Upregulation of mitochondrial Nox4 mediates TGF-β-induced apoptosis in cultured mouse podocytes

Abstract

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis

“…It has been reported recently that podocyte-specific overexpression of TGF-␤ induces segmental glomerulosclerosis with dysfunction in endothelial cells and podocyte depletion (30). We have also published previously that TGF-␤1 induces podocyte apoptosis through transcriptional up-regulation of NADPH oxidase 4 (Nox4) by activation of the Smad pathway (31). There is also evidence that high glucose-mediated inactivation of AMP-activated protein kinase activates mTOR in a TSC2-dependent manner, which accounts for Nox4-mediated podocyte depletion in diabetic mice (32).…”

“…Cell Culture and Drugs-Immortalized mouse podocytes were obtained from the laboratory of Prof. Peter Mundel (Harvard Medical School) and cultured as described previously (31). Cells were grown on collagen-coated 100-mm dishes using lowglucose Dulbecco's modified Eagles medium supplemented with 5% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin.…”

“…Measurement of Reactive Oxygen Species (ROS)-Measurement of reactive oxygen species in podocytes was performed as described previously (31). Intracellular ROS generation was detected by the fluorescent dye chloromethyl-H 2 -dichlorofluorescein diacetate (catalog no.…”

“…Cascade in Podocytes-Immortalized podocyte cell line expressing the differentiation marker synaptopodin at 37°C in the absence of interferon ␥ has been checked previously (31). Under similar culture conditions, podocytes were treated with TGF-␤1 (5 ng/ml) in a time-dependent manner to check the activation of mTOR signaling.…”

Evolution of the Mir-155 Family and Possible Targets in Cancers and the Immune System

“…As known, SMAD2 proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways, and is responsible for the transmission of extracellular signals from ligands of the Transforming Growth Factor beta (TGF-β) superfamily of growth factors into the cell nucleus. TGF-β is a pleiotropic cytokine taking part in the processes of fibrosis, angiogenesis, apoptosis and immunosupression (Louafi et al, 2010;Das et al, 2014). The TGF-β receptor-Smad2/3 pathway is essential for the cell apoptosis and proliferation (Zhi-Yong1 et al, 2011;DasXu et al, 2014).…”