Abstract: PO0500
Canonical TGF-β Signaling Mediates Renal Tubule Epithelial Cell Differentiation In Vitro
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
- Love, Harold D., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Humes, H. David, University of Michigan, University of Michigan, Ann Arbor, MI, US, academic, 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
Transforming growth factor-β (TGFβ) initiates multiple signaling pathways involved in the regulation of epithelial cell fate and cell plasticity. Here we identify canonical TGFβ signaling as a critical regulator of renal tubule epithelial cell membrane transporter expression in vitro.
Methods
Primary human renal tubule epithelial cells (HREC) were cultured on permeable supports on an orbital shaker. After two weeks in culture, cells were supplemented with TGFβ receptor I (TβR1) inhibitors SB431542 or A8301, Smad3 inhibitor SIS3, PI3K inhibitor LY294002, Rapamycin, p38 MAPK inhibitor SB203580, Takinib (2nM), Trametinib. After four weeks, apicobasal fluid transport and gene expression by RT-PCR was measured. Statistical differences were estimated by two-tailed Student’s t-test in MatLab.
Results
Canonical TGFβ inhibitors SB431542 and A8301 increase apicobasal fluid transport, while Smad3 inhibitor SIS3 does not. Non-canonical TGFβ inhibitors LY294002, Rapamycin, SB203580, Takinib, Trametinib do not increase apicobasal fluid transport. SB431542 and A8301 suppress Snail1 transcription, while SIS3 does not. SB431542 and A8301 increase AQP2 transcription, while SIS3 does not, and have a greater effect on NHE3 transcription.
Conclusion
Increased inhibitable transport by renal tubule cells in vitro appears to be mediated by canonical TGF-β signaling. The lack of response to SIS3 suggests that Smad2, ratehr than Smad3, is responsible.
Figure 1 Smad2-mediated TGFβ signaling mediates renal tubule epithelial cell differentiation A. Canonical apicobasal fluid transport; Data are mean ± SD (n=4). B. Non-canonical apicobasal fluid transport. Data are mean ± SD (n=3). C-E. Snail1, NHE3, and AQP2 transcription. Data are mean ± SEM (n=4). *p<0.05, **p<0.01, ***p<0.001.
Funding
- Private Foundation Support