Abstract

Aim: To investigate the biophysical mechanisms underlying proximal tubule fibrosis and the impact cell mechanics have on disease progression, we measured the mechanical stress applied to single cells treated with TGFβ1 as a function of time and quantified viscoelastic properties using stress-relaxation mathematical models.
Methods: Cytoskeletal reorganization was assessed, and single cell stress-relaxation testing performed by Atomic Force Microscopy (AFM) based force spectroscopy on model human proximal tubule cells (HK2) +/- TGF-β1 (10ng/mL) at 37oC. Fitted with a spherical bead, AFM cantilevers functioning under constant force mode measured mechanical forces over time and viscoelastic properties were calculated using the Maxwell model of viscoelasticity.
Results: Data suggest that both control and TGFβ1 treated cells exhibit transient (5sec) force relaxation of 0.7 and 0.3nN respectively, in response to physical deformation. The initial phase was followed by stabilization in force over a subsequent 30sec period. TGFβ1 treated cells demonstrated a reduction in relaxation characteristics, suggesting that complex viscoelastic components are strongly affected by reorganization of the actin cytoskeleton. Data indicate that viscosities after treatment vary up to 35% (12cells, n=3, p<0.001). Treated cells showed a three-fold reduction in experimental decay of the force time curve that leads to significant changes in the adhesive deformation behaviour.
Conclusion: TGFβ1 (10ng/mL) triggered complex nanomechanical changes in the viscous-elastic behaviour of single cells. Our research suggests that the progression of the disease instigates intricate physical changes that may in part, mediate altered cell-ECM interactions linked to altered cell phenotype in tubular injury.