Abstract: TH-OR037
Matrix Elasticity Regulates Multiple Podocyte Functions
Session Information
- Bioengineering
November 07, 2019 | Location: 146 A/B, Walter E. Washington Convention Center
Abstract Time: 05:42 PM - 05:54 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Sant, Snehal, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Schwager, Samantha C., Vanderbilt University, Nashville, Tennessee, United States
- Coode, Monica Ann, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Reinhart-King, Cynthia A., Vanderbilt University, Nashville, Tennessee, United States
- Ferrell, Nicholas J., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background
The extracellular matrix provides biomechanical signals to adherent cells. Chronic kidney disease is associated with changes in the structural and mechanical properties of the glomerular basement membrane (GBM) that may be relevant to disease progression. The aim of this work was to use polyacrylamide hydrogels as GBM analogs to determine how variations from physiological substrate stiffness regulate podocyte proliferation, migration, and traction force generation. In addition, YAP activation, an established regulator of cellular mechanotransduction was evaluated in podocytes grown on hydrogels with varying levels of stiffness.
Methods
Conditionally immortalized mouse podocytes were cultured on soft (0.5 kPa) and stiff (10-50 kPa) polyacrylamide hydrogels. Podocyte force generation was measured using traction force microscopy (TFM). Podocyte proliferation was evaluated based on nuclear EdU incorporation. Cell migration rates were measured by live-cell imaging. YAP nuclear localization was determined by quantitative immunofluorescence staining. Gene expression for downstream targets of YAP activation were evaluated by qRT-PCR.
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Results
TFM analysis showed that cell generated forces were orders of magnitude higher on stiff (10 kPa) compared to soft (0.5 kPa) hydrogels. Podocyte spreading was also significantly lower on soft gels. Tractions stresses were also higher on stiff hydrogel showing that increased force generation was not simply related to differences in cell spreading.. Proliferation rate was nearly doubled on stiff gels compared to soft and the rate of podocyte migration was approximately 25 μm/hr on stiff substrates compared to <5 μm/hr on soft gels. Stiff substrates induced nuclear localization of YAP and resulted in up-regulation of gene expression for CTGF, CYR61, ANKRD1, and BIRC5, all which are known downstream targets of YAP activation.
Conclusion
These data show that the elasticity of podocyte substrata is important in regulating multiple podocyte functions that may be relevant to maintenance of normal physiological function and/or progression of chronic kidney injury. Additional work is needed to understand the pathological significance of changes in GBM stiffness in different chronic kidney injuries and to elucidate the molecular mechanisms that regulate stiffness-induced changes in cell behavior.
Funding
- NIDDK Support