Fig. 4

ITSN1 and ITSN2 are GEFs for Cdc42, regulating NS-related podocyte function, and Itsn2^−/− mice develop incompletely penetrant NS upon LPS injection. a Myc-tagged ITSN1-WT increased active Cdc42 levels upon overexpression in HEK293T cells as compared to mock, whereas three out of five ITSN1 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. b Active Cdc42 level was measured in COS7 cells. Myc-tagged ITSN2-WT overexpression increased active Cdc42 levels as compared to Mock. However, all three ITSN2 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. Error bars are defined as the standard error of at least four independent experiments. One-way ANOVA with Dunnet’s post hoc test vs. wild-type (WT) ITSN2 expressing control. c Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN1 expressing control. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN2 expressing control. Data are presented as the mean ± SEM. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Kidneys of WT and Itsn2 ^L−/L− mice were stained with periodic acid Schiff. The glomeruli of Itsn2 ^L−/L− mice showed normal findings, similar to WT. f Urine albumin of Itsn2 ^L−/L−, Itsn2 ^+/L−, and WT mice was measured before and after LPS injection. Albuminuria level was increased in Itsn2 ^L−/L− mice from 12 to 48 h after LPS injection compared with WT and Itsn2 ^+/L− mice. Two-tailed Student’s t-tests. Data are presented as the mean ± SEM. *P < 0.05. g Representative electron microscopy images. LPS injection-induced foot process (FP) effacement in both WT and Itsn2 ^L−/L− mice within 24 h. At 48 h after LPS injection, FP effacement was still observed in Itsn2 ^L−/L− mice, although WT mice recovered from FP effacement