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Deferoxamine Reduces Cold-Ischemic Renal Injury in a Syngeneic Kidney Transplant Model

2003; Elsevier BV; Volume: 3; Issue: 12 Linguagem: Inglês

10.1046/j.1600-6135.2003.00264.x

1600-6143

Hong Huang, Zhi He, L. Jackson Roberts, Abdulla K. Salahudeen,

Liver Disease and Transplantation

American Journal of TransplantationVolume 3, Issue 12 p. 1531-1537 Free Access Deferoxamine Reduces Cold-Ischemic Renal Injury in a Syngeneic Kidney Transplant Model Hong Huang, Hong Huang MedicineSearch for more papers by this authorZhi He, Zhi He Pathology, University of Mississippi Medical Center, Jackson, MSSearch for more papers by this authorL. Jackson Roberts II, L. Jackson Roberts II Departments of Pharmacology and Medicine, Vanderbilt University Medical Center, Nashville, TNSearch for more papers by this authorAbdulla K. Salahudeen, Corresponding Author Abdulla K. Salahudeen Medicine *Corresponding author: Abdulla K. Salahudeen, asalahudeen@medicine.umsmed.eduSearch for more papers by this author Hong Huang, Hong Huang MedicineSearch for more papers by this authorZhi He, Zhi He Pathology, University of Mississippi Medical Center, Jackson, MSSearch for more papers by this authorL. Jackson Roberts II, L. Jackson Roberts II Departments of Pharmacology and Medicine, Vanderbilt University Medical Center, Nashville, TNSearch for more papers by this authorAbdulla K. Salahudeen, Corresponding Author Abdulla K. Salahudeen Medicine *Corresponding author: Abdulla K. Salahudeen, asalahudeen@medicine.umsmed.eduSearch for more papers by this author First published: 21 November 2003 https://doi.org/10.1046/j.1600-6135.2003.00264.xCitations: 48AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract In cell-culture models, addition of deferoxamine (DFO) to University of Wisconsin Solution (UW solution) reduces cold-storage injury. The efficacy of DFO was therefore tested in a kidney transplantation model employing inbred Wistar Furth rats. Donor left kidneys, cold stored for 18 h in UW solution with or without 0.125 mM or 0.625 mM DFO were transplanted to the recipients' left renal fosse. Deferoxamine dose-dependently and significantly increased glomerular filtration rate (GFR) and renal blood flow (RBF), and suppressed renal F2-isoprostanes (vasoactive lipid peroxidation products) and apoptotic and necrotic injury 3 days post-transplantation. In a second set of similar experiments, the remaining native kidneys of the recipient rats were removed on day 7 of transplantation. Transplanted kidneys' function assessed by serum creatinine was 75% higher in the cold-stored transplanted kidneys treated with DFO compared with untreated kidneys. Moreover, the DFO treatment was attended by a significant reduction in apoptotic and necrotic tubular injury. Thus, our consistent findings from two sets of studies in a transplant model suggest that a simple strategy of including DFO in the cold-storage solution reduces cold ischemia-associated renal transplant damage and improves renal function. Our findings have potentially important ramifications for cold preservation of kidneys, and possibly other organs, in clinical transplantation. Introduction Cadaver kidneys are routinely stored cold while awaiting transplantation. Based on recent United Network of Organ Sharing (UNOS) data, the average cold ischemia time (CIT) of cadaveric kidneys has remained unchanged over the decades, at approximately 20–25 h, with the majority of kidneys undergoing at least 12 h of cold storage (1). Cold ischemia time is a strong and independent predictor of short and long-term graft survival (2,3). The formulation and the introduction of University of Wisconsin (UW) solution had been an important step in reducing cold storage organ injury (4). However, extended cold storage of organs in UW solution is still associated with severe graft injury and dysfunction (2). Deferoxamine (DFO) has been shown in in-vitro experiments to suppress cold storage-associated renal tubular cell injury and F2-isoprostane formation (5, 6). Moreover, DFO administration has been shown to reduce injury of native kidneys in various models including ischemia-reperfusion (7, 8). Based on this, we hypothesized that fortifying UW solution with DFO could potentially be a simple but effective strategy to reduce the cold ischemic injury of transplanted kidneys. However, to our knowledge the effect of DFO's inclusion in UW solution on the outcome of kidneys transplanted after cold storage has not been reported. Using a syngeneic kidney transplantation model, thus avoiding alloreactivity and rejection, we tested whether inclusion of DFO to UW solution could reduce cold ischemia-associated renal damage and improve the function of transplanted kidneys. Methods and Protocols Kidney transplantation technique Kidney transplantation was performed between in-bred male Wistar Furth rats (Harlan Inc., Indianapolis, IN) weighing 250–300 g. Donor nephrectomies and transplantation surgeries were carried out under a surgical microscope (Applied Fiberoptics Inc., Southbridge, MA). The technique was perfected such that donor nephrectomy was consistently performed in 15 min and transplant surgery in 90 min, both from beginning to end. Donor nephrectomy The donor rat's left kidney with artery, vein and ureter, the latter with a bladder cuff of 0.25 cm, was carefully excised. The kidney was immediately flushed using a previously described perfusion chamber (5). Briefly, the perfusion chamber consisted of a polystyrene tissue culture dish (100 × 20 mm) pierced with a butterfly scalp-vein needle of 26 G. Under microscopic vision, the renal artery was threaded on to the needle and held in place with a 5-zero black thread-tie. The donor kidneys were first perfused with 10 mL of normal saline solution (4 °C) over 5 min followed by 3 mL of UW solution (4 °C) over 3 min. Kidneys were then stored at 4 °C in a temperature-regulated and -monitored refrigerator while awaiting transplantation. Transplantation surgery Following left nephrectomy, the cold preserved left donor kidney was transplanted into the left renal bed of the recipient. Before transplantation the cold-stored kidney was first flushed with 5 mL of normal saline. We used interrupted 10–0 black monofilament nylon (Flanagan Instruments Inc., Mandeville, LA) sutures for the arterial anastomosis, continuous 9–0 black monofilament nylon (Flanagan Instruments Inc., Mandeville, LA) sutures for venous anastomosis, and interrupted 6–0 black silk (Ethicone, Inc., Somerville, NJ) sutures for the bladder cuff anastomosis. At the end of surgery, the rats received 6% albumin in normal saline at a dose of 1% of body weight as a 10-min bolus through the tail vein to compensate for the surgical loss of fluids. The temperature was monitored through a rectal probe and was regulated to 36–37 °C throughout surgery. Using this method, we had established a stable and reproducible model such that we were able to hold the warm renal ischemia time (time from removing from cold storage to reperfusion) consistently to 30 min. Care was taken to minimize postoperative pain and sepsis by administering bupernorphine and enrofloxacin, respectively. The rat survival and renal transplantation success rates were 90% and 85%, respectively. Study protocol The donor left kidneys, cold-stored for 18 h in UW solution with or without 0.125 or 0.625 mM DFO, were transplanted to the left renal fosse and recipients were allowed to recover to unrestricted food and water. DFO was dissolved directly in the UW solution and kept in airtight containers at 4 °C and fresh batches were made once weekly. Acute study The four groups of transplant recipient rats were: recipients of kidneys not subjected to cold storage (no-cold group; n = 6), recipients of kidneys subjected to 18-h cold storage (cold group; n = 7), recipients of kidneys subjected to cold storage with 0.125 mM DFO (low-dose DFO group; n = 7) and of kidneys subjected to cold storage with 0.625 mM DFO (high-dose DFO group; n = 7). Renal clearance measurements were carried on the 3rd day of transplantation under euvolemic condition on a heat-regulated table as detailed in a previous study (9). Briefly, following anesthesia, rats underwent femoral arterial, internal jugular vein and ureteral catheterization. Fluid loss during the study was replaced by an initial i.v. infusion of normal saline (1% of body weight) for 30 min, followed by a maintenance infusion of 0.5 mL/h. Femoral arterial pressure via a pressure transducer (Micro-med Inc., Tustin, CA) and body temperature via a rectal probe were continuously monitored. Hematocrits (Hct) by microcapillary method and serum protein by refractometry method were determined at the start and the end of clearance periods. Glomerular filtration rate (GFR) was measured by creatinine clearance and renal blood flow (RBF) by ultrasonic flow probing of the renal artery (Transonic systems Inc., Ithaca, NY). Plasma and urine creatinines were measured using the Beckman creatinine autoanalyzer (Somerset, NJ) and renal vascular resistance (RVR) was calculated from mean arterial pressure and RBF. The means of two consecutive clearance periods were used for these calculations. At the end of the clearance measurements, native and transplanted kidneys were removed and perfused with 10 mL of heparinized, cold, normal saline using the perfusion chamber described earlier. Kidneys were sectioned coronally at 0.2 cm apart and sampled in a systematic manner such that for a given measurement, for example, F2-isoprostanes sections from the same location of the kidneys, were used across the groups. Subacute study To verify the results of the acute study, we undertook a subacute study in which renal function of the transplanted kidney was determined day 9 as opposed to day 3 as employed in the acute study. The groups (n = 6 in each) were as for the acute study, i.e. no-cold group, cold group, low-dose DFO group and high-dose DFO group, and the exact protocol for the acute study was followed except that the remaining right native kidney of the recipient was removed on day 7 of transplantation. Leaving one of the native kidneys intact thus far provided interim renal support while the transplanted kidney began to recover from cold ischemic injury. The study was completed on day 9 of transplantation by measuring serum creatinine and obtaining tissue samples of the transplanted kidneys in the manner described earlier. Measurement of renal F2-isoprostane levels For the measurement vasoactive lipid peroxidation product of F2-isoprostanes, a portion of kidney weighing 200 mg was excised from the transplanted kidneys of acute study, snap-frozen in liquid nitrogen and kept frozen at –80 °C until measurement. Levels of F2-isoprostanes esterified to kidney lipids were measured as free F2-isoprostanes following extraction of lipids from a kidney homogenate by saponification, purification and derivatization using gas chromatography/negative ion-chemical ionization-mass spectrometry with [2H4]-prostaglandin F2 as the internal standard, as described previously (10). Histological assessment of transplanted kidneys Histological assessment was carried out in kidneys from acute and subacute studies. Tubular necrosis was quantitated using computer-assisted image analysis of hematoxylin-eosin stained coronal sections of kidneys using the ImagePro® image analysis software (Mediacybernetics, Carlsbad, CA). Digital images were taken from five points spaced equally across the coronal section of the slides, all at ×200 magnification. Thus, for each kidney, five digital images were obtained. Tubular sections with frank necrosis (Figure 1A) were point-counted with a line-grid containing 30 squares and 80 points, which was created in ImagePro®. A final mean score obtained for each kidney was used for statistical analysis. Figure 1Open in figure viewerPowerPoint Histological profiles of kidneys transplanted after cold storage in the acute model. (A) Hematoxylin and eosin-stained section; the arrow points to one of several tubular sections exhibiting frank necrosis. (B) Cluster of tubular sections with cells strongly staining for apoptosis; the arrow points to one such tubular section. For in-situ detection of apoptosis, the 4-µm paraffin sections of the formaldehyde-fixed kidney tissue were deparaffinized, dehydrated and stained with an apoptosis detection kit (ApopTag®, Serological corporation Inc., Norcross, GA) following the manufacturer's instructions. Briefly, sections were incubated with 30% terminal deoxynucleotide transferase, proteins were digested, and endogenous peroxidase was blocked using 3% H2O2. Sections were then incubated with peroxidase-conjugated antibody, visualized with diaminobenzidine, and counterstained with methyl green. The specificity of the apoptotic staining was verified by including appropriate positive and negative controls. To quantify apoptosis, digital images were taken from five areas per kidney as described earlier. All images were captured at ×200 magnification. The area of apoptosis (Figure 1B) was measured using ImagePro® image analysis software and a mean score for each kidney was used for statistical analysis. Statistical analysis Results are presented as mean ± standard error of the mean. The differences between the groups were determined by anova followed by Fisher's (post-hoc) test for comparison of multiple means. The level of significance was p < 0.05. Results Acute study The body weight, Hct and serum protein levels of four recipient groups at baseline were similar, including the weights of the donor kidneys at harvest. However, weights of kidneys increased during cold storage resulting in significantly higher weights in cold-stored kidneys compared with kidneys not subjected to cold storage (Figure 2A). Inclusion of DFO in the storage solution was associated with a significantly lower kidney weights than those of kidneys cold stored without DFO (Figure 2A). These findings persisted even after transplantation (Figure 2B). Figure 2Open in figure viewerPowerPoint (A) Weights of kidneys after an 18-h cold storage. Compared with kidneys not subjected to cold storage (no-cold), kidney weights were significantly higher for cold-stored kidneys (cold) and cold + low-dose deferoxamine (LD DFO). *p < 0.05 vs. cold and cold + LD DFO. (B) Acute study: weights of kidneys 3 days after transplantation. Compared with kidneys transplanted without cold storage (no-cold), 3-day post-transplant kidney weights were significantly higher for cold-stored kidneys (cold), but not for cold + LD or HD DFO. *p < 0.05 vs. the rest. GFR and RBF measured in the immediate post-transplantation period were significantly lower in kidneys transplanted with cold storage than kidneys transplanted without cold storage (Figure 3A,B). Inclusion of DFO in the storage solution significantly and dose-dependently increased GFR and RBF in the cold-stored kidneys (Figure 3A,B). Consistent with improved RBF- in DFO-treated groups, the RVR was significantly lower in kidneys cold stored with DFO (Figure 3C). Figure 3Open in figure viewerPowerPoint Acute study: (A) GFR of transplanted kidneys. GFR was measured in the transplanted kidneys 3 days after transplantation and was significantly lower in the cold group than the rest (*p < 0.05 vs. the rest). GFR was significantly higher in the low-dose (LD) and high-dose (HD) deferoxamine (DFO) groups vs. the cold group, but was not different between the no-cold and HD DFO groups. (B) RBF of transplanted kidneys. Renal blood flow was measured in the transplanted kidneys 3 days after transplantation and was significantly lower in the cold group than the rest (*p < 0.05 vs. the rest). Renal blood flow was significantly higher in the LD and HD DFO groups vs. the cold group, but was lower than in the no-cold control, (**p < 0.05). (C) Renal vascular resistance (RVR) of transplanted kidneys. Renal vascular resistance in the transplanted kidneys obtained 3 days after transplantation was significantly and markedly elevated in the cold group compared with the no-cold control of the DFO-treated cold groups (*p < 0.05 vs. the rest). Renal vascular resistance in the LD and HD DFO groups was significantly higher than in the no-cold group. F 2-isoprostanes were significantly higher in the cold-stored transplanted kidneys than in kidneys transplanted without cold storage. The levels were reduced with DFO treatment, significantly so in the high-dose DFO group (Figure 4). Figure 4Open in figure viewerPowerPoint Acute study: F2-isoprostanes in kidneys removed 3 days after transplantation. Levels were significantly higher in kidneys cold-stored (cold) vs. not cold-stored (no-cold). Inclusion of deferoxamine (DFO) reduced the levels, significantly so for the high-dose (HD) DFO. *p < 0.05 vs. the rest except the low-dose (LD) DFO group. Subacute study The recipient rats' body weight, transplanted kidneys' weight, Hct, serum protein, and urine protein-creatinine ratios measured on day 9 after transplantation are given in Table 1. None of these measurements was different statistically except for the kidney weights. As in the acute study, 9-day post transplantation kidney weights were significantly higher in the cold-stored kidneys compared with kidneys not subjected to cold storage or cold-stored with DFO (Table 1). Table 1. Measurements in the subacute study on day 9 of transplantation Groups n Body wt (g) Kidney wt (g) Hct S protein (g/dL) U prot/cr No-cold 6 258 ± 6 2.01 ± 0.07 39 ± 1.0 6.3 ± 0.06 0.67 ± 0.07 Cold 6 256 ± 3 2.60 ± 0.15* 40 ± 0.4 6.2 ± 0.08 0.99 ± 0.10 LD DFO 6 264 ± 7 2.20 ± 0.08** 39 ± 0.4 6.2 ± 0.07 0.83 ± 0.11 HD DFO 6 249 ± 8 2.09 ± 0.06 40 ± 0.5 6.1 ± 0.09 0.88 ± 0.06 n = number of rats; s = serum; U prot/cr = urine protein-creatinine ratio; LD = low dose. *p < 0.05 vs. the rest; **p < 0.05 vs. no-cold and high-dose deferoxamine (HD DFO). Serum creatinine levels measured on day 9 after transplantation also confirmed the GFR findings in the acute study. The mean serum creatinine (Figure 5) of cold-stored kidneys was fivefold higher than kidneys transplanted without cold storage (3.6 ± 0.6 mg/dL vs. 0.7 ± 0.1 mg/dL, p < 0.05), and DFO treatment was attended by a significant reduction in serum creatinine (low-dose DFO: 1.7 ± 0.5 mg/dL; high-dose DFO: 0.9 mg/dL). Figure 5Open in figure viewerPowerPoint Subacute study: serum creatine 9 days after transplantation. Seven days after transplantation the contralateral native kidneys were removed and 2 days later serum creatinines were measured. Mean serum creatinine was significantly higher in the cold group compared with the no-cold group or deferoxamine (DFO)-treated groups (*p < 0.05 vs. the rest). Inclusion of DFO reduced the levels, significantly so for the high-dose (HD) DFO group. *p < 0.05 vs. the rest except the low-dose (LD) DFO group. The levels were not different between the no-cold and HD DFO group and did not reach significance (p = 0.06) between LD DFO and HD DFO. Renal histological staining for apoptosis and necrosis Transplanted kidneys from acute and subacute studies were subjected to a semiquantitative histomorphometric computer-assisted image analysis as described earlier. In the acute study, apoptotic staining was markedly higher in the cold-stored transplanted-kidneys than in kidneys transplanted without cold storage (Table 2). Inclusion of DFO in the cold-storage solution markedly and significantly reduced cold ischemia-associated apoptosis (Table 2). Table 2. Apoptotic and necrotic score in the transplanted kidneys of acute and subacute studies Acute study Subacute study Groups Apoptotic score1 Necrotic score2 Apoptotic score1 Necrotic score2 No-cold 56 ± 10 15 ± 1 61 ± 5 8 ± 1 Cold 662 ± 52* 65 ± 5* 1621 ± 222* 47 ± 7* + LD DFO 200 ± 90** 53 ± 2** 469 ± 95** 34 ± 4** + HD DFO 189 ± 47** 51 ± 1** 195 ± 50** 32 ± 2** 1Area, as pixel count, with apoptotic staining; 2% of tubular sections with necrosis. *p < 0.05 vs. the rest; **p < 0.05 vs. no-cold (kidneys transplanted without cold storage). Cold = kidneys transplanted after 18 h of cold storage; + LD DFO = kidneys transplanted after 18 h of cold storage in low-dose deferoxamine; + HD DFO = kidneys transplanted after 18 h of cold storage in high-dose deferoxamine. In the subacute study in which kidneys were examined 9 days after transplantation, the cold-stored transplanted kidneys exhibited even more apoptotic staining than in kidneys transplanted without cold storage (Table 2). This was effectively and significantly suppressed by including DFO in the cold-storage solution (Table 2). Cold-storage-associated tubular necrosis was more prominent in the acute model than in the subacute model and both were modestly but significantly suppressed with the inclusion of DFO (Table 2). Discussion This study demonstrates that DFO in a dose-dependent manner reduces cold ischemia-associated renal injury and improves renal function post-transplantation in a syngenic kidney transplant model. The objective of the acute model was to determine the effect of cold ischemia in the early stages of kidney transplantation. Using this model, we were able to directly demonstrate that inclusion of DFO in the storage solution was associated with better post-transplant renal function and structure. The use of two doses of DFO allowed us to demonstrate that the DFO-effect was dose-related. Our method has the simplicity of adding the water-soluble DFO to the UW solution at the time of organ storage. The design also allows flushing out of the DFO before vascular anastomosis, thus minimizing DFO administration to the recipient. The subacute model was designed to study the effect of DFO on the function and structure of transplanted kidneys several days after transplantation. Using serum creatinine, which is a reliable and clinically used measure of renal function, we were able to confirm that DFO's acute renoprotective effect was persistent and led to early and nearly complete recovery. Cold ischemia causes cell and organ swelling, and consistently, kidneys kept in the cold for 18 h in our study had a 15% increase in weights. That this weight increase was significantly reduced with the inclusion of DFO in the cold storage solution agrees with the functional and structural protection observed with DFO. In clinical transplantation, cold ischemic injury is considered as an important cause of delayed graft dysfunction and of increased acute and chronic graft loss (2, 3). Several studies have attempted to discern the mechanisms involved in cold ischemic injury (11, 12), and recently studies have focused on the specific cellular effects of cold temperature (13-16). Free radicals, particularly iron-catalyzed free-radical species, have been suggested to contribute to cold-storage injury. Consistently, cold storage is reported to be associated with intracellular free-iron release and mitochondrial swelling (5,17, 18). It is possible that iron-catalyzed membrane degradation can lead to the release of endoplasmic reticulum-based calcium. Calcium loading can activate mitochondrial transition pore resulting in mitochondrial swelling, disruption of ATP production as well generation of free radicals as byproducts of impaired electron transport (19-21). We have recently reported that cold-storage-associated mitochondrial swelling occurs as early as 4 h and that a series of mitochondrial events during cold storage, such as Bcl2-Bax ratio reduction and cytochrome-C translocation, can lead to caspase-3 activation and apoptosis during rewarming (18). Furthermore, a number of recent studies suggest that heme oxygenase-1 probaby through ferritin up-regulation protects against transplant injury (16,22, 23). Thus, iron-catalyzed reactions may be a crucial early step in the cold ischemic injury cascade. Earlier studies have indeed demonstrated that iron chelator DFO can markedly suppress injury of native kidneys (7,24) as well as injury of cold-preserved cells and organs (5, 6,8,14,17). The present study is the first to demonstrate that inclusion of DFO in the storage solution results in better post-transplant allograft function. Although the protective effect of DFO in the injury setting is generally ascribed to its iron-sequestrating antioxidant property, other less well recognized effects of DFO such as induction of vascular endothelial growth factor and hypoxia-inducible factor 1 may contribute to the protection against injury (25). In our study, necrosis was prominent at the first few days of acute transplant injury, but the injury profile had shifted predominantly to ‘acute tubular apoptosis’ in the 2nd week. The extent and exuberance of apoptosis in the cold-stored kidneys in this study were striking. Several recent studies have reported the occurrence of apoptosis during ischemia-reperfusion, including clinical renal transplantation (26-28). In our study, the DFO appeared to have more effect against necrosis than apoptosis, which could be partly owing to not counting the subnecrotic tubules. Furthermore, the functional protection with DFO was more evident than structural protection, possibly because of the lack of close correlation often observed between dysfunction and histological injury in acute renal failure. Although this study by design avoided the issue of alloreactivity and immunosuppression, any injury from cold ischemia has to be the same for a kidney destined for allotransplantation. Therefore, DFO inclusion in the storage solution can be expected to be protective. Furthermore, such DFO-afforded protection in theory can be expected to reduce alloreactivity and rejection episodes, which in turn may reduce allograft loss. 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Semin Nephrol 1998; 18: 505– 518.CASPubMedWeb of Science®Google Scholar Citing Literature Volume3, Issue12December 2003Pages 1531-1537 AST and ASTS members - please log in via your Society website for full journal access.AST Members >> ASTS Members >> FiguresReferencesRelatedInformation