The Role And Mechanism Of Sympathetic Nerves Regulating Cellular Senescence In The Renal Fibrosis

Objective Sympathetic activation is associated with the progression of chronic kidney disease(CKD).Chronic senescent cells accumulate in the damaged kidney,causing progressive fibrosis after renal injury.In this study,we sought to determine whether and how tubular cell senescence may be responsible for the event of renal sympathetic nerves driving tubulointerstitial fibrogenesis.Therefore,this research can provide a theoretical basis for exploring new therapeutic targets of CKD.Methods To research the relationship between renal sympathetic activation and tubulointerstitial fibrogenesis,a mouse model of renal denervation was established firstly.Unilateral ureteral obstruction(UUO)or Unilateral ischemia-reperfusion injury(UIRI)was conducted 2 days after the operation of renal denervation.For the sake of confirming whether renal sympathetic nerves can regulate cellular senescence,the sympathetic neurotransmitter norepinephrine(NE)was used to stimulate the renal tubular epithelial cells and the changes of senescence-related proteins and the senescence-associated secretory phenotype(SASP)were observed in vitro.Besides,for investigating the mechanism of the regulation of renal sympathetic nerves,a series of adrenergic receptor(AR)antagonists and a specific small interfering RNA(si RNA)were adopted to pretreat tubular epithelial cells.Results Here,we report that renal tubulointerstitial fibrogenesis,as well as senescence,were aggravated after the manipulation of UUO and UIRI,which were both reversed in the denervated kidney.In tubular epithelial cells,the stimulation of NE can induce cellular senescence and upregulate the expression of profibrogenic growth factors.Further data show that NE acts through the α2-AR,especially α2A-AR,because blocking these receptors prevented tubular epithelial cells into a senescent phenotype.Furthermore,the potential role of β-arrestin 2 was assessed in our next research,in which β-arrestin 2,as a target of α2A-AR,promotes the process of sympathetic nerves regulating cellular senescence through mitochondrial damage and NF-κB signaling pathways.Conclusion These data suggest that the sympathetic neurotransmitter NE accelerates renal tubular epithelial cell senescence through α2A-AR/β-arrestin 2/mitochondrial damage and the NF-κB pathways,eventually leading to renal tubulointerstitial fibrogenesis.Part Ⅰ Sympathetic activation promotes renal tubular epithelial cell senescence,driving the occurrence and development of renal tubulointerstitial fibrogenesisObjective This part mainly discusses the link between sympathetic nerve,cellular senescence,and renal tubulointerstitial fibrosis,providing experimental support for preventing and treating renal tubulointerstitial fibrogenesis from the perspective of sympathetic nerves regulating cellular senescence.Methods The model of Unilateral ureteral obstruction(UUO)or Unilateral ischemia-reperfusion injury(UIRI)was established in mice after the successful operation of renal denervation.Then,for further discussing the relationship between sympathetic activation and cellular senescence,the sympathetic neurotransmitter norepinephrine(NE)was used to stimulate the renal tubular epithelial cells and H2O2 was adopted as a positive control in vitro.Results Here,we report that renal tubulointerstitial fibrogenesis,as well as senescence,were aggravated after the manipulation of UUO and UIRI,which were both reversed in the denervated kidney.In vitro,the stimulation of NE can induce renal tubular epithelial cell senescence and upregulate the expression of profibrogenic growth factors.Conclusion These data suggest that sympathetic activation accelerates renal tubular epithelial cell senescence and increases the expression of the senescence-associated secretory phenotype(SASP).These molecules can recruit inflammatory cells and increase inflammation by paracrine action.Besides,senescent cells can increase profibrogenic growth factors and matrix synthesis molecules.All these together promote the occurrence and development of renal tubulointerstitial fibrogenesis.Part Ⅱ The process of sympathetic nerves regulating cellular senescence was mediated by α2-adrenergic receptor(α2-AR),especially α2A-ARObjective The purpose of this part is to determine which specific adrenergic receptor conducts the signal of the sympathetic neurotransmitter norepinephrine,providing experimental evidence for exploring the mechanism of sympathetic nerve regulating cellular senescence.Methods The expression of adrenergic receptors was detected in renal tubular epithelial cells.Based on NE stimulation,several kinds of selective adrenergic receptor(AR)antagonist(such as β2-AR antagonist Butoxamine and ICI118551,α2-AR antagonist Atipamezole,α2A-AR antagonist BRL44408,α2B-AR antagonist Imiloxan and α2C-AR antagonist JP1302)were used to pretreat tubular epithelial cells respectively under a different concentration gradient.Results All the five kinds of AR were expressed in renal tubular epithelial cells,but α2-AR and β2-AR were the highest expressed receptors.The expression of senescence-related proteins and senescence-related secretory phenotypes in the NE stimulation group were higher than the Control group.Compared with the NE stimulation group,only the pretreatment of α2-AR antagonist Atipamezole and the α2A-AR antagonist BRL44408 prevented tubular epithelial cells into a senescent phenotype.However,β2-AR,α2B-AR,and α2C-AR antagonist all did not improve the senescence of renal tubular epithelial cells induced by NE.Conclusion These data suggest that the process of sympathetic nerves regulating cellular senescence was mediated by α2-adrenergic receptor(α2-AR),especially α2A-AR.Part Ⅲ β-arrestin 2,as a downstream target of α2A-AR,promotes the process of sympathetic nerves regulating cellular senescence through mitochondrial damage and NF-κB signaling pathwaysObjective This part is to study the downstream mechanism of the sympathetic neurotransmitter norepinephrine(NE)acting on α2A-AR after sympathetic activation.It also provides more comprehensive experimental evidence for the role of mitochondrial dysfunction in the progression of CKD.Methods First,three specific small interfering RNAs(si RNAs)(si-Arrb2-1、si-Arrb2-2、si-Arrb2-3)were designed to specifically down-regulate the gene of β-arrestin 2 in TKPTS cells.Subsequently,NE stimulation continued 48 hours after knock-down of β-arrestin 2 in vitro.Moreover,the association between α2A-AR and β-arrestin 2 was researched by cell immunofluorescence test and immunoprecipitation test.Finally,for discussing the downstream mechanism of β-arrestin 2,the changes of mitochondria-related indicators and NF-κB signaling pathways were observed after renal tubular epithelial cells were pretreated with si RNAs.Results The expression of β-arrestin 2 increased after NE stimulation and β-arrestin 2 knockdowns can prevent tubular epithelial cells into a senescent phenotype caused by NE.There is Co-expressing of α2A-AR and β-arrestin 2 in renal tubular epithelial cells.Knockdown of β-arrestin 2 can reverse mitochondrial damage and the increase of NF-κB expression induced by NE.Conclusion β-arrestin 2,as a downstream target of α2A-AR,promotes the process of sympathetic nerves regulating cellular senescence through mitochondrial damage and NF-κB signaling pathways.