The negative feedback loop of NF-κB/miR-376b/NFKBIZ in septic acute kidney injury
Zhiwen Liu, Chengyuan Tang, Liyu He, Danyi Yang, Juan Cai, Jiefu Zhu, Shaoqun Shu, Yuxue Liu, Lijun Yin, Guochun Chen, Yu Liu, Dongshan Zhang, Zheng Dong
Zhiwen Liu, Chengyuan Tang, Liyu He, Danyi Yang, Juan Cai, Jiefu Zhu, Shaoqun Shu, Yuxue Liu, Lijun Yin, Guochun Chen, Yu Liu, Dongshan Zhang, Zheng Dong
View: Text | PDF
Research Article Nephrology

The negative feedback loop of NF-κB/miR-376b/NFKBIZ in septic acute kidney injury

Abstract

Sepsis is the leading cause of acute kidney injury (AKI). However, the pathogenesis of septic AKI remains largely unclear. Here, we demonstrate a significant decrease of microRNA-376b (miR-376b) in renal tubular cells in mice with septic AKI. Urinary miR-376b in these mice was also dramatically decreased. Patients with sepsis with AKI also had significantly lower urinary miR-376b than patients with sepsis without AKI, supporting its diagnostic value for septic AKI. LPS treatment of renal tubular cells led to the activation of NF-κB, and inhibition of NF-κB prevented a decrease of miR-376b. ChIP assay further verified NF-κB binding to the miR-376b gene promoter upon LPS treatment. Functionally, miR-376b mimics exaggerated tubular cell death, kidney injury, and intrarenal production of inflammatory cytokines, while inhibiting miR-376b afforded protective effects in septic mice. Interestingly, miR-376b suppressed the expression of NF-κB inhibitor ζ (NFKBIZ) in both in vitro and in vivo models of septic AKI. Luciferase microRNA target reporter assay further verified NFKBIZ as a direct target of miR-376b. Collectively, these results illustrate the NF-κB/miR-376b/NFKBIZ negative feedback loop that regulates intrarenal inflammation and tubular damage in septic AKI. Moreover, urinary miR-376b is a potential biomarker for the diagnosis of AKI in patients with sepsis.

Authors

Zhiwen Liu, Chengyuan Tang, Liyu He, Danyi Yang, Juan Cai, Jiefu Zhu, Shaoqun Shu, Yuxue Liu, Lijun Yin, Guochun Chen, Yu Liu, Dongshan Zhang, Zheng Dong

×

Figure 3

NF-κB mediates the downregulation of miR-376b in LPS-induced AKI.

Options: View larger image (or click on image) Download as PowerPoint
NF-κB mediates the downregulation of miR-376b in LPS-induced AKI. (A) Im...
(A) Immunofluorescence showing nuclear translocation of p65/NF-κB in renal tubule cells in LPS-treated mice. Images were collected by laser scanned confocal microscopy. Scale bar: 50 μm. (B) Immunoblots showing increases of p-p65 and p65 in kidney tissues of LPS-treated mice. GAPDH was used as internal control. (C) Immunoblots showing LPS-induced p-p65 and p65 in BUMPT cells. GAPDH was used as internal control. (D) qPCR analysis showing that LPS-induced decrease of miR-376b is prevented by TPCA-1. BUMPT cells were treated with 100 μM TPCA-1 and then treated with LPS. Total RNA samples were extracted after LPS treatment to quantify miR-376b by qPCR. The levels of miR-376b were normalized to the levels of sno202 of the same samples to determine the ratios. Data are expressed as mean ± SD (n = 4, 1-way ANOVA with Tukey’s multiple comparisons test), *P < 0.05. (E) ChIP analysis of NF-κB binding in miR-376b promoter. BUMPT cells were treated with or without LPS for 8 hours to collect the chromatin for immunoprecipitation with a specific anti–NF-κB antibody. The immunoprecipitated samples were subjected to qPCR analysis of miR-376b promoter sequence. Quantitative data are expressed as mean ± SD (n = 4, 2-tailed Student’s t test), *P < 0.05.