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
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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

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Figure 7

miR-376b targets NFKBIZ in LPS induced septic AKI.

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miR-376b targets NFKBIZ in LPS induced septic AKI. (A) Conserved miR-376...
(A) Conserved miR-376b target sequence in the 3′-UTR of NFKBIZ mRNA. (B) Immunofluorescence showing the repressive effect of miR-376b on NFKBIZ expression. BUMPT cells were transfected with 200 nM miR-376b mimic or NC for 24 hours and then treated with or without 10 μg/mL LPS for 8 hours. (C) Immunoblot analysis showing the repressive effect of miR-376b on NFKBIZ expression. BUMPT cells were treated as in B to collect lysate for immunoblot analysis. GAPDH was used as internal control in immunoblot analysis. (D) Immunofluorescence showing the repressive effect of miR-376b on NFKBIZ expression. miR-376b mimic (7 mg/kg) or NC oligonucleotides were delivered to male C57BL/6 mice through tail vein injection 1 day before LPS injection. The mice were then injected intraperitoneally with 10 mg/kg LPS, while control mice were injected with normal saline. Kidney tissues were collected at 24 hours after LPS injection. Scale bar: 100 μm. (E) Immunoblot analysis showing the repressive effect of miR-376b on NFKBIZ expression. Mice were subjected to the same treatment as in D to collect kidney tissues for immunoblot analysis. GAPDH was used as internal control. (F) MicroRNA target reporter assay of Nfkbiz 3′-UTR. The putative miR-376b target sequence of the Nfkbizc 3′-UTR was cloned into the pMIR-REPORT vector. This and empty vector were transfected with miR-376b mimic or NC oligonucleotides to analyze luciferase activity. miR-376b mimic specifically reduced luciferase expression by pMIR-REPORT Nfkbiz 3′-UTR. Quantitative data are expressed as mean ± SD (n = 4 or n = 5, 2-tailed Student’s t test), *P < 0.05.