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Exaggerated Store-Operated Calcium Entry: Mechanism Contributes to High Glucose-induced Podocyte Injury and Mitochondria Damage

Purpose: Podocyte injury induced by hyperglycemia is the key factor contributes to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction is involved in the pathogenesis of DN-induced podocyte injury. However, the mechanism of podocyte mitochondria injury in response to high glucose (HG) is poorly understood. Store-operated calcium entry (SOCE) is the Ca2+ signaling essential for multiple cell functions in both excitable and non-excitable cells. However, the role of this Ca2+ entry signaling in podocyte injury and mitochondria damage remains unknown. The aim of the present study was to determine if enhanced SOCE mediated HG induced podocyte injury and mitochondria damage.

Methods: All experiments were carried out using cultured immortalized human podocytes (HPCs). The conventional whole-cell voltage-clamp configuration was performed to examine if the Orai1 channel is functional in podocytes. Western blot was performed to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate the functional change of the podocytes Orai1 channels in response to HG. Confocal microscopy was used to visualize podocyte actin arrangement. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP). MitoSox Red mitochondrial Superoxide Indicator was used to probe podocyte mitochondria reactive oxygen species (ROS) generation. Podocyte ATP production was determined by ATP assay kit. Orai1 CRISPR-CAS 9 lentivirus was delivered to genetically inhibit SOCE, whereas BTP2 was used to pharmacologically inhibit SOCE.

Results: Thapsigargin (TG), a well-known activator of SOC induced robust inward currents which were greatly reversed by La3+ (2 µM), an inhibitor of SOC. Ang II treatment (1 µM) invoked marked inward currents, which can be significantly, but not completely blocked by BTP2 (10 µM), an SOC blocker, suggesting a component of SOC currents. Orai1, but not STIM1 protein abundance significantly increased in response to HG (25mM) for time periods ranging from 2 to 12 hours. This HG induced Orai1 increase was dose-dependent. Besides, Ca2+ imaging experiment showed that 25mM HG treatment for 12 hours significantly increased podocyte SOCE. In addition, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F-actin. This HG responses were significantly blunted by BTP2 (4 µM) and Orai1 CRISPR-CAS 9 lentivirus. Furthermore, HG treatment (25mM for 24 hours) decreased podocyte MMP, ATP production and increased ROS generation, which can be blunted by BTP2 (4 µM) treatment. SOCE activation by TG (10min) significantly increased podocyte ROS generation which can be prevented by BTP2 (10 µM).

Conclusion: The present study suggests that enhanced SOCE contributes to HG induced podocyte injury and mitochondria damage.