Figure 7. TNF causes NFAT activation and NFAT-mediated ABCA1 repression leading to podocyte apoptosis.

(A) Quantitative RT-PCR analysis of regulator of calcineurin 1 (RCAN1) expression in podocytes exposed to sera from individual patients with FSGS (n = 6) and healthy patient (C) controls (n = 3). One-way ANOVA; *P < 0.05 vs. C. (B) TNF increases luciferase activity in mouse podocytes stably transfected with a cDNA coding for the luciferase gene under the control of an NFAT response element compared with untreated controls (n = 3). Two-tailed Student’s t test; *P < 0.05. (C) Quantitative RT-PCR analysis of RCAN1 expression in normal human podocytes exposed to TNF (n = 3) shows increased RCAN1 expression in TNF-treated podocytes (TNF) compared with untreated podocytes (C). Two-tailed Student’s t test; *P < 0.05. (D) Quantitative RT-PCR analysis of ABCA1 expression demonstrating that pretreatment of human podocytes for 1 hour with CsA and continued during TNF treatment (TNF + CsA) prevents TNF-mediated ABCA1 repression compared with TNF-treated cells (n = 4). One-way ANOVA; **P < 0.01. (E) Bar graph analysis showing that pretreatment with CsA and continued during TNF treatment prevents TNF-induced cleaved caspase 3 activity compared with TNF-treated cells (n = 4). One-way ANOVA; *P < 0.05, **P < 0.01. (F) Injection of murine recombinant TNF increases glomerular NFAT-mediated luciferase activity in an NFAT-luciferase reporter mouse model compared with vehicle-treated controls (C) 6 hours after injection (n = 4 per group). Two-tailed Student’s t test; ***P < 0.001. (G) The urinary albumin-to-creatinine ratio was significantly higher in TNF-injected BALB/cJ mice compared with C mice 6 hours after treatment. Pretreatment with CsA 24 hours before TNF administration significantly reduces albuminuria compared with TNF-treated mice (n = 5 per group). One-way ANOVA; **P < 0.01, ***P < 0.001.