Volume 85, Issue 1, Pages 112-123 (January 2014) Early treatment with xenon protects against the cold ischemia associated with chronic allograft nephropathy in rats  Hailin Zhao, Xianghong Luo, Zhaowei Zhou, Juying Liu, Catherine Tralau-Stewart, Andrew J.T. George, Daqing Ma  Kidney International  Volume 85, Issue 1, Pages 112-123 (January 2014) DOI: 10.1038/ki.2013.334 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Prolonged cold ischemia (CI) induced early onset of chronic allograft nephropathy. The Fischer renal graft was stored in 4°C Soltran preserving solution for 0h (quick flush, immediate engraftment), 4h, or 16h (CI) and then transplanted into Lewis recipient. (a) Histology (H and E staining) of the allograft with CI 0–16h 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=4, *P<0.05 and ***P<0.001). Bar=50μm. Arrow indicates injury in renal tubules. NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

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 2h and then recovered in the normal cell incubator for 24h. Expression levels of (a) IGF-1 was assessed by western blotting and that of (b) IGF-1R was assessed by flow cytometry at 24h 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=4). *P<0.05; **P<0.01 and ***P<0.001. Bar=50μm. MFI, mean fluorescence intensity; NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Xenon treatment enhanced cell proliferation and decreased caspase-3 and nuclear factor (NF)-κB 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 2h and then recovered in the normal cell incubator for 24h. (a) Ki-67 (green) expression in HK-2 cells. (b) Percentage of Ki-67+ cells after gas exposure. (c) Immunofluorescent dual-labeling of hypoxia-inducible factor (HIF)-1α (Red) and Ki-67 (Green) in HK-2 cells is shown. HK-2 cells were subjected to 16h hypothermia-hypoxia insults (4°C Soltran preserving solution under 8% O2), followed by 24h reoxygenation (37°C culture medium in normal cell incubator). A duration of 2h xenon or nitrogen gas exposure was given either 24h 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-κB (red) expression patterns on HK-2 are shown. Analysis of fluorescence intensity of (f) caspase-3 and (g) NF-κB is shown. Data are means±s.d. (n=4), significance level is shown as *P<0.05; **P<0.01 and ***P<0.001. Bar=50μm. DAPI, 4,6-diamidino-2-phenylindole; NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Effects of human proximal tubular (HK-2) conditioned medium on monocytes and fibroblasts. (a) HK-2 cells were subjected to 16h hypothermia-hypoxia (4°C Soltran preserving solution under 8% O2), followed by 24h reoxygenation (37°C culture medium in normal cell incubator). A duration of 2h gas exposure (70% Xe or N2 and 5% CO2 balanced with O2) was given either 24h 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)-β expression on fibroblasts, assessed by immunofluorescence at 24h after conditioned medium (CM) challenge. Fluorescence intensity is expressed as means±s.d. (n=4), significance level is shown as *P<0.05. Bar=50μm. MFI, mean fluorescence intensity; NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Effect of rapamycin and hypoxia-inducible factor (HIF)-1α 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-1α siRNA (20nmol/l) for 6h, or rapa 50nm for 30min, followed by xenon gas (70% Xe and 5% CO2 balanced with O2) for 2h and then recovered in normal cell incubator for 24h. (a) Immunofluorescent dual-labeling of HIF-1α (Red) and Ki-67 (Green) in HK-2 cells after rapa–xenon treatment. (b) The percentage of Ki-67+ proliferating cells after rapa–xenon treatment. (c) Immunofluorescent dual-labeling of HIF-1α (Red) and Ki-67+ (Green) in HK-2 cells after siRNA–xenon treatment. (d) The percentage of Ki-67+ proliferating cells after siRNA–xenon treatment. Data are expressed as mean±s.d. (n=4); ***P<0.001. Bar=50μm. DAPI, 4,6-diamidino-2-phenylindole; NC, naive control; rapa, rapamycin; Vh, vehicle. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Effect of insulin growth factor-1 receptor (IGF-1R)-neutralizing antibody αIR3 on the xenon-treated human proximal tubular (HK-2) cells. HK-2 cells were given 16h hypothermia-hypoxia challenge (4°C Soltran preserving solution under 8% O2), followed by 24h reoxygenation (37°C culture medium in normal cell incubator). A duration of 2h gas exposure (70% Xe or N2, and 5% CO2 balanced with O2) was given either 24h 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 posttreatment). IGF-1R-neutralizing αIR3 antibody was coadministered with xenon. (a) Left panel: Sample images of immunofluorescent dual-labeling of hypoxia-inducible factor (HIF)-1α (Red) and Ki-67+ (Green) in HK-2 cells after xenon± αIR3 treatment. Right panel: Percentage of proliferating cells labeled with Ki-67 relative to total cell number, with pre- or post-xenon±αIR3 treatment, after hypoxia-reoxygenation. (b) Nuclear factor (NF)-κB expression on HK-2 after hypoxia-reoxygenation. Data are means±s.d. (n=4), significance level is shown as *P<0.05 and ***P<0.001. Bar=50μm. DAPI, 4,6-diamidino-2-phenylindole; NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

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°C Soltran preserving solution for 16h (cold ischemia 16h) and then transplanted into the Lewis recipient; the graft was harvested 24h after transplantation (warm reperfusion 24h). The xenon gas exposure (70% Xe balanced with 30% O2 for 2h) was given either to donor (XD, xenon-donor treatment) 24h 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=4); *P<0.05, **P<0.01, and ***P<0.001; bar=50μm. NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Effects of xenon treatment on tubular inflammation after grafting. The Fischer renal graft was stored at 4°C Soltran preserving solution for 16h (cold ischemia 16h) and then transplanted into Lewis recipient. The graft was harvested 24h after transplantation (warm reperfusion 24h). The xenon gas (70% Xe balanced with 30% O2 for 2h) was given either to donor (XD, xenon-donor treatment) 24h 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+ (red) macrophages in renal tubules; nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole; blue). (c) Serum tumor necrosis factor (TNF)-1α and interleukin (IL)-1β concentrations were analyzed by enzyme-linked immunosorbent assay and expressed as mean±s.d. (n=4); *P<0.05 and **P<0.01; bar=50μm. NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 Effects of xenon treatment on T-cell infiltration and renal fibrosis after grafting. The Fischer renal graft was stored at 4°C Soltran preserving solution for 16h (cold ischemia 16h) and then transplanted into the Lewis recipient; the graft was harvested 24h after transplantation (warm reperfusion 24h). The xenon gas (70% Xe balanced with 30% O2 for 2h) was given either to donor (XD, xenon-donor treatment) 24h 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+, (b) CD8+, and (c) CD4+ 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=4); *P<0.05 and **P<0.01; bar=50μm. NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Effect of xenon treatment on the development of chronic allograft nephropathy. Fischer renal grafts after 16h cold ischemia (through storage in 4°C Soltran preserving solution) were transplanted into the Lewis recipient. Xenon treatment (70% xenon balanced with 30% nitrogen for 2h) 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=6; P<0.05, XD vs. ND or XR vs. NR). (b) Histology of the renal graft on day 1 and 2 months after transplant surgery. *P<0.05, **P<0.01 and ***P<0.001. Bar=50μm. Arrow indicates injury in renal tubules. NC, naive control. Kidney International 2014 85, 112-123DOI: (10.1038/ki.2013.334) Copyright © 2014 International Society of Nephrology Terms and Conditions