关键词: 【关键词】 klotho蛋白, 继发性甲状旁腺功能亢进, 人脐静脉内皮细胞;凋亡, ERK, 一氧化氮

Abstract:

ABSTRACT Objective: To investigate the effect of secondary hyperparathyroidism (SHPT) patients’ serum on the endothelial cells and to explore the protection of Klotho and its possible mechanisms in this process.  Methodology: Three types of mixed serum from fifteen patients with SHPT scheduled for parathyroidectomy, 10 CKD patients at stage 5 without SHPT and 15 healthy volunteers were collected. Human umbilical vein endothelial cells (HUVECs) were incubated in vitro with SHPT serum or serum of stage 5 CKD patients without SHPT or healthy serum as control. First, the effects of the above three types serum on the proliferation of HUVEC after 24h treatment(CCK-8) were compared. Second, HUVECs were divided into 3 groups: control group (10% healthy serum medium)，SHPT group and Klotho treatment group(SHPT serum and Klotho). The proliferation and apoptosis of endothelial cells were evaluated respectively by CCK-8 and Flow Cytometry. The synthesis of NO was measured by nitrate reduction method. The levels of extracellular signal-regulated kinase (ERK1/2) and phosphorylated forms of ERK1/2 (p-ERK1/2) were detected by Western blotting (with or without ERK inhibitor PD98059). Results: The proliferation of HUVEC were both inhibited by the serum of SHPT patients and CKD-5 patients without SHPT, and the inhibition of SHPT serum was greater than that of the other (P<0.05). With the increase of concentration in a certain range (5%-20%), the proliferation of HUVEC was inhibited by SHPT serum in a concentration-dependent manner (P<0.05). Compared with HUVEC incubated with SHPT serum for 6h, 12 h, the proliferation of HUVEC was significantly decreased when incubated for 24 h (P<0.05).The proliferation was partly restored and the apoptosis was inhibited when added 50ng/ml-100ng/ml of Klotho into 10% SHPT serum (P<0.05). In the meantime, p-ERK was also upregulated and could be inhibited by PD98059. The synthesis of NO was decreased in SHPT group (P<0.05) and increased in the treatment of Klotho (P<0.05).  Conclusion: The proliferation of HUVEC was inhibited by the serum of SHPT patients, which could be partly antagonized by Klotho . The NO synthesis of the endothelial cells in the SHPT serum was also increased by klotho. The promotion of HUVEC proliferation by Klotho might be due to the inhibition of HUVEC apoptosis and upregulation of p-ERK.

Key words: [key words] klotho protein,  , SHPT,  , HUVEC,  , apoptosis, ERK,  , NO