Volume 87, Issue 4, Pages 738-748 (April 2015) Necroptosis and parthanatos are involved in remote lung injury after receiving ischemic renal allografts in rats  Hailin Zhao, Jiaolin Ning, Alexandre Lemaire, Foteini-Stefania Koumpa, James J. Sun, Anthony Fung, Jianteng Gu, Bin Yi, Kaizhi Lu, Daqing Ma  Kidney International  Volume 87, Issue 4, Pages 738-748 (April 2015) DOI: 10.1038/ki.2014.388 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 Renal graft ischemia-reperfusion injury led to remote lung injury. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation) or 24h (renal graft cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient; the renal graft and lung were harvested on day 1 after transplantation. The contralateral kidney was removed or kept (CLK group). (a) Histology (hematoxylin and eosin staining) of renal grafts and lung. (b) Injury scores of kidney morphology. (c) Injury scores of lung morphology, (d) serum concentration of tumor necrosis factor-α (TNF-α), and (e) lung tissue concentration of TNF-α assessed by enzyme-linked immunosorbent assay. (f) Nuclear release of high-mobility group protein B1 (HMGB-1) in lung. (g) CD 68+ cell infiltration in lung. (h) Dry/wet ratio of lung samples. Scale bar=50μm. Data are expressed as mean±s.d. (n=4), (*P<0.05, **P<0.01, and ***P<0.001). CLK, (with) contralateral kidney; NC, naive control; rCI, renal graft cold ischemia. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Inflammation in lung tissues of recipients receiving ischemic allografts. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation or 24h (cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient, and the lung was harvested on day 1 after transplantation. Expression of (a) Poly (ADP-ribose) polymerase 1 (PARP-1) (red fluorescence) and (b) Poly(ADP-ribose) (PAR) (green fluorescence) in lung epithelial cells after grafting ischemic and non-ischemic renal grafts; nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI); normal lung as naive control. (c) Western blot analysis of PARP-1, fluorescent intensity of (d) PARP-1 and (e) PAR in the lungs. Double labeling of (f) PARP-1/TLR-4, (g) PARP-1/TLR-2, and (h) PARP-1/NF-κB. Nuclei were counterstained with DAPI; normal lung as naive control. Fluorescent intensity of (i) TLR-4, (j) TLR-2, and (k) NF-κB in the lungs. Scale bar=50μm. Data are expressed as mean±s.d. (n=4), (**P<0.01 and ***P<0.001). NC, naive control; rCI, renal graft cold ischemia. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Necroptosis was found in lung tissues of recipients receiving ischemic allografts. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation) or 24h (cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient, and the lung was harvested on day 1 after transplantation. Expression of (a) tumor necrosis factor receptor 1 (TNFR1) (green fluorescence), (b) receptor-interacting protein kinase 1 (Rip1) (green fluorescence), and (c) Rip3 (red fluorescence) in lung epithelial cells after grafting with ischemic and non-ischemic renal grafts; nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI); normal lung as naive control. Fluorescent intensity of (d) TNFR1, (e) Rip1, and (f) Rip3 in the lungs. (g) Western blot of RIP-1 expression. (h) Tdt-mediated dUtp nick end labeling (TUNEL) staining of lung tissue after receiving non-ischemic or ischemic renal grafts; co-staining with DAPI confirmed that the TUNEL staining is within the nuclei of lung alveolar cells (arrows). (i) Number of TUNEL+ cells per × 20 field in the lungs. Dual labeling of (j) Poly (ADP-ribose) polymerase 1 (PARP-1)/Rip1 and in the lungs. Scale bar=50μm. Data are expressed as mean±s.d. (n=4), (**P<0.01 and ***P<0.001). NC, naive control; rCI, renal graft cold ischemia. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor 3-aminobenzamide (3-AB) or/and receptor-interacting protein kinase 1 (RIP-1) inhibitor necrostatin-1 (Nec-1) attenuated lung injury in vivo after receiving ischemic allografts. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation) or 24h (cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient, and the graft was finally harvested on day 1 after transplantation. 3-AB or/and Nec-1 was given to the recipient immediately after grafting. Expression of (a) PARP-1 (red), (b) PAR (green), and (c) Rip1 (green) in lung epithelial cells after grafting ischemic grafts; nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI); normal lung as naive control. (d) Histology (hematoxylin and eosin staining) of lung tissue. (e) Tdt-mediated dUtp nick end labeling (TUNEL) staining of lung tissue. Scale bar=50μm. (f) Lung injury score. (g) Number of TUNEL+ cells per × 20 field. Scale bar=50μm. Data are expressed as mean±s.d. (n=4), (*P<0.05, **P<0.01, and ***P<0.001). NC, naive control; rCI, renal graft cold ischemia; Ve, PBS vehicle. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 Acute immune rejection to renal allograft exacerbated the lung injury and enhanced Poly (ADP-ribose) polymerase 1 (PARP-1) activities in the lung. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation) or 24h (cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient, and the graft was finally harvested on day 4 after transplantation. (a) Histology (hematoxylin and eosin staining) of renal allograft and lung tissue. (b) CD3+ T cell (brown) infiltration in renal allograft, indicating development of immune rejection. Nuclei were counterstained with hematoxilin (blue). (c) Serum concentration of tumor necrosis factor-α (TNF-α) and (d) lung tissue concentration of TNF-α assessed by enzyme-linked immunosorbent assay. (e) Tdt-mediated dUtp nick end labeling (TUNEL) staining of lung tissue. (f) Number of TUNEL+ cells per × 20 field in the lungs. (g) Lung injury score. Expression of (h) PARP-1 (red) and (i) PAR (green) in lung epithelial cells after grafting with ischemic and non-ischemic grafts; nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI); normal lung as naive control. Fluorescent intensity of (j) PARP-1 and (k) PAR in the lungs. Scale bar=50μm. Data are expressed as mean±s.d. (n=4), (*P<0.05, **P<0.01, and ***P<0.001). NC, naive control; rCI, renal graft cold ischemia. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 Combined treatment of cyclosporine A and 3-aminobenzamide or/and necrostatin-1 attenuated the lung injury. Brown–Norway renal graft was stored in 4°C Soltran preserving solution for 0 (live transplantation) or 24h (cold ischemia rCI0 or 24h) and then transplanted into Lewis recipient, and cyclosporine A combined with 3-AB or/and nec-1 was given to the recipient for 4 days, and the renal graft and lung were harvested on day 4 after transplantation. (a) Histology (hematoxylin and eosin (H and E) staining) of renal allograft. (b) CD3+T cell (brown) infiltration in renal allograft, indicating suppression of immune rejection by cyclosporine A. Nuclei were counterstained with hematoxilin (blue). (c) Serum concentration of tumor necrosis factor-α (TNF-α) assessed by enzyme-linked immunosorbent assay. (d) Histology (H and E staining) of lung tissue. (e) Tdt-mediated dUtp nick end labeling (TUNEL) staining (green) of the lung tissue. Scale bar: 50μm. Data are expressed as mean±s.d. (n=4), (*P<0.05), CyA, cyclosporine A; Nec-1, necrostatin-1; rCI, renal graft cold ischemia; 3-AB, 3-aminobenzamide. Kidney International 2015 87, 738-748DOI: (10.1038/ki.2014.388) Copyright © 2015 International Society of Nephrology Terms and Conditions