The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

Abstract

Background: We previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.

Methods: We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLN_R431C and ANLN_G618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLN_WT ) or ANLN_R431C .

Results: Experiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLN_R431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLN_R431C -expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLN_R431C -expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLN_R431C . Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.

Conclusions: The ANLN_R431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

Keywords: AKT; ANILLIN; ER-STRESS; MTOR; RAC1; focal segmental glomerulosclerosis.