Introduction

Low-intensity pulsed ultrasound (LIPUS) has been widely accepted as a noninvasive therapeutic tool for treatment of bone fractures. Numerous clinical trials have shown that LIPUS therapy can accelerate radiographic healing of fractures.¹ Recently, LIPUS treatment has been reported to be effective in various disease models such as myocardial ischemia,^2–4 skin wounds,⁵ rheumatoid arthritis,⁶ muscle atrophy,⁷ Alzheimer’s disease,⁸ and erectile dysfunction^9,10 other than fractures. For these disease models, LIPUS treatment has been shown to be effective through various mechanisms. Notably, LIPUS treatment has been reported to exert anti-inflammatory effects. It suppressed lipopolysaccharide-induced inflammatory responses by inhibiting formation of the toll-like receptor 4-myeloid differentiation primary response 88 complex.¹¹ In addition, it has been shown that LIPUS treatment reduces lipopolysaccharide-induced inflammatory factors.¹² These anti-inflammatory effects of LIPUS may be effective against various inflammatory diseases.

Chronic kidney disease affects 753 million individuals globally, and many patients with chronic kidney disease eventually develop renal failure, requiring renal replacement therapy.¹³ The number of patients receiving renal replacement therapy worldwide was 2.6 million in 2010, and the number is expected to increase to 5.4 million in 2030.¹⁴ Thus, the development of new therapeutic strategies for preventing renal failure is crucial. In a clinical setting, chronic kidney disease occurs in a population with chronic diseases of various causes, such as hypertension, diabetes, and chronic glomerular nephritis. Pathologically, regardless of the primary disease, renal fibrosis is a histological manifestation of patients with chronic kidney disease during progression to the end stage of the disease. In addition, chronic interstitial fibrosis is exacerbated by the persistence of inflammation,¹⁵ and the progression of fibrosis promotes further inflammation.¹⁶

As LIPUS exhibits immunosuppressive abilities, it may be a useful therapy for preventing the progression of renal fibrosis. In this study, we used mouse models of hypertensive nephropathy and diabetic nephropathy to evaluate the effects of LIPUS treatment on inflammatory cell infiltration and renal fibrosis. We also evaluated the mechanism by which LIPUS stimulation directly suppresses the progression of fibrosis.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals

Seven-week-old male C57BL/6 mice and db/db heterozygote mice (BKS.Cg-Dock7^m+/+Lepr^db/J) were purchased from CLEA Japan, Inc (Tokyo, Japan). The mice were housed in a light- and temperature-controlled room in the Laboratory Animal Center of Hiroshima University (Hiroshima, Japan). All animal studies were conducted according to the Guide for the Care and Use of Laboratory Animals, eighth edition, 2010 (National Institutes of Health, Bethesda, MD) and approved by the Institutional Animal Care and Use Committee of Hiroshima University (Permit Numbers: A15–51 and A16–55). All efforts were taken to minimize pain and distress for the animals.

Mouse Model of Angiotensin II-Salt-Induced Hypertensive Nephropathy

C57BL/6 mice (9 weeks of age) underwent right nephrectomy with or without splenectomy under anesthesia with isoflurane (Mylan, Inc, PA) using an animal anesthesia machine (DS Pharma Biomedical Co, Ltd, Osaka, Japan; maintenance concentration: 2%; flow rate: 2.8 L/minute). Angiotensin II (Sigma-Aldrich, St Louis, MO) was dissolved in distilled water containing dimethyl sulfoxide, and the solution was administered to mice using osmotic mini pumps (Alzet model 2004, Cupertino, CA). After nephrectomy, pumps were subcutaneously implanted in mice, ensuring constant infusion of angiotensin II at a dose of 1.0 μg/kg per minute throughout the 4-week study period.¹⁷ Subsequently, all mice received 1.0% sodium chloride (NaCl) solution and 0.3% NaCl standard chow (Oriental Yeast Co, Ltd, Tokyo, Japan). Two investigators (S. Kishimoto and T. Maruhashi) who were not informed of experimental groups performed noninvasive measurement of blood pressure at the tail using a sphygmomanometer at the tail with a noninvasive sphygmomanometer (BP-98A; Softron Co, Ltd, Tokyo, Japan).¹⁸

Mouse Model of Diabetic Nephropathy

Nine-week-old db/db heterozygote mice underwent right nephrectomy under anesthesia as described in the previous section. After nephrectomy, the mice received 1.0% NaCl solution and 0.3 % NaCl standard chow. Twenty-four-hour urine samples were collected using metabolic cages. Mice were maintained in the metabolic cages for 1 day of acclimatization before initiating urine collection. Measurement of urine samples was outsourced to SRL, Inc (Tokyo, Japan). A previous study showed that mean blood glucose levels were ≈400 mg/dL in db/db heterozygote mice with or without a high salt intake.¹⁹

Ultrasound Stimulation of Mice

One day after nephrectomy, all mice were anesthetized with isoflurane and each mouse was held on a heated pad at 35 °C. Ultrasonic gel was applied to an ultrasound transducer (Nippon Sigmax Co, Ltd, Tokyo, Japan), and the transducer was placed over the skin of the back on the left kidney. Exposure to ultrasound (20 minutes per session) was repeated daily throughout the study period. The conditions were as follows: transducer effective area of 1.68 cm², intensity of 30 mW/cm² with a 20% duty cycle, and pulse frequency of 2.0 MHz with 1 kHz repetition rate. For control mice, the condition of exposure to ultrasound was pulse frequency of 0 MHz. Since menstruation may affect the effects of ultrasound exposure, male mice were randomly assigned to the experimental groups.

Ultrasound Stimulation of Human Proximal Tubular Cell Line Cells

Following starvation of human proximal tubular cell line cells (American Type Culture Collection, Manassas, VA) with serum-free DMEM for 24 hours, the cells were exposed to 2.5 ng/mL recombinant human TGF-β1 (transforming growth factor-β1; R&D Systems, Minneapolis, MN). Subsequently, the culture plate was placed on a solid ultrasound gel (Toshiba Medical Supply Co, Ltd, Tokyo, Japan) and exposed to ultrasound. Transducers were connected to the ultrasonic main controller (Nippon Sigmax Co, Ltd). The conditions of exposure to ultrasound were as follows: intensity of 30 mW/cm², transducer effective area of 6.5 cm², pulse frequency of 2.0 MHz with 200-μs pulse duration (20% duty cycle), 1 kHz repetition rate, and exposure time of 20 minutes. Following 1 hour of exposure to ultrasound, whole cell lysates were prepared in radioimmunoprecipitation assay buffer (Sigma-Aldrich) and analyzed. Following 24 hours of exposure to ultrasound, total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and subjected to polymerase chain reaction.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

Total RNA extraction, cDNA synthesis, and real-time reverse transcription polymerase chain reaction were performed as previously described.²⁰ Results of polymerase chain reaction experiments were analyzed by TaqMan Gene Expression Assays and 7500 Fast (Applied Biosystems, Foster City, CA). Specific oligonucleotide primers and probes for mice IL-1β (interleukin-1β; assay ID: Mm 00434228_m1), mice TNF-α (tumor necrosis factor α; assay ID: Mm00443258_m1), human α-SMA (α-smooth muscle actin; assay ID: Hs00426835_g1), human fibronectin (assay ID: Hs01549976_m1), and GAPDH rRNA (endogenous control) were obtained for TaqMan Gene Expression Assays (Applied Biosystems). The mRNA levels of the samples were normalized to those of GAPDH rRNA.

Western Blotting Analysis

Sample collection and Western blotting were performed as previously described.²¹ Primary antibodies used in this study were mouse monoclonal anti-α-SMA antibody (1:1000, A2547; Sigma-Aldrich), rabbit polyclonal anti-TGF-β1 antibody (1:1000, SAB4502954; Sigma-Aldrich), rat monoclonal anti-F4/80 antibody (1:100, ab6640; Abcam, Cambridge, United Kingdom), and rabbit monoclonal anti-GAPDH antibody (1:1000, No. 2118; Cell Signaling Technology, Beverley, CA). Secondary antibodies used in this study were horseradish peroxidase-linked donkey anti-rabbit IgG antibody (GE Healthcare, Buckinghamshire, United Kingdom) and horseradish peroxidase-linked sheep anti-mouse IgG antibody (GE Healthcare). Signals were detected using the Super Signal West Dura system (Thermo Fisher, Rockford, IL). The intensity of each band was analyzed with ImageJ software (version 1.47v; National Institutes of Health) and normalized to the levels of GAPDH.

Immunofluorescence Assay

Immunofluorescence staining was performed on frozen tissues. Briefly, frozen kidneys were cut into 5-μm-thick sections on a cryostat and dried in air. The sections were subsequently fixed in 4% paraformaldehyde, blocked with Block Ace (DS Pharma Biomedical), and incubated overnight at 4 °C with the rabbit polyclonal anti-TGF-β1 antibody (1:100, SAB4502954; Sigma-Aldrich). The sections were washed in PBS, incubated with Alexa Fluor 594 Donkey anti-Rabbit IgG (1:500, R37119; Thermo Fisher Scientific) for 3 hours, and mounted with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA). The sections were visualized with a fluorescence microscope (KEYENCE, Osaka, Japan; ×200).

Immunohistochemistry Analysis

Immunohistochemical staining was performed as previously described.²¹ The following primary antibodies were used: rat monoclonal anti-F4/80 antibody (1:100, ab6640; Abcam), rabbit polyclonal anti-CD3 antibody (1:100, ab5690; Abcam), and mouse monoclonal anti-α-SMA antibody (1:400, A2547; Sigma-Aldrich). All sections were visualized using 3,3′-diaminobenzidine (Sigma-Aldrich). F4/80- and CD3-positive cells and the positive area for α-SMA staining were assessed as the average of 10 randomly selected fields (×200) for each mouse with ImageJ software (National Institutes of Health). The scorer did not know the treatment group when scoring.

Histopathology

Masson’s trichrome staining was performed as previously described.²¹ The areas of interstitial fibrosis were assessed as the average of 10 randomly selected fields (×200) for each mouse with ImageJ software. Periodic Acid Schiff staining was performed according to the manufacturer’s instructions. Twenty glomeruli under a high-power field (original magnification ×600) were selected from sections of mouse kidneys. Glomerular volume was calculated by the average radius of glomeruli as previously described.²² Glomerular sclerosis index was evaluated as reported previously.²³ Briefly, glomeruli stained with Periodic Acid Schiff were graded on a scale of 0 to 4: 0, normal; 1, involvement of 1% to 25% of glomerular tufts; 2, involvement of 26% to 50% of glomerular tufts; 3, involvement of 51% to 75% of glomerular tufts; 4, involvement of 75% to 100% of glomerular tufts. The scorer did not know the treatment group when scoring.

Statistical Analysis

Results are expressed as the means±SE. Differences between 2 groups were analyzed using Student t test. P<0.05 denoted statistically significant differences. One investigator (Y. Aibara) was informed of the experimental groups to prevent errors, while another investigator (M. Kajikawa) was blinded to the group assignments when performing the statistical analyses.

Results

LIPUS Treatment Suppressed Tubulointerstitial Fibrosis in Mice With Angiotensin II-Salt-Induced Hypertensive Nephropathy

Chronic interstitial fibrosis is exacerbated by the persistence of inflammation.^11,12 As LIPUS inhibits the infiltration of inflammatory cells,¹⁵ we examined whether LIPUS may exert anti-inflammatory and antifibrotic effects in mice with angiotensin II-salt-induced hypertensive nephropathy.

The blood pressure of mice was measured using a noninvasive sphygmomanometer to confirm the successful creation of angiotensin II-salt-induced hypertensive nephropathy. Systolic blood pressure in mice with angiotensin II-salt-induced hypertensive nephropathy that were not exposed to LIPUS (control group) increased from day 3. In the LIPUS group, exposure to LIPUS was repeated every day throughout the study period. Systolic blood pressure of mice in the LIPUS group was similar to that of mice in the control group until day 14. However, systolic blood pressure of mice in the LIPUS group was significantly reduced compared with that of mice in the control group at day 21. Systolic blood pressures at day 28 were 9.0 mm Hg 153.6±7.7 mm Hg in the control and LIPUS groups, respectively (P<0.01; Figure 1A).

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At days 14 and 28 after the start of exposure to LIPUS, the protein levels of TGF-β1 and α-SMA (markers of fibrosis) were reduced (Figure 1B). Immunofluorescence staining of TGF-β1 and immunohistochemical staining of α-SMA were also performed to assess renal fibrosis. Similar to the results obtained from Western blotting, immunofluorescence staining revealed that the size of TGF-β1-positive area was decreased in the LIPUS group. Positive α-SMA staining was found mainly in the interstitium, and exposure to LIPUS significantly reduced the size of α-SMA-positive area (Figure 1B). In addition, kidneys at 28 days post–angiotensin II-infusion showed large areas stained with aniline blue after Masson’s trichrome staining, indicating fibrotic tissue. The size of interstitial fibrosis area was significantly decreased in the LIPUS group (Figure 1C).

Collectively, these findings indicate that LIPUS treatment can powerfully ameliorate tubulointerstitial fibrosis in mice with angiotensin II-salt-induced hypertensive nephropathy. The suppression of renal fibrosis by exposure to LIPUS may have moderated the increase in systolic blood pressure in mice in the LIPUS group.

LIPUS Treatment Inhibited Infiltration of Inflammatory Cells in Mice With Angiotensin II-Salt-Induced Hypertensive Nephropathy

Damaged cells release inflammatory mediators, and the persistence of inflammation leads to excessive accumulation of extracellular matrix and fibrotic changes. Therefore, we investigated the expression of F4/80 (a macrophage marker) and CD3 (a T lymphocyte marker) to evaluate the anti-inflammatory effects of LIPUS in mice with angiotensin II-salt-induced hypertensive nephropathy. After exposure to LIPUS, the protein levels of F4/80 significantly decreased, and immunohistochemical staining revealed that the number of F4/80-positive cells was decreased (Figure 1D). Similar to the results obtained for F4/80, immunohistochemical staining revealed that the number of CD3-positive cells was significantly decreased after exposure to LIPUS (Figure 1E). In addition, we investigated the IL-1β and TNF-α mRNA expression levels to evaluate the anti-inflammatory effects of LIPUS and found that these mRNA levels were significantly decreased by exposure to LIPUS (Figure 1F). The results for mice that underwent a sham operation (Figure 1S in the Data Supplement).

LIPUS Treatment Suppressed Tubulointerstitial Fibrosis and Inhibited Infiltration of Inflammatory Cells in Mice With Diabetic Nephropathy

Subsequently, we used a mouse model of diabetic nephropathy to evaluate the effect of LIPUS treatment on renal fibrosis and inflammatory cell infiltration. At 6 weeks after nephrectomy, the degree of proteinuria was significantly decreased in mice in the LIPUS group compared with that observed in the control group (Figure 2A). Eight weeks later, mice were sacrificed and injured kidneys were collected to evaluate fibrosis through Western blotting. The protein levels of TGF-β1 and α-SMA were decreased by exposure to LIPUS (Figure 2B). Similar to the results obtained by Western blotting, the positive area of TGF-β1 and α-SMA were decreased in the LIPUS group (Figure 2B). Masson’s trichrome staining also revealed that the size of interstitial fibrosis area was significantly decreased in the LIPUS group (Figure 2C). In addition, we performed Periodic Acid Schiff staining and found that many glomeruli from kidneys at 8 weeks after nephrectomy demonstrated marked glomerulosclerosis, adhesions to Bowman’s capsule, and hypertrophy. Exposure to LIPUS significantly reduced the glomerular volume and glomerular sclerosis index (Figure 2D).

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Western blotting showed that the protein levels of F4/80 were significantly decreased by exposure to LIPUS. Similarly, immunohistochemical staining revealed that the number of F4/80-positive cells was decreased by exposure to LIPUS (Figure 2E). Immunohistochemical staining also revealed that the number of CD3-positive cells was significantly decreased by exposure to LIPUS (Figure 2F).

LIPUS Treatment Also Suppressed Renal Fibrosis and Infiltration of Inflammatory Cells in Mice With Hypertensive Nephropathy in Which Splenectomy Was Performed

Exposure to ultrasound 24 hours before renal ischemia reperfusion has recently been reported to prevent infiltration of inflammatory cells into the injured kidney and maintain kidney function through the splenic cholinergic anti-inflammatory pathway.^24–26 Although this ultrasound treatment is different from the protocol of our LIPUS treatment, we investigated whether daily exposure to LIPUS exerts a therapeutic effect via the spleen.

In mice with angiotensin II-salt-induced hypertensive nephropathy in which splenectomy was performed, systolic blood pressure of the mice was increased at day 3 in both the control and LIPUS groups. Systolic blood pressure of mice in the LIPUS group was significantly lower than that recorded in the control group at days 21 and 28 (Figure 3A).

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At day 28, the protein levels of TGF-β1 and α-SMA were decreased by exposure to LIPUS (Figure 3B). Immunofluorescence staining revealed that the size of TGF-β1-positive area was decreased in the LIPUS group (Figure 3B). The protein levels of F4/80 were significantly decreased by exposure to LIPUS, and immunohistochemical staining also revealed that the number of F4/80-positive cells was decreased by this treatment (Figure 3C).

Exposure to LIPUS Directly Inhibits TGF-β1/Smad Signaling

The TGF-β1/Smad signaling pathway induces renal fibrosis. We investigated whether exposure to LIPUS could inhibit TGF-β1/Smad signaling in the human proximal tubular cell line cells. Western blotting showed that the protein levels of phosphorylated Smad2 (pSmad2) and Smad3 induced by TGF-β1 stimulation were significantly decreased by exposure to LIPUS (Figure 4A). In addition, exposure to LIPUS significantly decreased the mRNA expression levels of α-SMA and fibronectin (Figure 4B). These findings suggest that LIPUS treatment directly inhibits the TGF-β1/Smad signaling pathway.

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Discussion

This study provided the first evidence that daily LIPUS therapy ameliorates inflammation and tubulointerstitial fibrosis in experimental hypertensive nephropathy and diabetic nephropathy. It was also shown that exposure to LIPUS directly inhibits the TGF-β1/Smad signaling pathway. Finally, the therapeutic effect of LIPUS on injured kidneys of mice with hypertensive nephropathy in which splenectomy was performed indicated that LIPUS treatment is unlikely to exert a therapeutic effect via the splenic cholinergic anti-inflammatory pathway.

Hypertensive nephropathy and diabetic nephropathy are the main underlying diseases of renal failure. Therefore, we investigated the effect of daily LIPUS therapy on hypertensive nephropathy induced by unilateral nephrectomy and subcutaneous infusion of angiotensin II via osmotic mini-pumps and the effects of daily LIPUS therapy on diabetic nephropathy induced in db/db mice by unilateral nephrectomy. In both mouse models of hypertensive nephropathy and diabetic nephropathy used in the present study, kidney injury continued until the end of study. Our results suggest that daily LIPUS treatment is effective against ongoing kidney injury even in these models.

Numerous studies have shown that the TGF-β1/Smad signaling pathway plays a pivotal role in the induction of various types of fibrosis.²⁷ We showed that LIPUS treatment could directly inhibit the TGF-β1/Smad signaling pathway in the human proximal tubular cell line. On the other hand, the persistence of inflammatory cell infiltration also plays an important role in the process of fibrosis. Although we could not detect a direct anti-inflammatory effect of LIPUS treatment in vitro, Zhang et al²⁸ revealed that LIPUS stimulation could directly induce a change in the phenotype of macrophages to immunosuppressive M2. Indeed, daily LIPUS treatment decreased the number of F4/80-positive cells in mice with experimental hypertensive nephropathy and mice with diabetic nephropathy. In addition to the direct antifibrotic effects, these findings suggest that the suppression of renal fibrosis by LIPUS therapy may be partly due to the suppression of inflammatory cell infiltration by exposure to LIPUS.

The conditions used in the daily 20-minute exposure to ultrasound in this study were pulse frequency of 2.0 MHz with a 1 kHz repeat rate. These conditions are similar to the protocol of LIPUS used for treatment of bone fractures. Recently, Okusa et al showed that preexposure to ultrasound prevents renal ischemia reperfusion injury through the splenic cholinergic anti-inflammatory pathway.^24,25 Although the conditions of exposure to ultrasound in that study differ from the protocol of our daily LIPUS treatment, we investigated whether daily exposure to LIPUS exerts a therapeutic effect via the spleen. The results showing a therapeutic effect of daily LIPUS on the injured kidneys of mice with hypertensive nephropathy in which splenectomy was performed indicated that LIPUS treatment was unlikely to exert a therapeutic effect via the splenic cholinergic anti-inflammatory pathway.

In mice with angiotensin II-salt-induced hypertensive nephropathy, systolic blood pressure was significantly reduced in the LIPUS group compared with that in the control group after day 21. These findings suggest that the attenuation of renal fibrosis by LIPUS treatment led to decreased arterial pressure. The reduced arterial pressure may have contributed to further suppression of renal fibrosis. However, one limitation of this study is that blood pressure was measured noninvasively at the tail with a sphygmomanometer. This measurement technique is a suboptimal technique that is inferior to measurements of 24-hour blood pressure using a telemetry transmitter. Therefore, the possibility that mice in the LIPUS group had lower blood pressure than mice in the control group during the first 2 weeks of the study could not be ruled out completely.

The mouse models used in this study are another limitation. As the remaining unilateral kidney without LIPUS exposure made it difficult to clarify the direct effects of LIPUS treatment, we used unilaterally nephrectomized angiotensin II-salt models as hypertensive nephropathy models and unilaterally nephrectomized db/db mice with a high salt intake as diabetic nephropathy models. The unilaterally nephrectomized angiotensin II-salt-induced hypertensive nephropathy model was likely to have higher blood pressure and greater renal damage than those in mice with angiotensin II-infusion or a high-salt diet alone. However, we did not have results from mice with angiotensin II-infusion alone or a high-salt diet alone as a control group. In addition, we did not collect 24-hour urine samples from hypertensive nephropathy models. If a moderate model of hypertensive nephropathy was used, the findings of this study could be better translated to humans. Previous studies showed no significant difference in systolic blood pressures in db/db heterozygote mice with and without a high-salt diet. However, a high-salt diet has been shown to increase urinary albumin and kidney damage.^29,30 Unilateral nephrectomized db/db mice with a high-salt diet are likely to have higher blood pressure and greater kidney damage than db/db heterozygote mice with a high-salt diet. Indeed, Periodic Acid Schiff staining revealed a high glomerular sclerosis index in this model. Therefore, this model is more likely to reflect hypertensive diabetic nephropathy rather than diabetic nephropathy. However, we did not measure blood pressure or determine estimated glomerular filtration rate in these mice. Further studies on these models are needed.

Perspectives

LIPUS treatment is a noninvasive therapeutic tool without side effects. This modality may be useful for preventing the progression of renal fibrosis.

Acknowledgments

We wish to thank Ms Megumi Wakisaka and Ms Satoko Michiyama for sophisticated technical assistance. A. Nakashima and Y. Higashi designed the study; Y. Aibara, A. Nakashima, K.-i. Kawano, F.M. Yusoff, F. Mizuki, S. Kishimoto, and T. Maruhashi performed the experiments; Y. Aibara and M. Kajikawa analyzed and interpreted the data; A. Nakashima and Y. Higashi wrote the manuscript with comments from the co-authors.

Footnotes