Kidney transplantation (KTx) remains the optimal treatment of end-stage renal disease. However, ischemia-reperfusion (I/R) injury is unavoidable in KTx and plays a major role in delayed graft function and in the long-term changes (1). Furthermore, cold ischemia time is a potential risk factor for graft survival in recipients of kidneys from deceased cardiac death donors (2). A cold ischemia time of less than 12 hr is strongly associated with superior graft survival, but few kidneys from deceased cardiac death donors show such short cold ischemia times (3). An imbalance in metabolic supply and demand within the ischemic organ results in profound tissue hypoxia and microvascular dysfunction. Subsequent reperfusion further enhances the activation of innate and adaptive immune responses and cell death programs (4). Therefore, a better management of organ preservation could ameliorate I/R injury, improving graft viability and long-term outcome.
One of the major events in I/R-induced kidney injury is the generation of cytotoxic oxygen radicals (5). An increase in cytotoxic oxygen radicals leads to increased cellular injury, including DNA damage, protein oxidation and nitrosylation, lipid peroxidation, and apoptosis (6). Recently, Ohsawa et al. (7) demonstrated that hydrogen (H2) gas had a protective effect against cerebral I/R injury by reducing the levels of cytotoxic oxygen radical species. In addition, Shingu et al. (8) demonstrated that intravenous injection of a hydrogen-rich saline solution prepared by gassing with hydrogen attenuated renal I/R injury in a rat model. Previous studies reported several interventions using gaseous supplementation to the preservation solutions. Minor et al. (9) reported that gaseous oxygenation during cold preservation was highly effective in improving liver graft viability. Nakao and colleagues (10, 11) demonstrated that University of Wisconsin (UW) solution saturated with carbon monoxide provided protection against cold I/R injury in rat KTx models. In this study, we hypothesized that hydrogen-rich UW (HRUW) solution could attenuate renal cold I/R injury in a rat KTx model.
RESULTS
Hydrogen and Oxygen Concentrations of UW Solution
We immersed the centrifuge tubes containing UW solution into MiZ hydrogen-saturated water (1.60 mg/L; MiZ Co., Ltd., Kanagawa, Japan) maintained at 5°C (Fig. 1A) and confirmed that the dissolved hydrogen concentration of the UW solution increased in a time-dependent manner, reaching 1.32 mg/L after 48 hr of immersion (Fig. 1B). Thereafter, we immersed the kidney grafts into centrifuge tubes containing HRUW solution, which were maintained in MiZ hydrogen-saturated water at 5°C for 24 hr. This apparatus maintained the dissolved H2 concentration of the preservation solution at the same level as the initial HRUW solution (initial HRUW solution without kidney grafts vs. HRUW solution with kidney grafts at 24 hr after preservation, 1.32 [±0.09] vs. 1.39 [±0.05] mg/L, respectively; nonsignificant) (Fig. 1C). Immersion into MiZ hydrogen-saturated water did not affect the oxygen concentration levels (normal UW solution vs. HRUW solution, 9.6 vs. 9.5 [±0.40] mg/L, respectively; nonsignificant).
;)
FIGURE 1:
MiZ nondestructive hydrogen dissolver and the hydrogen and oxygen concentrations of University of Wisconsin (UW) solution in hydrogen-saturated water. A, Water circulating between the electrolyzer and the water tank was electrolyzed periodically and stably saturated with hydrogen (H2). When commercial fluids such as saline and organ preservation solution were immersed in saturated H2 water, hydrogen permeated through their containers, increasing the H2 concentration of liquid medicines in a time-dependent manner until equilibrium is reached. B, The centrifuge tubes containing UW solution in MiZ hydrogen-saturated water (1.60 mg/L) were maintained at 5°C, and the dissolved hydrogen concentration of the UW solution was shown to increase in a time-dependent manner, reaching 1.32 mg/L at 48 hr after immersion. C, The centrifuge tubes containing HRUW and kidney grafts in the MiZ hydrogen-saturated water confirmed that the dissolved hydrogen concentration of the preservation solution was maintained after 24 hr (hydrogen-rich UW [HRUW] vs. HRUW with kidney grafts, 1.32 [±0.09] vs. 1.39 [±0.05] mg/L, respectively).
Recipient Survival Rates and Renal Function
Syngeneic KTx was performed, and three groups of animals were examined. All the nonpreserved grafts (control group) survived for 100 days after KTx (Fig. 2A). There were no significant differences in graft survival between UW and HRUW groups under 24-hr cold preservation (UW vs. HRUW, 78.6% vs. 100%, respectively, at 100 days after KTx; P=0.174) (Fig. 2B). However, HRUW treatment significantly prolonged graft survival after 36 hr of cold preservation (UW vs. HRUW, 20.0% vs. 66.7%, respectively, at 100 days after KTx; P=0.031) (Fig. 2C). The protective effect of HRUW solution was not observed after 48 hr of preservation (Fig. 2D).
FIGURE 2:
Hydrogen-rich University of Wisconsin (HRUW) improved recipient survival rates and renal function. The survival rate of recipients with nonpreserved grafts (control group) (A) was 100% at 100 days after kidney transplantation (KTx). No significant differences were observed between UW and HRUW groups for 24-hr cold preservation (B) (UW vs. HRUW, 78.6% vs. 100%, respectively, at 100 days after KTx; P=0.174). In 36-hr cold preservation (C), HRUW treatment significantly prolonged the recipient survival rate (UW vs. HRUW, 20.0% vs. 66.7%, respectively, at 100 days after KTx; P=0.031). No long-term survival was observed after KTx with 48-hr-preserved kidney grafts (D) in UW and HRUW solutions. Renal function of recipients with 24-hr-preserved grafts were assessed by serum creatinine (E), creatinine clearance (F), and 24-hr urine protein (G) at 90 days after KTx. Data are expressed as mean (SD) (n=7 to 8 per group). Tukey test: *P<0.05, **P<0.01.
Although there were no significant differences in survival rates between UW and HRUW groups under conditions of 24 hr of cold preservation, HRUW treatment significantly improved renal function 90 days after KTx (Fig. 2E–G). HRUW solution significantly suppressed the increase in creatinine (Cr) (UW vs. HRUW, 0.66 [±0.20] vs. 0.45 [±0.09] mg/dL, respectively; P<0.05). Whereas Cr clearance was significantly decreased in the UW group 90 days after KTx compared with the control group (1.49 [±0.63] vs. 2.63 [±0.53] mL/min, respectively; P<0.01), treatment with HRUW solution maintained the Cr clearance levels (2.18 [±0.40] mL/min, P<0.05 vs. UW group). Furthermore, recipients in the UW group exhibited significantly higher levels of protein in urine (47.4 [±13.4] mg per 24 hr) compared with the control group (13.5 [±4.0] mg per 24 hr, P<0.01). In contrast, recipients in the HRUW group showed significantly lower protein excretion (21.4 [±10.6] mg per 24 hr, P<0.01 vs. UW group).
HRUW Treatment Alleviated Kidney Graft Tubular Injury and Interstitial Fibrosis
In parallel with renal function, periodic acid–Schiff staining revealed that 24 hr of cold preservation in UW solution induced characteristic histologic changes on day 100, including tubular atrophy and dilatation, loss of the brush border, inflammatory cell infiltration, and cast formation (Fig. 3B). In contrast, HRUW treatment significantly alleviated tubular injury scores compared with the UW group (Fig. 3C–D). We quantitatively estimated the effects of HRUW solution on interstitial fibrosis stained with Masson trichrome staining using a color image analyzer (Fig. 3E–H). Although the recipients of the UW group showed progression of interstitial fibrosis, HRUW treatment significantly ameliorated the development of fibrosis.
FIGURE 3:
Hydrogen-rich University of Wisconsin (HRUW) alleviated kidney graft tubular injury and interstitial fibrosis. Representative photomicrographs show the changes in renal morphology observed by means of periodic acid–Schiff staining (A–C) and Masson trichrome staining (E–G) in the control group (A and E), the UW group (24-hr-preserved grafts) (B and F), the HRUW group (24-hr-preserved grafts) (C and G) on day 100. HRUW treatment significantly alleviated tubular injury scores (D) and suppressed the development of interstitial fibrosis (H) compared with the UW group. Data are expressed as mean (SD) (n=5 to 8 per group). Tukey test: *P<0.05, **P<0.01.
Effects of HRUW Solution on Oxidative Stress
Next, we investigated the possible mechanisms underlying the cytoprotective effects of hydrogen in the early phases using recipients with 36-hr-preserved grafts. To assess the therapeutic effects of HRUW solution on oxidative stress, we examined graft malondialdehyde (MDA) and serum 8-Hydroxydeoxyguanosine (Fig. 4). Tissue MDA levels were significantly lower in kidney grafts of the 3 hr after KTx stored in HRUW solution for 36 hr compared with those stored in UW solution for 36 and 3 hr after KTx (UW vs. HRUW, 53.8 [±14.3] vs. 37.7 [±7.3] μM/g tissue, respectively; P<0.05). In contrast, there were no significant differences in serum 8-Hydroxydeoxyguanosine levels between UW and HRUW solution (UW vs. HRUW with kidney grafts, 0.34 [±0.08] vs. 0.29 [±0.03] mg/L, respectively; P=0.524).
FIGURE 4:
Hydrogen-rich University of Wisconsin (HRUW) reduces kidney graft oxidative injuries. A, Graft malondialdehyde (MDA) levels are shown (n=6 per group). Reperfusion of the kidney grafts, which were preserved for 36 hr, resulted in increased tissue MDA levels. Tissue MDA levels were significantly lower in kidney grafts stored in HRUW solution compared with those in UW solution at 3 hr after reperfusion. B, Serum levels of 8-Hydroxydeoxyguanosine at 3 hr after kidney transplantation are shown (n=3 per group). No significant differences were observed in 8-Hydroxydeoxyguanosine serum levels between UW and HRUW groups (UW vs. HRUW with kidney grafts, 0.34 [±0.08] vs. 0.29 [±0.03] mg/L, respectively; P=0.524). Data are shown as mean (SD).
HRUW Treatment Inhibited Tubular Apoptosis and Reduced Interstitial Macrophage Infiltration
Because HRUW treatment reduced oxidative stress in the kidney graft, we then evaluated tubular apoptosis and the interstitial infiltration of macrophages into the grafts. As shown in Figure 5, tubular apoptosis was stimulated 24 hr after KTx in the UW group, whereas HRUW treatment significantly inhibited tubular apoptosis. Moreover, immunohistochemistry staining revealed that HRUW treatment significantly decreased interstitial ED-1–positive macrophage infiltration at 24 hr after KTx compared with simple cold storage using UW solution.
FIGURE 5:
Hydrogen-rich University of Wisconsin (HRUW) protected tubular epithelial cells from apoptosis and reduced interstitial macrophage infiltration. Tubular apoptosis and macrophage infiltration in the kidney grafts were evaluated using recipients with 36-hr-preserved graft at 24 hr after kidney transplantation (KTx) and naive Lewis (LEW) rats. Although terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL)-positive cells were few in naive LEW kidney (A), tubular apoptosis was increased in the outer medulla after prolonged cold ischemia for 36 hr (B) and was suppressed by HRUW treatment (C). Although ED-1–positive macrophages were few in a naive LEW kidney (E), prolonged cold storage under UW solution induced marked infiltration of macrophages around the damaged tubules and within the interstitium (F). In contrast, the number of macrophages was decreased by HRUW treatment (G). TUNEL-positive cells and ED-1–positive macrophages were counted in the outer medulla at ×200 magnification in a minimum of 10 fields (D and H) (n=3 per group). HRUW decreases kidney graft cortical tissue inflammatory messenger RNA (mRNA) expression and inducible nitric oxide synthase (iNOS) protein expression. I, The relative levels of interferon (IFN)-γ, interleukin (IL)-6, tumor necrosis factor (TNF)-α, heme oxygenase (HO)-1, chemokine (C-C motif) ligand 2 (CCL2), IL-1β, and iNOS mRNA expression (I) in the 36-hr-preserved grafts at 24 hr after KTx are shown (n=3 per group). HRUW treatment significantly decreased CCL2, IL-1β, and iNOS mRNA levels at 24 hr after KTx compared with simple cold storage using UW solution. Western blotting analysis (J) also demonstrated that HRUW treatment decreased the expression of iNOS in the 36-hr-preserved grafts at 24 hr after KTx. Changes in mRNA expression versus one sample of the control group were calculated. The relative quantities are presented as the ratio of the comparative cycle threshold of the target genes against those of housekeeping gene 18. Data are expressed as mean (SD). Tukey test: *P<0.05, **P<0.01.
Kidney Grafts and Cortical Tissue Messenger RNA and Protein Expression
Real-time reverse-transcriptase polymerase chain reaction analysis was performed to examine cortical tissue inflammatory messenger RNA (mRNA) expression (Fig. 5I). There were no significant differences in mRNA expression of interferon γ, interleukin (IL)-6, tumor necrosis factor α, or heme oxygenase (HO)-1 between UW and HRUW groups. In contrast, HRUW treatment significantly decreased chemokine (C-C motif) ligand 2, IL-1β, and inducible nitric oxide synthase (iNOS) mRNA levels at 24 hr after KTx compared with simple cold storage using UW solution. Western blotting analysis also demonstrated that HRUW treatment decreased the expression of iNOS at 24 hr after KTx compared with the UW group (Fig. 5J).
DISCUSSION
The safety of hydrogen gas for the human body is recognized by its medical application to prevent decompression sickness in deep-sea divers (12). Although hydrogen is known to be highly flammable, it is noteworthy that it has no risk of flammability or explosion at concentrations of less than 4.7% in the air. Since Ohsawa et al. (7) discovered that hydrogen gas displays antioxidant properties that protect the brain against I/R injury and stroke by selectively neutralizing hydroxyl radicals hydrogen gas has come to the forefront of therapeutic medical gas research (13).
Hydrogen can be delivered by means of inhalation, which has been reported to reduce I/R injury of the heart (14, 15), lung (16), liver (17), and intestine (18). Although inhaled hydrogen gas may act more rapidly, this method of administration is not practical in daily life or suitable for continuous consumption for preventive or therapeutic use. In contrast, solubilized hydrogen may be beneficial because it represents a portable, easily administered, and safe means of delivering molecular hydrogen. Drinking hydrogen-rich water reduces dopaminergic neuron loss in mice with Parkinson disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (19) and prevents chronic allograft nephropathy in the rat KTx model (20). In addition, drinking hydrogen-saturated water results in reduced levels of several oxidative stress biomarkers in patients with type 2 diabetes (21) and potential metabolic syndrome (22). Although oral administration is safe and convenient, the hydrogen in water tends to evaporate over time, and hydrogen is lost in the stomach or intestine, making it difficult to control the concentration of hydrogen administered. Administration of hydrogen by means of an injectable hydrogen-rich vehicle may allow delivery of more accurate hydrogen concentrations and has been reported to reduce I/R injury of neonatal brain (23), heart (24), lung (25), liver (26), and kidney (8). In these studies, hydrogen-rich vehicle was prepared by exposure to high-pressure hydrogen or bubbling with hydrogen. In the present study, we developed a novel strategy of adding hydrogen into organ preservation solution. The “Nondestructive H2 adding apparatus” (MiZ Co., Ltd.) is a method of H2 production used for commercial liquid medicines, which does not require the container to be opened; thus, this method is free from concerns of contamination or microorganism infiltration. This represents an advantage for applications in a clinical setting. In addition, this strategy makes it possible to maintain a high concentration of dissolved hydrogen during the preservation of organ grafts. This is not possible with previous hydrogen-rich vehicle methods. Although, as the smallest molecule, hydrogen easily leaves the container in a time-dependent manner, this apparatus makes it possible to prepare hydrogen-saturated solutions, such as saline solution, organ preservation solution, and other commercial liquid medicines, according to a patient’s condition. We have already demonstrated that our method can saturate commercially available saline solution (500 mL; Otsuka Pharmaceutical Co., Ltd.) with hydrogen (data not shown). Our apparatus can be applied to large-animal study and also clinical human research.
First, we evaluated the long-term graft outcome after cold preservation using this new strategy in a rat KTx model. Whereas the graft survival rates of both UW and HRUW groups are high (UW vs. HRUW, 78.6% vs. 100%, respectively, at 100 days after KTx), there were no significant differences in survival rates between UW and HRUW groups under conditions of 24 hr of cold preservation. However, HRUW treatment significantly prolonged recipient survival rates compared with the UW group under conditions of 36 hr of cold preservation (P=0.031). No long-term survival was observed after KTx with 48-hr-preserved kidney grafts in UW and HRUW groups, suggesting that 48 hr of cold preservation might inflict too severe damage on the grafts and require more than 10 days to recover from acute tubular necrosis. Although there were no significant differences in survival rates between UW and HRUW groups under conditions of 24 hr of cold preservation, HRUW treatment significantly improved renal function, alleviated tubular injury, and suppressed development of interstitial fibrosis. Next, we investigated the possible mechanisms underlying the cytoprotective effects of hydrogen. Because hydrogen protects cells by reducing oxidative damage to DNA, lipids, and proteins, we evaluated oxidative stress by monitoring the tissue levels of MDA. Prolonged cold ischemia for 36 hr in UW solution resulted in increased tissue MDA levels at 3 hr after KTx, whereas preservation in HRUW solution significantly decreased tissue MDA level, suggesting that HRUW treatment reduced the levels of reactive oxygen species. In addition to the direct reduction of oxidative damage, we showed that HRUW treatment inhibited iNOS expression. Nitric oxide, generated by iNOS, reacts with superoxide radicals (O2−·) at a near diffusion-limited rate to produce another highly reactive species, peroxynitrite (ONOO−), which causes oxidative/nitrative modification of biomolecules, thus modulating physiologic and pathophysiologic processes. Several studies have demonstrated that inhibition of iNOS decreases I/R injury (27–29), suggesting an essential role of iNOS in this process.
Furthermore, our data showed that tubular apoptosis was stimulated after cold I/R injury, whereas HRUW treatment significantly inhibited tubular apoptosis. We also demonstrated that treatment with hydrogen inhibited the infiltration of macrophages. We evaluated the expression of inflammatory genes in the kidney grafts at 24 hr after KTx. There were no significant differences in mRNA expression of interferon γ, IL-6, and tumor necrosis factor α between groups. In contrast, HRUW treatment significantly decreased chemokine (C-C motif) ligand 2, IL-1β, and iNOS mRNA levels at 24 hr after KTx compared with simple cold storage using UW solution. These findings were compatible with the results of macrophage infiltration.
We additionally assessed the expression of HO-1, which plays a primary role in eliminating toxic-free heme and protects cells from heme-induced oxidative stress (30). Although endogenous HO-1 was upregulated in kidney grafts preserved with UW and HRUW solutions, no significant differences in HO-1 mRNA expression between UW and HRUW groups were observed. These results suggest that endogenous HO-1 expression is associated with renal graft injury and that the cytoprotective effect of hydrogen does not depend on HO-1 activation.
The results of the present study suggest that the application of hydrogen into UW solution decreases oxidative stress, resulting in the inhibition of apoptosis and macrophage infiltration. These cytoprotective effects of hydrogen suppressed tubular injury and interstitial fibrosis, leading to a superior long-term outcome of the renal grafts
In conclusion, HRUW improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.
MATERIALS AND METHODS
Preparation of HRUW Solution and Measurement of Dissolved Hydrogen and Oxygen Levels
HRUW solution was prepared using MiZ nondestructive hydrogen dissolver (MiZ Co., Ltd.) (Fig. 1A). Water circulating between the electrolyzer and the water tank was electrolyzed periodically and stably saturated with hydrogen (1.61 mg/L at 20°C). When commercial liquid medicines such as saline solution and organ preservation solution are immersed in H2 water, hydrogen permeates through their containers, resulting in the H2 concentration of the liquid gradually increasing in a time-dependent manner. We prepared HRUW solution using this apparatus by immersing 50-mL centrifuge tubes (Iwaki, Tokyo, Japan) containing UW solution (ViaSpan; DuPont, Wilmington, DE) for more than 48 hr. HRUW solution was then applied as a perfusion and preservation solution for kidney grafts. In addition, we immersed the centrifuge tubes containing HRUW solution and kidney grafts into MiZ H2-saturated water maintained at 5°C during preservation.
The hydrogen concentration was measured using a picoampere meter (PA2000; Unisense, Aarhus, Denmark). Dissolved oxygen levels were measured using CHEMets Kits R-7512 (CHEMetrics, Inc., Calverton, VA) according to the manufacturer’s protocol.
Animals
Inbred male Lewis (LEW) rats, weighing 250 to 350 g, were purchased from SLC Japan (Hamamatsu, Japan). All studies were performed in accordance with the principles of the Guidelines for Animal Experimentation at Osaka University and the National Research Institute for Child Health and Development.
Kidney Transplantation
Orthotopic KTx was performed using a technique described previously (31, 32). Briefly, the left kidney of a male donor LEW rat was flushed with ice-cold UW or HRUW solution through the abdominal aorta and removed. The excised graft was preserved in UW or HRUW solution for 24, 36, or 48 hr. After left-kidney nephrectomy of a recipient LEW rat, the kidney graft was transplanted orthotopically into the recipient by end-to-end anastomosis of the left renal vessels and ureter, with 10–0 sutures using microsurgical techniques. The remaining right native kidney was removed 10 days after KTx for assessment of renal function and survival rate as a life-supporting model (i.e., no remaining host kidney).
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
Experimental Protocols
Syngeneic KTx was performed, and three groups of animals were examined: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution (UW group), and recipients with grafts preserved in HRUW solution (HRUW group). Recipients in the control group were immediately transplanted after donation without cold preservation period, and the remaining right native kidney was removed 10 days after KTx as well as UW and HRUW groups. Blood and urine samples were obtained 90 days after KTx. Recipients were sacrificed at 3 and 24 hr and 100 days after KTx, and the kidneys were removed.
Renal Function
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
Morphologic Analysis
Tubular injury was scored by estimating the percentage of tubules in the outer medulla and corticomedullary junction, which showed epithelial necrosis or had necrotic debris or cast as follows: 0, none; 1+, <10%; 2+, 10% to 25%; 3+, 26% to 45%; 4+, 46% to 75%; or 5+, >75% (33). The interstitial fibrotic area was stained blue with Masson trichrome staining, and a color image analyzer was used to quantitatively estimate the area. For periodic acid–Schiff staining and Masson trichrome staining, 10 viewing fields randomly selected from the outer medulla and corticomedullary junction on each slide section were examined at ×200 magnification, and the scores were averaged. For ED-1 and terminal deoxynucleotide transferase–mediated dUTP nick-end labeling staining, the number of positive cell nuclei was counted in 10 random high-power fields (×400) of each slide and averaged.
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
Oxidative Damage Measurements
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
RNA Isolation and Quantitative Real-time Reverse-Transcriptase Polymerase Chain Reaction
Data are expressed as the comparative cycle threshold (Ct). The normalized Ct value of each gene was obtained by subtracting the Ct value of 18S ribosomal RNA. The fold change versus one sample of the control group was calculated as described previously (34).
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
Western Blotting Analysis
Detailed methods are provided (see Materials and Methods, SDC, https://links.lww.com/TP/A671).
Statistical Analyses
Data are expressed as mean (SD). The Kaplan-Meier method was used to calculate survival rates. Log-rank tests were used for comparisons between the two groups. Statistical analyses were performed using the Tukey test for parametric multiple comparisons. Differences were considered statistically significant at P<0.05.
ACKNOWLEDGMENTS
The authors thank Astellas Pharma Inc. for supplying the UW solution (Viaspan) and Mizuki Takeyama and Ryosuke Kurokawa for their invaluable technical assistance.
REFERENCES
1. Gueler F, Gwinner W, Schwarz A, et al.. Long-term effects of acute ischemia and reperfusion injury. Kidney Int 2004; 66 (2): 523.
2. Locke JE, Segev DL, Warren DS, et al.. Outcomes of kidneys from donors after cardiac death: implications for allocation and preservation. Am J Transplant 2007; 7 (7): 1797.
3. Summers DM, Johnson RJ, Allen J, et al.. Analysis of factors that affect outcome after transplantation of kidneys donated after cardiac death in the UK: a cohort study. Lancet 2010; 376 (9749): 1303.
4. Eltzschig HK, Eckle T. Ischemia and reperfusion—from mechanism to translation. Nat Med 2011; 17 (11): 1391.
5. Noiri E, Nakao A, Uchida K, et al.. Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol 2001; 281 (5): F948.
6. Rodrigo R, Bosco C. Oxidative stress and protective effects of polyphenols: comparative studies in human and rodent kidney. A review. Comp Biochem Physiol C Toxicol Pharmacol 2006; 142 (3–4): 317.
7. Ohsawa I, Ishikawa M, Takahashi K, et al.. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007; 13 (6): 688.
8. Shingu C, Koga H, Hagiwara S, et al.. Hydrogen-rich saline solution attenuates renal ischemia-reperfusion injury. J Anesth 2010; 24 (4): 569.
9. Minor T, Sitzia M, Dombrowski F. Kidney transplantation from non– heart-beating donors after oxygenated low-flow machine perfusion preservation with histidine-tryptophan-ketoglutarate solution. Transpl Int 2005; 17 (11): 707.
10. Faleo G, Neto JS, Kohmoto J, et al.. Carbon monoxide ameliorates renal cold ischemia–reperfusion injury with an upregulation of vascular endothelial growth factor by activation of hypoxia-inducible factor. Transplantation 2008; 85 (12): 1833.
11. Nakao A, Faleo G, Shimizu H, et al.. Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts. Kidney Int 2008; 74 (8): 1009.
12. Fontanari P, Badier M, Guillot C, et al.. Changes in maximal performance of inspiratory and skeletal muscles during and after the 7.1-MPa Hydra 10 record human dive. Eur J Appl Physiol 2000; 81 (4): 325.
13. Huang CS, Kawamura T, Toyoda Y, et al.. Recent advances in hydrogen research as a therapeutic medical gas. Free Radic Res 2010; 44 (9): 971.
14. Hayashida K, Sano M, Ohsawa I, et al.. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 2008; 373 (1): 30.
15. Nakao A, Kaczorowski DJ, Wang Y, et al.. Amelioration of rat cardiac cold ischemia/reperfusion injury with inhaled hydrogen or carbon monoxide, or both. J Heart Lung Transplant 2010; 29 (5): 544.
16. Kawamura T, Huang CS, Tochigi N, et al.. Inhaled hydrogen gas therapy for prevention of lung transplant-induced ischemia/reperfusion injury in rats. Transplantation 2010; 90 (12): 1344.
17. Fukuda K, Asoh S, Ishikawa M, et al.. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun 2007; 361 (3): 670.
18. Buchholz BM, Kaczorowski DJ, Sugimoto R, et al.. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury. Am J Transplant 2008; 8 (10): 2015.
19. Fujita K, Seike T, Yutsudo N, et al.. Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. PLoS One 2009; 4 (9): e7247.
20. Cardinal JS, Zhan J, Wang Y, et al.. Oral hydrogen water prevents chronic allograft nephropathy in rats. Kidney Int 2010; 77 (2): 101.
21. Kajiyama S, Hasegawa G, Asano M, et al.. Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr Res 2008; 28 (3): 137.
22. Nakao A, Toyoda Y, Sharma P, et al.. Effectiveness of hydrogen rich water on antioxidant status of subjects with potential metabolic syndrome—an open label pilot study. J Clin Biochem Nutr 2010; 46 (2): 140.
23. Cai J, Kang Z, Liu K, et al.. Neuroprotective effects of hydrogen saline in neonatal hypoxia-ischemia rat model. Brain Res 2009; 1256: 129.
24. Sun Q, Kang Z, Cai J, et al.. Hydrogen-rich saline protects myocardium against ischemia/reperfusion injury in rats. Exp Biol Med (Maywood) 2009; 234 (10): 1212.
25. Mao YF, Zheng XF, Cai JM, et al.. Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats. Biochem Biophys Res Commun 2009; 381 (4): 602.
26. Liu Q, Shen WF, Sun HY, et al.. Hydrogen-rich saline protects against liver injury in rats with obstructive jaundice. Liver Int 2010; 30 (7): 958.
27. Chatterjee PK, Patel NS, Kvale EO, et al.. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int 2002; 61 (3): 862.
28. He S, Rehman H, Wright GL, et al.. Inhibition of inducible nitric oxide synthase prevents mitochondrial damage and improves survival of steatotic partial liver grafts. Transplantation 2010; 89 (3): 291.
29. Shi Y, Rehman H, Wright GL, et al.. Inhibition of inducible nitric oxide synthase prevents graft injury after transplantation of livers from rats after cardiac death. Liver Transpl 2010; 16 (11): 1267.
30. Kumar S, Bandyopadhyay U. Free heme toxicity and its detoxification systems in human. Toxicol Lett 2005; 157 (3): 175.
31. Azuma H, Isaka Y, Li X, et al.. Superagonistic CD28 antibody induces donor-specific tolerance in rat renal allografts. Am J Transplant 2008; 8 (10): 2004.
32. Imamura R, Isaka Y, Sandoval RM, et al.. Intravital 2-photon microscopy assessment of renal protection efficacy of siRNA for p53 in experimental rat kidney transplantation models. Cell Transplant 2010; 19 (12): 1659.
33. Yamada K, Miwa T, Liu J, et al.. Critical protection from renal ischemia reperfusion injury by CD55 and CD59. J Immunol 2004; 172 (6): 3869.
34. Morita M, Fujino M, Jiang G, et al.. PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft. Am J Transplant 2010; 10 (1): 40.
