Mary S. Ametani

Regeneration of ATP in kidney slices after warm ischemia and hypothermic preservation

The current shortage of cadaveric kidneys may be alleviated to some degree by increasing our capabilities to use less than ideal donor-kidneys, such as those from non-heart-beating donors. These kidneys are often exposed to no flow (ischemia) for varying lengths of time. Full utilization of these kidneys may require better methods of organ preservation that could reverse existing ischemic injury. This may conceivably require that, during preservation, energy stores (ATP) lost during warm ischemia be recharged. This would require continuous perfusion. Using a kidney slice model, we investigated the effects of simulated hypothermic machine perfusion with the UW gluconate perfusate on the capability of rabbit kidneys exposed to warm ischemia to regenerate ATP. After 30 min of warm ischemia, ATP content was low (0.2 μmol/g wet weight) but increased to 0.7–0.9 μmol/g wet weight after 24 h of simulated machine perfusion at 4°C. After an additional 2 h of rewarming (37°C in oxygenated Krebs Henseleit buffer), the slice ATP content increased to about 1.0 μmol/g wet weight (similar to kidneys not exposed to warm ischemia) when the antioxidants desferrioxamine and N-2-(mercaptopropionyl) glycine were included in the preservation media. Significantly less ATP was present without the antioxidants. After 60 min of warm ischemia, less ATP was regenerated after 24 h of simulated machine perfusion (about 0.4 μmol/g wet weight) than after 30 min of warm ischemia. However, more ATP was regenerated when antioxidants were included in the perfusate (0.4 vs 0.8 μmol/g wet weight). This study shows that ATP can be regenerated in kidneys exposed to warm ischemia by continuous perfusion in the UW gluconate solution. Furthermore, oxygen free radicals appear to cause suppression of ATP regeneration since an iron and a hydroxyl radical scavenger improved ATP formation. The ATP content of kidneys exposed to 60 min of WI was less than after 30 min of warm ischemia, suggesting that better methods of preservation may be needed to improve our capability to utilize kidneys damaged by extensive warm ischemia (> 60 min). This may require the development of new methods and/or perfusates for kidney preservation.

Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO4

During continuous hypothermic perfusion of dog kidneys there occurs a gradual decrease in ATP from about 1.4 to 0.6 mumol/g wet wt after 5 days of preservation. The loss of ATP can be prevented by including both adenosine (10 mM) and PO4 (25 mM) in the perfusate. Under these conditions kidney cortex ATP levels were more than double control values--3.5 mumol/g wet wt. Both adenosine and PO4 were necessary since omission of one substance resulted in no net synthesis of ATP. Furthermore, these high levels of ATP were obtained only if adequate concentrations of adenosine were maintained during perfusion. Following 3 days of perfusion the adenosine level in the perfusate decreased to about 1 mM and under this condition ATP levels were low. Adenosine levels were maintained in the perfusate by two methods: (1) addition of fresh perfusate or (2) pretreatment of the kidney with the adenosine deaminase inhibitor--deoxycoformycin. The increased levels of ATP appear directly related to the availability of nucleotide precursors and the presence of inhibitors of the enzymes involved in the catabolism of nucleotides and nucleosides (PO4 and deoxycoformycin). Mitochondrial activity was similar in kidneys with high or low ATP levels following 5 days of preservation.

Brain death does not affect hepatic allograft function and survival after orthotopic transplantation in a canine model.

Background. Brain death has been shown to decrease graft function and survival in rodent models. The aim of this study was to evaluate how brain death affects graft viability in the donor and liver tolerance to cold preservation as assessed by survival in a canine transplant model. Methods. Beagle dogs were used for the study. Non-brain dead (BD) donors served as controls. Brain death was induced by sudden inflation of a subdural balloon catheter with continuous monitoring of arterial blood pressure and electroencephalographic activity. Sixteen hours after confirmation of brain death, liver grafts were retrieved. All livers were flushed in situ and preserved for 24 hr in cold University of Wisconsin solution before transplantation. Recipient survival rates, serum hepatic enzyme levels, coagulation, and metabolic parameters of the recipients were analyzed. Results. No significant changes were observed in serum aminotransferases (alanine and aspartate transaminases) and lactate dehydrogenase levels in the BD donor. After preservation, control (n=6) and BD livers (n=5) showed full functional recovery after transplant with 100% survival in both groups at day 7. There was no significant difference in peak serum alanine, aspartate transaminases, and lactate dehydrogenase after transplantation in recipients who received a liver from BD donor compared to control group. BD livers were functionally as capable as control livers in correcting metabolic acidosis during the first 24 hr posttransplantation. Coagulation profiles (index normalized ratio, activated partial thromboplastin time) after reperfusion were similar between groups. Conclusion. In contrast to previous reports in rodent models, our study shows that brain death does not cause significant liver dysfunction in the donor before organ removal. Donor brain death and prolonged liver graft preservation do not interact significantly to impair liver function and survival after transplantation.

Cytoskeletal involvement in hypothermic renal preservation injury.

Background. Cytoskeletal degradation occurs in warm renal ischemia and reperfusion and during hypothermia. The purpose of this study was to determine cytoskeletal changes during cold storage preservation injury in renal tubules and to determine whether these changes contribute to the injury. Methods. Isolated canine renal proximal tubules or their primary epithelial cultures were cold-stored in University of Wisconsin solution and reperfused in vitro to simulate renal cold storage preservation injury. Assays for cytoskeletal protein degradation and viability together with biologically active cytoskeletal agents and molecular interventions were used to test this hypothesis. Results. Progressively increasing the cold storage time of isolated renal tubules in University of Wisconsin solution caused proportional disassembly of both ezrin and Na/K adenosine triphosphatase proteins from their cytoskeletal attachments. The sublamellar structural protein, fodrin, was metabolized to products consistent with calpain hydrolysis during preservation. Time-dependent deterioration in tubule membrane function (organic cation transporter-1 transport activity) was observed during cold preservation, and this was mimicked in fresh tubules by chemically induced cytoskeletal disruption with cytochalasin-D treatment. Cold preservation decreased total tubulin content. Taxol slowed this loss by preventing tubulin depolymerization, which improved membrane function and tubule viability. The viability of primary renal epithelial cells was enhanced by overexpression of ezrin (transfection) and diminished with specific ezrin small interfering RNA knockdown during cold preservation injury. Conclusion. Hypothermic storage preservation causes disruption of key cytoskeletal elements in kidney tubules, which contributes causally to the injury at rewarming.

Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes.

BACKGROUND A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated. METHODS Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine). RESULTS There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis. CONCLUSION The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.

Hypothermic perfusion of rabbit livers: effect of perfusate composition (Ca and Lactobionate) on enzyme release and tissue swelling

Rabbit livers were preserved by continuous hypothermic (5 degrees C) perfusion at a flow rate of 1 ml/min-1 g-1 for as long as 72 hr. Cell swelling (total tissue water, TTW) and the rate at which intracellular enzymes were released into the perfusate were measured. Livers perfused with a simple NaCl-based solution containing hydroxyethyl starch as a colloid released relatively large amounts of aspartate aminotransferase (AST, 442 +/- 224 u/liter-1 100 g-1) and lactic dehydrogenase (LDH, 1580 +/- 688 u/liter-1 100 g-1) into the perfusate during 72 hr of perfusion. The addition of Ca (0.5 mmol/liter) to the perfusate reduced the leakage of enzymes into the perfusate (AST, 70 +/- 30 u; LDH, 450 +/- 50 u) and reduced cell swelling (TTW, 3.1 kg/kg dry mass vs 4.4 kg/kg dry mass without added Ca). But the use of a higher concentration of Ca (1.5 mmol/liter) caused membrane damage (AST, 4000 +/- 1500 u; LDH, 10,000 +/- 2222 u) and increased cell swelling (TTW, 3.7 kg/kg dry mass). The release of intracellular enzymes caused by continuous perfusion with a chloride-based perfusate also could be reduced by replacing the chloride with lactobionate (AST, 100 +/- 30 u; LDH, 400 +/- 100 u, at 72 hr). In the lactobionate-containing perfusate, the addition of Ca (0.5 or 1.5 mmol/liter) did not alter the rate at which intracellular enzymes were released. There was no tissue swelling after 72 hr of preservation with the lactobionate-containing perfusate, and the TTW (2.1 kg/kg dry mass) was similar to the TTW of freshly harvested rabbit livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hypothermic perfusion of kidneys on tissue and mitochondrial phospholipids

The changes in the level of phospholipids in kidney tissue and isolated mitochondria from dog kidneys perfused hypothermically (6-8 degrees C) for 1, 3, and 5 days were compared. Following 1 day of perfusion there was no change in total tissue phosphatidylserine (PS), a 25% decrease in the level of phosphatidylethanolamine (PE), and a 16% decrease in phosphatidylcholine (PC). No further decrease was observed with longer perfusion times. In fact, an increase in the level of PE occurred between the third and fifth days. Mitochondria isolated from perfused kidneys also showed a slight decrease in PE and PC following 1 day, no further change at 3 days, and an increase at Day 5. The loss of tissue phospholipids does not appear related to the viability of perfused kidneys. The major loss occurs within 1 day of perfusion and kidneys perfused up to 3 days are fully viable. Five-day perfused kidneys are nonviable, but show no greater loss of phospholipids than the viable 1- or 3-day perfused kidneys.

Role of peroxynitrite anion in renal hypothermic preservation injury

Background. Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. Methods. Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. Results. Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0–120h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). Conclusions. This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.