Short term exposure to high glucose negatively regulates store-operated calcium channel proteins in mesangial cells.




Jiang, Hui
Chaudhari, Sarika
Zou, Shubiao
Huang, Linjing
Ma, Rong


Journal Title

Journal ISSN

Volume Title



Purpose: Glomerular mesangial cells (MCs) are an important target of metabolic abnormalities in diabetic environment. The Orai1-mediated store-operated calcium entry (SOCE) is associated with many physiological processes in a variety of cells, including MCs. However, whether SOCE in MCs is involved in diabetic kidney disease is not clear. High glucose (HG) is the principal cause of MC pathogenesis in diabetes and altered MC function by HG is central to the pathogenesis of progressive diabetic glomerulopathy. The present study was carried out to determine if HG treatment altered the protein content of Orai1 and the protein-mediated SOCE in MCs. Methods: Western blot was conducted to estimate abundance of Orai1 protein and Fura-2 fluorescence ratiometry was used to analyze SOCE. Results: We found that treatment of rat MCs with HG (25 mM) for time periods ranging from 2 hours to 24 hours decreased abundance of Orai1 protein. A significant decrease was observed at the time point of 8 hours, which sustained at least for additional 16 hours. Consistently, HG treatment for 8 hours significantly reduced SOCE. HG treatment for the same time periods did not alter the level of Orai1 transcript. In the presence of cycloheximide, a protein synthesis inhibitor, the HG effects on the level of Orai1 protein still existed, suggesting posttranslational mechanisms involved. Furthermore, both MG132 (the ubiquitin-proteasome inhibitor) and NH4Cl (the lysosomal pathway inhibitor) significantly attenuated the HG-induced reduction of Orai1 protein abundance. Moreover, HG treatment for 8 hours stimulated ubiquitination of Orai1 protein. We further found that HG treatment elevated the level of cellular hydrogen peroxide (H2O2) in a time-dependent manner. Treatment of cells with PEG-catalase significantly blunted the HG-induced reduction of Orai1 protein abundance. In addition, H2O2 itself also significantly decreased abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Conclusion: Taking together, these results suggest that HG treatment for a short-term, decreased the abundance of Orai1 protein in MCs by promoting its degradation through the ubiquitination-proteosome and –lysosome mechanisms. This HG-stimulated posttranslational regulation of Orai1 protein was mediated by H2O2.