The Investigation On The Mechanisms Of Pannexin-1 Channel In Regulating Cisplatin-Induced Premature Senescence Of Renal Tubule Cells

Objective Cisplatin is a class of chemotherapeutic drugs widely used in clinical practice,but its clinical application is limited due to its toxic side effects,especially nephrotoxicity.Cellular senescence is one of the main phenotypes of renal tubular epithelial damage caused by cisplatin.Most studies have suggested that telomere shortening,DNA damage,mitogen or oncogene activation,and hypoxia/reoxygenation can cause G1/S cell cycle arrest and then induce cellular senescence through pathways dependent on p53 and p21 activation.Meanwile,some studies have shown that the TGFβ/PI3K-mediated pathway and p53 independent can also activate the p21 pathway to induce progressive cellular senescence.In the current discussion of the relevant mechanisms of cellular senecence,two major pathways that have been widely studied and recognized are:p53/p21^WAFI and Rb-p16^INK4A,In addition,we still know very little about other pathways related to cellular senescence.In recent years,more and more studies have proposed that calcium signal transduction is also one of the key ways to control cellular senescence.As an endoplasmic reticulum calcium release channel,Pannexin-1 has been shown to play an important role in regulating intracellular calcium ion concentration.However,in the model of renal tubular epithelial cell senescence induced by cisplatin,it is unknown whether Pannexin-1 can control cellular senescence by regulating calcium signals.Methods In a human renal tubular epithelial cell line HK-2 cell injury model induced by cisplatin,the expression of Pannexin-1 in HK-2 cells was detected by Q-PCR,Western blot,immunofluorescence and other methods,and the expression of cellular senescence was detected.After knocking down Pannexin-1 by small interfering RNA,we tested whether the senescent phenotype of HK-2 cells induced by cisplatin was reduced.At the same time,after overexpression of Pannexin-1 with adenovirus,we tested whether HK-2 cells were induced to become senescent.In HK-2 cell models using small interfering RNA knockdown of Pannexin-1 and adenovirus overexpressing Pannexin-1,we detected Pannexin-1 expression on the endoplasmic reticulum by Western blot and immunofluorescence,and recorded changes of calcium ions by Fluo4-AM and Mag Fluo4-AM in cytoplasm and endoplasmic reticulum separately.In a HK-2 cell model in which adenovirus overexpresses Pannexin-1,we applied BAPTA chelator to chelate calcium ions in the cytoplasm,and examined whether the senescent phenotype of HK-2 cells was reduced.In the HK-2 cell model using small interfering RNA knockdown of Pannexin-1 and BAPTA pretreated adenovirus overexpressing Pannexin-1,we used Mito Sox,Mito Tracker to detect changes in mitochondrial morphology and function.Results（1）Cisplatin induces senescence of HK-2 cells accompanied by increased expression of Pannexin-1;knockdown of Pannexin-1 by siRNA significantly reduces senescence of HK-2 cells induced by cisplatin;（2）Overexpression of Pannexin-1 by adenovirus alone can significantly induce senescence in HK-2 cells;（3）After cisplatin induced senescence in HK-2 cells or overexpression of Pannexin-1 by adenovirus alone,Western blot and immunofluorescence co-localization results both showed that Pannexin-1 was mainly overexpressed in the endoplasmic reticulum;at the same time,after cisplatin induced senescence in HK-2 cells or overexpression of Pannexin-1 by adenovirus alone,the calcium ion concentration in the cytoplasm of HK-2 cells increased significantly,while the calcium ion content in the endoplasmic reticulum decreased significantly.It was seen that the calcium ion accumulation in the cytoplasm of HK-2 cells stimulated by cisplatin was significantly reduced,and the calcium ion concentration in the endoplasmic reticulum was higher;（4）After BAPTA pretreatment chelate calcium ions in cytoplasm,it can significantly reduce the senescence of HK-2 cells induced by adenovirus overexpression Pannexin-1;（5）In cisplatin-induced cellular senescence of HK-2 cells or adenovirus alone overexpression of Pannexin-1 in HK-2 cell models,it can be detected that HK-2 cells have increased ROS production in mitochondria,increased oxidative stress,and severe mitochondrial damage.After knocking down Pannexin-1 by siRNA,we can see that oxidative stress of HK-2 cells reduced by cisplatin and mitochondrial damage is reduced.At the same time,after pretreatment of HK-2 cells by BAPTA,we can see the adenovirus over-expresses Pannexin-1 intracellular Oxidative stress is significantly suppressed,thus protecting mitochondrial morphology and function.Conclusions In the senescence model of renal tubular epithelial cells induced by cisplatin,Pannexin-1 acts as an endoplasmic reticulum calcium channel,which promotes the release of endoplasmic reticulum calcium ions,then causes calcium ion accumulation in the cytoplasm to promote oxidative stress in the mitochondria,and further induces cell cycle arrest,eventually leads the cell to senescence.