Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca²⁺-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca²⁺-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca²⁺-concentration ([Ca²⁺]_i) by Fura-2-fluorescence, and SOCE from increase of [Ca²⁺]_i following re-addition of extracellular Ca²⁺ after store depletion with thapsigargin (1 μM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 μM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca²⁺-entry and osteogenic signaling in HAoSMCs.

糖尿病和慢性肾脏病（CKD）都触发血管成骨信号和钙化，导致心血管事件导致早期死亡。成骨信号传导涉及转录因子CBFA1，MSX2和SOX9以及碱性磷酸酶（ALP）的上调，碱性磷酸酶是通过降解钙化抑制剂焦磷酸盐来促进钙化的酶。在CKD中，成骨信号由高磷血症触发，高磷血症可上调血清和糖皮质激素诱导的激酶SGK1，这是Ca ²⁺通道ORAI1的强刺激剂。该通道由STIM1激活并完成存储操作的Ca ²⁺-条目（SOCE）。本研究探讨了人主动脉平滑肌细胞（HAoSMCs）暴露于高细胞外葡萄糖浓度是否同样上调ORAI1和/或STIM1表达，SOCE和成骨信号。为此，HAoSMCs暴露于高的细胞外葡萄糖浓度（15 mM，24小时），而没有或另外暴露于磷酸盐供体β-甘油磷酸盐。使用qRT-PCR评估转录物水平，使用Western印迹法评估蛋白质丰度，使用比色测定试剂盒评估ALP活性，使用茜素红染色的钙沉积物，通过Fura-2-荧光检测胞浆中的Ca 2 ^+-浓度（[Ca ²⁺ ] _i）和[Ca ²⁺ ] _i的增加引起的SOCE用毒胡萝卜素（1μM）耗尽后重新添加细胞外Ca ²⁺之后。结果，葡萄糖提高了SGK1和ORAI1，ORAI2和STIM2的转录水平，ORAI1，SOCE的蛋白质丰度，CBFA1，MSX2，SOX9和ALPL的转录水平以及钙沉积物。此外，葡萄糖显着增强了β-甘油磷酸对SGK1和ORAI1转录水平的刺激作用，SOCE，成骨标记物的转录水平以及钙沉积物。ORAI1抑制剂MRS1845（10μM）显着减弱了葡萄糖诱导的CBFA1和MSX2转录水平的上调。总之，糖尿病的高血糖会刺激SGK1和ORAI1的表达，从而增强HAoSMCs的存储操纵性Ca ²⁺进入和成骨信号传导。