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

Yujiro Maeoka; Takumi Okamoto; Yan Wu; Atsushi Saito; Rie Asada; Koji Matsuhisa; Miho Terao; Shuji Takada; Takao Masaki; Kazunori Imaizumi; Masayuki Kaneko

doi:10.1016/j.bbrc.2019.04.168

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

Biochem Biophys Res Commun. 2019 Jun 25;514(2):436-442. doi: 10.1016/j.bbrc.2019.04.168. Epub 2019 May 1.

Authors

Yujiro Maeoka¹, Takumi Okamoto², Yan Wu², Atsushi Saito³, Rie Asada⁴, Koji Matsuhisa², Miho Terao⁵, Shuji Takada⁵, Takao Masaki⁶, Kazunori Imaizumi⁷, Masayuki Kaneko⁸

Affiliations

¹ Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Nephrology, Hiroshima University Hospital, Hiroshima, Japan.
² Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
³ Department of Stress Protein Processing, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
⁴ Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, MO, USA.
⁵ Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan.
⁶ Department of Nephrology, Hiroshima University Hospital, Hiroshima, Japan.
⁷ Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: imaizumi@hiroshima-u.ac.jp.
⁸ Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: mkaneko@hiroshima-u.ac.jp.

PMID: 31053298
DOI: 10.1016/j.bbrc.2019.04.168

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.

Keywords: CRISPR/Cas9; Furosemide; Hypertonic stress; NFAT5; RNF183; Ubiquitin ligase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Down-Regulation / drug effects
Female
Furosemide / pharmacology
Gene Expression Regulation* / drug effects
Gene Knock-In Techniques
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Kidney Medulla / physiology*
Kidney Tubules, Collecting / metabolism*
Male
Mice
Transcription Factors / metabolism*
Ubiquitin-Protein Ligases / metabolism*

Substances

Nfat5 protein, mouse
Transcription Factors
Green Fluorescent Proteins
Furosemide
RNF183 protein, mouse
Ubiquitin-Protein Ligases