Ultrasound in Med. & Biol., Vol. 40, No. 6, pp. 1273–1281, 2014 Copyright Ó 2014 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.2013.12.006
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Original Contribution ULTRASOUND-MEDIATED MICROBUBBLE DESTRUCTION INCREASES RENAL INTERSTITIAL CAPILLARY PERMEABILITY IN EARLY DIABETIC NEPHROPATHY RATS YI ZHANG,*y CHUAN YE,zx YALI XU,* XUEXIN DONG,y JIANPING LI,y RONG LIU,y and YUNHUA GAO* * Department of Ultrasound, Xinqiao Hospital of the Third Military Medical University, Chongqing, China; y Department of Ultrasound, Forty-Fourth Military Hospital, Guiyang, China; z Department of Orthopaedics, Affiliated Hospital of Guiyang Medical College, Guiyang, China; and x Center for Tissue Engineering and Stem Cells, Guiyang Medical College, Guiyang, China (Received 21 September 2012; revised 30 November 2013; in final form 9 December 2013)
Abstract—Diabetic nephropathy (DN) is defined as persistent proteinuria corresponding to a urinary albumin excretion rate .300 mg/mg in the absence of other non-diabetic renal diseases. The aim of this study was to determine if ultrasound (US)-mediated microbubble (MB) destruction could increase renal interstitial capillary permeability in early DN rats. Diabetes was induced with streptozotocin. DN rats presented with mild micro-albuminuria 30 d after onset of diabetes. DN rats (N 5 120) were divided into four groups that received Evans blue (EB) followed by: (i) no treatment (control group); (ii) continuous ultrasonic irradiation for 5 min (frequency 5 7.00 MHz, mechanical index 5 0.9, peak rarefactional pressure 5 2.38 MPa: US group); (iii) microbubble injection (0.05 mL/kg: MB group); and (iv) both ultrasound and microbubble injection (US 1 MB group). Another 8 DN rats were subjected to ultrasound and microbubbles and then injected with EB after 24 h (recovery group). EB content, EB extravasation and E-selectin mRNA and protein expression significantly increased, and interstitial capillary walls became discontinuous in the US 1 MB group. Neither hemorrhage nor necrosis was observed on renal histology. Urine samples were collected 24 h post-treatment. There was no hematuria, and the urinary albumin excretion rate did not increase after ultrasound-microbubble interaction detected by urinalysis. EB content returned to the control group level after 24 h, as assessed for the recovery group. In conclusion, ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability in DN rats, and should be considered a therapy for enhancing drug and gene delivery to the kidney in the future. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Diabetic nephropathy, Ultrasound, Microbubble, Capillary permeability.
et al. 2007). DN has been subdivided into two stages based on the urinary albumin excretion rate (UAER): micro-albuminuria (30–299 mg/mg creatinine) and macro-albuminuria ($300 mg/mg creatinine) (Maahs et al. 2007). Micro-albuminuria is an early sign of diabetic nephropathy. Kidney transplantation is the preferred cell replacement therapy for DN. However, the scarcity of transplantable donors and the need for lifelong immunosuppression limit widespread use of this curative therapy. New antifibrotic therapies aimed at inhibition of profibrotic mediators are in development, but require large amounts of antibodies and need to be tested on top of the existing therapy of renin-angiotensin system inhibition (Deelman et al. 2009, 2010). The sustained hyperglycemic state accelerates the progress of early diabetic nephropathy in both type 1 and type 2 diabetic patients. Meticulous control of blood glucose decreases the risk of developing
INTRODUCTION As one of the most detrimental long-term complications of diabetes mellitus (DM), diabetic nephropathy (DN) has evolved as a leading cause of end-stage renal disease worldwide and is highly associated with the premature morbidity and mortality of diabetic patients (Ezquer et al. 2009). Traditionally, DN is considered one of the microvascular complications of DM; advanced DN is also characterized by tubulointerstitial fibrosis (Bonegio and Susztak 2012). It is widely accepted that tubulo-interstitial damage correlates with the degree of renal dysfunction and is a reliable predictor of end-stage renal disease (Lindenmeyer Address correspondence to: Yunhua Gao, Department of Ultrasound, Xinqiao Hospital of the Third Military Medical University, 183 Xinqiaozheng Street, Chongqing 400037, China. E-mail: [email protected] 1273
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nephropathy, but is not always feasible because of the limitations of currently available drugs and therapeutic techniques (Babaei-Jadidi et al. 2003). There is now strong evidence that early intervention and tight control have the potential to slow, or perhaps even reverse, the progression of early diabetic nephropathy. Consequently, early prevention of DN is an urgent medical issue at present. Ultrasound microbubble contrast agents have been developed to enhance conventional ultrasound imaging in clinical ultrasonography, by destroying intravascular microbubbles to characterize refill kinetics. Furthermore, ultrasound-mediated microbubble destruction is a new and promising technique applied in many fields and has been used to deliver drugs and genes to the cardiovascular system (Ferrara et al. 2007; Mayer and Bekeredjian 2008), blood-brain barrier and blood-tumor barrier (Vlachos et al. 2011; Wang et al. 2011); to enhance capillary and vascular permeability enhancement (Bekeredjian et al. 2007; Stieger et al. 2007); and to home stem cells (Xu et al. 2010). In vitro, 1–2 3 107 microbubbles bearing 100 pg small inhibitory RNA were added to a 60-mm dish seeded with 1 3 105 mouse squamous cell carcinoma cells (SCC-VII). The dish was inverted to permit microbubble-cell contact via microbubble buoyancy and continuously insonified at 1.3 MHz with a mechanical index (MI) of 1.6 for 2 min. Epidermal growth factor receptor expression in SCC-VII cells and epidermal growth factor-dependent growth were significantly reduced. The rupture of microbubbles via ultrasonic irradiation resulted in the deposition of microbubble shell components and increased cell membrane permeability localized to the site of microbubble-ultrasound interaction (Carson et al. 2012). In vivo, transforming growth factor b signaling and renal fibrosis were blocked in a rat ureteral obstruction model by transferring a doxycycline-regulated Smad7 gene using an ultrasound-microbubble-mediated system (continuous-wave output of 1-MHz ultrasound at 5% power output, for a total of 60 s with thirty 30-s intervals). The mechanism may be ultrasound-mediated microbubble cavitation, which increases the permeability of capillary and tubular basement membranes, allowing local release of DNA across the capillary (and tubular) basement membrane and its entry onto into glomerular, interstitial and tubular epithelial cells (Lan et al. 2003). However, no studies have reported whether ultrasound-mediated microbubble destruction is feasible for increasing renal interstitial capillary permeability in DN. On the basis of these previous findings, we explored whether this technique can be implemented to increase interstitial capillary permeability in DN because, in theory, most drugs and antifibrotic genes would benefit from increased capillary permeability. It is possible to take advantage of this technique to selectively increase drug and gene delivery to the
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tubulo-interstitial area, to enhance the therapeutic effect, to slow disease progression or, perhaps in the future, even to reverse the progression of DN. Previous studies on ultrasound-mediated microbubble destruction in the kidney focused mostly on the bio-effect with a combination of low-frequency, highpower and interval irradiation (Johnson et al. 2012). Miller et al. (2009) found that glomerular capillary hemorrhage, intratubular obstruction and tubular injury contribute to peritubular fibrosis in rats kidneys exposed to diagnostic ultrasound (1.5 MHz, MI 5 1.9, intermittent image exposure, peak rarefactional pressure amplitude 5 2.3 MPa) for 5 min. However, other researchers did not observe the same phenomenon under different conditions. Wible et al. (2002) reported that higher-frequency (.5 MHz), low-output-power ultrasound causes minimal biological alterations, and there was little no hemorrhage on the renal surface during continuous ultrasound exposure at higher frequencies (4 and 6 MHz). Jimenez et al. (2008) confirmed that there was no evidence of renal tissue damage and no capillary bleeding in porcine kidneys exposed to ultrasonic irradiation with a high MI (1.9), PPRA of 2.1 MPa and spatialpeak temporal-average intensity of 607 mW/cm2. These studies indicated that microbubble insonation and inertial cavitation may be tolerated by the kidney. Hence, our objective in the present study was to investigate whether ultrasound-mediated microbubble destruction increases renal interstitial capillary permeability in early diabetic nephropathy rats that presented with microalbuminuria but no renal histopathological changes. If it does, the technique might be exploited to promote higher concentrations of drugs, genes or antifibrotic mediators in the tubulo-interstitial area and improve the therapeutic effect. In addition, tubular epithelial cells express a variety of renal transporter proteins, which are localized mainly in the kidney proximal tubules. We studied mainly ultrasound-microbubble interaction and its impact on renal peritubular capillaries. On the basis of existing information, we optimized the parameters by evaluating the variables (MI, frequency, irradiation duration and dosage of microbubbles), and then used a combination of temperate MI (0.9), high frequency (7 MHz), peak rarefactional pressure (PRP) of 2.38 MPa and continuous irradiation. In particular, E-selectin is found only in endothelium. So, changes of state of endothelial cells after ultrasonic irradiation were detected by E-selectin mRNA and protein expression. Renal interstitial capillary permeability was assessed by Evans blue (EB) content assay, confocal laser scanning microscopy and transmission electron microscopy (TEM). Possible injury to the renal structure was evaluated by renal histology. Capillary permeability recovery was assessed by EB content assay.
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METHODS Ultrasound system A Model S2000 color diagnostic ultrasound machine (Siemens, Munich, Germany) with a linear array probe (9 L4) was employed as the diagnostic ultrasound source. The parameters of the ultrasound equipment and probe were as follows: MI 5 0.9; frequency 5 7.00 MHz; continuous irradiation for 5 min; depth 5 3 cm; single focal point of 1 cm. The MI and frequency provide an estimate of PRP within the renal tissue. The PRP, the value of which is 2.38 MPa, was calculated according to the formula pffiffiffi MI 5 Pr= f (1) where Pr is peak rarefactional pressure and f is frequency. Contrast medium Zhifuxian, a novel ultrasound contrast agent made by encapsulating perfluoropropane with a lipid shell, was awarded a National Invention Patent by China in 2005. It was provided by the ultrasound department of Xinqiao Hospital. Its concentration and mean diameter were approximately 4–9 3 109/mL and 2.13 mm, respectively. Numerous animal experimental studies had revealed that Zhifuxian does not produce changes in right ventricular blood pressure, as monitored by right ventricular systolic pressure and right ventricular end-diastolic pressure, and has no obvious effects on kidneys in rats, as observed by pathologic examination—properties that make it suitable for application (Liu et al. 2011; Gao et al. 2004a, 2004b). Establishment of model of early diabetic nephropathy rats Adult male Sprague Dawley rats (180–200 g, n 5 128) were provided by the Center for Experimental Animals of the Third Military Medical University. The experiments were approved by Animal Care and Use Committee of the Third Military Medical University. Rats were housed in wire cages with free access to a standard diet and tap water. The temperature and relative humidity of the animal facility were maintained under conventionally controlled conditions (22 C, 55% humidity) with a day-night rhythm. All rats were lightly anesthetized by intraperitoneal injection of 2% pentobarbital sodium at a dose of 30 mg/kg and received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ, Sigma) immediately after it had been dissolved in 0.1 M citrate buffer at pH 4.5. Three days after streptozotocin injection, blood samples were collected from the tail vein of nonfasted rats, and blood glucose concentrations were determined with a glucometer (Accu-Chek Aviva,
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Roche Applied Science). Diabetes was confirmed by a random blood glucose concentration .16.7 mmol/L for 3 consecutive days. Four weeks after onset of diabetes, diabetic rats presented with mild microalbuminuria (an early sign of DN) and were used in the experiments. Experimental groups Experiments were performed in a thermostatically controlled room at an ambient temperature of about 24– 26 C. Rats were fixed in the left lateral position after satisfactory anesthesia was induced by intraperitoneal injection of 2% pentobarbital sodium at a dose of 40 mg/kg. Only the right kidney of each rat was treated. The right flank was shaved, and the remaining hair removed using a depilatory cream. In rats exposed to ultrasound, the right kidney was localized and irradiated using the 9 L4 linear array probe of the ultrasound imaging system for 5 min of continuous irradiation (42 frames/s, MI 5 0.9, frequency 5 7.00 MHz). The probe was positioned in a transducer holder at an appropriate place, and the image plane of the ultrasound field was such that the contralateral kidney could not be insonated in the same ultrasound image. The rats (N 5 120) were randomly assigned to four groups. All rats received EB (2% in saline, 50 mg/kg) first. (i) The control group received no treatment after EB. (ii) The ultrasound (US) group was subjected to ultrasonic irradiation and injected with 0.5 mL of phosphate-buffered saline. (iii) The MB group was injected with microbubbles without ultrasonic irradiation. (iv) The US 1 MB group was subjected to ultrasonic irradiation and injected with microbubbles. Before injection, the bottle of microbubbles was shaken 45 s in a custom-built oscillation apparatus to form a microbubble suspension. Microbubbles were injected into the lateral tail vein through a 26-gauge needle connected to a 1-mL syringe via a 15-cm-long catheter (0.45 3 15 round wall, long bevel, China), controlled by a syringe pump; the injection was completed within 30 s and was followed by 0.5 mL saline to wash the tube. Rats were then covered with blankets for 1 h. After 1 h, 8 rats from each group (a total of 32 rats) were placed in metabolic cages for urine sample collection 24 h later, and the remaining rats were heart-perfused with heparinized saline (10 U/mL) through the left ventricle until the perfusion fluid collected from the right atrium became clear. Rats were then killed by decapitation, and right kidneys were quickly removed for testing. Additionally, to assess the permeability recovery of kidneys after ultrasoundmediated microbubble destruction, another 8 rats were subjected to ultrasonic irradiation, injected with microbubbles and, after 24 h, injected with EB (recovery group).
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Evans blue assay Right kidneys (32 of 120 rats, 8/group) were weighed on a balance, photographed with a digital camera (Casio, EX-Z80 A) in surface and coronal sectional views, cut into pieces and incubated in formamide solution at 37 C for 24 h. Pure formamide was used as a negative control. Optical density was measured with a spectrophotometer (Shimadzu, Kyoto, Japan) at the wavelength of 620 nm. The concentration of eluted dye was calculated using a standard curve (y 5 32.782x – 0.113) generated through serial dilutions of EB in 0.9% sodium chloride. EB content was assessed as eluted EB concentration and expressed as micrograms of EB per gram of dry tissue. The percentage of blue staining in photographs was analyzed using Image Pro Plus 6.0 software (Media, Cybernetics). The general steps were: (1) selection and measurement of target area; (2) measurement of surface and coronal areas; (3) determination of percentage of blue staining of renal surface and coronal plane using target area-to-surface area and target area-to-coronal area ratios, respectively. Confocal laser scanning microscopy Right kidneys (12 of 120 rats, 3/group) were cut in cross section at approximately 1-cm thickness to ensure that the irradiated part was located within the section. Frozen sections (5 mm) were made rapidly, and nuclei were stained with 4’,6-diamidino-2-phenylindole (blue). Fluorescence spatial images were obtained with a confocal laser scanning microscope (Zeiss LSM510, laser type diode, R-HeNe 633). EB was excited at lexc 5 510 nm, and fluorescence was monitored at lem 5 595 nm, whereas 4’,6-diamidino-2-phenylindole was excited at lexc 5 365 nm, and fluorescence was measured at lem 5 455 nm.
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Real-time polymerase chain reaction Real-time polymerase chain reaction (PCR) analysis was performed to investigate E-selectin mRNA expression. The entire tissue preparation procedure was carried out at 4 C. Renal cortices were weighted, minced and homogenized in a glass homogenizer. DNA was extracted with chloroform and precipitated with ethanol, and total DNA was assayed by ultraviolet absorbance. The oligonucleotide primers were as follows: E-selectin: 50 -CTCTGCTCTCACCTTTGTTCTCC-30 (sense) 50 -GCCCAGTTCTTAGCTTCCTCC-30 (antisense) b-actin: 50 -CGTTGACATCCGTAAAGACCTC-30 (sense) 50 -TAGGAGCCAGGGCAGTAATCT-30 (antisense) Real-time PCR assay was performed with target DNA, E-selectin primers and a fluorescence probe using a real-time PCR instrument (Applied Biosystems). Western blot analysis Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. Membranes were blocked using 0.1% Tween-20 in Tris-buffered saline (TTBS) containing 5% bovine serum albumin at room temperature for 2 h. Then membranes were washed three times with TTBS and incubated for 2 h at room temperature with goat polyclonal anti-E-selectin (1:500 in TTBS, Santa Cruz Biotechnology, Dallas, TX, USA) and b-actin (stained with polyclonal anti-b-actin 1:500 dilution, Santa Cruz Biotechnology). The signal was detected by the chemiluminescence method (Pierce Chemical, Rockford, IL, USA).
Transmission electron microscopy Renal cortices of right kidneys (12 of 120 rats, 3/group) were cut into 1-mm pieces and fixed in 2% glutaraldehyde for 1 h. After being washed with phosphate-buffered saline three times, the samples were postfixed in 1% osmium tetroxide in cacodylate buffer (pH 7.2) for 1 h. Subsequently, the samples were dehydrated in ethanol and embedded in epoxy resin (Agar 100). Ultrathin sections (50 nm) were cut, double stained with uranyl acetate and Reynolds lead citrate and examined under a transmission electron microscope (TECNAI 10, Philips, USA).
Urinalysis To assess the impact of ultrasound-mediated microbubble destruction on kidneys, urine samples from 32 rats in metabolic cages were collected 24 h after treatment and measured for red blood cells, urinary albumin and urinary creatinine. Microscopic hematuria is defined as three or more red blood cells per high-power field in a centrifuged urine specimen (Thaller and Wang 1999). Urinary albumin and creatinine concentrations were determined in the clinical laboratory of Xinqiao Hospital using an automatic biochemical analyzer (Hitachi, Tokyo, Japan). The urinary albumin excretion rate (UAER) was determined with the urinary albumin-to-urinary creatinine ratio.
Histologic analysis Right kidneys (20 of 120 rats, 5/group) were fixed in 10% formaldehyde, embedded in paraffin and sectioned at 5-mm thickness for hematoxylin-eosin (HE) and Periodic acid-Schiff staining. All sections were evaluated by a pathologist who was blinded to treatment.
Statistical analysis Data are summarized as means 6 standard deviations for each group. Student’s t-test was used to determine significant differences between two groups. One-way analysis of variance and post hoc comparisons (Bonferroni test) were used to determine significant differences among
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Fig. 1. Seven-megahertz ultrasound image of the rat kidney (mechanical index 5 0.9, B-mode scans, gray-scale mapping). Images were obtained (a) 0 s, (b) 15 s, and (c) 5 min after injection of microbubbles.
multiple groups. A p-value , 0.05 was considered to indicate statistical significance. RESULTS Renal ultrasonography Figure 1a is a renal ultrasound image of a diabetic nephropathy rat. Renal opacification and subsequent microbubble destruction could be observed under ultrasonic irradiation, indicating sufficient renal perfusion (Fig. 1b, c). Evans blue extravasation Photographs of the surface and coronal plane of kidneys revealed that portions of renal tissue stained blue under the combined effect of ultrasound and microbubbles, and the blue staining corresponded to regions that received ultrasonic irradiation (Fig. 2a). There was
slight blue staining in the US group—9.37 6 3.45% and 10.10 6 3.24% of the renal surface and coronal plane, respectively—but no visible staining in the control and MB groups. The proportions of renal surface and coronal plane that stained blue in the US 1 MB group were 49.12 6 7.31% and 37.99 6 4.36%, respectively. Renal capillary permeability was quantitatively evaluated with the EB content assay. Compared with the control group (13.942 6 2.848 mg/g), there were no statistically significant differences in EB content in the US, MB and recovery groups: 14.126 6 2.570, 13.969 6 2.076 and 14.658 6 3.916 mg/g, respectively, p . 0.05 (Fig. 2b). EB content was significantly higher in the US 1 MB group (37.267 6 4.948 mg/g) than in the control group (13.942 6 2.848 mg/g, p , 0.01), US group (14.126 6 2.570 mg/g, p , 0.01) and MB group (13.969 6 2.076 mg/g, p , 0.01), indicating a significant
Fig. 2. (a) Representative photographs of rat kidneys and changes in Evans blue (EB) extravasation. (b) Histogram of changes in EB extravasation. *p , 0.01 compared with the control group. #p , 0.01 compared with the group subjected to ultrasound (US). Op , 0.01 compared with the group injected with microbubbles (MB). :p , 0.01 compared with the recovery group.
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Fig. 3. Red fluorescence representing Evans blue (EB) was obvious in the group subjected to ultrasound and injected with microbubbles (US 1 MB) (d), but was not observed in the control group (a), the group subjected to ultrasound (US) (b) or the group injected with microbubbles (MB) (c). Nuclei were labeled with 4’,6-diamidino-2phenylindole (DAPI) (blue).
increase in renal capillary permeability after ultrasoundmediated microbubble destruction. There was no difference in EB content between the control and recovery groups (13.942 6 2.848 and 14.658 6 3.916 mg/g, respectively, p . 0.05), suggesting that the increased permeability was recovered. Exudative change in Evans blue under confocal laser scanning microscopy Renal capillary permeability was visually evaluated by confocal laser scanning microscopy. Renal interstitial capillary structure maintained intact and clear, and there was no EB extravasation in the renal interstitium in the control, US and MB groups (Fig. 3a–c); however, compared with the other groups (Fig. 3d), extravasation of red EB fluorescence around the interstitial capillary was evident in the US 1 MB group, resulting in a ‘‘fishing net’’ graph (Fig. 3a–c). This phenomenon depicted intuitively that intravascular EB extravasated from the interstitial capillary lumen and penetrated the renal interstitium. Ultrastructural changes in renal interstitial capillary under TEM Transmission electron microscopy was used to observe ultrastructural changes in capillaries, and we found that portions (approximately 30%–50%) of inter-
stitial capillary walls became thinner, discontinuous and roughened in the US 1 MB group (Fig. 4a), but remained intact in the control (Fig. 4b), US (Fig. 4c) and MB (Fig. 4d) groups, suggesting that mild injury to capillary endothelial cells was responsible for the changes and that interstitial capillary permeability increased after ultrasound-mediated microbubble destruction. E-selectin mRNA and protein expression To further evaluate the state of capillary endothelial cells after ultrasonic irradiation, E-selectin mRNA and protein expression was detected by real-Time PCR and Western blot. Similar to the changes in EB content and EB extravasation, the increases in E-selectin mRNA and protein expression were apparently more pronounced in the US 1 MB group than in the control, US and MB groups (p , 0.01) (Figs 5, 6). There were no significant differences between the control and US groups, between the control and MB groups or between the US and MB groups (p . 0.05). Renal histology There were no gross abnormalities of the kidneys in any group at necropsy. Periodic acid-Schiff staining revealed that there were no marked glomerular or tubular histological changes of early diabetic nephropathy,
Fig. 4. Endothelial cells of a renal interstitial capillary appeared mildly injured under transmission electron microscopy (bar 5 1 mm, original magnification 5 8900) and parts of the interstitial capillary walls were thinner, discontinuous and roughened (arrows) in the group subjected to ultrasound and injected with microbubbles (US 1 MB) (a), whereas the endothelial cells and interstitial capillary walls remained intact in the control group (b), US group (c) and MB group (d).
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There was no significant difference in UAER between the US 1 MB and control groups (60.12 6 25.67 and 55.99620.55 mg/mg creatinine, respectively, p . 0.05) or between the US 1 MB and US groups (60.12 6 25.67 and 57.43 6 18.40 mg/mg creatinine, respectively, p . 0.05) or US 1 MB and MB groups (60.12 6 25.67 and 53.86 6 22.42 mg/mg creatinine, respectively, p . 0.05). Hematuria was not detected in any group. Fig. 5. Real-time polymerase chain analysis revealed differences in E-selectin mRNA expression between the group subjected to ultrasound and injected with microbubbles (US 1 MB) and the other groups. Values represent means 6 standard deviations. *p , 0.01 versus the control group. # p , 0.01 versus the group subjected to ultrasound (US); O p , 0.01 versus the group injected with microbubbles (MB).
including mesangial expansion, glomerular basement membrane thickening, extracellular matrix deposition and tubular dilation. Hematoxylin-eosin staining revealed no hemorrhage or necrosis in the renal structure in all groups. In Figure 7 are images of the glomeruli, adjacent cortical tubule-interstitium and peritubular capillary of four groups. Urinalysis Albumin and creatinine levels were measured, and the UAER was calculated 24 h after treatment.
Fig. 6. Western blot analysis revealed differences in E-selectin protein expression between the group subjected to ultrasound and injected with microbubbles (US 1 MB) and the other groups. (a) Representative protein banding illustrated expressional differences in E-selectin. Results from the representative experiment were normalized to b-actin expression. (b) E-Selectin expression was quantified by scanning densitometry. Values represent means 6 standard deviations. *p , 0.01 versus the control group. #p , 0.01 versus the group subjected to ultrasound (US). Op , 0.01 versus the group injected with microbubbles (MB).
DISCUSSION Although most studies of DN have focused on glomeruli, tubule-interstitial damage is a major feature of DN and is an important prognostic factor in renal dysfunction. Given the limitations of kidney transplantation and drug and antifibrotic therapy for DN, our goal was to provide a new, non-invasive method to maximize the therapeutic effect for patients with DN patients. Ultrasound-mediated microbubble destruction is a new appealing technique for site-specific drug and gene delivery and has been used successfully in different clinical situations. Evans blue conjugates to serum albumin and is a sensitive and convenient tracer for assessing alterations in vascular permeability (Patterson et al. 1992; Tremblay et al. 2006; Whalen et al. 1998). The blue staining in the pelvis was visible in all treatment groups. A possible reason is that the kidney has three filtration membranes (capillary fenetration, basement membrane and podocyte pedicel), and the podocyte pedicel is the key barrier, filtrating only substances ,70,000 in molecular weight. Evans blue is filtrated by the podocyte pedicel because its molecular weight is 960. Serum albumin has a molecular weight of 65,000; therefore, Evans blue-serum albumin is also filtrated. In this study, the significant increases in EB content and EB fluorescence intensity in the US 1 MB group confirmed that interstitial capillary permeability could be increased by the interaction of ultrasound and microbubbles. The underlying mechanism may be that interstitial capillary walls became thinner, discontinuous and roughened as a result of ultrasound-mediated microbubble destruction, and EB extravasates from the ruptured capillary and penetrates the renal interstitium. The proposed method may allow the locally released antifibrotic agent to cross the ruptured interstitial capillary and enter the renal interstitium. Miller et al. (2008) consider the interaction of ultrasound pulses with gas bodies to be a form of acoustic cavitation, and they also may act as inertial cavitation nuclei. This interaction produces mechanical perturbation and a potential for bio-effects on nearby cells or tissues (Miller et al. 2008). E-Selectin is unique to endothelium. We surmised that microbubble cavitation via ultrasonic irradiation mildly injured
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Fig. 7. Periodic acid-Schiff (PAS) staining revealed no mesangial expansion, glomerular basement membrane thickening, extracellular matrix deposition or tubular dilation. Hematoxylin and eosin (HE) staining confirmed no evidence of hemorrhage and necrosis in the renal structure. US 5 group subjected to ultrasound, MB 5 group injected with microbubbles, US 1 MB 5 group subjected to ultrasound and injected with microbubbles.
endothelial cells, induced endothelial activity and then produced an inflammatory response in endothelial cells, resulting in the upregulation of E-selectin expression. Only the ultrastructural changes were observed on TEM, there was no evidence of hemorrhage and necrosis in renal structure and EB content returned to the control group level after 24 h, suggesting that the cavitation intensity was moderate, the inflammatory response was mild and acceptable and the increased permeability was recovered within a relatively short time and did not cause greater damage to the endothelial cells. There was no difference in UAER between the control group and the other groups 24 h post-treatment, indicating that the technique did not alter glomerular filtration function. Hematuria was not detected in any group, suggesting that there was essentially no glomerular capillary hemorrhage. Two possible reasons for these findings have been postulated. First, continuous irradiation decreased the extent of microbubble destruction. Intermittent irradiation might produce a greater degree of microbubble destruction than continuous irradiation for microbubble reperfusion in a sufficient interval. Second, an injection longer (within 30 s) than the bolus injection was used in this experiment. Increasing the injection time might decrease microbubble concentration per capillary and, thus, reduce the occurrence of glomerular capillary hemorrhage. Both ultrasound and microbubbles increased capillary permeability when used in combination and had no effect on capillary permeability when used alone, as there were no statistically significant differences in EB content and E-selectin mRNA and protein expression between the US and MB groups. Stem cell therapy is becoming an attractive option and a more viable therapy for DN (Sun et al. 2011; Volarevic et al. 2011). Differentiation of stem cells is subject to the micro-environment, which could be altered by ultrasound-mediated microbubble destruction (Zhong
et al. 2012). In one study, the local and transient increases in chemo-attractants and micro-environmental changes induced by pulsed focused ultrasound lasted at least 3 d, which enhanced homing, permeability and retention of bone marrow stromal cells to the treated kidney (Ziadloo et al. 2012). In this experiment, the mild injury to the interstitial capillary after cavitation caused the upregulation of E-selectin, which perhaps promoted the homing of transplanted stem cells to DN. This technology perhaps provides a new idea—personalized cell therapy for DN using stem cells. The parameters used in this study may differ if applied to humans because of the (i) lack of direct transferability between small animal conditions and human patients and (ii) greater variability in anatomic variability between human patients and rats. We performed this preliminary study to prove that ultrasound-mediated microbubble destruction is a non-invasive and feasible technology for increasing renal interstitial capillary permeability and modulating the micro-environment in early-stage diabetic nephropathy in the rat. This technique may provide guidance for development of a clinically applicable method to enhance the delivery of drugs, genes, antifibrotic agents or stem cells to the renal interstitium and improve the therapeutic effect. Much work remains in this area. For example, we may be able to take advantage of this technique and targeted microbubbles incorporating drugs, genes or antibodies to locally aggregate these components attached to microbubble fragments when the targeted microbubbles are irradiated and release transported substances to target organs to repair damaged tissue. However, elucidation of a detailed mechanism is warranted. CONCLUSIONS Ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability
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in early diabetic nephropathy rats and should be considered a therapy enhancing delivery of drugs, genes, antifibrotic agents or stem cells to the kidney in the future. The underlying mechanism may be that ultrasound-mediated microbubble cavitation ruptures interstitial capillary walls, which causes therapeutic substances to cross through discontinuous walls, enter renal the interstitium and maximize the therapeutic effect. Acknowledgments—This work was supported by the National Natural Science Foundation of China (Grant 81071160, 81101062); the Department of Public Health of Guizhou Province (gzwkj2013-1-157); the key project of the Chinese Ministry of Education (2012156); the Department of Science and Technology (G20107015, 20103166); and the Department of Education (2009143) of Guizhou Province.
REFERENCES Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S, Thornalley PJ. Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Diabetes 2003;52:2110–2120. Bekeredjian R, Kroll RD, Fein E, Tinkov S, Coester C, Winter G, Katus HA, Kulaksiz H. Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas. Ultrasound Med Biol 2007;33:1592–1598. Bonegio R, Susztak K. Notch signaling in diabetic nephropathy. Exp Cell Res 2012;318:986–992. Carson AR, McTiernan CF, Lavery L, Grata M, Leng X, Wang J, Chen X, Villanueva FS. Ultrasound-targeted microbubble destruction to delivery siRNA cancer therapy. Cancer Res 2012;72: 6191–6199. Deelman LE, Decleves AE, Rychak JJ, Sharma K. Targeted renal therapies through microbubbles and ultrasound. Adv Drug Deliv Rev 2010;62:1369–1377. Deelman LE, Sharma K. Mechanisms of kidney fibrosis and the role of antifibrotic therapies. Curr Opin Nephrol Hypertens 2009;18:85–90. Ezquer F, Ezquer M, Simon V, Pardo F, Yanez A, Carpio D, Conget P. Endovenous administration of bone-marrow-derived multipotent mesenchymal stromal cells prevents renal failure in diabetic mice. Biol Blood Marrow Transplant 2009;15:1354–1365. Ferrara K, Pollard R, Borden M. Ultrasound microbubble contrast agents: Fundamentals and application to gene and drug delivery. Annu Rev Biomed Eng 2007;9:415–447. Gao YH, Tan KB, Liu P. Experimental study on a new long circulation ultrasound contrast agent enhancing heart imaging. Chin J Ultrasound Med 2004a;20:81–83. Gao YH, Zeng GJ, Tan KB. The effect of contrast-enhanced ultrasonography on experimental renal tumor using a self-made liposome perfluocarbon ultrasound contrast agent. J Ultrasound Clin Med 2004b;6:257–259. Jimenez C, Gracia RD, Aguilera A, Alonso S, Cirugeda A, Benito J, Regojo RM, Aguilar R, Warlters A, Gomez R, Largo C, Selgas R. In situ kidney insonation with microbubble contrast agents does not cause renal tissue damage in a porcine model. J Ultrasound Med 2008;27:1607–1615. Johnson K, Cianciolo R, Gessner RC, Dayton PA. A pilot study to assess markers of renal damage in the rodent kidney after exposure to 7 MHz ultrasound pulse sequences designed to cause microbubble translation and disruption. Ultrasound Med Biol 2012;38:168–172. Lan HY, Mu W, Tomita N, Huang XR, Li JH, Zhu HJ, Morishita R, Johnson RJ. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol 2003;14:1535–1548. Lindenmeyer MT, Kretzler M, Boucherot A, Berra S, Yasuda Y, Henger A, Eichinger F, Gaiser S, Schmid H, Rastaldi MP, Schrier RW, Schlondorff D, Cohen CD. Interstitial vascular rarefac-
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tion and reduced VEGF-A expression in human diabetic nephropathy. J Am Soc Nephrol 2007;18:1765–1776. Liu P, Wang X, Zhou S, Hua X, Liu Z, Gao Y. Effects of a novel ultrasound contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue. Ultrasonics 2011;51:210–214. Maahs DM, Snell-Bergeon JK, Kinney GL, Wadwa RP, Garg S, Ogden LG, Rewers M. ACE-I/ARB treatment in type 1 diabetes patients with albuminuria is associated with lower odds of progression of coronary artery calcification. J Diabetes Complications 2007;21: 273–279. Mayer CR, Bekeredjian R. Ultrasonic gene and drug delivery to the cardiovascular system. Adv Drug Deliv Rev 2008;60:1177–1192. Miller DL, Averkiou MA, Brayman AA, Everbach EC, Holland CK, Wible JH, Wu J. Bioeffects considerations for diagnostic ultrasound contrast agents. J Ultrasound Med 2008;27:611–632. Miller DL, Dou CY, Wiggins RC. Glomerular capillary hemorrhage induced in rats by diagnostic ultrasound with gas-body contrast agent produces intratubular obstruction. Ultrasound Med Biol 2009;35:869–877. Patterson CE, Rhoades RA, Garcia JG. Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol 1992;72:865–873. Stieger SM, Caskey CF, Adamson RH, Qin S, Curry FE, Wisner ER, Ferrara KW. Enhancement of vascular permeability with lowfrequency contrast-enhanced ultrasound in the chorioallantoic membrane model. Radiology 2007;243:112–121. Sun CK, Yen CH, Lin YC, Tsai TH, Chang LT, Kao YH, Chua S, Fu M, Ko SF, Leu S, Yip HK. Autologous transplantation of adiposederived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model. J Transl Med 2011;9:118. Thaller TR, Wang LP. Evaluation of asymptomatic microscopic hematuria in adults. Am Fam Physician 1999;60:1143–1152. Tremblay PL, Auger FA, Huot J. Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases. Oncogene 2006;25:6563–6573. Vlachos F, Tung YS, Konofagou E. Permeability dependence study of the focused ultrasound-induced blood-brain barrier opening at distinct pressures and microbubble diameters using DCE-MRI. Magn Reson Med 2011;66:821–830. Volarevic V, Arsenijevic N, Lukic ML, Stojkovic M. Concise review: Mesenchymal stem cell treatment of the complications of diabetetes mellitus. Stem Cells 2011;29:5–10. Wang JE, Liu YH, Liu LB, Xia CY, Zhang Z, Xue YX. Effects of combining low frequency ultrasound irradiation with papaverine on the permeability of the blood-tumor barrier. J Neurooncology 2011;102:213–224. Whalen MJ, Carlos TM, Kochanek PM, Heineman S. Blood-brain barrier permeability, neutrophil accumulation and vascular adhesion molecule expression after controlled cortical impact in rats: A preliminary study. Acta Neurochir 1998;71:212–214. Wible JH Jr, Galen KP, Wojdyla JK, Hughes MS, Klibanov AL, Brandenburger GH. Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats. Ultrasound Med Biol 2002;28:1535–1546. Xu YL, Gao YH, Liu Z, Tan KB, Hua X, Fang ZQ, Wang YL, Wang YJ, Xia HM, Zhuo ZX. Myocardium-targeted transplantation of mesenchymal stem cells by diagnostic ultrasoundmediated microbubble destruction improves cardiac function in myocardial infarction of New Zealand rabbits. Int J Cardiol 2010;138:182–195. Zhong SG, Shu SY, Wang ZG, Luo J, Zhong WY, Ran HT, Zheng YY, Yin YH, Ling ZY. Enhanced homing of mesenchymal stem cells to the ischemic myocardium by ultrasound-targeted microbubble destruction. Ultrasonics 2012;52:281–286. Ziadloo A, Burks SR, Gold EM, Lewis BK, Chaudhry A, Merino MJ, Frenkel V, Frank JA. Enhanced homing permeability and retention of bone marrow stromal cells by noninvasive pulsed focused ultrasound. Stem Cells 2012;30:1216–1227.
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Original Contribution ULTRASOUND-MEDIATED MICROBUBBLE DESTRUCTION INCREASES RENAL INTERSTITIAL CAPILLARY PERMEABILITY IN EARLY DIABETIC NEPHROPATHY RATS YI ZHANG,*y CHUAN YE,zx YALI XU,* XUEXIN DONG,y JIANPING LI,y RONG LIU,y and YUNHUA GAO* * Department of Ultrasound, Xinqiao Hospital of the Third Military Medical University, Chongqing, China; y Department of Ultrasound, Forty-Fourth Military Hospital, Guiyang, China; z Department of Orthopaedics, Affiliated Hospital of Guiyang Medical College, Guiyang, China; and x Center for Tissue Engineering and Stem Cells, Guiyang Medical College, Guiyang, China (Received 21 September 2012; revised 30 November 2013; in final form 9 December 2013)
Abstract—Diabetic nephropathy (DN) is defined as persistent proteinuria corresponding to a urinary albumin excretion rate .300 mg/mg in the absence of other non-diabetic renal diseases. The aim of this study was to determine if ultrasound (US)-mediated microbubble (MB) destruction could increase renal interstitial capillary permeability in early DN rats. Diabetes was induced with streptozotocin. DN rats presented with mild micro-albuminuria 30 d after onset of diabetes. DN rats (N 5 120) were divided into four groups that received Evans blue (EB) followed by: (i) no treatment (control group); (ii) continuous ultrasonic irradiation for 5 min (frequency 5 7.00 MHz, mechanical index 5 0.9, peak rarefactional pressure 5 2.38 MPa: US group); (iii) microbubble injection (0.05 mL/kg: MB group); and (iv) both ultrasound and microbubble injection (US 1 MB group). Another 8 DN rats were subjected to ultrasound and microbubbles and then injected with EB after 24 h (recovery group). EB content, EB extravasation and E-selectin mRNA and protein expression significantly increased, and interstitial capillary walls became discontinuous in the US 1 MB group. Neither hemorrhage nor necrosis was observed on renal histology. Urine samples were collected 24 h post-treatment. There was no hematuria, and the urinary albumin excretion rate did not increase after ultrasound-microbubble interaction detected by urinalysis. EB content returned to the control group level after 24 h, as assessed for the recovery group. In conclusion, ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability in DN rats, and should be considered a therapy for enhancing drug and gene delivery to the kidney in the future. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Diabetic nephropathy, Ultrasound, Microbubble, Capillary permeability.
et al. 2007). DN has been subdivided into two stages based on the urinary albumin excretion rate (UAER): micro-albuminuria (30–299 mg/mg creatinine) and macro-albuminuria ($300 mg/mg creatinine) (Maahs et al. 2007). Micro-albuminuria is an early sign of diabetic nephropathy. Kidney transplantation is the preferred cell replacement therapy for DN. However, the scarcity of transplantable donors and the need for lifelong immunosuppression limit widespread use of this curative therapy. New antifibrotic therapies aimed at inhibition of profibrotic mediators are in development, but require large amounts of antibodies and need to be tested on top of the existing therapy of renin-angiotensin system inhibition (Deelman et al. 2009, 2010). The sustained hyperglycemic state accelerates the progress of early diabetic nephropathy in both type 1 and type 2 diabetic patients. Meticulous control of blood glucose decreases the risk of developing
INTRODUCTION As one of the most detrimental long-term complications of diabetes mellitus (DM), diabetic nephropathy (DN) has evolved as a leading cause of end-stage renal disease worldwide and is highly associated with the premature morbidity and mortality of diabetic patients (Ezquer et al. 2009). Traditionally, DN is considered one of the microvascular complications of DM; advanced DN is also characterized by tubulointerstitial fibrosis (Bonegio and Susztak 2012). It is widely accepted that tubulo-interstitial damage correlates with the degree of renal dysfunction and is a reliable predictor of end-stage renal disease (Lindenmeyer Address correspondence to: Yunhua Gao, Department of Ultrasound, Xinqiao Hospital of the Third Military Medical University, 183 Xinqiaozheng Street, Chongqing 400037, China. E-mail: [email protected] 1273
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nephropathy, but is not always feasible because of the limitations of currently available drugs and therapeutic techniques (Babaei-Jadidi et al. 2003). There is now strong evidence that early intervention and tight control have the potential to slow, or perhaps even reverse, the progression of early diabetic nephropathy. Consequently, early prevention of DN is an urgent medical issue at present. Ultrasound microbubble contrast agents have been developed to enhance conventional ultrasound imaging in clinical ultrasonography, by destroying intravascular microbubbles to characterize refill kinetics. Furthermore, ultrasound-mediated microbubble destruction is a new and promising technique applied in many fields and has been used to deliver drugs and genes to the cardiovascular system (Ferrara et al. 2007; Mayer and Bekeredjian 2008), blood-brain barrier and blood-tumor barrier (Vlachos et al. 2011; Wang et al. 2011); to enhance capillary and vascular permeability enhancement (Bekeredjian et al. 2007; Stieger et al. 2007); and to home stem cells (Xu et al. 2010). In vitro, 1–2 3 107 microbubbles bearing 100 pg small inhibitory RNA were added to a 60-mm dish seeded with 1 3 105 mouse squamous cell carcinoma cells (SCC-VII). The dish was inverted to permit microbubble-cell contact via microbubble buoyancy and continuously insonified at 1.3 MHz with a mechanical index (MI) of 1.6 for 2 min. Epidermal growth factor receptor expression in SCC-VII cells and epidermal growth factor-dependent growth were significantly reduced. The rupture of microbubbles via ultrasonic irradiation resulted in the deposition of microbubble shell components and increased cell membrane permeability localized to the site of microbubble-ultrasound interaction (Carson et al. 2012). In vivo, transforming growth factor b signaling and renal fibrosis were blocked in a rat ureteral obstruction model by transferring a doxycycline-regulated Smad7 gene using an ultrasound-microbubble-mediated system (continuous-wave output of 1-MHz ultrasound at 5% power output, for a total of 60 s with thirty 30-s intervals). The mechanism may be ultrasound-mediated microbubble cavitation, which increases the permeability of capillary and tubular basement membranes, allowing local release of DNA across the capillary (and tubular) basement membrane and its entry onto into glomerular, interstitial and tubular epithelial cells (Lan et al. 2003). However, no studies have reported whether ultrasound-mediated microbubble destruction is feasible for increasing renal interstitial capillary permeability in DN. On the basis of these previous findings, we explored whether this technique can be implemented to increase interstitial capillary permeability in DN because, in theory, most drugs and antifibrotic genes would benefit from increased capillary permeability. It is possible to take advantage of this technique to selectively increase drug and gene delivery to the
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tubulo-interstitial area, to enhance the therapeutic effect, to slow disease progression or, perhaps in the future, even to reverse the progression of DN. Previous studies on ultrasound-mediated microbubble destruction in the kidney focused mostly on the bio-effect with a combination of low-frequency, highpower and interval irradiation (Johnson et al. 2012). Miller et al. (2009) found that glomerular capillary hemorrhage, intratubular obstruction and tubular injury contribute to peritubular fibrosis in rats kidneys exposed to diagnostic ultrasound (1.5 MHz, MI 5 1.9, intermittent image exposure, peak rarefactional pressure amplitude 5 2.3 MPa) for 5 min. However, other researchers did not observe the same phenomenon under different conditions. Wible et al. (2002) reported that higher-frequency (.5 MHz), low-output-power ultrasound causes minimal biological alterations, and there was little no hemorrhage on the renal surface during continuous ultrasound exposure at higher frequencies (4 and 6 MHz). Jimenez et al. (2008) confirmed that there was no evidence of renal tissue damage and no capillary bleeding in porcine kidneys exposed to ultrasonic irradiation with a high MI (1.9), PPRA of 2.1 MPa and spatialpeak temporal-average intensity of 607 mW/cm2. These studies indicated that microbubble insonation and inertial cavitation may be tolerated by the kidney. Hence, our objective in the present study was to investigate whether ultrasound-mediated microbubble destruction increases renal interstitial capillary permeability in early diabetic nephropathy rats that presented with microalbuminuria but no renal histopathological changes. If it does, the technique might be exploited to promote higher concentrations of drugs, genes or antifibrotic mediators in the tubulo-interstitial area and improve the therapeutic effect. In addition, tubular epithelial cells express a variety of renal transporter proteins, which are localized mainly in the kidney proximal tubules. We studied mainly ultrasound-microbubble interaction and its impact on renal peritubular capillaries. On the basis of existing information, we optimized the parameters by evaluating the variables (MI, frequency, irradiation duration and dosage of microbubbles), and then used a combination of temperate MI (0.9), high frequency (7 MHz), peak rarefactional pressure (PRP) of 2.38 MPa and continuous irradiation. In particular, E-selectin is found only in endothelium. So, changes of state of endothelial cells after ultrasonic irradiation were detected by E-selectin mRNA and protein expression. Renal interstitial capillary permeability was assessed by Evans blue (EB) content assay, confocal laser scanning microscopy and transmission electron microscopy (TEM). Possible injury to the renal structure was evaluated by renal histology. Capillary permeability recovery was assessed by EB content assay.
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METHODS Ultrasound system A Model S2000 color diagnostic ultrasound machine (Siemens, Munich, Germany) with a linear array probe (9 L4) was employed as the diagnostic ultrasound source. The parameters of the ultrasound equipment and probe were as follows: MI 5 0.9; frequency 5 7.00 MHz; continuous irradiation for 5 min; depth 5 3 cm; single focal point of 1 cm. The MI and frequency provide an estimate of PRP within the renal tissue. The PRP, the value of which is 2.38 MPa, was calculated according to the formula pffiffiffi MI 5 Pr= f (1) where Pr is peak rarefactional pressure and f is frequency. Contrast medium Zhifuxian, a novel ultrasound contrast agent made by encapsulating perfluoropropane with a lipid shell, was awarded a National Invention Patent by China in 2005. It was provided by the ultrasound department of Xinqiao Hospital. Its concentration and mean diameter were approximately 4–9 3 109/mL and 2.13 mm, respectively. Numerous animal experimental studies had revealed that Zhifuxian does not produce changes in right ventricular blood pressure, as monitored by right ventricular systolic pressure and right ventricular end-diastolic pressure, and has no obvious effects on kidneys in rats, as observed by pathologic examination—properties that make it suitable for application (Liu et al. 2011; Gao et al. 2004a, 2004b). Establishment of model of early diabetic nephropathy rats Adult male Sprague Dawley rats (180–200 g, n 5 128) were provided by the Center for Experimental Animals of the Third Military Medical University. The experiments were approved by Animal Care and Use Committee of the Third Military Medical University. Rats were housed in wire cages with free access to a standard diet and tap water. The temperature and relative humidity of the animal facility were maintained under conventionally controlled conditions (22 C, 55% humidity) with a day-night rhythm. All rats were lightly anesthetized by intraperitoneal injection of 2% pentobarbital sodium at a dose of 30 mg/kg and received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ, Sigma) immediately after it had been dissolved in 0.1 M citrate buffer at pH 4.5. Three days after streptozotocin injection, blood samples were collected from the tail vein of nonfasted rats, and blood glucose concentrations were determined with a glucometer (Accu-Chek Aviva,
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Roche Applied Science). Diabetes was confirmed by a random blood glucose concentration .16.7 mmol/L for 3 consecutive days. Four weeks after onset of diabetes, diabetic rats presented with mild microalbuminuria (an early sign of DN) and were used in the experiments. Experimental groups Experiments were performed in a thermostatically controlled room at an ambient temperature of about 24– 26 C. Rats were fixed in the left lateral position after satisfactory anesthesia was induced by intraperitoneal injection of 2% pentobarbital sodium at a dose of 40 mg/kg. Only the right kidney of each rat was treated. The right flank was shaved, and the remaining hair removed using a depilatory cream. In rats exposed to ultrasound, the right kidney was localized and irradiated using the 9 L4 linear array probe of the ultrasound imaging system for 5 min of continuous irradiation (42 frames/s, MI 5 0.9, frequency 5 7.00 MHz). The probe was positioned in a transducer holder at an appropriate place, and the image plane of the ultrasound field was such that the contralateral kidney could not be insonated in the same ultrasound image. The rats (N 5 120) were randomly assigned to four groups. All rats received EB (2% in saline, 50 mg/kg) first. (i) The control group received no treatment after EB. (ii) The ultrasound (US) group was subjected to ultrasonic irradiation and injected with 0.5 mL of phosphate-buffered saline. (iii) The MB group was injected with microbubbles without ultrasonic irradiation. (iv) The US 1 MB group was subjected to ultrasonic irradiation and injected with microbubbles. Before injection, the bottle of microbubbles was shaken 45 s in a custom-built oscillation apparatus to form a microbubble suspension. Microbubbles were injected into the lateral tail vein through a 26-gauge needle connected to a 1-mL syringe via a 15-cm-long catheter (0.45 3 15 round wall, long bevel, China), controlled by a syringe pump; the injection was completed within 30 s and was followed by 0.5 mL saline to wash the tube. Rats were then covered with blankets for 1 h. After 1 h, 8 rats from each group (a total of 32 rats) were placed in metabolic cages for urine sample collection 24 h later, and the remaining rats were heart-perfused with heparinized saline (10 U/mL) through the left ventricle until the perfusion fluid collected from the right atrium became clear. Rats were then killed by decapitation, and right kidneys were quickly removed for testing. Additionally, to assess the permeability recovery of kidneys after ultrasoundmediated microbubble destruction, another 8 rats were subjected to ultrasonic irradiation, injected with microbubbles and, after 24 h, injected with EB (recovery group).
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Evans blue assay Right kidneys (32 of 120 rats, 8/group) were weighed on a balance, photographed with a digital camera (Casio, EX-Z80 A) in surface and coronal sectional views, cut into pieces and incubated in formamide solution at 37 C for 24 h. Pure formamide was used as a negative control. Optical density was measured with a spectrophotometer (Shimadzu, Kyoto, Japan) at the wavelength of 620 nm. The concentration of eluted dye was calculated using a standard curve (y 5 32.782x – 0.113) generated through serial dilutions of EB in 0.9% sodium chloride. EB content was assessed as eluted EB concentration and expressed as micrograms of EB per gram of dry tissue. The percentage of blue staining in photographs was analyzed using Image Pro Plus 6.0 software (Media, Cybernetics). The general steps were: (1) selection and measurement of target area; (2) measurement of surface and coronal areas; (3) determination of percentage of blue staining of renal surface and coronal plane using target area-to-surface area and target area-to-coronal area ratios, respectively. Confocal laser scanning microscopy Right kidneys (12 of 120 rats, 3/group) were cut in cross section at approximately 1-cm thickness to ensure that the irradiated part was located within the section. Frozen sections (5 mm) were made rapidly, and nuclei were stained with 4’,6-diamidino-2-phenylindole (blue). Fluorescence spatial images were obtained with a confocal laser scanning microscope (Zeiss LSM510, laser type diode, R-HeNe 633). EB was excited at lexc 5 510 nm, and fluorescence was monitored at lem 5 595 nm, whereas 4’,6-diamidino-2-phenylindole was excited at lexc 5 365 nm, and fluorescence was measured at lem 5 455 nm.
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Real-time polymerase chain reaction Real-time polymerase chain reaction (PCR) analysis was performed to investigate E-selectin mRNA expression. The entire tissue preparation procedure was carried out at 4 C. Renal cortices were weighted, minced and homogenized in a glass homogenizer. DNA was extracted with chloroform and precipitated with ethanol, and total DNA was assayed by ultraviolet absorbance. The oligonucleotide primers were as follows: E-selectin: 50 -CTCTGCTCTCACCTTTGTTCTCC-30 (sense) 50 -GCCCAGTTCTTAGCTTCCTCC-30 (antisense) b-actin: 50 -CGTTGACATCCGTAAAGACCTC-30 (sense) 50 -TAGGAGCCAGGGCAGTAATCT-30 (antisense) Real-time PCR assay was performed with target DNA, E-selectin primers and a fluorescence probe using a real-time PCR instrument (Applied Biosystems). Western blot analysis Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. Membranes were blocked using 0.1% Tween-20 in Tris-buffered saline (TTBS) containing 5% bovine serum albumin at room temperature for 2 h. Then membranes were washed three times with TTBS and incubated for 2 h at room temperature with goat polyclonal anti-E-selectin (1:500 in TTBS, Santa Cruz Biotechnology, Dallas, TX, USA) and b-actin (stained with polyclonal anti-b-actin 1:500 dilution, Santa Cruz Biotechnology). The signal was detected by the chemiluminescence method (Pierce Chemical, Rockford, IL, USA).
Transmission electron microscopy Renal cortices of right kidneys (12 of 120 rats, 3/group) were cut into 1-mm pieces and fixed in 2% glutaraldehyde for 1 h. After being washed with phosphate-buffered saline three times, the samples were postfixed in 1% osmium tetroxide in cacodylate buffer (pH 7.2) for 1 h. Subsequently, the samples were dehydrated in ethanol and embedded in epoxy resin (Agar 100). Ultrathin sections (50 nm) were cut, double stained with uranyl acetate and Reynolds lead citrate and examined under a transmission electron microscope (TECNAI 10, Philips, USA).
Urinalysis To assess the impact of ultrasound-mediated microbubble destruction on kidneys, urine samples from 32 rats in metabolic cages were collected 24 h after treatment and measured for red blood cells, urinary albumin and urinary creatinine. Microscopic hematuria is defined as three or more red blood cells per high-power field in a centrifuged urine specimen (Thaller and Wang 1999). Urinary albumin and creatinine concentrations were determined in the clinical laboratory of Xinqiao Hospital using an automatic biochemical analyzer (Hitachi, Tokyo, Japan). The urinary albumin excretion rate (UAER) was determined with the urinary albumin-to-urinary creatinine ratio.
Histologic analysis Right kidneys (20 of 120 rats, 5/group) were fixed in 10% formaldehyde, embedded in paraffin and sectioned at 5-mm thickness for hematoxylin-eosin (HE) and Periodic acid-Schiff staining. All sections were evaluated by a pathologist who was blinded to treatment.
Statistical analysis Data are summarized as means 6 standard deviations for each group. Student’s t-test was used to determine significant differences between two groups. One-way analysis of variance and post hoc comparisons (Bonferroni test) were used to determine significant differences among
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Fig. 1. Seven-megahertz ultrasound image of the rat kidney (mechanical index 5 0.9, B-mode scans, gray-scale mapping). Images were obtained (a) 0 s, (b) 15 s, and (c) 5 min after injection of microbubbles.
multiple groups. A p-value , 0.05 was considered to indicate statistical significance. RESULTS Renal ultrasonography Figure 1a is a renal ultrasound image of a diabetic nephropathy rat. Renal opacification and subsequent microbubble destruction could be observed under ultrasonic irradiation, indicating sufficient renal perfusion (Fig. 1b, c). Evans blue extravasation Photographs of the surface and coronal plane of kidneys revealed that portions of renal tissue stained blue under the combined effect of ultrasound and microbubbles, and the blue staining corresponded to regions that received ultrasonic irradiation (Fig. 2a). There was
slight blue staining in the US group—9.37 6 3.45% and 10.10 6 3.24% of the renal surface and coronal plane, respectively—but no visible staining in the control and MB groups. The proportions of renal surface and coronal plane that stained blue in the US 1 MB group were 49.12 6 7.31% and 37.99 6 4.36%, respectively. Renal capillary permeability was quantitatively evaluated with the EB content assay. Compared with the control group (13.942 6 2.848 mg/g), there were no statistically significant differences in EB content in the US, MB and recovery groups: 14.126 6 2.570, 13.969 6 2.076 and 14.658 6 3.916 mg/g, respectively, p . 0.05 (Fig. 2b). EB content was significantly higher in the US 1 MB group (37.267 6 4.948 mg/g) than in the control group (13.942 6 2.848 mg/g, p , 0.01), US group (14.126 6 2.570 mg/g, p , 0.01) and MB group (13.969 6 2.076 mg/g, p , 0.01), indicating a significant
Fig. 2. (a) Representative photographs of rat kidneys and changes in Evans blue (EB) extravasation. (b) Histogram of changes in EB extravasation. *p , 0.01 compared with the control group. #p , 0.01 compared with the group subjected to ultrasound (US). Op , 0.01 compared with the group injected with microbubbles (MB). :p , 0.01 compared with the recovery group.
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Fig. 3. Red fluorescence representing Evans blue (EB) was obvious in the group subjected to ultrasound and injected with microbubbles (US 1 MB) (d), but was not observed in the control group (a), the group subjected to ultrasound (US) (b) or the group injected with microbubbles (MB) (c). Nuclei were labeled with 4’,6-diamidino-2phenylindole (DAPI) (blue).
increase in renal capillary permeability after ultrasoundmediated microbubble destruction. There was no difference in EB content between the control and recovery groups (13.942 6 2.848 and 14.658 6 3.916 mg/g, respectively, p . 0.05), suggesting that the increased permeability was recovered. Exudative change in Evans blue under confocal laser scanning microscopy Renal capillary permeability was visually evaluated by confocal laser scanning microscopy. Renal interstitial capillary structure maintained intact and clear, and there was no EB extravasation in the renal interstitium in the control, US and MB groups (Fig. 3a–c); however, compared with the other groups (Fig. 3d), extravasation of red EB fluorescence around the interstitial capillary was evident in the US 1 MB group, resulting in a ‘‘fishing net’’ graph (Fig. 3a–c). This phenomenon depicted intuitively that intravascular EB extravasated from the interstitial capillary lumen and penetrated the renal interstitium. Ultrastructural changes in renal interstitial capillary under TEM Transmission electron microscopy was used to observe ultrastructural changes in capillaries, and we found that portions (approximately 30%–50%) of inter-
stitial capillary walls became thinner, discontinuous and roughened in the US 1 MB group (Fig. 4a), but remained intact in the control (Fig. 4b), US (Fig. 4c) and MB (Fig. 4d) groups, suggesting that mild injury to capillary endothelial cells was responsible for the changes and that interstitial capillary permeability increased after ultrasound-mediated microbubble destruction. E-selectin mRNA and protein expression To further evaluate the state of capillary endothelial cells after ultrasonic irradiation, E-selectin mRNA and protein expression was detected by real-Time PCR and Western blot. Similar to the changes in EB content and EB extravasation, the increases in E-selectin mRNA and protein expression were apparently more pronounced in the US 1 MB group than in the control, US and MB groups (p , 0.01) (Figs 5, 6). There were no significant differences between the control and US groups, between the control and MB groups or between the US and MB groups (p . 0.05). Renal histology There were no gross abnormalities of the kidneys in any group at necropsy. Periodic acid-Schiff staining revealed that there were no marked glomerular or tubular histological changes of early diabetic nephropathy,
Fig. 4. Endothelial cells of a renal interstitial capillary appeared mildly injured under transmission electron microscopy (bar 5 1 mm, original magnification 5 8900) and parts of the interstitial capillary walls were thinner, discontinuous and roughened (arrows) in the group subjected to ultrasound and injected with microbubbles (US 1 MB) (a), whereas the endothelial cells and interstitial capillary walls remained intact in the control group (b), US group (c) and MB group (d).
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There was no significant difference in UAER between the US 1 MB and control groups (60.12 6 25.67 and 55.99620.55 mg/mg creatinine, respectively, p . 0.05) or between the US 1 MB and US groups (60.12 6 25.67 and 57.43 6 18.40 mg/mg creatinine, respectively, p . 0.05) or US 1 MB and MB groups (60.12 6 25.67 and 53.86 6 22.42 mg/mg creatinine, respectively, p . 0.05). Hematuria was not detected in any group. Fig. 5. Real-time polymerase chain analysis revealed differences in E-selectin mRNA expression between the group subjected to ultrasound and injected with microbubbles (US 1 MB) and the other groups. Values represent means 6 standard deviations. *p , 0.01 versus the control group. # p , 0.01 versus the group subjected to ultrasound (US); O p , 0.01 versus the group injected with microbubbles (MB).
including mesangial expansion, glomerular basement membrane thickening, extracellular matrix deposition and tubular dilation. Hematoxylin-eosin staining revealed no hemorrhage or necrosis in the renal structure in all groups. In Figure 7 are images of the glomeruli, adjacent cortical tubule-interstitium and peritubular capillary of four groups. Urinalysis Albumin and creatinine levels were measured, and the UAER was calculated 24 h after treatment.
Fig. 6. Western blot analysis revealed differences in E-selectin protein expression between the group subjected to ultrasound and injected with microbubbles (US 1 MB) and the other groups. (a) Representative protein banding illustrated expressional differences in E-selectin. Results from the representative experiment were normalized to b-actin expression. (b) E-Selectin expression was quantified by scanning densitometry. Values represent means 6 standard deviations. *p , 0.01 versus the control group. #p , 0.01 versus the group subjected to ultrasound (US). Op , 0.01 versus the group injected with microbubbles (MB).
DISCUSSION Although most studies of DN have focused on glomeruli, tubule-interstitial damage is a major feature of DN and is an important prognostic factor in renal dysfunction. Given the limitations of kidney transplantation and drug and antifibrotic therapy for DN, our goal was to provide a new, non-invasive method to maximize the therapeutic effect for patients with DN patients. Ultrasound-mediated microbubble destruction is a new appealing technique for site-specific drug and gene delivery and has been used successfully in different clinical situations. Evans blue conjugates to serum albumin and is a sensitive and convenient tracer for assessing alterations in vascular permeability (Patterson et al. 1992; Tremblay et al. 2006; Whalen et al. 1998). The blue staining in the pelvis was visible in all treatment groups. A possible reason is that the kidney has three filtration membranes (capillary fenetration, basement membrane and podocyte pedicel), and the podocyte pedicel is the key barrier, filtrating only substances ,70,000 in molecular weight. Evans blue is filtrated by the podocyte pedicel because its molecular weight is 960. Serum albumin has a molecular weight of 65,000; therefore, Evans blue-serum albumin is also filtrated. In this study, the significant increases in EB content and EB fluorescence intensity in the US 1 MB group confirmed that interstitial capillary permeability could be increased by the interaction of ultrasound and microbubbles. The underlying mechanism may be that interstitial capillary walls became thinner, discontinuous and roughened as a result of ultrasound-mediated microbubble destruction, and EB extravasates from the ruptured capillary and penetrates the renal interstitium. The proposed method may allow the locally released antifibrotic agent to cross the ruptured interstitial capillary and enter the renal interstitium. Miller et al. (2008) consider the interaction of ultrasound pulses with gas bodies to be a form of acoustic cavitation, and they also may act as inertial cavitation nuclei. This interaction produces mechanical perturbation and a potential for bio-effects on nearby cells or tissues (Miller et al. 2008). E-Selectin is unique to endothelium. We surmised that microbubble cavitation via ultrasonic irradiation mildly injured
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Fig. 7. Periodic acid-Schiff (PAS) staining revealed no mesangial expansion, glomerular basement membrane thickening, extracellular matrix deposition or tubular dilation. Hematoxylin and eosin (HE) staining confirmed no evidence of hemorrhage and necrosis in the renal structure. US 5 group subjected to ultrasound, MB 5 group injected with microbubbles, US 1 MB 5 group subjected to ultrasound and injected with microbubbles.
endothelial cells, induced endothelial activity and then produced an inflammatory response in endothelial cells, resulting in the upregulation of E-selectin expression. Only the ultrastructural changes were observed on TEM, there was no evidence of hemorrhage and necrosis in renal structure and EB content returned to the control group level after 24 h, suggesting that the cavitation intensity was moderate, the inflammatory response was mild and acceptable and the increased permeability was recovered within a relatively short time and did not cause greater damage to the endothelial cells. There was no difference in UAER between the control group and the other groups 24 h post-treatment, indicating that the technique did not alter glomerular filtration function. Hematuria was not detected in any group, suggesting that there was essentially no glomerular capillary hemorrhage. Two possible reasons for these findings have been postulated. First, continuous irradiation decreased the extent of microbubble destruction. Intermittent irradiation might produce a greater degree of microbubble destruction than continuous irradiation for microbubble reperfusion in a sufficient interval. Second, an injection longer (within 30 s) than the bolus injection was used in this experiment. Increasing the injection time might decrease microbubble concentration per capillary and, thus, reduce the occurrence of glomerular capillary hemorrhage. Both ultrasound and microbubbles increased capillary permeability when used in combination and had no effect on capillary permeability when used alone, as there were no statistically significant differences in EB content and E-selectin mRNA and protein expression between the US and MB groups. Stem cell therapy is becoming an attractive option and a more viable therapy for DN (Sun et al. 2011; Volarevic et al. 2011). Differentiation of stem cells is subject to the micro-environment, which could be altered by ultrasound-mediated microbubble destruction (Zhong
et al. 2012). In one study, the local and transient increases in chemo-attractants and micro-environmental changes induced by pulsed focused ultrasound lasted at least 3 d, which enhanced homing, permeability and retention of bone marrow stromal cells to the treated kidney (Ziadloo et al. 2012). In this experiment, the mild injury to the interstitial capillary after cavitation caused the upregulation of E-selectin, which perhaps promoted the homing of transplanted stem cells to DN. This technology perhaps provides a new idea—personalized cell therapy for DN using stem cells. The parameters used in this study may differ if applied to humans because of the (i) lack of direct transferability between small animal conditions and human patients and (ii) greater variability in anatomic variability between human patients and rats. We performed this preliminary study to prove that ultrasound-mediated microbubble destruction is a non-invasive and feasible technology for increasing renal interstitial capillary permeability and modulating the micro-environment in early-stage diabetic nephropathy in the rat. This technique may provide guidance for development of a clinically applicable method to enhance the delivery of drugs, genes, antifibrotic agents or stem cells to the renal interstitium and improve the therapeutic effect. Much work remains in this area. For example, we may be able to take advantage of this technique and targeted microbubbles incorporating drugs, genes or antibodies to locally aggregate these components attached to microbubble fragments when the targeted microbubbles are irradiated and release transported substances to target organs to repair damaged tissue. However, elucidation of a detailed mechanism is warranted. CONCLUSIONS Ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability
US-mediated microbubble destruction in early diabetic rats d Y. ZHANG et al.
in early diabetic nephropathy rats and should be considered a therapy enhancing delivery of drugs, genes, antifibrotic agents or stem cells to the kidney in the future. The underlying mechanism may be that ultrasound-mediated microbubble cavitation ruptures interstitial capillary walls, which causes therapeutic substances to cross through discontinuous walls, enter renal the interstitium and maximize the therapeutic effect. Acknowledgments—This work was supported by the National Natural Science Foundation of China (Grant 81071160, 81101062); the Department of Public Health of Guizhou Province (gzwkj2013-1-157); the key project of the Chinese Ministry of Education (2012156); the Department of Science and Technology (G20107015, 20103166); and the Department of Education (2009143) of Guizhou Province.
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