Signaling Cascade Mediating the Effect of FTY720P on the Na+/K+ ATPase in LLC-PK1

Abstract

Background/Aims: In renal ischemia, the Na+/K+ ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na+/K+ ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na+/K+ ATPase using its analogue FTY720P and LLC-PK1 cells. Methods: The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis. Results: FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na+/K+ ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca2+ chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na+/K+ ATPase in absence of free Ca2+ revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular. Conclusion: The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.