Abstract
Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.
Highlights
The intrinsic apoptotic process of programmed cell death is a defense mechanism against cancer and plays a role in various aspects of tissue homeostasis and late embryonic development of the brain and kidneys [1]
Exposure to a high glucose concentration triggers apoptosis of renal epithelial cells in a time- and dose-dependent manner This study was performed on rat primary proximal tubular cells (PTCs), which mainly take up glucose via the sodium-dependent glucose transporter SGLT2 [29]
Since members of the Bcl-2 family often show a high degree of functional redundancy, we investigated whether Bad is required for apoptosis induced by exposure to high glucose concentration by silencing its expression in PTCs using siRNA (Fig. 4A, B)
Summary
The intrinsic apoptotic process of programmed cell death is a defense mechanism against cancer and plays a role in various aspects of tissue homeostasis and late embryonic development of the brain and kidneys [1]. It is inappropriately activated in certain diseases such as neurodegenerative diseases, where excessive apoptosis results in loss of neurons [2], and in type 2 diabetes, where the loss of pancreatic β-cells results in insulin deficiency [3]. Experience from failed trials indicates that future anti-apoptotic drug candidates must interfere with the apoptotic process upstream of caspase activation and the point of no return
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