Klotho在盐敏感性高血压肾损伤中的作用及分子机制研究

doi:10.12114/j.issn.1007-9572.2023.0010

摘要/Abstract

摘要： 背景 Klotho与肾脏疾病的发生、发展密切相关，盐敏感性高血压（SSH）常伴随肾脏疾病的发生。目前klotho在SSH肾损伤中的作用及分子机制研究鲜见报道。目的探讨klotho在SSH肾损伤中的作用及分子机制。方法于2021年6月—2022年1月选取大鼠肾小球系膜细胞株HBZY1为实验细胞，将实验细胞分为对照组与造模组，采用NaCl 137 mmol/L和血管紧张素Ⅱ（Ang Ⅱ）10-6 mmol/L共同诱导的HBZY1细胞损伤模型模拟SSH肾损伤，收集细胞。实时荧光定量PCR（qRT-PCR）与蛋白质印记法（Western Blot）检测klotho mRNA和蛋白的表达。构建klotho干扰载体和过表达载体与血管紧张素Ⅱ1型受体（AT1R）过表达载体，klotho干扰实验分为5组，包括对照组、空载组、klotho-siRNA1组、klotho-siRNA2组、klotho-siRNA3组；klotho过表达实验分为3组，包括对照组、空载组、klotho过表达组；AT1R过表达实验分为3组，包括对照组、空载组、AT1R过表达组。将构建的载体转染至细胞中并验证转染效率。转染成功后将实验分两部分进行，第一部分实验验证klotho的肾脏保护作用，实验分为4组，包括对照组、造模组、klotho过表达组与klotho干扰组，第二部分实验探索klotho的肾脏保护作用是否与AT1R相关，实验分为3组，包括造模组、klotho过表达组、klotho+AT1R过表达组。转染成功后进行下列检测，细胞计数试剂8（CCK-8）法检测细胞活力，流式细胞术检测细胞活性氧（ROS）水平，酶联免疫吸附测定（ELISA）法检测细胞上清液中丙二醛（MDA）与超氧化物歧化酶（SOD）水平，免疫共沉淀（Co-IP）检测klotho与AT1R相互作用的影响。结果与对照组相比，造模组中klotho的mRNA水平与蛋白表达均下降（t=7.102、7.506，P=0.002、0.002）。与对照组相比，klotho-siRNA2干扰效果显著（P<0.001）；klotho过表达组的klotho蛋白表达升高（P<0.001）；AT1R过表达组的AT1R蛋白表达升高（P<0.001）。Klotho对细胞活力及氧化应激损伤的影响：与对照组相比，造模组细胞活力下降（P<0.001），细胞内ROS、MDA水平升高（P<0.001、P=0.004），细胞内SOD水平下降（P=0.041）；与造模组相比，klotho过表达组细胞活力升高（P<0.001），细胞内ROS、MDA水平下降（P<0.001、P=0.003），细胞内SOD水平上升（P=0.018）；与造模组相比，klotho干扰组细胞活力下降（P<0.001），细胞内ROS、MDA水平升高（P<0.001、P=0.002），细胞内SOD水平下降（P=0.001）。Klotho通过AT1R对细胞活力及氧化应激损伤的影响：与造模组相比，klotho过表达组细胞活力升高（P<0.001），细胞内ROS、MDA水平下降（P<0.001、P=0.024），细胞内SOD水平上升（P=0.007）；与klotho过表达组相比，klotho+AT1R过表达组细胞活力下降（P<0.001），klotho+AT1R过表达组细胞内ROS、MDA水平上升（P<0.001、P=0.001），细胞内SOD水平下降（P=0.002）。Co-IP确定klotho与AT1R之间存在交互作用。结论 Klotho通过与AT1R相互作用，抑制氧化应激损伤，从而在SSH肾损伤中发挥保护作用。

关键词: 高血压, 盐敏感性高血压, 肾损伤, 氧化应激, Klotho, 血管紧张素Ⅱ, 受体，血管紧张素，1型

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.

Key words: Hypertension, Salt-sensitive hypertension, Kidney injury, Oxidative Stress, Klotho, Angiotensin Ⅱ, Receptor, angiotensin, type 1

赵伟,唐荣杰,杨珊珊,等. Klotho在盐敏感性高血压肾损伤中的作用及分子机制研究[J]. 中国全科医学, 2023, 26(24): 3042-3049. DOI: 10.12114/j.issn.1007-9572.2023.0010.
ZHAO Wei,TANG Rongjie,YANG Shanshan, et al. The Role and Molecules Mechanism of Klotho in Renal Injury in Salt-sensitive Hypertension[J]. Chinese General Practice, 2023, 26(24): 3042-3049. DOI: 10.12114/j.issn.1007-9572.2023.0010.

图/表 9

图1 NaCl和AngⅡ处理对HBZY1细胞中klotho mRNA和蛋白的影响注：A为klotho的Western Blot分析图，B为klotho的mRNA表达量图，C为klotho的蛋白表达量图；a表示与对照组比较P<0.05；GADPH=甘油醛-3-磷酸脱氢酶。

Figure 1 Effect of NaCl and Ang Ⅱ treatment on klotho mRNA and protein in HBZY1 cells

图2 Klotho干扰验证结果注：A为klotho-siRNA的Western Blot分析图，B为klotho-siRNA的蛋白表达量图；a表示与对照组比较P<0.05。

Figure 2 Verification results of klotho interference

图3 Klotho过表达验证结果注：A为klotho过表达的Western Blot分析图，B为klotho过表达的蛋白表达量图；a表示与对照组比较P<0.05。

Figure 3 Verification results of klotho overexpression

图4 AT1R过表达验证结果注：A为AT1R过表达的Western Blot分析图，B为AT1R过表达的蛋白表达量图；a表示与对照组比较P<0.05；AT1R=血管紧张素Ⅱ-1型受体。

Figure 4 Verification results of AT1R overexpression

图5 Klotho对细胞活力的影响注：a表示与对照组比较P<0.05，b表示与造模组比较P<0.05。

Figure 5 Effect of klotho on cell viability

图6 Klotho对细胞氧化应激损伤的影响注：A为ROS的流式细胞图，B为流式细胞术测定ROS的表达量图，C为ELISA法测定MDA的表达量图，D为ELISA法测定SOD的表达量图；a表示与对照组比较P<0.05，b表示与造模组比较P<0.05；ROS=活性氧，SOD=超氧化物歧化酶，MDA=丙二醛。

Figure 6 Effect of klotho on oxidative stress injury of cells

图7 Klotho通过AT1R对细胞活力的影响注：a表示与造模组比较P<0.05，b表示与klotho过表达组比较P<0.05。

Figure 7 Effect of Klotho on cell viability via AT1R

图8 Klotho通过AT1R对细胞氧化应激损伤的影响注：A为ROS的流式细胞图，B为流式细胞术测定ROS的表达量图，C为ELISA法测定MDA的表达量图，D为ELISA法测定SOD的表达量图；a表示与造模组比较P<0.05，b表示与klotho过表达组比较P<0.05。

Figure 8 Effect of Klotho on oxidative stress injury of cells via AT1R

图9 免疫共沉淀实验证明klotho与AT1R相互作用

Figure 9 Interaction between klotho and AT1R proved by co-immunoprecipitation experiment

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