Lactate controls glomerular endothelial barrier integrity in lupus nephritis
Jiaxin Lei, Xingyu Zhai, Yixin Wang, Ying Li, Lei Li, Mengdi Liu, Jing Guo, Lingyi Li, Zhezhuyun Chen, Qinghua Cao, Zhichun Liu, Ting Liu, Lin Xu, Zhenke Wen
Jiaxin Lei, Xingyu Zhai, Yixin Wang, Ying Li, Lei Li, Mengdi Liu, Jing Guo, Lingyi Li, Zhezhuyun Chen, Qinghua Cao, Zhichun Liu, Ting Liu, Lin Xu, Zhenke Wen
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Research Article Nephrology

Lactate controls glomerular endothelial barrier integrity in lupus nephritis

Abstract

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease that affects multiple organs and tissues, with lupus nephritis (LN) as one of its most severe complications. Although LN progression is associated with compromised permeability of human renal glomerular endothelial cells (HRGECs), the underlying mechanisms are not fully defined. Herein, we demonstrate that aberrant glycolysis drives this glomerular endothelial barrier defect by suppressing the transcription of tight junction (TJ) genes. Mechanistically, circulating self-DNA in SLE plasma acts as a ligand that activates the cGAS/STING pathway in HRGECs, driving aberrant glycolytic adaption. The resulting glycolytic product, lactate, serves as a substrate for protein lactylation, leading to extensive lactylation and subsequent ubiquitination of enhancer of zeste homolog 2 (EZH2). In consequence, EZH2 deficiency results in reduced H3K27me3 levels, thereby suppressing the transcription of TJ genes. In a self-DNA–induced SLE model, inhibition of cGAS/STING signaling or lactate production effectively restored the integrity of TJs of HRGECs and concurrently alleviated key LN symptoms. Together, lactate programs lactylation and ubiquitination of EZH2 to impair the glomerular endothelial barrier in human SLE.

Authors

Jiaxin Lei, Xingyu Zhai, Yixin Wang, Ying Li, Lei Li, Mengdi Liu, Jing Guo, Lingyi Li, Zhezhuyun Chen, Qinghua Cao, Zhichun Liu, Ting Liu, Lin Xu, Zhenke Wen

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Figure 4

Lactate accumulation induces the ubiquitination of EZH2 in HRGECs.

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Lactate accumulation induces the ubiquitination of EZH2 in HRGECs. (A an...
(A and B) ECs were treated with 10% plasma from HCs or patients with SLE for 24 hours, with or without the addition of MG132 (2 μM) during the final 4 hours of culture. EZH2 protein levels (A) and mRNA levels of TJ-related proteins (B) were analyzed by Western blot and qPCR, respectively. Mean ± SEM from 6 individuals in each group (B). (C) ECs were treated with 10% plasma from HCs or patients with SLE for 24 hours, with or without bafilomycin A1 (BafA1, 100 nM, 4 hours). EZH2 protein levels were assessed by Western blot. (D) IP analysis of EZH2 ubiquitination was performed in ECs after 24 hours’ treatment with 10% plasma from HC or SLE individuals. (E) HA-EZH2 stable ECs were cultured with or without lactic acid (10 mM) for 24 hours, followed by IP analysis of EZH2 ubiquitination. (F) IP analysis of EZH2 ubiquitination was performed in HA-EZH2 stable ECs stimulated with 10% plasma from HCs or patients with SLE for 24 hours, with or without 40 μM GSK2837808A cotreatment. (G) Co-IP was performed to assess the interaction between EZH2 and RACK1 in HRGECs. (H) HA-EZH2 stable ECs were transfected with RACK1-targeting siRNA or control siRNA, then treated with 10% SLE plasma for 24 hours. Subsequent IP analysis evaluated EZH2 ubiquitination in these cells. (I and J) HRGECs were transfected with RACK1-targeting siRNA or control siRNA, followed by 24 hours’ treatment with 10% SLE plasma. Western blot analysis was used to determine the protein levels of EZH2 (I) and H3K27me3 (J). H3 was used as the loading control for normalization (J). (K and L) HRGECs were transfected with RACK1-targeting siRNA or control siRNA, followed by 24 hours’ stimulation with 10% SLE plasma. The expression levels of TJ-associated proteins were examined via qPCR (K) and Western blot (L). Data are mean ± SEM (5 individuals per group for K). (M) Co-IP assays were conducted in HA-EZH2 stable ECs treated with 10% plasma from HC or SLE individuals to evaluate the interaction between HA-EZH2 and RACK1. (N) Co-IP was performed to assess the interaction of EZH2 with RACK1 in HRGECs after culture with or without lactate (40 mM). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 with 2-way ANOVA plus Tukey’s method (B and K).