FSGS-associated α-actinin-4 (K256E) impairs cytoskeletal dynamics in podocytes

Abstract

Mutations in the ACTN4 gene, encoding the actin crosslinking protein alpha-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E alpha-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E alpha-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E alpha-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on alpha-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type alpha-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E alpha-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E alpha-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E alpha-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E alpha-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E alpha-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.

Nephropathy and Elevated BP in Mice with Podocyte-Specific NADPH Oxidase 5 Expression

“…5 Our in vitro studies show that de novo human Nox5 expression in mouse podocytes induces actin cytoskeleton rearrangement, Rac1 activation, and a lamellipodia-rich, ROS-dependent cellular phenotype reminiscent of the phenotype reported in the work by Hsu et al 5 It has been suggested that increased motility in vitro is analogous to podocyte effacement in vivo, an event closely associated with slit diaphragm deterioration and development of albuminuria. 40 In agreement with this hypothesis and our in vitro results, Nox5 pod+ tg mice develop albuminuria and podocyte effacement, highlighting a role for Nox5 both at the cellular level and in the broader context of filtration barrier dysfunction.…”

“…40 Briefly, cells were fixed with 4% paraformaldehyde and permeabilized in PBST. Cells were then incubated with Alexa Fluor 594 phalloidin (Molecular Probes) to visualize actin cytoskeleton.…”

Glomerular epithelial cell injury associated with mutant α-actinin-4

“…Normally, ␣-actinin-4 may be required for integrin-dependent adhesion of GEC (11), and GEC that express ␣-actinin-4 K256E show defective spreading and motility (22). Further studies, including in vivo models, will be required to ascertain the functional importance of the proteasome and ER stress-associated pathways in the pathogenesis of GEC injury and FSGS.…”

“…The human kidney expresses only ␣-actinin-4 (although mouse GEC also express ␣-actinin-1) (14,38). In cultured GEC, ␣-actinin-4 mediates adhesion and cytoskeletal dynamics (11,22). Knockout of ␣-actinin-4 in mouse podocytes in vivo leads to severe glomerular disease, renal failure, and early death (13,19,38).…”

Proteinuria: is it all in the foot?

“…Several pathomechanisms can affect the actin cytoskeleton. First, mutation of α-actinin 4, which causes hereditary focal segmental glomerulosclerosis (FSGS), impairs cytoskeletal dynamics (13). A second mechanism involves loss of integrity of the SD complex.…”