Kidney Week

Abstract: TH-PO0029

Hemagglutinin (HA)-Tagged Vasopressin 2 Receptor (V2R) Knock-In Mice Enable In Vivo Visualization of Transcellular Trafficking of V2R

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
  October 22, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Nagaoka, Kanako, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Kanako Nagaoka, MD

• Takami, Jun, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Jun Takami, MD

• Fujiki, Tamami, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Tamami Fujiki, MD

• Kikuchi, Hiroaki, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Hiroaki Kikuchi, MD, PhD

• Mandai, Shintaro, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Shintaro Mandai, MD, PhD, FASN

• Mori, Yutaro, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Yutaro Mori, MD, PhD, FASN

• Mori, Takayasu, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Takayasu Mori, MD, PhD

• Susa, Koichiro, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Koichiro Susa, MD, PhD

• Sohara, Eisei, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Eisei Sohara, MD, PhD

• Ando, Fumiaki, Tokyo Kagaku Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo, Tokyo, Japan

Fumiaki Ando, MD, PhD

Background

The vasopressin type 2 receptor (V2R) undergoes agonist-induced internalization from the basolateral membrane and forms a stable complex with β-arrestin, enabling sustained cAMP generation within endosomes. However, in vivo V2R internalization in renal collecting ducts has not been precisely characterized due to the lack of specific antibodies against V2R. We generated HA-tagged V2R knock-in (KI) mice to analyze endogenous V2R trafficking.

Methods

An HA-tag was inserted into the endogenous Avpr2 (V2R) locus using CRISPR/Cas9. Physiological receptor function was evaluated by blood chemistry and urinary concentrating response to dDAVP. V2R localization and internalization were assessed by sucrose density gradient fractionation, immunofluorescence microscopy, and electron microscopy. Candidate V2R-interacting proteins were identified by interactome profiling in mpkCCD cell lines stably expressing HA-V2R.

Results

HA-V2R KI mice were viable and showed no polyuria or weight loss. Blood chemistry and a dDAVP challenge test revealed preserved urinary concentrating ability without significant electrolyte abnormalities, indicating that the HA-tag did not impair V2R function. Immunofluorescence microscopy and sucrose density gradient fractionation showed dDAVP-dependent redistribution of V2R to Rab11-positive recycling endosomes. Electron microscopy revealed that HA-V2R was internalized beyond the perinuclear region and reached the subapical region of collecting duct cells. Interactome profiling using mpkCCD cell lines identified candidate V2R-interacting proteins, including vimentin and ARP3.

Conclusion

HA-V2R KI mice enable direct in vivo visualization of endogenous V2R trafficking and its distribution across membrane fractions in renal collecting ducts, thereby serving as a novel tool for investigating the pathophysiology of urinary concentration disorders.