Mutant Actn4 alters the distribution of intracellular stress within podocytes and contributes to their mechanical failure in response to stretch

Abstract

Alpha‐actinin‐4 (ACTN4 in humans, Actn4 in mice) crosslinks actin filaments in podocytes, providing them mechanical resilience as they help maintain the glomerular filtration barrier. Although human disease‐causing mutant ACTN4 leads to brittle reconstituted actin networks in vitro, how this affects the ability of podocytes to withstand the external mechanical challenges within the glomerulus remains unknown. We studied primary podocytes isolated from wild‐type (WT) and disease‐causing mutant Actn4 knock‐in mice. We previously reported that, after transient stretch, WT cells recovered their baseline contraction and actin cytoskeleton breakages, whereas mutant podocytes developed irrecoverable reductions in their contraction and irreparable disruptions in their cytoskeletons. We now report that after stretch, when challenged in a centrifugal detachment assay, mutant podocytes (11/23) had a higher probability of detachment than WT (2/19) (odds ratio =7.42, 95% CI 1.27, 80.75; p=0.017). Through an analysis of baseline biophysical properties, we found that the actin filaments of mutant Actn4 podocytes displayed a more uniform distribution of orientation (aligned more parallel to each other) compared with the filaments of WT podocytes (which displayed more random orientation). We then computed baseline intracellular stress in terms of two constituent components: the magnitude of mean principal stress as tension (σ), and the orientation of first principal stress as principal orientation (θ). The spatial distribution of intracellular stress within the podocytes was measured using the autocorrelation function C(r). The distance over which the C(r) decays to a value of 0.5 was extrapolated as a singular value from the autocorrelation curves and is termed correlation length, or r1/2. The greater the r1/2 value, the greater the distance over which intracellular stress maintains uniformity. We found that mutant podocytes displayed a more uniform distribution of intracellular stress across the cell, both in terms of magnitude (r1/2 σ mean ± SE: mutant 18.0 ± 0.8 μm vs. WT 13.4 ± 0.8 μm) and orientation (r1/2 θ mean ± SE: mutant 34.7 ± 2.2 μm vs. WT 25.9 ± 2.3 μm). Moreover, the more uniform the intracellular stress was distributed at baseline, the less likely mutant podocytes recovered their baseline contraction after periodic stretch. Taken together, our findings provide novel insight into the mechanism by which ACTN4 mutation alters the alignment of the cytoskeleton and impairs the resiliency of the podocyte, leaving it vulnerable to mechanical stretch and more prone to detachment in its glomerular environment.Support or Funding InformationR37DK059588 and T32DK007199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.