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Basic and Experimental Research

Naked Small Interfering RNA of Caspase-3 in Preservation Solution and Autologous Blood Perfusate Protects Isolated Ischemic Porcine Kidneys

Yang, Bin; Hosgood, Sarah A.; Nicholson, Michael L.

Author Information
doi: 10.1097/TP.0b013e318207949f

Abstract

Renal transplantation is the best treatment for patients with end-stage renal failure and has greatly improved the quality of life of these patients. However, still there are many tactical problems that need to be resolved such as the injuries occurring during the preservation and reperfusion of donor kidneys. Ischemia-reperfusion injury (IRI) is a common and inevitable injury in renal transplantation and is associated with acute and long-term allograft dysfunction. The causes of renal IRI are complex and linked to oxidative stress, apoptosis, and inflammation (1–3). Apoptosis is a cascade of events, which includes the up-regulation of caspases (cysteine proteases). Caspase-3, an effector caspase, can be up-regulated by IRI (4), providing a convergence point for a number of apoptotic pathways. Caspase-3 also plays a crucial role in inflammation and graft immunogenicity (4).

Pan caspase inhibitors and specific caspase-1 and -3 inhibitors have been shown to manipulate IRI-induced inflammation, apoptosis, and necrosis in a number of studies (4, 5). However, the potential toxicities of these inhibitors have thus far limited their clinical use. The development of specific caspase-3 inhibitors has also been hampered by the high degree of homology shared by different caspases in the family (6). Nevertheless, caspase-3 knockout (Casp3−/−) mice have been shown to be protected from developing diabetes because of their resistance to apoptosis and the absence of lymphocyte infiltration in pancreatic islets (7). Therefore, specifically targeting caspase-3 gene could be a promising strategy against inflammation and apoptosis-mediated injury.

The emergence of RNA interference has provided one such strategy for the targeted inhibition of caspase-3 (8). RNA interference using a 21-nucleotide small interfering RNA (siRNA) is a posttranscriptional gene silencing approach, in which the introduction of a double-stranded RNA elicits the selective degradation of homologous mRNA transcripts. The exogenous administration of siRNA is capable of blocking gene expression in mammalian cells (9) and has proved to be a potent and specific method of gene silencing (specific to 1 nucleotide mismatch) (10, 11). Administration of siRNA in vivo is relatively safe and can simultaneously target multiple genes (12–14).

So far, most studies using synthetic siRNA have been carried out in murine models. These models yield important results and are cost effective but may fail to mirror human diseases faithfully. Thus, large animal models of human disease complement murine studies, because they have greater similarity with humans, particularly with respect to porcine models that in common with humans have a diverse genetic background (15–17). siRNA-mediated suppression of antiapoptotic RNA-stabilizing protein human antigen R increases apoptosis during energy depletion in porcine proximal tubular cells (LLC-PK1, a porcine kidney proximal tubular cell line) (18). Our own experiments validated the sequences of caspase-3 siRNA in LLC-PK1 cells (19).

The effect of synthetic caspase-3 siRNA on isolated kidney preservation, however, has not been well defined. In this study, we investigated the effect of administering caspase-3 siRNA directly into the renal artery and autologous blood perfusate before 24-hr cold storage (CS) followed by a further reperfusion in an ex vivo isolated organ perfusion system for 3 hr. The level of caspse-3 mRNA, protein, and activity was monitored; and in particular, its downstream biologic events such as apoptosis and inflammation, and functional parameters were also evaluated.

RESULTS

Caspase-3 Protein Reduced by Its siRNA in the Kidney

The expression of caspase-3 protein subunits in postreperfusion kidneys was detected by western blot in the preserved kidneys. The 32-kDa precursor and 17-kDa active subunit of caspase-3 were demonstrated in kidney tissues, with different exposure times on the x-ray film because of saturated precursor or invisible active subunit of caspase-3 (Fig. 1A). The expression of caspase-3 protein was presented as optical volume density (×mm2) and corrected by a 42-kDa band of β-actin as loading control. Both caspase-3 precursor (6.2±0.9 vs. 8.8±0.4) and active subunit (1.7±0.4 vs. 3.8±0.4) were significantly down-regulated by the siRNA of caspase-3 in postreperfusion kidneys (Fig. 1B).

FIGURE 1.
FIGURE 1.:
Expression of caspase-3 protein in postreperfusion kidneys was measured by western blotting. The immunoblot was a representative figure of each group. The 32-kDa precursor and 17-kDa active subunit of caspase-3 were predominantly revealed (A) and significantly decreased by the small interfering RNA (siRNA) of caspase-3 (B). Data are expressed as corrected volume density (mean±standard error of the mean) against the loading control of 42-kDa β-actin (n=6). *P less than 0.05; **P less than 0.01.

Active Caspase-3+ Cells in Kidney Decreased by Caspase-3 siRNA

The distribution of active caspase-3+ cells in the kidneys was detected by immunohistochemistry. Active caspase-3+ cells were seen in the glomerular, tubular, interstitial areas, and tubular lumens (Fig. 2A–E). The majority of active caspase-3+ cells demonstrated morphologic features of apoptosis (Fig. 2A) or shed into tubular lumens with polymorphic nuclei (Fig. 2D). Semiquantitative analysis revealed that active caspase-3+ cells (per 400× field) in postreperfused kidneys were significantly reduced 40% by the siRNA of caspase-3 (7.5±0.9 vs. 12.6±1.9, P<0.05). There were more active caspase-3+ cells in reperfused kidneys (Fig. 2G) than that in pre-CS (3.3±0.4) and post-CS kidneys with or without caspase-3 siRNA (2.4±0.3 or 3.0±0.6), whereas there was no statistical difference between pre- and post-CS kidneys.

FIGURE 2.
FIGURE 2.:
Active caspase-3+ cells in the kidney were detected by immunohistochemistry. Active caspase-3+ cells were revealed in different compartments of the kidney (A–E), and the most of them demonstrated of apoptotic features or shed into tubular lumens. There was not any staining in the section of the negative control (F). Interestingly, active caspase-3+ cells were significantly reduced 40% by caspase-3 small interfering RNA (siRNA) in the postreperfused kidneys, in where active caspase-3+ cells were more than that in the pre-sold storage (CS) and post-CS kidneys (G). Data are expressed as mean number in the high power field of each group (mean±SEM; n=6). postR, postreperfusion. *P less than 0.05.

Apoptotic Cells Decreased by Caspase-3 siRNA

Apoptotic cells in pre- and post-CS and postreperfusion kidneys were examined by in situ end-labeling fragmented DNAs. Apoptotic cells were mainly located in the tubular and interstitial area (Fig. 3); some of them had polymorphic nuclei (Fig. 3D and F), and a few were shedding into the tubular lumen (Fig. 3A, D, and E). There were few apoptotic cells seen in the glomerular area. Semiquantitative analysis revealed that the number of apoptotic cells (per 400× field) in the renal cortex was greatly increased in postreperfusion kidneys (4.91±1.02, Fig. 3G) compared with that in pre-CS (0.04±0.01) and post-CS kidneys with or without caspase-3 siRNA (0.13±0.03 or 0.19±0.03). There was no significant difference between the two groups of post-CS kidneys, but apoptotic cells were significantly decreased by the siRNA of caspase-3 (2.62±0.67) in the reperfused kidneys.

FIGURE 3.
FIGURE 3.:
The change of apoptosis detected by in situ end-labeling fragmented DNAs in pre-cold storage (CS) and post-CS, and post-3–hr reperfusion kidneys. Apoptotic cells (black arrows) are showed in the tubular (A, D, and E) and interstitial (B and C) areas and the tubular lumens (F); some of them had polymorphic nuclei (D and F). The number of apoptotic cells was greatly increased by the reperfusion but significantly reduced by the treatment of caspase-3 small interfering RNA (siRNA) (G). Data are expressed as mean number in the high power field of each group (mean±SEM; n=6). postR, postreperfusion. *P less than 0.05.

MPO+ Cells Marginally Reduced by Caspase-3 siRNA

The distribution of myeloperoxidase (MPO)+ cells in pre-CS and post-CS, and postreperfusion kidneys was detected by immunohistochemistry. MPO+ cells were mainly seen in the tubular (Fig. 4A, B, E, and D) and interstitial areas (Fig. 4B, D, and E) and also revealed in the glomerular area and vascular lumens (data not shown). Some MPO+ cells demonstrated morphologic features of apoptosis such as condensed nuclei (Fig. 4A and B) or were shed into the tubular lumen (Fig. 4C, D and F). Semiquantitative analysis revealed that a more MPO+ cells (per 400× field) were located in the tubulointerstitial areas of reperfused kidneys (7.25±0.55) than that in pre-CS (0.15±0.03) and post-CS kidneys with or without caspase-3 siRNA (0.47±0.12 or 0.46±0.25). There was no significant difference between the two groups of post-CS, but MPO+ cells were marginally reduced by the siRNA of caspase-3 (5.38±1.19, P=0.09) after reperfusion.

FIGURE 4.
FIGURE 4.:
Myeloperoxidase (MPO)+ cells detected by immunohistochemistry in pre-cold storage (CS) and post-CS, and post-3–hr reperfusion kidneys. MPO+ cells were seen in tubular (A, B, E, and D) and interstitial areas (B, D, and E) and tubular lumens (C, D, and F); some of them had apoptotic features (A and B).

Renal Function

Renal blood flow (milliliter per minute per 100 g) was gradually increased from 5 to 180 min after reperfusion and was marginally improved at the end of 3-hr reperfusion by caspase-3 siRNA (78±17 vs. 44±16, P=0.08, Fig. 5A). In addition, oxygen consumption (milliliter per minute per gram) was significantly increased after 1 (46±7 vs. 24±7) and 3-hr reperfusion by caspase-3 siRNA (50±10 vs. 25±8, Fig. 5B). Caspase-3 siRNA also significantly neutralized perfusate pH (7.38±0.08 vs. 7.27±0.07, Fig. 6A). However, area under the curve creatinine clearance (257±47 vs. 400±104 [mL/min/100 g]·hr) and total urine output (2.5±0.8 vs. 2.3±1.3 mL) post-3–hr reperfusion were not affected by the siRNA of caspase-3.

FIGURE 5.
FIGURE 5.:
The renal blood flow was gradually increased from 5 min to 3 hr and marginally improved by the small interfering RNA (siRNA) of caspase-3 at the end of 3-hr reperfusion (A). The oxygen consumption was significantly increased by the siRNA of caspase-3 after both 1- and 3-hr reperfusion (B). n=6. *P less than 0.05.
FIGURE 6.
FIGURE 6.:
The small interfering RNA (siRNA) of caspase-3 significantly neutralized perfusate pH (A). However, area under the curve (AUC) creatinine clearance (B) and total urine output (C) were not affected by caspase-3 siRNA. n=6. *P less than 0.05; not significant (NS), P more than 0.05.

DISCUSSION

Because of the shortage of kidney donors and the poor control of donor quality, any approach that could prevent IRI occurring in the CS and reperfusion stages is in high clinical demand. Maintaining kidney viability during preservation is critical for a successful transplantation. This is the first study of preserving isolated large animal organs, porcine kidneys, using naked siRNA of caspase-3 during CS, and further assessing the quality of the kidney by reperfusion using caspase-3 siRNA-treated autologous blood. The caspase-3 siRNA administered directly into the renal artery and hemoperfusate for 24 hr at 4°C significantly improved IRI with reduced caspase-3 protein, active caspase-3+ cells, and apoptosis and better renal oxygenation and acid base homeostasis. These significant advances in mitigating cold preservation and reperfusion injury are derived from specifically interfering in a key molecule, caspase-3, that initiates and cascades other down-stream causal factors in IRI. Therefore, this proof of principle experiment using a novel siRNA therapy in large animal organs provides valuable and transferable data for the potential modulation of human kidney preservation.

It has been reported that delivery of caspase-8 and -3 siRNAs successfully decrease mice liver IRI (13). Treatment with Fas siRNA protects mice against renal IRI with less tubular apoptosis, atrophy, and hyaline damage, and an improved survival rate (20); and a rapid delivery of p53 siRNA to proximal tubule cells led to the attenuation of apoptotic signaling and improvement of improved renal histology and function yielding therapeutic benefits for ischemic kidney injury in mice (21). The siRNAs of complement 3, caspase-3 and -8, and complement 5a receptors prevented apoptotic damage, renal histology, and function of improved IRI in mice (14, 22, 23). In these studies, siRNAs were systemically delivered in their naked synthetic version (13, 20, 21) or by nonviral plasmid vectors (14, 22, 23) by hydrodynamic or a simple bolus intravenous injection. The siRNAs are small and only need to cross the cell membrane and not the nuclear membrane to be effective, so they are easier to deliver. However, three obstacles must be overcome for siRNA to be successful in vivo: selective delivery into the desired tissue, adequate protection from degradation en route to the target tissue, and protection of the siRNA from rapid excretion. The pharmacokinetics of siRNA and the concentration of siRNA entering the kidney by systemic delivery need to be further elucidated.

More recently, heart perfused with a Cy3-labeled siRNA solution and preserved at 4°C for 48 hr demonstrated a fluorescence uptake by myocardial cells (24). University of Wisconsin solution containing naked siRNAs targeting tumor necrosis factor-α, complement 3, and Fas genes has been applied to preserve mice heart grafts at 4°C for 48 hr, which were subsequently transplanted into syngeneic recipients. This siRNA solution knocked down the expression of targeted genes at mRNA and protein levels, improved histology, and retained strong myocardial contraction. Therefore, incorporation of siRNA into organ storage solution is a feasible approach to attenuate heart IRI, protect cardiac function, and prolong graft survival (24). In comparison with small animal models, the porcine kidney has similar size, weight, and immune system, and anatomic, physiologic, and genetic characteristics of human (25). Therefore, large animal models have become increasingly important despite the costs and the extensive clinical attention they require (16, 17). This study preserved short ischemic porcine kidneys using caspase-3 siRNA administered directly into the renal artery for 24 hr CS with the renal vein clamped. This method used a limited amount of siRNA for a local delivery, which is ideal for siRNA getting into desired tissues and remaining for a period of time without excretion. Furthermore, it is simple and adaptable in the clinical setting of human renal transplantation. The number of active caspase-3+ cells, apoptotic cells, and MPO+ cells, however, was not affected by caspase-3 siRNA at the CS stage because of the nature of these biologic processes, but low temperature might slow down the degradation of the siRNA.

The preserved kidneys were further assessed by 3 hr hemoreperfusion using caspase-3 siRNA treated autologous blood. This additional treatment of autologous blood might reduce resources of inflammation in the perfusate. Although the feasibility to treat the recipient in a similar way needs to be explored, both 32-kDa precursor and 17-kDa active subunit of casaspe-3 were significantly down-regulated by its siRNA. The protein level change is a step further to the mRNA level change that is commonly detected in the siRNA intervention. The alteration at the protein level of a molecule is more associated with its down-steam biologic events than its mRNA level. The immunostaining of active caspase-3+ cells in the kidneys not only further verified a reduction of 17-kDa caspase-3 subunit detected by western blotting but also provided extra information through its distribution and morphologic features in terms of positive cells with apoptotic morphologic features or positive cells with polymorphic nuclei located in the tubular lumens, which reflected the nature of biologic involvement of caspase-3 in apoptosis and inflammation. The efficacy of caspase-3 siRNA was also confirmed by subsequent down-stream biologic events in the kidneys after 3-hr reperfusion, such as a reduction of apoptosis, even through the change in inflammation did not reach a statistical significance. Finally and most importantly, renal functional parameters such as oxygen consumption and perfusate pH were improved by the intervention of caspase-3 siRNA. These outcomes might be a coordinated co-action from both treatment of caspase-3 siRNA directed into the renal artery and into autologous blood perfusate. The results achieved in small animals described earlier (13, 14, 20–23) were further confirmed in this isolated porcine kidney preservation model.

The safety, specificity, and potency of therapeutic siRNAs have now been firmly established. Up to date, siRNAs have been used in a wide range of biologic models including renal IRI mouse models (13, 20, 23). Delivery of siRNA in vectors can lead to a relatively stable and long-term silencing, but the side effects of vectors such as inflammation and immune modification, especially using viral vectors, are inevitable. Using synthetic siRNA represents a novel nonviral approach and offers a transient effect that could adequately prevent or treat acute injuries in different organ systems. Therefore, the efficacy of synthetic caspase-3 siRNA verified here in isolated porcine kidney preservation could facilitate further tests in porcine kidney transplantation that is a well-established model and a necessary procedure for preclinical studies of synthetic siRNA in renal transplant patients.

Clinical translation of this work, however, has certain hurdles that need to be overcome: the genome database of porcine species is much less well established compared with that of human and mouse. There is a need for development of scramble siRNA material under good manufacturing practices. The tissue distribution and washout of siRNA postreperfusion need to be tracked. The complementary impacts of gene silencing in the context of allografts require to be further evaluated. The optimized dose and delivery routes and maximized impacts need to be further investigated.

In conclusion, the administration of caspase-3 siRNA directly to the artery of the kidney and hemoperfusate during cold preservation improved IRI with reduced caspase-3 protein and apoptosis, better renal oxygenation, and acid base homeostasis. This proof of principle experiment using a novel siRNA therapy in large animal organs provides valuable and transferable data for the potential modulation of human kidney donors.

MATERIALS AND METHODS

Recovery of Organs

Experimental porcine kidneys (n=6) were retrieved after 10 min of warm ischemia from schedule 1 killed white pigs weighing 60 to 70 kg. The kidneys were flushed with 500 mL hyperosmolar citrate (Soltran; Baxter healthcare, Norfolk, United Kingdom) and infused at 100 cm H2O hydrostatic pressure. Caspase-3 siRNA was administered directly into the renal artery in hyperosmolar citrate solution (3 μg/ml) with the renal vein clamped and into autologous blood (0.15 μg/ml), both of which were stored for 24 hr on ice. Approximately 1 L blood was collected from each animal into a sterile receptacle containing 25,000 units heparin (Multiparin; CP Pharmaceuticals, Wrexham, United Kingdom).

Three pairs of double-stranded caspase-3 siRNA, targeting porcine caspase-3 mRNA (NCBI CoreNucleotide Accession No.AB029345), were designed and constructed by Applied Biosystems/Ambion (Warrington, United Kingdom). These caspase-3 siRNAs were first applied in LLC-PK1 cell culture. The dose and time responses of these sequences were determined, and the most effective pair of caspase-3 siRNA sequences, sense, 5′-GGGAGACCUUCACAAACUUtt-3′, and antisense, 5′-AAGUUUGUGAAGGUCUCCCtg-3′, was selected and applied in this ex vivo study. Other two sequences of caspase-3 siRNA also acted as relative controls to verify the specificity of caspase-3 siRNA (19). The down-regulated expression of caspase-3 protein was detected at 24 hr and remained up to 96 hr at least, which gave a decent time window for therapeutic intervention. The in vitro data facilitated a reduction in the number of animals and in the cost of ex vivo study.

Reperfusion

The isolated organ preservation system was designed using clinical-grade cardiopulmonary bypass technology (Medtronic, Watford, United Kingdom) and incorporated an oxygenator (Medtronic), heat exchanger (Grant, GD120, Cambridge, United Kingdom), and centrifugal pump. After cannulation of the renal artery, vein, and ureter, kidneys were flushed with saline (Baxter Healthcare) at 4°C and reperfused with 500 mL of circuit priming solution. Five hundred milliliter of heparinized autologous whole blood was then added to the circuit, supplemented with nutrients and antibiotics, oxygenated with 95% oxygen/5% carbon dioxide for 3 hr at 38°C, and set mean arterial pressure of 85 mm Hg. The system has been well established in a variety of our previous studies (15, 26, 27). Renal blood flow and urine output were recorded continuously. Urine output was replaced with Ringer's lactate solution. Creatinine (Sigma, Dorset, United Kingdom) was added to the perfusate to achieve an initial circulating concentration of 1000 μmol/L. Creatinine clearance was calculated as the following formula: urinary creatinine×urine volume per minute/plasma creatinine. Four needle core biopsies were taken from different areas of the kidney in each time point of pre- and post-CS and 3 hr postreperfusion and fixed in 4% (wt/vol) neutral buffered formalin or stored at −80°C for histologic and molecular analyses.

Measurement of Caspase-3 Protein

Twenty microgram of protein of kidney cortex homogenate were separated on 15% (wt/vol) polyacrylamide denaturing gels and electroblotted onto Hybond-C membranes (Amersham Life Science, Buckinghamshire, United Kingdom) (2). These membranes were blocked with 5% (wt/vol) milk and probed with anti–full-length caspase-3 (1:1000 dilution, Santa Cruz Biochemicals, Santa Cruz, CA) antibody and reprobed with anti β-actin antibody (1:5000 dilution, Abcam plc, Cambridge, United Kingdom) (28). The antibody binding was revealed using an peroxidase conjugate from a DAKO ChemMate EnVision Detection Kit (DAKO, Glostrup, Denmark) diluted at 1:10 and both the ECL chemiluminescent detection system (Amersham Life Science) and 3,3-diaminobenzidine. Developed films/membranes were semiquantitatively analyzed by scanning volume density using a Bio-Rad GS-690 densitometer (Bio-Rad Laboratories Ltd., Hertfordshire, United Kingdom). Results were expressed as optical volume density of caspase-3 corrected by β-actin for loading.

Immunostaining of Active Caspase-3

Immunohistochemical staining of active caspase-3, recognizing 17-kDa subunit, was undertaken on paraffin sections using a DAKO ChemMate EnVision Detection Kit (DAKO) (2). Antigen retrieval was performed by immersion of the sections in 10-mM sodium citrate buffer, pH 6.0, in a steam bath maintained by high power microwave for 20 min and blocked by peroxidase-blocking reagent. The sections were labeled by an antiactive caspase-3 antibody (1:100 dilution, R&D System, Abingdon, United Kingdom) at 4°C overnight or normal rabbit immunoglobulin G (IgG) at the same protein concentration of primary antibody as negative control. The antibody binding was revealed by 3′-amino-9-ethylcarbazole (AEC, dark red color). Active caspase-3+ cells in the cortex of kidneys were semiquantitatively scored in 20 fields at 400× magnification.

In Situ End-Labeling Apoptotic Cells

Four-micrometer paraffin sections were used to label fragmented DNAs in situ with digoxigenin-deoxyuridine by terminal deoxynucleotidyl transferase using an ApopTag peroxidase kit (Appligene Oncor, Illkirch, France) (3). Briefly, sections were digested by 40 μg/mL proteinase K for 15 min at 37°C, incubated with terminal deoxynucleotidyl transferase and digoxigenin-deoxyuridine at 37°C for 60 min, and transferred to “stop” buffer for 30 min. After adding antidigoxigenin-peroxidase complex for 30 min, these sections were developed by AEC substrate. Apoptotic cells were examined at 400× magnification over 20 fields of tubular and interstitial areas separately.

Immunostaining of MPO

Immunohistochemical staining of MPO, a marker mainly for neutrophil granulocytes, was undertaken on paraffin sections using a DAKO ChemMate EnVision Detection Kit (DAKO) (15). The sections were digested by 40 μg/mL proteinase K for 15 min at 37°C and blocked by peroxidase-blocking reagent. The sections were labeled by an anti-MPO antibody (1:600 dilution, DAKO) at 4°C overnight. The antibody binding was revealed by AEC. MPO+ cells in the tubular and interstitial areas were semiquantitatively scored in 20 fields at 400× magnification separately.

Statistical Analysis

Results are expressed as mean±standard error of the mean. Normality tests were carried out, and statistical differences between two groups were assessed by unpaired t test using GraphPad Prism (GraphPad Software Inc., San Diego, CA). P values less than 0.05 were considered to be statistically significant. Continuous variables such as serum creatinine were plotted as level versus time curves for each kidney, and the mean area under the curve was calculated using Microsoft Excel Software (Microsoft, Reading, United Kingdom).

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Keywords:

Ischemia-reperfusion injury; Caspase-3; Apoptosis; Inflammation; Small interfering RNA; Isolated porcine kidneys

© 2011 Lippincott Williams & Wilkins, Inc.