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Abstract: SA-PO017

Smad4 Knockout Increases NHE3 Expression in Renal Tubule Epithelial Cells In Vitro

Session Information

Category: Bioengineering

  • 400 Bioengineering

Authors

  • Hunter, Kuniko, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Love, Harold D., 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

Sodium-hydrogen antiporter 3 (NHE3) is a critical indicator of terminal renal proximal tubule epithelial cell (RPTEC) differentiation and accounts for 80% of tubular water reabsorption in vivo. In vitro, RPTEC have reduced NHE3 expression, which limits their functional fidelity. Transforming Growth Factor-β (TGF-β) is a pleiotropic cell signaling pathway, involved in the regulation of epithelial cell fate and plasticity. Canonical TGF-β signaling is mediated by the Smad proteins, Smad2/3/4. Here we observe that knockout of Smad4 increases NHE3 transcription in vitro.

Methods

HEK293 (ATCC, Manassas, VA) were plated at a density of 5x105 in 35mm plates to achieve 60% confluence the following day. Cells were co-transfected with CRISPR sgRNA with PiggyBac (Pb) hygromycin resistance and eGFP vectors and the m7pB hyperactive Pb transposase at a 3:1 ratio using Lipofectamine LTX Plus (Thermo Fisher). For control transfection, cells were transfected with a scramble sequence. 48 hours post-transfection, cells were supplemented with 200µg/mL hygromycin for one week. Cells were passaged once then harvested for RT-PCR analysis.

Results

Smad2, Smad3, and Smad4 knockouts significantly reduced expression of Smad2, Smad3, and Smad4, respectively. NHE3 expression significantly increased with only Smad4 knockout.

Conclusion

Smad4 modulates NHE3 expression in renal tubule epithelial cells.