Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury
Christopher E. Pedigo, … , Sandra Merscher, Alessia Fornoni
Christopher E. Pedigo, … , Sandra Merscher, Alessia Fornoni
Published August 2, 2016
Citation Information: J Clin Invest. 2016;126(9):3336-3350. https://doi.org/10.1172/JCI85939.
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Research Article Inflammation

Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury

Abstract

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol–dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1–mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol–dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol–dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.

Authors

Christopher E. Pedigo, Gloria Michelle Ducasa, Farah Leclercq, Alexis Sloan, Alla Mitrofanova, Tahreem Hashmi, Judith Molina-David, Mengyuan Ge, Mariann I. Lassenius, Carol Forsblom, Markku Lehto, Per-Henrik Groop, Matthias Kretzler, Sean Eddy, Sebastian Martini, Heather Reich, Patricia Wahl, GianMarco Ghiggeri, Christian Faul, George W. Burke III, Oliver Kretz, Tobias B. Huber, Armando J. Mendez, Sandra Merscher, Alessia Fornoni

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Figure 7

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

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TNF causes NFAT activation and NFAT-mediated ABCA1 repression leading to...
(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.