The key role of NLRP3 and STING in APOL1-associated podocytopathy
Junnan Wu, … , Andreas Linkermann, Katalin Susztak
Junnan Wu, … , Andreas Linkermann, Katalin Susztak
Published October 15, 2021
Citation Information: J Clin Invest. 2021;131(20):e136329. https://doi.org/10.1172/JCI136329.
View: Text | PDF
Research Article Cell biology Nephrology

The key role of NLRP3 and STING in APOL1-associated podocytopathy

Abstract

Coding variants in apolipoprotein L1 (APOL1), termed G1 and G2, can explain most excess kidney disease risk in African Americans; however, the molecular pathways of APOL1-induced kidney dysfunction remain poorly understood. Here, we report that expression of G2 APOL1 in the podocytes of Nphs1rtTA/TRE-G2APOL1 (G2APOL1) mice leads to early activation of the cytosolic nucleotide sensor, stimulator of interferon genes (STING), and the NLR family pyrin domain–containing 3 (NLRP3) inflammasome. STING and NLRP3 expression was increased in podocytes from patients with high-risk APOL1 genotypes, and expression of APOL1 correlated with caspase-1 and gasdermin D (GSDMD) levels. To demonstrate the role of NLRP3 and STING in APOL1-associated kidney disease, we generated transgenic mice with the G2 APOL1 risk variant and genetic deletion of Nlrp3 (G2APOL1/Nlrp3 KO), Gsdmd (G2APOL1/Gsdmd KO), and STING (G2APOL1/STING KO). Knockout mice displayed marked reduction in albuminuria, azotemia, and kidney fibrosis compared with G2APOL1 mice. To evaluate the therapeutic potential of targeting NLRP3, GSDMD, and STING, we treated mice with MCC950, disulfiram, and C176, potent and selective inhibitors of NLRP3, GSDMD, and STING, respectively. G2APOL1 mice treated with MCC950, disulfiram, and C176 showed lower albuminuria and improved kidney function even when inhibitor treatment was initiated after the development of albuminuria.

Authors

Junnan Wu, Archana Raman, Nathan J. Coffey, Xin Sheng, Joseph Wahba, Matthew J. Seasock, Ziyuan Ma, Pazit Beckerman, Dorottya Laczkó, Matthew B. Palmer, Jeffrey B. Kopp, Jay J. Kuo, Steven S. Pullen, Carine M. Boustany-Kari, Andreas Linkermann, Katalin Susztak

×

Figure 3

APOL1-induced cytotoxicity of high-risk human podocytes is inflammasome and STING dependent.

Options: View larger image (or click on image) Download as PowerPoint
APOL1-induced cytotoxicity of high-risk human podocytes is inflammasome ...
(A) Experimental design: Low- (G0/G0) and high-risk (G1/G2) HUPECs treated with IFN-γ. (B) Representative Western blots and (C) densitometric quantification of APOL1, NLRP3, cleaved caspase-1, STING, phosphorylated STING, and actin of G0/G0 and G1/G2 cells treated with vehicle or 2 ng/mL IFN-γ for 24 hours (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (D) Cytotoxicity, measured by LDH release, was normalized to calcein absorbance as an indicator of live cell count. G0/G0 and G1/G2 cells were treated for 24 hours with vehicle (n = 5), 0.2 ng/mL IFN-γ (n = 6), or 2 ng/mL IFN-γ (n = 6). ***P < 0.001 vs. control. (E) Cytotoxicity in G1/G2 HUPECs. G1/G2 HUPECs were treated with 2 ng/mL IFN-γ for 24 hours in the presence of inhibitors of NLRP3 (MCC950 [MCC]), caspase-1 (Ac-YVAD-CHO [Cho] and VX765 [VX]), caspase-9 (LEHD [Leh]), necroptosis (NEC1s [Nec]), ferroptosis (Liproxstatin [Lip]), p38 MAPK (SB 203580 [p38]), autophagy (choloroquine [CQ]), and inducers of autophagy (STF66247 [STF] and rapamycin [Rapa]). n = 3. ###P < 0.001 vs. control; **P < 0.01, ***P < 0.001 vs. IFN-γ. (F) Change in intracellular Ca2+ (measured by FURA-2 AM fluorescence) presented as percentage change from baseline. Cells were treated with 0.2 ng/mL, 2 ng/mL, or 20 ng/mL IFN-γ for 8 hours (n = 3). *P < 0.05 vs. control-treated cells. (G) Relative calcineurin activity of G0/G0 and G1/G2 HUPECs treated with sham or the indicated concentrations of IFN-γ. **P < 0.01, ***P < 0.001 vs. control (n = 6); ###P < 0.001 vs. indicated group. (H) Cytotoxicity of G1/G2 HUPECs. G1/G2 cells were treated for 24 hours with 2 ng/mL or 20 ng/mL IFN-γ with or without pretreatment with 0.5 μM BAPTA in Ca2+-free HBSS for 2 hours (n = 6). ***P < 0.001 vs. control-treated cells; red-colored, *P < 0.05 vs. indicated group. Significance was determined by 1-way ANOVA and SNK post hoc test. Data are expressed as the mean ± SEM.