The Study Of Simple Cold Storage Of Canine Kidneys With Self-Designed Multiple Organ Preservation Solution

Organ preservation is major issue in transplantation because of the protective effect exerted by the solution used on ischemic-oxidative damage during storage and on reperfusion injury. UW (University of Wisconsin) solution is widely accepted as the the gold standard for organ preservation solution. Since that important landmark development in preservation, several other solutions have been synthesized to further improve the impact of cold storage on ischemia and reperfusion injury of the graft, i.e. Celsior solution, HTK(Histidine-Tryptophan-Ketoglutarate)solution. These solutions are designed for multiple organ transplantation, and used mostly in abdominal organ transplantation.There isn't any kind of mutiple organ preservation solution produced in China. it is a pity to accept the fact that we have no choice but to use expensive foreign preservation solution in our clinical organ transplantation. According to the important philosophies of hypothermic organ preservation and combining the clinical experience of preservation solution used in the organ transplantation, we designed a new organ preservation solution-SMO solution, and study the efficacy and safety of the SMO solution in the isolated canine kidney cold preservation model.Objective:1 , To established simple cold storage of isolated canine kidney model. The kidney was flushed perfused with and stored in the coId(0-4℃)organ preservation solution. Kidney was sampled during the storage, and morphologic analysis was undergone with scanning electric microscopy and light microscopy. The degree of kidney cortex damage was determined by pathologist blinded to the treatment with a histologic classification including: cell swelling, mitochondrial swelling, tubular cell necrosis, and so on. The apoptosis of cortex cell during cold storage should be examined too. Apoptosis index (AI) should be used to define the degree of cell death.2, To study the changes of mitochondria function in canine kidney cortex during cold storage and the protective effects of self-designed organ preservation solution. The mitochondria function index include respiratory control ratio(RCR) andP/O(addition of ADP:O ratio), which reflect the integrity of cortex mitochondria. The damage induced by reactive oxygen species during cold storage should be measured by the determination of MDA and SOD.3 -. To determine the changes of adenine nucleotides (ATP> ADPx AMP) in canine renal cortex during simple cold storage with high performance liquid chromatography( HPLC). To establish the standard-quantitative-calculations by peak-height measurement of adenine nucleotides standard solution in HPLC system. ATP, ADP, AMP, total adenine nucleotides(TAN) and Energy Charge(EC) should be calculated to analysis the energy metabolism during the cold storage. ATP/ADP and ATP /AMP ratio should also be calculated.Methods:K We used adult male or female dogs(15±kg body mass). Dogs were anesthetized with sodium pentobarbital(30mg/kg) and heparin sodium(500 IU) was given intraperitoneally. We harvested kidneys through a midline incision. Bilateral nephrectomy was carried out at the same time, kidney was flushed perfused with cold organ transplantation solution, the flushed-out volume was about 200ml, the pressure of perfusion was about 100cmH2O. During the cold storage kidney was sampled for morphologic analysis, and the apoptosis was assessed with TUNEL assay. Apoptosis index was defined as the number of stained positive cells in cortex cells per view under 400X magnification microscope. Five fields from each sectionwere selected randomly.2> Kidney tissue was sampled during cold storage at 2,24 ^ 48 ^ 72hrs during cold storage. Mitochondria in cortex were isolated from cortex homogenization by differential centrifugation. Mitochondria respiratory function were measured polarographically with Clark oxygen electrode. Mitochondrial four-state respiratory were measured: state2: oxygen consumption stimulated by the addition of succinate; State 3 : oxygen consumption stimulated by the addition of ADP; State 4: oxygen consumption after completion of the addition of ADP. RCR=The rate of state 3 oxygen comsuption/ the rate of state 4 oxygen comsuption. P/O=ratio between the nanomoles of ADP phosphorylated and the nanomoles of oxygen consumed duringstate 3. Contents of superoxide dismutase(SOD) and malondialdehyde (MDA) in the cortex homogenization were observed at the same time with the test of surf-barbituric acid method and xanthine oxidase method respectively.3 -. Canine kidney cortex were sampled during the cold storage. After homogenization, 0.3mol/L HCLO4 was added and mixed well to precipitate the protein. The mixture was centrifuged at 3000 rpm for 5 min and a sample of the resulting supernatant was neutralized with 0.5mol/L KOH and centrifuged at 3000 rpm for 5 min. The adenine nucleotides in the supernatant were determined by HPLC. Chromatographic condition is single 100% buffer A running at 1 ml/min. The eluent is monitored at 254 run with UV1000 detector. According to standard curve, quantitive calculation were based on peak-height measurement. The contents of ATP, ADP and AMP, the changes of Energy Charge[EC =(ATP+0.5ADP)/ATP+ADP+AMP], TAN (= ATP+ADP+AMP) and the ratio of ATP/ADP ATP/AMP was calculated and compared between three groups. Results:1 .. Pathologic assessments indicated kidney damage was similar in three groups within 24 hrs, kidney tubular cell swelling and necrosis was more heavier in HC-A group beyond 48 hrs; Mitochondria changes(swelling or rupture) under electric microscopy was more extensive and serious in HC-A group than in SMO or UW group after 24 hrs cold storage. Apoptosis was occurred in cortex after 2 hours' cold-storage, and the apoptosis index is about 2.3% in three groups. AI was increased faster in HC-A group during the first day in cold storage. AI was higher in HC-A group than in the other two groups during 1-3 days cold storage (p<0.05), which is about 22% in HC-A group, 17% in SMO group after 3 days' storage; AI was similar in SMO or UW group during the cold storage, the difference has no statistical significance.2 > Cortex mitochondria oxygen consumption of state III, respiratory control ratio and ADP/O ratio was decreased during the cold storage. The rate of oxygen consumption in state III in HC-A group was lower than that in SMO group duringthe 1-3 day of cold preservation(p<0.05). The rate of oxygen consumption in state IV was slightly increased during the cold storage, but there is no statistical difference between HC-A group, SMO group or UW group. During cold storage RCR decreased as the consequence of the decrement in state III, with no significant change in state IV. These finding suggested that cold ischemia produced uncoupling of the cortex respirotary chain and oxidative phosphorylation. RCR> P/O in SMO group were statistically higher than that in HC-A group (p<0.05) through the storage. RCR in SMO group was about 3.12 at the 0 day of storage, and decreased to about 2.05 three days later; RCR in HC-A group was below 3 at the 0 day, and decrease to 1.6 three days later. P/O in SMO group is higher than HC-A group, it indicated that the efficiency of ATP synthesis in SMO group was higher than HC-A group. Mitochondria oxygen consumption of state HI, respiratory control ratio(RCR) and ADP/O ratio(P/O) of UW group were similar with SMO group during the cold storage(P>0.05). Concentration of MDA in cortex homogenization was increased in three groups during the storage. MDA in HC-A group was higher than SMO group(p<0.05); It indicated membrane degradation caused by reactive oxygen species formation that are likely of mitochondria origin was worse in HC-A group than in SMO group. Content of cortex SOD in three groups had no statistical difference over the duration of the experiment (P>0.05).3 n ATP, ADP, AMP concentration and the total concentration of adenine nucleotides in cortex were decreased during the cold storage. In SMO group ATP concentration decreased to 3.9 + 0.4 nmol/mgpro after 2 hours cold storage, and after 72 hours storage it dropped to 2.3 + 0.5 nmol/mgpro. The ATP and TAN concentration in SMO group after 12, 24, 36, 48, 72 hours preservation is significantly higher than that in HC-A group(p<0.05), and was similar to that in UW group during storage. ATP concentration in HC-A group after 2 hour and 72 hour storage is 3.4 +1.1 nmol/mgpro and 1.6 + 0.2 nmol/mgpro respectively. The ADP> AMP concentration in SMO group were similar to that in control groups(P>0.05). Energy charge which is a sensitive parameter of cell energy metabolism was greater in SMO group than that in HC-A group at most time-point during preservation(p<0.05). But it was similar with that ofUW group(P>0.05). ATP/ADP ratio was similar in three groups; ATP/AMP ratio in SMO group was higher than HC-A group at most check point in cold storage(p<0.05).Conclusion:1 > SMO solution should protect kidney cortex cell from injury by attenuated cell swelling and necrosis during cold storage, and decrease cold-induced apoptosis in cortex cells, compared with HC-A group.2n The results suggested that SMO solution should protect kidney cortex mitochondria function from injury induced by hypothermia and hypoxia during cold storage. SMO solution protect cortex cell from MPT caused by calcium overload, ATP depletion and reactive oxygen species originated from mitochondria during cold storage.3 ^ SMO preservation solution protected kidney cortex from hypothermic ischemic injury by maintenance of higher ATP and TAN levels and energy charge compared with HC-A solution.