Abstract: TH-PO376

The Role of Chaperones and ROS in Trafficking of the mitoBK Channel in Response to Renal Cold Storage and Transplantation

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Shrum, Stephen A, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
  • MacMillan-Crow, Lee Ann, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
  • Parajuli, Nirmala, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
  • Tobacyk, Julia, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
Background

Patients with renal failure require a kidney transplant in order to avoid dialysis with high mortality. Deceased donor kidneys regularly undergo cold storage (CS) preservation while a recipient is matched. However, the process of CS damages kidneys and greatly increases the chance of transplant failure. Our lab and others have previously reported that renal CS induces oxidative stress, mitochondrial dysfunction, and renal dysfunction. The mitochondrial isoform of the large-conductance Ca2+-activated K+ channel (mitoBK) is dynamically involved with ROS and is a promising therapeutic target in ischemia-reperfusion injury. Mitochondrial chaperone proteins, such as mortalin, are involved in protein trafficking, thus our goal was to investigate how CS+Tx alters the mitoBK channel and if ROS and chaperones are involved.

Methods

Rat kidneys or rat renal proximal tubular (NRK) cells were exposed to CS for 18h, followed by transplantation into syngeneic recipient rats (in vivo; CS+Tx) or rewarming (in vitro; CS+RW). Protein expression of mitoBK and chaperones was evaluated by western blot. Immunocytochemistry and confocal microscopy were used to assess intracellular localization of mitoBK with co-labeled organelle-specific dyes. The mechanistic relationships between mitoBK, chaperone proteins, and ROS during CS were examined using knockdown (siRNA), overexpression (plasmid), and relevant pharmacological agents (activators, inhibitors).

Results

Results show that the mitoBK channel is expressed in rat kidney in vivo and in NRK cells in vitro and that CS reduces its expression. Interestingly, CS substantially alters the intracellular localization of mitoBK. In addition, mortalin expression was decreased after CS+RW or CS+Tx. Molecular studies suggest that mortalin and ROS are involved with trafficking of mitoBK due to CS. Ongoing experiments are further examining these relationships in the context of CS+Tx.

Conclusion

Our results suggest that CS-/+Tx alters expression and trafficking of mitoBK and that ROS and mortalin are involved. Future studies will address whether mitochondrial targeted antioxidants restore mitoBK channel expression which may be informative for investigating therapeutic interventions for renal CS+Tx.

Funding

  • Other NIH Support