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.

背景我们之前曾报道过，阿里林（ANLN）基因的突变会导致家族形式的FSGS。ANLN是一种F-肌动蛋白结合蛋白，可调节足细胞的运动，并通过缝隙隔膜衔接蛋白CD2相关蛋白（CD2AP）与磷酸肌醇3激酶（PI3K）途径相互作用。但是，尚不清楚ANLN突变如何导致FSGS表型。我们假设R431C突变通过将ANLN从CD2AP解偶联来发挥其致病作用。

方法我们在斑马鱼中进行了体内互补测定，以确定先前鉴定的错义ANLN变体，ANLN _R431C和ANLN _G618C在发育过程中的作用。我们还使用稳定表达野生型ANLN（ANLN _WT）或ANLN _R431C的人足细胞细胞系进行了体外功能测定。

结果在缺乏anln的斑马鱼胚胎中进行的实验表明，每种ANLN变体的功能丧失效应。在人足细胞系中，ANLN _R431C的表达增加了细胞迁移，增殖和凋亡。表达ANLN _R431C的足细胞的生化特征表明PI3K / AKT / mTOR / p70S6K / Rac1信号轴过度活化，_而表达ANLN _R431C的足细胞中mTOR驱动的内质网应激被_激活。mTOR的抑制，GSK-3 β，Rac1的，或钙调磷酸酶改善的效果ANLN _R431C。此外，抑制钙调神经磷酸酶/ NFAT途径可降低内源性ANLN和mTOR的表达。

结论该ANLN _R431C突变通过PI3K / AKT / mTOR的/的p70S6K / Rac1的信令的过度活化引起足细胞功能的多个紊乱。我们的发现表明，钙调神经磷酸酶在FSGS中的抑制作用可能部分归因于ANLN和mTOR的抑制。此外，这些研究表明，家族性FSGS的合理治疗目标可通过失调的足细胞表型的生化特征来确定。