Kidney Week

Abstract: FR-PO602

Regulation of Rhcg Protein in the Intercalated Cells of the Outer Medullary Collecting Duct by Aldosterone

Session Information

• Fluid and Electrolytes: Basic - I
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Izumi, Yuichiro, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Yuichiro Izumi,

• Eguchi, Koji, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Koji Eguchi,

• Ono, Makoto, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Makoto Ono,

• Hiramatsu, Akiko, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Akiko Hiramatsu,

• Nakayama, Yushi, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Yushi Nakayama,

• Inoue, Hideki, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Hideki Inoue,

• Nonoguchi, Hiroshi, Kitasato University Medical Center, Kitamoto, SAITAMA, Japan

Hiroshi Nonoguchi,

• Kakizoe, Yutaka, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Yutaka Kakizoe,

• Kuwabara, Takashige, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Takashige Kuwabara,

• Mukoyama, Masashi, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

Masashi Mukoyama,

Background

Rhcg, an ammonia transporter, is expressed mainly in the intercalated cells (ICs) of the collecting duct. Serum potassium level regulates the expression of Rhcg. Aldosterone level is increased in metabolic acidosis; however, direct effects of aldosterone on the Rhcg expression remain obscure.

Methods

C57BL/6J mice were divided into four groups: 1) sham-operation, 2) sham and NH₄Cl drinking (Sham-NH₄Cl), 3) adrenalectomy and NH₄Cl drinking (ADX-NH₄Cl), and 4) adrenalectomy with continuous administration of aldosterone and NH₄Cl drinking (ADX-Aldo-NH₄Cl). Mice were sacrificed three days after NH₄Cl drinking. Urinary ammonium excretion and localizations of Rhcg and ubiquitin were examined. For in vitro experiments, IN-IC cells, which we previously established as an intercalated cell-derived cell line, that stably express flag-tagged Rhcg (Rhcg-flag) were generated. The effects of aldosterone, spironolactone, Go6983 (a PKC inhibitor) and MG132 (a proteasome inhibitor) on the expression of Rhcg-flag were examined.

Results

NH₄Cl-induced increase in urinary ammonium excretion was less in ADX-NH₄Cl mice than that in Sham-NH₄Cl mice. Aldosterone increased ammonium excretion in ADX-Aldo-NH₄Cl mice as much as that in Sham-NH₄Cl mice. Immunohistochemical study revealed that NH₄Cl drinking increased the accumulation of Rhcg at apical membrane of the ICs of the outer medullary collecting duct in Sham-NH₄Cl mice. Adrenalectomy and aldosterone decreased and increased the accumulation of Rhcg at apical membrane of the ICs in ADX-NH₄Cl and ADX-Aldo-NH₄Cl mice, respectively. Ubiquitin expression was not changed between in PCs and ICs of Sham-NH₄Cl mice, while it was less in ICs than in PCs in ADX-NH₄Cl mice. Aldosterone restored the ubiquitin expression in ICs. In in vitro experiment, Western blotting showed that aldosterone increased the expression of Rhcg-flag in the membrane fraction of IN-IC cells. Spironolactone and Go6983 each inhibited the expression of Rhcg-flag. Treatment with MG132 increased the expression of Rhcg-flag in whole cell lysate.

Conclusion

Aldosterone could directly regulate Rhcg expression through the mineralocorticoid receptor-PKC pathway. Furthermore, aldosterone could modulate Rhcg expression through the ubiquitin-proteasome pathway.

Funding

• Government Support - Non-U.S.