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Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences (F.M.v.d.W., A.C.W., J.G.P.P., F.G.M.R., R.M.), and Department of Nuclear Medicine (O.C.B.), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
(Received February 1, 2006; accepted May 16, 2006)
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Abstract |
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). Because of the potential application of antisense oligonucleotides as therapeutic agents, several research groups investigated the biodistribution and pharmacokinetics of antisense oligonucleotides (reviewed by Levin, 1999). After i.v. administration, antisense oligonucleotides are rapidly cleared from the plasma and distributed to the peripheral tissues. In rats, phosphorothioate antisense oligonucleotides are mainly taken up by the kidney (Bijsterbosch et al., 1997; Lendvai et al., 2005). Studies on the intrarenal distribution of antisense oligonucleotides showed that oligonucleotide uptake is most prominent in the proximal tubule (Oberbauer et al., 1995; Carome et al., 1997). This specific uptake takes place via filtration by the glomeruli, followed by tubular reabsorption, as well as by direct uptake at the capillary side of the tubules (Sawai et al., 1996).
More recently, siRNA was discovered as a promising gene silencing tool in research and in the clinic. The molecular mechanism of RNA interference was first described by Fire et al. (1998), who observed a sequence-specific gene silencing at the post-transcriptional level in the nematode Caenorhabditis elegans after exposure to double-stranded RNA (dsRNA). In the RNA interference pathway, long dsRNA sequences are cleaved by the enzyme dicer into short 21-base pair stretches of dsRNA, the siRNA. These siRNA duplexes associate with a multiprotein complex known as the RNA-induced silencing complex. After unwinding, siRNA strands bind to complementary mRNA, causing a targeted degradation of mRNA and, thus, a translational block. The finding that exposure to siRNA duplexes shorter than 30 base pairs circumvent activation of the interferon pathway and overall suppression of gene expression enabled the successful use of this technique in mammalian cells (Elbashir et al., 2001; Tuschl and Borkhardt, 2002). Compared with antisense oligonucleotides, siRNA duplexes seem to be more resistant to biodegradation in cell culture and more efficient in silencing gene expression (Bertrand et al., 2002; Grunweller et al., 2003).
Besides the applications of siRNA in vitro, a great interest exists to apply this technique as an experimental or therapeutic tool in vivo. To achieve this, different experimental methods have been used to enhance effective delivery of siRNA to the target organs. For example, hydrodynamic injection of siRNA and cationic liposome-mediated delivery of siRNA in mice were effective in silencing gene expression in different organs in vivo (Lewis et al., 2002; Sioud and Sorensen, 2003; Song et al., 2003; Sorensen et al., 2003). However, despite the successful silencing of genes by siRNA in vivo, the effective delivery of siRNA to the target organs still forms a major obstacle, and little is known about the in vivo behavior of synthetic siRNA duplexes.
The objective of the present study was to investigate whether siRNA duplexes, like antisense oligonucleotides, are delivered spontaneously to the kidney and whether siRNA can silence genes in vivo. Our results showed that siRNA accumulates rapidly in rat kidneys after i.v. injection. In addition, we showed effective silencing of a transporter gene in the kidney proximal tubule.
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Materials and Methods |
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Radiolabeling of siRNA. Duplex 3'-amino siRNAMrp4 was conjugated with cDTPA (Sigma, St. Louis, MO) and subsequently labeled with Indium-111 (111In; half-life, 2.8 days) under strict RNase-free conditions. All the solutions were supplemented with 0.2% (v/v) diethylpyrocarbonate (Fluka, Buchs, Switzerland). All the solutions and pipet tips were autoclaved. The siRNA (20 nmol) was solubilized in 10 µl of suspension buffer and prepared as described above. The pH of the solution was adjusted to 8.2 by adding 10 µl of 0.2 M NaHCO3. A 50-fold molar excess of cDTPA solubilized in 36 µl of dimethylsulfoxide was added to the siRNA solution, and the reaction mixture was incubated for 45 min at room temperature. The reaction was stopped by adding 180 µl of 0.1 M citrate buffer, pH 5.0, and the fraction mixture was dialyzed extensively against 0.1 M citrate buffer, pH 5.0, in a Slide-A-Lyzer with a molecular cutoff of 3000 Da (Pierce, Rockford, IL). The siRNA-DTPA conjugate was stored in aliquots at –20°C until use.
For radiolabeling, we used 10 nmol of siRNA-DTPA conjugate in 100 µl of 0.1 M citrate buffer, pH 5.0. Next, 500 µCi 111InCl3 (Tyco Medical, Petten, The Netherlands) was added, and the mixture was incubated for 30 min at room temperature. The 111In-labeled DTPA-siRNA was purified by gel filtration on a disposable G25M Sephadex column (PD10, Pharmacia, Woerden, The Netherlands) eluted with phosphate-buffered saline. The specific activity of the final preparation was 30 µCi/nmol. The radiochemical purity of the preparation was tested by eluting a sample of the preparation on a PD10 column and exceeded 95%.
In Vivo Experiments. The Animal Experimental Committee of the Radboud University Nijmegen approved all the procedures involving animals. Specific pathogen free-bred Male Wistar Hannover (WH) rats (10–12 weeks) were obtained from Harlan (CPB, Zeist, The Netherlands). WH rats and Mrp2 transport-deficient (TR–) rats (de Vries et al., 1989) were housed under routine laboratory conditions at the Central Animal Facility Nijmegen.
Biodistribution of Radiolabeled siRNA. To study the biodistribution of 111In-labeled DTPA-siRNA, three rats were injected i.v. with 50 µCi 111Inlabeled DTPA-siRNA diluted in 200 µl of phosphate-buffered saline. A control group of three rats was injected i.v. with 50 µCi 111In-labeled DTPA. The rats were placed on a gamma camera (Siemens Orbitor, Hoffmann Estates, IL) equipped with a medium energy collimator, and scintigraphic images were acquired at 5, 30, and 60 min postinjection. Rats were euthanized at 1 h postinjection, and the biodistribution of the radiolabel was determined. A blood sample was drawn by cardiac puncture, and normal tissues (muscle, lung, spleen, kidney, liver, small intestines, and brain) were dissected, weighed, and counted in a gamma counter (Wizard, Pharmacia-LKB, Uppsala, Sweden). To correct for radioactive decay, injection standards were counted simultaneously. The activity in samples was expressed as a percentage of the injected dose per gram tissue (% ID/g).
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). TR– rats, lacking Mrp2 expression, were used to compare siRNA effects on Mrp2 transport function. Data Analysis. Data were analyzed using GraphPad Prism version 4.02 for windows (GraphPad Software, San Diego, CA). Data are given as mean ± S.E.M. Mean values were considered to be significantly different when p < 0.05 by use of a Student's t test, one-way analysis of variance (ANOVA), or two-way ANOVA, both followed by Bonferroni's multiple comparison test.
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Results |
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Functional Assessment of siRNA Treatment. The effects of siRNA duplexes were tested through assessment of the Abcc2/Mrp2 function. As a control, siRNA against rat Mrp4 (Abcc4) was used. MRP2 and MRP4 are members of the MRP family (MRP1-9), belonging to the subfamily C of the ATP-binding cassette transporters. These two transport proteins are expressed in the brush-border membrane of the kidney proximal tubule (Schaub et al., 1997; van Aubel et al., 2002), where they excrete a broad range of organic anionic compounds into the urine (van de Water et al., 2005). We showed previously that Mrp2 function can be monitored using an isolated perfused rat kidney model and calcein as a substrate (Masereeuw et al., 2003). Isolated kidneys were perfused with medium containing calcein-AM, which was converted intracellularly by esterases into the fluorescent Mrp2 substrate calcein. In TR– rats, the urinary calcein excretion was found to be highly impaired because of the lack of a functional Mrp2 (Masereeuw et al., 2003).
Four days after exposure of rats to a single dose of siRNAMrp2, a significant inhibition of the calcein excretion was found as compared with normal WH rats, indicating reduced Mrp2 function (Fig. 3). With the excretion in Mrp2-deficient TR– rats set as a baseline, a 35% inhibition of Mrp2 function was observed in siRNAMrp2-treated rats over the period 80 to 150 min of perfusion. Inhibition of calcein excretion was already observed 3 days postinjection (p < 0.001 at t = 60–70 min) (data not shown), but the functional knockdown of Mrp2 was highest 4 days after injection. Injection of rats with siRNAMrp4 did not significantly reduce Mrp2 function. This shows that other siRNA sequences, which are not complementary to Mrp2 mRNA, do not result in a nonspecific silencing of Mrp2.
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The present study clearly shows that after i.v. administration of even small amounts, siRNA preferentially accumulates in the kidney. After tail-vein injection, the sequences rapidly distributed throughout the body, and a large amount was delivered to the kidneys and excreted into the urine. One hour after injection, the amount of siRNA present in the kidneys was about 40 times higher than in the other organs. Although the biodistribution was studied with an siRNA sequence against Mrp4, we expect to find a similar biodistribution pattern with siRNA duplexes against any other gene. The functional down-regulation of Mrp2 in the kidney supports this.
Braasch et al. (2004) showed previously the biodistribution of siRNA in mice at 0 to 72 h after i.v. injection. In contrast to our findings, they found that 125I-labeled siRNA accumulated in the liver in addition to the kidney. This difference may be caused by the siRNA sequence and the radiolabeling method used and/or by interspecies differences. Because of its hydrophilic character, the DTPA linker we used for radioactive labeling of siRNA is easily filtered by the glomerulus, after which the complexes are specifically taken up by the renal proximal tubules. In accordance with Braasch et al., we detected very low siRNA concentrations in the brain, most likely reflecting a poor ability of siRNA to penetrate the blood-brain barrier.
The siRNA biodistribution pattern is similar to that found after i.v. injection of antisense oligonucleotides (Bijsterbosch et al., 1997; Lendvai et al., 2005), which is most likely independent of the sequence studied (Rifai et al., 1996).
For siRNA duplexes, the intrarenal distribution has not been investigated yet. However, because of the high reabsorption capacity of the proximal tubule, duplexes may, like antisense oligonucleotides, accumulate in this part of the nephron. Previous studies showed that antisense oligonucleotides predominantly accumulate in the proximal tubule cells after i.v. administration (Oberbauer et al., 1995; Carome et al., 1997). Uptake in these cells takes place via the capillary side and via reabsorption of antisense oligonucleotides from the tubular lumen. Compared with the capillary side, uptake at the lumenal side is much more efficient (Sawai et al., 1996). The uptake at both sites is saturable, indicating the involvement of receptor-mediated processes (Rappaport et al., 1995; Sawai et al., 1996). Although the receptors responsible for the uptake at both sites of the proximal tubule have not been identified yet, different groups showed the presence of oligonucleotide receptor proteins (Rappaport et al., 1995; Sawai et al., 1996; Bijsterbosch et al., 1997). In addition, two receptors expressed on the brush-border membrane of proximal tubule cells, cubulin and megalin, may play a role in the reabsorption of oligonucleotides from the glomerular filtrate (Sawai et al., 1996; Birn et al., 2000; Zhai et al., 2000).
Besides the application of synthetic siRNA duplexes of 21 nucleotides, RNA silencing can be induced by siRNA-expressing viral and nonviral vectors. Because of their different physical properties, biodistribution patterns of these vectors may be different from those of short 21-nucleotide siRNA duplexes. With a few successful exceptions, the kidney has been difficult to transduce with the various vectors available (Favre et al., 2000). DNA vectors can transduce proximal tubular cells by filtration through the glomerulus and reabsorption by the proximal tubule. DNA complexes that are unable to pass the glomerular basement membrane are most likely not effective to transduce proximal tubular cells via the basolateral site (Favre et al., 2000; Foglieni et al., 2000).
A research area in which siRNA can provide a useful tool in the characterization of gene function and the development of new therapies is that of drug transport proteins. Mrp2 and P-glycoprotein (MDR1, ABCB1), for example, play an important role in the excretion of many compounds, and multidrug resistance in cancer is associated with high expression levels of these transporters. Also, siRNA can be useful in the identification of new substrates of these transporters, as well as in therapy. An excellent overview about the application of siRNA in the study of drug transport proteins has been published recently by Tian et al. (2005).
The present study shows that after i.v. administration, siRNA accumulation in the kidney was sufficient to knock down transporter gene function. The i.v. administration of siRNAMrp2 resulted in a specific inhibition of Mrp2 function. We used an isolated rat kidney model perfused with calcein-AM to study Mrp2 function. This lipophilic compound diffuses into the cells, where it is converted by esterases into calcein. This fluorescent compound is excreted into urine by Mrp2 and not by Mrp4, and can be used as a model substrate to monitor Mrp2 function. We showed previously that calcein is an excellent model substrate for monitoring Mrp2 function in the isolated perfused rat kidney (Masereeuw et al., 2003), but we cannot exclude completely the involvement of other Mrp, viz. Mrp1, Mrp3, Mrp5, and Mrp6, in calcein excretion. In contrast to Mrp2 and Mrp4, Mrp1, Mrp3, Mrp5, and Mrp6 are localized in the basolateral membrane, of which only Mrp6 is expressed in the proximal tubule. Therefore, changes in Mrp1, Mrp3, and Mrp5 expression may not have influenced the urinary calcein excretion. Mrp6 may contribute to overall renal calcein handling; however, the substrate specificity of Mrp6 is very narrow. So far, calcein has not been identified as an Mrp6 substrate. In addition, exposure to siRNAMrp4 did not affect calcein excretion, suggesting that there were no aspecific effects on other Mrp transporters.
The effects of siRNA on protein expression largely depend on siRNA concentrations and stability, as well as the turnover rate of the protein of interest. Compared with antisense oligonucleotides, siRNA duplexes are relatively stable, and degradation of siRNA in serum is slow (Bertrand et al., 2002; Braasch et al., 2004). Expression of Mrp2 in the apical membrane of the proximal tubule is dependent on de novo protein synthesis, insertion into the apical membrane, and retrieval from the membrane followed by degradation. Jones et al. (2005) investigated the rate of Mrp2 protein synthesis and degradation in rat liver. Using in vivo metabolic labeling and autoradiography, they estimated the degradation half-life of Mrp2 as 27 h. With a half-life of 27 h for Mrp2, siRNA silencing effects on this protein can be detectable within several days after siRNA administration. Using the isolated perfused rat kidney model, we observed a clear inhibition of Mrp2 function on days 3 and 4 after injection. Unfortunately, we did not detect an inhibition of transporter expression after administration of siRNA using real-time reverse transcriptase-polymerase chain reaction or semiquantitative Western blot analysis (data not shown). Although mRNA and protein expression must be down-regulated as well, in our hands these two methods were not sensitive enough to significantly detect small changes in expression levels.
To test whether siRNA administration does not alter kidney function, different parameters were analyzed during the perfusion experiments. In siRNA-treated rats, basic renal functional parameters, like glomerular filtration rate, diuresis, fractional reabsorption of water, and renal perfusion pressure, were not different from control values, indicating that siRNA administration is safe and without side effects.
The use of siRNA has an advantage over the use of chemical inhibitors to reduce Mrp2 transport function because it shows a specific down-regulation. To date, no inhibitors have been identified that exclusively affect Mrp2. In addition, chemical inhibitors may have side effects. The spontaneous accumulation of siRNA duplexes in the kidney after i.v. administration has potential therapeutic implications. The i.v. administration of siRNA duplexes seems a simple but effective method in research and therapy to silence gene expression in the kidney. A more site-directed administration, like injection of siRNA into the renal artery, may even further enhance the accumulation of siRNA duplexes in the kidney.
In summary, the present study shows that after i.v. injection in rats, siRNA duplexes are distributed throughout the body within minutes. Most of the i.v. administered siRNA accumulates rapidly in the kidneys and is excreted via the urine over time. Moreover, we show for the first time successful suppression of Mrp2 in the renal proximal tubule. Therefore, i.v. administration of siRNA duplexes provides a novel research and potential therapeutic tool for gene silencing in the kidney.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: siRNA, short interfering RNA; dsRNA, double-stranded RNA; Mrp/Abcc, multidrug resistance protein; WH, Wistar Hannover; TR–, Mrp2 transport deficient; calcein-AM, calcein-acetoxymethylester; ANOVA, analysis of variance.
Address correspondence to: R. Masereeuw, Department of Pharmacology and Toxicology 149, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: r.masereeuw{at}ncmls.ru.nl
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, n = 16), treated for 4 days with siRNAMrp2 (
, n = 5), treated with siRNAMrp4 (
, n = 6), or in TR– rats (
, n = 4). Data are given as mean ± S.E.M. Statistical comparisons were performed by two-way ANOVA. *, significantly different from untreated WH rats (p < 0.001).