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Abstract: SA-PO016

AMP-Activated Protein Kinase (AMPK) and TGF-β Alter Cell Size and Morphology in Renal Tubule Epithelial Cells

Session Information

Category: Bioengineering

  • 400 Bioengineering

Authors

  • Hunter, Kuniko, Vanderbilt University, Nashville, Tennessee, United States
  • Fissell, William Henry, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Roy, Shuvo, University of California San Francisco, San Francisco, California, United States

Group or Team Name

  • The Kidney Project.
Background

Epithelial barrier function is regulated by tight junction protein complexes (TJ) between adjacent cell membranes. TJ may develop non-linear ruffled or spiked architectures which in turn alter cell morphology. Here we observe that AMP-activated protein kinase (AMPK) and Transforming Growth Factor-β (TGF-β) alter renal tubule epithelial cell (RTEC) morphology and induce a ruffled appearance when administered together.

Methods

Primary human RTEC were obtained from Innovative Biotherapies (Ann Arbor, MI). Cells were maintained in DMEM/F12 and seeded onto polycarbonate cell culture inserts at 100,000 cells/cm2. After two weeks, cells were apically supplemented with AMPK activator Metformin (200μM, MET), TGF-β receptor I inhibitor SB431542 (10μM, SB), or both. After four weeks, cells were stained for DAPI and ZO-1, and imaged using conventional methods. CellProfiler (Broad Institute) was used to threshold and segment nuclei and cell perimeters. Form factor (FF) was calculated as 4*pi*area/perimeter2. Compactness index (CI) was calculated as the mean2 distance between the objects pixels from the centroid divided by area. Statistical significance was assessed by performing 2-way ANOVA using Prism 9 (GraphPad).

Results

In comparison to controls, all treatments increased cell perimeter and cell area. MET increased cell perimeter and cell area, in comparison to SB and combination treatments. All treatments significantly reduced FF and increased CI, respectively.

Conclusion

All treatments increase cell perimeter and area, suggesting cell swelling. All treatments reduce FF, suggesting reduced circularity. All treatments increase CI, which suggests increased irregularity of shape, such as increased cell interdigitation or plasma membrane ruffling.