF-β1. TGF-β1 also phosphorylated mTOR and downstream target p70S6K. Rapamycin, an inhibitor of mTOR complex I, prevented TGF-β1-induced ROS generation, total Nox activity, mitochondrial membrane potential depolarization and caspase activation indicating the role of mTOR activation in TGF-β1-induced podocyte apoptosis. In addition, rapid phosphorylation of ERK1/2 by TGF-β1 was observed. Pharmacological inhibition of ERK1/2 protected from TGF-β1-induced mTOR and p70S6K phosphorylation, enhanced Nox activity, loss of mitochondrial membrane potential, and casapase-3 activation. Taken together, these data suggest that TGF-β1-induced oxidative stress and podocyte apoptosis are mediated by TGF-β receptor-Smad2/3-mitochondrial Nox4 axis and/or activation of ERK1/2-mTORC1 cascade also co-operates to enhanced Nox activity which may contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.