EFFECT OF MANNITOL AND POLYETHYLENE GLYCOL ON THE ACTION OF FRUSEMIDE DURING RENAL STORAGE AND TRANSPLANTATION1 : Transplantation

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Experimental Transplantation

EFFECT OF MANNITOL AND POLYETHYLENE GLYCOL ON THE ACTION OF FRUSEMIDE DURING RENAL STORAGE AND TRANSPLANTATION1

Lane, Nick J.2,3; Thorniley, Maureen S.2; Manek, Sanjiv2,4; Fuller, Barry J.5; Green, Colin J.2

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Abstract

Ischemia-reperfusion injury is a major cause of acute tubular necrosis following renal storage and transplantation (1). Damage may result from hypoxia alone or from free radical generation upon reperfusion. Different sections of the nephron vary in their susceptibility to hypoxic or free-radical-mediated injury (2). The medullary thick ascending tubules (mTAT*) and the pars recta (S3) segments of the proximal tubules are particularly susceptible to hypoxia alone(3-5). This susceptibility stems from their high requirement for oxygen to carry out the work of active tubular reabsorption in an environment on the brink of hypoxia even under normal conditions(3-5). Inhibition of active transport by ouabain(a specific inhibitor of Na-K-ATPase) and frusemide (a specific inhibitor of Na-K-2Cl-cotransporter in the mTAT) has been shown to ameliorate hypoxic injury to both the mTAT and the pars recta in the isolated perfused kidney(5, 6).

Inhibition of active tubular reabsorption by frusemide is reflected in changes in the redox state of cytochrome oxidase (cyt aa3), the terminal member of the respiratory chain, and in renal oxygenation(7). These parameters can be measured in vivo using near-infrared spectroscopy (NIRS) (8). NIRS in transmission mode measures concentration changes in oxyhemoglobin, deoxyhemoglobin, and the oxidized form of cyt aa3 across the entire kidney (8 and Methods). It has been shown in the isolated perfused kidney that agents that alter metabolism or active transport primarily in the renal cortex have little or no effect on the redox state of cyt aa3, which suggests that the rate of oxygen consumption is not a critical determinant of cyt aa3 redox status in these sections of the tubule (7). In contrast, inhibition of active reabsorption in the mTAT in this model caused an increase in oxygen tension and a corresponding increase in the redox state of cyt aa3(7). Thus, the effect of frusemide on metabolic activity in the mTAT can be monitored noninvasively by measurements of changes in the redox state of cyt aa3, using NIRS in transmission mode.

During renal storage for transplantation, hypothermic swelling of the mTAT results in mechanical constriction of the peritubular capillaries and vasa recta (9, 10). Upon reperfusion, outflow of blood from the medulla is blocked (10). Medullary hemostasis results in profound hemoglobin desaturation and secondary ischemia in the surrounding parenchyma (11). Under these circumstances, inhibition of active tubular reabsorption by frusemide may be less effective at preventing cellular injury than in the isolated perfused kidney: first, medullary hemostasis may prevent frusemide from reaching the mTAT; second, the severity of ischemia-reperfusion injury during renal transplantation may cause an irreversible loss of mTAT integrity, such that frusemide could have at best a limited effect on cellular metabolism. We hypothesize that amelioration of cellular swelling during the storage period by use of more effective impermeants could improve cellular integrity during reperfusion, increase medullary blood flow, and maximize the potential therapeutic effects of frusemide.

These effects should be detectable using NIRS to monitor changes in both the redox state of cyt aa3 and renal hemoglobin oxygenation. We have previously shown that the responsiveness of cyt aa3 to respiratory chain inhibitors, such as sodium pentobarbitone (Amytal), in the first few minutes of reperfusion, is a good indicator of long-term renal viability(12). As frusemide primarily affects ion pump function, its physiological effects over a period commensurate with its duration of action in vivo (about 30 min) may provide information on the pathophysiology of renal ischemia-reperfusion injury and the relative value of impermeant solutes, both during the short period measured and, by extrapolation, on long-term viability. Therefore, we have compared the effect of two impermeants(mannitol and polyethylene glycol [PEG]) on renal responsiveness to frusemide, using NIRS, during the first 35 min of reperfusion of rabbit kidneys stored hypothermically for 72 hr before transplantation.

MATERIALS AND METHODS

All animal procedures were carried out according to home office regulations, Animals (Scientific Procedures) Act, 1986.

Preservation solutions. Two solutions were compared: (1) standard hypertonic citrate solution (HCA), from Baxter Health Care (Thetford, Norfolk, UK), containing mannitol as an impermeant, and (2) HCA-PEG, in which mannitol was replaced by PEG (average m.w., 600). There were no other differences between the two solutions. PEG was originally included at the same concentration as mannitol, but is not an ideal solute in terms of its colloidal osmotic pressure. The concentration of PEG added was therefore lowered to maintain a final measured osmolarity of 485 mOsm/L, to compare with standard HCA solution (see Table 1).

Surgical procedures. Thirty adult female New Zealand White rabbits (2.5-3 kg), in six groups of five animals (described below), were anesthetized with an intramuscular injection of fentanyl fluanisone (hypnorm; 0.2 ml/kg) followed by slow injection of diazepam (1.0 mg/kg) and heparin (300 U/kg). Oxygen (2 L/min) was supplied via an open face mask. The right kidney was exposed through a midline abdominal incision and removed after careful dissection and ligation of the vessels and ureter. The renal artery was cannulated and flushed with 40 ml of ice-cold (0-2 °C) HCA solution from a bag suspended at a height of 1.5 m. At the time of autografting, the left kidney was carefully removed after the renal vessels had been clamped and the right kidney was autografted into the left renal bursa using standard microsurgical techniques. Upon reperfusion, 60 ml of isotonic saline were infused intravenously to the animal over 35 min. After 5 min of reperfusion, groups 2, 4, and 6 received an intravenous bolus of frusemide (0.6 mg/kg in 1 ml). Controls did not receive vehicle alone (as they were already receiving continuous slow saline infusion).

Experimental groups. In group 1, freshly nephrectomized left kidneys were flushed with 40 ml of cold (0-2 °C) HCA containing mannitol, via the renal artery, and autografted immediately into the left renal bursa using standard microsurgical techniques. Kidneys were monitored continuously in situ during reperfusion by NIRS for 35 min. After 35 min, reperfusion was terminated by lethal infusion of sodium pentobarbitone (200 mg/kg). The experimental protocol for group 2 was identical to that used for group 1, except that group 2 recipients received an intravenous bolus of frusemide (0.6 mg/kg) after 5 min of reperfusion. In group 3 animals, kidneys were flushed as in group 1 and then stored in HCA containing mannitol (see Table 1), surrounded by ice to maintain a temperature of 0-2 °C for 72 hr before autografting. Recipients did not receive frusemide. The protocol for group 4 was identical to that for group 3, except that group 4 recipients received an intravenous bolus of frusemide (0.6 mg/kg) after 5 min of reperfusion. In group 5, kidneys were flushed and stored in HCA containing PEG (m.w. 600; see Table 1) surrounded by ice to maintain a temperature of 0-2 °C for 72 hr before autografting. Recipients did not receive frusemide. The protocol for group 6 was identical to that for group 5, except that the recipients received an intravenous bolus of frusemide (0.6 mg/kg) after 5 min of reperfusion.

NIRS measurements of oxyhemoglobin, deoxyhemoglobin, and cytochrome oxidase. NIRS measurements were made using a NIRO-500 monitor (Hamamatsu, Kyoto, Japan). This technique has been used for measurements of cerebral oxygenation for many years (13, 14); however, its use in renal physiology has been limited. Near-infrared transmission spectroscopy through tissues up to 10 cm is possible because of the relative transparency of biological tissues to near-infrared light (700-1000 nm). Absorption due to oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and cytochrome oxidase (cyt aa3) can be quantified using a modified form of the Beer-Lambert law, which is applicable to a homogenous scattering medium in which absorption changes are linearly related to concentration, and which may be expressed as: Equation where OD is the optical density, Σ is the extinction coefficient of the chromophore(mM-1cm-1), c is concentration (mM), L is the difference between the points where light enters and leaves the tissue(cm), B is a path-length factor that takes account of the scattering of light in the tissue (which causes the optical path length to be greater than L), and G is a factor related to the geometry of the tissue. If L, B, and G remain constant, then changes in chromophore concentration can be obtained from the expression:Equation

Concentration changes in HbO2, Hb, and cyt aa3 across the kidney were calculated by linear summation of absorption changes (measured in absorbance units) at 772 nm, 830 nm, 842 nm, and 909 nm, and multiplied by factors obtained by spectroscopic and biochemical measurements of a wide range of hemoglobin concentrations in a scattering medium (15). The multiplier coefficients used in the estimation of concentration changes of cyt aa3 were obtained from experimental procedures involving fluorocarbon transfusion studies in anesthetized animals(16). The multiplication factors are shown in Table 2. One indication of the reliability of these algorithms is the independence of the cyt aa3 response compared with HbO2, in that the changes in cyt aa3 oxidation do not mirror the changes in HbO2.

The optical path length (B) has been determined for the cerebellum by time of flight measurements of an ultra-short pulse of light across the head (17). However, as far as we know, there have been no estimates of B in kidneys. An estimate of B was not made in the present study. Nevertheless, since we have compared two groups of kidneys subjected to the same treatments, and the optical path length (and differential path length factor) has been shown to be constant within the near-infrared region despite gross changes in oxygenation and perfusion before and after death (14, 17), the relative changes in chromophore concentration determined in this study are directly comparable between groups.

The near-infrared probes were placed on either side of the kidney at a distance (L) of 1 cm and immobilized using a retort stand, to give an indication of hemoglobin oxygenation across both the cortex and medulla. Baseline measurements of HbO2, Hb, and cyt aa3 were made every 5 sec in situ for 5 min before reperfusion (i.e., before removal of the clamps but after completion of the anastomoses). Changes in these parameters were then measured during 35 min of reperfusion. These values were used to calculate the hemoglobin oxygenation index (hemoglobin oxygenation index = [HbO2] - [Hb]).

Histology. Slices were taken from all kidneys soon after termination of reperfusion and fixed in 10% formal saline for processing to paraffin wax. Sections were stained with hematoxylin and eosin for light microscopical analysis. Ischemia-reperfusion damage was assessed using several histological features, including edema, congestion, hemorrhage, inflammation, and necrosis in the cortex, corticomedullary junction, and medulla.

Statistics. In Figures 2 and 3, data are presented as mean ± SEM. Statistical significance within and between groups was tested using an unpaired Student's t test. InFigure 4, data are presented as mean ± range. Statistical differences in assessments of histology between groups were tested using Kruskal-Wallis one-way analysis of variance to test the medians of all groups for equality, followed by the Mann-Whitney U test to compare each group against group 1 controls (unstored transplanted).

RESULTS

Figure 1, A and B, shows representative time course plots of changes in concentration of HbO2, Hb, and cyt aa3 in group 1 (unstored, untreated; Fig. 1A) and group 2(unstored, frusemide treated; Fig. 1B) transplanted kidneys during 35 min of reperfusion. In untreated kidneys, reperfusion resulted in rapid increases in both [HbO2] and [Hb] and a large reduction in cyt aa3 within 2 min. In this case, there was a pronounced hyperemia in the first 2 min, followed by a modest hemoglobin desaturation that persisted throughout the monitoring period. Reperfusion was continued for 30 min in 5/5 group 1 and 5/5 group 2 animals, before termination by lethal infusion of sodium pentobarbitone. Sodium pentobarbitone infusion resulted in falls in both [HbO2] and [Hb] and no apparent change in the oxidation state of cyt aa3. In the group 2 kidney, frusemide (0.6 mg/kg) was rapidly infused intravenously to the recipient after 5 min of reperfusion, at which point the major hemodynamic changes occurring during initial reperfusion had taken place and a relatively stable baseline could be monitored. Frusemide infusion resulted in an increase in [HbO2] and a decrease in [Hb], but no apparent changes in oxidation of cyt aa3. Lethal infusion of sodium pentobarbitone caused a large fall in [HbO2], a large rise in [Hb], and a large oxidation of cyt aa3.

Figure 2 shows the mean and SEM changes in total hemoglobin concentration ([HbT]) and oxygenation index ([HbO2] - [Hb]) in groups 1 and 2, which took place from a zero point 2 min before frusemide infusion (i.e., after 3 min of reperfusion). There were no significant differences in [HbT] between the two groups. In group 2 kidneys, frusemide infusion resulted in a significant increase in oxygenation index by 5 min(P<0.05) compared with group 1 kidneys, which did not receive frusemide. The oxygenation index remained significantly higher in frusemide-treated kidneys for the duration of reperfusion.

In kidneys stored in the mannitol-based solution (groups 3 and 4,Fig. 3), frusemide infusion resulted in a significant increase in the hemoglobin oxygenation index compared with untreated kidneys(P<0.05), but this was delayed by 15 min compared with group 1(Figs. 2 and 3). As in unstored autografted kidneys, there were no significant differences in [HbT] (Fig. 3).

In groups 5 and 6 (stored in the PEG-based solution), there were no significant differences in either the oxygenation index or [HbT] between the two groups during the 30 min of reperfusion monitored (Fig. 4).

Termination of reperfusion by lethal infusion of sodium pentobarbitone (200 mg/kg) resulted in falls in both [HbT] and the oxygenation index in all groups as expected. The fall in oxygenation index was significantly greater(P<0.005) in group 2 than in any other group, indicating of greater hemoglobin saturation in this group immediately before pentobarbitone infusion.

There was a tendency for cyt aa3 to become reduced during reperfusion in all groups (Figs. 5-7), but only in groups 2 (unstored, treated with frusemide) and 4 (stored for 72 hr in the mannitol-based solution and treated with frusemide) did the changes achieve significance (P<0.05). However, although group 2 cyt aa3 became significantly reduced compared with time 0, it was not significantly different from untreated controls (group 1) at any time point. It was notable that changes in cyt aa3 oxidation in unstored kidneys were greater than those occurring in stored kidneys, and changes in the oxidation state of cyt aa3 in kidneys stored in the PEG-based solution (groups 5 and 6,Fig. 7) were minimal. Upon termination of reperfusion, by lethal infusion of sodium pentobarbitone, there were significant increases in oxidation of cyt aa3 in groups 2 and 4 only (P<0.05), but a tendency toward oxidation in all groups.

There was minimal to mild medullary congestion in both unstored groups of kidneys, and this was exacerbated with storage regardless of treatment(Fig. 8). However, there was a tendency for congestion to be more severe in kidneys stored in the PEG-based solution compared with the mannitol-based solution. Frusemide infusion significantly reduced cortical edema in unstored kidneys (P<0.05), and had a tendency to do this in stored kidneys; however, the changes here did not achieve significance. As would be expected, there was no inflammatory infiltrate, no frank necrosis, no hemorrhage, and very little cortical congestion in any specimen.

DISCUSSION

In the isolated perfused kidney, inhibition of the Na-K-2Cl cotransporter in the mTAT by frusemide causes oxidation of cyt aa3, which has been detected using dual wavelength white light spectroscopy(7). Inhibition of active reabsorption not only increases viability of mTAT cells, but, by increasing oxygen tension in the corticomedullary junction, also has a positive effect on survival of the pars recta of the proximal tubule (5). However, when Atkins and Lankford (18) measured the oxidation of cyt aa3 in vivo, they found that, surprisingly, frusemide infusion caused a reduction in cyt aa3 in the outer medulla. Using laser Doppler flowmetry, they demonstrated that the reduction in cyt aa3 correlated with decreased blood flow through the outer medulla (18).

Frusemide has several documented properties, in addition to inhibiting active reabsorption in the mTAT: it (1) acts as a vasodilator, probably by increasing the rate of breakdown of arachidonic acids for eicosanoid synthesis(19-21); (2) inhibits tubuloglomerular feedback (22); (3) increases plasma renin activity(19); (4) is a weak inhibitor of carbonic anhydrase in the cortical proximal tubules (23); and (5) has a direct inhibitory effect on electron transport in the mitochondrial respiratory chains (24).

These various properties of frusemide are likely to have diverse effects on renal physiology, depending on the timing and dosage of frusemide infusion, and the model studied. Because frusemide is routinely administered after renal transplantation, its precise effects in this setting deserve closer scrutiny.

In the present study, the effect of frusemide on cyt aa3 oxidation in unstored transplanted kidneys appeared to be analogous to the study of Atkins and Lankford (18). However, the reduction in cyt aa3 upon frusemide infusion was coupled with an increase in hemoglobin oxygenation (Fig. 2). This seemingly paradoxical effect could be explained by the following mechanisms: (1) frusemide infusion stimulated cortical vasodilation and a corresponding decrease in medullary blood flow (18, 25); (2) the expected oxidation of cyt aa3, as a consequence of inhibition of the Na-K-2Cl cotransporter in the mTAT by frusemide, was overshadowed by the fall in medullary blood flow, leading to the observed reduction in cyt aa3; and (3) cortical vasodilation, coupled with inhibition of active sodium reabsorption in the mTAT, and possibly of mitochondrial electron transfer in the cortex(24), led to diminished cortical oxygen extraction, hence the observed increase in the hemoglobin oxygenation index.

A prime factor in the preservation of cellular integrity during organ storage for transplantation is the selection of osmotically active impermeant solutes in the preservation solution (26). The beneficial effect of the University of Wisconsin solution seems to be a result of amelioration of cell swelling and edema during cold storage by the impermeants raffinose and lactobionate (26). The effectiveness of an impermeant species appears to correlate with its molecular weight(26, 27). Mannitol has been used in Marshall's hypertonic citrate solution with good clinical results for many years, but its efficacy during prolonged hypothermia may to be limited by its relatively low molecular weight, allowing it to gradually permeate cells(27). In contrast, impermeant PEGs of medium to high molecular weight have been shown to be highly effective at ameliorating cellular swelling by means of a combination of colloidal osmotic pressure and unexplained interactions with the cell membrane (28).

The effect of frusemide on cellular metabolism in organs stored prior to transplantation has not been previously studied. Therefore, we determined the effects of frusemide on cyt aa3 oxidation and renal hemodynamics in kidneys stored for 72 hr in solutions containing mannitol or PEG prior to transplantation, using NIRS. Data obtained may help to explain the relationship between cellular swelling, and ion pump and respiratory chain function soon after reperfusion. We have previously shown that respiratory chain function during the first few minutes of reperfusion provides a good indication of long-term renal viability (12). The responsiveness of the respiratory chain to frusemide infusion, over a period commensurate with its physiological duration of action (about 30 min), may therefore be considered to reflect not only early renal function, but also, by extrapolation, long-term renal viability.

Infusion of frusemide after 72 hr of storage in the mannitol-based solution resulted in a significant (P<0.05) increase in oxygenation index, but this was significantly delayed compared with group 2 (unstored) kidneys(P<0.005; Fig. 3). The increase in oxygenation index was synchronous with a significant reduction in cyt aa3 (P<0.05; Fig. 6), suggesting that, as in unstored kidneys, frusemide infusion stimulated cortical vasodilation.

The delay in response of these parameters to frusemide, in comparison with unstored transplanted kidneys, may be related to the medullary perfusion defect. We have previously shown that medullary congestion begins to clear 30-60 min after reperfusion in the renal autograft model used here(11), and that its clearance is associated with a significant rise in serum levels of 6-keto-prostaglandin F1a (a breakdown product of the vasodilator prostacyclin), but not with an increase in oxygenation index (11, 29). Prostacyclin modulates cyclic AMP levels in both endothelial cells and mTAT cells, leading to decreases in, respectively, endothelial cell permeability and sodium reabsorption (30, 31). This should abate the process of hemoconcentration in the peritubular capillaries and ameliorate swelling of the mTAT.

Thus, it is possible that stimulation of eicosanoid synthesis by frusemide could facilitate clearance of medullary congestion in an energy-dependent manner, leading to a reduction in cyt aa3, despite increased blood flow. This premise is supported to some extent by histological analysis of the severity of medullary congestion: there was a tendency toward less congestion in kidneys stored in mannitol prior to frusemide treatment, compared with untreated, stored kidneys (groups 3 and 4; Fig. 8). Although this did not attain significance, the numbers studied were small, and clearance of medullary congestion was in any case at an early stage.

In contrast, there was a surprisingly poor response to frusemide infusion in the kidneys stored in the PEG-based solution, in terms of both the oxygenation index and cyt aa3 oxidation, and of histology(Figs. 4 and 7). There are several possible explanations for this.

First, PEG is nonideal in terms of its colloidal osmotic pressure. Inclusion of PEG in the preservation solution at an equivalent concentration(in mmol/L) to mannitol resulted in a measured osmolarity of 700 mOsm/L, as compared with 485 mOsm/L with mannitol (unpublished results). Accordingly, the concentration of PEG added to the solution was lowered to produce a final measured osmolarity of 485 mOsm/L. It is possible that this maneuver diminished the efficacy of PEG in ameliorating cell swelling.

Second, Marsh and co-workers used high molecular weight PEGs, as their effect seemed to derive partly from unexplained interactions with cell membranes, rather than being simply a function of their colloidal osmotic pressure (28). In the present experiments, relatively low molecular weight PEGs were used (average m.w. 600), as this is the lowest weight compatible with impermeability (32), and should produce the highest colloidal osmotic pressure, allowing comparison with mannitol. It is possible that lower molecular weight PEGs do not interact with cell membranes in the same fashion as their larger cousins.

Third, PEG may actually be toxic to renal cells. Although PEG can be taken up by renal lysosomes (33), and this may eventually be deleterious, the process occurs only slowly, and is unlikely to have occurred during the preservation period. However, Kopolovic et al.(34) have shown in the isolated perfused kidney that PEG can cause ultrastructural damage to different sections of the tubule, depending on their degree of water permeability. PEG was found to cause particular damage to the mTAT-a pertinent finding in view of the specificity of frusemide for the mTAT.

Finally, the relatively high efficacy of mannitol compared with PEG may be related to other reported properties of mannitol: in addition to acting as an impermeant colloid, mannitol can stimulate vasodilation(35) and act as a scavenger of reactive oxygen metabolites (36).

We conclude that the beneficial effects of frusemide on renal viability after storage and transplantation depend in part on the degree of cellular damage in the mTAT. As hemoglobin oxygenation in normally functioning kidneys is maintained in an equilibrium, whereby increases in blood flow (and therefore oxygenation) stimulate greater sodium reabsorption (and therefore an increase in oxygen consumption), it follows that increases in renal hemoglobin oxygenation cannot occur unless sodium reabsorption is inhibited. Thus, despite the reduction of cyt aa3 in unstored kidneys, or those stored in the mannitol-based solution, the increase in hemoglobin oxygenation must have reflected inhibition of the Na-K-2Cl cotransporter by frusemide. The fact that kidneys stored in the PEG-based solution did not respond to frusemide suggests that cellular damage was too progressed to allow a physiological response to frusemide.

The reduction of cyt aa3 may have been a consequence of decreased medullary blood flow, but, in the case of stored kidneys at least, it may also reflect clearance of medullary congestion via a frusemide-stimulated, prostacyclin-mediated mechanism. As medullary congestion causes secondary ischemia, and may lead ultimately to necrosis of large parts of the corticomedullary junction, the beneficial effects of frusemide following renal storage and transplantation may be exerted partly through this mechanism.

F1-6
Figure 1:
Representative time course plots of changes in HbO2, Hb, and cyt aa3 in a group 1 kidney (A, unstored untreated) and a group 2 kidnet (B, unstored + frusemide) during 35 min of reperfusion. Reperfusion of the autografted kidney took place at time 0. Reperfusion was terminated after 35 min by lethal sodium pentobarbitone infusion, which inhibits complex 1 of the respiratory chain.
F2-6
Figure 2:
Mean and SEM changes (n=5) in [HbT] and the hemoglobin oxygenation index (HbO2 - Hb) upon frusemide infusion after 5 min of reperfusion in group 1 (▴, unstored, no frusemide) and group 2 (▪, unstored + frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone. * P<0.05 between groups at equivalent time points.
F3-6
Figure 3:
Mean and SEM changes (n=5) in [HbT] and the hemoglobin oxygenation index (HbO2 - Hb) upon frusemide infusion after 5 min of reperfusion in group 3 (▴, stored for 72 hr in HCA-mannitol; untreated) and group 4 (▪, stored for 72 hr in HCA-mannitol; + frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone. ** P<0.005, * P<0.05 between groups at equivalent time points.
F4-6
Figure 4:
Mean and SEM changes (n=5) in [HbT] and the hemoglobin oxygenation index (HbO2 - Hb) upon frusemide infusion after 5 min of reperfusion in group 5 (▴, stored for 72 hr in HCA-PEG; untreated) and group 6 (▪, stored for 72 hr in HCA-PEG; + frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone.
F5-6
Figure 5:
Mean and SEM changes (n=5) in cyt aa3 oxidation upon frusemide infusion after 5 min of reperfusion in group 1 (▴, unstored, no frusemide) and group 2 (▪, unstored + frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone.+ P<0.05 within group 2 from time 0 min;× P<0.05 within group 2 from time 30 min.
F6-6
Figure 6:
Mean and SEM changes (n=5) in cyt aa3 oxidation upon frusemide infusion after 5 min of reperfusion in group 3 (▴, stored for 72 hr in HCA-mannitol; untreated) and group 4 (▪, stored for 72 hr in HCA-mannitol; + frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone. * P<0.05 between groups at equivalent time points; + P<0.05 within group 4 from time 0 min; × P<0.05 within group 4 from time 30 min.
F7-6
Figure 7:
Mean and SEM changes (n=5) in cyt aa3 oxidation upon frusemide infusion after 5 min of reperfusion in group 5 (▴, stored for 72 hr in HCA-PEG; untreated) and group 6 (▪, stored for 72 hr in HCA-PEG;+ frusemide). Reperfusion was terminated at 30 min by lethal infusion of sodium pentobarbitone.
F8-6
Figure 8:
Degree of medullary congestion and edema (as assessed by light microscopy) in the renal cortex after termination of reperfusion. Results are presented as mean and range scores (n=5) in groups 1 (unstored), 2 (unstored + frusemide), 3 (stored for 72 hr in HCA-mannitol), 4 (stored for 72 hr in HCA-mannitol + frusemide), 5 (stored for 72 hr in HCA-PEG), and 6 (stored for 72 hr in HCA-PEG + frusemide). Tissue was scored blind by an independent pathologist on a scale of 0 (absent), 1 (minimal), 2 (mild), 3 (moderate), and 4 (severe). * P<0.05, Mann Whitney U test compared with group 1.

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