The Protective Mechanism Of Dexmedetomidine Against Acute Kidney Injury In Septic Rats By Regulating The Activation Pathway Of NLRP3 Inflammasome

ObjectiveTo investigate the effects of dexmedetomidine（DEX）on acute kidney injury（AKI）in septic rats and its mechanism,and to determine whether DEX can protect AKI in septic rats by regulating the activation pathway of NLRP3 inflammasome.Methods1.Animals and grouping:Sixty male SD rats were selected as experimental animals.Grouping by random number table.Four groups:sham operation（Sham）group,Sham+DEX group,CLP group and CLP+DEX group,with 15 rats in each group.Each group was subdivided into 3 subgroups at 6h,12h and 24h after surgery,with 5 rats in each group.2.Pretreatment:Each group was given pretreatment by continuous injection with a micro-injection pump 1 hour before model making.The Sham+DEX group and the CLP+DEX group were pumped into DEX at a rate of 5ug·kg^-1·h^-1through the caudal vein,and the pumping time was 1 hour.The Sham group and the CLP group were pumped with the equal amount of normal saline（NS）through the caudal vein.3.Animal model:Modified CLP was used to prepare a sepsis model.4.Specimen collection:Five rats in each group were sacrificed at 6h,12h,and 24h after operation,blood was collected from the abdominal aorta,and kidney tissue specimens were obtained.5.To observe the histopathological changes and morphological changes of the kidney in rats:（1）The morphological changes of the kidney were observed roughly;（2）Light microscopy was used to observe renal histopathological changes.Renal tubule injury score was calculated.（3）The ultrastructural changes of renal tissue in each group were observed.6.The changes of renal function in rats were observed:Scr and BUN levels were detected by biochemical analyzer.7.The changes of serum markers in the early stage of kidney injury in rats were observed:ELISA was used to detect the levels of serum NGAL and KIM-1.8.The expression of Claudin-5 and ZO-1 in kidney tissue of rats were observed:（1）Immunohistochemical method was used to detect the expression and localization of Claudin-5and ZO-1 in kidney tissue.（2）Western Blot was used to detect the expression of Claudin-5and ZO-1 protein in kidney tissue.9.The expression of cytokines related to the activation pathway of NLRP3inflammasome in renal tissues of rats was observed.The detection factors were NLRP3,caspase-1,IL-1βand IL-18:（1）The localization and expression of these cytokines were detected by immunohistochemical method.（2）The protein expression of these factors was detected by Western Blot.（3）The m RNA expression of these factors was detected by RT-PCR.Results1.The pathological and morphological changes of kidney tissue in rats1.1 Gross observation:The kidneys in the Sham group and the Sham+DEX group were normal in color and morphology.The kidneys in the CLP group were dark red,swollen and congested,and the renal capsule were tense.The degree of swelling and congestion in the CLP+DEX group was reduced.1.2 The pathological changes of kidney tissue were observed under a light microscope（1）HE staining:The structure of kidney tissue in Sham group and Sham+DEX group was normal with clear outline.In the CLP group,glomerular edema and congestion were observed in the early stage and atrophy in the later stage,renal tubular epithelial cells（TECs）were swollen,vacuolar degeneration,loss of brush border,renal tubule expansion,interstitial inflammatory cell infiltration.Kidney damage tends to be aggravated with the postoperative time prolonged.The degree of renal injury in the CLP+DEX group was less than that in the CLP group at each time point.（2）Renal tubular injury score:At each time point,the scores of the CLP group were higher than those of the Sham group（all P<0.01）.Compared with CLP group,the scores of the CLP+DEX group were all lower（all P<0.01）.1.3 Transmission electron microscopy was used to observe the ultrastructural changes of renal tissue in rats（1）Ultrastructural changes of the glomerulus:No obvious abnormalities were seen in the Sham group and the Sham+DEX group.The glomeruli in the CLP group showed pathological damage,mainly manifested as focal lodging and fusion of the foot process,uneven basement membrane thickness,and the degree of damage varies with the prolongation of postoperative time showed a trend of aggravation.After pretreatment with DEX,the glomerular damage at each time point was less than that of the CLP group.（2）Ultrastructural changes in renal tubules:The Sham group and the Sham+DEX group were almost normal.Renal tubular damage in the CLP group was mainly manifested as vacuolar degeneration of renal TECs,microvilli rupture and shedding,and mitochondria were damaged to varying degrees,and even renal TECs were seen apoptosis.After pretreatment with DEX,the degree of renal tubular damage at each time point was less than that of the CLP group.2.Changes of renal function indexes of rats:Serum Scr and BUN levels of rats in all groups showed no significant differences at 6h after surgery,but at 12h and 24h after surgery,these two indexes in CLP group were higher than those in Sham group（all P<0.01）,and the two indexes in CLP+DEX group were lower than those in CLP group（all P<0.01）.3.Changes of serum markers NGAL and KIM-1 in the early stage of renal injury in rats:Compared with the Sham group,serum NGAL and KIM-1 levels in the CLP group were increased at each time point（all P<0.01）.After pretreatment with DEX,serum NGAL and KIM-1 levels decreased（all P<0.01）.4.Immunohistochemistry and Western Blot results showed that the expression of claudin-5 and zo-1 in renal tissue at each time point decreased in the CLP group compared with the Sham group,and the longer the injury time was,the more obvious the decrease was（all P<0.01）,and the two indexes in the CLP+DEX group were higher than those in the CLP group（all P<0.01）.5.Immunohistochemistry,Western Blot and RT-PCR results showed that the expressions of NLRP3,Caspase-1,IL-1βand IL-18 in renal tissues at each time point were increased in the CLP group compared with Sham group,and the expressions showed an increasing trend with the extension of postoperative time（all P<0.01）,while the expressions in the CLP+DEX group were decreased compared with the CLP group（all P<0.05）.Conclusion1.DEX pretreatment has a protective effect on AKI in septic rats.2.The protective mechanism of DEX pretreatment on AKI in septic rats may be related to the inhibition of activation of NLRP3 inflammasome.