Influence of retrieval conditions on renal medulla injury: evaluation by proton NMR spectroscopy in an isolated perfused pig kidney model

BACKGROUND: Delayed graft function (DGF) has remained an important complication after renal transplantation. The exact causes of DGF remain to be clarified, particularly the impact of retrieval conditions and preservation factors. In the present investigation, (1)HNMR spectroscopy of urine was assessed in order to detect the influence of retrieval condition on renal medulla damage. METHODS: The isolated perfused pig kidney (IPK) was used to assess initial renal function from multiorgan donors (MOD) or single organ donors (SOD) after in situ cold flush and 24-h cold storage (CS) preservation with two standard preservation solutions: Euro-Collins (EC) and University of Wisconsin (UW) solutions. Kidneys flushed with cold heparinized saline and immediately perfused were used as the control group. Kidneys were perfused for 90 min at 37.5 degrees C for functional evaluation. During reperfusion, renal perfusion flow rate (PF) was measured. Glomerular filtration rate (GFR), tubular reabsorption of Na(+), and lactate dehydrogenase (LDH) and N-acetyl-beta-d-glucosaminidase (NAG) excretions were determined. Ischemia-reperfusion impairment was also determined by histological techniques and (1)HNMR spectroscopy. RESULTS: PF, GFR, and tubular reabsorption of Na(+) were significantly decreased in experimental groups when compared to the control group but there was no significant difference between experimental SOD groups. GFR was significantly greater in UW-MOD than in EC-MOD and tubular reabsorption of Na(+) was significantly greater in UW-MOD than in EC-MOD after 45 min of reperfusion. The release of LDH in the effluent and the urinary excretion of NAG were not significantly different after 24-h CS in the various experimental groups. The most relevant resonances determined by (1)HNMR spectroscopy were citrate, trimethylamine-N-oxide, lactate, acetate, and amino acids. Excretion of these markers was significantly different when compared to biochemical markers. A resonance (P) detected particularly in EC-MOD after 24-h CS was identified and well correlated to renal dysfunction. Histological study showed that ultrastructural damage and mitochondrial injury were more pronounced in the EC-MOD group. CONCLUSION: These results show that retrieval condition influences renal medullary damage. NMR spectroscopy, which is a noninvasive and nondestructive technique, is more efficient in assessing renal damage than conventional histology and biochemical analysis.     

Lipid peroxidation after cold storage and normothermic reperfusion: the effect of trimetazidine

Abstract Ischemia reperfusion injury is still a leading cause of early graft dysfunction after transplantation. Trimetazidine (TMZ) has been postulated to be protective against renal damage from oxygen free radicals. The aim of this study was to assess the effect TMZ during cold storage (CS) and normothermic reperfusion in an isolated perfused pig kidney model. Three groups were studied: control group, immediately perfused (GO), 48 h CS in Euro-Collins solution (G1), and 48 h CS in Euro-Collins solution plus TMZ (G2). Glomerular filtration rate (GFR) and fractional sodium reabsorption (FRNa⁺) were calculated during reperfusion from urine and perfusate samples. Lipid peroxidation was determined by the renal tissue level of Schiff bases (SB) and malondialdehyde (MDA) after reperfusion. A histological evaluation was performed after reperfusion. Renal function was significantly improved and lipid peroxidation reduced after preservation in Euro-Collins solution plus TMZ. Functional data were closely related to histological damage. In conclusion, TMZ is a useful protective agent against renal damage induced by CS.     

Evaluation of a preservation solution containing fructose-1,6-diphosphate and mannitol using the isolated perfused rat kidney. Comparison with Euro-Collins and University of Wisconsin solutions

The renal preservation ability of a flushing solution (F-M)with fructose-1,6-diphosphate (1 g/dl) and mannitol (2 g/dl)during cold ischaemia was studied with the isolated perfusedrat kidney model and compared with the Euro-Collins (EC) andUniversity of Wisconsin (UW) solutions. Kidneys were storedin hypothermia for 4 and 18 h after initial flushing with thesolution being tested, and then reperfused at 37°C in anisolated perfusion circuit for 90 min with a Krebs-Henseleitsolution containing 4.5% albumin. Forty-four kidneys were studied and divided in a control groupand six study groups according to the cold ischaemia time andflushing solution used. Renal functional parameters of plasmaflow rate (PFR), renal vascular resistance (RVR), urine flowrate (UFR) glomerular filtration rate (GFR), fractional (FRNa)and net (TNa) sodium reabsortion were assessed during reperfusion.Conventional histology and malon-dialdehyde tissue levels (MDA)were also evaluated. Our results show that PFR, RVR, and UFR were similar in allstudy groups. After 4 and 18 h of cold ischaemia, GFR, FRNaand TNa were better, and conventional histology worse in F-Mthan in EC flushed kidneys. After 4 and 18 h of cold ischaemia,GFR, FRNa and TNa, in fact, were not different between F-M andUW flushed kidneys. After 4 h of cold ischaemia, conventionalhistology was similar in F-M and UW flushed kidneys. Nevertheless,after 18 h of cold ischaemia, UW flushed kidneys showed worsehistological parameters than F-M flushed kidneys. After 4 hof cold ischaemia, MDA was similar in kidneys flushed with thethree solutions. After 18 h of cold ischaemia MDA was higherin EC than in F-M or UW flushed kidneys. In summary, our newly developed cold storage solution showspromising results in renal preservation and its ability to preserveis at least as good as UW solution assessed in the isolatedperfused rat kidney.     

Iron chelators do not reduce cold-induced cell injury in the isolated perfused rat kidney model.

BACKGROUND: In vitro, cold-induced injury is an important contributor to renal tubular cell damage. It is mediated by iron-dependent formation of reactive oxygen species and can be prevented by iron chelation. We studied whether iron chelators can prevent cold-induced damage in the isolated perfused rat kidney (IPK) model both after cold perfusion (CP) and after cold storage (CS). We hypothesized that in the CP model iron-dependent cold-induced injury is more pronounced, since oxygen is constantly provided. METHODS: The IPK was either flushed with University of Wisconsin (UW) solution and stored for 4, 18 or 24 h at 4 degrees C or perfused during 4 h at 4 degrees C with UW for machine perfusion. The iron chelators 2,2'-dipyridyl or desferal, or the negative control 4,4'-dipyridyl were added during the cold perfusion. Kidney function was measured during 2 h reperfusion at 37.5 degrees C and compared to a control group (without cold preservation). RESULTS: Compared to control perfusion, kidney function was decreased in all experimental protocols. glomerular filtration rate and FR(H2O) were significantly decreased, while FE(gluc) and FE(Na) were higher after 4 h CS and CP. After 4 h CP, also renal vascular resistance was increased. Addition of 2,2'-dipyridyl did not improve kidney function after either CS or CP. Prolonged periods of CS worsened kidney function. The addition of 2,2'-dipyridyl or desferal did not improve kidney function after longer periods of CS. CONCLUSIONS: Addition of an iron chelator to the preservation solution UW did not improve kidney function after both CS and CP. Iron chelation is not able to prevent cold-induced damage in the isolated perfused rat kidney.     

Evaluation of preservation solutions by ESR-spectroscopy: superior effects of University of Wisconsin over Histidine-Tryptophan-Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.     

Nifedipine improves recovery function of kidneys preserved in a high- sodium, low-potassium cold-storage solution: study with the isolated perfused rat kidney technique

BACKGROUND: Extracellular types (high-Na) of cold-storage solution (CSS) have been shown to be more effective in preserving kidneys than intracellular CSS (high-K). On the other hand, calcium entry blockers (CEB) have been demonstrated to improve graft function when administered after and/or prior to transplantation. The ischaemia reperfusion syndrome involves, in part, an alteration in intracellular calcium metabolism that induces an increase in renal vascular resistances (RVR) and other cellular dysfunction, and high-K CSS per se are vasoconstrictive. Since CEB act via a modification in intracellular calcium metabolism on vascular smooth muscle, glomerular, and tubular cells, we evaluated the actual benefit on CEB on kidneys preserved in Belzer's CSS (K-UW) and a high-Na version of Belzer's CSS (Na-UW). METHOD: The isolated perfused rat kidney (IPK) was used, first as a vascular bed to test the effects of CSS on RVR, and the influence of nifedipine. Second, the recovery function of the IPK was assessed by GFR and tubular Na reabsorption, after 24 h preservation in K-UW and Na- UW, with or without nifedipine. Results were compared with a control group in which renal function was measured without prior cold-storage. RESULTS: K-UW but not Na-UW induced an increase in RVR when flushed into the kidney. This vasoconstriction is prevented by nifedipine. K-UW CSS was more deleterious to renal function than Na-UW. Addition of nifedipine to the flush, the CSS for 24 h, and to the normothermic reperfusate further improved recovery function of the IPK cold stored in Na-UW but not in K-UW, without any modification of RVR. CONCLUSION: Nifedipine may be of potential effect in attenuating ischaemic injury by a mechanism which does not involve its vasodilatory properties.      

Evaluation of a high sodium-low potassium cold-storage solution by the isolated perfused rat kidney technique

The isolated perfused rat kidney (IPK) model was used to assessinitial renal function after 24 h preservation in 3 differentcold storage solutions: EuroCollins (EC), a solution preparedaccording to the formulation of Belzer's solution (High-K⁺ UW)and a high Na⁺-low K⁺ Belzer UW solution (High Na⁺ UW). GFR and FR_Na were measured after 24 h cold storage in each ofthe solutions during 60 min, and were compared to values obtainedin a control group in which renal function was measured immediatelyafter the kidneys had been harvested. ATP and CP were measuredin fresh renal tissue, in kidneys preserved for 24 h in eachsolution, in control IPK, and in reperfused IPK after they hadbeen preserved for 24 h. Main results showed that preservationin either solution caused a dramatic decrease in GFR and inFR_Na within the first 60 min following reperfusion of cold-storedkidneys. However FR_Na was significantly higher in the High-Na⁺UW group. ATP and CP content were decreased to 10% of basalvalues in all experimental groups after cold-storage. Normothermicreperfusion of IPK after cold-storage induced a restorationof ATP levels, but CP content decreased further. There was nosignificant difference in ATP and CP content between cold-storagesolutions, nor any correlation between metabolic and functionalparameters.     

Histological evaluation of proximal tubule cell injury in isolated perfused pig kidneys exposed to cold ischemia

BACKGROUND. Ischemic injury of the renal allograft before transplantation is a major cause of impaired graft function. Proton nuclear spectroscopy provides a useful technique for evaluating proximal tubular activity. In addition to this technique, we proposed a histological grading system for quantifying proximal tubule alterations. METHODS. The aim of this study was to evaluate the histological lesions of tubule epithelial cells in the model of isolated perfused pig kidneys following 48 to 72 h of cold storage in Euro-Collins solution. Normothermic isolated perfused pig kidneys were randomized in three experimental groups : Group 1, control group; cold flush with cold heparinized solution followed by immediate reperfusion (n = 6); Group 2, 48 h of cold storage in Euro-Collins followed by reperfusion (n = 6); Group 3, 72 h of cold storage in Euro-Collins followed by reperfusion (n = 6). Proton nuclear spectroscopy of urine and biochemical studies were performed for whole renal functional evaluation during reperfusion. Optical and electron microscopy analyses of the reperfused kidneys were performed by four investigators and the degree of cell injury was assessed using 8 different criteria in a 5-scale numerical score. RESULTS. Numerical scores corroborate the results from NMR spectroscopy and differed significantly between the three groups studied. The degree of proximal tubule cell damage was increased with prolonged cold ischemia as shown particularly in Group 3. CONCLUSION. The results from this study showed that analysis of cell injury based on an histological grading system in the model of isolated perfused kidney allows the quantification of the degree of proximal tubule injury. Thus, such morphological system analysis could be a useful method for quantifying tubule cell injuries observed under various physiopathological conditions.     

Cold storage sensitizes hepatocytes to oxidative stress injury

Liver cold storage leads to oxygen free radical production and reperfusion injury. Antioxidants are effective in suppressing reperfusion injury in rat livers when used in the reperfusion medium. However, in clinical liver transplantation their effectiveness is not clear, which may be due to the way they are used (in the recipient). In this study we compare the effectiveness of antioxidants when used in the reperfusion medium versus the cold storage solution in isolated hepatocytes and the isolated perfused liver. Hepatocytes were cold stored in UW solution for 24 h. Oxidative stress, induced by t-butyl hydroperoxide (tBHP), was measured in the presence of one of five different antioxidants – deferoxamine (DFO), dithiothreitol (DTT), trolox, tocopherol, dimethylthiourea (DMTU) – in the reperfusion buffer or UW solution. Efficacy was judged by reduction in membrane damage (LDH release) during rewarming. Also, rat livers were cold stored for 48 h in UW solution ( ± antioxidant) and reperfused ( ± antioxidants). Efficacy was judged by the effect on enzyme release and bile production. Cold storage of hepatocytes for 24 h sensitized them to oxidative stress. The concentration of tBHP required to induce maximal cell death (80 %–90 % LDH release) was reduced from 1.3 mM (fresh cells) to 0.37 mM (LD-50 values). All antioxidants except DMTU suppressed oxyradical-induced LDH release when used in the reperfusion medium, but only DFO was effective when used in the UW solution. In the isolated perfused liver, DFO, DTT, and trolox were effective and suppressed enzyme release when added to the reperfusion buffer, but none were effective when used in the UW solution. We conclude that cold storage sensitizes liver cells to oxidative stress. The most effective antioxidant was the iron chealator, DFO, which was effective in the reperfusion buffer (isolated perfused liver or hepatocytes) but not in the UW solution when tested in the isolated perfused liver. Suppression of reperfusion injury in liver transplantation could be obtained by antioxidant therapy. However, it is unclear how best to deliver the antioxidants to the site of oxyradical generation. Received: 23 December 1996 Received after revision: 14 April 1997 Accepted: 13 May 1997     

A non-heart-beating donor model to evaluate functional and morphologic outcomes in resuscitated pig hearts.

A non-heart-beating donor model was considered to examine whether pig hearts from the abattoir could be resuscitated by whole blood reperfusion. For preservation, machine perfusion using University of Wisconsin (UW) solution was compared with storage on ice. Nineteen hearts from abattoir pigs, harvested 25 +/- 3 min after exsanguination, were harvested and transported to the laboratory. Controls (n = 7) were immediately reperfused with homologous whole pig blood in an isolated heart model for 60 min with monitoring of left ventricular developed pressure (LVDP), contractility, and coronary flow. UW solution hearts (UW, n = 6) were perfused for 4 h with 10 degrees C cold UW solution before blood reperfusion. In the cold storage group (CS, n = 6), the organs were stored for an additional 4 h on ice before blood reperfusion. In all hearts, histology was performed after 60 min of blood reperfusion to evaluate myocardial reperfusion injury. All three groups showed significant increases in LVDP (p <.001), although this functional recovery was earliest in the control group and latest in the UW group. Significant declines were observed for both LVDP and contractility from the peak values in each group to the end of blood reperfusion. Coronary flow increased steadily over the time course for the UW group, whereas in the control and CS groups flow increased during the first 15 min of blood reperfusion and then decreased. In the UW and CS groups, there were significant positive correlations between coronary flow and LVDP (p <.001). Microscopic examination revealed no differences between the three groups. Thus, hearts from an abattoir with 25 min of warm ischemic time can be resuscitated. For storage of these organs, continuous machine perfusion with UW solution is superior to cold storage on ice.     

Comparison of HTK- and UW-solution for liver preservation tested in an orthotopic liver transplantation model in the pig

The aim of this experimental study was to compare the preservation potency of University of Wisconsin (UW) and HTK (Bretschneider) solutions in an orthotopic liver transplantation (OLT) model in pigs. Livers were harvested using an in situ perfusion technique, where organs were flushed with the solution being tested, stored on ice — cold storage (CS) — for 2 or 24 h and then transplanted. Parameters monitored were liver enzymes in serum, hepatic water content, high energy phosphates, nuclear magnetic resonance (NMR) relaxation time T2, light microscopy and bile production. CS for 24 h is an extreme in pig liver preservation and is not compatible with animal survival. Biopsies showed drastic morphological changes and grafts did not produce bile in either group. (Bile production 2 h CS: HTK, 5.6 ± 1.8 ml/h; UW, 4.7 ± 2.3 ml/h) Enzyme release after reperfusion (ASGOT, ?LDH) was higher in long-term preservation. Hepatic tissue water content significantly decreased during CS in UW preserved livers. Edema alter reperfusion (?H₂0: HTK 24 h = + 5.6%, UW 24 h= + 4.8%) and regeneration capacity after reperfusion (UW 2 h = 63%, HTK 2 h = 55%, UW 24 h = 30%, HTK 24 h = 30%) were not significantly different. However, we did not observe major differences in preservation potency between the solutions tested. Differences were correlated, rather, with length 9 time of CS, than with the solution used. Therefore, HTK solution seemed to be a low potassium containing alternative to UW solution.     

Prevention of cold ischaemia-reperfusion injury by an endothelin receptor antagonist in experimental renal transplantation.

BACKGROUND: Endothelin (ET) is known to play a role in the pathogenesis of warm ischaemic renal damage, however, little is known about its involvement in renal cold ischaemia. This study was designed to investigate the response of ET after kidney cold ischaemia, and to assess the potential protective effect of bosentan, a dual, non-selective ET(A)/ET(B) receptor antagonist, against cold ischaemia reperfusion injury in a rat model of syngeneic renal transplantation. METHODS: Kidneys from Lewis rats were transplanted, either immediately or after 5 h of cold preservation. After 48 h, contralateral nephrectomy was performed. Rats were organized into three groups: Tr-NoISC, no cold ischaemia; Tr-ISC, 5 h cold ischaemia; and Tr-BOS, 5 h cold ischaemia plus bosentan (100 mg/kg/day, from the day before transplantation until the seventh day post-transplantation). On day 7, plasma and tissue immunoreactive ET (irET), as well as ET mRNA tissue expression, were evaluated. Renal function was measured by means of serum creatinine on days 3, 4, 5 and 7, and by creatinine clearance on day 7. Conventional histology was performed. RESULTS: The ischaemic group had significantly higher plasma irET levels than the non-ischaemic group and significantly lower levels than the bosentan group. Tissue irET levels and ET mRNA expression were similar in the ischaemic and bosentan groups and were higher than in the non-ischaemic group. Throughout the follow-up, serum creatinine was significantly higher in the ischaemic group than in the bosentan group. Moreover, creatinine decreased rapidly in the bosentan group after nephrectomy, whereas it continued to increase for 48 h in the ischaemic group. Kidneys from the ischaemic group showed a higher degree of tubular-cell necrosis and epithelial-cell detachment than kidneys from the bosentan group. CONCLUSIONS: We conclude that cold ischaemia and preservation damage induces an increase in renal ET mRNA and irET expression in the reperfusion phase, contributing both to the deterioration of renal function and to tubular necrosis. Bosentan is effective in protecting kidneys from this cold ischaemia reperfusion damage. Non-selective ET(A)/ET(B) receptor antagonists might be potentially useful in clinical renal transplantation.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

目的 比较Celsior(CS)液与UW液对大鼠无心跳供者(NHBD)供肝的保存效果.方法 选取健康雄性SD大鼠作为肝移植的供、受者.通过阻断大鼠主动脉和膈上下腔静脉10 min的方法,制备和获取NHBD供肝,并采用不同的器官保存液灌注和冷保存供肝.随机将受者分为4组.CS8 h组:受者采用经CS液灌注和冷保存8 h的供肝移植;UW8 h组:受者采用经UW液灌注和冷保存8 h的供肝移植;CS16 h组:受者采用经CS液灌注和冷保存16 h的供肝移植;UW16 h组:受者采用经UW液灌注和冷保存16 h的供肝移植.受者门静脉开放前、开放后1、3及6 h,取各组受者的静脉血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、内皮素1(ET-1)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平;观察和比较各组受者的胆汁生成量、移植肝组织病理学改变及术后7 d内的存活率.结果 NHBD供肝经UW液灌注后呈"花斑"状,肝叶边缘灌注不良,经CS液灌注后肝叶边缘灌注良好.CS8 h组和UW8 h组受者的胆汁生成量分别为(0.21±0.01)ml和(0.10±0.02)ml(P<0.05).门静脉开放后1、3及6 h,CS8 h组受者的血清ALT及AST水平明显低于UW8 h组(P<0.05),门静脉开放后1、3h,CS8 h组受者的血清ET-1、IL-1及TNF-α水平均明显低于UW8 h组(P<0.05);CS8 h组受者移植肝肝窦扩张、门静脉充血及炎症细胞浸润等病理学改变明显轻于UW8 h组,CS8 h组和UW8 h组受者术后7 d的存活率分别为58.3%和25.0%(P<0.05).CS16 h组和UW16 h组受者各时点的胆汁分泌量、血清ALT、AST、ET-1、IL-1及TNF-α水平的比较,差异均无统计学意义(P>0.05),两组受者均在术后3 d内死亡,两组受者移植肝组织病理学改变无明显差异.结论 CS液对大鼠NHBD供肝的保存效果优于UW液,这可能与UW液较CS液粘稠及CS液能够减少枯否细胞的激活有关;NHBD供肝的冷保存时间不宜超过16 h.     

A Non-Heart-Beating Donor Model to Evaluate Functional and Morphologic Outcomes in Resuscitated Pig Hearts

A non-heart-beating donor model was considered to examine whether pig hearts from the abattoir could be resuscitated by whole blood reperfusion. For preservation, machine perfusion using University of Wisconsin (UW) solution was compared with storage on ice. Nineteen hearts from abattoir pigs, harvested 25 &#45 3 min after exsanguination, were harvested and transported to the laboratory. Controls ( n = 7) were immediately reperfused with homologous whole pig blood in an isolated heart model for 60 min with monitoring of left ventricular developed pressure (LVDP), contractility, and coronary flow. UW solution hearts (UW, n = 6) were perfused for 4 h with 10°C cold UW solution before blood reperfusion. In the cold storage group (CS, n = 6), the organs were stored for an additional 4 h on ice before blood reperfusion. In all hearts, histology was performed after 60 min of blood reperfusion to evaluate myocardial reperfusion injury. All three groups showed significant increases in LVDP ( p < .001), although this functional recovery was earliest in the control group and latest in the UW group. Significant declines were observed for both LVDP and contractility from the peak values in each group to the end of blood reperfusion. Coronary flow increased steadily over the time course for the UW group, whereas in the control and CS groups flow increased during the first 15 min of blood reperfusion and then decreased. In the UW and CS groups, there were significant positive correlations between coronary flow and LVDP ( p < .001). Microscopic examination revealed no differences between the three groups. Thus, hearts from an abattoir with 25 min of warm ischemic time can be resuscitated. For storage of these organs, continuous machine perfusion with UW solution is superior to cold storage on ice.     

Comparative study of the University of Wisconsin solution versus Eurocollins in kidney transplant

UW (University of Wisconsin) solution, formulated by Belzer's team in Madison, has already been proved to increase cold ischemia time in liver and pancreas preservation. A multicentre clinical trial is being conducted to compare renal preservation in human transplantation using two different solutions: UW and Eurocollins (EC). This paper, whose results will be included in the multicentre trial, reports local comparative results between UW and EC perfused Kidneys. The two donor populations UW (28 cases) and EC (47 cases) were not randomized. They were however comparable in renal function prior to harvesting but not in age (35 +/- 13.4 years EC versus 27.7 +/- 12.4 years UW). The two recipient populations (48 EC versus 48 UW) were more homogeneous. Comparative results were significant with better graft function in the UW group: creatinine at one week: 499.2 +/- 296.3 EC versus 277.6 +/- 226.2 mumol/l, p less than 0.0001; creatinine at one month: 228.7 +/- 135 EC versus 159.7 +/- 135.6 mumol/l, p less than 0.02 and a decrease in acute tubular necrosis (39.5% EC versus 14.5% UW) and hospital stay. These results justify the use of UW solution by intraaortic flush especially during multi-organ procurement.     

Celsior solution compared with University of Wisconsin solution (UW) and histidine–tryptophan–ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia–reperfusion injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.     

UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury.

Celsior solution (CS), a new preservation solution in thoracic organ transplantation, was evaluated for its efficacy in cold preservation of human liver endothelial cells (HLEC) and was compared to University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). HLEC cultures were preserved at 4 degrees C in CS, UW, and HTK, for 2, 6, 12, 24, and 48 hours, with 6 hours of reperfusion. Levels of lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine 5'-triphosphate (ATP) were measured after each interval of ischemia and the respective phase of reperfusion. Preservation injury of HLEC as measured by LDH release, intracellular ATP level, and MTT reduction were overall significantly (P CS > HTK.     

The enhancement of cellular cAMP with olprinone protects autotransplanted rat kidney against cold ischemia-reperfusion injury

The administration of a cyclic nucleotide analog improves cold ischemia/reperfusion injury in several organs. The type 3 phosphodiesterase inhibitor olprinone is a potent stimulus that enhances cellular cAMP levels. The present study was performed to investigate the protective effects of enhanced intracellular cAMP levels by olprinone in rat orthotopic kidney transplantation. Autotransplantation and immediate contralateral nephrectomy were performed in Lewis rats after 18 hours of graft storage at 4 degrees C in University of Wisconsin (UW) solution with or without 25 microg/mL olprinone hydrochloride. At 2 hours after reperfusion, serum and urinary biochemical indicators of renal dysfunction and injury were measured: serum creatinine, fractional excretion of Na+ and urinary N-acetyl-D-glucosaminidase. Additionally, intracellular cAMP in kidney tissues was measured by a radioimmunology method. Compared to the only UW solution group, olprinone hydrochloride significantly reduced the increased in serum creatinine, FENa and NAG caused by renal ischemia/reperfusion injury, after 2 hours of reperfusion. The content of cAMP at the endpoint of 18 hours cold preservation was significantly greater in the UW plus olprinone hydrochloride group than that in the UW group. Two hours after reperfusion, the content of cAMP in the UW plus olprinone hydrochloride group was still significantly higher than that in the UW group without containing olprinone hydrochloride. These results support a beneficial effect of olprinone against cold ischemia and reperfusion injury via an increased intracellular cAMP levels.     

Hepatic energy metabolism during hypothermic storage and reperfusion using different protecting solutions.

Effects of 5 cold storage solution on hepatic high energy phosphate metabolism and metabolic function were examined using the isolated perfused rat liver. University of Wisconsin (UW), Euro-Collins (EC), and 2 cardioplegic solutions, Bretschneider's HTK and St. Thomas Hospital solution, were studied for their protective capacity. Krebs-Henseleit bicarbonate buffer (KHB) was used to point out the effect of simple hypothermia. Liver ATP, total adenine nucleotides and energy charge losses were significantly lower during 21 h of storage in UW-preserved livers. Also, only UW-protected livers were able to complete regeneration of ATP and total adenine nucleotides after 1 h of reperfusion, whereas EC, HTK, St. Thomas and KHB stored livers only showed minimal regeneration. Concerning metabolic function, UW protected livers liberated significantly less LDH and sGOT as well in the 21-hour storage solution as into the perfusate under reperfusion conditions. This study demonstrates the capability of UW solution in liver preservation by its ability to maintain and restore high energy phosphates.     

Successful 72-hour cold storage of dog kidneys with UW solution

Effects of three cold-storage solutions on kidney function in dogs were examined with the isolated perfused (IPK) kidney model and the autotransplant model. EuroCollins' (EC) solution, phosphate-buffered sucrose solution, and a new solution developed at the University of Wisconsin (UW) were studied. Kidneys were cold-stored for 48 hr or 72 hr. With the IPK model, cold storage for 48 hr or 72 hr in each of the three solutions caused creatinine clearance to decrease by 80%-90%. More protein was excreted by kidneys stored for 48 hr in PBS solution than by kidneys stored in EC or UW solution; protein excretion after 72 hr of storage was similar for kidneys stored in EC or UW solution. Sodium reabsorption decreased after 48 hr or 72 hr of storage, but was higher in kidneys stored in UW solution (83% and 56%, respectively) than in EC solution (52% and 22%, respectively). With the autotransplant model, 40% of the kidneys were viable after 48-hr storage in PBS solution, but 80% viable when stored in EC solution and 100% were viable when stored in UW solution. All kidneys were viable when stored for 72 hr in UW solution; none were viable when stored for 72 hr in EC solution. These results suggest that UW solution effectively preserves kidneys for 72 hr. We previously reported successful 72-hr pancreas preservation. Recently UW solution was able to preserve canine livers for 30 hr. Thus, this single solution appears to be effective for preserving all intraabdominal organs and may simplify cold storage of organs for transplantation.