Early treatment with xenon protects against the cold ischemia associated with chronic allograft nephropathy in rats

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http://www.kidney-international.org & 2013 International Society of Nephrology

Early treatment with xenon protects against the cold ischemia associated with chronic allograft nephropathy in rats Hailin Zhao1, Xianghong Luo1,2, Zhaowei Zhou1, Juying Liu2, Catherine Tralau-Stewart3, Andrew J.T. George4 and Daqing Ma1,2 1

Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK; 2Department of Anesthesiology, Taihe Hospital, Hubei University of Medicine, Hubei, China; 3Drug Discovery Centre, Department of Medicine, Imperial College London, London, UK and 4Section of Molecular Immunology, Department of Medicine, Imperial College London, Hammersmith Hospital, London, UK

Chronic allograft nephropathy (CAN) is a common finding in kidney grafts with functional impairment. Prolonged hypothermic storage–induced ischemia-reperfusion injury is associated with the early onset of CAN. As the noble gas xenon is clinically used as an anesthetic and has renoprotective properties in a rodent model of ischemiareperfusion injury, we studied whether early treatment with xenon could attenuate CAN associated with prolonged hypothermic storage. Exposure to xenon enhanced the expression of insulin growth factor-1 (IGF-1) and its receptor in human proximal tubular (HK-2) cells, which, in turn, increased cell proliferation. Xenon treatment before or after hypothermia-hypoxia decreased cell apoptosis and cell inflammation after reoxygenation. The xenon-induced HK-2 cell proliferation was abolished by blocking the IGF-1 receptor, mTOR, and HIF-1a individually. In the Fischer-toLewis rat allogeneic renal transplantation model, xenon exposure of donors before graft retrieval or recipients after engraftment enhanced tubular cell proliferation and decreased tubular cell death and cell inflammation associated with ischemia-reperfusion injury. Compared with control allografts, xenon treatment significantly suppressed T-cell infiltration and fibrosis, prevented the development of CAN, and improved renal function. Thus, xenon treatment promoted recovery from ischemia-reperfusion injury and reduced susceptibility to the subsequent development of CAN in allografts.

Transplantation remains the preferred treatment for patients suffering from end-stage renal failure. Despite improved postoperative immunosuppression regimens, the majority of late graft failures are attributable to chronic allograft nephropathy (CAN).1 Histologically, renal graft with CAN is characterized by intimal thickening of arteries, glomerulosclerosis, tubular interstitial fibrosis, and renal atrophy. The clinical course of CAN is manifested as a progressive deterioration in renal function, in combination with proteinuria and aggravation of de novo hypertension.2 The etiology of CAN remains to be elucidated, but it is widely believed to be the end result of cumulative damage to the renal grafts associated with immune and nonimmune factors.3 A wide range of clinical evidence has demonstrated that ischemia-reperfusion injury (IRI) is one of the vital events ultimately leading to the development of CAN.4,5 Investigations into an effective renoprotective strategy are therefore needed in order to enhance early graft functional performance. Consequently, the development of CAN could be prevented. The noble gas xenon is clinically used as an anesthetics6 and has recently been shown to possess therapeutic value as an organo-protectant against IRI.7–11 The aim of the present study was to evaluate the efficacy of xenon in preventing IRI and CAN after allogeneic kidney transplantation in rats.

Kidney International (2013) 85, 112–123; doi:10.1038/ki.2013.334; published online 11 September 2013

The relationship between the duration of cold ischemia and development of CAN was first investigated in the Fisher-toLewis renal transplantation model. Renal grafts were stored in cold preserving solution for up to 16 h and then transplanted into the recipient. As demonstrated in Figure 1a, more severe tubular injury was found in ischemic allografts than in non-ischemic allografts on day 1 after surgery. In allografts with 16 h cold ischemia, severe tubular damage, interstitial fibrosis, and cell infiltration were observed. Two months after transplantation, tubular, vascular, and glomerular lesions were more evident in

KEYWORDS: chronic allograft nephropathy; cold ischemia; delayed graft function; graft protection; xenon

Correspondence: Daqing Ma, Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK. E-mail: [email protected] Received 31 January 2013; revised 1 June 2013; accepted 20 June 2013; published online 11 September 2013 112

RESULTS Prolonged cold ischemia led to an earlier onset of CAN

Kidney International (2014) 85, 112–123

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H Zhao et al.: Xenon and chronic allograft nephropathy

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Figure 1 | Prolonged cold ischemia (CI) induced early onset of chronic allograft nephropathy. The Fischer renal graft was stored in 4 1C Soltran preserving solution for 0 h (quick flush, immediate engraftment), 4 h, or 16 h (CI) and then transplanted into Lewis recipient. (a) Histology (H and E staining) of the allograft with CI 0–16 h on day 1, 1 month, 2 months, or 6 months after surgery. (b) Serum creatinine and urea concentration on days 40 and 180 after surgery. Data are expressed as mean ±s.d. (n ¼ 4, *Po0.05 and ***Po0.001). Bar ¼ 50 mm. Arrow indicates injury in renal tubules. NC, naive control.

ischemic allografts than in non-ischemic allografts. No grafts with 16 h cold ischemia were able to survive beyond 3 months. Six months after surgery, tubular atrophy and vascular obliteration were more widely observed in ischemic grafts than in non-ischemic grafts. On day 40 after transplantation, significantly higher serum creatinine or urea levels were recorded in recipients with ischemic grafts than in nonischemic grafts. Renal function deteriorated more rapidly in ischemic grafts than in non-ischemic grafts 6 months after transplantation (Figure 1b).

and the potential role of xenon treatment in relation to IGF-1 in this respect was then explored. The cell cycle transition regulatory proteins cyclin D and cyclin E were found to be increased in HK-2 cells after xenon exposure (Figure 2c and d); this suggested that xenon treatment promotes progression of the cell cycle through enhanced IGF-1/IGF-1R signaling, and that cyclin D and and cyclin E are possibly involved in the signaling network of IGF-1.

Xenon exposure caused upregulation of insulin growth factor-1 (IGF-1) and IGF-1R

Xenon treatment induced continuous increase in the number of Ki-67 þ cells (a marker of proliferation) up to 24 h after exposure (Figure 3a and b). Hypoxia-inducible factor-1a (HIF-1a) is the established downstream mediator of trophic stimuli of IGF-1 and promotes cell survival and cell proliferation.14 To confirm the increased proliferation of HK-2 cells as a direct consequence of HIF-1a activation, the localization of HIF-1a and Ki-67 on HK-2 cells was assessed

Increased production of IGF-1 was observed 24 h after xenon exposure on human proximal tubular cell line HK-2 (Po0.05; Figure 2a). Flow cytometry analysis demonstrated that the expression of IGF-1R increased 24 h after gas exposure (Po0.05; Figure 2b). Activation of the IGF-1 signaling pathway is involved in cell cycle progression,12,13 Kidney International (2014) 85, 112–123

Xenon exposure reduced cell death and cell inflammation after hypoxia-reoxygenation

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Figure 2 | Insulin growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), cyclin D, and cyclin E expression patterns in human kidney proximal tubular cells (HK-2) after xenon (Xe) exposure. HK-2 cells were treated with 70% Xe or N2 and 5% CO2 balanced with O2 for 2 h and then recovered in the normal cell incubator for 24 h. Expression levels of (a) IGF-1 was assessed by western blotting and that of (b) IGF-1R was assessed by flow cytometry at 24 h after gas exposure. (c) Cyclin D and (d) cyclin E were assessed by immunofluorescent staining (nuclei counterstained with DAPI (4,6-diamidino-2-phenylindole)) and western blotting. Expression level was evaluated through western blotting. Data are expressed as means±s.d. (n ¼ 4). *Po0.05; **Po0.01 and ***Po0.001. Bar ¼ 50 mm. MFI, mean fluorescence intensity; NC, naive control.

by fluorescence staining (Figure 3c). In naive control cells and cells with nitrogen exposure, weak cytoplasmic HIF-1a staining was associated with the absence of Ki-67 expression; however, the majority of cells with strong nuclei HIF-1a expression are Ki-67 positive at 24 h after xenon exposure. This colocalization indicated the essential role of HIF-1a in regulating cellular proliferation. To verify the cytoprotective effects of xenon on HK-2 cells, caspase-3 expression of HK-2 cells after hypoxia-reoxygenation was evaluated. Xenon pretreatment or xenon post treatment reduced caspase-3 expression after insults (Figure 3d and f). IGF-1/IGF-1R suppresses apoptosis, which may reduce nuclear factor (NF)kB activities. A significant reduction in NF-kB activation after insults was found with xenon treatment (Figure 3e and g). To assess whether xenon treatment could affect the 114

activation of macrophage and fibroblasts through reduced tubular cell death and inflammation, the conditioned medium of HK-2 cells at the end of 24 h reoxygenation was collected and used to stimulate human monocyte/macrophage cell line U937 and human fibroblasts. After being challenged with this conditioned media for 24 h, the expression levels of TLR-4 and CD86 on macrophages were significantly increased in the nitrogen-treated group but was decreased in the xenon-treated group (Figure 4a). This indicated that macrophage was less inflammatory, and the interactions between renal tubular cells and macrophages through TLR-4 and that between macrophages and T cells through costimulatory CD86 were decreased. Interestingly, the upregulation of TLR-4 expression in macrophages after the HK-2 cell–conditioned medium challenge was Kidney International (2014) 85, 112–123

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Figure 3 | Xenon treatment enhanced cell proliferation and decreased caspase-3 and nuclear factor (NF)-jB expression in human proximal tubulor (HK-2) cells after hypoxia-reoxygenation. Gas exposure (70% Xe or N2 and 5% CO2 balanced with O2) was given to HK-2 for 2 h and then recovered in the normal cell incubator for 24 h. (a) Ki-67 (green) expression in HK-2 cells. (b) Percentage of Ki-67 þ cells after gas exposure. (c) Immunofluorescent dual-labeling of hypoxia-inducible factor (HIF)-1a (Red) and Ki-67 (Green) in HK-2 cells is shown. HK-2 cells were subjected to 16 h hypothermia-hypoxia insults (4 1C Soltran preserving solution under 8% O2), followed by 24 h reoxygenation (37 1C culture medium in normal cell incubator). A duration of 2 h xenon or nitrogen gas exposure was given either 24 h before hypothermia-hypoxia challenge (Xe or N2 pre: Xe or N2 pretreatment) or immediately after hypothermia-hypoxia challenge (Xe or N2 post, Xe or N2 post treatment). (d) Caspase-3 (red) and (e) NF-kB (red) expression patterns on HK-2 are shown. Analysis of fluorescence intensity of (f) caspase-3 and (g) NF-kB is shown. Data are means±s.d. (n ¼ 4), significance level is shown as *Po0.05; **Po0.01 and ***Po0.001. Bar ¼ 50 mm. DAPI, 4,6-diamidino-2phenylindole; NC, naive control.

significantly decreased by xenon pretreatment but not by xenon post treatment. When fibroblasts were challenged with HK-2–cell conditioned medium, xenon treatment resulted in less production of transforming growth factor (TGF)-b (Figure 4b), which is essential for the fibrogenic transformation of fibroblasts. Xenon-induced cell proliferation was attenuated by blocking IGF-1 signaling cascade

The mammalian target of rapamycin (mTOR) and HIF-1a are the downstream mediators of the IGF-1/IGF-1R signaling pathway.14 To assess their roles in xenon-enhanced cell proliferation, mTOR inhibitor rapamycin was administered to HK-2 cells at a dose of 50 nmol/l for 30 min before the xenon gas pretreatment. Compared with the vehicle treatment, rapamycin administration significantly decreased the expression of HIF-1a, and cell proliferation was abolished (percentage of Ki-67 þ cells: 9.75±3.9% vs. 60.25±14.0%, rapamycin vs. vehicle, Po0.01; Figure 5a and b). To further Kidney International (2014) 85, 112–123

confirm the role of HIF-1a on xenon-mediated cell proliferation, we examined the effect of HIF-1a small interfering RNA (siRNA) on HK-2 cells treated with xenon. A dosage of 20 nmol/l of HIF-1a siRNA was administered to HK-2 cells, followed by xenon preconditioning treatment. HIF-1a siRNA administration significantly decreased the expression of HIF1a, and cell proliferation was abolished (Figure 5c and d). These data suggested the pivotal involvement of IGF-1/IGF1R and its downstream mediators m-TOR and HIF-1a in xenon-enhanced cell proliferation. To further assess the causal role of IGF-1/IGF-1R in the proliferation of renal cells after hypoxia insults, we used the neutralizing antibody aIR3 to block IGF-I receptor. The expression of HIF-1a and Ki-67 was evaluated by immunofluorescence 24 h after gas exposure. aIR3 abolished HIF-1a upregulation and cell proliferation induced by xenon treatment (Figure 6a). Administration of IGF-1R-neutralizing antibody restored NF-kB nuclear activities after hypoxiareoxygenation (Figure 6b). 115

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Figure 4 | Effects of human proximal tubular (HK-2) conditioned medium on monocytes and fibroblasts. (a) HK-2 cells were subjected to 16 h hypothermia-hypoxia (4 1C Soltran preserving solution under 8% O2), followed by 24 h reoxygenation (37 1C culture medium in normal cell incubator). A duration of 2 h gas exposure (70% Xe or N2 and 5% CO2 balanced with O2) was given either 24 h before hypothermia-hypoxia challenge (Xe or N2 pre: Xe or N2 pretreatment) or immediately after hypothermia-hypoxia challenge (Xe or N2 post, Xe or N2 post treatment). After hypoxia-reoxygenation, HK-2-conditioned medium was collected and was used to stimulate monocyte and fibroblasts. (a) Expression levels of Toll-like receptor 4 (TLR-4) and CD86 on U937 monocytes, assessed by flow cytometry, and (b) transforming growth factor (TGF)-b expression on fibroblasts, assessed by immunofluorescence at 24 h after conditioned medium (CM) challenge. Fluorescence intensity is expressed as means±s.d. (n ¼ 4), significance level is shown as *Po0.05. Bar ¼ 50 mm. MFI, mean fluorescence intensity; NC, naive control.

Xenon treatment decreased IRI in the renal allograft and improved renal function

The efficacy of xenon treatment on IRI was then examined in the Fischer-to-Lewis transplantation model, renal grafts underwent 16 h cold storage, and renal histology was assessed 24 h after grafting. Xenon treatment significantly enhanced IGF-1R expression (Figure 7a), which further confirmed the in vitro observation. The number of TUNEL þ (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling-positive) cells was significantly reduced in the kidneys from xenon-treated animals (Figure 7b). Ki-67 þ cells were increased after xenon treatment (Figure 7c). The renal function was preserved 24 h after surgery with xenon treatment, with significantly lower serum levels of creatinine (154.3±58.1 vs. 301.6±113.2 mmol/l, Xe vs. N2 in donor treatment, Po0.05; 196.9±52 vs. 358.1±72.6 mmol/l, Xe vs. N2 in recipient treatment, Po0.05) and urea (31.8± 5.6 vs. 14.8±6.2 mmol/l, Xe vs. N2 in donor treatment, Po0.05; 40.7±4.2 vs. 20.3±9.6 mmol/l, Xe vs. N2 in recipient treatment, Po0.05; Figure 7d and e). Intense staining pattern of ICAM-1 was seen in the renal cortex 24 h 116

after grafting. The ICAM-1 expression was markedly decreased with treatment of xenon (Figure 8a). Concurrently, with the upregulated ICAM-1 on graft tissue, infiltration of macrophages (Figure 8b) and release of proinflammatory cytokines tumor necrosis factor (TNF)-1a and interleukin (IL)-1b (Figure 8c) were significantly reduced with xenon treatment. This indicated that the robust inflammation was attenuated with xenon treatment.

Xenon treatment reduced T-cell infiltration and fibrosis

T-cell infiltration and renal fibrosis were then examined in the renal cortex, which are the pathological hallmarks of the development of CAN. Large numbers of infiltrating CD3 þ (Figure 9a), CD8 þ (Figure 9b), and CD4 þ (Figure 9c) T cells were found in grafts with 16-h cold ischemia at 1 month after surgery. In contrast, in grafts treated with xenon, infiltration of T cells was markedly reduced. Accumulation of collagens was found particularly in the tubular interstitium; however, renal fibrosis was suppressed in xenon-treated grafts (Figure 9d). Kidney International (2014) 85, 112–123

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Figure 5 | Effect of rapamycin and hypoxia-inducible factor (HIF)-1a small interfering RNA (siRNA) on the xenon-mediated cell proliferation. Human proximal tubular (HK-2) cells were treated with either scrambled siRNA (scr siRNA) or HIF-1a siRNA (20 nmol/l) for 6 h, or rapa 50 nm for 30 min, followed by xenon gas (70% Xe and 5% CO2 balanced with O2) for 2 h and then recovered in normal cell incubator for 24 h. (a) Immunofluorescent dual-labeling of HIF-1a (Red) and Ki-67 (Green) in HK-2 cells after rapa–xenon treatment. (b) The percentage of Ki-67 þ proliferating cells after rapa–xenon treatment. (c) Immunofluorescent dual-labeling of HIF-1a (Red) and Ki-67 þ (Green) in HK-2 cells after siRNA–xenon treatment. (d) The percentage of Ki-67 þ proliferating cells after siRNA–xenon treatment. Data are expressed as mean±s.d. (n ¼ 4); ***Po0.001. Bar ¼ 50 mm. DAPI, 4,6-diamidino-2-phenylindole; NC, naive control; rapa, rapamycin; Vh, vehicle.

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Figure 6 | Effect of insulin growth factor-1 receptor (IGF-1R)-neutralizing antibody aIR3 on the xenon-treated human proximal tubular (HK-2) cells. HK-2 cells were given 16 h hypothermia-hypoxia challenge (4 1C Soltran preserving solution under 8% O2), followed by 24 h reoxygenation (37 1C culture medium in normal cell incubator). A duration of 2 h gas exposure (70% Xe or N2, and 5% CO2 balanced with O2) was given either 24 h before hypothermia-hypoxia challenge (Xe or N2 pre: Xe or N2 pretreatment) or immediately after hypothermiahypoxia challenge (Xe or N2 post, Xe or N2 posttreatment). IGF-1R-neutralizing aIR3 antibody was coadministered with xenon. (a) Left panel: Sample images of immunofluorescent dual-labeling of hypoxia-inducible factor (HIF)-1a (Red) and Ki-67 þ (Green) in HK-2 cells after xenon± aIR3 treatment. Right panel: Percentage of proliferating cells labeled with Ki-67 relative to total cell number, with pre- or post-xenon±aIR3 treatment, after hypoxia-reoxygenation. (b) Nuclear factor (NF)-kB expression on HK-2 after hypoxia-reoxygenation. Data are means±s.d. (n ¼ 4), significance level is shown as *Po0.05 and ***Po0.001. Bar ¼ 50 mm. DAPI, 4,6-diamidino-2-phenylindole; NC, naive control. Kidney International (2014) 85, 112–123

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Figure 7 | Insulin growth factor-1 receptor (IGF-1R) expression, cell death, and cell proliferation in cortical tubular cells after grafting. The Fischer renal graft was stored at 4 1C Soltran preserving solution for 16 h (cold ischemia 16 h) and then transplanted into the Lewis recipient; the graft was harvested 24 h after transplantation (warm reperfusion 24 h). The xenon gas exposure (70% Xe balanced with 30% O2 for 2 h) was given either to donor (XD, xenon-donor treatment) 24 h before donor organ retrieval or to recipient (XR, xenon-recipient treatment) immediately after organ engraftment. Animals receiving nitrogen gas (70% N2 balanced with 30% O2) in donor stage (ND, nitrogen-donor treatment) or recipient (NR, nitrogen-recipient treatment) served as treatment controls. (a) DAB (3,3-diaminobenzidine tetrahydrochloride) staining (brown) of IGF-1R in renal tubules; nuclei were counterstained with hematoxylin (blue). (b) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling) staining (brown) of renal tubules; nuclei were counterstained with methyl green (green). (c) Immunoflorescence staining of Ki-67 (red) in renal tubules; nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole; blue). (d) Serum creatinine and (e) urea concentrations were analyzed and expressed as mean±s.d. (n ¼ 4); *Po0.05, **Po0.01, and ***Po0.001; bar ¼ 50 mm. NC, naive control.

Xenon treatment attenuated the development of CAN and preserved the renal function

On day 40 after surgery, the mean serum creatinine concentration for surviving recipients in the nitrogen-treated group was significantly higher compared with that of xenontreated rats (312±84 vs. 174±46 mmol/l in donor treatment, Po0.001and 338±65 vs. 197±51 mmol/l in recipient treatment, Po0.01). Similarly, a lower urea level was found in the xenon-treated group (33.8±7.7 vs. 15.75±5.6 mmol/l in donor treatment, Po0.05 and 30.3±5.0 vs. 21.3±4.2 mmol/l in recipient treatment, Po0.05; Figure 10a). In nitrogentreated control grafts, marked destruction of renal tissue was found on day 1 after transplantation. Renal proximal tubules demonstrated severe structural deformity and collapse, with infiltration of mononuclear cells. In contrast, allografts from xenon-treated recipients were well preserved, and cell infiltration was reduced. Tubular structure remained mostly 118

normal, with no apparent protein deposition throughout the graft tissue. At 2 months after transplantation, renal allografts from the nitrogen-treated group demonstrated the histological evidence of CAN when compared with those receiving xenon. Substantially, less tissue damage, such as tubulitis, glomerulitis, vasculitis, fibrosis, and tubular atrophy, was found in renal grafts treated with xenon (Figure 10b).

DISCUSSION

In the current study, we have demonstrated that early treatment with xenon reduced the IRI and prevented pathological progression associated with CAN in the Fischer-to-Lewis allografts. Enhanced cellular proliferation and elevated IGF-1/IGF-1R signaling cascade after xenon exposure might be responsible for the rapid recovery of renal grafts from initial injury and suppression of the later CAN. Kidney International (2014) 85, 112–123

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Figure 8 | Effects of xenon treatment on tubular inflammation after grafting. The Fischer renal graft was stored at 4 1C Soltran preserving solution for 16 h (cold ischemia 16 h) and then transplanted into Lewis recipient. The graft was harvested 24 h after transplantation (warm reperfusion 24 h). The xenon gas (70% Xe balanced with 30% O2 for 2 h) was given either to donor (XD, xenon-donor treatment) 24 h before donor organ retrieval or to recipient (XR, xenon-recipient treatment) immediately after organ engraftment. Animals receiving nitrogen gas (70% N2 balanced with 30% O2) in donor stage (ND, nitrogen-donor treatment) or recipient (NR, nitrogen-recipient treatment) served as a treatment control. (a) DAB (3,3-diaminobenzidine tetrahydrochloride) staining (brown) of intercellular adhesion molecule 1 (ICAM-1) in renal tubules; nuclei were counterstained with hematoxylin. (b) Immunoflorescence staining of CD68 þ (red) macrophages in renal tubules; nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole; blue). (c) Serum tumor necrosis factor (TNF)-1a and interleukin (IL)-1b concentrations were analyzed by enzyme-linked immunosorbent assay and expressed as mean±s.d. (n ¼ 4); *Po0.05 and **Po0.01; bar ¼ 50 mm. NC, naive control.

In the data presented here, signs of CAN such as glomerulosclerosis, interstitial fibrosis, arterial obliteration, and tubular atrophy became apparent after ischemic insult; all these features were attenuated by xenon treatment through mitigating IRI. IRI induced acute tubular apoptosis and/or necrosis. Restoration of the renal function relies on the proliferation and differentiation of surviving tubular cells in the region of damage.15,16 In vitro, xenon treatment enhanced the production of IGF-1 and the upregulation of IGF-1R. HK-2 cell cycle progression was accelerated, indicated by high expression levels of cyclin D and cyclin E. IGF-1 signaling regulates renal cell growth and proliferation; in addition, IGF-1 signaling blocks apoptosis by inhibition of proapoptotic proteins, such as BAX and BAD.17,18 IGF-1 receptor is a tyrosine kinase receptor, and its ligation with IGF-1 promotes cell survival and cell proliferation, which accelerate the recovery from IRI. Activation of the IGF-1/ IGF-1R signaling pathway after xenon treatment might be responsible for the functional recovery of kidney from acute insults. Previous studies have shown that the IGF-1/IGF-1R signaling pathway acted as an important mediator of cardiac and kidney regeneration.19,20 IGF-1/IGF-1R autocrine production has also been detected in post-ischemic kidney, Kidney International (2014) 85, 112–123

which mediates self-repair in the proximal tubular cells,21 and, in addition, it was shown that IGF-I administered before or after acute ischemic injury to rats accelerates the recovery of renal function and the regeneration of damaged proximal tubular epithelium.22 Signaling through IGF-1R activated mTOR and HIF-1a, which enhanced DNA synthesis and facilitated cell cycle progression.23 In our study, IGF-1Rneutralizing antibodies, rapamycin, and HIF-1a siRNA attenuated HK-2 proliferation enhanced by xenon, which confirmed their involvement in xenon-mediated cytoprotection. However, whether xenon promotes IGF-1 or its receptor synthesis or how xenon interacts with IGF-1 receptor warrants further investigation in the future studies. The blood–gas partition coefficient of xenon is generally accepted as 0.14, with a recent measurement of 0.115,24 and therefore xenon is the least soluble gas used for anesthesia.25 This property leads to rapid fall in the body at the end of gas exposure, with xenon relative to other gas.26 Xenon treatment at 70% was well established in rodents8,11,27 without compromising oxygenation,28 which is also true according to our pilot studies (see Methods). Our data indicated that a strategy targeting HIF-1 in transplantation might be a potential organo-protective 119

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CD8

CD3

CD4

XD

ND

XD

ND

XD

NR

XR

NR

XR

NR

XR

*

100 50 0 NC

ND

XD

NR

XR

**

200 150 100 50 0 NC

1 Month after transplantation

***

Number of CD4+ T cells in renal cortex

**

150

Number of CD8+ T cells in renal cortex

Number of CD3+ T cells in renal cortex

ND

ND

XD

NR

100

*

80 60 40 20 0

XR

NC

1 Month after transplantation

ND

NR

XR

MTS-stained blue area (%)

MTS XD

NR

XR

1 Month after transplantation 50

ND

XD

**

*

40 30 20 10 0 NC

ND

XD

NR

XR

1 Month after transplantation

Figure 9 | Effects of xenon treatment on T-cell infiltration and renal fibrosis after grafting. The Fischer renal graft was stored at 4 1C Soltran preserving solution for 16 h (cold ischemia 16 h) and then transplanted into the Lewis recipient; the graft was harvested 24 h after transplantation (warm reperfusion 24 h). The xenon gas (70% Xe balanced with 30% O2 for 2 h ) was given either to donor (XD, xenon-donor treatment) 24 h before donor organ retrieval or to recipient (XR, xenon-recipient treatment) immediately after organ engraftment. Animals receiving nitrogen gas (70% N2 balanced with 30% O2) in donor stage (ND, nitrogen-donor treatment) or recipient (NR, nitrogen-recipient treatment) served as treatment control. DAB (3,3-diaminobenzidine tetrahydrochloride) staining (brown) of (a) CD3 þ , (b) CD8 þ , and (c) CD4 þ in renal tubules; nuclei were counterstained with hematoxylin (blue). (d) Masson trichrome staining (MTS) of collagen (blue) at 1 month after surgery. Data were expressed as mean±s.d. (n ¼ 4); *Po0.05 and **Po0.01; bar ¼ 50 mm. NC, naive control.

strategy. Recent elegant work by Bernhardt et al.29 showed that donor pretreatment with a PHD (prolyl hydroxylase domain) inhibitor, FG-4497, resulted in HIF-1 accumulation and induction of HIF-1 downstream proteins, which were detected beyond cold storage. The frequency of delayed graft function was significantly reduced, and a long-term graft survival was markedly improved. In line with that study, our data also indicated that the long-term outcomes of organ transplantation could be improved by inducing activation of the HIF-1 system by xenon before organ retrieval. Although the exact mechanisms of enhanced inflammatory and fibrogenic changes in allografts after IRI remain incompletely understood, insufficient nephron numbers might have a significant role. Several clinical reports support the concept that insufficient renal mass is linked with the early development of CAN.30–32 Proinflammatory cytokines released by infiltrating cells and resident cells promote the fibroblast to adapt to a fibrogenic phenotype and produce basement membrane and extracellular matrix molecules, such as collagens and fibronectins.33 All these pathological 120

events lead to the initiation of CAN. Recovery from initial insults with xenon treatment may preserve more functional nephrons in the long term. In addition, xenon treatment decreased the release of proinflammatory cytokines, inhibited the fibroblast transformation to myofibroblasts and subsequent formation of fibrotic lesions, and, therefore, it prevented the development of CAN. CD3 þ , CD4 þ , and CD8 þ T-cell infiltrations in the renal grafts were significantly reduced in xenon-treated allografts, indicating that alloimmune response was attenuated by this treatment. The anti-inflammatory effects might be associated with anti-apoptotic effects of xenon, mediated by IGF-1/IGF-1R. In our studies, both pretreatment and post treatment with xenon conferred protection in the renal grafts, and it might be reasonable in future studies to evaluate whether combined therapy of pre- and post-xenon treatment could achieve even better protection than either alone. Given the profound impact of CAN for transplant patients and the largely ineffective therapeutic interventions, the development of efficient therapeutic strategies is required to Kidney International (2014) 85, 112–123

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*

400 300 200 100

40 30 20 10

NC

ND

NC

XD

ND

300 200 100 NR

40 Days after transplantation

40 Days after transplantation

30 20 10

XR

NC

40 Days after transplantation

NR

XR

40 Days after transplantation

ND

XD

NR

XR

ND

XD

NR

XR

Day 60

Day 1

b

40

0 NC

XD

*

50

400

0

0

0

**

500 Creatinine (umol/l)

50

Urea (mmol/l)

***

500

Urea (mmol/l)

Creatinine (μmol/l)

a

Figure 10 | Effect of xenon treatment on the development of chronic allograft nephropathy. Fischer renal grafts after 16 h cold ischemia (through storage in 4 1C Soltran preserving solution) were transplanted into the Lewis recipient. Xenon treatment (70% xenon balanced with 30% nitrogen for 2 h) was given either to donor before organ retrieval (XD) or to recipient immediately after engraftment (XR). Nitrogen gas to donor (ND, nitrogen-donor treatment) or to recipient (NR, nitrogen-recipient treatment) served as treatment control. Contralateral kidney was removed day 4 after transplant surgery. (a) Serum creatinine and urea concentrations 40 days after surgery were analyzed, and data are expressed as mean±s.d. (n ¼ 6; Po0.05, XD vs. ND or XR vs. NR). (b) Histology of the renal graft on day 1 and 2 months after transplant surgery. *Po0.05, **Po0.01 and ***Po0.001. Bar ¼ 50 mm. Arrow indicates injury in renal tubules. NC, naive control.

prolong the allograft survival. Xenon is protective against IRI when given before or immediately after the insults, which could potentially delay the onset of CAN. Xenon has a safety profile and has been used as anesthetics clinically,25 and, therefore, if our preclinical study setting can be extrapolated at a clinical level, then xenon could be readily exploited for the clinical treatment of renal graft ischemia reperfusion. At present, several multicenter randomized clinical trials are ongoing to assess neuroprotective and cardioprotective properties of xenon. On the basis of our experimental data, the potential therapeutic value of xenon in preventing the onset of CAN necessitates for further preclinical studies toward initiating clinical trials. MATERIALS AND METHODS In vitro cell culture, hypothermia-hypoxia challenge, and conditioned medium Human proximal tubular HK-2 cells and human monocyte/ macrophage U937 cells (European Cell Culture Collection) were cultured in RPMI 1640 medium, and human fibroblasts (Cambridge bioscience, UK) were cultured in Dulbecco’s modified Eagle’s medium; the culture medium was supplemented with 10% fetal bovine serum (Invitrogen, Paisley, UK), 2 mmol/l L-glutamine (Invitrogen), and 100 U/ml penicillin–streptomycin (Invitrogen). HK-2 were then incubated at 4 1C for 16 h in Soltran preserving solution (Baxter Healthcare, Newbury, UK) in a closed and purposebuilt airtight chamber containing 8% O2 and 5% CO2 balanced with N2. Cells were then recovered for 24 h at 37 1C in RPMI 1640 medium in normal cell incubator. After hypoxia-reoxygenation, HK-2 cell medium was collected and was used to stimulate macrophage and fibroblasts. Kidney International (2014) 85, 112–123

Xenon exposure in vitro Xenon treatment was given to HK-2 cells either 24 h before (pretreatment) or after hypoxia (post treatment). Cells were incubated in the RPMI medium pre-bubbled 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 37 1C in the gas chamber mentioned above. The treated cells were recovered in RPMI medium in normal incubator at 37 1C for 24 h. Determination of protein expression in vitro by flow cytometry HK-2 cells were incubated with fluorochrome-conjugated anti-IGF1R antibody (e-Bioscience, Cambridge, UK). U937 cells were incubated with fluorochrome-conjugated anti-TLR-4(e-Bioscience) and anti-CD86 (e-Bioscience) or their isotype controls for 30 min at 4 1C, washed with fluorescence-activated cell sorting buffer. Immunofluorescence intensity was acquired and analyzed using flow cytometry (FACSCalibur; Becton Dickinson, Sunnyvale, CA). Each assay included at least 20,000 gated events. Cell treatments in vitro To assess the role of mTOR and HIF-1a, some cohort cultures were treated with rapamycin (50 nmol/l) or vehicle (Tocris, Abingdon, UK) for 30 min before xenon treatment, whereas some cohort cultures were treated with human HIF-1a siRNA or scrambled siRNA (Qiagen, Crawley, West Sussex, UK). In vitro HIF-1a siRNA transfections were carried out using lipofectamine (Invitrogen). siRNA-targeting human HIF-1a (Qiagen; sense strand: 50 -GAAGAACUAUGAACAUAAATT-30 and antisense strand: 50 -UUUAUGUUCAUAGUUCUUCCT-30 ) was dissolved in siRNA suspension buffer and administered to HK-2 cells at a dose of 20 nmol/l; scrambled siRNA served as a negative control. Cells were incubated 121

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with siRNA for 6 h at 37 1C in humidified air containing 5% carbon dioxide, after which it was removed and replaced with experimental medium followed by xenon gas treatment. To assess the role of the IGF-1 pathway in cell recovery after hypoxia-hypothermia, IGF-1Rneutralizing antibodies aIR3 (Millipore, Livingston, UK) at a dose of 1 mg/ml was administered to cell culture together with xenon gas exposure before (xenon pretreatment) or after (xenon post treatment) the hypothermia-hypoxia challenge. Animals Inbred adult male Lewis rats and Fischer Rats weighing 225–250 g were purchased from Harlan, Bicester, UK, and bred in temperatureand humidity-controlled cages in a specific pathogen-free facility at Chelsea-Westminster Campus, Imperial College London, London, UK. This study was approved by the Home Office, UK, and all animal procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Renal transplantation Fisher (F344, RT11vr)-to-Lewis (LEW, RT11) rat renal transplantation was used as the CAN model. Rat donor kidneys were transplanted orthotopically into recipients using standard microvascular techniques. In brief, the donor left kidney, aorta, and inferior vena cava were carefully exposed, and the kidney graft was then extracted, flushed, and stored in 4 1C heparinized Soltran Preserving Solution (Baxter Healthcare UK). After the specified period of cold ischemia, the recipient’s left kidney was extracted, and anastomosis of donor and recipient renal artery, vein, and ureter was performed with 8-0 sutures. The total surgical ischemia time was restricted to o45 min. The contralateral native kidney was excised either immediately after surgery (acute model, animal killed on day 1 after surgery) or 4 days later (long-term model). All allograft recipients were treated with low-dose cyclosporine A (5 mg/kg/day) for 10 days after surgery to prevent allograft loss from acute rejection. Gas exposure in vivo Rats were exposed to either 70% xenon or 70% nitrogen balanced with 30% O2 for 2 h via an anesthetic chamber with a closed-circle system reported previously.7,8 Animals had good oxygenation with these gas mixture treatment found from our pilot studies (xenon vs. nitrogen mean±s.d. (n ¼ 4): PaO2 170±15 mm Hg vs. 175±17; PaCO2 37±4 mm Hg vs. 42±4 and PaO2 180±9 mm Hg vs. 179±18; PaCO2 41±5 mm Hg vs. 43±6 after 1 and 2 h exposure, respectively). Gas exposure was given either to donor 24 h before organ retrieval (donor pretreatment) or to recipient immediately after grafting (recipient post treatment). The 2-h duration of xenon exposure and 24-h gap between exposure and organ retrieval were well established in the previous studies.7,8,11 Gas concentrations of xenon and oxygen were constantly monitored by xenon monitor (Air Products Model No. 439Xe, Bradford, UK) and Datex monitor (Datex-Ohmeda, Bradford, UK). Hematoxylin and eosin staining The animals were perfused with 4% paraformaldehyde, and the transplanted kidneys were bisected along the long axis and were fixed in 4% parafamydehyde and paraffin. Sections of 5-mm thickness were taken from the kidney specimens, and hematoxylin and eosin staining was performed. 122

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TUNEL staining Apoptosis of tubular epithelial cells was detected by the in situ TUNEL assay (Millipore, UK) according to the manufacturer’s instructions. The fixed paraffin sections were deparaffinized and washed with phosphate-buffered saline (PBS). The sections were labeled with terminal deoxynucleotidyl transferase (TDT enzyme) at 37 1C for 1 h. Thereafter, the slides were incubated with antidigoxygenin conjugated to horseradish peroxidase for 30 min at room temperature. TUNEL þ nuclei were visualized by 3,3-diaminobenzidine tetrahydrochloride (DAB), followed by counterstaining with methyl green. Ten areas of renal cortex (with at least four renal tubules) under 40 magnification were randomly selected, and the numbers of TUNEL þ cells per renal tubule were counted by an investigator who was unaware of the treatment. The tubular necrosis level is expressed as the percentage of TUNEL þ cells per tubule. Masson trichrome staining (MTS) MTS was performed on paraffin-embedded tissue according to the manufacturer’s instructions (MTS kit, Sigma-Aldrich, Poole, UK). The amount of collagen deposition (percentage of MTS-stained blue areas) was then digitally quantified using the Image J software (NIH, US National Institutes of Health, Bethesda, MD). Immunohistochemistry For in vivo DAB staining, 5-mm-thick paraffin sections were first dewaxed and subjected to heat-mediated antigen retrieval. Sections were exposed to 3% H2O2 in methanol for 10 min to quench endogenous peroxidases and then incubated with donkey serum followed by the primary antibody: rabbit anti-IGF-1R (1:200, Abcam, Cambridge, UK), mouse anti-ICAM-1 (1:200, Abcam), rabbit anti-CD3 (1:200, Abcam), mouse anti-CD4 (1:200, Abcam), and mouse anti-CD8 (1:200, Abcam) at 4 1C overnight. For in vivo fluorescence staining, 25-mm-thick frozen sections were incubated with rabbit anti-Ki-67 (1:200, Abcam) and mouse anti-CD68 (1:200, Abcam) at 4 1C overnight. After washing with PBS-Tween 20, the slides were incubated with biotin- or fluorochrome-conjugated secondary antibodies (Millipore, UK). The antibody staining patterns were visualized with DAB or fluorescence. Sections were counterstained with hematoxylin (DAB staining) or DAPI (4,6diamidino-2-phenylindole; fluorescence staining). For in vitro fluorescence staining, cells were fixed in paraformaldehyde in 0.1 mol/l PBS solution. Cells were then incubated in 10% normal donkey serum in 0.1 mol/l PBS-Tween 20 and then incubated overnight with rabbit anti-cyclin D (1:200, Abcam), rabbit anticyclin E (1:200, Abcam), rabbit anti-HIF-1a (1:200, Novus, Littleton, CO), mouse anti-Ki-67 (1:200, DAKO, Cambridge, UK), rabbit anti-p65 NF-kB (1:200, Abcam), rabbit anti-TGF-b (1:200, Abcam), and rabbit anti-cleaved caspase-3 (1:200, Cell Signalling, Danvers, MA), followed by incubation with secondary antibody for 1 h. HIF-1a/Ki-67 was used for double-labeling immunofluorescence. HK-2 cells were incubated with the first primary antibody overnight, followed by the first secondary antibody and then the second primary antibody and the second secondary antibody. The slides were counterstained with nuclear dye DAPI and mounted with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA). Sections were examined using an Olympus (Watford, UK) BX4 microscope. Immunofluorescence or DAB staining was quantified using ImageJ (National Institutes of Health). Ten representative regions per section (in vivo) or field (in vitro) were randomly selected by an assessor blinded to the treatment groups. Values were then Kidney International (2014) 85, 112–123

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calculated as percentages of the mean value for naive controls and expressed as the percentage of fluorescence or DAB intensity. Western blotting Cultured cells were mechanically homogenized in the lysis buffer. The cell lysates were centrifuged and then supernatant was collected and total protein concentration in the supernatant was quantified by the Bradford protein assay (Bio-Rad, Hemel Hempstead, UK). The protein extracts (40 mg/sample) were heated, denatured, and loaded on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) for electrophoresis and then transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was treated with blocking solution (5% dry milk in TBS with 0.1% Tween-20 (TBS-T)) for 2 h and probed with the following primary antibodies: rabbit anti-IGF-1 (1:1000, Abcam), rabbit anticyclin D (1:1000, Abcam), and rabbit anti-cyclin E (1:1000, Abcam) in TBS-T overnight at 4 1C, followed by horseradish peroxidase-conjugated secondary antibody for 1 h. The loading control was the constitutively expressed protein a-tubulin (1:10000, Sigma-Aldrich). The blots were visualized with the enhanced chemiluminescence system (Santa Cruz Biotechnology, Santa Cruz, CA) and analyzed with GeneSnap (Syngene, Cambridge, UK). Protein band intensity was normalized with a-tubulin and expressed as ratio of control for data analysis. Enzyme-linked immunosorbent assay (ELISA) Rat TNF-a and IL-1b were measured by ELISA (Rat TNF-a and IL1b ELISA kits, Invitrogen). 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±s.d. Comparison between the treatment groups was analyzed by one-way analysis of variance, followed by the post hoc Student–Newman–Keuls test (GraphPad Prism 5.0 software, San Diego, CA). A P-value o0.05 was considered to be statistically significant. DISCLOSURE

All the authors declared no competing interests.

8. 9.

10.

11.

12.

13.

14. 15. 16. 17.

18.

19.

20.

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23.

24. 25. 26.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Council (MRC)Developmental Pathway Funding Scheme (DPFS) (G802392).

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28.

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