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Short Hairpin RNA Knockdown of Connective Tissue Growth Factor by Ultrasound-Targeted Microbubble Destruction Improves Renal Fibrosis
Ultrasound in Med. & Biol., Vol. -, No. -, pp. 1–12, 2016 Copyright Ó 2016 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2016.07.022
Original Contribution
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SHORT HAIRPIN RNA KNOCKDOWN OF CONNECTIVE TISSUE GROWTH FACTOR BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION IMPROVES RENAL FIBROSIS SHUPING WEI,* CHAOLI XU,* JOSHUA J. RYCHAK,y ALICE LUONG,y YU SUN,z ZHIJIAN YANG,z MINGXIA LI,* CHUNRUI LIU,* NINGHUA FU,* and BIN YANG* y
* Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China; Targeson, Inc., San Diego, California, USA; and z Department of Pharmacological Study, Origin Biosciences, Inc., Nanjing, Jiangsu Province, China (Received 5 March 2016; revised 14 July 2016; in final form 25 July 2016)
Abstract—The purpose of this study was to evaluate whether ultrasound-targeted microbubble destruction transfer of interfering RNA against connective tissue growth factor (CTGF) in the kidney would ameliorate renal fibrosis in vivo. A short hairpin RNA (shRNA) targeting CTGF was cloned into a tool plasmid and loaded onto the surface of a cationic microbubble product. A unilateral ureteral obstruction (UUO) model in mice was used to evaluate the effect of CTGF knockdown. Mice were administered the plasmid-carrying microbubble intravenously, and ultrasound was applied locally to the obstructed kidney. Mice undergoing a sham UUO surgery and untreated UUO mice were used as disease controls, and mice administered plasmid alone, plasmid with ultrasound treatment and microbubbles and plasmid without ultrasound were used as treatment controls. Mice were treated once and then evaluated at day 14. CTGF in the kidney was measured by quantitative reverse transcription polymerase chain reaction and Western blot. Expression of CTGF, transforming growth factor b1, a smooth muscle actin and type I collagen in the obstructed kidney was evaluated by immunohistochemistry. The cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA and protein expression of CTGF (p , 0.01). Furthermore, CTGF gene silencing decreased the interstitial deposition of transforming growth factor b1, a smooth muscle actin and type I collagen as assessed in immunohistochemistry, as well as reduced renal fibrosis in pathologic alterations (p , 0.01). No significant changes in target mRNA, protein expression or disease pathology were observed in the control cohorts. A single treatment of ultrasound-targeted microbubble destruction is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in disease severity. This technique may be a potential therapy for treatment of renal fibrosis. (E-mail: yb12yx@ hotmail.com) Ó 2016 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound-targeted microbubble destruction, Sonoporation, Cationic microbubbles, Renal fibrosis, Unilateral ureteral obstruction, Connective tissue growth factor, Transforming growth factor-b, Gene delivery, RNA interfering, Short hairpin RNA.
INTRODUCTION
health, but also a severe social and economic problem (Ackland 2014; Essue et al. 2013). Renal fibrosis, which is characterized by excessive accumulation of extracellular matrix (ECM) and proliferation of myofibroblasts and fibroblasts, is widely regarded as the common histologic hallmark of CKD progressing to end-stage renal diseases (Klein et al. 2011; Tampe and Zeisberg 2014). This progression is considered to be effectively suppressed by anti-fibrotic treatment. a Smooth muscle actin (a-SMA) is a hallmark of myofibroblast differentiation and enables myofibroblasts to cause contraction of ECM (Rao et al. 2014). ECM production and cell proliferation are induced by several factors
There is a high incidence of chronic kidney disease (CKD) worldwide, and a trend toward increasing frequency has been noted. Approximately 10–13% of the general population is affected by some degree of CKD (Coresh et al. 2007; de Jong et al. 2008; Zhang et al. 2012), which is not only a serious threat to human
Address correspondence to: Bin Yang, Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, 305 Zhongshan East Road, Nanjing, Jiangsu Province 210002, China. E-mail: yb12yx@ hotmail.com 1
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including transforming growth factor-b1 (TGF-b1). TGF-b1 is generally considered to be the key profibrotic mediator in progressive renal fibrosis (Boor et al. 2010; Leask and Abraham 2004; Verrecchia and Mauviel 2002). Therapeutic strategies that involve blockage of TGF-b1 have resulted in attenuation of renal fibrosis in animal models (Feger et al. 2015). However, the multifunctional role of TGF-b1, which includes both anti-inflammatory and anti-proliferative effects, makes therapies using systemic blockade vulnerable to undesired adverse effects (Ihn 2008). Connective tissue growth factor (CTGF) was found to be an important downstream mediator of TGF-b1, and controls pro-fibrotic activities in renal fibrosis (Grotendorst 1997; Nguyen and Goldschmeding 2008). CTGF induced by TGF-b1 also enhances the biological activity of the latter (Crean et al. 2004; Riser et al. 2003). The expression of CTGF is low in normal renal tissue, whereas it is strongly upregulated both in experimental models of CKD and in human patients with a variety of chronic renal disease (de las Heras et al. 2006; Ito et al. 1998; Kanemoto et al. 2004). Because of its unique function in mediating fibrogenic activity, this novel modulator is considered to be a more suitable therapeutic target than direct inhibition of TGF-b1. Multiple animal and clinical studies have confirmed that inhibition of CTGF, by either blocking antibodies or gene therapy, reduces renal fibrosis (Adler et al. 2010; Guha et al. 2007; Yokoi et al. 2004). Gene therapy holds enormous potential in the treatment of renal disease. Use of a tissue-specific delivery technology would enable therapeutic genes to be targeted selectively to the kidney, potentially increasing efficacy and reducing off-target effects (van der Wouden et al. 2004). Existing methods of gene delivery generally suffer from low efficacy or poor tolerability in the kidney, which motivated our investigation of a novel kidney-specific approach to gene delivery. Ultrasound is widely used in clinical diagnostic imaging, both with and without microbubble contrast agents. Low-frequency ultrasound, in combination with some microbubble formulations, has been explored as a means for targeted delivery of biomolecules through a mechanism known as sonoporation (Delalande et al. 2015; Fan et al. 2014; Sirsi and Borden 2012) or ultrasound-targeted microbubble delivery (UTMD). Although the precise mechanism underlying sonoporation is not known, it is believed that oscillation of the microbubble in the ultrasound field induces transient poration in adjacent cells. The size of the pores can be large, on the order of 5 mm (Hu et al. 2013), which enables cytosolic delivery of a wide range of bio-active molecules. Various payloads have been delivered, including small molecules (Zhang et al. 2014), antibodies
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(Togtema et al. 2012), plasmid DNA (Tlaxca et al. 2013), viral particles (Warram et al. 2012) and small interfering RNA (Li et al. 2013). Sonoporation provides a method for localized and non-invasive intracellular delivery of therapeutic payloads. In addition, the microbubbles used for sonoporation are generally detectable using contrast ultrasound imaging, which provides a noninvasive means for precise guidance of the payload delivery (Carson et al. 2011). In the present study, we sought to determine whether UTMD could be used to deliver a short hairpin RNA (shRNA) specifically to the kidney and mediate knockdown of CTGF sufficiently to induce a meaningful therapeutic response. We used a commercially available sonoporation microbubble and designed an acoustic treatment protocol using a conventional ultrasound scanner to deliver a shRNA against CTGF in a mouse model of renal fibrosis. METHODS Construction of shRNA expression plasmids Three shRNAs targeting CTGF were synthesized and cloned into GV102 tool plasmid (Genechem, Shanghai, China). The sequences used were: shRNA1: 50 - CTTCCAAAGCAGTTGCAAA-30 shRNA2: 50 - ATACCTTCTGCAGGCTGGA-30 shRNA3: 50 -AAGCTGACCTAGAGGAAAA-30 . Expression was driven by the U6 promoter. All inserted sequences were verified through sequencing. The plasmids were amplified in Escherichia coli and purified using a plasmid DNA purification kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The plasmid concentration was measured by photometric absorption at 260 nm using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Knockdown efficiency of three CTGF-targeted shRNAs was verified in NRK-49 F cells (Cell Resource Center of Shanghai Life Sciences Institute, Chinese Academy of Sciences, Shanghai, China). shRNA sequence 1 had the highest silencing efficiency and was selected for subsequent use with UTMD. Microbubble preparation Cationic microbubbles (Targesphere) were purchased from Targeson (San Diego, CA, USA; distributed by Origin Biosciences in China). Targesphere is a dispersion of lipid/polymer microspheres encapsulating a core of decafluorobutane gas. The microbubble shell contains a slight positive charge to enable electrostatic binding of nucleic acids (Tlaxca et al. 2010). All microbubble products used here were from a single Targesphere lot. The size distribution of the microbubble product was
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measured by electrozone sensing using a Coulter IV Multisizer (Beckman-Coulter, Indianapolis, IN). A 50-mm aperture was used, providing histograms with 400 bins over 1.0–15 mm (diameter). The plasmid was incubated with the Targesphere microbubbles according to the manufacturer’s instructions, and fresh samples were prepared for each experiment. Briefly, 400 mg of CTGF-shRNA expression plasmid was injected into the Targesphere vial and incubated at room temperature for about 20 min with gentle shaking to keep the Targesphere agents suspended. Unconjugated plasmid was removed by three rounds of centrifugal washing at a speed of 1500 rpm for 5 min. Microbubbles were then re-suspended at a final concentration of 1 3 108/mL. The final concentration of the plasmid was measured as 667 mg/mL by absorbance at 260 nm. The presence of shRNA plasmid on the microbubble surface was confirmed by flow cytometry and epifluorescence microscopy, as described by Tlaxca et al. (2010). Plasmid-bearing microbubbles were prepared as described above, then diluted to 5 3 107/mL. Aliquots of 100 mL were fluorescence labeled with the green nucleotide-avid fluorophore YoYo-1 (Lot 1449939, Life Technologies [Thermo Fisher Scientific]) (3 mg per 1 3 108 MBs), and incubated at room temperature for 25 min. Unbound fluorophore was removed by three rounds of centrifugal washing in phosphate-buffered saline (PBS). Flow cytometry (Accuri C6; BD Biosciences, San Jose, CA, USA) was performed on the microbubbles, with unlabeled microbubbles used as a compensation control. Plasmid binding to the microbubble surface was also confirmed by epifluorescence microscopy (Olympus BX53, Tokyo, Japan). Mouse model of renal fibrosis Six-week-old female C57/B6 mice weighing 18– 25 g were purchased from the Beijing Kelihua Laboratory Animal Center (Beijing, China). Animals were maintained in a HEPA-filtered environment, where the room temperature was 24 C–25 C and the humidity was 50%–60%. Animals were fed an autoclaved laboratory rodent diet. The protocol for animal experiments was approved by the institutional animal care and use committee at our institution. A unilateral ureteral obstruction (UUO) model was induced as previously described (Nagae et al. 2008; Yokoi et al. 2004; Zhang et al. 2010). In brief, each mouse was anesthetized by intraperitoneal injection of 10% chloral hydrate (4 mL/kg) and prepared on a heated stage for aseptic surgery. The right ureter was exposed and separated after a right-side abdominal incision, then ligated with 4-O silk at two points and cut between the ligatures to prevent retrograde infection. The abdominal wound was closed in layers and sutured. A
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sham operation, in which the ureters of mice were exposed and manipulated without ligation, was performed in control animals. The animals were underwent UTMD at day 3 after UUO or sham operation. Experimental groups A total of 36 mice were randomly divided into six experimental groups as follows (6 mice per experimental group): In group 1, the sham group, animals underwent a sham operation as a disease control (Sham). In group 2, the UUO group, animals underwent UUO and received no treatment. In group 3, UUO models received CTGFshRNA expression plasmid alone without UTMD (P). In group 4, UUO models received plasmid and microbubble complexes without ultrasound exposure (P 1 MB). In group 5, UUO models received a plasmid injection (without microbubbles) followed by local ultrasound exposure (P 1 US). And in group 6, UUO models received plasmid and microbubbles followed by local ultrasound exposure (P 1 UTMD). Ultrasound imaging and UTMD Mice were anesthetized and then placed in a supine position on a heated stage. Ultrasound imaging was performed using a Siemens Sequoia 512 ultrasound scanner with a 15 L-8 linear transducer (Siemens, Mountain View, CA, USA) to orient the kidney for subsequent UTMD. The transducer was coupled to the mouse skin in the right kidney region with acoustic coupling gel, and a conventional B-mode ultrasound scan without contrast agents was first conducted to obtain the location, size and baseline echogenicity of the kidney. Color Doppler flow imaging was then performed to visualize blood flow in the kidney. The scanner was placed in contrast imaging mode (Cadence CPS), and gain settings were optimized to enable non-destructive detection of microbubble agents. A mechanical index (MI) of 0.14 was used. Plasmid and microbubble complexes (containing 40 mg of plasmid) were administered in 60 mL (approximately 1.2 3 107 microbubbles per dose) as a bolus by retroorbital injection. For animals receiving plasmid alone, microbubbles were replaced by PBS, and a dose of 40 mg plasmid in 60 mL was administered by retroorbital injection. The microbubble dose and plasmid dose were the same for each group. Non-destructive contrast ultrasound imaging was performed both before and after UTMD treatment to verify the arrival and complete clearance of microbubbles within the kidney. Ultrasound-targeted microbubble delivery was performed using a hand-held sonoporator (Sonitron 2000, Artison, OK, USA). This device consists of a piezoelectric transducer with a center frequency of 1 MHz. The transducer diameter was 20 mm. The settings used were as follows: duty cycle 5 25%, treatment
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duration 5 30 s, acoustic intensity 5 2 W/cm2 (peak negative pressure of approximately 600 kPa). The sonoporation probe was positioned at the right kidney region, as verified by B-mode ultrasound imaging. The sonoporator was cycled on and off in 30-s intervals to enable the microbubbles to fully reperfuse the kidney after each 30-s cycle of UTMD. This process was repeated for a total duration of 5 min, at which time clearance of all circulating microbubbles was observed by contrast ultrasound imaging. A second dose of microbubbles was then administered, and the UTMD treatment repeated for a total of three times. Histology and immunohistochemistry All mice were euthanized 14 d post-treatment. The right kidney was carefully excised, and tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Twomicrometer-thick sections were stained with Masson’s trichrome, The slides were evaluated by two blinded pathologists without knowledge of the origin. The fibrotic area was measured quantitatively using IMAGE-PRO PLUS software. For immunohistochemical analyses, the slices were rinsed with PBS for 3 min and blocked with 10% goat serum for 15 min at room temperature. Slices were then incubated overnight at 4 C with the following antibodies: rabbit anti-mouse CTGF polyclonal antibody (ab6992, Abcam, Cambridge, MA, USA); anti-TGFb1 (V) antibody (sc-146, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-a-SMA antibody [E184] (ab32575, Abcam); and anti-collagen I antibody (ab21286, Abcam). Samples were visualized using horseradish peroxidaselabeled goat anti-rabbit secondary antibody (HAF008, R&D Systems, Minneapolis, MN, USA) at 1:200 dilution. The staining intensity of CTGF, a-SMA, TGF-b1 and type I collagen was quantified using IMAGE-PRO PLUS software. Three fields of view selected randomly from each of two sections were examined by two pathologists without knowledge of the origin of the slides. Quantitative RT-PCR assay Total RNA was extracted from the different groups using Trizol reagent (Invitrogen; Carlsbad, CA, USA), per the manufacturer’s instructions. Briefly, approximately 20 mg of tissue was collected into an EP tube. One milliliter of Trizol reagent was added, and the tissue was homogenized and centrifuged at 12,000 rpm for 15 min. The supernatant was added to 200 mL of chloroform and shaken, then centrifuged at 12,000 rpm for 10 min. The supernatant was then placed in a new EP tube to which an equal volume of isopropanol was added, followed by mixing and centrifugation. The RNA was added to 1 mL of 75% ethanol, washed and centrifuged. The RNA was diluted in water, and concentration and pu-
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rity were measured. Reverse transcription was carried out using the PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc., Shiga, Japan) according to the standard protocol. A 20-mL reaction mixture containing 2 mg RNA and the following primers were used: CTGF Forward: 50 - CCCTGACCCAACTATGATGC-30 Reverse: 50 - CCTTACTCCCTGGCTTTACG-30 GADPH Forward: 50 - GGAAGGTGAAGGTCGGAGTC-30 Reverse: 50 - AATGAAGGGGTCATTCATGG-30 Fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) was performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions with the ABI StepOne plus FQ-RT-PCR system (Version 2.3, StepOne Software, Thermo Fisher Scientific). The housekeeping gene GAPDH was used as an internal control. The primers of CTGF and GAPDH were synthesized by Shanghai Generay Biotech (Shanghai, China), Primers were used at concentration of 0.2 mmol/L per reaction. Amplification conditions were as follows: 95 C for 5 min, followed by 40 cycles of 5 s at 95 C and 30 s at 60 C. For data analysis, the raw threshold cycle (Ct) value was normalized to the negative shRNA for each sample to obtain DCt. The normalized DCt was then calibrated to control samples to calculate DDCt. The relative quantitative results of mRNA were calculated with the equation 2–DDCt. Each assay was performed in triplicate. Western blot analysis Kidney tissue proteins were extracted using a Total Protein Extraction Kit (KeyGEN BioTECH, Nanjing, China). The protein concentration was determined using the Bradford (1976) method. Approximately 20 mg protein from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (5% stacking, 10% separating), at stacking 80 V for 60 min and separating 100 V for 90 min, and then transferred to an Immobilon polyvinylidene fluoride membrane. After being blocked with 5% skim milk blocking buffer overnight at room temperature, the membranes were incubated with CTGF antibody or b-actin antibody (AF0003, Beyotime Biotechnology, Shanghai, China) at a 1:200 dilution overnight at 4 C. After being washed, the membranes were hybridized with a horseradish peroxidaseconjugated secondary antibody at room temperature for 2 h and developed. The protein signals were normalized to b-actin levels. Antigen–antibody complexes were visualized with the enhanced chemiluminescence (ECL) system. The bands were analyzed with ImageJ (Version 6.0, National Institutes of Health, Bethesda, MD, USA).
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Statistical methods The data analysis software SPSS 16.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data are expressed as means 6 standard deviations. Statistical analysis of values was performed using the Mann–Whitney U-test. A p value ,0.05 was considered to indicate statistical significance. RESULTS Microbubble characterization The mean number and volume-weighted diameter of the Targesphere microbubbles used in this study were 2.1 mm and 4.4 mm, respectively. Figure 1a and b illustrate the averaged size distributions for two vials from the product lot used in this study. A payload of 384 mg of plasmid per 1 3 108 cationic microbubbles was measured by spectroscopy. Successful coupling of the plasmid to the microbubble surface was verified by flow cytometry and epifluorescence microscopy (Fig. 1c–e). Ultrasound imaging and gene delivery At day 3 after UUO, on conventional ultrasound imaging, the obstructed kidney was noticeably larger than the
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control or sham group kidney. On B-mode ultrasound, the parenchyma was progressively atrophic and surrounded a dilated collecting system, and slight hydronephrosis appeared in the obstructed kidney of all UUO animals (Fig. 2a, b). Blood flow to the kidney was confirmed for all animals by color Doppler imaging (Fig. 2c, d). Before microbubble administration, the contrast mode gain was set such that the pre-contrast noise was barely visible (Fig. 3b). After injection of Targesphere microbubbles, the kidney parenchyma was significantly enhanced (Fig. 3c). After each 30-s cycle of UTMD, the microbubbles were destroyed (Fig. 3d) and then allowed to reperfuse the kidney over 30 s (Fig. 3e). After 5 min of UTMD, no further replenishment of the kidney with contrast was observed (Fig. 3f).
Silencing of CTGF in vivo Knockdown was assessed at the messenger level by RT-PCR. CTGF mRNA expression of obstructed kidneys in the UUO group was significantly elevated relative to that of the sham surgery group (p , 0.01), as expected. A significant (p , 0.05) reduction in CTGF mRNA was observed in the UTMD treatment group relative to the
Fig. 1. Characterization of microbubble sonoporation reagents. Microbubble size distribution in (a) number and (b) volume mode. Histograms illustrate the fluorescence intensity of (c) unlabeled microbubbles (background) and (d) plasmidloaded microbubbles labeled with YoYo1. (e) Epifluorescence micrograph of plasmid-loaded microbubbles labeled with YoYo1.
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Fig. 2. Conventional ultrasound and color Doppler flow images of kidney at day 3 after unilateral ureteral obstruction (UUO) and sham operation. (a) B-Mode image of UUO kidney (arrow). (b) B-Mode image of sham kidney (arrow). (c) Color Doppler flow image of UUO kidney. (d) Color Doppler flow image of sham kidney.
untreated group. Groups receiving partial treatment (plasmid alone, plasmid 1 ultrasound and plasmid 1 microbubbles without ultrasound) exhibited a trend toward reduced CTGF mRNA, although this was not statistically significant (Fig. 4a). Western blot analysis of protein expression revealed a pattern similar to that of the RT-PCR results. Increased
CTGF protein expression was observed in the UUO group compared with the sham group (p , 0.01). UTMD treatment significantly (p , 0.01) reduced CTGF expression relative to the untreated group. There was no significant difference in CTGF expression between the three partial treatment groups and the untreated group (Fig. 4b, c).
Fig. 3. Conventional ultrasound and contrast-enhanced ultrasound images of obstructed kidney at day 3 after unilateral ureteral obstruction (UUO). (a) B-Mode image of obstructed kidney at day 3 after UUO, before microbubble administration. (b) Contrast-enhanced ultrasound image of obstructed kidney before microbubble injection (baseline). (c) Contrastenhanced ultrasound imaging of microbubble wash-in, approximately 10 s after injection. (d) Contrast-enhanced ultrasound image immediately after destruction of microbubbles UTMD. (e) Contrast-enhanced ultrasound image of microbubble replenishment within kidney, 30 s after UTMD. (f) Contrast-enhanced ultrasound image 5 min after UTMD, revealing the absence of microbubbles.
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Immunohistochemistry Immunohistochemical staining of the untreated group revealed marked upregulation in expression of CTGF, TGF-b1, a-SMA and type I collagen after UUO (p , 0.01) relative to sham-operated mice. The expression of each of these markers was significantly decreased in the UTMD treatment group (p , 0.01). In contrast, expression of CTGF, TGF-b1, a-SMA and type I collagen was unchanged in the partial treatment groups (Fig. 5). EFFECT OF CTGF SILENCING ON FIBROSIS HISTOPATHOLOGY In the UUO group, the obstructed kidneys developed tubular atrophy and epithelial flattening. Abundant mature collagen fibers with blue staining were observed by standard histologic assessment with Masson’s trichrome. Significant attenuation of fibrosis was observed in the UUO kidneys treated with UTMD (p , 0.01). However, there was no significant disease resolution in the other three treated groups compared with the UUO group (Fig. 6). DISCUSSION
Fig. 4. Silencing of CTGF in vivo (n 5 6 per group). (a) The mRNA of CTGF was detected by quantitative real-time polymerase chain reaction. (b) Expression of CTGF protein was determined using Western blot analysis. (c) Band intensities were measured and protein signals were normalized to b-actin levels. **p , 0.01, compared with the sham group. # p , 0.05, ##p , 0.01, compared with the UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
In the present study, we investigated the ability of UTMD to mediate silencing of CTGF and commensurate amelioration of renal fibrosis in mice after UUO. Successful knockdown of CTGF was observed at the messenger and protein levels in mice treated with UTMD after a single treatment. Mice treated with plasmid alone (without microbubbles) or microbubbles and plasmid in the absence of ultrasound exhibited a trend toward CTGF reduction, although this was not statistically significant. Reduced expression of pro-fibrotic markers was observed by immunohistochemistry in the UTMD-treated group. Finally, we noted a marked resolution of renal fibrosis by conventional histopathology in the UTMD-treated group at day 14. Unilateral ureteral expression is a widely used experimental model for inducing progressive renal tubulo-interstitial fibrosis. As its obstructed kidney is characterized by cellular proliferation, increased interstitial fibrosis, accumulation of collagen and decreased renal function, the induced pathogenetic events in this model are similar to those observed in human renal fibrosis (Moon et al. 2006; Voelkl et al. 2013). This model is likely to reveal useful biomarkers of key molecular pathways of progression of renal disease, as well as for evaluating new therapies (Feger et al. 2015; Ucero et al. 2014). In addition, UUO itself occurs in a wide variety of clinical disease settings. The prolonged insult will lead to irreversible renal
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Fig. 5. Immunocytochemical staining of CTGF, TGF-b1, a-SMA and type I collagen of the obstructed kidney in each group (n 5 6). (a) Representative microphotographs of CTGF, TGF-b1, a-SMA and type I collagen immunohistochemical staining in the kidney (3400). Quantitative evaluation of (b) CTGF, (c) TGF-b1, (d) a-SMA and (e) type I collagen with immunohistochemical staining using Image-Pro Plus software. Data are representative of three randomly selected microscopic fields from two samples. **p , 0.01, compared with the sham group. ##p , 0.01, compared with the UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; TGF-b1 5 transforming growth factor b1; a-SMA 5 a smooth muscle actin; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
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Fig. 6. Histopathology (n 5 6 per group). Renal fibrosis in each group was assessed by (a) H&E staining (3200) and (b) Masson’s trichrome staining (3200). (c) Semiquantitative analysis of the area of blue staining in Masson’s trichrome. Data are representative of three randomly selected microscopic fields from two samples. *p , 0.05, compared with the sham group. ##p , 0.01, compared with UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
injury, which may be prevented or reversed if the fibrotic response is halted. Treatments to address renal fibrosis are therefore warranted to ameliorate renal injury (Chevalier et al. 2009; Miyajima et al. 2000). Transforming growth factor b1 plays an important role in the formation and development of renal fibrosis. Previous experimental gene therapy techniques have involved blocking of TGF-b signaling or other targets in the TGF-b pathway using UTMD with microbubble contrast agents (Optison) to prevent renal fibrosis (Hou et al. 2005; Lan et al. 2003). Compared with TGF-b1, CTGF is considered to be a more attractive anti-fibrotic therapeutic target in renal disease because of its more restricted biological role. Several previous studies have reported that gene therapy targeting CTGF can effectively inhibit renal fibrosis (Liu et al. 2014; Ren et al. 2015). In this study, we used microbubbles to deliver a shRNA plasmid specifically to the kidney. When bound to the microbubble surface, plasmid was protected against degradation by circulating endonucleases in the blood. Additionally, the microbubble formulation used here is heavily PEGylated, which reduces unwanted interactions of the microbubbles with the reticuloendothelial system. This could further enhance the site-specific delivery and could provide a higher
plasmid concentration close to the site of cell membrane perforation (Lentacker et al. 2006; Wang et al. 2012). Finally, the cationic microbubble formulation enables a relatively high payload density (Borden et al. 2007; Vandenbroucke et al. 2008) relative to typical microbubble contrast agents to which DNA cannot be electrostatically coupled. Release of the plasmid from the microbubble and subsequent delivery into adjacent cells can be mediated by sonoporation, using locally applied ultrasound energy. This mechanism enables accumulation of the therapeutic gene in the target region at higher concentrations relative to competing techniques, thereby increasing therapeutic efficacy and potentially reducing side effects. This process can be monitored by contrast ultrasound imaging, which provides a convenient means for monitoring delivery in real time. Previous studies reported that after unilateral ureteral obstruction, TGF-b1 and CTGF mRNA expression in the obstructed kidney was upregulated from the early stage of interstitial fibrosis (at day 3), further increased at day 6 and peaked at day 14 (Yokoi et al. 2001). Similar findings were observed in a pilot experiment in our lab (data not shown). Our objective in this study was to determine whether a single UTMD treatment was sufficient to alter the course of renal fibrosis, and therefore, we
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Fig. 6. (Continued)
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decided to treat at day 3 when CTGF was first detectable. In our pilot experiment, we also found that CTGF mRNA expression in the obstructed kidney declined gradually after day 14 and then remained nearly constant. We therefore decided to use 14 d post-treatment as the study endpoint. Our partial treatment groups exhibited only negligible effects on molecular target expression and disease progression. This is in line with previous studies indicating that the plasmid-carrying microbubbles have essentially no ability to deliver their payload in the absence of applied ultrasound energy (Tlaxca et al. 2013). There are several limitations to our study. We did not directly investigate delivery of the shRNA payload to the kidney after UTMD. A pilot study suggested that a single dose of microbubbles was not sufficient to achieve sufficient knockdown (data not shown); for this reason, three successive cycles of UTMD were used. Exact measurement of the delivered mass dose of shRNA, and further optimization, is a topic for a subsequent study. The specific renal cell type or types to which the therapy was delivered were not directly assessed. Given the intravascular nature of the microbubble agent, treatment of endothelial and perivascular cells is most likely, although previous studies have reported delivery within the tubules after UTMD (Hou et al. 2005; Lan et al. 2003). In conclusion, our study indicates that delivery of CTGF shRNA by UTMD is able to inhibit the expression of CTGF and that a single treatment is able to ameliorate progressive renal fibrosis. UTMD provides a noninvasive and non-viral method for efficient and localized application of gene therapy and may be suitable for use in a variety of disease settings. Acknowledgments—This study was funded by grant from the National Natural Science Foundation of China (No. 81271592) to B.Y. Assistance from Targeson is gratefully acknowledged.
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microbubble destruction-mediated hVEGF-siRNA transfection in human PC-3 cells in vitro. Int J Mol Med 2013;31:163–171. Liu Y, Li W, Liu H, Peng Y, Yang Q, Xiao L, Liu Y, Liu F. Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of extracellular matrix molecules in cultured human renal proximal tubular cells. Renal Failure 2014; 36:278–284. Miyajima A, Chen J, Lawrence C, Ledbetter S, Soslow RA, Stern J, Jha S, Pigato J, Lemer ML, Poppas DP, Vaughan ED, Felsen D. Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 2000;58: 2301–2313. Moon JA, Kim HT, Cho IS, Sheen YY, Kim DK. IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy. Kidney Int 2006;70:1234–1243. Nagae T, Mori K, Mukoyama M, Kasahara M, Yokoi H, Suganami T, Sawai K, Yoshioka T, Koshikawa M, Saito Y, Ogawa Y, Kuwabara T, Tanaka I, Sugawara A, Kuwahara T, Nakao K. Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis. Kidney Int 2008;74:70–80. Nguyen TQ, Goldschmeding R. Bone morphogenetic protein-7 and connective tissue growth factor: Novel targets for treatment of renal fibrosis? Pharm Res 2008;25:2416–2426. Rao KB, Malathi N, Narashiman S, Rajan ST. Evaluation of myofibroblasts by expression of alpha smooth muscle actin: A marker in fibrosis, dysplasia and carcinoma. J Clin Diagn Res 2014;8: ZC14–ZC17. Ren Y, Du C, Yan L, Wei J, Wu H, Shi Y, Duan H. CTGF siRNA ameliorates tubular cell apoptosis and tubulointerstitial fibrosis in obstructed mouse kidneys in a Sirt1-independent manner. Drug Design Dev Ther 2015;9:4155–4171. Riser BL, Cortes P, DeNichilo M, Deshmukh PV, Chahal PS, Mohammed AK, Yee J, Kahkonen D. Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: Preliminary report. Kidney Int 2003;64:451–458. Sirsi SR, Borden MA. Advances in ultrasound mediated gene therapy using microbubble contrast agents. Theranostics 2012;2:1208–1222. Tampe D, Zeisberg M. Potential approaches to reverse or repair renal fibrosis. Nat Rev Nephrol 2014;10:226–237. Tlaxca JL, Anderson CR, Klibanov AL, Lowrey B, Hossack JA, Alexander JS, Lawrence MB, Rychak JJ. Analysis of in vitro transfection by sonoporation using cationic and neutral microbubbles. Ultrasound Med Biol 2010;36:1907–1918. Tlaxca JL, Rychak JJ, Ernst PB, Konkalmatt PR, Shevchenko TI, Pizarro TT, Rivera-Nieves J, Klibanov AL, Lawrence MB. Ultrasound-based molecular imaging and specific gene delivery to mesenteric vasculature by endothelial adhesion molecule targeted microbubbles in a mouse model of Crohn’s disease. J Control Release 2013;165:216–225. Togtema M, Pichardo S, Jackson R, Lambert PF, Curiel L, Zehbe I. Sonoporation delivery of monoclonal antibodies against human
Volume -, Number -, 2016 papillomavirus 16 E6 restores p53 expression in transformed cervical keratinocytes. PLoS One 2012;7:e50730. Ucero AC, Benito-Martin A, Izquierdo MC, Sanchez-Nino MD, Sanz AB, Ramos AM, Berzal S, Ruiz-Ortega M, Egido J, Ortiz A. Unilateral ureteral obstruction: beyond obstruction. Int Urol Nephrol 2014;46:765–776. van der Wouden EA, Sandovici M, Henning RH, de Zeeuw D, Deelman LE. Approaches and methods in gene therapy for kidney disease. J Pharmacol Toxicol Methods 2004;50: 13–24. Vandenbroucke RE, Lentacker I, Demeester J, De Smedt SC, Sanders NN. Ultrasound assisted siRNA delivery using PEGsiPlex loaded microbubbles. J Control Release 2008;126: 265–273. Verrecchia F, Mauviel A. Transforming growth factor-beta signaling through the Smad pathway: Role in extracellular matrix gene expression and regulation. J Invest Dermatol 2002; 118:211–215. Voelkl J, Mia S, Meissner A, Ahmed MS, Feger M, Elvira B, Walker B, Alessi DR, Alesutan I, Lang F. PKB/SGK-resistant GSK-3 signaling following unilateral ureteral obstruction. Kidney Blood Pressure Res 2013;38:156–164. Wang DS, Panje C, Pysz MA, Paulmurugan R, Rosenberg J, Gambhir SS, Schneider M, Willmann JK. Cationic versus neutral microbubbles for ultrasound-mediated gene delivery in cancer. Radiology 2012;264:721–732. Warram JM, Sorace AG, Saini R, Borovjagin AV, Hoyt K, Zinn KR. Systemic delivery of a breast cancer-detecting adenovirus using targeted microbubbles. Cancer Gene Ther 2012;19: 545–552. Yokoi H, Mukoyama M, Nagae T, Mori K, Suganami T, Sawai K, Yoshioka T, Koshikawa M, Nishida T, Takigawa M, Sugawara A, Nakao K. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 2004;15:1430–1440. Yokoi H, Sugawara A, Mukoyama M, Mori K, Makino H, Suganami T, Nagae T, Yahata K, Fujinaga Y, Tanaka I, Nakao K. Role of connective tissue growth factor in profibrotic action of transforming growth factor-beta: a potential target for preventing renal fibrosis. Am J Kidney Dis 2001;38:S134–S138. Zhang C, Huang P, Zhang Y, Chen J, Shentu W, Sun Y, Yang Z, Chen S. Anti-tumor efficacy of ultrasonic cavitation is potentiated by concurrent delivery of anti-angiogenic drug in colon cancer. Cancer Lett 2014;347:105–113. Zhang D, Sun L, Xian W, Liu F, Ling G, Xiao L, Liu Y, Peng Y, Haruna Y, Kanwar YS. Low-dose paclitaxel ameliorates renal fibrosis in rat UUO model by inhibition of TGF-beta/Smad activity. Lab Invest 2010;90:436–447. Zhang L, Wang F, Wang L, Wang W, Liu B, Liu J, Chen M, He Q, Liao Y, Yu X, Chen N, Zhang JE, Hu Z, Liu F, Hong D, Ma L, Liu H, Zhou X, Chen J, Pan L, Chen W, Wang W, Li X, Wang H. Prevalence of chronic kidney disease in China: A cross-sectional survey. Lancet 2012;379:815–822.
http://dx.doi.org/10.1016/j.ultrasmedbio.2016.07.022
Original Contribution
d
SHORT HAIRPIN RNA KNOCKDOWN OF CONNECTIVE TISSUE GROWTH FACTOR BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION IMPROVES RENAL FIBROSIS SHUPING WEI,* CHAOLI XU,* JOSHUA J. RYCHAK,y ALICE LUONG,y YU SUN,z ZHIJIAN YANG,z MINGXIA LI,* CHUNRUI LIU,* NINGHUA FU,* and BIN YANG* y
* Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China; Targeson, Inc., San Diego, California, USA; and z Department of Pharmacological Study, Origin Biosciences, Inc., Nanjing, Jiangsu Province, China (Received 5 March 2016; revised 14 July 2016; in final form 25 July 2016)
Abstract—The purpose of this study was to evaluate whether ultrasound-targeted microbubble destruction transfer of interfering RNA against connective tissue growth factor (CTGF) in the kidney would ameliorate renal fibrosis in vivo. A short hairpin RNA (shRNA) targeting CTGF was cloned into a tool plasmid and loaded onto the surface of a cationic microbubble product. A unilateral ureteral obstruction (UUO) model in mice was used to evaluate the effect of CTGF knockdown. Mice were administered the plasmid-carrying microbubble intravenously, and ultrasound was applied locally to the obstructed kidney. Mice undergoing a sham UUO surgery and untreated UUO mice were used as disease controls, and mice administered plasmid alone, plasmid with ultrasound treatment and microbubbles and plasmid without ultrasound were used as treatment controls. Mice were treated once and then evaluated at day 14. CTGF in the kidney was measured by quantitative reverse transcription polymerase chain reaction and Western blot. Expression of CTGF, transforming growth factor b1, a smooth muscle actin and type I collagen in the obstructed kidney was evaluated by immunohistochemistry. The cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA and protein expression of CTGF (p , 0.01). Furthermore, CTGF gene silencing decreased the interstitial deposition of transforming growth factor b1, a smooth muscle actin and type I collagen as assessed in immunohistochemistry, as well as reduced renal fibrosis in pathologic alterations (p , 0.01). No significant changes in target mRNA, protein expression or disease pathology were observed in the control cohorts. A single treatment of ultrasound-targeted microbubble destruction is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in disease severity. This technique may be a potential therapy for treatment of renal fibrosis. (E-mail: yb12yx@ hotmail.com) Ó 2016 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound-targeted microbubble destruction, Sonoporation, Cationic microbubbles, Renal fibrosis, Unilateral ureteral obstruction, Connective tissue growth factor, Transforming growth factor-b, Gene delivery, RNA interfering, Short hairpin RNA.
INTRODUCTION
health, but also a severe social and economic problem (Ackland 2014; Essue et al. 2013). Renal fibrosis, which is characterized by excessive accumulation of extracellular matrix (ECM) and proliferation of myofibroblasts and fibroblasts, is widely regarded as the common histologic hallmark of CKD progressing to end-stage renal diseases (Klein et al. 2011; Tampe and Zeisberg 2014). This progression is considered to be effectively suppressed by anti-fibrotic treatment. a Smooth muscle actin (a-SMA) is a hallmark of myofibroblast differentiation and enables myofibroblasts to cause contraction of ECM (Rao et al. 2014). ECM production and cell proliferation are induced by several factors
There is a high incidence of chronic kidney disease (CKD) worldwide, and a trend toward increasing frequency has been noted. Approximately 10–13% of the general population is affected by some degree of CKD (Coresh et al. 2007; de Jong et al. 2008; Zhang et al. 2012), which is not only a serious threat to human
Address correspondence to: Bin Yang, Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, 305 Zhongshan East Road, Nanjing, Jiangsu Province 210002, China. E-mail: yb12yx@ hotmail.com 1
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including transforming growth factor-b1 (TGF-b1). TGF-b1 is generally considered to be the key profibrotic mediator in progressive renal fibrosis (Boor et al. 2010; Leask and Abraham 2004; Verrecchia and Mauviel 2002). Therapeutic strategies that involve blockage of TGF-b1 have resulted in attenuation of renal fibrosis in animal models (Feger et al. 2015). However, the multifunctional role of TGF-b1, which includes both anti-inflammatory and anti-proliferative effects, makes therapies using systemic blockade vulnerable to undesired adverse effects (Ihn 2008). Connective tissue growth factor (CTGF) was found to be an important downstream mediator of TGF-b1, and controls pro-fibrotic activities in renal fibrosis (Grotendorst 1997; Nguyen and Goldschmeding 2008). CTGF induced by TGF-b1 also enhances the biological activity of the latter (Crean et al. 2004; Riser et al. 2003). The expression of CTGF is low in normal renal tissue, whereas it is strongly upregulated both in experimental models of CKD and in human patients with a variety of chronic renal disease (de las Heras et al. 2006; Ito et al. 1998; Kanemoto et al. 2004). Because of its unique function in mediating fibrogenic activity, this novel modulator is considered to be a more suitable therapeutic target than direct inhibition of TGF-b1. Multiple animal and clinical studies have confirmed that inhibition of CTGF, by either blocking antibodies or gene therapy, reduces renal fibrosis (Adler et al. 2010; Guha et al. 2007; Yokoi et al. 2004). Gene therapy holds enormous potential in the treatment of renal disease. Use of a tissue-specific delivery technology would enable therapeutic genes to be targeted selectively to the kidney, potentially increasing efficacy and reducing off-target effects (van der Wouden et al. 2004). Existing methods of gene delivery generally suffer from low efficacy or poor tolerability in the kidney, which motivated our investigation of a novel kidney-specific approach to gene delivery. Ultrasound is widely used in clinical diagnostic imaging, both with and without microbubble contrast agents. Low-frequency ultrasound, in combination with some microbubble formulations, has been explored as a means for targeted delivery of biomolecules through a mechanism known as sonoporation (Delalande et al. 2015; Fan et al. 2014; Sirsi and Borden 2012) or ultrasound-targeted microbubble delivery (UTMD). Although the precise mechanism underlying sonoporation is not known, it is believed that oscillation of the microbubble in the ultrasound field induces transient poration in adjacent cells. The size of the pores can be large, on the order of 5 mm (Hu et al. 2013), which enables cytosolic delivery of a wide range of bio-active molecules. Various payloads have been delivered, including small molecules (Zhang et al. 2014), antibodies
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(Togtema et al. 2012), plasmid DNA (Tlaxca et al. 2013), viral particles (Warram et al. 2012) and small interfering RNA (Li et al. 2013). Sonoporation provides a method for localized and non-invasive intracellular delivery of therapeutic payloads. In addition, the microbubbles used for sonoporation are generally detectable using contrast ultrasound imaging, which provides a noninvasive means for precise guidance of the payload delivery (Carson et al. 2011). In the present study, we sought to determine whether UTMD could be used to deliver a short hairpin RNA (shRNA) specifically to the kidney and mediate knockdown of CTGF sufficiently to induce a meaningful therapeutic response. We used a commercially available sonoporation microbubble and designed an acoustic treatment protocol using a conventional ultrasound scanner to deliver a shRNA against CTGF in a mouse model of renal fibrosis. METHODS Construction of shRNA expression plasmids Three shRNAs targeting CTGF were synthesized and cloned into GV102 tool plasmid (Genechem, Shanghai, China). The sequences used were: shRNA1: 50 - CTTCCAAAGCAGTTGCAAA-30 shRNA2: 50 - ATACCTTCTGCAGGCTGGA-30 shRNA3: 50 -AAGCTGACCTAGAGGAAAA-30 . Expression was driven by the U6 promoter. All inserted sequences were verified through sequencing. The plasmids were amplified in Escherichia coli and purified using a plasmid DNA purification kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The plasmid concentration was measured by photometric absorption at 260 nm using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Knockdown efficiency of three CTGF-targeted shRNAs was verified in NRK-49 F cells (Cell Resource Center of Shanghai Life Sciences Institute, Chinese Academy of Sciences, Shanghai, China). shRNA sequence 1 had the highest silencing efficiency and was selected for subsequent use with UTMD. Microbubble preparation Cationic microbubbles (Targesphere) were purchased from Targeson (San Diego, CA, USA; distributed by Origin Biosciences in China). Targesphere is a dispersion of lipid/polymer microspheres encapsulating a core of decafluorobutane gas. The microbubble shell contains a slight positive charge to enable electrostatic binding of nucleic acids (Tlaxca et al. 2010). All microbubble products used here were from a single Targesphere lot. The size distribution of the microbubble product was
CTGF knockdown by UTMD improves renal fibrosis d S. WEI et al.
measured by electrozone sensing using a Coulter IV Multisizer (Beckman-Coulter, Indianapolis, IN). A 50-mm aperture was used, providing histograms with 400 bins over 1.0–15 mm (diameter). The plasmid was incubated with the Targesphere microbubbles according to the manufacturer’s instructions, and fresh samples were prepared for each experiment. Briefly, 400 mg of CTGF-shRNA expression plasmid was injected into the Targesphere vial and incubated at room temperature for about 20 min with gentle shaking to keep the Targesphere agents suspended. Unconjugated plasmid was removed by three rounds of centrifugal washing at a speed of 1500 rpm for 5 min. Microbubbles were then re-suspended at a final concentration of 1 3 108/mL. The final concentration of the plasmid was measured as 667 mg/mL by absorbance at 260 nm. The presence of shRNA plasmid on the microbubble surface was confirmed by flow cytometry and epifluorescence microscopy, as described by Tlaxca et al. (2010). Plasmid-bearing microbubbles were prepared as described above, then diluted to 5 3 107/mL. Aliquots of 100 mL were fluorescence labeled with the green nucleotide-avid fluorophore YoYo-1 (Lot 1449939, Life Technologies [Thermo Fisher Scientific]) (3 mg per 1 3 108 MBs), and incubated at room temperature for 25 min. Unbound fluorophore was removed by three rounds of centrifugal washing in phosphate-buffered saline (PBS). Flow cytometry (Accuri C6; BD Biosciences, San Jose, CA, USA) was performed on the microbubbles, with unlabeled microbubbles used as a compensation control. Plasmid binding to the microbubble surface was also confirmed by epifluorescence microscopy (Olympus BX53, Tokyo, Japan). Mouse model of renal fibrosis Six-week-old female C57/B6 mice weighing 18– 25 g were purchased from the Beijing Kelihua Laboratory Animal Center (Beijing, China). Animals were maintained in a HEPA-filtered environment, where the room temperature was 24 C–25 C and the humidity was 50%–60%. Animals were fed an autoclaved laboratory rodent diet. The protocol for animal experiments was approved by the institutional animal care and use committee at our institution. A unilateral ureteral obstruction (UUO) model was induced as previously described (Nagae et al. 2008; Yokoi et al. 2004; Zhang et al. 2010). In brief, each mouse was anesthetized by intraperitoneal injection of 10% chloral hydrate (4 mL/kg) and prepared on a heated stage for aseptic surgery. The right ureter was exposed and separated after a right-side abdominal incision, then ligated with 4-O silk at two points and cut between the ligatures to prevent retrograde infection. The abdominal wound was closed in layers and sutured. A
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sham operation, in which the ureters of mice were exposed and manipulated without ligation, was performed in control animals. The animals were underwent UTMD at day 3 after UUO or sham operation. Experimental groups A total of 36 mice were randomly divided into six experimental groups as follows (6 mice per experimental group): In group 1, the sham group, animals underwent a sham operation as a disease control (Sham). In group 2, the UUO group, animals underwent UUO and received no treatment. In group 3, UUO models received CTGFshRNA expression plasmid alone without UTMD (P). In group 4, UUO models received plasmid and microbubble complexes without ultrasound exposure (P 1 MB). In group 5, UUO models received a plasmid injection (without microbubbles) followed by local ultrasound exposure (P 1 US). And in group 6, UUO models received plasmid and microbubbles followed by local ultrasound exposure (P 1 UTMD). Ultrasound imaging and UTMD Mice were anesthetized and then placed in a supine position on a heated stage. Ultrasound imaging was performed using a Siemens Sequoia 512 ultrasound scanner with a 15 L-8 linear transducer (Siemens, Mountain View, CA, USA) to orient the kidney for subsequent UTMD. The transducer was coupled to the mouse skin in the right kidney region with acoustic coupling gel, and a conventional B-mode ultrasound scan without contrast agents was first conducted to obtain the location, size and baseline echogenicity of the kidney. Color Doppler flow imaging was then performed to visualize blood flow in the kidney. The scanner was placed in contrast imaging mode (Cadence CPS), and gain settings were optimized to enable non-destructive detection of microbubble agents. A mechanical index (MI) of 0.14 was used. Plasmid and microbubble complexes (containing 40 mg of plasmid) were administered in 60 mL (approximately 1.2 3 107 microbubbles per dose) as a bolus by retroorbital injection. For animals receiving plasmid alone, microbubbles were replaced by PBS, and a dose of 40 mg plasmid in 60 mL was administered by retroorbital injection. The microbubble dose and plasmid dose were the same for each group. Non-destructive contrast ultrasound imaging was performed both before and after UTMD treatment to verify the arrival and complete clearance of microbubbles within the kidney. Ultrasound-targeted microbubble delivery was performed using a hand-held sonoporator (Sonitron 2000, Artison, OK, USA). This device consists of a piezoelectric transducer with a center frequency of 1 MHz. The transducer diameter was 20 mm. The settings used were as follows: duty cycle 5 25%, treatment
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duration 5 30 s, acoustic intensity 5 2 W/cm2 (peak negative pressure of approximately 600 kPa). The sonoporation probe was positioned at the right kidney region, as verified by B-mode ultrasound imaging. The sonoporator was cycled on and off in 30-s intervals to enable the microbubbles to fully reperfuse the kidney after each 30-s cycle of UTMD. This process was repeated for a total duration of 5 min, at which time clearance of all circulating microbubbles was observed by contrast ultrasound imaging. A second dose of microbubbles was then administered, and the UTMD treatment repeated for a total of three times. Histology and immunohistochemistry All mice were euthanized 14 d post-treatment. The right kidney was carefully excised, and tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Twomicrometer-thick sections were stained with Masson’s trichrome, The slides were evaluated by two blinded pathologists without knowledge of the origin. The fibrotic area was measured quantitatively using IMAGE-PRO PLUS software. For immunohistochemical analyses, the slices were rinsed with PBS for 3 min and blocked with 10% goat serum for 15 min at room temperature. Slices were then incubated overnight at 4 C with the following antibodies: rabbit anti-mouse CTGF polyclonal antibody (ab6992, Abcam, Cambridge, MA, USA); anti-TGFb1 (V) antibody (sc-146, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-a-SMA antibody [E184] (ab32575, Abcam); and anti-collagen I antibody (ab21286, Abcam). Samples were visualized using horseradish peroxidaselabeled goat anti-rabbit secondary antibody (HAF008, R&D Systems, Minneapolis, MN, USA) at 1:200 dilution. The staining intensity of CTGF, a-SMA, TGF-b1 and type I collagen was quantified using IMAGE-PRO PLUS software. Three fields of view selected randomly from each of two sections were examined by two pathologists without knowledge of the origin of the slides. Quantitative RT-PCR assay Total RNA was extracted from the different groups using Trizol reagent (Invitrogen; Carlsbad, CA, USA), per the manufacturer’s instructions. Briefly, approximately 20 mg of tissue was collected into an EP tube. One milliliter of Trizol reagent was added, and the tissue was homogenized and centrifuged at 12,000 rpm for 15 min. The supernatant was added to 200 mL of chloroform and shaken, then centrifuged at 12,000 rpm for 10 min. The supernatant was then placed in a new EP tube to which an equal volume of isopropanol was added, followed by mixing and centrifugation. The RNA was added to 1 mL of 75% ethanol, washed and centrifuged. The RNA was diluted in water, and concentration and pu-
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rity were measured. Reverse transcription was carried out using the PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc., Shiga, Japan) according to the standard protocol. A 20-mL reaction mixture containing 2 mg RNA and the following primers were used: CTGF Forward: 50 - CCCTGACCCAACTATGATGC-30 Reverse: 50 - CCTTACTCCCTGGCTTTACG-30 GADPH Forward: 50 - GGAAGGTGAAGGTCGGAGTC-30 Reverse: 50 - AATGAAGGGGTCATTCATGG-30 Fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) was performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions with the ABI StepOne plus FQ-RT-PCR system (Version 2.3, StepOne Software, Thermo Fisher Scientific). The housekeeping gene GAPDH was used as an internal control. The primers of CTGF and GAPDH were synthesized by Shanghai Generay Biotech (Shanghai, China), Primers were used at concentration of 0.2 mmol/L per reaction. Amplification conditions were as follows: 95 C for 5 min, followed by 40 cycles of 5 s at 95 C and 30 s at 60 C. For data analysis, the raw threshold cycle (Ct) value was normalized to the negative shRNA for each sample to obtain DCt. The normalized DCt was then calibrated to control samples to calculate DDCt. The relative quantitative results of mRNA were calculated with the equation 2–DDCt. Each assay was performed in triplicate. Western blot analysis Kidney tissue proteins were extracted using a Total Protein Extraction Kit (KeyGEN BioTECH, Nanjing, China). The protein concentration was determined using the Bradford (1976) method. Approximately 20 mg protein from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (5% stacking, 10% separating), at stacking 80 V for 60 min and separating 100 V for 90 min, and then transferred to an Immobilon polyvinylidene fluoride membrane. After being blocked with 5% skim milk blocking buffer overnight at room temperature, the membranes were incubated with CTGF antibody or b-actin antibody (AF0003, Beyotime Biotechnology, Shanghai, China) at a 1:200 dilution overnight at 4 C. After being washed, the membranes were hybridized with a horseradish peroxidaseconjugated secondary antibody at room temperature for 2 h and developed. The protein signals were normalized to b-actin levels. Antigen–antibody complexes were visualized with the enhanced chemiluminescence (ECL) system. The bands were analyzed with ImageJ (Version 6.0, National Institutes of Health, Bethesda, MD, USA).
CTGF knockdown by UTMD improves renal fibrosis d S. WEI et al.
Statistical methods The data analysis software SPSS 16.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data are expressed as means 6 standard deviations. Statistical analysis of values was performed using the Mann–Whitney U-test. A p value ,0.05 was considered to indicate statistical significance. RESULTS Microbubble characterization The mean number and volume-weighted diameter of the Targesphere microbubbles used in this study were 2.1 mm and 4.4 mm, respectively. Figure 1a and b illustrate the averaged size distributions for two vials from the product lot used in this study. A payload of 384 mg of plasmid per 1 3 108 cationic microbubbles was measured by spectroscopy. Successful coupling of the plasmid to the microbubble surface was verified by flow cytometry and epifluorescence microscopy (Fig. 1c–e). Ultrasound imaging and gene delivery At day 3 after UUO, on conventional ultrasound imaging, the obstructed kidney was noticeably larger than the
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control or sham group kidney. On B-mode ultrasound, the parenchyma was progressively atrophic and surrounded a dilated collecting system, and slight hydronephrosis appeared in the obstructed kidney of all UUO animals (Fig. 2a, b). Blood flow to the kidney was confirmed for all animals by color Doppler imaging (Fig. 2c, d). Before microbubble administration, the contrast mode gain was set such that the pre-contrast noise was barely visible (Fig. 3b). After injection of Targesphere microbubbles, the kidney parenchyma was significantly enhanced (Fig. 3c). After each 30-s cycle of UTMD, the microbubbles were destroyed (Fig. 3d) and then allowed to reperfuse the kidney over 30 s (Fig. 3e). After 5 min of UTMD, no further replenishment of the kidney with contrast was observed (Fig. 3f).
Silencing of CTGF in vivo Knockdown was assessed at the messenger level by RT-PCR. CTGF mRNA expression of obstructed kidneys in the UUO group was significantly elevated relative to that of the sham surgery group (p , 0.01), as expected. A significant (p , 0.05) reduction in CTGF mRNA was observed in the UTMD treatment group relative to the
Fig. 1. Characterization of microbubble sonoporation reagents. Microbubble size distribution in (a) number and (b) volume mode. Histograms illustrate the fluorescence intensity of (c) unlabeled microbubbles (background) and (d) plasmidloaded microbubbles labeled with YoYo1. (e) Epifluorescence micrograph of plasmid-loaded microbubbles labeled with YoYo1.
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Fig. 2. Conventional ultrasound and color Doppler flow images of kidney at day 3 after unilateral ureteral obstruction (UUO) and sham operation. (a) B-Mode image of UUO kidney (arrow). (b) B-Mode image of sham kidney (arrow). (c) Color Doppler flow image of UUO kidney. (d) Color Doppler flow image of sham kidney.
untreated group. Groups receiving partial treatment (plasmid alone, plasmid 1 ultrasound and plasmid 1 microbubbles without ultrasound) exhibited a trend toward reduced CTGF mRNA, although this was not statistically significant (Fig. 4a). Western blot analysis of protein expression revealed a pattern similar to that of the RT-PCR results. Increased
CTGF protein expression was observed in the UUO group compared with the sham group (p , 0.01). UTMD treatment significantly (p , 0.01) reduced CTGF expression relative to the untreated group. There was no significant difference in CTGF expression between the three partial treatment groups and the untreated group (Fig. 4b, c).
Fig. 3. Conventional ultrasound and contrast-enhanced ultrasound images of obstructed kidney at day 3 after unilateral ureteral obstruction (UUO). (a) B-Mode image of obstructed kidney at day 3 after UUO, before microbubble administration. (b) Contrast-enhanced ultrasound image of obstructed kidney before microbubble injection (baseline). (c) Contrastenhanced ultrasound imaging of microbubble wash-in, approximately 10 s after injection. (d) Contrast-enhanced ultrasound image immediately after destruction of microbubbles UTMD. (e) Contrast-enhanced ultrasound image of microbubble replenishment within kidney, 30 s after UTMD. (f) Contrast-enhanced ultrasound image 5 min after UTMD, revealing the absence of microbubbles.
CTGF knockdown by UTMD improves renal fibrosis d S. WEI et al.
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Immunohistochemistry Immunohistochemical staining of the untreated group revealed marked upregulation in expression of CTGF, TGF-b1, a-SMA and type I collagen after UUO (p , 0.01) relative to sham-operated mice. The expression of each of these markers was significantly decreased in the UTMD treatment group (p , 0.01). In contrast, expression of CTGF, TGF-b1, a-SMA and type I collagen was unchanged in the partial treatment groups (Fig. 5). EFFECT OF CTGF SILENCING ON FIBROSIS HISTOPATHOLOGY In the UUO group, the obstructed kidneys developed tubular atrophy and epithelial flattening. Abundant mature collagen fibers with blue staining were observed by standard histologic assessment with Masson’s trichrome. Significant attenuation of fibrosis was observed in the UUO kidneys treated with UTMD (p , 0.01). However, there was no significant disease resolution in the other three treated groups compared with the UUO group (Fig. 6). DISCUSSION
Fig. 4. Silencing of CTGF in vivo (n 5 6 per group). (a) The mRNA of CTGF was detected by quantitative real-time polymerase chain reaction. (b) Expression of CTGF protein was determined using Western blot analysis. (c) Band intensities were measured and protein signals were normalized to b-actin levels. **p , 0.01, compared with the sham group. # p , 0.05, ##p , 0.01, compared with the UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
In the present study, we investigated the ability of UTMD to mediate silencing of CTGF and commensurate amelioration of renal fibrosis in mice after UUO. Successful knockdown of CTGF was observed at the messenger and protein levels in mice treated with UTMD after a single treatment. Mice treated with plasmid alone (without microbubbles) or microbubbles and plasmid in the absence of ultrasound exhibited a trend toward CTGF reduction, although this was not statistically significant. Reduced expression of pro-fibrotic markers was observed by immunohistochemistry in the UTMD-treated group. Finally, we noted a marked resolution of renal fibrosis by conventional histopathology in the UTMD-treated group at day 14. Unilateral ureteral expression is a widely used experimental model for inducing progressive renal tubulo-interstitial fibrosis. As its obstructed kidney is characterized by cellular proliferation, increased interstitial fibrosis, accumulation of collagen and decreased renal function, the induced pathogenetic events in this model are similar to those observed in human renal fibrosis (Moon et al. 2006; Voelkl et al. 2013). This model is likely to reveal useful biomarkers of key molecular pathways of progression of renal disease, as well as for evaluating new therapies (Feger et al. 2015; Ucero et al. 2014). In addition, UUO itself occurs in a wide variety of clinical disease settings. The prolonged insult will lead to irreversible renal
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Fig. 5. Immunocytochemical staining of CTGF, TGF-b1, a-SMA and type I collagen of the obstructed kidney in each group (n 5 6). (a) Representative microphotographs of CTGF, TGF-b1, a-SMA and type I collagen immunohistochemical staining in the kidney (3400). Quantitative evaluation of (b) CTGF, (c) TGF-b1, (d) a-SMA and (e) type I collagen with immunohistochemical staining using Image-Pro Plus software. Data are representative of three randomly selected microscopic fields from two samples. **p , 0.01, compared with the sham group. ##p , 0.01, compared with the UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; TGF-b1 5 transforming growth factor b1; a-SMA 5 a smooth muscle actin; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
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Fig. 6. Histopathology (n 5 6 per group). Renal fibrosis in each group was assessed by (a) H&E staining (3200) and (b) Masson’s trichrome staining (3200). (c) Semiquantitative analysis of the area of blue staining in Masson’s trichrome. Data are representative of three randomly selected microscopic fields from two samples. *p , 0.05, compared with the sham group. ##p , 0.01, compared with UUO group. CTGF 5 connective tissue growth factor; UUO 5 unilateral ureteral obstruction; UTMD 5 ultrasound-targeted microbubble delivery; P 5 plasmid; MB 5 microbubbles; US 5 ultrasound.
injury, which may be prevented or reversed if the fibrotic response is halted. Treatments to address renal fibrosis are therefore warranted to ameliorate renal injury (Chevalier et al. 2009; Miyajima et al. 2000). Transforming growth factor b1 plays an important role in the formation and development of renal fibrosis. Previous experimental gene therapy techniques have involved blocking of TGF-b signaling or other targets in the TGF-b pathway using UTMD with microbubble contrast agents (Optison) to prevent renal fibrosis (Hou et al. 2005; Lan et al. 2003). Compared with TGF-b1, CTGF is considered to be a more attractive anti-fibrotic therapeutic target in renal disease because of its more restricted biological role. Several previous studies have reported that gene therapy targeting CTGF can effectively inhibit renal fibrosis (Liu et al. 2014; Ren et al. 2015). In this study, we used microbubbles to deliver a shRNA plasmid specifically to the kidney. When bound to the microbubble surface, plasmid was protected against degradation by circulating endonucleases in the blood. Additionally, the microbubble formulation used here is heavily PEGylated, which reduces unwanted interactions of the microbubbles with the reticuloendothelial system. This could further enhance the site-specific delivery and could provide a higher
plasmid concentration close to the site of cell membrane perforation (Lentacker et al. 2006; Wang et al. 2012). Finally, the cationic microbubble formulation enables a relatively high payload density (Borden et al. 2007; Vandenbroucke et al. 2008) relative to typical microbubble contrast agents to which DNA cannot be electrostatically coupled. Release of the plasmid from the microbubble and subsequent delivery into adjacent cells can be mediated by sonoporation, using locally applied ultrasound energy. This mechanism enables accumulation of the therapeutic gene in the target region at higher concentrations relative to competing techniques, thereby increasing therapeutic efficacy and potentially reducing side effects. This process can be monitored by contrast ultrasound imaging, which provides a convenient means for monitoring delivery in real time. Previous studies reported that after unilateral ureteral obstruction, TGF-b1 and CTGF mRNA expression in the obstructed kidney was upregulated from the early stage of interstitial fibrosis (at day 3), further increased at day 6 and peaked at day 14 (Yokoi et al. 2001). Similar findings were observed in a pilot experiment in our lab (data not shown). Our objective in this study was to determine whether a single UTMD treatment was sufficient to alter the course of renal fibrosis, and therefore, we
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Fig. 6. (Continued)
CTGF knockdown by UTMD improves renal fibrosis d S. WEI et al.
decided to treat at day 3 when CTGF was first detectable. In our pilot experiment, we also found that CTGF mRNA expression in the obstructed kidney declined gradually after day 14 and then remained nearly constant. We therefore decided to use 14 d post-treatment as the study endpoint. Our partial treatment groups exhibited only negligible effects on molecular target expression and disease progression. This is in line with previous studies indicating that the plasmid-carrying microbubbles have essentially no ability to deliver their payload in the absence of applied ultrasound energy (Tlaxca et al. 2013). There are several limitations to our study. We did not directly investigate delivery of the shRNA payload to the kidney after UTMD. A pilot study suggested that a single dose of microbubbles was not sufficient to achieve sufficient knockdown (data not shown); for this reason, three successive cycles of UTMD were used. Exact measurement of the delivered mass dose of shRNA, and further optimization, is a topic for a subsequent study. The specific renal cell type or types to which the therapy was delivered were not directly assessed. Given the intravascular nature of the microbubble agent, treatment of endothelial and perivascular cells is most likely, although previous studies have reported delivery within the tubules after UTMD (Hou et al. 2005; Lan et al. 2003). In conclusion, our study indicates that delivery of CTGF shRNA by UTMD is able to inhibit the expression of CTGF and that a single treatment is able to ameliorate progressive renal fibrosis. UTMD provides a noninvasive and non-viral method for efficient and localized application of gene therapy and may be suitable for use in a variety of disease settings. Acknowledgments—This study was funded by grant from the National Natural Science Foundation of China (No. 81271592) to B.Y. Assistance from Targeson is gratefully acknowledged.
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