Journal of the American Society of Nephrology
Basic Research
The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes
Hall, Gentzon1,2,3; Lane, Brandon M.1,2; Khan, Kamal4; Pediaditakis, Igor4; Xiao, Jianqiu4; Wu, Guanghong1,3; Wang, Liming3; Kovalik, Maria E.1,2,3; Chryst-Stangl, Megan1,2; Davis, Erica E.1,4; Spurney, Robert F.3; Gbadegesin, Rasheed A.1,2,3
Departments of 1Pediatrics and
3Medicine, Duke University School of Medicine, Durham, North Carolina;
2Duke Molecular Physiology Institute, Durham, North Carolina; and
4Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina
G.H. and B.M.L. contributed equally to this work.
Correspondence: Dr. Rasheed A. Gbadegesin, Department of Pediatrics, Duke University Medical Center, Duke University, T-Level RM0909 CHC, BOX 3959, Durham, NC 27710. Email: [email protected]
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, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.
Results
Experiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.
Conclusions
The ANLNR431C 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.