The Advantage And Mechanism Of Hypothermia Machine Perfusion On Marginal Kidney Preservation

Objective:The purpose of this experiment was to establish a autologous kidneytransplantation model in rabbit. To investigate the advantages of HypothermiaMachine Perfusion compare to static cold storage in marginal kidney preservation andexplore its possible mechanism.This experiment is divided into two parts.PartⅠ: Establish model of living rabbit kidney hypothermia machine perfusion andcold storageMethods:Taking healthy10adult rabbits with unlimited sex, weight (3.0±0.3) Kg. Therabbits were randomly divided into2groups: Hypothermia Machine Perfusion group(group MP)(n=5) and cold storage group (Group CS)(n=5). Group MP and GroupCS were took blood samples before conventional laparotomy surgery, and anatomizedthe renal artery and vein, and blocked the renal blood flow for35min, taking renalartery catheterization after open the blood flow for60min and irrigate kidney withcold solution(HCAⅡ), put the kidneys into the compound of ice and water. GroupMP was connected with Lifeport (Organ Recovery Systems company of America,serial number:0919703) and continuously perfused for4hours with the pressure of20mmHg. Group CS was only been preserved in compound of ice and water for thesame long time, while the temperature of preserve solution of two group was recordedevery half-hour. Removed the catheters and stitched the gaps of the renal artery andvein after4hours and than opened blood flow and rewarm the kidney immediatelywith hot water. Resected the left kidney and did cystostomy and indwelling catheter.Made blood gas analysis during the period of Pre-operation（B1）、During operation（B2）and Post-operation（B3）. Record anal temperature every5min during theperiod of renal blood flow blocked. Group MP was recorded the flow, display on themachine, every60min. Take loop rehydration and anti-infection after operation. Record the survival rate and urine for24hours.Part Ⅱ: Investigate the advantages and mechanisms of Hypothermia MachinePerfusion compare with static cold storage.Methods:Taking healthy15adult rabbits with unlimited sex, weight (3.0±0.3) Kg. Therabbits were randomly divided into sham operation group (Group SO), andHypothermia Machine Perfusion group (Group MP) and cold storage group (GroupCS)(n=5). Group SO made conventional laparotomy surgery with anatomized therenal artery and vein. Group MP copy the model of hypothermia machine perfusion,Group CS copy the model of cold storage. Take renal tissue and blood samples after24hours. Test the indicators of renal function, serum creatinine (CRE) and ureanitrogen (BUN). Detected serum interleukin-6(IL-6) by enzyme-linkedimmunosorbent assay (ELISA). By the means of reverse transcriptase polymerasechain reaction (RT-PCR), test ICAM-1mRNA transcription level in kidney tissue.Exam the expression of NF-κB p65protein in kidney tissues byimmunohistochemical techniques and observe the Hematoxylin eosin (HE) stainingchanges on kidney biopsy.Results Ⅰ:1. Temperature: on the whole the anal temperature was stable during the time ofrenal blood flow blocked.There are no significant differences among3groups andevery groups’interior (P>0.05).2. Flow of perfusion: The flow increase sharply during the first hour ofhypothermia machine perfusion, the flow at0min is significant changed comparewith that at60min(P<0.001). And the flow of other point-in-times are significantchanged compare with the point of0min (P>0.05).3. Survival rate: The rate of24hours survival of either Group MP or Group CSare100%, there are no difference between them.ResultsⅡ:1. Urine volume: The24hours urine volume of Group SO and Group MP aresuperior to Group CS (p<0.05). Group MP is superior to Group SO (p<0.05).2. CRE and BUN: Group CS and Group MP increased significantly compare with Group SO (p<0.05); Group CS increased significantly compare with Group MP(p<0.05). There are no significant differences among all the3groups (P>0.05).3. IL-6: Group CS and Group MP increase significantly compare with Group SO(p<0.05); Group CS increase significantly compare with Group MP (p<0.05). Thereare no significant differences in all3groups (P>0.05).4. NF-κB p65protein expression: Quantitative analysis of the quantitative imageanalysis software to measure the average optical density (OD) of the results ofimmunohistochemistry. The expression levels of Group CS increased significantlycompare with Group SO and Group MP (p<0.05); There is no significant differencebetween Group SO and Group MP (P>0.05).5. ICAM-1mRNA: The expression ICAM-1mRNA of transcription levelsmeasured by RTFQ-PCR in Group CS and Group MP were significantly higher thanGroup SO (p<0.05); Group CS increased significantly compare with Group MP(p<0.05).6. Histological examination (Hematoxylin eosin)(HE): The kidney tissuestructure is normal in Group SO, The renal tubular epithelial cell expressed mildedema in Group MP while the diameters of renal tubular expand especially inproximal convoluted tubule. There was also mild edema in the renal tubular epithelialcell in Group CS with the diameters of proximal convoluted tubule and distalconvoluted tubule become smaller. The renal tubular epithelial cell expressed mildedema in both Group MP and Group CS while Group CS is even more serious.Conclusion:1. The models of living rabbit kid hypothermia machine perfusion and coldstorage were stable and could be used in other studies.2. Comparing with cold storage, hypothermia machine perfusion can decreasethe levels of CRE、BUN and TNF-α in serum and decrease the expression ofICAM-1and NF-κB in kidney tissue after the kidney “transplanted”，it can alsoincrease the urine volume and survival rate. Hypothermia machine perfusion it betterthan cold storage at kidney preserve.. 3. Histological examination show the hypothermia machine perfusion canimprove the microcirculation of the donor kidneys and it could improve the renalfunction after kidney transplantation.4. Hypothermia machine perfusion can remove the metabolites during preservingprocess and decrease nonspecific inflammatory injury after rewarm.