Protective Effects And Mechanisms Of Dexmedetomidine/Noble Gas Modified UW Solution On The Marginal Donor Kidney From Porcine DCD And In Vitro Human Lung Tissue After Warm Ischemia

Background and purposeOrgan transplantation is an effective treatment for patients with end-stage organ failure.However,the shortage of donor organs is becoming increasingly severe.Although the number of kidney transplants has increased annually,it has not maintained pace with the growth of the waiting lists.It has been reported that only about 25%of transplant patients received a kidney from a deceased donor within five years and only 20%of the donor lungs qualify for transplants of the highest quality.While living donors and donation after brain death（DBD）donors cannot meet clinical demand,it is important to expand the use of marginal donors such as donation after cardiac death（DCD）donors.This could,to some extent,alleviate the shortage of donor graft sources and help to expand the donor pool.However,the warm ischemia time of DCD donor grafts was prolonged,and the proportion of Primary non-function（PNF）and Delayed graft function（DGF）was higher in receipents.Consequently,enhancing the preservation quality of marginal donors,enhancing the function of grafts after transplantation,and implementing these techniques effectively in the clinic are among the most important methods for addressing the organ shortage.It is evident that expanding the application of marginal donors is conducive to expanding the donor pool,allowing more patients with organ failure access to organ transplantation opportunities,which has significant clinical value.Dexmedetomidine（Dex）,a highly selectiveα₂ adrenergic receptor agonist,has sedative and analgesic effects and is commonly used in clinical anesthesia and ICU.Dex protects cells from injury by reducing inflammatory responses and activating anti-apoptotic signaling pathways,and has organ protective effects on nervous system,heart,lungs,kidneys,liver and small intestine.Additionally,noble gases xenon（Xe）and argon（Ar）also have organ protection effects.Studies have shown that Xe can reduce the expression of Caspase-3 and inhibit the activation of TLR-4/NF-κB in renal tubule cells,as well as reduce the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α,thereby protecting renal function,improving the survival rate of grafts,and extending their survival time.Ar plays a protective role in ischemia reperfusion injury of the heart,brain,and kidneys.In porcine kidney transplant models,Ar stimulates antioxidant defense and limits inflammation,thereby reducing graft injury and improving graft survival.These results suggest that Dex,Xe,and Ar have protective effects on a variety of organs,including grafts.Based on this,we hypothesize that the addition Dex,Xe,Ar to University of Wisconsin（UW）organ preservation solutions may improve the quality of preservation quality and graft function of DCD marginal donors following transplantation.In this study,the UW organ preservation solution was saturated with different concentrations of Dex/noble gases to construct the porcine DCD marginal donor model and obtain the marginal donor kidney.In addition,to confirm the graft protective effects of Dex,the UW solution was supplemented with Dex and was used to cold preserve human lung tissue samples that were collected clinically.Then the porcine DCD kidneys and human lung tissues were then cold preserved in modified UW solution to mimic clinic static cold storage（SCS）.HE staining,WB,TUNEL,immunofluorescence,transmission electron microscopy and additional techniques were used to verify whether Dex,Xe,or Ar can improve the quality of preservation of porcine DCD marginal donor kidneys and human lung tissues.The modified preservation solutions on different programmed cell death patterns such as apoptosis,necroptosis and ferroptosis will also be investigated.Materials and methods（a）Protective effect and mechanism of dexmedetomidine/noble gas modified UW solution on the marginal donor kidneys from DCD porcine1.Preparation of modified UW solutions based on the supplementation of Dex/Noble gasesModified solutions formula:solution 1:30%N₂+5%CO₂+65%O₂+UW solution;solution 2:30%N₂+5%CO₂+65%O₂+0.1nM Dex+UW solution;solution 3:30%Xenon+5%CO₂+65%O₂+0.1nM Dex+UW solution;solution 4:30%Argon+5%CO₂+65%O₂+0.1nM Dex+UW solution;solution 5:15%Xenon+15%Argon+5%CO₂+65%O₂+0.1nM Dex+UW solution;solution 6:50%Xenon+5%CO₂+45%O₂+0.1nM Dex+UW solution;solution 7:50%Argon+5%CO₂+45%O₂+0.1nM Dex+UW solution;solution 8:25%Xenon+25%Argon+5%CO₂+45%O₂+0.1nM Dex+UW solutionAccording to the formula of the modified solution designed in the experiment,with or without 0.1nM Dex,30%nitrogen（N₂）and different concentrations of noble gases（Xe and/or Ar）were filled into the UW solution at the rate of 0.5L/min for 20 minutes to complete the preparation of eight modified preservation solutions.2.To establish porcine DCD donor model and obtain marginal donor kidneysAfter sedation,mini pigs were monitored for vital signs and administered heparin.Intravenous injection of excessive anesthetic drugs propofol and cisatracurium to complete the construction of the porcine DCD donor model.Thoracotomy was performed,followed by heparin-containing saline cyclic lavage.The kidneys were then extracted after 30 minutes of warm ischemia to complete the acquisition of the marginal donor kidneys.3.To verify the protective effect of the modified solutions on the DCD porcine kidneys and the effects of modified solutions on apoptosis,necroptosis and ferroptosis.After obtained the porcine DCD kidneys,HE staining was performed on the above 8modified solution SCS for 24h or 72h,and tissue damage score was performed on the sections by double-blind method,to observe and evaluate the pathological changes of the preserved porcine DCD kidneys.Renal cell apoptosis was detected by TUNEL,the expression of phosphorylation mixed lineage kinase domain like protein（pMLKL）and glutathione peroxidase 4（GPX4）were detected by immunofluorescence to evaluate necroptosis and ferroptosis.（b）Investigation into the protective effect and mechanism of dexmedetomidine modified UW solution on ex vivo human lungs after warm ischemiaIn clinical cases of pulmonary lobectomy,normal human lung tissues（ex vivo human lungs after warm ischemia）were obtained from the lobelectomy tissues according to the collection standard.Dex modified UW solution SCS 16h human lung tissues were used to simulate the cold preservation state of the donor lungs.HE staining,TUNEL,WB,immunofluorescence and electron microscopy were used to evaluate the effect of Dex on cold preservation of human lung tissues.The effects of Dex on apoptosis,necroptosis and ferroptosis were detected.Results（1）After intravenous injection of excessive anesthetic drugs propofol and cisatracurium,cardiac arrest was achieved in all 5 mini pigs,and the porcine DCD donor model was successfully constructed with a success rate of 100%.（2）Dex combined with noble gases improved the morphology and structure of porcine DCD marginal donor kidneys SCS after 24h and 72h,and reduced the kidney injury.Compared with NC group（non-cold preservation group）,cold ischameia for 24 and 72 hours（CI24h and CI72h）groups significantly reduced the mean ratio of healthy cells in renal tubules and glomeruli and increased kidney injury score（P<0.001～0.0001）.Compared with CI24h group,except CI24h+Dex group,all Dex with Xe and/or Ar groups increased the percentage of healthy renal tubules（P<0.05）;Dex+50%Xe/50%Ar/25%Xe+25%Ar group increased the percentage of healthy glomerular cells（P<0.01）;Dex+50%Xe/50%Ar/15%Xe+15%Ar/25%Xe+25%Ar group reduced the kidney injury score of SCS at 24h（P<0.05）.Compared with Dex+15%Xe+15%Ar group,kidney injury score in Dex+25%Xe+25%Ar group decreased（P<0.05）.In the CI72h groups,the kidney tissues were severely damaged.Compared with CI72h group,the Dex+50%Xe/50%Ar/25%Xe+25%Ar group increased the percentage of healthy cells in renal tubules（P<0.05）;the Dex+30%Ar/15%Xe+15%Ar/50%Xe/50%Ar/25%Xe+25%Ar group increased the percentage of healthy glomerular cells（P<0.05）;Dex+50%Xe/50%Ar/25%Xe+25%Ar group reduced the kidney injury score of SCS at 72h（P<0.05）.Compared with Dex+30%Ar group,Dex+50%Ar group increased the percentage of healthy renal tubule cells and decreased the renal injury score（P<0.05）,Dex+50%Ar group showed a better protective effect on DCD kidneys.（3）Dex and/or noble gases reduced the apoptosis of porcine DCD marginal kidney donor SCS at 24h and 72h.Compared with NC group,the number of TUNEL positive cells increased in CI24 and CI72 groups（P<0.05）.Compared with CI24h and CI72h groups,the number of TUNEL positive cells decreased in all groups supplemented with Dex/Dex+Xe/Dex+Ar/Dex+Xe+Ar（P<0.05）.The number of TUNEL positive cells in CI24h+Dex+50%Ar group was lower than that in CI24h+Dex+30%Ar group and CI24h+Dex+50%Xe group（P<0.05）.（4）Dex and/or noble gases decreased the expression of pMLKL at SCS 24h of porcine DCD marginal kidneys.Compared with NC group,the expression of pMLKL was increased in CI24 and CI72h groups（P<0.05）.Compared with CI24h group,the expression of pMLKL decreased in all groups supplemented with Dex and noble gases（P<0.05）.（5）Dex and/or noble gases had no significant effect on GPX4 expression of porcine DCD marginal kidneys after SCS.Compared with NC group,GPX4 expression was increased in CI24h and CI72 groups（P<0.05）.Compared with CI24h and CI72h groups,there was no significant change in GPX4 expression in all groups supplemented with Dex/Dex+Xe/Dex+Ar/Dex+Xe+Ar.（6）Dex improved the morphology and structure of human lung tissues after warm after SCS for 16h,reduced the injury of lung epithelial cells and endothelial cells,and reduced the lung injury score（P<0.01）;Dex also decreased the number of TUNEL+apoptotic cells and the expression of pMLKL（P<0.05）during SCS.（7）Dex did not showed any changes on GPX4 expression of SCS human lung tissues.Conclusions1.Intravenous injection of excessive propofol and cisatracurium effectively establish a porcine DCD marginal donor model.2.The combination of Dex and noble gases Xe/Ar supplemented in the UW solution provided protection to the marginal donor kidneys from porcine DCD with long-term SCS,and its mechanism involved the reduction of apoptosis and necroptosis.3.Dex+50%Ar supplemented in the UW solution showed the best protective effect.4.Ferroptosis may occur in the constructed porcine DCD marginal kidneys,the UW solution supplemented with Dex or noble gases had no protective effect on ferroptosis.5.Dex reduces the damage of ex vivo human lung tissues after SCS for 16h,and its mechanism was related to the inhibition of apoptosis and necroptosis,but did not involve the regulation of ferroptosis.