Abstract:
The kidneys play a pivotal role in the regulation of blood composition and osmolarity. This regulatory role is dependent heavily on the activity of the Na+-K+ ATPase, a pump that provides the driving force for the movement of various electrolytes and solutes. Renal ischemia reperfusion injury (IRI) was shown to be associated with a decrease in the expression of the ATPase and was reduced by sphingosine-1-phosphate (S1P). Because the sphingolipid and the Na+-K+ ATPase are both implicated in renal IRI, a cause effect relationship may exist. This work aims at investigating and determining the effect of S1P, through its analogue FTY720P, on the Na+-K+ ATPase activity, and at unraveling the signaling pathway involved, using the proximal tubule cells LLC-PK1 as a model. 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. The expression of S1P receptors (S1PR) in LLC-PK1 cells was studied using Western Blot analysis and revealed that all S1PRs were expressed. FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and at a dose of 80 nM. The activation of the Na+-K+ ATPase completely disappeared 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. The involvement of S1PR2 was confirmed by treating the cells with CYM 5520, a S1PR2 agonist that mimicked FTY720P’s activation. 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 both in presence and absence of wortmannin, an inhibitor of PI3K, indicating that PI3K is upstream NO. However, glyco-SNAP-1 and 8-bromo-
Description:
Thesis. M.S. American University of Beirut. Department of Biology, 2019. T:7088.
Advisor : Dr. Sawsan Kuraydiyyah, Professor, Biology ; Members of Committee : Dr. Colin Andrew Smith, Professor, Biology ; Dr. Khouzama Knio, Professor, Biology.
Includes bibliographical references (leaves 67-89)