Dexmedetomidine Ameliorates Renal Ischemia-reperfusion Injury Through The Cholinergic Anti-inflammatory Pathway In Mice

Background and objective:Inflammatory response plays a critical role in the pathophysiology of renal ischemia-reperfusion(r I/R)induced acute kidney injury(AKI).Local and systemic inflammatory response can be regulated via vagus nerve and nicotinic acetylcholine receptors(n ACh Rs),which constitute the cholinergic anti-inflammatory pathway(CAP).Pretreatment with pharmacologic or electrical vagus nerve stimulation(VNS)may suppress the proinflammatory cytokines release and limit the excessive inflammatory response.Dexmedetomidine(Dex),a novel α2 adrenergic receptor(α2AR)agonist,exerts potent anti-inflammatory and organoprotective effects in addition to its sedative,analgesic and anxiolytic properties.Our previous studies demonstrated that Dex attenuated both kidney and remote lung injury induced by the bilateral r I/R in a mice model.In this study,we found that pretreatment of Dex could significantly strengthen the discharge frequency of cervical vagus nerves.Moreover,Dex remarkably promoted the ACh release as well as reduced the inflammatory mediators levels.Consequently the kidney cells apoptosis were significantly attenuated following Dex treatment.These effects were abolished by pretreatment of atipamezole(Atip),an α2AR antagonist.It implies that anti-inflammatory effect of Dex is associated with α2AR-dependent activation of CAP.Similarly,VNS has the same anti-inflammatory effects.However,Dex failed to reduce the inflammatory mediators after vagotomy.Therefore,through this study we intend to confirm that the renoprotection of Dex via anti-inflammatory effect is associated with CAP as well as depends on the integrity of CAP.Methods:1.Vagus Nerve Discharge Activity Analysis1.1 A pair of bipolar stainless steel electrodes were used to hook the mice’s right cervical vagal nerve and to record the activity by using the BL-420 F data acquisition and analysis system.1.2 Cervical vagus nerve discharge activity was recorded during the r I/R period or administration of Dex.1.3 Changes of vagus nerve discharge activity were detected by pretreatment of Dex on r I/R period.1.4 Changes of vagus nerve discharge activity were detected when pretreatment of Atip prior to Dex.2.Serum ACh and CA Level Analysis2.1 Mouse ACh ELISA Kit and Mouse CA ELISA Kit were used for the serum ACh and CA analysis.2.2 Mice serum ACh and CA levels of different groups were detected,respectively.3.Serum Inflammatory Mediators Analysis3.1 Aimplex? Mouse Inflammation 8-Plex assay kits are used for the serum inflammatory mediators detection.3.2 The sample beads fluorescence signals of antigen-antibody complex were acquired by a flow cytometer and the inflammatory mediators level were analyzed by using FCAP Array 3.0.4.Kidney Injury Histological Evaluation4.1 The renal tissue sections were stained with H&E for morphologic assessments and histologic scoring.4.2 The histopathological evaluations were assessed by an experienced pathologist who was blinded to the groups and treatments.5.Kidney Cells Apoptosis TUNEL Staining Analysis5.1 The apoptosis of tubular epithelial cells were detected by In Situ Cell Death Detection Kit,POD.5.2 The sum of the TUNEL positive cells in an objective grid from 10 areas of randomly selected sections were counted using Image J software under a 20 × objective lens by an investigator who was blinded to the experimental protocol.Results:1.Both Dex and r I/R could increase the mice cervical vagus nerve activity.Pretreatment of atipamezole could abolish the increasing discharge frequency effect of Dex but did not have any significant influences on r I/R.2.The serum ACh and CA level did not change in r I/R,but Dex or VNS could significantly increase the serum ACh level and decrease the CA level,no matter if it is subjected to splenectomy or not,and preemptive injection of atipamezole or vagotomy before Dex,the ACh level decreased.3.Renal I/R could remarkably increase the serum IL-1β,IL-6,TNF-α,IL-10,VEGF,s ICAM-1 and s P-selectin levels,and pretreatment of Dex or VNS before r I/R significantly decreased all above inflammatory mediators,but it did not have any obvious effect on s VCAM-1.This anti-inflammatory effect could be abolished by pretreatment of splenectomy,vagotomy or atipamezole.4.In the H&E staining,a severe and extensive acute tubular dilatation,flattened renal epithelial cells and loss of nuclear staining were observed in the cortex and outer medulla in r I/R,and it remarkably increased the histopathological scoring when compared with Sham control or Dex only,meanwhile pretreatment with Dex or VNS could significantly attenuated the kidney damage when compared to the r I/R.However,splenectomy,atipamezole or vagotomy prior to Dex could result in the protective effects to vanish.5.Apoptotic cells nuclei were observed in all kidney slices,and r I/R remarkably increased the TUNEL positive cells,meanwhile the quantitative analysis confirmed that the proportion of apoptotic nuclei was significantly higher in the r I/R group than the Sham control group,and pretreatment of Dex or VNS could significantly decrease the apoptotic cells.Similarly,pretreatment of atipamezole or vagotomy prior to Dex resulted in Dex ’s cytoprotective effects vanish and splenectomy could even exacerbate the apoptosis.Conclusions:1.Dex can promote ACh release as well as down-regulates serum CA level through directly strengthening of cervical vagus nerve activity.2.Dex can decrease the proinflammatory cytokines level through activating the CAP to provide renoprotection.3.The anti-inflammatory effects of Dex depend on the integrity of vagus nerve.