Mechanism Of Renal Protective Effect Of Dexmedetomidine After Renal Allograft Transplantation And Renal Ischemia/Reperfusion Injury

Background:Kidney transplantation has been recognized as the most appropriate treatment for most patients with end-stage renal disease.Renal dysfunction after transplantation is associated with hemodynamic alterations,immune rejection,ischemia/reperfusion injury(IRI),inflammatory responses,and sympathetic activity,etc.Early renal tubular injury begins with ischemia/reperfusion(I/R)and is usually accompanied by progressive tubulointerstitial fibrosis(TIF).Early IRI is one of the important factors leading to the loss of graft function,and the most common pathological changes of progressive allograft failure are TIF and tubular atrophy.Therefore,inhibition of I/R induced tubular injury and TIF may improve the outcome of renal transplantation and the survival rate of renal transplantation.As a selective adrenergic receptor agonist,dexmedetomidine can not only reduce the excitability of the sympathetic adrenal medulla system to maintain hemodynamics stability,but also play an important role in organ protection by inhibiting inflammatory reaction and decreasing apoptosis in recent studies.However,little is known about the effect and mechanism of antifibrosis induced by dexmedetomidine.Objective:This study aims to evaluate the effects of dexmedetomidine on perioperative stress response,and to clarify the effect of dexmedetomidine on tubular injury and TIF after renal transplantation and I/R,and to investigate the role of HIF-1 a and transforming growth factorβ1(TGF-β1)/Smads signaling pathways in antifibrosis mediated by dexmedetomidine.This study is divided into five parts:Part Ⅰ:Effect of dexmedetomidine on perioperative stress response in kidney transplantation.Methods:Two recipients who received from the same brain death donor were randomly divided into two groups:dexmedetomidine group(Dex,n=39)and control group(Con,n=39).The mean arterial pressure and heart rate during operation were recorded at different time points.The use of vasoactive drugs and anesthetics were compared.The incidence of pain,nausea,vomiting,irritability and shivering were compared between two groups after operation to appraise the impact of dexmedetomidine on perioperative stress response.Results:The mean arterial pressure after tracheal intubation(P=0.012)and skin incision(P=0.018)in Dex group was lower than that in Con group.The heart rate after tracheal intubation in Dex group was also less than Con group(P=0.021).The dosage of sufentanil in Con group was obviously more than Dex group(P=0.039),and the use of atropine in Con group was significantly less than that in Dex group(P=0.027).Patients in the Dex group had lower VAS pain scores at 6 h(P=0.01)and 12 h(P=0.002)after surgery.The incidence of postoperative shivering in Dex group was significantly lower than Con group(P=0.037).Body temperature at the end of operation,the time of extubation,the incidence of nausea,vomiting and irritability within 30 minutes after operation have no significant difference between groups.Summary:The perioperative use of dexmedetomidine can reduce the hemodynamics fluctuation during renal transplantation,reduce the dosage of analgesic in the operation,improve postoperative shivering and pain.Dexmedetomidine can relieve the perioperative stress reaction.Part Ⅱ:Effect of dexmedetomidine on postoperative complications and prognosis of renal transplantation.Methods:The incidence of DGF,hospital stay,postoperative infection,nephrectomy rate and mortality were compared between Dex Group and Con group.The rate of complications was assessed by Clavien-Dindo complication grading system.The concentration of serum creatinine(Cr),urea and urine output were compared between groups within 6 days after operation.Serum Cr,urea,β2-MG,CysC and eGFR were tested during 18-month follow-up to evaluate renal function.Results:The incidence of Clavien-Dindo complications graded Ⅰ and Ⅱ in Dex group was significantly lower than Con group(P=0.046 and P=0.025).The hospital stay in Dex group were evidently shorter than Con group.The occurrence rate of DGF in Dex group(12.8%)was less than Con group(17.9%),but the difference was not statistically significant.The difference of serum Cr,urea and urine output between the two groups in the first 6 days after operation was also not statistically significant.During the 18-month follow-up,there was no statistic significance in renal function between the two groups.But compared with the 6th day after operation,the Cr reduction rate of 2M,3M,12M and 18M in Dex group were significantly higher than Con group.Summary:Dexmedetomidine can reduce the incidence of complications after renal transplantation,shorten the days of hospitalization,and increase the Cr reduction rate in 18month follow-up.Dexmedetomidine reduced the incidence of postoperative DGF,but there was no statistical significance.Part Ⅲ:Effect of dexmedetomidine on renal injury and TIF after renal transplantation.Methods:Detection of TGF-β1,Smad2 and Smad3 mRNA in peripheral blood monocytes after 7 and 21 days of renal transplantation using RT-PCR method.Determination of IL-6 and TNF-α in serum,TGF-β1 and kidney injury molecule-1(KIM-1)in urine using ELISA.Results:The mRNA level of TGF-β1,Smad2 and Smad3 in Dex Group was obviously down-regulated on the 7th and 21st day after operation compared with Con group.The relative concentration of TGF-β1 in urine(corrected by creatinine)and the concentration of serum IL-6 and TNF-α were significantly decreased in Dex Group.The concentration of KIM-1 in Dex group was lower than Con group at 2h 24h 48h and 72h after reperfusion.Summary:Perioperative use of dexmedetomidine can ameliorate the renal injury,alleviate the inflammatory reaction and inhibit the activation of TGF-β1/Smads signaling pathway after renal transplantation.Part Ⅳ:Effects of dexmedetomidine on HIF-1α and TGF-β1/Smads after I/R in mice.Methods:Three different concentrations of dexmedetomidine were intraperitoneally injected 30 min before the I/R model in male C57BL/6J mice(ischemia for 40 min),kidneys and blood were obtained 24h after reperfusion.HE and PAS staining were used to evaluate the effect of different doses of dexmedetomidine on renal tubular injury after I/R.Immunohistochemistry and TUNEL staining were used to evaluate the apoptosis.ELISA was used to detect the concentration of serum Cr,Urea and TGF-β1.The effects of dexmedetomidine on the protein and mRNA expression levels of HIF-1α,TGF-β1/Smads orα-SMA were detected by western blot and RT-PCR after I/R.Results:Dexmedetomidine significantly alleviated renal tubular injury,decreased cell apoptosis,and improved renal function in a dose-dependent manner.The level of HIF-1αpeaked at D1 after I/R,and the expression of TGF-β1,Smad2 and Smad3 peaked at D7.Expression level of α-SMA was obviously increased at D7 after reperfusion,peaked at D14,and then lasted until D21,Intraperitoneal injection of 50μg/kg dexmedetomidine reduced renal HIF-1α,TGF-β1,p-Smad2 and p-Smad3 and also TGF-β1 in serum at 7 and 21 days after I/R.Summary:Dexmedetomidine can alleviate renal tubular injury,reduce apoptosis,downregulate overexpression of HIF-1α induced by I/R,and inhibit the activation of TGFβ1/Smads signaling pathway.Part V:Dexmedetomidine ameliorates IRI-induced TIF by inhibiting HIF-1α and TGFβ1/Smads pathway.Methods:Male C57BL/6J mice were randomly divided into Sham group,I/R group,Dex+I/R group,YC-1+I/R group and Yohinbin(Yoh)+Dex+I/R group.Western blot was used to detect the protein expressions of HIF-1α,TGF-β1,p-Smad2,Smad2,p-Smad3,Smad3,αSMA and Collagen I.The mRNA expressions of HIF-1α,TGF-β1,Smad2 and Smad3 were detected by RT-PCR.The degree of tubular injury and the area of TIF was estimated by HE,PAS and Masson staining.ELISA was used to detect the expression of serum IL-6,TNF-α,Cr and urea.Results:HIF-1α antagonist YC-1 significantly decreased the expression of HIF-1α in kidney,and the expression of TGF-β1/Smads protein and mRNA also decreased.α2 adrenergic receptor antagonist Yoh counteracted the down-regulation effect of dexmedetomidine on HIF-1α,the overexpression of HIF-1α induced up-regulated of TGF-β1,p-Smad2 and p-Smad3.The level of α-SMA,Collagen I,and the area of TIF was obviously increased by activation of TGF-β1/Smads pathway.Inhibition of HIF-1α,could down-regulate the expression level of TGF-β1/Smads,decrease the level of α-SMA and Collagen I,reduce the area of TIF,alleviate the concentration of serum IL-6,TNF-α,Cr and Urea.Summary:The overexpression of HIF-1α induced by IRI can activate TGF-β1,promote the phosphorylation of Smad2 and Smad3,and aggravate the occurrence and development of TIF induced by IRI.Part of the mechanism of the renoprotective effect of dexmedetomidine may be that it restrains the activation of TGF-β1/Smads signaling pathway by down-regulate the level of HIF-1α in kidney after I/R.Therefore,the expression of α-SMA and collagen Ⅰwere decreased and TIF was relieved.The inhibitory effect of dexmedetomidine on TIF after I/R was also related to the reduction of renal tubular injury and the alleviation of inflammatory reaction.Conclusion:1.Dexmedetomidine can reduce the intraoperative hemodynamics,the dosage of analgesic used in the operation,the postoperative shivering and pain,decrease the incidence of postoperative complications and the length of hospital stay,and also increase the creatinine reduction rate during the 18-month follow-up.The protective effect of dexmedetomidine on renal allograft may be related to the inhibition of perioperative stress response,the reduction of I/R induced inflammatory reaction and renal injury,and the decreases of TGF-β1/Smads signal pathway.2.Dexmedetomidine can alleviate renal tubular injury induced by I/R in mice,reduce apoptosis,decrease inflammatory factors,downregulate the overexpression of HIF-1αinduced by IRI and inhibit the activation of TGF-β1/Smads signal pathway.3.IRI induced upregulation of HIF-1α could activate TGF-β1,promote the phosphorylation of Smad2 and Smad3,and exacerbate the development of TIF induced by I/R.Partial mechanism of renal protection induced by dexmedetomidine may be down-regulating the level of renal HIF-1α and restraining the up-regulation of TGF-β1/Smads signal pathway.Therefore,the expression of α-SMA and collagen Ⅰ were decreased,and TIF was relieved.