Xenon Treatment Protects Against Cold Ischemia Associated Delayed Graft Function and Prolongs Graft Survival in Rats

Prolonged hypothermic storage causes ischemia-reperfusion injury (IRI) in the renal graft, which is considered to contribute to the occurrence of the delayed graft function (DGF) and chronic graft failure. Strategies are required to protect the graft and to prolong renal graft survival. We demonstrated that xenon exposure to human proximal tubular cells (HK-2) led to activation of range of protective proteins. Xenon treatment prior to or after hypothermia–hypoxia challenge stabilized the HK-2 cellular structure, diminished cytoplasmic translocation of high-mobility group box (HMGB) 1 and suppressed NF-κB activation. In the syngeneic Lewis-to-Lewis rat model of kidney transplantation, xenon exposure to donors before graft retrieval or to recipients after engraftment decreased caspase-3 expression, localized HMGB-1 within nuclei and prevented TLR-4/NF-κB activation in tubular cells; serum pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were reduced and renal function was preserved. Xenon treatment of graft donors or of recipients prolonged renal graft survival following IRI in both Lewis-to-Lewis isografts and Fischer-to-Lewis allografts. Xenon induced cell survival or graft functional recovery was abolished by HIF-1α siRNA. Our data suggest that xenon treatment attenuates DGF and enhances graft survival. This approach could be translated into clinical practice leading to a considerable improvement in long-term graft survival.


Introduction
As a mainstream therapeutic method, kidney transplantation has drastically transformed the health quality of patients suffering from end-stage renal diseases. However, improvement of renal graft life-span is far from satisfactory despite of advances in surgical techniques and more powerful immunosuppressants (1), with severe functional impairment occurring in the majority of recipients by 10 years posttransplantation (2). Hypothermic preservation is routinely used to enable long-distance transport and to improve the histocompatibility match of the grafts; however, accumulating laboratory and clinical evidence highlights the importance of ischemia-reperfusion injury (IRI) as a contributing factor in the early delayed graft function (DGF) and the later graft loss (3,4). During prolonged cold ischemia, the absence of blood perfusion to the graft results in severe metabolic imbalance, which promotes tissue necrosis. Reinstitution of blood flow and concomitant reoxygenation is frequently associated with an exacerbation of tissue injury (5). The consequences of IRI are not only the initial ablation of functioning nephrons, but also the activation of the immune response (4,6). This immunity against the post-ischemic graft involves signaling events through damage associated molecular pattern molecules (DAMPs) such as HMGB-1, their binding to toll-like receptors (TLRs), subsequent initiation of NF-kB downstream signaling cascades and the induction of robust inflammatory cytokine production (7), consequently leading to an increased alloresponse and graft injury.
Xenon is used as anesthetic gas due to its narcotic effects and remarkable safety profile (8). Its organoprotective effects on vital organs have been documented in the literature (9)(10)(11)(12)(13); in particular, xenon administered prior to warm ischemia-reperfusion injury attenuated kidney injury and also prolonged survival in mice (14). The current work further extends this finding to explore whether xenon, administered to either the donor before grafting or the recipient following grafting, ameliorates early graft injury associated with cold ischemia in a rat model of kidney transplantation. The aims of this study were to evaluate the efficacy of xenon administration in preventing cold ischemia reperfusion injury and to explore the underlying mechanisms of its action both in vitro and in vivo. Longterm graft outcome through preserving graft quality by xenon was also investigated in the Lewis-to-Lewis and Fischer-to-Lewis kidney transplant models.

Materials and Methods
In vitro cell culture and hypothermia-hypoxia challenge Human kidney-2 (HK-2) cells (European Cell Culture Collection) were cultured at 378C in RPMI 1640 medium and then incubated at 48C for 24 h in Soltran preserving solution (Baxter Healthcare, Berkshire, UK) in a closed and purpose-built airtight chamber containing 8% O 2 and 5% CO 2 balanced with N 2 . Cells were then recovered for 24 h at 378C in RPMI 1640 medium in normal cell incubator.

Xenon exposure in vitro
Xenon treatment was given either 24 h prior to (pretreatment) or after hypoxia (posttreatment). Cells were incubated with the RPMI medium prebubbled with gas mixtures of 70% xenon and 5% carbon dioxide balanced with oxygen, and then exposed to the same gas concentrations for 2 h at 378C in the gas chamber mentioned above. The treated cells were recovered in RPMI medium in normal incubator at 378C for 24 h.

Cell viability in vitro
The viability of cells was assessed by using a colorimetric MTT assay (Merck KGaA, Darmstadt, Germany).

In vitro siRNA Transfection
In vitro Hypoxia inducible factor-1a (HIF-1a) siRNA transfections were carried out using lipofectamine (Invitrogen, Paisley, UK). siRNA targeting Human HIF-1a (Qiagen, Crawley, West Sussex, UK, Sense strand; 5 0 -GAAGAACUAUGAACAUAAATT-3 0 and antisense strand: 5 0 -UUUAUGUU-CAUAGUUCUUCCT-3 0 ) were dissolved in siRNA suspension buffer and administered to HK-2 cells in a dose of 20 nM, scrambled siRNA served as negative control. Cells were incubated with siRNA for 6 h at 378C in humidified air containing 5% carbon dioxide, after which it was removed and replaced with experimental medium followed by xenon gas treatment.

Animals
Inbred adult male Lewis rats and Fischer Rats weighing 225-250 g were purchased from Harlan, UK and bred in temperature-and humidity-controlled cages in a specific pathogen-free facility at Chelsea-Westminster Campus, Imperial College London. This study was approved by the Home Office, United Kingdom, and all animal procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986.

Renal transplantation
Lewis rat (LEW, RT1 1 ) to Lewis (LEW, RT1 1 ) rat and Fisher (F344, RT1 1vr ) to Lewis rat (LEW, RT1 1 ) rat renal transplantations were used as the isograft and allograft model, respectively. Rat donor kidneys were transplanted orthotopically into recipients using standard microvascular techniques. Briefly, the donor left kidney, aorta and inferior vena cava were carefully exposed and the kidney graft was then extracted, flushed and stored in 48C heparinized Soltran Preserving Solution (Baxter Healthcare). After the specified period of cold ischemia, the recipient's left kidney was extracted and the donor renal vein was connected to the recipient renal vein through end-to-end anastomosis. The arterial anastomosis between the donor aortic patch was connected to recipient aorta in an end-to-side manner. Urinary reconstruction was performed by ureter-to-bladder anastomosis. The total surgical ischemia time was restricted to <45 min.
Animal models were as follows: Lewis-Lewis isografts short-term: the contralateral kidney was removed immediately after surgery. Grafts were harvested 24 h after transplantation, at which the delayed graft function (DGF) occurs.
Lewis-Lewis isografts and Fischer-Lewis allografts long-term survival: The contralateral kidney was removed 4 days after surgery to enable the animal to survive through the acute DGF period. Fischer-Lewis allografts received once daily intramuscular doses of cyclosporine A in 5 mg/kg for 10 days. All animals were monitored on a daily basis with a scoring assay based on body weight, activity, general appearance and behavior (14). Any animal that scored over 7 was killed.
Gas exposure in vivo Rats were exposed to either 70% xenon or 70% nitrogen balanced with 30% O 2 for 2 h via an anesthetic chamber. Gas was given either to donor 24 h prior to organ retrieval (donor pretreatment), or immediately after grafting (recipient posttreatment). Gas concentrations of xenon and oxygen were constantly monitored by xenon monitor (Air Products Model No. 439 Xe, Bradford, UK) and Datex monitor (Datex-Ohmeda, Bradford, UK).

Hematoxylin and eosin staining
The kidney graft was fixed in 4% paraformaldehyde and embedded with paraffin. Sections of 5-mm thickness were taken and hematoxylin & eosin (H&E) staining was carried out. , followed by fluorescently-conjugated secondary antibodies. Slides were counterstained with DAPI. For DAB staining, kidney paraffin sections were incubated with rabbit anti-rat CD3 (1:200, Abcam) and then with biotinconjugated secondary antibody. The slides were visualized with 3,3diaminobenzidine tetrahydrochloride (DAB) followed by counterstaining with hematoxylin. Immunofluorescence was quantified using Image J (National Institutes of Health, Bethesda, MD). Ten high-power fields at 20Â magnification were photographed. Fluorescent intensity was calculated as percentages of the mean value for na€ ıve controls.

Renal function
Blood samples were collected when animals were killed. After centrifugation, serum urea and creatinine concentrations were measured using an Olympus AU2700 analyzer (Diamond Diagnostics, Watford, UK).

Statistical analysis
All numerical data were expressed as mean AE standard deviation (SD). Comparison between treatment groups was analyzed by one-way ANOVA followed by post hoc Student Newman-Keuls test (GraphPad Prism 5.0 Software). Animal survival analysis was performed using Kaplan-Meier survival estimates, and statistical significance was analyzed by the log-rank test (GraphPad Prism 5.0; GraphPad Software). A p value <0.05 was considered to be a statistical significance.

Results
Xenon treatment enhanced the expression of HIF-1a, VEGF and Bcl-2 in HK-2 cells Hypoxia inducible factor-1a (HIF-1a), vascular endothelial growth factor (VEGF) and Bcl-2 in human proximal tubular cell line (HK-2) were measured via immunofluorescent staining after exposure to 70% Xe and 5% CO 2 balanced with O 2 for 2 h (Figure 1). HIF-1a expression was increased 16 h after gas exposure ( Figure 1A and E). Similarly, its downstream effector VEGF was enhanced ( Figure 1B and F). A significant increase in Bcl-2 expression was observed 16 h after gas exposure ( Figure 1C and G). Co-localization of HIF-1a and VEGF was evident in HK-2 cells 24 h after exposure to xenon gas ( Figure 1D). These data indicated that xenon exposure was associated with the downstream activation of prosurvival proteins which enhance the cell survival.
Xenon treatment preserved cell structural integrity and reduced the cellular inflammation after hypothermia-hypoxia challenge Extracellular high-mobility group box-1 (HMGB1) is a potent innate ''danger signal'' for the initiation of inflammatory response. Cellular stress and injury during ischemiareperfusion normally causes necrotic nuclear disintegration, translocation of HMGB-1 into the cytoplasm and its subsequent release into extracellular space. Rapid repair and replication promoted by persistent HIF-1a up-regulation could restore the integrity of cellular structures and contain HMGB-1 within the nucleus, thus preventing the activation of innate immunity in the extracellular milieu. To determine if xenon could prevent release of HMGB-1, we performed an immunofluorescence analysis of HMGB-1 and cellular structural changes. HMGB-1 in na€ ıve HK-2 cells is universally distributed inside the nucleus (Figure 2A). Exposure to nitrogen gas (70% N 2 , 5% CO 2 balanced with O 2 ) did not alter the cytoplasmic translocation of HMGB-1 after hypothermia-hypoxia challenge but surprisingly, with xenon (70% Xe, 5% CO 2 balanced with O 2 ) pretreatment, HMGB-1 remained to a large extent within the nuclei ( Figure 2B) after hypothermia-hypoxia challenge. In addition, a similar effect with xenon posttreatment to HK-2 cells was found with HMGB-1 restricted to the nucleus in the majority of the cells ( Figure 2C). Notably, in contrast to na€ ıve controls, the nitrogen treated cells given a hypothermiahypoxia challenge showed more chaotic distribution of cytoskeleton network structure; while HK-2 cells treated with xenon before or after hypothermia-hypoxia challenge tended to have a more organized cytoskeletal network and better integrity of the nuclei, which correlates well with nuclear HMGB-1 localization ( Figure 2E-G). The HK-2 cell viability after insults was enhanced after xenon exposure ( Figure 2D).

Xenon induced renoprotective protein expression in renal cortex
To answer the question of whether the PI3K-Akt-mTOR pathway is transmitting cell survival and proliferation signaling in renal tubular cells in vivo, the expression levels of PI-3K, phospho-Akt and phospho-mTOR in the kidney were examined ( Figure 3A-C). Animals were exposed to 70% Xe balanced with 30% O 2 . Western blot analysis revealed a significant increase in this pathway in the renal cortex compared with naive control 16 h after gas exposure. PI-3K was increased up to twofold relative to the na€ ıve control (2.35 AE 0.5 vs. 1.00 AE 0.01 of control, p < 0.01) and both p-Akt and p-mTOR were increased nearly three times (p-Akt 2.4 AE 0.63 vs. 1.00 AE 0.02 of control, p < 0.01, and p-mTOR 2.18 AE 0.22 vs. 1.00 AE 0.02 of control, p < 0.01). The up-regulation of these proteins was maintained up to 24 h post-exposure (p < 0.05 for PI-3k, p < 0.01 for p-Akt and p-mTOR respectively, at 24 h vs. control). To confirm whether the expression of Akt-mTOR pathway leads to productions of HIF-1a and Bcl-2, the expression patterns of HIF-1a, VEGF and Bcl-2 were evaluated ( Figure 3D Xenon treatment prevented translocation of HMGB-1 and activation of TLR-4 in kidney graft after transplantation To further confirm the in vitro finding shown (Figure 2), the effects of xenon on the release of HMGB-1 were also examined in vivo. Twenty-four hours after renal transplantation, HMGB-1 was translocated from the nucleus into the cytosol, and then secreted into the extracellular space making cellular structure become undistinguishable after up to 24 h cold ischemia. Both donor and recipient xenon treatments stabilized and preserved the integrity of tubular cell nuclei and decreased the translocation of HMGB-1 into the cytoplasm ( Figure 4A). Consistent with these findings, serum HMGB-1 was also decreased with xenon donor (N 2 vs. Xe in CI 24 h: 125.5 AE 15.1 ng/ml vs. 46.4 AE 15.5 ng/ml, p < 0.01) and with xenon recipient (N 2 vs. Xe in CI 24 h: 108.3 AE 13.6 ng/ml vs. 64.7 AE 10.6 ng/ ml, p < 0.01) treatment ( Figure 4B). Release of DAMP such as HMGB-1 is associated with inflammatory response, this response is mediated via a signaling cascade which is TLR-4 dependent. Immunofluorescent studies indicated that , normal HK-2 cells exposed to normal air. Middle panel: nitrogen exposure, HK-2 cells were treated with nitrogen gas (70% N 2 and 5% CO 2 balanced with O 2 ) for 2 h and then recovered in the normal cell incubator for 24 h. Bottom panel: HK-2 cells were treated with xenon gas (70% Xe and 5% CO 2 balanced with O 2 ) for 2 h and then recovered in the normal cell incubator for 24 h, HIF-1a (Red) and VEGF (Green) co-expressed on xenon-treated cells 24 h after gas exposure. Scale bar: 50 mm. Mean fluorescence intensity (% of na€ ıve control) of HIF-1a (E), VEGF (F), and Bcl-2 (G) after gas exposure shows the expression levels of these proteins were significantly increased 16 h after Xe exposure when compared with N 2 control. NC, na€ ıve control. Bars represent means AE SD (n ¼ 4). *p < 0.05, **p < 0.01 and ***p < 0.001 Xe versus N 2 treatment. Scale bar: 50 mm.
naive kidney tissue expressed TLR-4 at a basal level. Twenty-four hours after grafting, kidney grafts subjected to 16 or 24 h ischemia and receiving nitrogen gas displayed high immunoreactivity for TLR-4, notably on the surface of renal tubules and infiltrating leukocytes. Both xenon donor and recipient treatment decreased TLR-4 expression ( Figure 4D and E), and this result was verified through western blotting of renal graft subjected to 24 h cold ischemia followed by 24 h reperfusion ( Figure 4C).
Xenon treatment decreased NF-kB and caspase-3 activation in kidney graft after transplantation We next examined the level of tubular apoptosis and inflammation after ischemia-reperfusion in the Lewis-to-Lewis renal transplantation model. The grafts underwent 16 or 24 h of cold ischemia and were harvested at Day 1 after transplantation. Signaling through TLR-4 increases NF-kB activity which is essential for induction of proinflammatory cytokines genes (15). As shown in Figure 5B and C, in the na€ ıve kidney, cytoplasmic expression of NF-kB was maintained at a minimal basal level. In nitrogen treated animals, activated NF-kB was detected mainly in cortical tubular epithelial cells and sparsely in some glomerular cells. Both xenon donor or xenon recipient treatment decreased the NF-kB signaling intensity, in contrast, with nitrogen treatment, NF-kB expression was enhanced and translocation into nuclei was intensified. The xenon-induced decrease of NF-kB expression was confirmed by Western blot study on grafts O 2 ) and then followed by 24 h reoxygenation (378C culture medium in normal cell incubator). Two hours gas exposure (70% Xe or N 2 , and 5% CO 2 balanced with O 2 ) was given either 24 h prior to hypothermia-hypoxia challenge (pretreatment) or immediately after hypothermiahypoxia challenge (posttreatment). (A-C)The cytoplasm and nucleus were immunolabeled with anti a-tubulin (Green) and DAPI nuclear stain (Blue). HMGB-1 (Red) translocated from nucleus to cytoplasm in nitrogen-treated groups, while xenon treatment restricted the translocation. Scale bar: 50 mm. (D) Cell viability after hypoxia-reoxygenation was assessed by MTT and the ratio against the NC was used as the arbitrary unit. Percent of HK-2 cells with cytoskeleton disorganization (E), nuclear fragmentation (F), HMGB-1 cytoplasmic translocation (G). Data are means AE SD (n ¼ 4), significance level is shown as **p < 0.01 and ***p < 0.001. NC, na€ ıve control. with 24 h cold ischemia followed by 24 h warm reperfusion ( Figure 5A). Caspase-3 immunoreactivity was measured in the renal cortex, which is involved in execution of apoptosis. Xenon treatment of donor or recipient significantly decreased the intensity of cytoplasmic staining compared with nitrogen treatment (Figure 5E and F). Reduction of caspase-3 and expression was confirmed by Western blot analysis of grafts with 24 h cold ischemia followed by 24 h warm reperfusion ( Figure 5D).
Xenon treatment decreased proinflammatory cytokine release and prevented delayed graft function (DGF) after kidney engraftment Serum levels of proinflammatory cytokines, including IL-1b, TNF-a and IL-6, were reduced by xenon treatment to donor or to recipient but not affected by nitrogen treatment (Figure 6A-C). We then evaluated the effect of xenon treatment on graft function after severe ischemia-reperfusion. The function of kidney grafts receiving nitrogen gas before or after ischemia deteriorated rapidly, treatment with xenon preserved renal function after grafting ( Figure 6D and E).

HIF-1a siRNA attenuated xenon mediated protection
To further confirm the role of HIF-1a on xenon mediated protection, 20 nM of HIF-1a siRNA was administered to HK-2 cells followed by xenon pretreatment (Figure 7). Compared with the scramble siRNA, HIF-1a siRNA administration attenuated the up-regulation of HIF-1a and VEGF induced by xenon treatment (Figure 7A-C). After hypothermia-hypoxia challenge, considerably high level of cytoplasmic staining of HMGB-1 was detected with HIF-1a siRNA treatment compared with that with scramble siRNA, together with increased nuclear and cytoskeleton damages ( Figure 7D-G). Enhanced cell viability by xenon treatment was abolished by HIF-1a siRNA treatment after hypothermia-hypoxia challenge ( Figure 7H). These observations were also supported by In vivo data obtained by Figure 3: Activation of PI3K-Akt-mTOR pathway, and enhanced expression of HIF-1a, VEGF and Bcl-2 expression on renal cortex by xenon gas. Lewis rats were exposed to xenon gas (70% Xe balanced with 30% O 2 ) for 2 h and then room air for 24 h. Expressions of PI-3K (A), p-Akt (B), p-mTOR (C), HIF-1a (D), VEGF (E), and Bcl-2 (F) were assessed by Western blot at 0-24 h after gas exposure. Normal kidney graft serves as na€ ıve control (NC). PI3K-AKT-mTOR pathways were activated 16 h after gas exposure compared with normal control and significant up-regulation of HIF-1a, VEGF and Bcl-2 was found 16 h after gas exposure compared naive control. Data are means AE SD (n ¼ 4). *p < 0.05, **p < 0.01 and ***p < 0.001. Xe exposure versus na€ ıve control.
using the hydrodynamic injection of HIF-1a techniques ( Figure 8A). Enhanced NF-kB expression ( Figure 8B) and Caspase-3 ( Figure 8C) expression were found in HIF-1a siRNA treated grafts. The anti-inflammatory effect of xenon was abolished with HIF-1a siRNA injection ( Figure 8D-G). Grafts received HIF-1a siRNA developed delayed graft function, with high level of creatinine and urea ( Figure 8H and I).

Donor xenon pretreatment and recipient posttreatment prolonged the survival of isografts and allografts
The survival of animals receiving isografts that were stored at 48C in Soltran for 24 h was significantly prolonged by pretreatment with xenon, as compared with nitrogen treated groups ( Figure 9). All nitrogen treated groups (whether to donor or to recipient) developed signs of acute renal failure within 3 weeks posttransplant. The survival for xenon donor group was significantly greater than that for the N 2 donor group (p < 0.001; Figure 9A) A similar pattern of survival was found in the posttreatment to recipients ( Figure 9B; p < 0.001, Xe vs. N 2 ).
In the Fischer to Lewis allogeneic transplantation, Fisher kidney grafts were stored in 48C Soltran solution for 16 h. Xenon treatment preserved renal morphology on Day 20 after transplantation ( Figure 10A). CD3 þ T cell infiltration was significantly reduced by xenon treatment on Day 20 after surgery ( Figure 10B). The allograft survival was significantly prolonged by xenon donor or recipient treatment ( Figure 10C and D). . NC, na€ ıve control. Both xenon donor and xenon recipient treatment reduced the concentration of proinflammatory cytokines compared with nitrogen control grafts (*p < 0.05, **p < 0.01 and ***p < 0.001).

Discussion
Our findings represent the first report on the renoprotective effects of xenon against cold ischemia associated delayed graft function in rodent transplant model. Xenon given either to graft donors (pretreatment) or to recipients (posttreatment) prolonged graft survival. The underlying mechanism is very likely to be due to sustained upregulation of HIF-1a and its down-stream products which led to protection from tubular cell injury induced by IRI and subsequently enhanced tubular cell survival. Ultimately, these effects in turn dampened the inflammatory response via inhibition of the HMGB1/TLR4 signaling pathways.
Previous studies have shown that inhibiting HIF degradation via hydroxylases protected kidneys from ischemiareperfusion injury and enhanced long-term kidney graft survival (16,17). Our previous study would suggest that xenon treatment does not activate HIF-1 via the HIF-1a degradation pathway but activated the up-stream mTOR pathways to accelerate HIF-1a synthesis (14). Moreover, targeting HIF results in the broad spectrum of protective effects through the master switch of several sets of downstream genes such as erythropoietin (EPO) (18), VEGF (19) and heme oxygenase-1(HO-1) (20). Thus, manipulation of the HIF-1 pathway could be a promising approach to improve kidney transplant outcome.
Shortly after transplantation, expression of both HMGB-1 (21) and its receptor TLR-4 (22) are greatly enhanced in grafts with long cold preservation time, indicating that the full development of graft ischemia-reperfusion was dependent upon signaling through the HMGB-1/TLR-4 pathway (23,24) which promotes production of pro- inflammatory cytokines (TNF-a, IL-6 and IL-1b) and inflammation. Involvement of the HMGB-1/TLR-4 pathway is supported by evidence that TLR-4 deficient (TLR4À/À) mice are spared from kidney IRI and that the administration of a neutralizing antibody to HMGB-1 either before or soon after ischemia-reperfusion conferred significant protection (24). Our data demonstrate that HIF-1a upregulation could stabilize the nuclear and cytoskeletal structure of tubular cells in response to oxidative and inflammatory stress and that this cellular structural stabilization restricts the HMGB-1 within the nuclei. This is consistent with a previous report that inhibition of HMGB-1 cytoplamic translocation protects cells from further damage (25). Deactivation of TLR-4/NF-kB would prevent the formation of inflammatory milieu, reflected by decreased systemic release of proinflammatory cytokines, such as TNF-a, IL-6 and IL-1b, and the further exacerbation of renal injury would be attenuated as shown in our study.
Our data clearly demonstrate that xenon counteracted the IRI-associated deleterious consequences of renal function-al disturbance, which prevented the occurrence of delayed graft function. The long-term transplant survival is prolonged in both syngeneic and allogeneic grafts. Despite a wealth of clinical and laboratory evidence showing that transplanted kidneys with prolonged cold ischemia time are more susceptible to long-term deterioration (26,27), the precise mechanism underlying this correlation is still unclear. A likely explanation is that the initial ischemiareperfusion injury severely diminishes the functional nephron reserves, Increasing workload on the remaining nephrons would result in suboptimal efficiency of blood filtration and gradual accumulation of toxic metabolites and harmful oxidative byproducts within the cells (28). This renders nephrons more susceptible to injury and accelerates the cell aging, resulting in further reduction of functional renal mass (29). In addition, the vigorous cell death initiated by IRI causes the massive release of DAMPs such as HMGB-1, and induces heavy influx of infiltrating immunocompetent cells, such as neutrophils and macrophages. This in turn aggravates the injury to the tubular cells and produces more cell components readily presented by Figure 6: Serum TNF-a, IL-1b and IL-6, serum creatinine and urea concentration after engraftment. The Lewis renal graft was stored in 48C Soltran preserving solution for 16 h (Cold Ischemia CI 16 h) or for 24 h (Cold Ischemia CI 24 h) and then transplanted into Lewis recipient, the graft was finally harvested 24 h after transplantation (Warm Reperfusion, WR 24 h). The xenon gas (70% Xe and 30% O 2 ) was given either to donor (XD, Xenon-Donor treatment) 24 h before donor organ retrieval or given to recipient (XR, Xenon-Recipient treatment) immediately after organ engraftment. Animals receiving nitrogen gas (70% N 2 balanced with 30% O 2 ) in donor stage (ND, Nitrogen-Donor treatment) or recipient (NR, Nitrogen-Recipient treatment) served as treatment control, normal kidney serves as na€ ıve control (NC; A-C) Serum cytokine TNF-a, IL-1b and IL-6 concentration was assessed by ELISA. Data is expressed as mean AE SD (n ¼ 4). (D and E) Serum creatinine and urea concentration were analyzed and expressed as Mean AE SD (n ¼ 4). Both xenon donor and xenon recipient treatment reduced the concentration of pro-inflammatory cytokines and restore renal function compared with nitrogen control grafts (*p < 0.05, **p < 0.01 and ***p < 0.001). (30), accelerating antigen processing and presenting to T cells, which mount the alloresponse (31). As chronic reduction of nephron populations further progresses, the compensatory changes would eventually fail to preserve homeostasis, proteinuria could prevail until the graft fails. Based on this, we could conclude that xenon could terminate this selfperpetuating cycle of nephron ablation by enhancing cell survival and proliferation and constraining the release of DAMPs, which could potentially elevate the immunogeneity of the post-ischemic kidney and agitate adaptive immunity. Indeed, our data indicated that the xenon treatment attenuates the T cell response ( Figure 10B), possibly through its cytoprotection, which in turn decreases graft immunogenicity. This warrants further study. The renal functional reserve could be raised and sufficient functioning nephrons could survive through the subsequent incoming immune and nonimmune insults.

MHC molecules on dendritic cells and macrophages
Our study is not without limitations. First, the PI3K/Akt/HIF-1a pathways after engraftment were not explored in this study and hence the further impact of HIF1a siRNA on those pathways and xenon-induced protection during IRI is open to a question. This warrants further study. Second, HIF-1a up-regulation is not unique to xenon as such effect has been reported with other anesthetics, for example, isoflurane (32)(33)(34). However, the profound depressing effects of isoflurane on cardiovascular and respiratory system can be an obstacle for clinical use in vulnerable transplanted patients.
Xenon has been used as an anesthetic gas clinically for several decades and is known to have a remarkable safety  Xe HIF-1a siRNA-treated rats were significantly higher than in Xe scrambled siRNA-treated rats (n ¼ 3). (H and I) Serum creatinine and urea levels in Xe HIF-1a siRNA-treated rats were significant higher than in Xe scrambled siRNA-treated rats (n ¼ 3). Data is expressed as mean AE SD, **p < 0.01 and ***p < 0.001. Figure 9: Xenon treatment prolonged the syngeneic grafts survival. Xenon treatment (70% xenon balanced with 30% oxygen) was given either to Lewis donor animals before organ retrieval (XD) or Lewis recipient immediately after engraftment (XR). Grafts were kept in 48C Soltran preserving solution (cold ischemia; CI) for 24 h. Donor or recipient exposed to nitrogen (70% N 2 balanced with 30% O 2 ) served as treatment control. Contralateral kidney was removed 4 days after transplant surgery. Kaplan-Meier survival analysis of recipient animals in Lewis-to-Lewis isografts. Xenon gas was given to donor Lewis animals (A) before organ retrieval or to recipient immediately (B) after engraftment (n ¼ 10). profile (8). All these suggest that xenon is readily suitable for clinical trial in the kidney transplant setting. Although the high cost may be an obstacle for its wide use, this can be overcome by using a recycle system (35) and hence, the cost to benefit ratio is very likely to be decreased when compared to the high cost of dialysis. Arguably, our data reported here indicated that xenon pretreatment could provide the optimal protection in terms of timing and efficacy while posttreatment may be more feasible with cadaveric donor graft. If our data can be extrapolated to the clinical setting, then translation of this strategy into clinic may likely improve the long-term transplant perspectives through minimizing the early graft injury. Figure 10: Xenon treatment prolonged the allogeneic grafts survival. Xenon treatment (70% xenon balanced with 30% oxygen) was given either to Fischer donor animals before organ retrieval (XD) or Lewis recipient immediately after engraftment (XR). Grafts were kept in 48C Soltran preserving solution (cold ischemia; CI) for 16 h. Donor or recipient exposed to nitrogen (70% N 2 balanced with 30% O 2 ) served as treatment control. Contralateral kidney was removed 4 days after transplant surgery. Allograft recipient received cyclosporin A (5 mg/kg) for 10 days. (A) Histology of renal graft on Day 20 after transplant surgery. (B) CD3 þ T cells assessed by immunohistochemistry on Day 20 after transplantation. Kaplan-Meier survival analysis of recipient animals in Fischer-to-Lewis allografts. Xenon gas was given to donor Fischer animals (C) before organ retrieval or to recipient immediately (D) after engraftment (n ¼ 10). Scale bar: 50 mm.