The Effect Of SHPT Patients’serum On The Endothelial Cells, And The Intervention Of Klotho

Objective:To investigate the effect of SHPT patients’ serum on the endothelial cells and to explore the protection of Klotho and its possible mechanisms in this process.Methods:Three types of mixed serum from15patients with SHPT scheduled for parathyroidectomy,10CKD patients at stage5without SHPT and15healthy volunteers were collected. Human umbilical vein endothelial cells (HUVECs) were incubated in vitro with SHPT serum or serum of stage5CKD patients without SHPT or healthy serum as control. First, we compared the effects of the above three types serum on the proliferation of HUVEC after24hincubation(CCK-8). Second, HUVECs were divided into3groups: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-8and Flow Cytometry. The synthesis of NO was measured by nitrate reduction method. The secretion of MCP-1by endothelial cells was evaluated by ELISA. The activity of caspase-3was measured by spectrophotometry. The levels of extracellular signal-regulated kinase(ERK1/2), phosphorylated forms of ERK1/2(p-ERKl/2)(with or without ERK inhibitor PD98059) and AKT, phosphorylated forms of AKT (with or without AKT inhibitor LY294002).were detected by Western blotting.Results:The proliferation of HUVEC were both inhibited by the serum from SHPT patients and CKD-5patients without SHPT, and the inhibiton of SHPT serum was greater than 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 for6h,12h, the proliferation of HUVEC was significantly decreased when incubated for24h(P<0.05).The proliferation was partly restored when added50ng/ml-100ng/ml of Klotho into10%SHPT serum (P<0.05). Apoptosis of HUVEC was significantly increased when incubated in10%SHPT serum for24h,and inhibited when added50ng/ml-100ng/ml of Klotho(P<0.05). In the meantime, p-ERK and p-AKT were also upregulated and could be inhibited by PD98059or LY294002. The synthesis of NO was decreased in SHPT group (P<0.05) and increased in the treatment of Klotho (P<0.05). The secretion of MCP-1was increased in SHPT group(P<0.05) and decreased in the treatment of Klotho (P<0.05). The activity of caspase-3was upregulated by treatment of SHPT serum and inhibited when added Klotho.Conclusion:The Klotho protein can antagonize the toxic effects of the serum from SHPT patients on endothelial cells by a variety of mechanisms,The proliferation of HUVEC was inhibited and apoptosis was increased by the serum from 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 secretion of MCP-1and activity of caspase-3of the endothelial cells in the SHPT serum were also decreased by klotho.The promotion of HUVEC proliferation by Klotho might be due to upregulation of p-ERK. The inhibition of HUVEC apoptosis by Klotho might be due to upregulation of p-AKT.