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Abstract: FR-PO597

Insulin Stimulates V-ATPase on Renal Proximal Tubules via the Akt/mTORC2 Pathway

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic


  • Nakamura, Motonobu, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Satoh, Nobuhiko, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Tsukada, Hiroyuki, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Mizuno, Tomohito, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Fujii, Wataru, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Horita, Shoko, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Nangaku, Masaomi, The University of Tokyo hospital, Bunkyoku, Tokyo, Japan
  • Suzuki, Masashi, Tokyo Gakugei University, Koganei, Tokyo, ToKyo, Japan

Maintaining an acid-base balance is essential for homeostasis. Acid-base transport in renal proximal tubules (PTs) is mainly sodium dependent and conducted coordinately by apical Na+/H+ exchanger (NHE)3, vacuolar H+-adenosine triphosphatase (V-ATPase) and basolateral Na+/HCO3- cotransporter (NBC)e1. V-ATPase on the PTs well-known to play an important role in proton excretion. Previously, we reported stimulation of PT sodium transport by insulin was mediated via Akt2 mTORC2 pathway. However, it is unclear whether insulin is involved in acid-base balance on the renal PTs. We hypothesized insulin may regulate V-ATPase on PTs.


We measured luminal V-ATPase activity in freshly isolated, split-opened mouse PTs by using a pH-sensitive dye BCECF. To uncover the signaling mechanism, we examined the effect of bafilomycin, Akt1/2 inhibitor VIII, an mTORC1 inhibitor rapamycin and an mTORC1/2 inhibitor, PP242. The measurement of V-ATPase activity was as follows; Freshly isolated and split-opened PTs in the chamber were first perfused in HEPES, and then the perfusate was switched to Na+- free HEPES. The intracellular pH recovery rates during perfusing with Na+-free HEPES were measured, and intracellular pH change was calculated during the initial 30 seconds as V-ATPase activity. To confirm the protein expression, protein phosphorylation was analyzed by Western blotting.


V-ATPase activity in PTs was markedly stimulated by insulin, and this stimulation was almost completely inhibited by bafilomycin, Akt inhibitor VIII, and PP242, but not by rapamycin. In freshly isolated mouse PTs, V-ATPase activity was increased approximately 20% by 1nM insulin above baseline and this stimulation was completely suppressed by Akt 1/2 inhibitor VIII. While PP242 completely suppressed the insulin-mediated V-ATPase stimulation in mouse PTs, rapamycin failed to affect the insulin effect. Insulin-induced phosphorylation of Akt in mouse renal cortex was completely suppressed by Akt1/2 inhibitor VIII and PP242, but not rapamycin.


Our results indicated that stimulation of V-ATPase activity by insulin in PTs is mediated via Akt2/mTORC2pathway. These results implicate the complex signaling in the proximal tubule acid-base balance, providing treatment targets for renal disease.


  • Government Support - Non-U.S.