Volume 22, Issue 10, Pages 1817-1828 (October 2014) Serum-stabilized Naked Caspase-3 siRNA Protects Autotransplant Kidneys in a Porcine Model  Cheng Yang, Tian Zhao, Zitong Zhao, Yichen Jia, Long Li, Yufang Zhang, Mangen Song, Ruiming Rong, Ming Xu, Michael L Nicholson, Tongyu Zhu, Bin Yang  Molecular Therapy  Volume 22, Issue 10, Pages 1817-1828 (October 2014) DOI: 10.1038/mt.2014.111 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Caspase-3 mRNA and protein in the post-CS and post-transplant kidneys and caspase-3 mRNA in WBC. With caspase-3 siRNA treatment, the level of caspase-3 mRNA was significantly decreased in the post-CS and post-transplant kidneys compared to those with negative siRNA treatment or the IR control. (a) In addition, the expression of caspase-3 mRNA was significantly increased by the negative siRNA in the post-CS kidneys compared with the IR control. (b) The expressions of caspase-3 mRNA in WBC fluctuated, without significant difference between groups from day 1 to 14. (c) The caspase-3 siRNA directed caspase-3 mRNA degradation intermediates were detected in the caspase-3 siRNA treated post-CS and post-transplant kidneys by 5′-RACE PCR. The schematic picture showed the complementary binding sites of caspase-3 siRNA, its specific cleavage position between nucleotide 9 and 10 of the sense strain and the sequencing result of 5′-RACE PCR products. The numbers indicate the nucleotides of caspase-3 mRNA and the red characters indicate the sequence of 5′-RACE PCR products complementary to caspase-3 siRNA. (d) A total of 15 clones were sequenced and the frequency of each amplicon among 15 clones was listed at the end of the sequence. (e,f) The 32 kD caspase-3 precursor was significantly decreased in the caspase-3 siRNA group compared to the IR group in the post-CS kidneys with no significant difference among three groups in the post-transplant kidneys. (e,g) The 17 kD caspase-3 active subunit was significantly decreased in the caspase-3 siRNA-treated post-transplant kidneys when compared with either the negative siRNA group or the IR group. The expression of mRNA was presented as 2−ΔΔCt normalized with β-actin and related to the original level before transplantation and the protein level was expressed as corrected volume density against the loading control of 42 kD β-actin (mean ± SEM, n = 5). C3 siR, caspase-3 siRNA; IR, ischemia reperfusion; Neg siR, negative siRNA; Post-CS, post-cold storage; Post-Tx, post-transplant; WBC, white blood cell. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Apoptotic cells in the kidney detected by in situ end-labeling fragmented DNAs at ×400 magnification. (a) After transplantation, apoptotic cells were increased and mainly located in tubular and interstitial areas compared with the post-CS kidneys, some of which were shedding into tubular lumens. (b) With the treatment of caspase-3 siRNA, apoptotic cells were significantly decreased in both the post-CS and post-transplant kidneys in comparison with either the negative siRNA or IR group. Data were expressed as mean number in the high power field of each group (mean ± SEM; n = 5). C3 siR, caspase-3 siRNA; IR, ischemia reperfusion; Neg siR, negative siRNA; Post-CS, post-cold storage; Post-Tx, post-transplant. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Myeloperoxidase (MPO)+cells in the kidney examined by immunostaining at ×400 magnification. MPO+ cells were scattered in the post-CS kidneys, while most MPO+ cells in the post-transplant kidneys located in vascular lumens and interstitial areas. (a) It was also found that MPO+ cells penetrated through tubular areas, or demonstrated morphologic features of apoptosis such as condensed nuclei. (b) The number of MPO+ cells in the post-CS and post-transplant kidneys was remarkably decreased by caspase-3 siRNA treatment against both the negative siRNA and IR groups. Data were expressed as mean number in the high power field of each group (mean ± SEM, n = 5). C3 siR, caspase-3 siRNA; IR, ischemia reperfusion; Neg siR, negative siRNA; Post-CS, post-cold storage; Post Tx, post-transplant. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Cytokine and transcription factor mRNA in the kidney assessed by real-time quantitative polymerase chain reaction. (a–f) With caspase-3 siRNA treatment, the level of IL-1β, IL-6, NF-κB, IFN-α, IFN-β, IFN-γ, IRF3, IRF7, and IFITI mRNA was significantly decreased in the post-CS and post-transplant kidneys compared to those with negative siRNA treatment and IR alone. The expression of mRNA was presented as 2−ΔΔCt normalized with β-actin and related to the original level before transplantation (mean ± SEM, n = 5). C3 siR, caspase-3 siRNA; IR, ischemia reperfusion; Neg siR, negative siRNA; Post-CS, post-cold storage; Post Tx, post-transplant. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Peripheral blood IL-1β, IL-6, IL-8, TGF-β, IL-4, and IL-10 determined by multiplex Luminex technology and IFN-α and IFN-β detected by enzyme-linked immunosorbant assay. (a) The level of IL-1β was significantly decreased by caspase-3 siRNA from day 6 to 12 compared with the negative siRNA group, from day 7 to 14 compared to the ischemia reperfusion (IR) group. (b) A similar change trend was observed in IL-6 with significant differences between the caspase-3 siRNA group and other two groups from day 12–13. (c,d,g,h) The overall level of IL-8, TGF-β, IFN-α, and IFN-β was lower in the caspase-3 siRNA group with significant differences from day 6 to 13 and day 11 to 13 for IL-8; from day 5 to 6 and day 11 to 13 for TGF-β; from day 6 to 14 and day 5 to 14 for IFN-α; from day 5 to 14 and day 7 to 14 for IFN-β compared with the negative siRNA or IR group. (e,f) The trends in IL-4 and IL-10 were reversed with significantly higher levels in the caspase-3 siRNA group from day 4 to 8 and day 4 to 9 for IL-4; from day 3 to 6 and day 1 to 9 for IL-10 compared with the negative siRNA and IR group respectively. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Improvement of renal function and renal tissue damage with reduced extracellular matrix deposition. (a,b) SCr and urea nitrogen were gradually increased after transplantation, but significantly decreased by caspase-3 siRNA from day 5 or 6 to 14 compared to the negative siRNA group, and from day 11 to 14 compared to the IR group. The TID score was assessed in H&E sections at ×200 magnification (c) in both post-CS and post-transplant kidneys was significantly improved by caspase-3 siRNA (e). The Sirius Red staining (×100 magnification) mainly located in the tubulointerstitial areas (d), with significant less staining in the caspase-3 siRNA treated post-transplant kidneys compared with both the negative siRNA treated kidneys and the IR kidneys (f). Data were expressed as mean number of each group (mean ± SEM, n = 5). C3 siR, caspase-3 siRNA; Neg siR, negative siRNA; Post-CS, post-cold storage; Pre N, prenephrectomy; Post Tx, post-transplant. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Correlation between 17 kD caspase-3, HMGB1, inflammation, apoptosis, renal function, and structure. The positive correlations were seen between HMGB1 protein and 32 kD or 17 kD caspase-3, MPO+ cells, ISEL+ cells, IL-1β, IL6, or NF-κB mRNA in kidneys, and IL-1β, IL6 IL-8, or TGFβ in serum (a–d); SCr and17 kD caspase-3, HMGB1, MPO+ cells, ISEL+ cells and TID score (e,f). Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 The schematic pictures demonstrate the Toll-like receptor (TLR) pathways detected in the current study (a) and design of the experiment: orange arrows indicating renal tissue collection points (b). Immune recognition of siRNA is mainly via TLR3 (adaptor TRIF) and TLR7 (adaptor MyD88). HMGB1 is released from apoptotic or necrotic cells and recognized by TLR2/4 or RAGE. NF-κB, IRF3, and IRF7 are located in convergent points of different signaling pathways and mediate the production of type I interferons and inflammatory cytokines. Molecular Therapy 2014 22, 1817-1828DOI: (10.1038/mt.2014.111) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions