| Part Ⅰ Dexmedetomidine reduces delayed graft function following donation after cardiac death kidney transplant:a randomized doubleblind placebo-controlled trialObjective:Delayed graft function(DGF)is an important risk factor for acute rejection and graft failure after kidney allotransplantation.This randomized controlled trial aimed to investigate whether perioperative dexmedetomidine infusion could reduce DGF after donation after cardiac death(DCD)kidney transplant and whether it could improve postoperative graft function compared with normal saline.Methods:In this randomized,double-blind,placebo-controlled clinical study,a total of 114 patients who diagnosed with end-stage renal disease and planned to undergo kidney transplant under general anesthesia were selected.This study was conducted between September 2019 and January 2021.Recipients who were≥18 years old,and scheduled for DCD kidney transplant under general anesthesia were eligible for inclusion.Patients who had sick sinus syndrome,atrioventricular block,a left ventricular ejection fraction<30%,or who were receiving multi-organ transplantation were excluded.The patients were randomly divided into the dexmedetomidine group(intravenous injection of dexmedetomidine 0.4 μg/kg/h immediately after induction of anesthesia until the end of the operation,and continued infusion of dexmedetomidine after the removal of the tracheal tube,then 0.1 μg/kg/h maintained for 24 hours)and normal saline control group according to the ratio of 1:1.General anesthesia was maintained by inhalation of 1%-3%sevoflurane in both groups,titrated to maintain the BIS value in the range of 40-60.Additional sufentanil was given for intraoperative analgesia.Additional cisatracurium was given for intraoperative muscle relaxation.At the end of the surgery,a sufentanil-based patient-controlled intravenous analgesia was initiated and continued until 48 hours after surgery.After surgery,the endotracheal tube was removed,and the patient was transferred to a designated transplant ward.The primary outcome was the incidence of DGF(need for dialysis during the first posttransplant week).The secondary outcomes included repeated dialysis during the first posttransplant week,in-hospital acute rejection,and 30-day serum creatinine,serum cystatin C,eGFR,need for dialysis,and patient survival.The perioperative data were graft functionrelated parameters during hospitalization(serum creatinine,serum cystatin C,creatinine clearance rate,and urine output),visual analogue scale pain scores at 30 min,24 hours,and 48 hours postoperatively,and perioperative bradycardia and hypotension.Several in-hospital and long-term outcomes were assessed post hoc:creatinine reduction ratio on post-transplant day 2(CRR2),the proportion of CRR2<30%,long-term serum creatinine,serum cystatin C,eGFR,allograft failure,and patient survival.All patients were followed up postoperatively at regular intervals,with the data updated on July 31,2021.Results:The remaining 111 patients received the scheduled transplantation and had available primary outcome data.The modified intent-to-treat population had 56 patients in the dexmedetomidine group and 55 in the normal saline group.DGF occurred in 10 of the 56(17.9%)patients receiving dexmedetomidine and 19 of the 55(34.5%)patients receiving normal saline(OR=0.41;95%CI,0.17 to 0.98;P=0.045).Dexmedetomidine reduced the need for repeated dialysis during the first post-transplant week(12.5%vs.30.9%;OR=0.32;95%CI,0.13 to 0.88;P=0.019),without significant between-group difference after multiple testing corrections.In-hospital biopsy-proven acute rejection occurred in 5 patients in the dexmedetomidine group and 7 in the normal saline group,without significant between-group difference(8.9%vs.12.7%;OR,0.67;95%Cl,0.22 to 2.14;P=0.519).Compared with the normal saline group,the dexmedetomidine group had no significant difference in serum creatinine,serum cystatin C and eGFR 30 days after transplantation.One patient in the dexmedetomidine group and 3 patients in the normal saline group needed dialysis at posttransplant day 30.No patient died within 30 post-transplant days.Compared with the normal saline group,the dexmedetomidine group had no significant difference in serum creatinine,serum cystatin C during hospitalization.The creatinine clearance rate was higher in the dexmedetomidine group than in the normal saline group on POD 1(9.9[4.9,21.2]vs.7.9[2.0,10.4]mL/min;difference=2.0 mL/min;95%CI,0.5 to 6.8 mL/min)and POD 2(29.6[9.7,67.4]vs.14.6[3.8,45.1]mL/min;difference=15 mL/min;95%CI,0.4 to 18.5 mL/min).The urine output was higher in the dexmedetomidine group than in the normal saline group during POD 2(106.5[66.3,175.6]vs.82.9[27.1,141.9]mL/h;difference=23.6 mL/h;95%CI,0.17 to 59 mL/h),POD 7(126.1[98.0,151.3]vs.107.0[82.5,137.5]mL/h;difference=19.1 mL/h;95%CI,1.7 to 36.3 mL/h),and at hospital discharge(110.4[92.8,121.9]vs.97.1[77.5,113.8]mL/h;difference=13.3 mL/h;95%CI,4.2 to 22.5 mL/h).The VAS scores of the dexmedetomidine group at 30 minutes and 24 hours after operation were significantly lower than those in the normal saline group.The incidences of bradycardia were 16.1%and 9.1%in the dexmedetomidine and normal saline groups,respectively,without significant between-group difference.The incidences of hypotension were 14.3%and 10.9%in the dexmedetomidine and normal saline groups,respectively,without significant between-group difference.Compared with normal saline group,the dexmedetomidine group had a higher median CRR2(36.5%vs.23.6%)and a lower incidence of CRR2<30%(46.4%vs.56.4%),but without significant between-group difference.Patients were followed up for a median duration of 13(IQR,9-19)months.Long-term follow-up results showed that the dexmedetomidine group had no significant difference in serum creatinine,serum cystatin C while compared with normal saline group.The dexmedetomidine group had a higher eGFR compared to the normal saline group(difference=8.4 mL/min,P=0.051).Of the 4 patients with 30-day need for dialysis,3 patients in the normal saline group were still on regular dialysis at the point of the last follow-up,and 1 patient in the dexmedetomidine group did not require dialysis until the last follow-up.No patient died until the last follow-up.Conclusion:The 24-hour perioperative dexmedetomidine infusion can reduce delayed graft function and improve early graft function after donation after cardiac death kidney transplant.Part Ⅱ Dexmedetomidine attenuates renal ischemia-reperfusion injury through activating α2/PI3K/Akt/eNOS signaling and suppressing inflammation in miceObjective:To determine whether dexmedetomidine(DEX)treatment exerts protective effects against ischemia/reperfusion(I/R)-induced renal injury through regulatingα2/PI3K/Akt/eNOS signaling and suppressing inflammation in a mice renal I/R injury model.Methods:(1)Effects of dexmedetomidine pretreatment and posttreatment on renal function during renal I/R injury in mice.Adult healthy male mice were randomly divided into 3 groups(n=6):sham group,I/R group,and DEX+I/R group.The bilateral renal pedicles occlusion for ischemia 45min was performed using microvascular clamps,and then the artery clamps were removed for reperfusion 48h.Sham group mice underwent the same surgical procedures but with no occlusion of the renal pedicles.Besides,the mice in DEX+I/R group were received intraperitoneal injection of DEX(50μg/kg)at two different time points.The first injection was 30 min prior to ischemia when anesthesia was performed,and the second injection was at the end of the ischemia,then bilateral arterial clips were released.After reperfusion for 48h,animals were sacrificed and bilateral kidney tissue and the blood samples were collected to observe the histological and renal functional changes.(2)The whole-transcriptome mRNA sequencing was used to investigate the biological processes,signaling pathways and protein interactions of differentially expressed genes(DEGs)after renal I/R injury.Six healthy adult mice were randomly divided into 2 groups(n=3),sham group and I/R group.After 48 hours of reperfusion,bilateral kidney tissues were collected,the whole-transcriptome mRNA sequencing was performed to investigate the biological processes,signaling pathways and protein interactions of DEGs between two groups.(3)qPCR verified DEGs in damaged-related signaling pathways.Twelve healthy adult mice were randomly divided into 2 groups(n=6),sham group and I/R group.We further verified the relevant DEGs changes on damaged-related signaling pathways using qPCR method.(4)Effects of dexmedetomidine pretreatment and posttreatment on phosphatidylinositol 3 kinases/protein serine threonine kinase(PI3K/Akt)signaling pathway,expression of the endothelial nitric oxide synthase(eNOS)and nitric oxide(NO).Twelve healthy adult mice were randomly divided into 3 groups(n=4),sham group,I/R group and DEX+I/R group.After 48 hours of reperfusion,the protein expression levels of PI3K,phospho-PI3K(p-PI3K),Akt,phospho-Akt(p-Akt)and eNOS in the kidney tissues of the three groups were detected by western blotting.The expression of NO was detected by Nitric Oxide Assay Kit.(5)Effects of dexmedetomidine pretreatment and posttreatment on inflammatory cytokines.Eighteen healthy adult mice were randomly divided into 3 groups(n=6),sham group,I/R group and DEX+I/R group.After 48 hours of reperfusion,the kidney tissue and serum of the mice were collected,the inflammatory cytokine tumor necrosis factor(TNF-α),IL-6,intercellular adhesion molecule-1(ICAM-1),monocyte chemoattractant protein-1(MCP-1)were detected by qPCR and Enzyme Linked Immunosorbent Assay(ELISA)respectively.(6)Effects of α2 receptor blocker atipamezole(Atip)intervention on the renoprotective effect of dexmedetomidine.Twenty healthy adult mice were randomly divided into 4 groups(n=5),sham group,I/R group,DEX+I/R group and Atip+DEX+I/R group.Atip+DEX+I/R group were simultaneously given DEX(50 μg/kg)and atipamezole(250 μg/kg)during anesthesia.After 30 minutes of anesthesia,the bilateral renal pedicles were clipped,DEX(50 μg/kg)and atipamezole(250 μg/kg)were administered again after 45 minutes of ischemia,then bilateral arterial clips were released.After 48 hours of reperfusion,the kidney tissues of the mice were collected,and the histological changes of the kidneys were observed and histological damage scores were performed.(7)DEX acts via α2 receptor on PI3K/Akt pathway,eNOS and NO.Sixteen healthy adult mice were randomly divided into 4 groups(n=4),sham group,I/R group,DEX+I/R group and Atip+DEX+I/R group.After 48 hours of reperfusion,the kidney tissues were collected and western blotting was used to detect the protein expression levels of PI3K,pPI3K,Akt,p-Akt and eNOS in the kidney tissues.The expression of NO was detected by Nitric Oxide Assay Kit.(8)DEX acts via α2 receptor on inflammatory cytokines.Twenty healthy adult mice were randomly divided into 4 groups(n=5),sham group,I/R group,DEX+I/R group and Atip+DEX+I/R group.After 48 hours of reperfusion,the kidney tissues were collected and the inflammatory cytokines in the kidney tissues were detected by qPCR.(9)Both α2 receptor blocker and PI3K agonist(740Y-P)intervention were performed to determine the effect of DEX on PI3K/Akt pathway,eNOS and NO.Twenty healthy adult mice were randomly divided into 5 groups(n=4),sham group,I/R group,DEX+I/R group,Atip+DEX+I/R group and Atip+740Y-P+DEX+I/R group.Atip+740Y-P+DEX+I/R group were simultaneously given DEX(50 μg/kg),atipamezole(250 μg/kg)and 740Y-P(2.5 mg/kg)during anesthesia.After 30 minutes of anesthesia,the bilateral renal pedicles were clipped,DEX(50 μg/kg),atipamezole(250 μg/kg)and 740Y-P(2.5 mg/kg)were administered again after 45 minutes of ischemia,then bilateral arterial clips were released.After 48 hours of reperfusion,the kidney tissues of the mice were collected,western blotting was used to detect the protein expression levels of PI3K,p-PI3K,Akt,p-Akt and eNOS in the kidney tissues.The expression of NO was detected by Nitric Oxide Assay Kit.(10)Both α2 receptor blocker and PI3K agonist intervention were performed to determine the effect of DEX on inflammatory cytokines.Twenty healthy adult mice were randomly divided into 5 groups(n=4),sham group,I/R group,DEX+I/R group,Atip+DEX+I/R group and Atip+740Y-P+DEX+I/R group.After 48 hours of reperfusion,the kidney tissues were collected and the inflammatory cytokines in the kidney tissues were detected by qPCR.(11)Both α2 receptor blocker and PI3K agonist intervention were performed to determine the effect of DEX on renal histology.Twenty-five healthy adult mice were randomly divided into 5 groups(n=5),sham group,I/R group,DEX+I/R group,Atip+DEX+I/R group and Atip+740Y-P+DEX+I/R group.After 48 hours of reperfusion,the kidney tissues were collected and the histological changes of the kidneys were observed and histological damage scores were performed.Results:(1)Compared with the sham group,the I/R group could significantly increase the histological damage score,and the serum creatinine and blood urea nitrogen levels were significantly increased(P<0.01),while DEX treatment could significantly reduce the increased histopathological score and decrease serum creatinine and blood urea nitrogen level during renal I/R injury.(2)Our RNA-seq analysis showed that a total of 2486 DEGs were discovered,including 1901 up-regulated genes and 585 down-regulated genes in the heatmaps.The first three biological processes in GO enrichment analysis were:stress response,unicellular and multicellular biological processes,and cellular component organization.KEGG enrichment analysis showed that the top three signaling pathways related to tissue damage were PI3K/Akt signaling pathway,TNF-α signaling pathway and mitogen-activated protein kinase(MAPK)signaling pathway.(3)q-PCR verified that the mRNA levels of PI3K,Akt,eNOS,TNF-α and nuclear transcription factor(NF-κB)in the I/R group were significantly higher than those in the sham group(P<0.01).There was no significant difference in the mRNA levels of P53,FOXO4,Bax/Bcl2 between the two groups.(4)Western blotting analysis showed that I/R injury significantly increased the p-PI3K,p-Akt,eNOS protein expression.Moreover,DEX treatment further enhanced these above protein expressions(P<0.01).Besides,there was no significant change in total PI3K and Akt after I/R injury or DEX treatment.Nitric Oxide Assay Kit showed that I/R injury significantly increased the NO expression,and DEX treatment further enhanced NO expressions.(5)Both qPCR and ELISA showed that renal I/R injury could significantly increase the expression of inflammatory cytokines TNF-α、IL-6、ICAM-1 and MCP-1 on the mRNA and protein levels(P<0.01),while DEX treatment could significantly reduce the increased expression of inflammatory cytokines TNF-α、IL-6、ICAM-1 and MCP-1 after renal I/R injury.(6)Compared with DEX+I/R group,α2 receptor blocker intervention significantly increased renal histological damage score(P<0.01).(7)Compared with DEX+I/R group,the renal expression of p-PI3K/PI3K,p-Akt/Akt,eNOS and NO in Atip+DEX+I/R group were significantly decreased(P<0.01).(8)Compared with DEX+I/R group,the renal expression of inflammatory cytokines TNF-α、IL-6、ICAM-1 and MCP-1 in Atip+DEX+I/R group were significantly increased(P<0.01).(9)Compared with Atip+DEX+I/R group,the renal expression of p-PI3K/PI3K,pAkt/Akt,eNOS and NO in Atip+740Y-P+DEX+I/R group were significantly increased(P<0.01).(10)Compared with Atip+DEX+I/R group,the renal expression of inflammatory cytokines TNF-α、IL-6、ICAM-1 and MCP-1 in Atip+740Y-P+DEX+I/R group were significantly decreased(P<0.01).(11)Compared with Atip+DEX+I/R group,the renal histological score in Atip+740YP+DEX+I/R group were significantly lower(P<0.01).Conclusion:Dexmedetomidine pretreatment and posttreatment can alleviate renal ischemia-reperfusion injury in mice.Dexmedetomidine attenuates renal ischemiareperfusion injury through activating α2/PI3K/Akt/eNOS signaling and suppressing inflammation in mice. |
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