Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5

https://doi.org/10.1016/j.bbrc.2019.04.168Get rights and content

Highlights

  • RNF183-green fluorescent protein knock-in mice were generated by CRISPR/Cas9.

  • RNF183 is predominantly expressed in the renal medullary collecting ducts.

  • Decreased tonicity by furosemide reduces RNF183 expression via NFAT5 downregulation.

Abstract

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.

Introduction

In mammals, the renal medulla is constantly under hypertonic conditions to concentrate urine through water reabsorption [1]. Hyperosmolality of interstitial fluid formed by increased sodium chloride and urea concentrations regulates water reabsorption [2]. Nuclear factor of activated T-cells 5 (NFAT5), a tonicity-responsive enhancer binding protein, plays a key role in renal medulla homeostasis by adapting to hypertonic conditions [[3], [4], [5]]. NFAT5 promotes the transcription of aldose reductase (AR/AKR1B1) [5,6], sodium- and chloride-dependent betaine-gamma-aminobutyric acid (GABA) transporter (BGT1/SLC6A12) [4,5], sodium myo-inositol co-transporter (SMIT2/SLC5A3) [3,5], and heat shock protein 70 (HSP70/HSPA1B) [7,8], which mediate intracellular accumulation of sorbitol, betaine, and myo-inositol and function as intracellular molecular chaperones. Enhanced transcription of these genes is accompanied by hypertonicity-induced NFAT5 activation, which is a combination of its protein expression and nuclear translocation [4,9]. Therefore, NFAT5 levels in the renal medulla are extremely high under normal physiological conditions [10,11]. The loop diuretic furosemide can downregulate NFAT5 levels by inhibiting the Nasingle bondKsingle bondCl cotransporter type 2 (NKCC2), an important transporter controlling renal medullary interstitial salinity, in the thick ascending limb of Henle's loop (TALH) cells [11]. Thus, NFAT5 target gene expression is decreased by furosemide-induced NFAT5 downregulation [[11], [12], [13]].

Although ubiquitin ligase is involved in various cellular events, its role under hypertonic conditions remains unclear. RING finger protein 183 (RNF183) is a kidney-specific ubiquitin ligase [14]. We demonstrated that NFAT5 binds directly to the RNF183 promoter and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells [15]. Another study revealed that microRNA-7 binds directly to the 3′-untranslated region (3′-UTR) of RNF183 mRNA, resulting in its degradation and translational inhibition in human colon adenocarcinoma cells and colon tissues of colitic mice [16]. Of the two regulators, NFAT5 is considered to be a more important regulator of RNF183 in the normal kidney because microRNA-7 expression is extremely low in the kidney [17]. However, to date, whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney remain unclear. In this study, we generated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice using the CRISPR/Cas9 system and investigated NFAT5-regulated RNF183 expression and localization.

Section snippets

Construction of a donor plasmid

RNF183 homology arms were amplified from C57BL/6 murine genomic deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR) using the following primer sets:

  • arm1, 5′-GGATCCGAAGTAGGAAACTGTCACCC-3′ (forward) and 5′-AAAGTATACCCATGGAGTGC-3′ (reverse)

  • arm2, 5′-ACAGGGCCAGGAGCTCAGA-3′ (forward) and 5′-GAATTCTAGTTGGATGTCTGGGTGGT-3′ (reverse)

GFP and the linker region were amplified using the pEGFP-C1 vector (Clontech, CA, USA) by PCR using the 5′single bond

Generation of RNF183-GFP knock-in mice

Renal localization of RNF183 remains unclear because there is no specific anti-RNF183 antibody. Fig. 1A and B shows the generation of CRISPR/Cas9-mediated RNF183-GFP knock-in mice. Figs. 1A and S1 show the introduction of GFP with linker sequences upstream of the RNF183 open-reading frame in exon 3 by homologous recombination through the donor plasmid containing 1 kb of homology arms flanking GFP. PCR-based genotyping analysis of F1 mice tails demonstrated that the knock-in allele was detected

Discussion

In this study, we demonstrated that the loop diuretic furosemide decreases RNF183 and NFAT5 target gene mRNA expression (AKR1B1, BGT1, and SGK1) by altering the high salinity in the renal medulla in vivo. Moreover, we excluded the possibility that furosemide affects RNF183 mRNA levels and promoter activities in vitro. However, dehydration-induced hyperosmolality significantly increased AKR1B1 mRNA expression, whereas the upregulation of RNF183 and BGT1 mRNA expression was relatively weak. Based

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (KAKENHI: 18K06685, 17H06416, 17H01424, and 18H06105) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We appreciate the advice and expertise of Isao Naguro, Shigehiro Doi, Ayumu Nakashima, Toshiki Doi, Shuma Hirashio, and Kensuke Sasaki. Sincere appreciation is extended to Ayumu Nakashima for handling metabolic cage. We are grateful to Takeshi Ike, Yui Tanita, and Satoshi Hara for their assistance.

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