The Role And Molecular Mechanism Of Klotho Protein In Salt-Sensitive Hypertensive Renal Injury

Background:Klotho protein is closely related to the occurrence and development of kidney disease.Salt-sensitive hypertension（SSH）is often associated with kidney disease.At present,there are few reports on the role and molecular mechanism of Klotho protein in renal injury caused by SSH.Objective:To investigate the role and molecular mechanism of Klotho protein in renal injury induced by SSH.Methods:First,to observe the expression of Klotho protein in SSH-induced renal injury.Rat Mesangium cell line HBZY1 was used in this experiment.The cells were divided into control group and model group,in the model group,Na Cl 137mmol/L and Angiotensin II（Ang II）10^-6 mmol/L were costimulated for 24h,and cells were collected.Quantitative real time PCR（q RT-PCR）and Western Blot were used to detect the expression of Klotho m RNA and protein.Second,to verify the protective effect of Klotho protein on SSH-induced renal injury.Klotho interference vector and Klotho overexpression vector were constructed and transfected into cells to verify the transfection efficiency,the control group,model group,Klotho overexpression group and Klotho interference group were included.Cell viability was measured by cell counting reagent 8（CCK-8）and reactive oxygen species（ROS）was measured by flow cytometry,the contents of malondialdehyde（MDA）and superoxide dismutase superoxide dismutase（SOD）in the supernatant were measured by enzyme-linked immunosorbent assay（ELISA）.Finally,to explore whether the renal protective effect of Klotho protein is related to the inhibition of angiotensinⅡtype 1 receptor（AT1R）mediated oxidative stress injury.The experiment was divided into five parts:the first part was to verify the interaction between Klotho protein and AT1R;the second part is divided into control group and model group to detect the expression of AT1R in renal injury;the third part was divided into model group and Klotho overexpression group to detect the effect of Klotho protein on AT1R;the fourth part constructs AT1R overexpression vector and verifies the transfection efficiency;the fifth part is divided into three groups:model group,Klotho overexpression group,Klotho+AT1R overexpression group,to verify whether the overexpression of AT1R reduces the antioxidant capacity and renal cytoprotective effect of Klotho protein.Co-immunoprecipitation（Co-IP）was used to detect the interaction between Klotho and AT1R.Western Blot was used to detect the expression of AT1R protein.Cell viability was measured by CCK-8.ROS content was measured by flow cytometry method,the contents of MDA and SOD in cell supernatant were detected by ELISA.Results:（1）Compared with the control group,the m RNA level and protein expression of Klotho decreased in the model group（P<0.05）.（2）Compared with the control group,the Klotho-si RNA2 interference effect was significant（P<0.05）;the Klotho protein expression in the Klotho overexpression group was significantly increased（P<0.05）;compared with the control group,the cell viability of the model group decreased（P<0.05）,the content of ROS and MDA in the model group increased,and the content of SOD decrease（P<0.05）,compared with the model group,the cell viability of the Klotho overexpression group was increased（P<0.05）,while that of the Klotho interference group was decreased（P<0.05）,the content of ROS and MDA decreased and the content of SOD increased in Klotho overexpression group（P<0.05）,while the content of ROS and MDA increased and the content of SOD decreased in Klotho interference group（P<0.05）.（3）Co-IP confirmed the interaction between Klotho and AT1R;compared with the control group,the expression of AT1R protein was increased in the model group（P<0.05）;while the expression of AT1R protein was decreased in the Klotho overexpression group（P<0.05）;the expression of AT1R protein in AT1R overexpression group increased significantly（P<0.05）;compared with the model group,the content of ROS and MDA in the cells of the Klotho overexpression group decreased,the content of SOD increased（P<0.05）,and the cell viability increased（P<0.05）,compared with the Klotho overexpression group,the content of ROS and MDA in the cells of the Klotho+AT1R overexpression group increased,the content of SOD decreased（P<0.05）,and the cell viability decreased（P<0.05）.Conclusion:（1）There is a decrease in the expression of Klotho in renal injury induced by SSH.（2）Klotho protein can improve cell viability,inhibit oxidative stress damage,and play a certain protective role in SSH renal injury.（3）The protective effect of Klotho protein in SSH-induced renal injury is related to the inhibition of AT1R-mediated oxidative stress injury.