Wang Shu, Wu Mingyu, Qin Ling, Song Yaxiang, Peng Ai
Nephrology and Rheumatology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
PeerJ. 2020 Jun 19;8:e9203. doi: 10.7717/peerj.9203. eCollection 2020.
Hyperphosphatemia, which is a high inorganic phosphate (Pi) level in the serum, promotes endothelial cells dysfunction and is associated with cardiovascular diseases in patients with chronic kidney diseases (CKD). However, the underlying mechanism of high Pi-induced endothelia cell apoptosis remains unclear.
Human umbilical vein endothelial cells (HUVECs) were treated with normal Pi (1.0 mM) and high Pi (3.0 mM), and then cell apoptosis, abnormal gene expression and potential signaling pathway involvement in simulated hyperphosphatemia were examined using flow cytometry, quantitative PCR (qPCR) and western blot analysis. A two-step 5/6 nephrectomy was carried out to induce CKD and biochemical measurements were taken.
The rat model of CKD revealed that hyperphosphatemia is correlated with an increased death-domain associated protein (DAXX) expression in endothelial cells. In vitro, high Pi increased the mRNA and protein expression level of DAXX in HUVECs, effects that were reversed by additional phosphonoformic acid treatment. Functionally, high Pi resulted in a significantly increased apoptosis in HUVECs, whereas DAXX knockdown markedly repressed high Pi-induced cell apoptosis, indicating that DAXX mediated high Pi-induced endothelial cell apoptosis. High Pi treatment and DAXX overexpression induced the activation of extracellular regulated protein kinases (ERKs), while DAXX knockdown inhibited high Pi-induced ERKs activation. Finally, we demonstrated that DAXX overexpression induced HUVECs apoptosis in the presence of normal Pi, whereas additional treatment with U0126 (a specific ERK inhibitor) reversed that effect.
Upregulated DAXX promoted high Pi-induced HUVECs apoptosis by activating ERK signaling and indicated that the DAXX/ERK signaling axis may be served as a potential target for CKD therapy.
高磷血症是指血清中无机磷（Pi）水平升高，它会促进内皮细胞功能障碍，并与慢性肾脏病（CKD）患者的心血管疾病相关。然而，高磷诱导内皮细胞凋亡的潜在机制仍不清楚。
将人脐静脉内皮细胞（HUVECs）分别用正常磷（1.0 mM）和高磷（3.0 mM）处理，然后使用流式细胞术、定量聚合酶链反应（qPCR）和蛋白质免疫印迹分析检测模拟高磷血症时的细胞凋亡、异常基因表达及潜在信号通路参与情况。进行两步5/6肾切除术诱导CKD，并进行生化检测。
CKD大鼠模型显示，高磷血症与内皮细胞中死亡结构域相关蛋白（DAXX）表达增加相关。在体外，高磷增加了HUVECs中DAXX的mRNA和蛋白质表达水平，而膦甲酸额外处理可逆转这些作用。在功能上，高磷导致HUVECs凋亡显著增加，而DAXX基因敲低明显抑制高磷诱导的细胞凋亡，表明DAXX介导了高磷诱导的内皮细胞凋亡。高磷处理和DAXX过表达诱导细胞外调节蛋白激酶（ERKs）激活，而DAXX基因敲低抑制高磷诱导的ERKs激活。最后，我们证明在正常磷存在的情况下，DAXX过表达诱导HUVECs凋亡，而用U0126（一种特异性ERK抑制剂）额外处理可逆转该效应。
DAXX上调通过激活ERK信号促进高磷诱导的HUVECs凋亡，表明DAXX/ERK信号轴可能作为CKD治疗的潜在靶点。
