Joaquin V. Rodriguez

Organ Preservation: Current Concepts and New Strategies for the Next Decade.

Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation. This review sets out the history of organ preservation, how applications of hypothermia have become central to the process, and what the current status is for the range of solid organs commonly transplanted. The science of organ preservation is constantly being updated with new knowledge and ideas, and the review also discusses what innovations are coming close to clinical reality to meet the growing demands for high quality organs in transplantation over the next few years.

Rat kidney function related to tissue glutathione levels

Rat renal function was evaluated during acute depletion of glutathione (GSH) produced by different doses of diethyl-maleate (DEM). Significant alterations in renal function were observed when the GSH level diminished. The replenishment of GSH and the restoration of renal function were also investigated at various times after the injection. Similar time courses were observed of both the GSH level and renal functions, but the former was shortest. This suggests that the restoration to normal of GSH renal content was necessary in order to regain appropriate kidney function. Furthermore, the fact that impairment of sodium excretion occurred simultaneously with GSH depletion may be considered as evidence of the first event in GSH protective action. It may be hypothetized that the thick ascending limb is the principal renal target for this deficiency.

Glyceraldehyde-3-phosphate dehydrogenase exerts different biologic activities in apoptotic and proliferating hepatocytes according to its subcellular localization

Recent evidences indicate new roles for the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in essential mammalian cell processes, such as apoptosis and proliferation. To clarify the involvement of this protein in growth and programmed cell death in the liver, cell models of hepatocytes in culture were used to study GAPDH expression, localization and enzymatic activity in hepatocyte proliferation and apoptosis. GAPDH expression in cell compartments was studied by Western blot. Nuclear expression of GAPDH increased in apoptosis, and cytoplasmic expression was elevated in apoptosis and proliferation. Subcellular localization was determined by GAPDH immunostaining and confocal microscopic analysis. Quiescent and proliferating hepatocytes showed cytoplasmic GAPDH, while apoptotic cells showed cytoplasmic but also some nuclear staining. The glycolytic activity of GAPDH was studied in nuclear and cytoplasmic cell compartments. GAPDH enzymatic activity increased in the nucleus of apoptotic cells and in cytoplasms of apoptotic and proliferating hepatocytes. Our observations indicate that during hepatocyte apoptosis GAPDH translocates to the nucleus, maintaining in part its dehydrogenase activity, and suggest that this translocation may play a role in programmed hepatocyte death. GAPDH over-expression and the increased enzymatic activity in proliferating cells, with preservation of its cytoplasmic localization, would occur in response to the elevated energy requirements of dividing hepatocytes. In conclusion, GAPDH plays different roles or biological activities in proliferating and apoptotic hepatocytes, according to its subcellular localization.

Uptake of [3H]bilirubin in freshly isolated rat hepatocytes: role of free bilirubin concentration

Hepatocytic transport of physiological concentrations of unconjugated bilirubin (UCB) has not been determined in isolated liver cells. Initial uptake of highly purified [3H]UCB was measured in rat hepatocytes in the presence of human serum albumin at various free, unbound UCB concentrations, [UCB]. At [UCB]=42 nM (below aqueous solubility of 70 nM), uptake was strictly temperature dependent; this was much less evident at [UCB]=166 nM (supersaturated). At low, physiological UCB concentrations, specific UCB uptake showed saturative kinetics with an apparent K m of 41 nM, indicating carrier‐mediated transport. With aqueous supersaturation, UCB entered hepatocytes mainly by passive diffusion.

Engraftment and function of intrasplenically transplanted cold stored rat hepatocytes.

Hepatocellular transplant may potentially be efficacious for the treatment of selected liver metabolic disorders and acute hepatic failure. On the other hand, the use of hepatocyte cold preservation techniques in these transplantation protocols would allow to have available cells at the right time and place and, consequently, make an optimal use of scarce human hepatocytes. In our experiments we evaluated the biodistribution and functionality of cold preserved hepatocytes transplanted in the spleen of syngeneic rats. Isolated hepatocytes were labeled with the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl-ester, cold-preserved in modified University of Wisconsin (UW) solution for 48 or 96 h, and then transplanted into the spleen. Recipient animals were euthanized at 0 and 3 h, and at 1, 2, 3, 5, 10, and 14 days after transplantation for tissue analysis. Labeled hepatocytes were clearly identifiable in the recipient tissues up to 14 days later. Fluorescence microscopy also showed no significant differences in biodistribution when either cold stored or freshly isolated hepatocytes were transplanted. In addition, functional activity of transplanted cells was demonstrated by immunohistochemical detection of albumin at levels comparable to those found in normal hepatocytes. Our findings establish that cold preserved hepatocytes appear morphologically and biochemically normal after intrasplenic transplantation. Consequently, it indicates that modified UW solution makes it possible to safety preserve hepatocytes for up to 96 h before transplantation, perhaps providing sufficient time for hepatocyte allocation and potential recipient preparation, if applicable clinically.

Effect of ethinylestradiol and epomediol on bile flow and biliary lipid composition in rat

Epomediol (1,3,3-trimethyl-2-oxabicyclo(2.2.2.)octan-6,7-endo,endo-diol) (EPO) is a terpenoid compound shown to reverse 17 alpha-ethinylestradiol (EE)-induced cholestasis in rat. The effect is related to the restoration of normal liver plasma membrane fluidity values. To further characterize the effect of EPO, bile flow and biliary lipid composition were measured in rats treated either with EE or EE associated with EPO. EE significantly reduced the bile flow; this reduction was prevented by concomitant treatment with EPO with an increase in the bile salt secretion rate. EPO alone showed a choleretic effect. The biliary secretion rate of cholesterol was also significantly reduced by EE while being comparable to controls in EE-EPO-treated animals. Phospholipid (PL) biliary excretion was significantly (P less than 0.002) increased by EE either alone or combined with EPO. After EE treatment, the biliary PL composition showed a reduction in phosphatidylcholine (PC) concentration with a parallel increase in lyso-phosphatidylcholine (LPC) when compared to control animals (PC:LPC ratio 5.0 +/- 2.5 vs 26.8 +/- 9.9, mean +/- SD, P less than 0.005). EPO administration to EE-treated rats restored the biliary PC:LPC ratio to control values (27.6 +/- 10.6). EPO alone did not show any appreciable effect as compared to both control and EE-EPO treated animals. As increased concentrations of LPC have been reported to induce an alteration in the function of membrane lipids and membrane-associated proteins, such as regulatory enzymes for bile acid, cholesterol and phospholipid metabolism, these results suggest that the protective effect of EPO in EE-induced cholestasis may be related to the reversal of the alterations in membrane lipid composition and function induced by EE.

Importance of pH in resuspension media on viability of hepatocytes preserved in University of Wisconsin solution

The effect of different pH of resuspension media on the viability of hepatocytes preserved (for 96 h at 4 degrees C) in University of Wisconsin solution (UW solution) was analyzed. After this cold resuspension media storage, we evaluated the rewarming step (incubation time 120 min at 37 degrees C) using different pH levels (6.80, 7.00, 7.20, and 7.40). Cell viability assessed by trypan blue exclusion (TBE) showed a significant difference (p < 0.05) for cells incubated at pH = 7.20. For instance, TBE expressed as percent of change was 78.1 +/- 1.4 compared with cells tested at other pH (pH = 6.80, TBE = 44.2 +/- 9.5; pH = 7.00, TBE = 66.5 +/- 1.1 and pH = 7.40, TBE = 62.0 +/- 1.4). We also evaluated the capacity of these cells both to maintain potassium content (0.509 +/- 0.230 microEq. K+/10(6) cells) and to synthesize urea (5.36 +/- 1.81 mumol Urea/10(6) cells). These results were compared with those obtained from freshly isolated non preserved hepatocytes (0.518 +/- 0.060 microEq. K+/10(6) cells and 5.91 +/- 0.43 mumol Urea/10(6) cells). The results show that viability is pH dependent and suggest that when resuspension media were used, the viability of hepatocytes was improved after 96 h of cold storage.

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model.

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h-4 °C) in UW; and UWS: IPRL was performed in livers preserved (48 h-4 °C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H₂S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H₂S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H₂S donor showed an enhanced capacity for BSP uptake (k(A) CON = 0.29 min⁻¹; k(A) UW = 0.29 min⁻¹ ; k(A) UWS = 0.36 min ⁻¹). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H₂S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.

A simple and effective method to improve intrasplenic rat hepatocyte transplantation.

Transplanted hepatocytes integrate, survive, and express their specific functions in the liver parenchyma. The aim of this study was to determine whether a large number of hepatocytes could move from the spleen to the liver when the cells are injected together with sodium nitroprusside, and if the improved hepatocyte migration may be related with portal vein dilatation. Wistar rats were transplanted in the spleen with fluorescent-labeled hepatocytes alone or together with sodium nitroprusside. At 1, 3, 6, and 24 h after the transplant, the liver from recipient animals was removed and morphometric analyses were performed. Portal and arterial pressures were also measured immediately after intrasplenic injection of a solution of sodium nitroprusside, hepatocytes alone, or hepatocytes plus sodium nitroprusside. Intrasplenically injected sodium nitroprusside produced a transient drop in arterial pressure and a sustained reduction in portal pressure. During hepatocyte transplantation it increased the number of transplanted cells migrating to the liver after 3 h. Sodium nitroprusside simultaneously injected with hepatocytes in the spleen allowed more cells to migrate into the liver of the host animal without risk in animal survival.

Protective effect of glutathione (GSH) over glutathione monoethyl-ester (GSH-E) on cold preservation of isolated rat liver cells

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. We investigated whether reduced Glutathione (GSH) inclusion into a modified University of Wisconsin (UW) solution, has a protective effect over Glutathione derivatives, such as Glutathione-monoethylester (GSH-E), when suspensions of hepatocytes are cold stored for several days. Isolated rat liver cells were cold preserved 96 h in UW, UW plus 3 mM GSH and UW plus 3 mM GSH-E. During the cold storage, not significant changes in cell viability were observed, but the total Glutathione content was higher in systems with extracellular GSH over those with GSH-E or without. After cold storage, the liver cells were gently resuspended in Krebs-Henseleit-1% Albumin and used for 120 min of normothermic (37 degrees C) incubation. We evaluate the functional response of the cells measuring the exclusion of Trypan Blue (TBE). This response was clearly different in preserved cells in presence of GSH. These results indicate a protective role of extracellular Glutathione, due to an accumulation of it, rather than the derivative, for hepatic cell during the cold storage in UW solutions. And also, it is possible to extend experiments with hepatocytes from a single cell isolation over 4 or more consecutive days.