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

BACKGROUND

We previously reported that mutations in the anillin () 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 mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.

METHODS

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

RESULTS

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

CONCLUSIONS

The 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.