Stimulatory effect of insulin on H+-ATPase in the proximal tubule via the Akt/mTORC2 pathway

Abstract

PurposeAcid-base transport in renal proximal tubules (PTs) is mainly sodium-dependent and conducted in coordination by the apical Na+/H+ exchanger (NHE3), vacuolar H+-adenosine triphosphatase (V-ATPase), and the basolateral Na+/HCO3- cotransporter. V-ATPase on PTs is well-known to play an important role in proton excretion. Recently we reported a stimulatory effect of insulin on these transporters. However, it is unclear whether insulin is involved in acid-base balance in PTs. Thus, we assessed the role of insulin in acid-base balance in PTs.MethodsV-ATPase activity was evaluated using freshly isolated PTs obtained from mice, and specific inhibitors were then used to assess the signaling pathways involved in the observed effects.ResultsV-ATPase activity in PTs was markedly enhanced by insulin, and its activation was completely inhibited by bafilomycin (a V-ATPase-specific inhibitor), Akt inhibitor VIII, and PP242 (an mTORC1/2 inhibitor), but not by rapamycin (an mTORC1 inhibitor). V-ATPase activity was stimulated by 1 nm insulin by approximately 20% above baseline, which was completely suppressed by Akt1/2 inhibitor VIII. PP242 completely suppressed the insulin-mediated V-ATPase stimulation in mouse PTs, whereas rapamycin failed to influence the effect of insulin. Insulin-induced Akt phosphorylation in the mouse renal cortex was completely suppressed by Akt1/2 inhibitor VIII and PP242, but not by rapamycin.ConclusionOur results indicate that stimulation of V-ATPase activity by insulin in PTs is mediated via the Akt2/mTORC2 pathway. These results reveal the mechanism underlying the complex signaling in PT acid-base balance, providing treatment targets for renal disease.

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via PPARγ-dependent pathway

“…Effects of azPC on luminal NHE activity in isolated rat PTs Next, we focused on luminal NHE activity in freshly isolated rat PTs. Luminal NHE activity was measured using lumen-opened PTs, as previously described (22,(31)(32)(33). The stimulatory effects of azPC on luminal NHE activity were observed in a dose-dependent manner (Figures 2A and S2), similar to NBCe1 activity.…”

“…Luminal NHE activity was determined as previously described (22,31,33). Briefly, the PT (S2 was not certified by peer review) is the author/funder.…”

“…Luminal NHE activity was determined as previously described (22,31,33). Briefly, the PT (S2 segment) fragment was freshly isolated in the same way as the measurements of NBCe1 activity, and they were attached to a glass coverslip with Cell-tak glue (Corning, One Riverfront Plaza, NY, USA).…”

“…The lumenopened PT was incubated with BCECF/AM in HEPES-buffered solution (144 mM Na + , 5 mM K + , 1.5 mM Ca 2+ , 1 mM Mg 2+ , 137 mM Cl -, 2 mM H 2 PO 4 -, 1 mM SO 4 2-, 5.5 mM glucose, 25 mM HEPES, adjusted to pH 7.4) (31,55) for 10 minutes, and pH i was monitored with MetaFluor 7.7 software. A pre-warmed (38 °C) HEPES-buffered solution was used for the bath perfusate, and 200 nM bafilomycin A1 (FUJIFILM Wako Pure Chemical) was added to block the effect of V-ATPase on PT transport (31,33). The perfusate was repeatedly switched from HEPES-buffered solution to an isotonic Na + -free solution (144 mM N-methyl-D-glucamine, 5 mM K + , 1.5 mM Ca 2+ , 1 mM Mg 2+ , 137 mM Cl -, 2 mM H2PO4 -, 1 mM SO4 2-, 5.5 mM glucose, 25 mM HEPES, adjusted to pH 7.4) in the absence and presence of azPC or other chemical agents such as two NHE inhibitors EIPA (Research Biochemicals Incorporated, Natick, MA, USA) at 100 µM and cariporide (Santa Cruz Biotechnology, Dallas, TX, USA) at 1 µM and GW9662 at 5 µM and PD98059 at 10 µM.…”

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway

“…Measurements of luminal NHE activity in renal PTs from rats, mice, and humans Luminal NHE activity was determined as previously described (22,31,33). Briefly, the PT (S2 segment) fragment was freshly isolated in the same way as the measurements of NBCe1 activity, and they were attached to a glass coverslip with Cell-tak glue (Corning, One Riverfront Plaza, NY, USA).…”