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Exaggerated Store‐Operated Calcium Entry: A Mechanism for High Glucose‐induced Podocyte Injury and Mitochondria Damage
Author(s) -
Tao Yu,
Chaudhari Sarika,
Yazdizadeh Shotorbani Parisa,
Ma Rong
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r2114
Subject(s) - podocyte , orai1 , microbiology and biotechnology , mitochondrion , chemistry , thapsigargin , calcium signaling , voltage dependent calcium channel , medicine , calcium , biology , endocrinology , signal transduction , stim1 , extracellular , kidney , endoplasmic reticulum , organic chemistry , proteinuria
Purpose and hypothesis Podocyte injury induced by hyperglycemia is the key factor contributing to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction leads to DN‐associated podocyte injury. However, the mechanism of podocyte mitochondria injury by high glucose (HG) is poorly understood. Store‐operated calcium entry (SOCE) is essential for multiple cell functions in both excitable and non‐excitable cells. However, the role of this Ca2+ signaling in podocytopathy remains unknown. The present study was carried out to test the hypothesis that enhanced SOCE mediated HG‐induced podocyte injury and mitochondria damage. Methods All experiments were carried out using cultured immortalized human podocytes. The conventional whole‐cell patch‐clamp was performed to measure Orai1 channel currents. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Mitochondrial function was evaluated by measuring: 1) the mitochondria membrane potential (MMP) using TMRE fluorescence; 2) generation of mitochondria reactive oxygen species (ROS) using MitoSox Red Mitochondrial Superoxide Indicator; 3) ATP production using ATP assay kit. In addition, CRISPR‐CAS 9 approach was used to delete Orai1 in podocytes. Results Thapsigargin (TG, 1 µM), the activator of Orai1 channel induced robust inward currents which were blocked by La3+ (2 µM), an inhibitor of Orai1 channel. Ang II (1 µM) also evoked inward currents, which were significantly blunted by BTP2 (10 µM), an Orai1 channel blocker. Orai1 protein abundance significantly increased by HG (25 mM) treatment for the time periods ranging from 2 to 12 h. This HG induced Orai1 increase was dose‐dependent. Consistently, Ca2+ imaging experiment showed that HG treatment for 12 h significantly increased podocyte SOCE. Thus, the biochemical and functional data suggested an enhancement of SOCE by HG. Furthermore, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F‐actin, an indication of podocyte injury. This HG response was significantly blunted by BTP2 and Orai1 CRISPR‐CAS 9 lentivirus. Moreover, HG treatment significantly decreased podocyte MMP, ATP production and increased mitochondrial ROS generation, all of which were significantly inhibited by BTP2. Furthermore, activation of SOCE by TG significantly increased mitochondrial ROS generation, which was prevented by pre‐treatment of BTP2. Conclusion Enhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.