Novel CRISPR/Cas9 mouse knock‐in model containing an alpha‐actinin 4 mutation associated with focal segmental glomerulosclerosis recapitulated human kidney injury and provides insight into how aberrations in cytoskeleton lead to podocyte dysfunction

Alpha‐actinin 4 (ACTN4) is a cytoskeleton protein that crosslinks actin filaments, helps anchor the actin cytoskeleton to focal adhesions, and provides structural support for cells. Mutations in ACTN4 cause a highly penetrant form of focal segmental glomerulosclerosis (FSGS) in humans, a type of kidney injury characterized by proteinuria and podocyte dysfunction. We previously found that disease‐causing mutant K255E ACTN4 increases podocyte contractility in response to injurious circulating stimuli and increased extracellular matrix stiffness. We hypothesize that this increased contractility is pathologic, rendering mutant podocytes unable to adapt to the dynamic mechanical stresses that they experience in the glomerulus while maintaining the filtration barrier. Here we test this hypothesis by subjecting primary podocytes derived from a novel mutant mouse model to repetitive stretch and measuring their response. Using CRISPR/Cas9 technology, we first developed a new mouse model Actn4 K256E (a mutation analogous to the FSGS‐causing K255E mutation in humans) in a pure FVB background. Compared with WT littermates, homozygous mutant mice developed significant proteinuria consistent with the human phenotype. Traction force microscopy was then used to study the contractile forces exerted by mutant and WT podocytes on their underlying substrates in response to stretch. Each podocyte was subjected to five intermittent 4s stretches, with a 3 min recovery period between stretches during which contraction force was continuously measured. Whereas WT podocytes were able to consistently recover their initial contraction forces after each stretch, mutant podocytes on average demonstrated higher initial contraction forces that drastically deteriorated without recovery over time. Additionally, mutant podocytes developed obvious cracks in their cytoskeletons as visualized by immunofluorescence staining – a finding largely absent in WT podocytes. These results represent the net effect of aberrant cytoskeleton remodeling contributed by mutant ACTN4, leading to abnormalities of podocyte structure and function that may underlie the development of FSGS. Support or Funding Information R37DK059588 and T32DK007199