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A Biphasic Response to Nitric Oxide Donation in an Ex Vivo Model of Donation After Cardiac Death Renal Transplantation
Journal of Surgical Research 175, 316–321 (2012) doi:10.1016/j.jss.2011.03.073
A Biphasic Response to Nitric Oxide Donation in an Ex Vivo Model of Donation After Cardiac Death Renal Transplantation Phillip J. Yates, B.Sc., M.D.,1 Sarah A. Hosgood, B.Sc., and Michael L. Nicholson, M.D., D.Sc Department of Infection, Immunity and Inflammation, Transplant Group, University of Leicester, Leicester General Hospital, Leicester, United Kingdom Originally submitted November 18, 2010; accepted for publication March 28, 2011
Background. Donation after cardiac death (DCD) donors are vital to maximize the organ donor pool. Reperfusion injury (RI) is an important sequela in DCD organs due to warm and cold ischemia. RI manifests clinically as a high incidence of delayed graft function (DGF) and primary non-function (PNF) compared with donation after brain death organs. The importance of nitric oxide (NO) in the generation of reperfusion injury is pivotal. Methods. Using an ex vivo porcine model of kidney transplantation the effects of reperfusion with and without NO supplementation on initial renal blood flow and function were compared. Real-time hemodynamic measurements were recorded and biochemical samples taken at set time-points. Molecular markers of reperfusion injury were also measured. Sodium nitroprusside was chosen as the NO donor. Results. Results showed that NO donation initially improved renal blood flow significantly over controls; at the end of reperfusion this benefit was lost. In addition, there was an improvement in creatinine clearance, fractional excretion of sodium and renal oxygen consumption. There were observed to be higher levels of urinary nitrite/nitrate excretion, but no difference in isoprostane levels. Conclusion. This study represents a good model for the initial reperfusion period in large animal renal transplantation. The improvement in renal blood flow observed in the NO supplemented groups represents NO mediated vasodilatation. Later in reperfusion, accumulation of nitrogenous free radicals impairs renal blood flow. Clinically, NO supplementation during initial reperfusion of DCD kidneys improves renal blood
1 To whom correspondence and reprint requests should be addressed at Department of Infection, Immunity, and Inflammation, Transplant Group, University of Leicester, Leicester General Hospital, Gwendolen Road, Leicester LE2 8JB, UK. E-mail: [email protected].
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
flow but should be considered with caution due to potential deleterious effects of nitrogenous compound accumulation. Ó 2012 Elsevier Inc. All rights reserved. Key Words: nitric oxide; transplantation; kidney; donation after cardiac death; reperfusion injury.
INTRODUCTION
Donation after cardiac death (DCD) kidneys are an important source of donor organs for transplantation. Unfortunately, DCD kidneys demonstrate increased levels of delayed graft function (DGF), 40%–85% and primary non-function (PNF), 9%–21% [1–4]. Although the pathophysiologic processes underlying this observation are incompletely understood, they are undoubtedly dependent upon the warm ischemic, hypothermic, and reperfusion injury [5–7]. Initial ischemic injury and hypothermic damage during transportation are unavoidable in DCD organs, however, reperfusion injury (RI) is potentially ameliorable and is thus of vital importance. Nitric oxide (NO) is an important monovalent gas that under normal physiologic circumstances and during reperfusion injury regulates vascular tone, inhibits platelet aggregation, and leukocyte adhesion, scavenges reactive oxygen species, and maintains vascular homeostasis [8, 9]. NO is synthesized by the nitric oxide synthase (NOS) group of enzymes. Of particular importance in reperfusion injury are endogenous NOS (eNOS), which also produces NO constitutively, and inducible NOS (iNOS), which produces NO during tissue injury. Importantly, NOS also synthesizes deleterious superoxide especially following bouts of prolonged ischemia [10, 11]. The aim of this study was to identify what immediate effect NO donation would have on reperfusion and
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oxidative stress, and subsequently to assess any functional changes over the reperfusion period. METHODS An ex vivo porcine model of DCD transplantation utilising cardiopulmonary bypass technology, as previously described, was used [12]. This model allows the real time measurement of functional parameters during initial reperfusion, as well as the collection of tissue and fluid samples for biochemical and histologic analysis. Furthermore, the urinary levels of NO and 8-isoprostane were measured to assess tissue damage and oxidative stress.
Kidney Retrieval and Blood Collection Large white pigs (60–70 kg) were killed by electrocution followed by exsanguination. Approximately 1 L of blood was collected into a sterile receptacle containing 25,000 units of heparin (Multiparin; CP Pharmaceuticals, Wrexham, UK). The blood was then transferred into CPDA-1 blood bags (Baxter Healthcare, Thetford, UK) and stored at 4 C. Leukocyte depletion was performed immediately prior to reperfusion by passing whole blood through a LeukoGuard RS white cell filter (Pall Medical, Portsmouth, UK). The kidneys were retrieved after 25 min of in situ warm ischemia (WI) and then flushed with 250 mL hyperosmolar citrate solution (Soltran; Baxter healthcare, Norfolk, UK) at a temperature of 4 C and a hydrostatic pressure of 100 cm H2O. The organs were placed on ice in hyperosmolar citrate solution, transported to the perfusion laboratory, and stored for 18 h.
Reperfusion Following hypothermic preservation, kidneys were prepared for ex vivo reperfusion. The renal artery, vein and ureter were cannulated and kidneys flushed with 100 mL of Ringer’s lactate solution (B. Braun, Sheffield, UK) at 4 C. Organs were then reperfused using the isolated organ preservation system (IOPS). The kidneys were reperfused for 3 h at a temperature of 37 C and a mean arterial pressure of 85 mmHg. The IOPS has been described previously. Briefly, a priming solution and nutrient supplements were added to the IOPS and kidneys reperfused with autologous blood under simulated physiologic conditions. Blood temperature and oxygenation is regulated by an in-line heat exchanger (GD120; Grant, Cambridge, UK) and membrane oxygenator (Minimax Plus membrane oxygenator; Medtronic, Tolochenaz, Switzerland). Creatinine (Sigma-Aldrich, Steinheim, Germany) was added to the perfusate to achieve an initial circulating concentration of 1000 mmol/L.
Groups Porcine kidneys were randomized into two groups. Kidneys in both groups received 25 min of WI. Kidneys in the control group were reperfused with whole blood only. Experimental group organs were reperfused with whole blood supplemented with Sodium Nitroprusside (SNP). SNP powder (Sigma-Aldrich, Steinheim, Germany) was reconstituted at a concentration of 100 mg/mL of 5% glucose solution (Baxter Healthcare, Thetford, UK). The SNP was added to the IOPS as a continuous infusion at a rate of 1.5 mg/h for the full duration of the reperfusion period; n ¼ 6 in both groups. Real-time measurement of renal blood flow (RBF) and intra-renal resistance (IRR) was performed. Urine output was measured at hourly intervals following reperfusion. Biochemical analysis of serum and urine samples was carried out at hourly intervals during reperfusion. Creatinine clearance (urinary creatinine 3 urinary volume/plasma creatinine) and fractional excretion of sodium [(urinary sodium 3 urine volume)/(glomerular filtration rate 3 plasma sodium) 3 100)] were calculated.
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Hourly arterial and venous blood gas analysis was performed to assess PaO2, PvO2 and acid-base homeostasis. Oxygen consumption [(PaO2 – venous PvO2) 3 flow rate/weight] was calculated. The hemoglobin, hematocrit, and leukocyte counts were measured before and after reperfusion.
Histology Wedge biopsies were taken at various time points. Biopsies were fixed in 10% formal saline, dehydrated, and embedded in paraffin wax. Sections of 4 mm were cut and stained with hematoxylin and eosin for evaluation under light microscopy. Sections were scored over five fields, assessing changes in four morphological variables; tubular dilation, tubular debris, vacuolation, and interstitial infiltration. Three trained assessors blinded to the experimental group scored samples from 0 to 3 according to the level of damage; 0 ¼ normal, 1 ¼ mild, 2 ¼ moderate, and 3 ¼ severe morphological changes.
Total Urinary Nitrite/Nitrate (NO2/NO3) Quantification Urine samples, taken at 60 and 180 min post-reperfusion were immediately snap-frozen in liquid nitrogen and stored at 80 C. Urine levels of nitrite/nitrate (NO2/NO3) were quantified using a total NO2/NO3 assay kit (Assay Designs, Ann Arbor, MI) according to the manufacturer’s instructions. This assay relies on the conversion of nitric oxide to nitrate and the subsequent conversion of nitrates to nitrite by the enzyme nitrate reductase. Nitrite is detected colorimetrically at 540 nm as an azodye product of the Griess reaction.
Urine 8-Isoprostane Quantification Levels of urine 8-isoprostane were determined by enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical Co., Ann Arbor, MI). Urine samples were centrifuged at 10,000 g for 2 min and the supernatant taken for analysis. Samples were diluted 10-fold prior to analysis. The sample and standards were added in duplicate to the ELISA plate together with an 8-isoprostane-acetylcholinesterase (AChE) conjugate and incubated for 18 h at 4 C. The plate was then washed and developed by the addition of the substrate to AChE. The plate was read at 405 nm after colour development for 90 min.
Statistical Analysis Values are presented as mean 6 SD. Continuous variables were plotted against time and the area under the curve (AUC) for individual perfusion experiments calculated using Excel (Microsoft, Reading, UK) and GraphPad Prism (GraphPad Software, San Diego, CA) software. Mean AUC values and individual values were compared using the Mann-Whitney test (GraphPad InStat ver. 3.00 for Windows 95, GraphPad Software, San Diego CA). P < 0.05 was considered as statically significant.
RESULTS Hemodynamics
In both groups, initial renal blood flow (RBF) was low. After 10 min of reperfusion RBF improved in both groups, and continued for the remainder of the reperfusion period. RBF in the SNP group improved throughout the first 30 min of reperfusion to a greater degree than in the control group. Beyond 30 min of reperfusion,
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the improvement in RBF in the SNP group was attenuated, whilst there was a persistent improvement in the control group. The divergence in RBF between groups became statistically significant at 30 min of reperfusion. After 120 min of reperfusion the RBF between groups began to converge and lost statistical significance. These data are summarized in Figure 1. AUC RBF for the 5–30 and 30–180 min of reperfusion were analyzed separately. AUC RBF 5–30 min of reperfusion were 705 6 190, 1072 6 383 in the control and SNP groups, respectively (Mann-Whitney test P ¼ 0.051). AUC 30–180 min of reperfusion were 4727 6 2327 and 8085 6 970 in the control and SNP groups respectively (Mann-Whitney test P ¼ 0.035). During the initial 30 min of perfusion AUC RBF in the SNP group, though higher, did not reach statistical significance compared with the control group. The AUC RBF was, however, significantly higher throughout the remainder of the perfusion period in the SNP group. These data are summarized in Figure 1. The gradient of the curves for RBF 5–30 min of reperfusion were 2.0 and 11.8 in the control and SNP groups, respectively. For 30–180 min of reperfusion the gradients were 3.5 and 1.1 in the control and SNP groups, respectively. Intra-renal resistance demonstrated reciprocal characteristics. During the initial 30 min of reperfusion IRR was higher in both groups than during the remainder of reperfusion. The IRR in the SNP group fell markedly at 30 min and remained at a low level for the remainder of reperfusion. IRR was analyzed separately during the period 5–30 and 30–180 min of reperfusion. AUC IRR for 5–30 min post-reperfusion was 43 6 16, 25 6 13 in the control and SNP groups, respectively (Mann-Whitney test P ¼ 0.035). AUC IRR for 30–180 min post-reperfusion was 192 6 149 and 66 6 6 in the control and SNP groups respectively (Mann-Whitney test P ¼ 0.035). During the entire period of reperfusion the IRR was statistically higher in the control group than the SNP group.
Filtration Function
At 60 min post-reperfusion, creatinine clearance (CrCl) was significantly lower in the control group than the SNP group. However, by 120 min of reperfusion, CrCl in the SNP group had fallen to the same level as in the control group and remained at this level for the remainder of the reperfusion period. These data are summarized in Table 1. Tubular Function
The fractional excretion of sodium (FENa) was lower in the SNP group throughout the reperfusion period. This difference did not, however, reach statistical significance. These data are shown in Table 1. Acid-Base Homeostasis
There was no difference in pre-perfusion pH between the groups. The SNP group was marginally more acidotic than controls but this did not reach statistical significance. These data are shown in Table 1. Oxygen Consumption
There was increased oxygen consumption throughout the reperfusion in all groups. The Oxygen consumption was higher in the SNP group compared wih the control group at both 60 and 180 min of reperfusion, although this was only statistically significant at 60 min reperfusion. Data are summarized in Table 1. Hematology
There was no difference between control and SNP group leukocyte, platelet, or hematocrit levels either before or after the reperfusion period. Histology
Histologic assessment did not demonstrate any significant difference in damage between groups pre- or post-reperfusion. These data are shown in Table 2. Total Urinary NO2/NO3 Quantification
Total NO2/NO3 levels were significantly different between control and SNP groups at both 60 and 180 min post-reperfusion, with P ¼ 0.045 and P¼ 0.014, respectively. These data are shown in Figure 2. 8-Isoprostane Quantification
FIG. 1. Mean RBF 6 SD in Control and SNP groups during 180 min of reperfusion. *P < 0.05.
Urinary levels of 8-isoprostane were measured at 180 min post-reperfusion. Although the levels of 8isoprostane were lower in the SNP group, this did
YATES, HOSGOOD, AND NICHOLSON: NO DONATION IN DCD TRANSPLANTATION
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TABLE 1 Difference in Creatinine Clearance, Fractional Excretion of Sodium, Acid-Base Balance, and Oxygen Consumption Between the Control and SNP Groups at Different Time-Points Group Creatinine Clearance (mL/min/100 mg) 60 min 120 min 180 min Fractional excretion of sodium (%) 60 min 120 min 180 min pH Pre 60 min 180 min Oxygen consumption (kPa.mL/min/100 g) 60 min 180 min
Control
SNP
P value
0.5 (0.2) 0.4 (0.2) 0.5 (0.4)
2.2 (1.3) 0.7 (0.6) 0.4 (0.4)
0.013* 0.927 0.432
121 (61) 82 (44) 84 (53)
66 (18) 51 (20) 48 (25)
0.066 0.235 0.133
7.43 (0.03) 7.32 (0.04) 7.3 (0.08)
7.5 (0.07) 7.26 (0.05) 7.28 (0.04)
0.074 0.074 0.775
19.3 (6.8) 28 (13.9)
44.1 (11.7) 40.9 (16.9)
0.003* 0.225
All values are mean (SD). * P < 0.05 (Mann-Whitney test).
not reach statistical significance (P ¼ 0.12). These data are shown in Figure 3.
DISCUSSION
DCD kidneys are important in expanding the organ donor pool. For this reason, strategies that improve their initial function and ameliorate the degree of ischemic and reperfusion injury are vital. This model used a combination of 25 min warm ischemia and 18 h cold storage to replicate the injury observed in uncontrolled human DCD kidneys. These ischemic times have previously been shown to impart a significant degree of injury in this model [12].
FIG. 2. Histogram showing total urinary NO2/NO3 levels 6 SD at 180 min reperfusion in the control and SNP group.
NO is a diatomic free radical, synthesized from Larginine by a group of enzymes called nitric oxide synthases. NO is produced constitutively by eNOS and, during RI, by iNOS. NOS not only catalyzes the formation of NO but also superoxide that reacts with NO to produce injurious peroxynitrite [13, 14]. The renal expression of eNOS and iNOS differs in response to ischemia. The expression of iNOS may increase 4-fold during warm ischemia, 8-fold during the first 30 min of reperfusion, and up to a 16-fold at 60 min post-reperfusion [15]. Conversely, eNOS expression is reduced by half during ischemia and remains suppressed for up to 3 h post-reperfusion, iNOS expression may increase 4-fold during warm ischemia, and 8-fold during the first 30 min of reperfusion up to a 16-fold maximal increase at 60 min postreperfusion [15]. There are many possible NO donors that could have been used in this study. SNP was chosen as it is easy to administer, has rapid action, is water soluble, and has limited toxicity [16]. Furthermore, SNP is a well established NO donor in clinical usage in humans, is often the benchmark to which other NO donors are compared, and there is a wealth of published literature demonstrating its potent NO donor action [16]. SNP has previously been used in this model, but because of its short half-life, a continuous infusion was necessary.
TABLE 2 Histologic Differences Between Pre-reperfusion and Post-Reperfusion Renal Biopsies Control
Tubular dilatation Tubular debris Tubular cell Vacuolation Interstitial infiltration
SNP
Pre
Post
Pre
Post
0.9 (0.7) 1.4 (0.5) 0.5 (0.8) 1.3 (0.5)
1.6 (0.6) 1.1 (0.7) 1.1 (1.1) 1.3 (0.5)
1.4 (0.8) 1.8 (0.6) 0.4 (0.5) 0.6 (0.5)
1.1 (0.9) 1.6 (0.6) 0.3 (0.6) 0.8 (0.8)
Values are means (SD) of a semiquantitative scoring system (0 ¼ normal, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe morphological changes).
FIG. 3. Histogram showing urinary 8-isoprostane levels 6 SD at 180 min reperfusion in the control and SNP group.
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There is a clear improvement in RBF in the SNP group between 5 and 30 min of reperfusion, this is associated with improved creatinine clearance, FENa, and oxygen consumption. However, from 30–180 min of reperfusion there is noted to be a convergence of the RBF curves, noted by a plateau in the SNP curve, and a continuing increased gradient in the control curve. This finding is associated with a loss of improvement in functional parameters, other than oxygen consumption, throughout the remainder of the reperfusion period. The improvement in RBF and function seen between 5 and 30 min reperfusion in the SNP group is explained by direct NO vasodilatation. However, the fall in RBF between 30 and 180 min requires further explanation. During ischemia and reperfusion, there is a progressive loss of L-arginine and the cofactors required for NO synthesis, and a progressive up-regulation of iNOS expression [10, 11, 15]. Thus, following reperfusion there is an increase in superoxide production that reacts with both endogenous and exogenous NO to form peroxynitrite [17]. High concentrations of peroxynitrite and superoxide have been shown to reduce renal blood flow, CrCl, and FENa [18]. Histologic evaluation of pre-perfusion and postperfusion biopsies did not demonstrate any statistical difference between groups. This finding is related to the degree of damage imparted by the duration of the warm ischemic time, and that after three h of reperfusion there is little opportunity for the tissues to demonstrate any histological improvement/deterioration. This suggests that staining of biopsies for specific markers of injury, in addition to immunohistochemical and in-situ hybridization techniques, warrant consideration for future study in this model. It is well established that NO is produced by the vascular endothelium both constitutively and in response to injury. It has also been shown experimentally that the renal tubule and glomerulus have the ability to produce NO in response to injury [19–21]. Measurement of plasma levels of NO2/NO3 would not detect alterations in NO production in the glomerulus or renal tubule in response to injury; thus urinary quantification was chosen as the assay of choice in this study. The utilization of the Griess reaction in this assay ensures that the total NO/NO2/NO3 burden during reperfusion is quantified. The finding that total urinary NO2/NO3 is significantly higher in the SNP group at 60 and 180 min explains the leveling of RBF in the SNP group. 8-Isoprostane, formed by the oxidation of arachidonic acid by reactive oxygen species, is a urinary marker of oxidative stress [22]. At 180 min post-reperfusion, the level of 8-isoprostane expression seen in the SNP group was not significantly lower than in the control group. This is suggestive that although initial flow is much improved, by the end of the reperfusion period the
oxidative stress is approaching the level seen in the control group, mirroring the functional data and demonstrating the effects of the increased NO2/NO3 burden. Our group has previously used SNP supplementation to assess the effects of carbon monoxide during reperfusion using carbon monoxide releasing molecule-3 (CORM-3) [23]. This study used kidneys with shorter WI time and the SNP was administered as a short infusion prior to and during the first hour of reperfusion only. Results in the SNP group showed that initial RBF was significantly higher than the control groups, but this benefit plateaued at 30 min, and converged with the RBF in the control group by 120 min postreperfusion. Despite the methodological differences that preclude direct comparison of this and the present study the effect on RBF of the pre-reperfusion instillation of SNP, coupled with the observed immediate improvement in RBF, does reinforce the hypothesis that NO vasodilatation is responsible for the improvement in flow early during reperfusion. Furthermore, the convergence of RBF latterly during reperfusion reinforced the observations made in the present study. There are a number of limitations with this study. This model of DCD organs used a warm ischemic period of 25 min. The most current published recommendations state that organs with a warm ischemic period of up to 60 min should be considered suitable for transplantation [24, 25]. Thus, extending this model to assess the effects of NO donation in organs with longer periods of warm ischemia would be appropriate and may demonstrate an even stronger benefit for NO donation. The reperfusion period was limited to 3 h; beyond 3 h, normothermic perfusion renal viability is markedly impaired [26]. As an alternative to prolonged ex vivo reperfusion, retrieval of kidneys from live donors followed by autotransplantation after the period of ex vivo perfusion allows assessment of the long-term impact of NO donation. The feasibility of this model has previously been demonstrated by our group [27]. Furthermore, an ex vivo reperfusion/autotransplant model could be compared with an in vivo reperfusion renal ischemia model to demonstrate potential benefits of ex vivo reperfusion and NO donation per se. Only a single NO donor, SNP, was used in this study. SNP was chosen due to its use clinically in humans for the control of hypertension, however, there is a plethora of alternate NO donors with different pharmacologic characteristics that may make them more efficacious in this model and would thus warrant further investigation. This study measured the overall nitrogenous burden by quantification of total urinary NO2/NO3 levels. Clearly, this method does not discriminate between nitrogenous compounds formed due to exogenously or endogenously produced NO. In order to provide this data assessment of eNOS and iNOS expression would be necessary at different time-points
YATES, HOSGOOD, AND NICHOLSON: NO DONATION IN DCD TRANSPLANTATION
post-reperfusion. This would be best achieved by utilizing immunohistochemistry and in-situ hybridization to quantify the presence of NOS isoforms and mRNA. In conclusion, this study demonstrates that the effects of NO supplementation are biphasic in nature, initially beneficial due to vasodilatation, but later deleterious due to oxidative stress and accumulation of toxic NO metabolites. The effects of this are seen functionally by a slight improvement in initial CrCl, FENa, and metabolic requirement, but a fall-back to control levels after the first hour of reperfusion. Further investigations into the importance of NO at different timings during reperfusion are pivotal in fully elucidating its importance in ameliorating RI. The significance of the NO:superoxide axis, generated by post-ischemic iNOS and eNOS, and its relationship with cofactor depletion undoubtedly requires further delineation. This study suggests that there may be a clinical role for NO donation in renal transplantation, certainly during early reperfusion, but that NO donation is not a panacea for RI. REFERENCES 1. Brook NR, White SA, Waller JR, et al. Non-heart beating donor kidneys with delayed graft function have superior graft survival compared with conventional heart-beating donor kidneys that develop delayed graft function. Am J Transplantation 2003;3:614. 2. Gok MA, Buckley PE, Shenton BK, et al. Long-term renal function in kidneys from non-heart-beating donors: A single-center experience. Transplantation 2002;74:664. 3. Kokkinos C, Antcliffe D, Nanidis T, et al. Outcome of kidney transplantation from non-heart-beating versus heart-beating cadaveric donors. Transplantation 2007;83:1193. 4. Nicholson ML, Metcalfe MS, White SA, et al. A comparison of the results of renal transplantation from non-heart-beating, conventional cadaveric, and living donors [erratum appears in Kidney Int 2001;59:821]. Kidney Int 2000;58:2585. 5. Quiroga I, McShane P, Koo DD, et al. Major effects of delayed graft function and cold ischemia time on renal allograft survival. Nephrol Dialysis Transplantation 2006;21:1689. 6. Boom H, Mallat MJ, de Fijter JW, et al. Delayed graft function influences renal function, but not survival. Kidney Int 2000;58:859. 7. Simpkins CE, Montgomery RA, Hawxby AM, et al. Cold ischemia time and allograft outcomes in live donor renal transplantation: Is live donor organ transport feasible? [see comment]. Am J Transplantation 2007;7:99. 8. Khanna A, Cowled PA, Fitridge RA, et al. Nitric oxide and skeletal muscle reperfusion injury: Current controversies (research review). J Surg Res 2005;128:98. 9. Hallstrom S, Gasser H, Neumayer C, et al. S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release. Circulation 2002; 105:3032.
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10. Pinsky DJ, Oz MC, Koga S, et al. Cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway. J Clin Invest 1994;93:2291. 11. Hillinger S, Schoedon G, Walter R, et al. Reduction of lung allograft ischemia/reperfusion injury by tetrahydrobiopterin coenzyme of NOS. Transplantation Proc 1999;31:183. 12. Hosgood SA, Bagul A, Yang B, Nicholson ML. The relative effects of warm and cold ischemic injury in an experimental model of non-heart-beating donor kidneys. Transplantation 2008; 85:88. 13. Cosentino F, Patton S, d0 Uscio LV, et al. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest 1998;101:1530. 14. Xia Y, Roman LJ, Masters BS, et al. Inducible nitric-oxide synthase generates superoxide from the reductase domain. J Biol Chem 1998;273:22635. 15. Li Volti G, Sorrenti V, Acquaviva R, et al. Effect of ischemiareperfusion on renal expression and activity of N(G)-N(G)dimethylarginine dimethylaminohydrolases. Anesthesiology 2008;109:1054. 16. Butler AR, Pearson RJ. NO donors’ applications in biological research. In: Wang PG, Cai TB, Taniguchi N, Eds. Nitric oxide donors: For pharmaceutical and biological applications. Weinheim: Wiley-VCH, 2005:201–25. 17. Ferdinandy P, Schulz R, Ferdinandy P, et al. Nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury and preconditioning. Br J Pharmacol 2003;138:532. 18. Matavelli LC, Kadowitz PJ, Navar LG, et al. Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats. Am J Physiol Renal Physiol 2009;296:F170. 19. Valdivielso JM, Crespo C, Alonso JR, et al. Renal ischemia in the rat stimulates glomerular nitric oxide synthesis. Am J Physiol Regul Integr Comp Physiol 2001;280:R771. 20. Liang M, Knox FG. Production and functional roles of nitric oxide in the proximal tubule. Am J Physiol Regul Integr Comp Physiol 2000;278:R1117. 21. Ortiz PA, Garvin JL, Ortiz PA, et al. Cardiovascular and renal control in NOS-deficient mouse models. Am J Physiol Regulatory Integrative Comp Physiol 2003;284:R628. 22. Wang Z, Ciabattoni G, Creminon C, et al. Immunological characterization of urinary 8-epi-prostaglandin F2 alpha excretion in man. J Pharmacol Exp Ther 1995;275:94. 23. Hosgood SA, Bagul A, Kaushik M, et al. Application of nitric oxide and carbon monoxide in a model of renal preservation. Br J Surg 2008;95:1060. 24. Reich DJ, Mulligan DC, Abt PL, et al. ASTS recommended practice guidelines for controlled donation after cardiac death organ procurement and transplantation. Am J Transplantation 2009; 9:2004. 25. Bernat JL, D0 Alessandro AM, Port FK, et al. Report of a National Conference on donation after cardiac death. Am J Transplantation 2006;6:281. 26. Grosse-Siestrup C, Unger V, Fehrenberg C, et al. A model of isolated autologously hemoperfused porcine slaughterhouse kidneys. Nephron 2002;92:414–21. 27. Hosgood SA, Barlow AD, Yates PJ, et al. A pilot study assessing the feasibility of a short period of normothermic preservation in an experimental model of non heart beating donor kidneys. J Surg Res 2010 (in press).
A Biphasic Response to Nitric Oxide Donation in an Ex Vivo Model of Donation After Cardiac Death Renal Transplantation Phillip J. Yates, B.Sc., M.D.,1 Sarah A. Hosgood, B.Sc., and Michael L. Nicholson, M.D., D.Sc Department of Infection, Immunity and Inflammation, Transplant Group, University of Leicester, Leicester General Hospital, Leicester, United Kingdom Originally submitted November 18, 2010; accepted for publication March 28, 2011
Background. Donation after cardiac death (DCD) donors are vital to maximize the organ donor pool. Reperfusion injury (RI) is an important sequela in DCD organs due to warm and cold ischemia. RI manifests clinically as a high incidence of delayed graft function (DGF) and primary non-function (PNF) compared with donation after brain death organs. The importance of nitric oxide (NO) in the generation of reperfusion injury is pivotal. Methods. Using an ex vivo porcine model of kidney transplantation the effects of reperfusion with and without NO supplementation on initial renal blood flow and function were compared. Real-time hemodynamic measurements were recorded and biochemical samples taken at set time-points. Molecular markers of reperfusion injury were also measured. Sodium nitroprusside was chosen as the NO donor. Results. Results showed that NO donation initially improved renal blood flow significantly over controls; at the end of reperfusion this benefit was lost. In addition, there was an improvement in creatinine clearance, fractional excretion of sodium and renal oxygen consumption. There were observed to be higher levels of urinary nitrite/nitrate excretion, but no difference in isoprostane levels. Conclusion. This study represents a good model for the initial reperfusion period in large animal renal transplantation. The improvement in renal blood flow observed in the NO supplemented groups represents NO mediated vasodilatation. Later in reperfusion, accumulation of nitrogenous free radicals impairs renal blood flow. Clinically, NO supplementation during initial reperfusion of DCD kidneys improves renal blood
1 To whom correspondence and reprint requests should be addressed at Department of Infection, Immunity, and Inflammation, Transplant Group, University of Leicester, Leicester General Hospital, Gwendolen Road, Leicester LE2 8JB, UK. E-mail: [email protected].
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
flow but should be considered with caution due to potential deleterious effects of nitrogenous compound accumulation. Ó 2012 Elsevier Inc. All rights reserved. Key Words: nitric oxide; transplantation; kidney; donation after cardiac death; reperfusion injury.
INTRODUCTION
Donation after cardiac death (DCD) kidneys are an important source of donor organs for transplantation. Unfortunately, DCD kidneys demonstrate increased levels of delayed graft function (DGF), 40%–85% and primary non-function (PNF), 9%–21% [1–4]. Although the pathophysiologic processes underlying this observation are incompletely understood, they are undoubtedly dependent upon the warm ischemic, hypothermic, and reperfusion injury [5–7]. Initial ischemic injury and hypothermic damage during transportation are unavoidable in DCD organs, however, reperfusion injury (RI) is potentially ameliorable and is thus of vital importance. Nitric oxide (NO) is an important monovalent gas that under normal physiologic circumstances and during reperfusion injury regulates vascular tone, inhibits platelet aggregation, and leukocyte adhesion, scavenges reactive oxygen species, and maintains vascular homeostasis [8, 9]. NO is synthesized by the nitric oxide synthase (NOS) group of enzymes. Of particular importance in reperfusion injury are endogenous NOS (eNOS), which also produces NO constitutively, and inducible NOS (iNOS), which produces NO during tissue injury. Importantly, NOS also synthesizes deleterious superoxide especially following bouts of prolonged ischemia [10, 11]. The aim of this study was to identify what immediate effect NO donation would have on reperfusion and
316
YATES, HOSGOOD, AND NICHOLSON: NO DONATION IN DCD TRANSPLANTATION
oxidative stress, and subsequently to assess any functional changes over the reperfusion period. METHODS An ex vivo porcine model of DCD transplantation utilising cardiopulmonary bypass technology, as previously described, was used [12]. This model allows the real time measurement of functional parameters during initial reperfusion, as well as the collection of tissue and fluid samples for biochemical and histologic analysis. Furthermore, the urinary levels of NO and 8-isoprostane were measured to assess tissue damage and oxidative stress.
Kidney Retrieval and Blood Collection Large white pigs (60–70 kg) were killed by electrocution followed by exsanguination. Approximately 1 L of blood was collected into a sterile receptacle containing 25,000 units of heparin (Multiparin; CP Pharmaceuticals, Wrexham, UK). The blood was then transferred into CPDA-1 blood bags (Baxter Healthcare, Thetford, UK) and stored at 4 C. Leukocyte depletion was performed immediately prior to reperfusion by passing whole blood through a LeukoGuard RS white cell filter (Pall Medical, Portsmouth, UK). The kidneys were retrieved after 25 min of in situ warm ischemia (WI) and then flushed with 250 mL hyperosmolar citrate solution (Soltran; Baxter healthcare, Norfolk, UK) at a temperature of 4 C and a hydrostatic pressure of 100 cm H2O. The organs were placed on ice in hyperosmolar citrate solution, transported to the perfusion laboratory, and stored for 18 h.
Reperfusion Following hypothermic preservation, kidneys were prepared for ex vivo reperfusion. The renal artery, vein and ureter were cannulated and kidneys flushed with 100 mL of Ringer’s lactate solution (B. Braun, Sheffield, UK) at 4 C. Organs were then reperfused using the isolated organ preservation system (IOPS). The kidneys were reperfused for 3 h at a temperature of 37 C and a mean arterial pressure of 85 mmHg. The IOPS has been described previously. Briefly, a priming solution and nutrient supplements were added to the IOPS and kidneys reperfused with autologous blood under simulated physiologic conditions. Blood temperature and oxygenation is regulated by an in-line heat exchanger (GD120; Grant, Cambridge, UK) and membrane oxygenator (Minimax Plus membrane oxygenator; Medtronic, Tolochenaz, Switzerland). Creatinine (Sigma-Aldrich, Steinheim, Germany) was added to the perfusate to achieve an initial circulating concentration of 1000 mmol/L.
Groups Porcine kidneys were randomized into two groups. Kidneys in both groups received 25 min of WI. Kidneys in the control group were reperfused with whole blood only. Experimental group organs were reperfused with whole blood supplemented with Sodium Nitroprusside (SNP). SNP powder (Sigma-Aldrich, Steinheim, Germany) was reconstituted at a concentration of 100 mg/mL of 5% glucose solution (Baxter Healthcare, Thetford, UK). The SNP was added to the IOPS as a continuous infusion at a rate of 1.5 mg/h for the full duration of the reperfusion period; n ¼ 6 in both groups. Real-time measurement of renal blood flow (RBF) and intra-renal resistance (IRR) was performed. Urine output was measured at hourly intervals following reperfusion. Biochemical analysis of serum and urine samples was carried out at hourly intervals during reperfusion. Creatinine clearance (urinary creatinine 3 urinary volume/plasma creatinine) and fractional excretion of sodium [(urinary sodium 3 urine volume)/(glomerular filtration rate 3 plasma sodium) 3 100)] were calculated.
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Hourly arterial and venous blood gas analysis was performed to assess PaO2, PvO2 and acid-base homeostasis. Oxygen consumption [(PaO2 – venous PvO2) 3 flow rate/weight] was calculated. The hemoglobin, hematocrit, and leukocyte counts were measured before and after reperfusion.
Histology Wedge biopsies were taken at various time points. Biopsies were fixed in 10% formal saline, dehydrated, and embedded in paraffin wax. Sections of 4 mm were cut and stained with hematoxylin and eosin for evaluation under light microscopy. Sections were scored over five fields, assessing changes in four morphological variables; tubular dilation, tubular debris, vacuolation, and interstitial infiltration. Three trained assessors blinded to the experimental group scored samples from 0 to 3 according to the level of damage; 0 ¼ normal, 1 ¼ mild, 2 ¼ moderate, and 3 ¼ severe morphological changes.
Total Urinary Nitrite/Nitrate (NO2/NO3) Quantification Urine samples, taken at 60 and 180 min post-reperfusion were immediately snap-frozen in liquid nitrogen and stored at 80 C. Urine levels of nitrite/nitrate (NO2/NO3) were quantified using a total NO2/NO3 assay kit (Assay Designs, Ann Arbor, MI) according to the manufacturer’s instructions. This assay relies on the conversion of nitric oxide to nitrate and the subsequent conversion of nitrates to nitrite by the enzyme nitrate reductase. Nitrite is detected colorimetrically at 540 nm as an azodye product of the Griess reaction.
Urine 8-Isoprostane Quantification Levels of urine 8-isoprostane were determined by enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical Co., Ann Arbor, MI). Urine samples were centrifuged at 10,000 g for 2 min and the supernatant taken for analysis. Samples were diluted 10-fold prior to analysis. The sample and standards were added in duplicate to the ELISA plate together with an 8-isoprostane-acetylcholinesterase (AChE) conjugate and incubated for 18 h at 4 C. The plate was then washed and developed by the addition of the substrate to AChE. The plate was read at 405 nm after colour development for 90 min.
Statistical Analysis Values are presented as mean 6 SD. Continuous variables were plotted against time and the area under the curve (AUC) for individual perfusion experiments calculated using Excel (Microsoft, Reading, UK) and GraphPad Prism (GraphPad Software, San Diego, CA) software. Mean AUC values and individual values were compared using the Mann-Whitney test (GraphPad InStat ver. 3.00 for Windows 95, GraphPad Software, San Diego CA). P < 0.05 was considered as statically significant.
RESULTS Hemodynamics
In both groups, initial renal blood flow (RBF) was low. After 10 min of reperfusion RBF improved in both groups, and continued for the remainder of the reperfusion period. RBF in the SNP group improved throughout the first 30 min of reperfusion to a greater degree than in the control group. Beyond 30 min of reperfusion,
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the improvement in RBF in the SNP group was attenuated, whilst there was a persistent improvement in the control group. The divergence in RBF between groups became statistically significant at 30 min of reperfusion. After 120 min of reperfusion the RBF between groups began to converge and lost statistical significance. These data are summarized in Figure 1. AUC RBF for the 5–30 and 30–180 min of reperfusion were analyzed separately. AUC RBF 5–30 min of reperfusion were 705 6 190, 1072 6 383 in the control and SNP groups, respectively (Mann-Whitney test P ¼ 0.051). AUC 30–180 min of reperfusion were 4727 6 2327 and 8085 6 970 in the control and SNP groups respectively (Mann-Whitney test P ¼ 0.035). During the initial 30 min of perfusion AUC RBF in the SNP group, though higher, did not reach statistical significance compared with the control group. The AUC RBF was, however, significantly higher throughout the remainder of the perfusion period in the SNP group. These data are summarized in Figure 1. The gradient of the curves for RBF 5–30 min of reperfusion were 2.0 and 11.8 in the control and SNP groups, respectively. For 30–180 min of reperfusion the gradients were 3.5 and 1.1 in the control and SNP groups, respectively. Intra-renal resistance demonstrated reciprocal characteristics. During the initial 30 min of reperfusion IRR was higher in both groups than during the remainder of reperfusion. The IRR in the SNP group fell markedly at 30 min and remained at a low level for the remainder of reperfusion. IRR was analyzed separately during the period 5–30 and 30–180 min of reperfusion. AUC IRR for 5–30 min post-reperfusion was 43 6 16, 25 6 13 in the control and SNP groups, respectively (Mann-Whitney test P ¼ 0.035). AUC IRR for 30–180 min post-reperfusion was 192 6 149 and 66 6 6 in the control and SNP groups respectively (Mann-Whitney test P ¼ 0.035). During the entire period of reperfusion the IRR was statistically higher in the control group than the SNP group.
Filtration Function
At 60 min post-reperfusion, creatinine clearance (CrCl) was significantly lower in the control group than the SNP group. However, by 120 min of reperfusion, CrCl in the SNP group had fallen to the same level as in the control group and remained at this level for the remainder of the reperfusion period. These data are summarized in Table 1. Tubular Function
The fractional excretion of sodium (FENa) was lower in the SNP group throughout the reperfusion period. This difference did not, however, reach statistical significance. These data are shown in Table 1. Acid-Base Homeostasis
There was no difference in pre-perfusion pH between the groups. The SNP group was marginally more acidotic than controls but this did not reach statistical significance. These data are shown in Table 1. Oxygen Consumption
There was increased oxygen consumption throughout the reperfusion in all groups. The Oxygen consumption was higher in the SNP group compared wih the control group at both 60 and 180 min of reperfusion, although this was only statistically significant at 60 min reperfusion. Data are summarized in Table 1. Hematology
There was no difference between control and SNP group leukocyte, platelet, or hematocrit levels either before or after the reperfusion period. Histology
Histologic assessment did not demonstrate any significant difference in damage between groups pre- or post-reperfusion. These data are shown in Table 2. Total Urinary NO2/NO3 Quantification
Total NO2/NO3 levels were significantly different between control and SNP groups at both 60 and 180 min post-reperfusion, with P ¼ 0.045 and P¼ 0.014, respectively. These data are shown in Figure 2. 8-Isoprostane Quantification
FIG. 1. Mean RBF 6 SD in Control and SNP groups during 180 min of reperfusion. *P < 0.05.
Urinary levels of 8-isoprostane were measured at 180 min post-reperfusion. Although the levels of 8isoprostane were lower in the SNP group, this did
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TABLE 1 Difference in Creatinine Clearance, Fractional Excretion of Sodium, Acid-Base Balance, and Oxygen Consumption Between the Control and SNP Groups at Different Time-Points Group Creatinine Clearance (mL/min/100 mg) 60 min 120 min 180 min Fractional excretion of sodium (%) 60 min 120 min 180 min pH Pre 60 min 180 min Oxygen consumption (kPa.mL/min/100 g) 60 min 180 min
Control
SNP
P value
0.5 (0.2) 0.4 (0.2) 0.5 (0.4)
2.2 (1.3) 0.7 (0.6) 0.4 (0.4)
0.013* 0.927 0.432
121 (61) 82 (44) 84 (53)
66 (18) 51 (20) 48 (25)
0.066 0.235 0.133
7.43 (0.03) 7.32 (0.04) 7.3 (0.08)
7.5 (0.07) 7.26 (0.05) 7.28 (0.04)
0.074 0.074 0.775
19.3 (6.8) 28 (13.9)
44.1 (11.7) 40.9 (16.9)
0.003* 0.225
All values are mean (SD). * P < 0.05 (Mann-Whitney test).
not reach statistical significance (P ¼ 0.12). These data are shown in Figure 3.
DISCUSSION
DCD kidneys are important in expanding the organ donor pool. For this reason, strategies that improve their initial function and ameliorate the degree of ischemic and reperfusion injury are vital. This model used a combination of 25 min warm ischemia and 18 h cold storage to replicate the injury observed in uncontrolled human DCD kidneys. These ischemic times have previously been shown to impart a significant degree of injury in this model [12].
FIG. 2. Histogram showing total urinary NO2/NO3 levels 6 SD at 180 min reperfusion in the control and SNP group.
NO is a diatomic free radical, synthesized from Larginine by a group of enzymes called nitric oxide synthases. NO is produced constitutively by eNOS and, during RI, by iNOS. NOS not only catalyzes the formation of NO but also superoxide that reacts with NO to produce injurious peroxynitrite [13, 14]. The renal expression of eNOS and iNOS differs in response to ischemia. The expression of iNOS may increase 4-fold during warm ischemia, 8-fold during the first 30 min of reperfusion, and up to a 16-fold at 60 min post-reperfusion [15]. Conversely, eNOS expression is reduced by half during ischemia and remains suppressed for up to 3 h post-reperfusion, iNOS expression may increase 4-fold during warm ischemia, and 8-fold during the first 30 min of reperfusion up to a 16-fold maximal increase at 60 min postreperfusion [15]. There are many possible NO donors that could have been used in this study. SNP was chosen as it is easy to administer, has rapid action, is water soluble, and has limited toxicity [16]. Furthermore, SNP is a well established NO donor in clinical usage in humans, is often the benchmark to which other NO donors are compared, and there is a wealth of published literature demonstrating its potent NO donor action [16]. SNP has previously been used in this model, but because of its short half-life, a continuous infusion was necessary.
TABLE 2 Histologic Differences Between Pre-reperfusion and Post-Reperfusion Renal Biopsies Control
Tubular dilatation Tubular debris Tubular cell Vacuolation Interstitial infiltration
SNP
Pre
Post
Pre
Post
0.9 (0.7) 1.4 (0.5) 0.5 (0.8) 1.3 (0.5)
1.6 (0.6) 1.1 (0.7) 1.1 (1.1) 1.3 (0.5)
1.4 (0.8) 1.8 (0.6) 0.4 (0.5) 0.6 (0.5)
1.1 (0.9) 1.6 (0.6) 0.3 (0.6) 0.8 (0.8)
Values are means (SD) of a semiquantitative scoring system (0 ¼ normal, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe morphological changes).
FIG. 3. Histogram showing urinary 8-isoprostane levels 6 SD at 180 min reperfusion in the control and SNP group.
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There is a clear improvement in RBF in the SNP group between 5 and 30 min of reperfusion, this is associated with improved creatinine clearance, FENa, and oxygen consumption. However, from 30–180 min of reperfusion there is noted to be a convergence of the RBF curves, noted by a plateau in the SNP curve, and a continuing increased gradient in the control curve. This finding is associated with a loss of improvement in functional parameters, other than oxygen consumption, throughout the remainder of the reperfusion period. The improvement in RBF and function seen between 5 and 30 min reperfusion in the SNP group is explained by direct NO vasodilatation. However, the fall in RBF between 30 and 180 min requires further explanation. During ischemia and reperfusion, there is a progressive loss of L-arginine and the cofactors required for NO synthesis, and a progressive up-regulation of iNOS expression [10, 11, 15]. Thus, following reperfusion there is an increase in superoxide production that reacts with both endogenous and exogenous NO to form peroxynitrite [17]. High concentrations of peroxynitrite and superoxide have been shown to reduce renal blood flow, CrCl, and FENa [18]. Histologic evaluation of pre-perfusion and postperfusion biopsies did not demonstrate any statistical difference between groups. This finding is related to the degree of damage imparted by the duration of the warm ischemic time, and that after three h of reperfusion there is little opportunity for the tissues to demonstrate any histological improvement/deterioration. This suggests that staining of biopsies for specific markers of injury, in addition to immunohistochemical and in-situ hybridization techniques, warrant consideration for future study in this model. It is well established that NO is produced by the vascular endothelium both constitutively and in response to injury. It has also been shown experimentally that the renal tubule and glomerulus have the ability to produce NO in response to injury [19–21]. Measurement of plasma levels of NO2/NO3 would not detect alterations in NO production in the glomerulus or renal tubule in response to injury; thus urinary quantification was chosen as the assay of choice in this study. The utilization of the Griess reaction in this assay ensures that the total NO/NO2/NO3 burden during reperfusion is quantified. The finding that total urinary NO2/NO3 is significantly higher in the SNP group at 60 and 180 min explains the leveling of RBF in the SNP group. 8-Isoprostane, formed by the oxidation of arachidonic acid by reactive oxygen species, is a urinary marker of oxidative stress [22]. At 180 min post-reperfusion, the level of 8-isoprostane expression seen in the SNP group was not significantly lower than in the control group. This is suggestive that although initial flow is much improved, by the end of the reperfusion period the
oxidative stress is approaching the level seen in the control group, mirroring the functional data and demonstrating the effects of the increased NO2/NO3 burden. Our group has previously used SNP supplementation to assess the effects of carbon monoxide during reperfusion using carbon monoxide releasing molecule-3 (CORM-3) [23]. This study used kidneys with shorter WI time and the SNP was administered as a short infusion prior to and during the first hour of reperfusion only. Results in the SNP group showed that initial RBF was significantly higher than the control groups, but this benefit plateaued at 30 min, and converged with the RBF in the control group by 120 min postreperfusion. Despite the methodological differences that preclude direct comparison of this and the present study the effect on RBF of the pre-reperfusion instillation of SNP, coupled with the observed immediate improvement in RBF, does reinforce the hypothesis that NO vasodilatation is responsible for the improvement in flow early during reperfusion. Furthermore, the convergence of RBF latterly during reperfusion reinforced the observations made in the present study. There are a number of limitations with this study. This model of DCD organs used a warm ischemic period of 25 min. The most current published recommendations state that organs with a warm ischemic period of up to 60 min should be considered suitable for transplantation [24, 25]. Thus, extending this model to assess the effects of NO donation in organs with longer periods of warm ischemia would be appropriate and may demonstrate an even stronger benefit for NO donation. The reperfusion period was limited to 3 h; beyond 3 h, normothermic perfusion renal viability is markedly impaired [26]. As an alternative to prolonged ex vivo reperfusion, retrieval of kidneys from live donors followed by autotransplantation after the period of ex vivo perfusion allows assessment of the long-term impact of NO donation. The feasibility of this model has previously been demonstrated by our group [27]. Furthermore, an ex vivo reperfusion/autotransplant model could be compared with an in vivo reperfusion renal ischemia model to demonstrate potential benefits of ex vivo reperfusion and NO donation per se. Only a single NO donor, SNP, was used in this study. SNP was chosen due to its use clinically in humans for the control of hypertension, however, there is a plethora of alternate NO donors with different pharmacologic characteristics that may make them more efficacious in this model and would thus warrant further investigation. This study measured the overall nitrogenous burden by quantification of total urinary NO2/NO3 levels. Clearly, this method does not discriminate between nitrogenous compounds formed due to exogenously or endogenously produced NO. In order to provide this data assessment of eNOS and iNOS expression would be necessary at different time-points
YATES, HOSGOOD, AND NICHOLSON: NO DONATION IN DCD TRANSPLANTATION
post-reperfusion. This would be best achieved by utilizing immunohistochemistry and in-situ hybridization to quantify the presence of NOS isoforms and mRNA. In conclusion, this study demonstrates that the effects of NO supplementation are biphasic in nature, initially beneficial due to vasodilatation, but later deleterious due to oxidative stress and accumulation of toxic NO metabolites. The effects of this are seen functionally by a slight improvement in initial CrCl, FENa, and metabolic requirement, but a fall-back to control levels after the first hour of reperfusion. Further investigations into the importance of NO at different timings during reperfusion are pivotal in fully elucidating its importance in ameliorating RI. The significance of the NO:superoxide axis, generated by post-ischemic iNOS and eNOS, and its relationship with cofactor depletion undoubtedly requires further delineation. This study suggests that there may be a clinical role for NO donation in renal transplantation, certainly during early reperfusion, but that NO donation is not a panacea for RI. REFERENCES 1. Brook NR, White SA, Waller JR, et al. Non-heart beating donor kidneys with delayed graft function have superior graft survival compared with conventional heart-beating donor kidneys that develop delayed graft function. Am J Transplantation 2003;3:614. 2. Gok MA, Buckley PE, Shenton BK, et al. Long-term renal function in kidneys from non-heart-beating donors: A single-center experience. Transplantation 2002;74:664. 3. Kokkinos C, Antcliffe D, Nanidis T, et al. Outcome of kidney transplantation from non-heart-beating versus heart-beating cadaveric donors. Transplantation 2007;83:1193. 4. Nicholson ML, Metcalfe MS, White SA, et al. A comparison of the results of renal transplantation from non-heart-beating, conventional cadaveric, and living donors [erratum appears in Kidney Int 2001;59:821]. Kidney Int 2000;58:2585. 5. Quiroga I, McShane P, Koo DD, et al. Major effects of delayed graft function and cold ischemia time on renal allograft survival. Nephrol Dialysis Transplantation 2006;21:1689. 6. Boom H, Mallat MJ, de Fijter JW, et al. Delayed graft function influences renal function, but not survival. Kidney Int 2000;58:859. 7. Simpkins CE, Montgomery RA, Hawxby AM, et al. Cold ischemia time and allograft outcomes in live donor renal transplantation: Is live donor organ transport feasible? [see comment]. Am J Transplantation 2007;7:99. 8. Khanna A, Cowled PA, Fitridge RA, et al. Nitric oxide and skeletal muscle reperfusion injury: Current controversies (research review). J Surg Res 2005;128:98. 9. Hallstrom S, Gasser H, Neumayer C, et al. S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release. Circulation 2002; 105:3032.
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