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Abstract: FR-PO602

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

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Izumi, Yuichiro, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Eguchi, Koji, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Ono, Makoto, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Hiramatsu, Akiko, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Nakayama, Yushi, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Inoue, Hideki, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Nonoguchi, Hiroshi, Kitasato University Medical Center, Kitamoto, SAITAMA, Japan
  • Kakizoe, Yutaka, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Kuwabara, Takashige, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
  • Mukoyama, Masashi, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
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.