Abstract

Aims: Loss of cell-to-cell adhesion is critically important for the progression of fibrosis in diabetic nephropathy. Research has focussed on the loss of cell surface molecular binding such as ligation of epithelial (E)-cadherin. Here, we examine the effects of cellular rigidity on cell-to-cell tethering in early proximal tubular injury.
Methods: Transforming Growth Factor-beta (TGF-β1) is the major pro-fibrotic cytokine associated with diabetic nephropathy. Membrane deformation and cell-to-cell coupling were assessed using Atomic Force Microscopy Single Cell Force Spectroscopy (AFM-SCFS) on single epithelial cells of the human proximal tubule (HK2) +/- TGF-β1 (10ng/mL) at 37oC. AFM-FS was fitted with a bead-attached cantilever to indent single cells and and thereby calculate single cell elasticity from force-displacement (F-d) curves.
Results: Single cell indentation of control and TGF-β1 treated HK-2 cells indicated a significant increase in the rigidity of the treated cells (42%, 12 cells, n=3, P<0.001), while the retraction force-displacement measurements indicated a decrease in the number of surface ligation rupture events by 35%. However, results demonstrated a variant/unequal decrease of the adhesion parameters between two cells. Although the maximum unbinding force decreased by 19%, the energy of detachment decreased by 53%, consistent with a reduction of 46% in distance of separation between two cells (>30cells, n=5, p<0.001)..
Discussions: The loss of structural integrity in a cell cluster in response to TGF-beta1 reflects differences in the mechanical properties of single cells, as well as our previously reported changes in E-cadherin ligation. This study correlates alterations in morphology, and adhesion with fibrosis, by elucidating a mechanism in which structural mechanics associated with TGF-beta induced tubular injury affect cell-to-cell adhesion.