Kidney Week

Abstract: FR-PO570

Development of Direct AQP2 Water Channel Inhibitors

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, 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

• Takahashi, Chisato, Dokkyo Ika Daigaku, Shimotsuga-gun, Tochigi, Japan

Chisato Takahashi, MD

• Harada, Shinya, Dokkyo Ika Daigaku, Shimotsuga-gun, Tochigi, Japan

Shinya Harada, MD

• Michizoe, Shotarou, Dokkyo Ika Daigaku, Shimotsuga-gun, Tochigi, Japan

Shotarou Michizoe, MD

• Abe, Makoto, Dokkyo Ika Daigaku, Shimotsuga-gun, Tochigi, Japan

Makoto Abe, MD

• Noda, Yumi, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Yumi Noda, MD, PhD

• Sasaki, Sei, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Sei Sasaki, MD

• Rai, Tatemitsu, Dokkyo Ika Daigaku, Shimotsuga-gun, Tochigi, Japan

Tatemitsu Rai, MD, PhD

Background

The regulation of urinary output is essential for the maintenance of body fluid homeostasis. Loss-of-function mutations of aquaporin-2 (AQP2) water channel cause renal diabetes insipidus and severe dehydration. In contrast, gain of function of AQP2 is implicated in the pathogenesis of congestive heart failure, liver cirrhosis, and SIADH. Inhibitors of AQP2 are expected to be potent pure water diuretics with few side effects, but their development has not been successful so far.

Methods

The objective of this study is to establish a drug screening system for the development of direct inhibitors of AQP2.
1. mpkCCDcl4 cells, which are immortalized cultured cells derived from renal collecting duct epithelial cells of SV40 large T cell antigen transgenic mouse, were utilized. The expression of AQP2 in mpkCCDcl4 cells and its cellular localization upon vasopressin stimulation were confirmed by Western blotting and fluorescent immunostaining.
2. mpkCCDcl4 cells were seeded on semipermeable filters (96-well polycarbonate Transwell plate, 0.4-µm pore size) to form an epithelial cell layer. Sucrose was added to the basolateral side to apply high osmotic pressure (Δ 850 mOsm). Water permeability was evaluated by the decrease in volume of the apical compartment.
3. Small molecule compounds in the compound library were applied to the apical compartment of this system to screen for the water permeability inhibitory effect of each compound. DMSO was applied as a negative control.

Results

1. mpkCCDcl4 cells expressed AQP2, which showed polarized trafficking to the apical plasma membrane in response to vasopressin stimulation.
2. In mpkCCDcl4 cells cultured in a 96-well Transwell plate, the volume of fluid transported from the apical to the basolateral side were 27.2±1.6% of the initial volume (mean±SD, n=8) in the presence of osmotic gradient. Osmotic water flux across the grown cells was inhibited by 0.3 mM HgCl₂.
3. Among the 156 compounds applied for initial screening, 13 compounds exerted inhibitory effect on osmotic water flux by under -2SD of the negative control.

Conclusion

An effective screening system was established to evaluate the water permeability of AQP2 using cultured cells. Initial screening of small-molecule compounds revealed several compounds with possible water permeability inhibitory effects.