FIG. 7.
Nephrin-mediated PI3K/AKT activation is associated with inhibition of apoptotic cell death. (A to D) The MDCK cell model of anoikis was used to test whether nephrin-stimulated AKT activity was associated with an inhibition of apoptosis. MDCK cells were retrovirally transduced to express nephrin and subjected to apoptosis assays (DNA laddering [A] and quantitative caspase 3 activity [B to D]) as described in Materials and Methods. (A) DNA-laddering assay. Lane 1, DNA marker; lane 2, control cells without detachment; lane 3, anoikis in cells transduced with control virus; lane 4, anoikis in cells transduced with nephrin retrovirus. (B) Time course of quantitative caspase 3 activity in cells transduced with control virus (n = 3; shaded circles) and nephrin-transduced cells (n = 3; solid circles). The inset shows that these cells express nephrin. FLAG-tagged full-length nephrin (Nephrin.F), expressed in 293T cells, served as a positive control for the blot. The error bars indicate SEM. (C) Statistical analysis of the nephrin effect on quantitative caspase 3 activity after induction of anoikis (n = 6; **, P < 0.01). (D) Effect of wortmannin treatment (100 nM; 120 min) (+) on quantitative caspase 3 activity in control and nephrin-expressing cells (n = 4; *, P < 0.05). (E) Nephrin expression and constitutively active AKT inhibit detachment-induced apoptosis in differentiated mouse podocytes. Mouse podocytes were retrovirally transduced to stably express nephrin or a constitutively active mutant of AKT. The differentiated mouse podocytes were detached and kept in suspension culture for 4 h. Equal amounts of protein from cell lysates were subjected to caspase 3 activity assays as described in Materials and Methods. Depicted is a statistical analysis of six (control [open column] versus nephrin [solid column]) and four (control [open column] versus constitutively active AKT [shaded column]) independent experiments (**, P < 0.01). (F) Nephrin expression maintains AKT and Bad phosphorylation after induction of anoikis. Differentiated control or nephrin-expressing podocytes were detached and kept in suspension culture for 4 h. Phosphorylation of AKT at serine-473 (pSer473; top) and Bad at serine-112 (middle) were monitored with phosphospecific antibodies; equal loading of proteins was confirmed by reprobing the membrane with an antitubulin antibody (bottom). (G) Increased susceptibility of cd2ap−/− podocytes to detachment-induced apoptosis. cd2ap+/+ and cd2ap−/− podocytes were differentiated, detached from the cell culture dish, and kept in suspension culture (2 h [left]; 4 h [right]). Programmed cell death was evaluated by annexin V binding as described in Materials and Methods (**, P < 0.01, and ***, P < 0.001 compared to cd2ap+/+ cells; n = 3). +, present; −, absent.