TGF-β1 stimulates mitochondrial oxidative phosphorylation and generation of reactive oxygen species in cultured mouse podocytes, mediated in part by the mTOR pathway.
Transforming growth factor (TGF)-β has been associated with podocyte injury; we have examined its effect on podocyte bioenergetics. We studied transformed mouse podocytes, exposed to TGF-β1, 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-β1 for 24, 48, and 72 h. TGF-β1 (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-β1 increased palmitate-driven OCR by 49%. Thus, TGF-β1 increases mitochondrial oxygen consumption and ATP generation in the presence of diverse energy substrates. TGF-β1 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-β1 exposure for 48 h. TGF-β activated the mammalian target of rapamycin (mTOR) pathway, and rapamycin reduced the TGF-β1-stimulated increases in OCR, ECAR, ATP generation, cellular metabolic activity, and protein generation. Our data suggest that TGF-β1, acting, in part, via mTOR, increases mitochondrial MMP and OCR, resulting in increased ROS generation and that this may contribute to podocyte injury.
10 Center Drive, NIH, Bethesda, MD 20892-1268. jbkopp@nih.gov.
Yoshifusa
Y
Abe
Toru
T
Sakairi
Craig
C
Beeson
Jeffrey B
JB
Kopp
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015374:Biosensing Techniques; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D050199:Podocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D000952:Antigens, Polyomavirus Transforming; D005227:Fatty Acids; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D000255:Adenosine Triphosphate; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases
TGF-β1; bioenergetics; extracellular acidification rate; mitochondria; oxygen consumption rate; podocyte