Zhongguo quanke yixue (Aug 2023)
The Role and Molecules Mechanism of Klotho in Renal Injury in Salt-sensitive Hypertension
Abstract
Background Klotho is closely related to the occurrence and development of kidney disease. Salt-sensitive hypertension (SSH) is often accompanied by kidney disease. At present, there are few reports on the role and molecules mechanism of klotho in renal injury in SSH. Objective To investigate the role and molecules mechanism of klotho in renal injury in SSH. Methods The rat glomerular mesangial cell line HBZY1 was selected as the experimental cells from June 2021 to January 2022, and the experimental cells were divided into the control group and the model group. The model of HBZY1 cell injury induced by NaCl 137 mmol/L and angiotensin Ⅱ (Ang Ⅱ) 10-6 mmol/L was used to simulate the renal injury in SSH, and the cells were collected. The differences in the expression of klotho mRNA and protein were detected by real-time fluorescent quantitative PCR (qRT-PCR) and Western Blot. The interference vector and overexpression vector of klotho and the overexpression vector of angiotensin Ⅱ type 1 receptor (AT1R) were constructed. The klotho interference experiments were divided into five groups, including the control group, empty group, klotho-siRNA1 group, klotho-siRNA2 group and klotho-siRNA3 group; the klotho overexpression experiments were divided into three groups, including the control group, empty group and klotho overexpression group; the AT1R overexpression experiments were divided into three groups, including the control group, empty group and AT1R overexpression group. The constructed vectors were transfected into cells with verified transfection efficiency. After successful transfection, the experiment was divided into two parts. The first part of the experiment was to verify the renal protective effect of klotho, the experiment subjects were divided into four groups, including the control group, model group, klotho overexpression group and klotho interference group. The second part of the experiment was to explore whether the renal protective effect of klotho was related to AT1R, the experiment subjects were divided into three groups, including the model group, klotho overexpression group and klotho+AT1R overexpression group. After successful transfection, the tests including cell viability detected by cell counting kit-8 (CCK-8) method, reactive oxygen species (ROS) content detected by flow cytometry, malondialdehyde (MDA) and superoxide dismutase (SOD) content in cell supernatant detected by enzyme-linked immunosorbent assay (ELISA) , interaction effect between kltho and AT1R detected by co-immunoprecipitation (Co-IP) . Results Compared with the control group, mRNA level and protein expression of klotho in the model group decreased in model group (t=7.102, 7.506; P=0.002, 0.002) , klotho-siRNA2 interference effect was more significant (P<0.001) , the expression of klotho protein in the klotho overexpression group increased significantly (P<0.001) , the expression of AT1R protein in the overexpression group increased significantly (P<0.001) . Effects of klotho on cell viability and oxidative stress injury: compared with the control group, cell viability in the model group decreased (P<0.001) , intracellular ROS and MDA content increased (P<0.001, P=0.004) , and SOD content decreased (P=0.041) ; compared with the model group, cell viability in the klotho overexpression group increased (P<0.001) , intracellular ROS and MDA content decreased and SOD content increased (P<0.001, P=0.003, P=0.018) ; compared with the model group, cell viability in the klotho interference group decreased (P<0.001) , while intracellular ROS and MDA content increased and SOD content decreased (P<0.001, P=0.002, P=0.001) . Effects of klotho on cell viability and oxidative stress injury through AT1R: compared with the model group, cell viability increased (P<0.001) , intracellular ROS and MDA content decreased and SOD content increased (P<0.001, P=0.024, P=0.007) in the klotho overexpression group; compared with the klotho overexpression group, cell viability decreased (P<0.001) , ROS and MDA content increased and SOD content decreased (P<0.001, P=0.001, P=0.002) in the klotho+AT1R overexpression group. Co-IP determined that there was an interaction between klotho and AT1R. Conclusion Klotho plays a protective role in renal injury in SSH by inhibiting oxidative stress injury through interaction with AT1R.
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