Abstract: TH-PO0038
ALK5 Inhibition Improves Transport and Respiration in Primary Renal Proximal Tubule Cells at One Year in Culture
Session Information
- Bioengineering: MPS, Flow, and Delivery
November 06, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 400 Bioengineering
Authors
- Ice, Alissa Angelica, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, 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
Cultured epithelial cells tend to undergo phenotypic atrophy in artificial culture. In renal tubule cells, loss of transporter expression and a shift to glycolysis instead of oxidative phosphorylation undermines applicability of cell culture to disease modeling, in vitro toxicity screening, or cell therapy of renal failure. We showed that ALK5 inhibition with SB431542 improved in vitro proximal tubule cell transporter expression and function, but the durability of this effect is unclear.
Methods
Primary human renal proximal tubule cells (Lonza, Basel) were seeded at 10^5 cells per cm^2 on permeable supports and grown in 50:50 DMEM/F12 with ITS, hydrocortisone, T3, and ascorbic acid in humidified 5% CO2/Air. 24 hours after seeding the cell culture plates were placed on an orbital shaker to generate 1 dyne/cm2 average fluid shear stress. 1 week after confluence, media was supplemented with metformin, SB431542, or both. Media was changed every 48-72 hours. Apicobasal leak was assessed using TRITC-labelled dextrans, and apicobasal transport was measured by media mass change at week 46. Cells were then trypsinized and cellular respiration was measured by high-resolution respirometry.
Results
At 46 weeks, metformin, SB- and combination treated cells had less apicobasal dextran leak and greater apicobasal transport than control cells (p < 10^-5 for leak and p < 10^-4 for transport). Transport was inhibitable with tenapanor but not furosemide or hydrochlorothiazide. Combination-treated cells had higher maximum oxygen consumption rates and higher spare capacity than control cells (n=3 per group, fig 1).
Conclusion
Inhibiting ALK5, a receptor for TGFβ, a cytokine associated with substrate elasticity and cell transformation, improved functions associated with healthy proximal tubule cells after 47 weeks in culture. The effect was potentiated by addition of metformin, an inhibitor of Complex I and a potent activator of AMP-associated kinase. This suggests that prolonged function of a tubule cell bioreactor for cell therapy is feasible. Further investigation of the mechanisms by which ALK5 inhibition improves cell function are warranted.
Funding
- Private Foundation Support