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. 2014 Jul 15;22(10):1817–1828. doi: 10.1038/mt.2014.111

Serum-stabilized Naked Caspase-3 siRNA Protects Autotransplant Kidneys in a Porcine Model

Cheng Yang 1, Tian Zhao 1, Zitong Zhao 1, Yichen Jia 1, Long Li 1, Yufang Zhang 2, Mangen Song 1,3, Ruiming Rong 1,4, Ming Xu 1, Michael L Nicholson 5,*, Tongyu Zhu 1,6,*, Bin Yang 1,2,*
PMCID: PMC4428396  PMID: 24930602

Abstract

The naked small interfering RNA (siRNA) of caspase-3, a key player in ischemia reperfusion injury, was effective in cold preserved and hemoreperfused kidneys, but not autotransplanted kidneys in our porcine models. Here, chemically modified serum stabilized caspase-3 siRNAs were further evaluated. The left kidney was retrieved and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 or negative siRNA into the renal artery for 24-hour cold storage (CS). After an intravenous injection of 0.9 mg siRNA and right-uninephrectomy, the left kidney was autotransplanted for 2 weeks. The effectiveness of caspase-3 siRNA was confirmed by caspase-3 knockdown in the post-CS and/or post-transplant kidneys with reduced apoptosis and inflammation, while the functional caspase-3 siRNA in vivo was proved by detected caspase-3 mRNA degradation intermediates. HMGB1 protein was also decreased in the post-transplanted kidneys; correlated positively with renal IL-1β mRNA, but negatively with serum IL-10 or IL-4. The minimal off-target effects of caspase-3 siRNA were seen with favorable systemic responses. More importantly, renal function, associated with active caspase-3, HMGB1, apoptosis, inflammation, and tubulointerstitial damage, was improved by caspase-3 siRNA. Taken together, the 2-week autotransplanted kidneys were protected when caspase-3 siRNA administrated locally and systemically, which provides important evidence for future clinical trials.

Introduction

Renal transplantation is the best treatment for end-stage renal failure patients. Ischemia reperfusion (IR) injury, inevitable in transplantation, is associated with delayed graft function and allograft rejection,1,2 and affects donor viability and graft survival.3,4 Caspase-3, upregulated by IR injury, is a major effector enzyme that initiates serial downstream reactions including apoptosis5 and inflammation.6 High-mobility group box 1 (HMGB1) protein regulates gene expression and acts as a nuclear transcription factor after nucleocytoplasmic translocation. Caspase-3 activation in apoptotic cells promotes HMGB1 secretion, while remaining apoptotic cells transformed to secondary necrotic cells also releases HMGB1 that triggers inflammation.7 Blocking HMGB1 either before or soon after IR injury inhibited apoptosis and inflammation,8 through a Toll-like receptor (TLR) 4 (HMGB1 receptor)-dependent pathway.9,10,11,12 Knockdown of TLR4 also inhibited HMGB1, TNF-α and myeloperoxidase positive (MPO)+) neutrophil accumulation in myocardial IR injury.13

Optimizing donor organ preservation is a necessary strategy to improve IR injury and donor quality.14 Beneficial effects were achieved via modifying caspase-3 activity using protein inhibitors15 and gene therapy,16,17 but potential toxicities of these reagents have limited their clinical application. Recently, RNA interference using a 21-nucleotide small interfering RNA (siRNA) has been proved to be a potent, specific, and safe method for gene silencing. So far, most siRNA studies have been conducted in cellular18 and murine models,19 but these may fail to faithfully mirror the effects in human diseases. Large animal models such as porcine models provide good alternatives given their similarities to humans with respect to anatomy, physiology, and immunology.5,20,21,22

Synthetic siRNAs, often delivered with vehicles, could activate innate immune responses, mediated by TLR signaling pathways, and subsequently produce inflammatory cytokines such as IL-1β and IFN-γ.23,24 The RNA-sensing TLR3 and TLR7, predominantly located inside the cell,25 possibly affect cell division, growth, and apoptosis.26 The adaptor molecule MyD88 is necessary for responses to all Toll-like receptors, except TLR3 that is mediated by the adaptor molecule TRIF.27 Cells from MyD88 knockout mice showed no responses to the TLR9 and TLR7 ligands.28,29 In addition, interferon regulatory factors (IRF)3, IRF7 and interferon-induced proteins with tetratricopeptide repeats (IFIT)1, which are located upstream of IFN, are more sensitive for monitoring IFN activation, as their upregulation can be observed prior to IFN production.30,31,32

In our previous study, naked caspase-3 siRNA, which is less likely to activate immune responses, was administered into both isolated kidneys and autologous blood during 24-hour cold storage (CS) prior to reperfusion suppressed caspase-3 expression, reduced apoptosis and inflammation, and partially improved renal function after hemoreperfusion.5 However, it is not known whether caspase-3 siRNA only, in local preservation fluid, was effective enough or an additional systemic treatment was essential in vivo. The effect of a short-acting caspase-3 siRNA administered only during kidney CS was first investigated previously in a 2-day porcine autotransplant model. Unfortunately, local delivery of siRNA did not protect autotransplanted kidneys, as levels of caspase-3 mRNA and protein were further increased.20 This might be due to the poor stability of naked siRNA and/or systemic compensative responses, suggesting that additional systemic treatment is required.

Chemical modification is one of the methods used to prolong the half-life of naked siRNA in serum, retaining its potency and low toxicity. In this study, the effects of a novel chemically modified naked caspase-3 siRNA with super serum stability were assessed in the porcine autotransplant model using a negative siRNA as the control, focusing on apoptosis, inflammation, immune responses, and potential toxicities. A single dose of caspase-3 siRNA was injected intravenously prior to transplantation in addition to infusing caspase-3 siRNA locally in preservation fluid. The period of observation was also extended to 2 weeks post-transplantation.

Results

Caspase-3 mRNA in the kidney and white blood cells (WBC)

The expression of caspase-3 mRNA was assessed by quantitative polymerase chain reaction (qPCR). The level of caspase-3 mRNA in the post-CS and post-transplant kidneys was significantly decreased by caspase-3 siRNA compared with negative siRNA treatment or IR alone. The caspase-3 mRNA in the post-CS kidneys was also significantly increased by negative siRNA in comparison to IR alone (Figure 1a). The level of caspase-3 mRNA in WBC collected daily pre- and post-transplantation fluctuated with an increased trend at day 2 to 3 post-transplantation, but there was no significant difference between groups (Figure 1b).

Figure 1.

Figure 1

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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.

To further confirm the function of caspase-3 siRNA in vivo, 5′-rapid amplification of cDNA ends (RACE) PCR was performed to determine the siRNA directed cleavage fragments of caspase-3 mRNA. DNA products corresponding to the mRNA fragments of different length were only detected in caspase-3 siRNA treated post-CS and post-transplant kidneys (Figure 1c). The DNA fragments were then cloned and 15 of them were sequenced. Ten of the 15 clones had 5′ ends; 2/15 were exactly at the cleavage site of siRNA; 3/15 clones 1 nucleotide away; 4/15 clones 2 nucleotides away; and 1/15 clone 3 nucleotides away (Figure 1d). These 5′-RACE PCR products were siRNA directed 3′ cleavage fragments of caspase-3 mRNA with different extents of decay from the 5′ end.

Caspase-3 protein in the kidney

The expression of caspase-3 protein was detected by western blotting (Figure 1e). The 32 kD caspase-3 precursor in the post-CS kidneys was significantly decreased by caspase-3 siRNA compared to the IR group, but there was no significant difference between groups in post-transplant kidneys (Figure 1f). More interestingly, the 17 kD caspase-3 active subunit in post-transplant kidneys was significantly decreased by caspase-3 siRNA compared with that in the negative siRNA or IR group (Figure 1g).

Reduced apoptotic cells

There were more apoptotic cells, detected by in situ end-labeling (ISEL) fragmented DNAs, in post-transplant kidneys, mainly located in tubular and interstitial areas, some of which were shedding into tubular lumens (Figure 2a). The number of apoptotic cells in the post-CS and post-transplant kidneys were significantly decreased by caspase-3 siRNA compared with the negative siRNA or IR group (Figure 2b).

Figure 2.

Figure 2

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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.

Decreased MPO+ cells

MPO+ cells in the kidney were determined by immunostaining. MPO+ cells were scattered in the post-CS kidneys, increased in the post-transplant kidneys. These were mostly located in vascular lumens and interstitial areas, some of which penetrated through tubular areas, or demonstrated morphologic features of apoptosis such as condensed nuclei (Figure 3a). The number of MPO+ cells in the post-CS and post-transplant kidneys was dramatically decreased by caspase-3 siRNA compared to both the negative siRNA and IR groups (Figure 3b).

Figure 3.

Figure 3

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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.

Cytokine and transcription factor mRNA in the kidney

The expression of proinflammatory cytokine and transcription factor mRNA was assessed by qPCR. The level of IL-1β, IL-6, NF-κB, IFN-α, IFN-β, IFN-γ, IRF3, IRF7, and IFIT1 mRNA was significantly decreased by caspase-3 siRNA in the post-CS and post-transplant kidneys compared to those treated by negative siRNA or IR alone (Figure 4a–i).

Figure 4.

Figure 4

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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.

Cytokines in the peripheral blood

IL-1β, IL-6, IL-8, TGF-β, IL-4, and IL-10 in the peripheral blood collected daily pre- and post-transplantation were determined by multiplex Luminex technology or IFN-α and IFN-β by enzyme-linked immunosorbant assay. The level of IL-1β was significantly decreased by caspase-3 siRNA from day 6 to 12 and day 7 to 14 compared with the negative siRNA and IR group respectively (Figure 5a). A similar trend was observed in IL-6 with significant differences between caspase-3 siRNA and the other two groups from day 12 to 13 (Figure 5b). 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 5 to 11 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 other groups (Figure 5c,d,g,h). However, the levels of IL-4 and IL-10 were raised by caspase-3 siRNA, and were significantly different from either negative siRNA or IR alone 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 (Figure 5e,f).

Figure 5.

Figure 5

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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.

Renal function

Serum creatinine (SCr) and urea nitrogen in animals were examined daily prenephrectomy, and pre- and post-transplantation. SCr and urea nitrogen were gradually increased after transplantation, reach a plateau between days 3 and 5, then fell off in all three groups. However, the level of SCr was significantly decreased by caspase-3 siRNA from day 5 to 14 and day 11 to 14 compared to the negative siRNA and IR group, with a maximal difference at day 5 (Figure 6a). A similar trend in urea nitrogen was seen with significant differences from day 6 to 14 and day 11 to 14 between the caspase-3 siRNA group and other two groups (Figure 6b).

Figure 6.

Figure 6

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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.

Renal tissue damage

Renal tubulointerstitial damage (TID) was assessed in H&E stained sections. There was mild tubular dilation, vacuolation, and interstitial edema in the post-CS kidneys. In contrast, more severe tubular vacuolation, cell detachment (and luminal cell debris), protein casts, interstitial expansion, and inflammatory cell infiltration were seen in the post-transplant kidneys (Figure 6c). Semiquantitative analysis revealed that the TID score in both post-CS and post-transplant kidneys was significantly improved by caspase-3 siRNA compared to the negative siRNA and IR groups (Figure 6e).

Extracellular matrix deposition in the kidney

Extracellular matrix deposition in the kidney was evaluated by Sirius Red staining (The dye molecules intercalate between tertiary grooves in the structure of collagen I and III and present with a pink color under white light). Sirius Red staining was located mainly in the tubulointerstitium rather than the glomerular interstitium (Figure 6d). Significantly reduced staining was observed in the caspase-3 siRNA group compared with both the negative siRNA and IR groups in post-transplant kidneys (Figure 6f).

Interaction between caspase-3, HMGB1, inflammation, apoptosis, renal function, and structure

The expression of 17 kD caspase-3 was associated with the number of MPO+ or ISEL+ cells (r = 0.597 or 0.521, P < 0.05). There were positive correlations between HMGB1 and the 32 and 17 kD subunits of caspase-3, MPO+ cells, ISEL+ cells, IL-1β, IL-6 and NF-κB mRNA, serum IL-1β, IL6 IL-8, and TGF-β respectively (Figure 7a–d). SCr was positively correlated to 17 kD caspase-3, HMGB1, MPO+ cells, ISEL+ cells, and TID (Figure 7e,f). IFN-α, IFN-β, IFN-γ mRNA was also positively associated with HMGB1 (r = 0.629, 0.738, and 0.584, P < 0.05) and SCr (r = 0.890, 0.760, and 0.782, P < 0.01), while serum IL-4 and IL-10 were negatively related to HMGB1 (r = −0.960 and −0.928, P < 0.001) and SCr (r = −0.583; −0.523, P < 0.05). Furthermore, multiple regression analyses showed that 17 kD caspase-3 and HMGB1 was most positively related to IL-1β mRNA (standardized coefficients-β (SC-β) = 1.083 or 1.011, P < 0.001) among the detected mRNAs in the kidney; and negatively related to IL-10 and IL-4 (SC-β = −0.612 and −0.985, P < 0.01) among the detected serum cytokines. More interestingly, SCr was strongly associated with ISEL+ cells (SC-β = 0.808, P < 0.05) more so than 17 kD caspase-3, HMGB1, renal and serum IL-1β, and MPO+ cells.

Figure 7.

Figure 7

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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).

HMGB1, TLR3, TLR7, MyD88, or TRIF protein in the kidney

The expression of HMGB1, TLR3, TLR7, MyD88, and TRIF protein in the post-CS and post-transplant kidneys was detected by western blotting. HMGB1 was significantly decreased by caspase-3 siRNA in both post-CS and post-transplant kidneys (Supplementary Figure S1a). There was no significant difference in TLR3, TLR7, MyD88 and TRIF protein (Supplementary Figure S1b–e).

Discussion

Optimized protection in 2-week autotransplanted kidneys was achieved for the first time via delivering serum-stabilized naked caspase-3 siRNA locally and systemically in a porcine model. The level of caspase-3 mRNA and protein, HMGB1 protein, as well as apoptotic cells and MPO+ cells, was reduced in the kidneys due to functional caspase-3 siRNA directed degradation of caspase-3 mRNA. The serum concentration of pro- and anti-inflammatory cytokines was favorably regulated. Active caspase-3 and HMGB1 were positively correlated to IL-1β mRNA, but negatively correlated to IL-10 and IL-4. More importantly, renal function, associated with active caspase-3, HMGB1, IL-1β, apoptosis, inflammation, and TID, was improved, with less extracellular matrix deposition, minimal off-target actions and favorable systemic responses. This proof-of-principle study in a large animal model demonstrates that caspase-3 siRNA therapy is a promising tool for clinical trials in humans.

First of all, the protective effects of caspase-3 siRNA on transplanted kidneys may be due to improvement in siRNA stability. Given that the putative half-life of serum-stabilized siRNA is about 1 week, caspase-3 siRNA treatment proved to be very effective compared with the negative siRNA in this study. The caspase-3 mRNA was decreased in both post-CS and post-transplant kidneys, caspase-3 precursor was reduced in the post-CS kidneys and the 17 kD active subunit was inhibited in the post-transplant kidneys. This indicates that siRNA silencing of caspase-3 mRNA affected its protein synthesis and subsequent activation, both of which were maintained for at least 2 weeks, implying prolonged stability of the siRNA. Furthermore, the 5′-RACE PCR products matching different positions of caspase-3 mRNA were only visible in the both post-CS and post-transplant kidneys treated by casapase-3 siRNA. Two out of three of siRNA-directed caspase-3 mRNA cleavage fragments, partially containing the 3′ half of the caspase-3 siRNA sense strand sequence, were exactly at, or close to, the cleavage site of siRNA, but not all as some of them might have been degraded. This evidence proves not only definitively functional caspase-3 siRNA in vivo, but also the anti-inflammatory and antiapoptotic effects were due to degenerated caspase-3 mRNA.

It should also be noted that the protective effects of caspase-3 siRNA on transplanted kidneys may be due to the simultaneous systemic treatment. The direct systematic efficacy and compensative responses of caspase-3 siRNA were further investigated. The overall unchanged caspase-3 mRNA in the peripheral WBC did not indicate any direct systemic effects induced by caspase-3 siRNA, although a transient increase was seen at the early stage which might reflect natural inflammatory processes after surgery and IR injury. Nevertheless, the changes of serum cytokines provided evidence of compensative responses. Reduced IL-1β, IL-6 IL-8, and TGF-β, as well as IFN-α and IFN-β, in the caspase-3 siRNA-treated group at different time periods suggested a restrained systemic inflammation apart from natural post-transplantation recovery in all groups. Furthermore, IL-10 and IL-4, typical anti-inflammatory cytokines, were remarkably increased in the caspase-3-treated group at day 4–8 and 3–6 post-transplantation, during which the injury was maximized. The beneficial effects of IL-10 and IL-4 on renal IR injury were supported by the fact that adoptive transfer of IL-10-expressing macrophages to ischemic kidneys blunted acute kidney injury;33 IL-4 blocking antibody reversed its protection against renal IR injury.34

The possible mechanism of caspase-3 siRNA treatment was further evaluated through its downstream effects on apoptosis and inflammation. After transplantation, apoptotic cells were increased, mainly located in tubulointerstitial areas, but significantly decreased in both the post-CS and post-transplant kidneys preserved by caspase-3 siRNA. The inflammation was assessed not only by MPO+ cell infiltration, but also by the mRNA levels of inflammatory mediators such as IL-1β, IL-6, NF-κB, IFN-α, IFN-β, IFN-γ, IRF3, IRF7, and IFIT1 in the kidney, all of which were significantly lower in the caspase-3 siRNA treated post-CS and post-transplant kidneys, as well as decreased IL-1β, IL-6, IL-8, TGF-β, IFN-α, and IFN-β, and increased IL-4 and IL-10 in serum. The changes in apoptosis and inflammation were positively correlated with reduced active 17 kD caspase-3, which was attributed to caspase-3 mRNA knockdown and decreased activation.

HMGB1 protein was reduced in the post-transplant kidneys by caspase-3 siRNA. HMGB1 is secreted from apoptotic cells after caspase-3 activation or passively released from necrotic cells including vascular and tubular cells when IR injury is excessive.35 HMGB1 promotes inflammatory responses such as activating NF-κB and releasing IL-1β, INF-α, and IFN-β;7,36,37,38 and also inducing apoptosis via caspase-3 activation.39,40 The level of HMGB1 protein in this study was positively associated with both caspase-3 precursor and its active subunits, MPO+ and apoptotic cells. It was positively correlated to renal IL-1β mRNA, but negatively related to serum IL-4. A recent study identified that HMGB1 had a chemoattractant effect on polymorphonuclear neutrophils through IL-8 production.41 These data confirmed a close interaction between caspase-3, HMGB1, apoptosis, and inflammation, all of which were related to the changes in renal function and structure.

SCr and blood urea nitrogen in all groups were decreased rapidly from day 5 post-transplantation, but they were still significantly higher in the negative siRNA and IR groups compared with the caspase-3 siRNA group, in which the renal function almost approached baseline levels at 2 weeks. These important results indicate that an early or timely intervention using caspase-3 siRNA was able to dramatically improve functional outcome of autotransplanted kidneys. Moreover, the TID score was also significantly ameliorated by caspase-3 siRNA, showing reduced extracellular matrix deposition. These results might be attributed to the effectiveness of caspase-3 siRNA and subsequently reduced caspase-3 activity and apoptosis, as well as reduced HMGB1, IL-1β and inflammation.

The side effects of caspase-3 siRNA, in terms of inflammation and innate immune activation, were identified against negative siRNA. These off-target actions may occur through the activation of cellular sensors to the delivery of viral or liposomal vehicles or foreign RNA with a specific sequence24 and lead to the release of type I interferon and proinflammatory cytokines.25,42 These have been the major limitations of synthetic siRNA, especially delivered by vehicle.43 In this study, caspase-3 mRNA expression was significantly increased in the post-CS kidneys by negative siRNA compared to IR alone, without changes in other measured parameters such as type I interferons and HMGB1. In the post-transplanted kidneys, there was no significant difference induced by negative siRNA, although numerical increases were seen in caspase-3, INF-γ, apoptosis, renal function, and histology. These phenomena imply that off-target effects caused by siRNA cannot be completely ruled out, but could be very limited. In addition, there were no significant differences in TLR3 and TLR7 proteins between the groups. There were also no significant differences in MyD88 and TRIF proteins between the groups. In addition, the negative siRNA did not significantly change the level of IRF3, IRF7, and IFIT1 mRNA in the kidney, or that of IFN-α and IFN-β protein in the serum, compared to the IR group. These findings, taken together, indicate that the novel siRNAs used in this study might not stimulate MyD88- or TRIF-dependent TLR activation in innate immunity to cause extra damage (Figure 8a). Any changes that might have occurred in the early stages may have recovered by the end of the 2-week period post-transplantation. This serum-stabilized naked siRNA appears to be relatively safe.

Figure 8.

Figure 8

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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.

Gene silencing therapy based on RNAi has demonstrated a great potential in translational medicine. Since the first RNAi-related clinical trial that introduced naked siRNA targeting vascular endothelial growth factor intravitreally into patients with age-related macular degeneration and diabetic macular edema, many approaches attempting to deliver naked siRNAs locally have been conducted.44 So far, there are over 20 registered clinical trials using siRNAs (www.ClinicalTrials.gov), but only I5NP, targeting p53, has been validated for renal injury. The serum-stabilized siRNA targeting caspase-3 used in this study could be another candidate for future clinical trials as the data obtained from the porcine kidney autotransplant model are more applicable to humans than any other rodent models. To translate siRNA therapy from the bench to bedside, however, the following aspects of siRNA treatment such as sequence design (in terms of nucleotide component and modification), delivery route and timing, off-target effects, and systemic compensative responses, etc. have to be taken into account and fully assessed for optimized outcome.

In conclusion, local and systemic administration of serum-stabilized naked caspase-3 siRNA effectively silenced caspase-3, favorably changed serum cytokines, alleviated HMGB1, reduced apoptosis and inflammation, and protected 2-week autotransplanted kidneys in a porcine model. These results suggest that this novel caspase-3 siRNA with prolonged serum stability and minimal toxicities is promising in limiting IR injury in kidney preservation and transplantation and provides invaluable data for potential human application.

Materials and Methods

Caspase-3 siRNA preparation. Three pairs of siRNA, targeting porcine caspase-3 mRNA (NCBI CoreNucleotide Accession No. AB029345), were designed (Life Technologies, Paisley, UK). The most effective sequences: 5′-GGGAGACCUUCACAAACUUtt-3′ and 5′-AAGUUUGUGAAGGUCUCCCtg-3′, were selected in LLC-PK1 cells.45 The in vivo Ready custom caspase-3 siRNA (Silencer Select) was then verified in our ex vivo study.5 The serum stable caspase-3 siRNA (Ambion In Vivo, catalogue number: 4457309) was based on the same sequence; and the negative siRNA (Ambion In Vivo, catalogue number: 4457289) was also provided by the Life Technologies and used in the same dosage of caspase-3 siRNA for negative control. The sequences of negative siRNA were 5′ UAACGACGCGACGACGUAAtt-3′ and 5′-UUACGUCGUCGCGUCGUUAtt-3′. Ambion In Vivo siRNAs were designed using the Silencer Select algorithm and incorporated chemical modifications for superior serum stability with in vivo delivery. The approach used for chemical modification is locked nucleic acid, organized along the siRNA backbone in a very specific format to drive serum stability and eliminate off-target phenotypes.32,46,47,48

Animals. Male mini pigs weighing 25–30 kg were used. They were housed with air-conditioned, straw-saw dust beds with free access to water; and fed with wetted, granulated full-fodder. All animal work was performed under the regulation layout by Chinese animal welfare authority.

Anesthetic protocol. The animals were premedicated with 0.5 mg/kg diazepam and 5 mg/kg ketamine hydrochloride i.m., followed by general anesthesia using 1 mg/kg propofol (Fresenius Kabi, Bad Homburg, Germany) i.v. and maintained with a mixed solution of 0.25 mg/kg/hour diazepam, 2.5 mg/kg ketamine hydrochloride and 0.0125 ml/kg/hour compound detomidine hydrochloride, or 0.5 mg/kg/h propofol i.v. in turn. Respiration was supported by a ventilator (Dräger, Shanghai, China) through an inserted trachea cannula. Five-hundred microliters of 5% glucose and 0.9% sodium chloride and 500 ml of hydroxyethyl starch 130/0.4 and sodium chloride injection (Fresenius Kabi) were also administered i.v. In addition, 100 ml of 0.3 g levofloxacin lactate and 2 million units of benzylpenicillin were given i.v. 30 minutes before surgery. The same anesthetic protocol was used for donor retrieving and transplantation.

Donor kidney retrieving and preservation. The left kidney was isolated and removed with minimal warm ischemic time, after which the renal artery was ligated near the abdominal aorta and the renal vein was ligated near the inferior vena cava and the ureter. The isolated kidney was flushed immediately with 200 ml of precooled Ringer's solution with 1,000 IU heparin at 100 cm H2O hydrostatic pressure until the kidney became pale. This was then followed by 200 ml of University of Wisconsin (UW, Bristol-Myers Squibb, New York) solution. Finally, half of the 40 ml precooled UW solution with 0.3 mg of caspase-3 siRNA or negative siRNA, or without siRNA (n = 5) was infused into the renal artery to flush out the remaining UW solution. The renal vein was then clamped with another 20 ml solution infused, and the renal artery was finally clamped. The kidney was then preserved on ice for 24 hours.

Right kidney nephrectomy and autotransplantation. The following day, the right kidney was resected after the right renal artery and vein (postnephrectomy kidneys), and the ureter was ligated close to the renal hilum. A dose of 0.9 mg of caspase-3 siRNA or negative siRNA was administrated i.v. The left kidney was then orthotopically autotransplanted into the right renal fossa for 2 weeks (Figure 8b). In addition, a double lumen cuffed silicone vascular access catheter (Arrow International, Reading) was placed in the left internal jugular vein. The lumens of the central line were fixed behind the ear and blocked with heparin.

Sample collection. Blood samples were taken before donor retrieval, pretransplant and daily post-transplant to isolate serum and WBC. Renal biopsies were also taken 24-hour post-CS. At 2 weeks post-transplant, the animal was anesthetized, sacrificed, and the graft was harvested. Some renal tissues were fixed with 10% buffered formalin and the others were snapping frozen.

The expression of mRNAs. Total RNA was extracted from renal tissues and WBC using Trizol reagent (Invitrogen, Carlsbad, CA). One microgram of total RNA was transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD). Real-time qPCR was performed using the TaqMAN premix (Takara Bio, Otsu, Japan), primers and probes for porcine caspase-3 and β-actin (Life Technologies)45 in a MasterCycler RealPlex4 system (Eppendorf, Hamburg, Germany). After a hot start (2 minutes at 50 °C, 10 minutes at 95 °C), amplification was performed for 45 cycles (15 seconds at 95 °C, 60 seconds at 60 °C). The IL-1β, IL-6, NF-κB, IFN-α, IFN-β, IFN-γ, IRF3, IRF7, and IFIT1 were performed using SYBR Green RT-PCR Kit (Takara Bio). The primer sequences are shown in Supplementary Table S1. The expression of mRNA in kidneys normalized with β-actin was calculated against relative non-IR injured kidneys (randomly selected 6 out of 12 postnephrectomy kidneys) using a 2−ΔΔCt method. The expression of caspase-3 mRNA in WBC was calculated against the baseline level in the pre-transplantation samples.

5′-RACE PCR. To identify the caspase-3 siRNA-directed degradation intermediates, 5′-RACE PCR was carried out by using a SMART RACE cDNA Amplification kit (Clontech, Madison, WI) following manufacturer's protocol. The 5′ adaptor GSP and caspase-3 reverse primer (Cas3–R) were used for RT; and adaptor forward primer (Ada-F) and Cas3-R were used for PCR amplification. The sequences of primers are shown in Supplementary Table S2. The detailed methods are available in the Supplementary Materials and Methods.

Western blotting analysis. Twenty microgram protein from kidney homogenate were separated on 15% (wt/vol) poly acrylamide denaturing gels and electro-blotted onto polyvinylidene fluoride membranes. The semiquantitative analysis results were expressed as optical volume density (OD × mm2) normalized to β-actin (AlphaView Software 3.3, Cell Biosciences, Santa Clara, CA). The details are available in the Supplementary Materials and Methods.

ISEL apoptotic cells. Paraffin embedded kidney tissue sections were used for ISEL-fragmented DNAs with digoxigenin-deoxyuridine (dUTP) detected by terminal deoxynucleotidyl transferase (TdT) using an Apoptosis Detection Kit (Millipore, Billerica, MA). The details are available in the Supplementary Materials and Methods.

MPO immunostaining. Immunostaining of MPO, a marker mainly for neutrophil granulocytes, was undertaken on paraffin sections using a DAKO ChemMate EnVision Detection Kit (DAKO, Carpinteria, CA). The details are available in the Supplementary Materials and Methods.

Cytokine multiplex analysis. Serum samples were analyzed for six cytokines using a customized Porcine Cytokine 6-Plex Antibody Bead Kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol. The details are available in the Supplementary Materials and Methods.

Enzyme-linked immunosorbant assay. Serum samples were analyzed for IFN-α and IFN-β using Porcine ELISA Kits (Abcam) according to the manufacturer's protocol. The concentration was determined using a standard curve according to the kit's instructions.

Histological assessment. The renal TID was assessed in hematoxylin and eosin (H&E)-stained sections by three researchers blinded to the coding. The details are available in the Supplementary Materials and Methods.

Extracellular matrix deposition assay. The deposition of extracellular matrix was evaluated by Sirius red (collagen-specific dye) staining, as previously described.49 The details are available in the Supplementary Materials and Methods.

Statistical analysis. Results are expressed as mean ± standard error of the mean. Normality tests were carried out and statistical analysis of the data was performed using one-way analysis of variance. The correlation between parameters was determined by linear correlation and multiple regression analyses using SPSS 18.0 software (SPSS, Armonk, NY). P < 0.05 was considered as statistically significant.

SUPPLEMENTARY MATERIAL Figure S1. Protein expression of HMGB1, TLR3, TLR7, MyD88 and TRIF in the kidney detected by western blotting. Table S1. Sequences of primers used for QPCR. Table S2. Sequences of primers used for 5'-RACE PCR. Materials and Methods

Acknowledgments

This study was supported by the UK-China Fellowship for Excellence, Department for Business Innovation and Skills (to B.Y.); University Hospitals of Leicester NHS Trust, UK, and National Nature Science Foundation of China (81170689 to B.Y., 81270832 to R.R., 81270833 to T.Z.); Science and Technology Commission of Shanghai Municipality (12ZR1405500 to R.R.); Zhongshan Hospital, Fudan University, China. We also thank Izabella Pawluczyk and Meeta Patel for their help in critically proof reading this manuscript. The authors have declared no conflict of interest.

Supplementary Material

Supplementary Figure S1

Protein expression of HMGB1, TLR3, TLR7, MyD88 and TRIF in the kidney detected by western blotting.

Click here for additional data file. (3.6MB, tiff)
Supplementary Table S1

Sequences of primers used for QPCR.

Click here for additional data file. (40KB, doc)
Supplementary Table S2

Sequences of primers used for 5'-RACE PCR.

Click here for additional data file. (26.5KB, doc)
Materials and Methods
Click here for additional data file. (32.5KB, doc)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

Protein expression of HMGB1, TLR3, TLR7, MyD88 and TRIF in the kidney detected by western blotting.

Click here for additional data file. (3.6MB, tiff)
Supplementary Table S1

Sequences of primers used for QPCR.

Click here for additional data file. (40KB, doc)
Supplementary Table S2

Sequences of primers used for 5'-RACE PCR.

Click here for additional data file. (26.5KB, doc)
Materials and Methods
Click here for additional data file. (32.5KB, doc)

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