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

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.

Risk and Resilience Factors During the COVID-19 Pandemic: A Snapshot of the Experiences of Canadian Workers Early on in the Crisis.

Research highlights several risk and resilience factors at multiple ecological levels that influence individuals' mental health and wellbeing in their everyday lives and, more specifically, in disaster or outbreak situations. However, there is limited research on the role of these factors in the early days of the COVID-19 crisis. The present study examined if and how potential risk factors (i.e., reduction in income, job insecurity, feelings of vulnerability to contracting the virus, lack of confidence in avoiding COVID-19, compliance with preventative policies) and resilience factors (i.e., trait resilience, family functioning, social support, social participation, and trust in healthcare institutions) are associated with mental health and well-being outcomes, and whether these resilience factors buffer (i.e., moderate) the associations between risk factors and said outcomes. One to two weeks after the government recommended preventative measures, 1,122 Canadian workers completed an online questionnaire, including multiple wellbeing outcome scales in addition to measures of potential risk and resilience factors. Structural equation models were tested, highlighting that overall, the considered risk factors were associated with poorer wellbeing outcomes, except social distancing which was associated with lower levels of stress. Each of the potential resilience factors was found to have a main effect on one or more of the wellbeing outcomes. Moderation analysis indicated that in general these resilience factors did not, however, buffer the risk factors. The findings confirm that the COVID-19 crisis encompasses several stressors related to the virus as well as to its impact on one's social, occupational, and financial situation, which put people at risk for lower wellbeing as early as one to two weeks after the crisis began. While several resilience factors emerged as positively related to wellbeing, such factors may not be enough, or sufficiently activated at that time, to buffer the effects of the numerous life changes required by COVID-19. From an ecological perspective, while mental health professionals and public health decision-makers should offer/design services directly focused on mental health and wellbeing, it is important they go beyond celebrating individuals' inner potential for resilience, and also support individuals in activating their environmental resources during a pandemic.