Beneficial effects of supplemental buffer and substrate on energy metabolism during small bowel storage

Successful preservation of small bowel (SB) is closely correlated with the maintenance of cellular energetics. This study was designed to assess the ability of a modified UW solution supplemented with buffer and glucose to facilitate ATP production during cold storage. In part A, rats SB (n = 4) were flushed vascularly as follows: Group 1, UW solution (control); Group 2, HUW solution (UW+90 mM histidine). Inclusion of histidine resulted in a >3-fold increase in buffering capacity over the pH range 7.4-6.8. Positive effects of histidine on ATP and energy charge were apparent after 4-10h storage. Examination of the key regulatory enzyme, Phosphofructokinase (PFK), reflected a sustained activation was over 1-4h in the HUW group only. In part B, groups were vascularly flushed as follows: Group 1, HUW solution (control); Group 2, Group 1+20mM glucose; and Group 3, Group 2+luminal flush. Elevated ATP and total adenylates over 2-10h in Group 3 compared to control were a direct consequence of improved glycolytic activity. This data supports the hypothesis that tissue energetics can be significantly improved during cold storage using a histidine-buffered UW solution supplemented with carbohydrate substrate.     

Comparing the effects of Dextran 70 and Hydroxyethyl starch in an intestinal storage solution

Introduction

Our lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw = 70 kDa) and Hydroxyethyl starch (HES; Mw = 2200 kDa).

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘backtable’ intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n = 6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n = 4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran.

Results

Over the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production.

Conclusion

The results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not.     

Comparing the effects of Dextran 70 and Hydroxyethyl starch in an intestinal storage solution

IntroductionOur lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw = 70 kDa) and Hydroxyethyl starch (HES; Mw = 2200 kDa).MethodsRat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘backtable’ intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n = 6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n = 4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran.ResultsOver the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production.ConclusionThe results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not.     

Optimizing the concentration of hydroxyethylstarch in a novel intestinal-specific preservation solution

Introduction

Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage.

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘back table’ intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n = 6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24 h time-course of cold storage.

Results

Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P < 0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P < 0.05). After 12 h, median modified Parks’ grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P < 0.05.

Conclusion

Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal.     

Noninvasive monitoring of citrate, acetate, lactate, and renal medullary osmolyte excretion in urine as biomarkers of exposure to ischemic reperfusion injury

Injury during the transplant process affects the alloantigen-dependent factors and the alloantigen-independent processes of "chronic" rejection. Consequently, the determination of reliable parameters for the assessment of ischemic damage is essential for the prediction of renal changes after ischemia/reperfusion injury. The aim of this study was to assess the ability of (1)H NMR spectroscopy to predict the early graft dysfunction in an ischemia/reperfusion model after preservation in two standard preservation solutions, Euro-Collins (EC) and University of Wisconsin (UW). The second aim was to specify the role of the UW solution in preventing renal medullary injury. Urine and plasma samples from three experimental groups were examined during 2 weeks: control group (n = 5), EC group (cold flushed and 48-h cold storage of kidney in EC and autotransplantation, n = 12), and UW group (cold flushed and 48-h cold storage of kidney in UW and autotransplantation; n = 12). We also examined these kidneys 30-40 min after implantation and on the sacrifice day. Creatinine clearance was significantly reduced in the EC group during the second week. Fractional excretion of sodium and urine N-acetyl-beta-d-glucosaminidase activity were improved but not significantly different in the preserved groups. Urinary concentrations of the alpha-class glutathione S-transferase were significantly greater in the EC group during the first week after transplantation. The most relevant resonances for evaluating renal function after transplantation determined by (1)H NMR spectroscopy were those arising from citrate, dimethylamine (DMA), lactate, and acetate in urine and trimethylamine-N-oxide (TMAO) in urine and plasma. These findings suggest that graft dysfunction is associated with damage to the renal medulla determined by TMAO release in urine and plasma associated with DMA and acetate excretion. Citrate is also a urinary marker that can discriminate kidneys with a favorable evolution. Our results suggest that (1)H NMR spectroscopy is an efficient technique for detecting ischemic damage when accurate and precise data on graft injury is required. In addition, this study outlines the specific impact of the UW solution against injury to the renal medulla.     

Cold storage of rabbit thoracic aorta in University of Wisconsin solution attenuates P2Y(2) purine receptors

Post-transplantation thrombosis may occur in donor segments of iliac arteries and livers following surgical removal and storage in University of Wisconsin (UW) solution for transplantation. We have previously suggested that purine receptors are vulnerable to denaturation after UW storage. The aims of the present study were to determine what particular subtypes of purine P2Y receptors in rabbit thoracic aorta deteriorate after 8 days of UW storage by studying vascular reactivity to acetylcholine, ATP, 2MeSATP and UTP. Ring segments of aortae from male New Zealand White rabbits were mounted upon fine-wire myographs and vasodilatation to the above agents tested on fresh tissue, and after 8 days of UW storage. Vasodilatation to ATP was attenuated by 100 microM L-NAME in fresh tissue suggesting that the relaxant response was, in part, due to nitric oxide (NO). P2Y-mediated relaxation to ATP was significantly attenuated by UW storage and cholinergic responses were not. This attenuated relaxation to ATP was not further attenuated by L-NAME, suggesting a loss of the NO-dependent mechanism. De-endothelialisation indicated that UTP-mediated vasorelaxation, via P2Y(2) receptors, was endothelium-dependent. Any residual endothelium-independent relaxation to UTP was abolished by UW storage and endothelium-dependent UTP relaxation was reduced to the same level as that seen in fresh, de-endothelialised tissue. In contrast responses to 2MeSATP, via P2Y(1) receptors, were predominantly endothelium-independent and were only partially attenuated by UW storage. Responses to pyridoxalphosphate-6-azophenyl-2('),4(')-disulphonic acid (PPADS) and L-NAME suggested that vasorelaxation to 2MeSATP and UTP was mediated by P2Y(1) and P2Y(2) receptors, respectively. It is therefore concluded that UW storage predominantly decreases P2Y(2) receptor-mediated vascular reactivity.     

Trophic factor supplemented UW solution reduces intimal hyperplasia in the rat aortic transplant model

BACKGROUND: Chronic allograft nephropathy (CAN) is associated with delayed graft function, cold ischemic injury, and is an important cause of premature graft loss. A characteristic vascular lesion of CAN is intimal hyperplasia (IH). The goal of this study was to evaluate the effect of cold storage in University of Wisconsin solution supplemented with trophic factors (UW-TF) on IH in rat aortic isograft (IG) and allograft (AG) models. METHODS: F344 --> F344 and Lewis --> F344 orthotopic abdominal aortic transplants were performed after 48 h of cold storage in either UW or UW-TF solution with and without immunosuppression. RESULTS: Significant reduction in IH was observed when IG were stored in UW-TF solution compared to UW solution. A significant reduction in intimal inflammation was observed in UW-TF stored, nonimmunosuppressed AG. In immunosuppressed recipients, AG stored in UW-TF solution evidenced significantly less IH compared to those stored in UW alone. CONCLUSIONS: UW-TF solution decreased IH in both alloindependent and dependent models.     

Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation

Previous studies have indicated that pyruvate is able to reduce ischaemia/reperfusion (I/R) injury in a variety of tissues, but a full understanding of the effects is lacking. In this current preliminary study, magnetic resonance spectroscopy (MRS) was used to investigate the biochemical effects of differing concentrations of pyruvate (3 and 15mM) on liver metabolism during the cold hypoxic preservation period itself, in order to gain insight into possible mechanisms. Hepatic lactate, alanine, and succinate levels were increased in livers preserved with 15mM pyruvate added to the University of Wisconsin (UW) solution and were generally elevated (but to a lesser degree) in livers flushed with 3mM pyruvate, compared to those cold stored in UW alone. Further, from enzymatic assays of adenine nucleotides, 15mM levels of pyruvate were found to maintain higher ATP levels during short periods (up to 4h) of cold hypoxic storage than in UW stored livers, whilst energy charge ratios (after 4 and 24h) were also higher (P<0.01 in each case). This may arise from enhanced glycolysis secondary to an improved redox status in the pyruvate-treated livers, as evident by the increase in the levels of lactate.     

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.     

Cold preservation of rat osteochondral tissues in two types of solid organ preservation solution, culture medium and saline

We compared Dulbecco’s modified Eagle’s medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days’ cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.     

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.     

Improvement of renal preservation by adding lidoflazine to University of Wisconsin solution. An experimental study in the rat.

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.     

Factors affecting flavonoids absorption

In experiments on rats, some of the factors affecting flavonoids absorption (solubility, glycosylation and nutritional status: fasted and not-fasted animals) were examined. Administration of quercetin with different solubilization degree showed no direct correlation between the quercetin absorption extent and solubility, i.e. despite 3 orders of difference in solubilization degree, the extent of absorption varied only about 4-fold. Absorption comparison of genistein and its glycoside genistin showed no difference in the extent of absorption; however, aglycone, in contrast to glycoside, was absorbed already from the rat stomach. Conjugation patterns (sulfation and glucuronization) of genistein metabolites demonstrated that the plasma of animals fasted prior to isoflavone administration contained significantly more sulfates and less glucuronides and mixed sulfates/glucuronides conjugates than the plasma of non-fasted animals.     

The Efficacy of Antioxidants Administered during Low Temperature Storage of Warm Ischemic Kidney Tissue Slices

Accumulation of products of lipid peroxidation (malondialdehyde, conjugated dienes, lipid peroxides, and Schiff bases) was evaluated in rabbit kidney cortex slices made ischemic for 60 min followed by 18 h storage at 5°C in UW Na gluconate solution and 210 min normothermic reoxygenated incubation. In addition, the effect of adding Trolox (1 mM), deferoxamine (1 mM), and ascorbate (1 mM) as supplemental antioxidants to the UW gluconate solution was evaluated. Lipid peroxidation was slightly increased after hypothermic storage compared to slices subjected to ischemia alone but was not significantly different than ischemic slices during subsequent incubation at normothermia. The addition of either deferoxamine or Trolox to the storage solution substantially reduced lipid peroxidation both during hypothermic storage and subsequent to normothermic incubation. Ascorbate had a mild prooxidant effect as a sole additive to the UW gluconate solution but was clearly prooxidant when combined with either deferoxamine or Trolox. These results suggest that supplemental antioxidants added to the UW gluconate solution under conditions analogous to machine perfusion preservation have a potential role in reducing oxidative stress in kidney tissues harvested after warm ischemia and that hypothermia may be a valuable adjunct to resuscitative therapeutic regimens developed for salvage of ischemic kidneys for transplantation.     

A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

The influence of fasting on liver sulfhydryl groups, glutathione peroxidase and glutathione-S-transferase activities in the rat

Sulfhydryl groups, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) are important elements of the antioxidant defence in the organism. The efficacy of their antioxidant action is influenced by many factors. In this work, the effect of fasting on total, protein-bound and nonprotein sulfhydryl groups and on the activity of liver and serum GPx and GST in rats were determined. Male Wistar rats were divided into two groups: non-fasted and 18-hour fasted. In fasted animals liver content of nonprotein sulfhydryl groups (represented predominantly by reduced glutathione; GSH) was diminished by 22% in comparison to non-fasted group, whereas total and protein-bound -SH groups were unaffected. The activity of liver and serum GPx was unchanged in food deprived rats. In these animals the activity of GST in serum was reduced by 26%. Fasting had no significant effect on the activity of GST in the liver. Our results demonstrate that in rats deprived of food for 18 hours liver and serum GPx and GST are not involved in protection against action of reactive oxygen species formed during fasting. The observed drop in the content of liver nonprotein sulfhydryl groups without concomitant rise in the activity of GPx and GST indicates that this effect may be due to augmented degradation of GSH, its potentiated efflux from hepatocytes and formation of conjugates with intermediates arising as a result of reactive oxygen species action.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Suppression of cold ischemic injury in stored kidneys by the antimicrobial peptide bactenecin

BACKGROUND: Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys. METHODS: Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro. RESULTS: Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics. CONCLUSIONS: These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.     

Respiratory activity of isolated rat hepatocytes following cold storage and subsequent rewarming: a comparison of sucrose-based and University of Wisconsin solutions

Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 +/- 3 and 23 +/- 3 nmol O(2)/min/10(6) cells, respectively) compared to control cell values (31 +/- 3 and 33 +/- 5 nmol O(2)/min/10(6) cells; P < 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 +/- 6 and 40 +/- 5 nmol O(2)/min/10(6) cells; for control cells: 43 +/- 5 and 46 +/- 6 nmol O(2)/min/10(6) cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended.