The Effects Of Tropomodulin1 On AQP2 Expression And Targeting In Renal Collecting Duct Cells And Its Mechanisms

The ability of the kidneys to reabsorb water is essential for the survival of the human body.Changes in water balance and related diseases are closely related to the activation state of Vasopressin-Aquaporin2(VP-AQP2).Studies have found that many factors participate in the process of water reabsorption by the renal collecting duct through the regulation of AQP2 membrane transport.Among them,the regulation of the AQP2 transport mechanism by the cytoskeleton has gradually attracted people's attention.This article will investigate whether Tropomodulin 1(Tmod1),a cytoskeleton protein expressed in the kidney,can participate in the process of AQP2 penetration and water reabsorption by the collecting duct in some way.Objective:The subject mainly explored whether Tmod1 regulates the reabsorption of water by the renal collection tube by affecting the shuttle of AQP2.If so,is the way of participation achieved through direct interaction with AQP2 or through interaction with other proteins?Methods:We first gave hypertonic treatment to the primary inner medullary collecting duct cells of mouse kidney to simulate the hypertonic environment of the medulla after water deprivation,and detected the changes of Tmod1;and through the primary inner medullary collecting duct cells and M-1 that stably express AQP2-1 After overexpression or knockout of Tmod1 by adenovirus in cell line,the total AQP2 and its phosphorylation changes at s256 were detected;after overexpression or knockout of Tmod1 by adenovirus,hypotonic and hypertonic treatment were given respectively,and Tmod1 was observed The effect on cell membrane permeability changes;after treatment with Forskolin,an agonist of adenylate cyclase,Observe the transport of AQP2 under a confocal microscope;After overexpression or knockout of Tmod1,detect the changes in AQP2 expression on the cell membrane;We use the method of immunoprecipitation to verify proteins that may interact with Tmod1,such as CDK1.CDK1 expression changes were detected after overexpression or knockout of Tmod1 in primary inner medullary collecting duct cells;and verified in TFK and its TF mouse kidney total protein.Results:In the primary cells after hypertonic treatment,the expression of Tmod1 increased.The use of adenovirus overexpression of Tmod1 in kidney primary inner medullary collecting duct cells significantly increased AQP2 and its phosphorylation at S256 and cell membrane permeability.The combined treatment with forskolin can significantly promote the depolymerization of F-actin and the accumulation of AQP2 on the membrane;we used biotin to label membrane proteins and found that the expression of AQP2 on the membrane increased after overexpression of Tmod1.On the contrary,after knocking down Tmod1,the opposite phenomenon occurred.Co-immunoprecipitation,it is found that there may be an interaction between Tmod1 and CDK1,and overexpression or knockout After Tmod1,the level of CDK1 protein also changes,and its changes are positively correlated.In vivo experiments show that the expression of CDK1 in the kidney of TFK mice is significantly reduced compared with TF mice.Conclusion:Tmod1 can affect the transport of aquaporin AQP2,Play a role in the regulation of kidney water.The mechanism may be that Tmod1 affects the stability of CDK1 that interacts with AQP2 and then affects the phosphorylation of AQP2.This indicates that Tmod1 can be used as a new target for regulating the body's water homeostasis.