Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5
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 NaKCl 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:
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arm1, 5′-GGATCCGAAGTAGGAAACTGTCACCC-3′ (forward) and 5′-AAAGTATACCCATGGAGTGC-3′ (reverse)
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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′
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.
References (30)
- et al.
The role of the kidney in the maintenance of water balance
Baillieres Clin. Endocrinol. Metab.
(1989) - et al.
Transcription of the sodium/myo-inositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5'-flanking region
J. Biol. Chem.
(1998) - et al.
Identification and characterization of multiple osmotic response sequences in the human aldose reductase gene
J. Biol. Chem.
(1997) - et al.
Interstitial tonicity controls TonEBP expression in the renal medulla
Kidney Int.
(2009) - et al.
NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells
J. Biol. Chem.
(2019) - et al.
Expression of betaine transporter mRNA: its unique localization and rapid regulation in rat kidney
Kidney Int.
(1996) - et al.
Disruption of aldose reductase gene (Akr1b1) causes defect in urinary concentrating ability and divalent cation homeostasis
Biochem. Biophys. Res. Commun.
(2000) - et al.
Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat
Kidney Int.
(1998) - et al.
Urinary Concentrating Mechanism: Structure and Function
(1982) - et al.
Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity
Proc. Natl. Acad. Sci. U.S.A.
(1999)
Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression
Proc. Natl. Acad. Sci. U.S.A.
TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity
Mol. Cell Biol.
The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress
Exp. Mol. Med.
Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP
Am. J. Physiol. Cell Physiol.
Hydration status affects nuclear distribution of transcription factor tonicity responsive enhancer binding protein in rat kidney
J. Am. Soc. Nephrol.
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