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Abstract: SA-PO043

Activation of AMPK and Inhibition of TGFß Stimulate In Vitro Transport in Human Renal Epithelial Cells

Session Information

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Fissell, William Henry, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Roy, Shuvo, UCSF, San Francisco, California, United States
  • Harris, Raymond C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Zent, Roy, Vanderbilt Medical Center, Nashville, Tennessee, United States
  • Wilson, Matthew H., Vanderbilt University, Nashville, Tennessee, United States
  • Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Love, Harold D., Vanderbilt Univ Med Ctr, Nashville, Tennessee, United States
Background

Qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart is critical to a wide variety of organs-on-a-chip applications including drug discovery, physiology-based pharmacokinetics, toxicity screens, as well as therapeutic applications for ex vivo or implanted engineered organs. Investigators have shown several differentiated functions of renal tubule cells in vitro including Vitamin D hydroxylation, ammoniagenesis, and specific drug uptake and excretion, but the tubule cells' primary role in the kidney, electrolyte and water transport, has
been elusive.

Methods

Primary human renal tubule epithelial cells (RPTEC) harvested from transplant discards were seeded at passage 1-3 onto Costar permeable supports and cultured at 37 on an orbital shaker. 3 weeks post-confluence a TGF-b inhibitor, SB431542, and metformin were added to hormonally-defined media. Apicobasal transport was measured by weighing apical volumes before and after culture, and by measuring the change in apical inulin concentration.

Results

RPTEC in hormonally defined media showed negligible transport (14 μL/cm2/day), but addition of SB431542 increased transport 5.2-fold; addition of metformin increased transport 5.6-fold; addition of both increased transport 10-fold. Addition of 10nM ouabain reduced transport by about 45% in all groups. Inulin leak rate did not increase.

Conclusion

Using simple modifications to cell culture protocols, we were able to attain inhibitable apicobasal transport, a key attribute of differentiated RPTEC.

Funding

  • Other NIH Support