Specific BK Channel Activator NS11021 Protects Rat Renal Proximal Tubular Cells from Cold Storage—Induced Mitochondrial Injury In Vitro

Abstract

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K+ (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury. We used normal rat kidney proximal tubular epithelial (NRK) cells as an in vitro model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 μM NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021–treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.

Pathological Roles of Mitochondrial Oxidative Stress and Mitochondrial Dynamics in Cardiac Microvascular Ischemia/Reperfusion Injury

“…Mitochondria are dynamic organelles, not static entities. Mitochondria can undergo replication, fission, and fusion; move from one location to another within cells; and form networks with other mitochondria or cellular structures to increase efficiency of ATP production as well as providing for intracellular signaling in response to physiological and pathological stimuli [115,116]. Increased fusion or reduced fission promotes the formation of elongated mitochondrial networks, whereas increased fission or reduced fusion causes mitochondrial fragmentation [117].…”

Mitochondrial Potassium Channels as Druggable Targets

“…NS13558, which is a derivative of NS11021, and to which BK Ca channels are insensitive, has the same property to uncouple the inner mitochondrial membrane and activate the respiratory chain. It is also interesting that NS11021 has a protective effect, despite its uncoupling properties on renal proximal tubular cells from cold storage [108]. It seems that some synergistic effects, in addition to the activation of mitochondrial potassium channels, also play a significant role.…”

The BK activator NS11021 partially protects rat kidneys from cold storage and transplantation-induced mitochondrial and renal injury

“…The protein expression of the pore-forming α subunit of the BK channel (BKα) was detected in rat kidney mitochondrial fraction protein lysates as a doublet of 100 kDa and 90 kDa bands ( Figure 1A ). Our recent study using rat renal cells and other reports have focused on the higher MW mitoBKα band observed (100 kDa, indicated by arrow) [ 18 – 19 , 24 ]. The lower MW 90 kDa mitoBKα band is of unknown identity and has not been observed in isolated mitochondria to our knowledge, though it may be a splice variant or a cleaved, incomplete form of mitoBKα.…”

“…Considering the endogenously-protective role that mitoBK channels have during IR-related injuries [ 19 , 27 – 28 ], we propose the loss of mitoBK channels contributes to CS+Tx-induced mitochondrial and renal injury. Accordingly, we anticipated that pharmacologically targeting the mitoBK channel to enhance its activity during CS with the use of NS11021 would be similarly protective in vivo as we recently reported using our renal cell CS+RW model [ 18 ].…”

“…Excitingly, we recently reported using an in vitro model of renal CS followed by rewarming (CS+RW) that pharmacological activation of the mitochondrial large (or Big) conductance Ca 2+- activated K + channel (mitoBK) during CS, by adding the specific BK activator NS11021 to the CS solution, protected rat kidney proximal tubular cells against CS+RW -induced mitochondrial injury (including respiratory dysfunction, superoxide production, and depolarization) and cell death in a mitoBK-dependent manner [ 18 ]. The mitoBK channel resides in the inner mitochondrial membrane (for review, see Balderas et al [ 19 ]) and has been identified in many cell types using in vitro and in vivo models.…”