Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5
Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5
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DOI：
10.1016/j.bbrc.2019.04.168
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发表时间：
2019-06-25
影响因子：
3.1
通讯作者：
Kaneko, Masayuki
中科院分区：
文献类型：
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作者：
Maeoka, Yujiro;Okamoto, Takumi;Kaneko, Masayuki
活化T细胞核因子5(NFAT5)直接与环指蛋白183(RNF183)基因启动子结合，并在高渗条件下诱导其转录。然而，目前还没有特异性的抗RNF183抗体用于免疫染色；因此，尚不清楚NFAT5是否调节RNF183在体内的表达，以及RNF183在肾脏中的定位。本研究利用CRISPR/Cas9介导的RNF183-绿色荧光蛋白(RNF183-GFP)敲入小鼠，研究了NFAT5对RNF183的体内表达和定位的调控作用。通过供体质粒的同源重组，将带有接头序列的绿色荧光蛋白引入RNF183外显子3开放阅读框的上游。GFP抗体免疫荧光染色显示，GFP信号从外髓向内髓逐渐增强，并与水通道蛋白-2共存。速尿治疗显著降低肾髓质中RNF183的表达，这与NFAT5蛋白和靶基因mRNA表达的降低一致。速尿对mIMCD-3细胞的mRNA表达和RNF183启动子活性没有影响。这些结果表明，RNF183主要在肾髓质集合管表达，速尿降低肾髓质张力可通过NFAT5下调RNF183的表达。(C)2019 Elsevier Inc.保留所有权利。
Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation. (C) 2019 Elsevier Inc. All rights reserved.