TGF-β1 stimulates mitochondrial oxidative phosphorylation and generation of reactive oxygen species in cultured mouse podocytes, mediated in part by the mTOR pathway

TGF-beta 1 stimulates mitochondrial oxidative phosphorylation and generation of reactive oxygen species in cultured mouse podocytes, mediated in part by the mTOR pathway. Am J Physiol Renal Physiol 305: F1477-F1490, 2013. First published September 18, 2013; doi:10.1152/ajprenal.00182.2013.-Transforming growth factor (TGF)-beta has been associated with podocyte injury; we have examined its effect on podocyte bioenergetics. We studied transformed mouse podocytes, exposed to TGF-beta 1, using a label-free assay system, Seahorse XF24, which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Both basal OCR and ATP generation-coupled OCR were significantly higher in podocytes exposed to 0.3-10 ng/ml of TGF-beta 1 for 24, 48, and 72 h. TGF-beta 1 (3 ng/ml) increased oxidative capacity 75%, and 96% relative to control after 48 and 72 h, respectively. ATP content was increased 19% and 30% relative to control after a 48- and 72-h exposure, respectively. Under conditions of maximal mitochondrial function, TGF-beta 1 increased palmitate-driven OCR by 49%. Thus, TGF-beta 1 increases mitochondrial oxygen consumption and ATP generation in the presence of diverse energy substrates. TGF-beta 1 did not increase cell number or mitochondrial DNA copy number but did increase mitochondrial membrane potential (MMP), which could explain the OCR increase. Reactive oxygen species (ROS) increased by 32% after TGF-beta 1 exposure for 48 h. TGF-beta activated the mammalian target of rapamycin (mTOR) pathway, and rapamycin reduced the TGF-beta 1-stimulated increases in OCR, ECAR, ATP generation, cellular metabolic activity, and protein generation. Our data suggest that TGF-beta 1, acting, in part, via mTOR, increases mitochondrial MMP and OCR, resulting in increased ROS generation and that this may contribute to podocyte injury.