Introduction

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Transplantation of a solid organ from a donor has emerged as a treatment option for many diseases. Both the interruption and subsequent restoration of blood flow are often the cause of tissue injury in any transplanted organ. The impact is most dramatic in the heart, which led to development of experimental procedures to protect and preserve the myocardium (Woolfson et al., 1994). Some of these strategies may be of potential usefulness in transplanted kidneys, where ischemia and reperfusion contribute to the frequently observed nonimmunological damage. The organs are usually obtained from brain-dead donors or from living related or living unrelated donors. Hypothermia (4°C) remains the essence of preservation today. The methods of preservation are basically two: hypothermic storage and hypothermic pulsatile perfusion. Hypothermic storage remains the most common technique used because of its practicality and reduced cost. EC and UW solutions are the mainstay of therapy for hypothermic storage protection, particularly for kidney preservation (Belzer, 1993). These solutions are high K⁺ formulation. All of the Cl in EC are replaced by the organic anion lactobionate in UW. Impermeant sugars are also included in these solutions (glucose in EC and raffinose in UW). Hydroxyethyl starch is used as colloid in UW, and pharmacological additions include glutathione, adenosine, allopurinol, insulin, dexamethasone and antibiotic. Glutathione, allopurinol and impermeants are important components in the UW solution (Southard et al., 1990). The exact role of the other components remains in conflict (Bonventre and Weinberg, 1992). For renal preservation, it has been demonstrated that UW solution was more efficient compared with EC solution, particularly after 24-hr CS, in reducing the occurrence of delayed graft function, improving graft function and extending graft survival (Ploeg et al., 1992). However, despite the progress in surgical techniques and preservation conditions, every transplantation starts with an inevitable insult on the graft: the ischemia-reperfusion syndrome. Ischemia-reperfusion injury may be considered an inflammatory and vasomotor phenomenon. In renal transplant, delayed graft function is a relatively common complication of this syndrome. Delayed graft function usually occurs in between 20% and 60% of kidney cadaveric transplantations and makes the diagnosis of rejection more difficult, prolongs hospital stay and reduces short- and long-term graft survival rates (Cecka et al., 1992; Gaston and Schlessinger,1994; Shoskes and Halloran, 1996). Although reperfusion is crucial for oxygen delivery to ischemically injured tissues, tissue reoxygenation is known to be detrimental because it allows the generation of reactive oxygen metabolites such as superoxide anions, hydroxyl radicals and hydrogen peroxides.

ROS, which are generated during ischemia and reperfusion, play a key role with cellular constituents, including proteins, lipids and DNA (Woolfson et al.,1994; Lehr and Messmer, 1996). ROS stimulate the release and the formation of inflammatory mediators with powerful chemotactic potential (Lehr and Messmer, 1996). These molecules are a distress signal for the immune system. They up-regulate adhesion molecules on leukocytes and endothelial cells and recruit leukocytes on to the site of injury (Linas et al., 1992; Granger and Kubes, 1994; Halliwell, 1994; Hansen, 1995). Moreover, ischemia and reperfusion up-regulate the expression of major histocompatibility complex molecules in clinical and experimental studies, rendering postischemic allografts more immunogenic and rejectable (Shackleton et al., 1990; Sischer et al., 1993; Lu 1996; Shoskes et al., 1990; Shoskes and Halloran, 1996). Thus, drugs or techniques that stabilize or reverse ischemia-reperfusion injury should improve organs to be transplanted.

The aim of the present study was to analyze the effect of an anti-ischemic agent developed by Servier Research Institute, the 1-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (TMZ) (fig. 1). Previous studies have demonstrated that the mechanism of action of TMZ involves a cellular effect demonstrated by the restoration of ATP and PCr levels in myocardial fibers after experimental ischemia (Lavanchy et al., 1987) and an improved viability of isolated rat myocytes when submitted to both hypoxia and high Ca⁺⁺ extracellular levels (Harpey et al., 1989). Moreover, other studies concerning isolated heart or isolated cardiomyocytes models demonstrated that TMZ improves mitochondrial functions during normothermic ischemic damage (Guarnieri and Muscari, 1993) and increases cell resistance to hypoxic stress (Fantini et al., 1994). An antioxidant activity was also reported (Maupoil et al., 1990). However, the beneficial effect of TMZ has never been assessed during simple cold ischemia and reperfusion. Our hypothesis is that TMZ could improve EC and, in particular, elaborated UW solution, which already contains additional cytoprotective components such as allopurinol. The aim of the present investigation in an IPK model was to determine (1) whether TMZ limits the lipid peroxidation during preservation with EC and, in particular, UW solutions and the consequence during normothermic reperfusion, (2) whether TMZ improves renal intracellular high-energy phosphorus metabolites during cold ischemia and reperfusion and (3) whether TMZ acts on intracellular homeostasis, particularly on intracellular pH, during preservation and reperfusion. The concentration of TMZ used was 10⁶ M. This concentration was selected as that effective to restore ATP synthesis of isolated mitochondria previously decreased by Ca⁺⁺ overload (for a review, see Harpey et al., 1989; Salducci et al., 1996). In addition, previous study has demonstrated that a higher concentration of TMZ exerted no protective effect (Boucher et al., 1994).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   TMZ chemical structure.

	Materials and Methods

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Animals and IPK preparation. Male Wistar rats weighing 350 to 400 g were used. Animals were fasted overnight before the experiments. They were anesthetized with urethane (16.8 µmol/kg i.p.), and the kidneys were removed.

Preservation solutions. Standard UW and EC solution, supplemented or not with TMZ (10⁶ M), were used. The EC solution was manufactured by Pharmacie Centrale des Hôpitaux (Paris, France) and supplied in 1-liter bottles. The composition of EC solution was (in mM): glucose (198), Na⁺ (10), K⁺ (115), Cl (15), HCO₃ (10) and phosphate (50), and osmolality was 355 mOsm/kg. UW was purchased from Dupont-Pharma (Paris, France). The composition of UW solution was (in mM): lactobionic acid (100), Na⁺ (30), K⁺ (125), magnesium (5), phosphate (25), rafinose (30), glutathione (3), adenosine (5), allopurinol (1) and hydroxyethyl starch (50 g/l), and osmolality was 320 mOsm/kg. TMZ was gift from Servier (Neuilly, France) and was added to the preservation solutions before cold flush and preservation.

Experimental protocols. In the control group (G1; n = 6), kidneys (right kidney) were perfused with the Kreb's solution immediately in situ before they were harvested. In experimental groups, kidneys were randomably distributed into four experimental groups for either biochemical dosages or NMR observations as follows: G2 (48-hr CS with EC, n = 12), G3 (48-hr CS with EC + TMZ, n = 12), G4 (48-hr CS with UW, n = 12) and G5 (48-hr CS with UW + TMZ, n = 12). Kidneys were catheterized and isolated without interruption of blood flow and flushed in situ with the CS solution at 4°C at a maximum pressure of 100 mm Hg. The kidneys were then placed with their catheter in a small beaker containing preservation solution (50 ml) at 4°C. At the end of the storage period (48 hr), kidneys were reperfused on the isolated perfusion circuit with perfusion medium (without TMZ). The first 20 ml of reperfusion fluid was discarded to wash preservation solution. After starting the perfusion, an equilibration period of 30 min was allowed before hemodynamic and energetic measurements.

Hemodynamic study. The PFR was adjusted to maintain the renal arterial perfusion pressure at 100 mm Hg. PP was measured by a pressure transducer (Statham P23; Grass, Quincy, MA) at the arterial outflow site. PFR was measured after 30, 60 and 90 min during reperfusion.

Measurement of the percentage of tissue water content. To test whether TMZ affects tissue edema in the kidney during the preservation period and subsequent reperfusion, renal water content was determined. The tissue water content was determined in fresh tissue (n = 6) soon after laparotomy; in the control group after reperfusion; at 12-, 24- and 48-hr CS; and after reperfusion. The kidney tissues were measured initially for the wet weight and then after 48 hr in an oven at 100°C for the dry weight, thus allowing the determination of the water content. The percentage of renal water content was calculated as follows: renal water content = (1  dry weight/wet weight) × 100 (%).

Measurement ofMDA tissue level. At the end of CS and reperfusion, a slice from whole kidney (left kidney from G1 and experimental groups after CS and right kidney after reperfusion) was uniformly minced and then homogenized in an ice-cold solution of 1.15% KCl. Fatty acid peroxidation was evaluated by tissue levels of MDA and measured with the thiobarbituric acid test. An aliquot of renal tissue homogenate was heated with thiobarbituric acid under acidic conditions (95°C for 60 min). The absorbance of the organic layer was measured at 532 nm at room temperature. The level of lipid peroxidases was expressed as nmol/mg of protein.

NMR measurement. After the equilibration period, isolated perfused kidneys were placed into 20-mm NMR tubes and introduced into the vertical magnet. During the experiment, the venous effluent was removed from the NMR tube by an aspiration line and recycled. ³¹P NMR experiments were performed on a Bruker AM400WB spectrometer at 92 MHz. Field homogeneity was achieved by shimming the water signal. Spectra were acquired with 4K data points for a spectral width of 6000 Hz and a 20-µsec (90°) pulses on before Fourier transformation. In each experiment, spectra were acquired after 30, 60 and 90 min of reperfusion (total acquisition time, 6 min). Intracellular pH (pH_i) was estimated by using the chemical shift of the inorganic phosphate (Pi) according to the formula: pH_i = 6.7 + log [( Pi  3.148)/(5.695   Pi)] (Bauza et al., 1995). The signal intensities of ATP, Pi and PCr were estimated by measuring the peak intensity on plotted spectra. The pH_i were also determined after 12, 24 and 48 hr of cold ischemia. The ATP/Pi ratio was calculated as a bioenergetic index during perfusion.

Statistical analysis. Results are expressed as the mean ± S.E.M. The data were analysed using a two-way variance analysis and the t test. Differences at a P < .05 were considered to be significant.

	Results

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PFR. As shown in figure 2, TMZ caused a significant rise of PFR during normothermic reperfusion after 48-hr CS in EC and, in particular, UW groups. PFR was not different in EC + TMZ and UW groups during reperfusion.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effect of addition of TMZ on PFR expressed as ml/min/g of tissue of 48-hr cold stored kidneys. ** P < .01 EC vs. EC plus TMZ and UW vs. UW plus TMZ.

Changes in the percentage of renal water content. While the water content in EC group increased gradually during preservation and reperfusion, that in the EC + TMZ group decreased significantly during preservation and increased rapidly during reperfusion. The water content decreased rapidly during preservation in the UW group and increased during reperfusion. However, the water content in UW + TMZ group was significantly lower than that in the UW and EC + TMZ groups (fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Changes in renal tissue water content. A, Water content was determined at different times: on fresh tissue, after 12, 24 and 48 hr of cold preservation (* P < .05 EC vs. EC plus TMZ and UW vs. UW plus TMZ; ** P < .01 EC vs. EC plus TMZ). B, Water content was determined immediately before (time 0) or after a 90-min period of reperfusion in the same experimental groups and control group (** P < .01 EC vs. EC plus TMZ and UW vs. UW plus TMZ).

Changes in MDA tissue levels. After 48-hr cold ischemia, MDA tissue levels were higher in groups without TMZ than in groups supplemented with TMZ. This difference was more significantly higher after 48-hr cold ischemia than after 12 or 24 hr (fig. 4). Reperfusion caused a significant increase in tissue MDA levels in all preserved groups. However, the increase after reperfusion in the TMZ groups was significantly lower than that in the EC and UW groups (fig. 4).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Changes in MDA tissue levels. A, MDA tissue levels were determined at different times: on fresh tissue (open ellipse, time 0), after 12, 24 and 48 hr of cold preservation with EC () EC plus TMZ (), UW (), UW plus TMZ (), (** P < .01 EC vs. EC plus TMZ and UW vs. UW plus TMZ). B, MDA tissue levels were determined immediately before (time 0) and after a 90-min period of reperfusion in the same experimental groups and control group () (** P < .01 EC vs. EC plus TMZ and UW vs. UW plus TMZ).

Changes in ATP/Pi ratio in the kidneys. Figure 5 depicts the values of energetic ratio in control IPK during reperfusion after 48-hr CS in the different solutions. PCr was detected in the control group and slightly in the TMZ groups. The ATP/Pi ratio, in the control group, during reperfusion was 1.31 ± 0.18 after 30 min, 1.28 ± 0.2 after 60 min and 1.27 ± 0.2 after 90 min. The ATP/Pi ratio was greater in the TMZ groups than groups without TMZ during reperfusion. Representative spectra from each experimental group after 60 min during reperfusion are illustrated in figure 6.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Changes in bioennergetic ratio. Bioenergetic ratio was determined during reperfusion (ATP/Pi) in experimental groups and control group (* P < .05 EC vs. EC plus TMZ and UW vs. UW plus TMZ; ** P < .01 EC vs. EC plus TMZ). The different solutions used were the same as described in Materials and Methods.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Typical spectra determined by 31P NMR spectroscopy from each experimental group during reperfusion (t60). ppm, parts per million.

Changes in pH_i in the kidneys. Cold ischemia reduced pH_i in all groups but in G5, pH_i was significantly greater during cold ischemia than in G3 (table 1). In EC groups (G2 and G3), the pH_i was not significantly different after 24-hr CS. During normothermic reperfusion, the kidneys preserved with TMZ showed a significantly higher pH_i after 48-hr CS than in kidneys preserved without TMZ. The highest shift in pH_i were observed by using UW + TMZ solution (table 1).

	Discussion

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Hypoperfusion related to the hemodynamic status of the donor and recipient and the interval of hypothermic preservation contribute to increase the ischemia-reperfusion injury. Recently, the transplant community has focused increased attention on identifying drugs that stabilize or reverse ischemia-reperfusion injury. The reperfusion of previously ischemic tissue occasionally potentiates release of intracellular enzymes, influx of Ca⁺⁺, breakdown of phospholipids and disruption of cell membranes, which either alone or in combination result in ultimate cell death (Paller and Greene, 1994). Current evidence leads to at least three major hypotheses concerning the mediators of reperfusion injury: (1) the generation of oxygen-derived free radicals, (2) calcium overloading and (3) degradation of phospholipids. The primary events that can be considered responsible for the cascade of metabolic, functional and structural alterations that develop in the cause of ischemia are energy imbalance and alterations of cellular homeostasis. Hypothermia does not stop metabolism but slows reaction rates and cell death. In this situation, the energy imbalance caused by ischemia is considerably attenuated by the use of hypothermia and preservation solution. The impact of TMZ during normothermic and cold ischemia and reperfusion has been already described in cardiac protection and during preservation. Mitochondria function and pathophysiology of ischemia-reperfusion vary from organ to another. However, the concept of ischemia-reperfusion injury is spread, and any tissue or cell undergoing ischemia may be subject to reperfusion injury. Consequently, TMZ with its anti-ischemic properties may also have a use in the attenuation of ischemia-reperfusion injury. In this study, the possibility of further improving the preservation capacity of standard preservation solutions (EC and particularly UW) by the addition of TMZ was evaluated.

The more recent and complete studies of hemodynamic effects of TMZ have demonstrated that TMZ did not modify hemodynamic parameters in the systemic circulation (Harpey et al., 1989). However, the present study demonstrates an increased PFR in kidneys preserved with TMZ, particularly in UW solution. Because TMZ is known to be devoid of hemodynamic effect and the initial 20 ml of reperfusion fluid is discarded to wash preservation solution, this result is partly related to less severe interstitial and cellular edema in kidneys preserved with TMZ during cold ischemia and particularly reperfusion. Consequently, TMZ might be preferred because it is devoid of adverse hemodynamic effects compared with Ca⁺⁺ blockers or other vasodilatators. These results are supported by tissue water content data. Tissue water content is thought to be an indicator of tissue edema, related to the disturbance of microvascular circulation due to membrane damage and depolarization of epithelial cell membrane. The lactobionate anion and the impermeant trisaccharide raffinose were added in UW solution as principal agents that suppress cell swelling during CS (Belzer and Southard, 1988). In the present study, tissue water content is significantly different during CS between UW and UW + TMZ groups. In contrast, preservation with EC resulted in subsequent cell swelling and damage. However, with EC + TMZ, tissue edema during cold ischemia was significantly less than that in the EC group. After reperfusion, the water content in TMZ groups was significantly lower than that in the UW and EC solutions. The kidney, particularly the medulla, is capable of synthesizing a range of prostaglandins, including thromboxane and prostaglandin F₂ and prostacyclin and prostaglandin E₂ (Robak and Sobanska, 1976). Hypoxia and ischemia have been shown to stimulate the production of some of these metabolites in whole kidneys subjected to ischemia. The result is an imbalance in eicosanoids in favor of vasoconstriction during reperfusion (Lelcuk et al., 1985). In addition, there is evidence to suggest that prostaglandins are involved in the development of edema after ischemia (Ianotti et al., 1981). Experimental study has shown that TMZ reduces intracellular accumulation of Na⁺ and Ca⁺⁺, edema and activation of thromboxane synthetase (Tsimoyiannis et al., 1993). Moreover, TMZ reduces leakage of intracellular potassium, which is a vasoconstrictor, into the extracellular space that occurs during ischemia (Maridonneau-Parini and Harpey, 1985).

A pivotal feature of ischemia-reperfusion injury is the formation of ROS. ROS are generated during ischemia and reperfusion and cause the recruitment of inflammatory cells and the subsequent microvascular dysfunction. The production of ROS after organ preservation in cold ischemia has been documented (Koyama et al., 1985). The main source of ROS are endothelial cells and leukocytes, which play an important role through their adherence to endothelial cells. ROS are capable of reacting with proteins, lipids and nucleic acids, leading to lipid peroxidation of biological membranes and MDA production. Our IPK model is free of leukocytes, and consequently, it is easy to discard a leukocyte-dependent mechanism because the ischemia-reperfusion injury is only due to the endothelial cell, which is the main source of oxygen free radicals (Herrero et al., 1995). The kidneys preserved without TMZ exibited high MDA levels after a long cold ischemia period, particularly with EC preservation. On the contrary, preservation with TMZ can attenuate MDA production related to ischemia-reperfusion injury, including with EC solution. Previous study has demonstrated an antioxidant activity of TMZ during normothermic ischemia and oxygenated reperfusion (Maupoil et al., 1990). Recently, Fantini et al. (1994) demonstrated that pretreatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress that was related to a modification of lipid metabolism. MDA data of the present study suggest a synergistic effect of TMZ and glutathione and allopurinol, which are other protective antioxidant included in UW solution. Consequently, TMZ seems to be particularly efficient as a free radical scavenger during reperfusion after cold ischemia.

³¹P NMR spectroscopy is a technique that uses the magnetic properties of nuclei, such as phosphorus, to produce a chemical shift spectrum. The signal gives a measure of the number of mobile nuclei contributing to the peak, which allows ratios of specific high energy phosphorus metabolites to be calculated. A ³¹P NMR spectrum of an ex vivo kidney during hypothermic storage or reperfusion shows relative concentrations of mobile adenine nucleotides and other phosphorus metabolites (Bretan et al., 1989). Recent study has demonstrated that the ATP/Pi ratios correlated very well with kidney viability and were related to the bioenergetic status (Bretan et al., 1987). The bioenergetic status is a measure of the energy state of the cell, reflecting the overall balance between metabolic energy supply and demand. During reperfusion, ATP levels remained very low compared with intact kidneys, probably because free ATP was rapidly used by the reperfused kidneys after the prolonged ischemia period. However, the ATP/Pi ratio was significantly higher in the kidneys preserved with TMZ than those preserved without TMZ during reperfusion. This increase in the ATP/Pi ratio is due to a decrease in Pi level resulting from an improvement in oxidative phosphorylation. In addition, previous study have demonstrated that TMZ reverses Ca⁺⁺ accumulation during ischemia (Guarnieri and Muscari, 1993). Consequently, the limitation of increase in intracellular calcium by TMZ is related both to less severe ATP consumption to buffer this intracellular calcium and impairment of phosphorylation during both cold ischemia and reperfusion. This result is a direct experimental support for the prevention of the intracellular calcium accumulation and, consequently, the dysfunction of the other metabolic pathways during cold ischemia-reperfusion. In addition, TMZ in UW solution, which contains adenine to provide purine substrate for resynthesis of ATP, improves energetic status during reperfusion. Otherwise, our results agree with the hypothesis that the postischemic recovery of ATP is a function not only of the residual nucleotide pool at the end of ischemia but also of the rate at which the kidney restores its energetic ratio after reperfusion (Vigués et al., 1993).

Intracellular acidosis is also known to affect cell viability, but its contribution to ischemic injury remains controversial (Bonventre and Weinberg, 1992). The development of ischemic acidosis is a multifactorial process that results in both excessive proton production and reduced proton extrusion from the cell and leads to a reduction of pH_i, particularly during prolonged cold ischemia. In agreement with previous study (Renaud, 1988), pH_i from kidneys preserved without TMZ was lower during both preservation and reperfusion. Thus, the mild reduction in pH_i from the physiological level to slightly more acidic values should provide protection to kidney cells, a phenomenon that has been proposed to be related to the decrease in phospholipase A₂ activity (Bonventre and Cheung, 1985). In addition, the difference between acidotic pH_i during preservation and pH_i during reperfusion is greater in groups preserved without TMZ than those preserved with TMZ. Consequently the "pH paradox," which precipitates lethal reperfusion injury (Currin et al., 1991), is reduced by TMZ.

In conclusion, TMZ added to the cold flush and during CS resulted in a significant degree of protection during cold ischemia and during normothermic reperfusion (fig. 7). Our experiments demonstrate that TMZ administration in a rodent model of prolonged cold ischemia and 2-hr reperfusion provided a significant renal protection when added to conventional intracellular preservation solutions. This effect is maximal with UW solution, which contains additional antioxidant components, and TMZ is a potential agent that could be efficient against initial ischemia-reperfusion injury and delayed graft function, which increase the immunogenicity of the transplanted organ.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Summary diagram of the mechanisms of ischemic reperfusion injury and the effects of TMZ (*).