Abstract: PO0501
TGF-β Mediates In Vitro Renal Tubule Cell Fatty Acid Oxidation
Session Information
- Bioengineering: Organoids and Organs-on-a-Chip
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Hunter, Kuniko, Vanderbilt University, Nashville, Tennessee, United States
- Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Love, Harold D., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Humes, H. David, University of Michigan, Ann Arbor, Michigan, United States
- Roy, Shuvo, University of California San Francisco, San Francisco, California, United States
- Fissell, William Henry, Vanderbilt University Medical Center, Nashville, Tennessee, United States
Group or Team Name
- The Kidney Project
Background
Renal tubule cells (HREC) are energetically demanding due to their role transporting solutes., but undergo a shift toward glycolysis and away from oxidative phosphorylation of fatty acids in vitro. We identified Transforming Growth Factor-β (TGFβ) as a critical modulator of HREC differentiation. Here, we find that TGFβ inhibition increases HREC fatty acid oxidation.
Methods
Primary HREC were seeded on polycarbonate Transwells or polystyrene Seahorse XFe96 tissue culture plates. Cells were supplemented with AMPK activator metformin, TGFβ receptor I inhibitor SB431542, or a combination of both. After four weeks, cell oxygen consumption (OCR) and extracellular acidification rates (ECAR) were assessed using a Seahorse XFe96. Glutamine oxidation inhibitor BPTES, fatty acid oxidation inhibitor etomoxir, and glucose oxidation inhibitor UK5099 were used to probe HREC substrate utilization. Gene expression was measured using RT-PCR . Statistical differences were estimated by two-tailed Student’s t-test. Results are considered significant at p<0.05.
Results
MET and SB43 stimulate transcription of electron transport chain Complexes I, II, IV, and V. Control and MET OCR did not respond to inhibitors. MET cells have diminished basal OCR and decreased ECAR in response to BPTES. SB43 increases basal OCR and cellular responses to UK5099 and etomoxir, implying increased glucose and fatty acid oxidation. SB43 increases transcription of fatty acid transporter CD36 and fatty acid oxidation genes FABP1, CPT1, and CPT2.
Conclusion
Inhibition of TGFβ increases in vitro transcription of mitochondrial genes and oxidative phosphorylation of fatty acids by HREC.
Metformin and SB431542 module substrate oxidation dependency A-D. RNA expression of NDUFB8, mt-CytB, mt-CO2, and mt-ATP6. Data are mean ± SEM (n=4). E-G. OCR and H-J. ECAR of substrate inhibition assays. Data are mean ± SD (n=5). K-N. RNA expression of CD36, FABP1, CPT1/2. *p<0.05, **p<0.01, ***p<0.001.
Funding
- Private Foundation Support