Study Of Simple Cold Storage Of Rabbit Kindneys With Self-Designed Multiple Organ Preservation Solution

Background:Organ preservation is one of the three techniques in transplantation. In order to relieve the contradiction of organ shortage and improve the long-term surviving rate of patients,it is faced with higher requirement in clinical transplantation.Simple staic hypothermy preservation remains the simplest and the most common organ preservation method.The most common organ preservation solutions used including Histidine-Tryptophan-Ketoglutarate(HTK)solution, Celsior solution and University of Wisconsin (UW)solution.UW solution is widely accepted as the gold standard for organ preservation solution,because it can preservating canine kidneys beyond 72 hours and liver for about 30 hours.However, its ingredients are complex and expensive,and there are two problems(high potassium and high viscosity)which makes it not fully satisfied with the clinical demands.Nowadays big organ transplantation and multiple-organ combination transplantation are being carried out rapidly,but there are no long acting multiple organ preservation solutions produced in our country,so on the basis of merits of domestic and abroad organ preservation solutions,we designed the new kind of organ preservation solution-AMU solution.By observing the morphology changes,oxygen free radical injury and energy metabolism of kidney cortex during cold preservation,we studied the efficacy and safety of the solution in the cold preservaton of isolated rabbit kidney.Objectives:1.Preparation of AMU solution:Taking the advantages of domestic and abroad organ preservation solutions and following the preparation principles of organ preservation solution,we determined the ingredients and contents of AMU.The powerful phosphate buffer system could effectively suppress the acidification of cells and tissues in cold and hypoxia situations.Reduced Glutathione,Verapamil and MgCI2 could relief the injuries of mitochondrion induced by hypothermy and ischemia and suppress intracellular calcium overloading and oxygen-radical injury so that the preservation time could be prolonged.The supply of ATP with a shape of ATP-MgCI2 raised multiple-organ surviving activity of pro-transplantation and ATP contain of the tissues in pre-transplantation.it could maintain the cell energy metabolism during cold preservation. Urokinase passed through to dissolve the emboluslus and to guarantee the microcirculation of the storage organ.2.To established simple cold storage of isolated rabbit kidney model.The kidney was flushed perfused with and stored in the cold(040C)organ preservation solution.At the each storage time point, morphologies of kidney cortex cells and mitochondrial,kytop;asm vacuolization,cell swelling,tubular cell necrosis and so on were observed with light microscope and electron microscope in order to comparing the microstructure and ultrastructure changes of cortex3.To detect the changes of MDA and superoxide dismutase's reactive of kidney cortex homogenate and to study the severity degree of cell attacked by free radical and the scavenging free radical capability of every preservation solution at each cold storage time point.4.Indirect assay of energy metabolism:To study the changes of mitochondria function in rabbit kidney cortex during cold storage,the mitochondria functions including respiratory control ratio(RCR)and P/O(addition of ADP:O ratio)which reflect the whole activity of cortex mitochondria respiratory chain and the efficiency of ATP synthesis by cell oxidative phosphorylation were measured.Meantime the activity of Na+-K+ATPase in renal cortex were measured.These reflected the protection of different solutions with respect to kidney energy metabolism during cold storage and combined the morphologic and function changes after injuries induced by hyperthermy and hypoxiaMethods:1.Proparation of AMU:Absorbing the merits of domestic and abroad organ preservation solution and following the principles of organ preservation,we determined the ingredient and contents of AMU. Phosphateserved as the buffer system. Reduced Glutathione,Verapamil and ATP-MgCI2 served as the cell protective ingredients.AMU containd exogenous ATP with a shape of ATP-MgCI2.It easied to permeate the cell membrane of the hyperthermy and hypoxia and raised multiple-organ surviving activity of pro-transplantation and ATP contain of the tissues in pre-transplantation.AMU solution removed expensive hydroxyethyl starch and meltose with cheap low molecular dextran-60 and cane sugar replacement. Urokinase dissolved the emboluslus and guaranteed the microcirculation of the storage organ was filled.Its osmotic pressure was designed to be 343±5mOsm/L and the PH was about 7.4±0.1.AMU prepared was packed by 500ml polythene plastic and sterilized by megatemperature.After these AMU solution was limpid,colorless,transparent and sterile solution.(This task was completed by Yanbian hospital praeparatum center).2.To establised simple cold storage of isolated rabbit kidney model:We used adult male or female rabbits.Rabbits were anesthetized with an introvenous injection of ketamine(2mg/kg) and heparin sodium(500IU) were also given intravenously.We harvested kidneys through a midline incision of abdomen.kidneys were flushed with two types of cold organ preservation solution(0-4℃) that the temperature of kidneys was reduced in the cold organ preservation solution immediately.The flushed-out volume was about 300ml and the pressure of perfusion was about 100cmH2O.During the cold storage(0-4℃),at 24h,48h and 72h kidneys were sampled for morphologic analysis by experienced kidney pathology expertThe swelling and necrosis of glomcrulus and tube cells and the changes of mitochondrials and nuclears were observed by light microscope and electric microscope respectively.3.Kidney tissue was sampled at 24,48h,72hours during cold storage After measuring the proteins in kidney cortex homogenating(1%) by BIADFOID,we caculated the contents of malondialdenhyde(MDA) according to Maleic Dialdehyde(TBA).The enzymatic activity of superoxide dismutase(SOD) was assayed according to Xnthinoxidase method. 4.Kidney tissue was sampled during cold storage at 24,48,72hours during cold storage.Mitochondria in cortex were isolated from cortex homogenization by centrifugation.Mitochondria respiratory function were measured with Clark oxygen electrode.Mitochondrial four-state respiratory were measured:state2:oxygen consumption stimulated by the addition of succinate,state3:oxygen consumption stimulated by the addition of ADP;state4:oxygen consumption after completion of the addition of ADP.RCE=the rate of state 3 oxygen consumption/the rate of state 4 oxygen consumption.ADP/O2=ratio between the nanomoles of ADP phosphorylated and the nanomoles of oxygen consumed during state 3.5. Kidney tissue was sampled at 24,48h,72hours during cold storage After measuring the proteins in kidney cortex homogenating(1%) by BIADFOID, we caculated the contents of Na+-K+ATPase according to Maleic Dialdehyde(TB A).Results:1.Pathologic results:The morphologic changes of renal tubule were more obvious than those of glomcrulus, especilly the proximal convoluted.The pathologic changes of AMU group was similar with that of UW group and there was no statistical difference at 24h,48h,72hrs during cold storage.Under light microscope in two groups,at 24 hrs the structures of the glomcrulus and renal tubule were almost integrated except light swelling of tubule epithelium At 48hrs the structureof the glomcrulus was fundamental normality,but renal tubule epithelium cells were micro-focal necrosis,cell nucleus light pynkosis,interstitial swelling,brush border partly impairment,basal lamina exposure appeared.At 72hrs above-mentioned pathological changes becomed further heavier,extensive spot or lamellar necrosis of tubule epithelium and vague tube structure appearedAt 96hrs the level of pathologic changes becomed the most heavier,but AMU groupe was severer than UW group.Electron microscope:At 24 hrs nuclear alteration and mitochondrion swelling were similar among the two groups.At 48 hrs in AMU and UW groupslight deformation of nuclei,pyknosis of caryoplasm marguin,mitochondrion swelling appeared.At 72 hrs deformation of nuclei,caryoplasm,severe swelling and rupture of mitochondrion appeared. At 96hrs the level of pathologic changes of the electron microscope in AMU groupe was severer than in UW group.These indicated that AMU solution was similar to UW solution with respect to extenuating cell morphology changes.but at 96 hrs UW solution was superior to AMU solution.2.During the cold storage, the activity of SOD in two groups all decreased.At 24h and 48h,the activity of SOD in AMU group was similar with that in HCA group and there was no statistical difference(P>0.05).At 72h the activity of SOD in AMU group was highter than that in HCA group and there was statistical difference(P<0.05).With the prolongation of storage time,content of MDAin cortex homogenization increased in all two groups.The content of MDA in cortex homogenization in AMU group was highter than that in HCA group at each time point and there was statistical difference (P<0.05).These indicated that AMU solution was superior to HCA solution with respect to extenuationg oxygen free radical injury.3.The activity change of mitochondria With the prolongation of storage time,mitochondria oxygen consumption of state III storage in all two groups. Except 2h,24h,The rate of oxygen consumption in state III AMU group was higher than in HCA group at each time point of cold preservation and there was statistical difference(P<Ⅳ(the cold storage,but there was no statistical difference between AMU group and HCA group.Especially at 72h,RCR in AMU group were significantly higher than that in HCA group(P<0.01).Except 2h,ADP/O in AMU group was higher than that in HCA group(P<0.05).These indicated that AMU was superior to HCA with respect to keeping mitochondria allomeric function and the efficiency of ATP synthesis.4. The activity of Na+-K+ATPase in renal cortex were decreased both in AMU group and HCA group.At each time point,there was no significant difference in the activity of the activity of Na+-K+ATPase between AMU group and HCA group at 24h and 48h(P>0.05):The activity of Na+-K+ATPase in AMU group was higher than that of HCA group and there was significant difference at 72h(P<0.05).Conclusion:AMU solution was similar to UW soluton with respect to extenuating cell morphology change to the cold storage 72h and there was no statistical difference At 96 hrs the level of pathologic changes of AMU group was severer than in UW group.AMU solution was superior to HCA solution with relieving oxygen free radical injury,keeping mitochondria allomeric function,the efficency of ATP synthesis,keeping cell energy metabolism and reliving the damage of cellular membrane of kidney cortex.These indicated AMU solution was superior to HCA solution with respect to biochemical function and similar to UW solution with respect to morphology change in protecting cell cold-induced damage.At the same time AMU solution removed expensive hydroxyethyl starch and meltose with cheap low molecular dextran-60 and cane sugar replacement.It was simple,effective,convenient,cheap and has clinical application prospect...