Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury
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
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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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 5

Reduced ABCA1-mediated cholesterol efflux and reduced SOAT1 activity cause free cholesterol–mediated podocyte apoptosis.

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Reduced ABCA1-mediated cholesterol efflux and reduced SOAT1 activity cau...
(A) Cholesterol efflux is increased in empty vector podocytes (EV) in the presence of ApoA1. TNF treatment of EV results in reduced ABCA1-mediated cholesterol efflux to ApoA1. ABCA1-overexpressing podocytes (ABCA1OE) have increased cholesterol efflux to ApoA1 compared with EV, which remains unaffected by TNF treatment (n = 3). One-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. (B) TNF treatment of ABCA1OE does not result in increased caspase 3 activity compared with TNF-treated EV controls (n = 4). One-way ANOVA; *P < 0.05, **P < 0.01. (C) siABCA1 podocytes have reduced cholesterol efflux to ApoA1 compared with siCO (n = 3). One-way ANOVA; **P < 0.01, ***P < 0.001. (D) siABCA1 podocytes have increased esterified cholesterol mass compared with siCO (n = 3). Two-tailed Student’s t test; *P < 0.05. (E) Representative confocal image (original magnification, ×20) using Opera HCS to analyze neutral lipid droplet staining (right panels) and cell segmentation (left panels) in siCO (top panels) and siABCA1 podocytes (bottom panels). (F) Bar graph of the quantitative lipid droplet analysis using Acapella demonstrating increased spots per cell in siABCA1 cells compared with controls (n = 5). Two-tailed Student’s t test; *P < 0.05. (G) Bar graph of the quantitative analysis demonstrating that siABCA1 podocytes treated with SOAT1 inhibitor (SI) have a reduced number of lipid droplet spots per cell (n = 4) when compared with untreated control (C). Two-tailed Student’s t test; *P < 0.05. (H) SI increases cleaved caspase 3 activity in siABCA1 but not siCO podocytes (n = 3). One-way ANOVA; *P < 0.05. (I) Pretreatment with CD prevented SI-induced caspase 3 activity in siABCA1 podocytes (n = 4). One-way ANOVA; *P < 0.05, **P < 0.01.