Fig. 7: Ouabain elicits anti-apoptotic effects through CaMKK1 signaling.

A CaMKK1 was silenced in rat proximal tubular cells (PTCs). RT-qPCR shows that mRNA expression of CaMKK1 is reduced by about 40% after siRNA treatment. N = 3. B Western blot of transfected cells showing downregulation of CaMKK1 by 35%. N = 3. C TUNEL assay of PTCs challenged with high glucose (HG) with or without ouabain and CaMKK1-targeting siRNA. Ouabain abolishes HG-induced apoptosis in control cells but not in those transfected with CaMKK1-targeting siRNA. N = 3. *p < 0.05 and **p < 0.01 vs. controls. D Schematic presentation of the proposed signaling cascade by which ouabain protects against apoptosis. Binding of ouabain to Na⁺,K⁺-ATPase results in concerted activation of the inositol 1,4,5-trisphosphate receptor (IP3R) and store-operated calcium channels regulated by STIM [34]. Calcium oscillations are subsequently generated by the endoplasmic reticulum. This activates CaM/Ca²⁺, which in turn binds to and activates CaMKK1 and/or CaMK2G. CaMKK1 and CaMK2G activate Akt via phosphorylation (T308 [23] and S473 [32], respectively). Akt phosphorylates Bad at S136, resulting in sequestration of Bad by 14-3-3 protein and inactivation of its pro-apoptotic properties.