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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: J Hypertens. 2019 Oct;37(10):2074–2082. doi: 10.1097/HJH.0000000000002158

Improved renal outcomes after revascularization of the stenotic renal artery in pigs by prior treatment with low-energy extracorporeal shockwave therapy

Xiao-Jun Chen 1,3, Xin Zhang 1, Kai Jiang 1, James D Krier 1, Xiangyang Zhu 1, Amir Lerman 2, Lilach O Lerman 1,2
PMCID: PMC7304646  NIHMSID: NIHMS1595725  PMID: 31246892

Abstract

Background:

Revascularization does not restore renal function in most patients with atherosclerotic renal artery stenosis (RAS), likely due to inflammation and fibrosis within the stenotic kidney. Low-energy shockwave therapy (LE-SWT) stimulates angiogenesis in the stenotic kidney, but its ability to improve renal function and structure after revascularization remains unexplored. We tested the hypothesis that a LE-SWT regimen before percutaneous transluminal renal angioplasty (PTRA) would enable PTRA to restore renal function in hypercholesterolemic pigs with RAS (HC+RAS), and that this would be associated with attenuation of renal inflammation and fibrosis.

Methods and Results:

Twenty-six pigs were studied after 16 weeks of HC+RAS, HC+RAS treated with PTRA with or without a preceding LE-SWT regimen (bi-weekly for 3 weeks), and controls. Single-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and oxygenation were assessed in vivo using imaging 4 weeks after PTRA, and then inflammation and fibrosis ex vivo.

Four weeks after successful PTRA, blood pressure fell similarly in both revascularized groups. Yet, stenotic-kidney GFR remained lower in HC+RAS and HC+RAS+PTRA (p<0.01 vs. normal), but was improved in HC+RAS+PTRA+SW (p>0.05 vs. normal). Furthermore, reduced inflammation, medullary fibrosis, and cortical hypoxia were only shown in swine stenotic kidneys pre-treated with LE-SWT before PTRA 4 weeks later.

Conclusions:

LE-SWT delivery before revascularization permitted PTRA to improve function and decrease cortical and medullary damage in the stenotic swine kidney. This study therefore supports the use of an adjunct SW pre-treatment to enhance the success of PTRA in blunting loss of kidney function in experimental HC+RAS.

Keywords: extracorporeal shockwave, renal artery stenosis, PTRA

Introduction

Atherosclerotic renal artery stenosis (RAS) remains a major contributor to secondary hypertension and renal ischemic disease, and may cause progressive renal dysfunction and cardiovascular disease[1]. Randomized clinical trials of renal artery revascularization showed that percutaneous transluminal renal angioplasty (PTRA) alone offered no benefit over medical treatment[2], probably due to ongoing inflammation, irreversible interstitial fibrosis, and renal microvascular loss, which are complicated by concurrent atherosclerosis[3]. Potentially, adjunct therapies targeted to revitalize the renal parenchyma may allow revascularization and restore function in kidneys with RAS.

Low-energy ultrasound extracorporeal shockwave (SW) therapy non-invasively evokes neovascularization and improves regional blood flow and function in various ischemic tissues[4-6]. We have also demonstrated its capability to restore the renal microcirculation in a hypercholesterolemic swine model with unilateral RAS (HC+RAS) [7] through mechanotransduction-mediated upregulation of angiogenic factor expression. However, the ability of SW to attenuate stenotic kidney inflammatory and fibrotic injury remains unexplored. Hypothetically, the ability of a SW regimen to target directly the post-stenotic kidney and attenuate detrimental processes may enable PTRA to more effectively reverse kidney damage. Yet, whether delivery of a low-energy SW regimen prior to PTRA would preserve the stenotic kidney structure and enable PTRA to restore renal function remains unknown.

Thus, this study was designed to test the hypothesis that in HC+RAS, delivery of low-energy SW before PTRA would improve kidney outcomes compared to PTRA alone, and that this is associated with attenuation of renal inflammation and fibrosis..

Materials and Methods

Animals and Experimental Design

Twenty-six domestic female pigs (50-60 Kg) were studied for 16 weeks after approval by the Institutional Animal Care and Use Committee. Pigs were randomized to Normal (n=7), untreated HC+RAS (n=7), HC+RAS treated with PTRA (n=6), or HC+RAS treated with PTRA preceded by SW (n=6).

Normal pigs were fed isocaloric diets of standard chow, and HC+RAS pigs with a high-fat diet (a surrogate for early atherosclerosis) for the entire course of the study. Unilateral RAS was induced after 6 weeks of diet by placing a local irritant coil in the right main renal artery, as previously described[8].

Three weeks after RAS induction, six HC+RAS pigs were treated with bi-weekly sessions of low-energy SW for 3 consecutive weeks (a total of 6 sessions). Guided by an Acuson SC2000 ultrasound system (Global Siemens Healthcare, Erlangen, Germany), SW was delivered through an ultrasound probe placed at the lateral aspect of the stenotic kidney, using Omnispec Vetspec Model (Medispec® LTD, Germantown, MD, USA; spark voltage 10-24 KV; energy density 0.09 mJ/mm2; frequency 120 pulse/minute)[7].

Six weeks after RAS induction, the degrees of stenosis in all the HC+RAS pigs were determined by renal angiography, and those assigned to renal revascularization were further treated with PTRA, while the others were sham-treated. The sham procedure included selective renal angiography, during which the renal artery was engaged using a catheter and contrast media were injected. Four weeks after PTRA or sham, pigs were again anesthetized, and blood samples collected from the inferior vena cava for creatinine measurements. PRA was measured in blood collected from veins draining stenotic or normal kidneys. Renal function and oxygenation were assessed using multidetector computed tomography (MDCT) and blood oxygen-level dependent (BOLD) magnetic resonance imaging (MRI), respectively. Animals were euthanized three days after in vivo studies with a lethal intravenous dose of sodium pentobarbital (100 mg/kg). The kidneys were removed and shock-frozen in liquid nitrogen, or preserved in formalin for histology.

In Vivo Studies

Blood Pressure and Renal Function

Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed using MDCT, as described previously [8,9]. Briefly, 160 consecutive kidneys scans were performed following a venous bolus injection of iopamidol (0.5 ml·kg−1·2s−1). Then, reconstructed MDCT images were displayed with the Analyze® software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN). For data analysis, the aorta, renal cortex, and medulla were traced on tomographic images to produce time-attenuation curves in each region and obtain estimates of renal function [8,9]. These curves were fitted by algorithms to obtain measures of renal function, including RBF and GFR. BP was measured in all animals using an arterial catheter during MDCT.

Renal Oxygenation

BOLD-MRI (Signa Echo Speed; GE Medical Systems, Milwaukee, WI) was performed 2 days before MDCT to assess intra-renal oxygenation (evaluated as R2*)[10,11]. Sixteen T2*-weighted images were acquired with echo times from 3.3 to 27.4 ms. For data analysis, the cortex and medulla were manually traced on the 7-ms echo time images, and the change in MR signals vs. echo time plotted to compute R2*.

Ex vivo studies

Fibrosis was evaluated by Masson’s trichrome staining and quantified semi-automatically as percent area staining (using AxioVision 4.8, ZEISS). Tubular injury (tubular dilation, atrophy, cast formation, sloughing tubular epithelial cells, or thickening of basement membrane) was assessed in sections stained with in Periodic acid–Schiff slides on a 1–5 scale (1: <10%, 2:10–25%, 3:26–50%, 4:51–75% and 5: >75% injury)[12]. Renal inflammation was assessed in sections stained for antibodies against CD68+/inducible nitric oxide synthase (iNOS)+ (M1) (1:100, Abcam), CD68+/Arginase-1+ (M2) (1:100, Abcam), and monocyte chemoattractant protein (MCP-1) (1:100, Abcam)[13]. The numbers of M1 (pro-inflammatory) or M2 (pro-repair) macrophages were manually quantified in randomly chosen 10-15 fields per slide. Renal vein levels of TNF-α (ELISA, Invitrogen) were also measured.

To assess the renal microcirculation, peritubular capillaries were identified in 5μm slides stained for H&E by the presence of lumen, red blood cells, and/or an endothelial cell lining[14,15]. Capillaries were counted at 100× magnification using an ApoTome microscope (Carl ZEISS SMT, Oberkochen, Germany), and the ratio of capillary number to tubules calculated. To evaluate endothelial-to-mesenchymal transition (EndoMT), a mechanism that might contribute to fibrosis and microvascular loss, kidneys were stained with anti-vimentin (1:100, Abcam) and CD31 (1:100, Bio-Rad) antibodies[16]. Renal expression of the angiogenic factor vascular endothelial growth factor (VEGF) (1:200 Santa-Cruz) and the mechanotransducers phosphate -focal adhesion kinase (p-FAK) (1:50 Cell Signaling) and FAK (1:50 Cell Signaling), was assessed by western blotting. Microvascular remodeling was assessed by measuring media-to-lumen ratio in α-smooth muscle actin (α-SMA, 1:50, Sigma) staining in small and medium-size arteries under 500μm in diameter[17].

Statistical methods

Statistical analysis was performed using JMP software package version 10.0 (SAS Institute Inc. Cary, NC). Based on our previous study[7], power calculations indicated that 6 animals per group will be required to detect differences among groups in GFR with power of 80% ( standard deviation of 10 mL/min, and an expected effect size of 20 mL/min. Results were expressed as mean±SEM for normally distributed variables, and median (range) for nonparametric variables. Parametric (ANOVA/Student’s t-test) and nonparametric (Wilcoxon/Kruskal-Wallis) tests were used as appropriate. Statistical significance for all tests was accepted for p<0.05.

Results

PTRA+SW Stabilized Renal Function in HC+RAS

Before PTRA or sham, the degree of stenosis in the HC+RAS, HC+RAS+PTRA, and HC+RAS+PTRA+SW groups was comparable (86±16, 71±19, and 81±19%, respectively, p=0.29, ANOVA). Four weeks after the procedure, no residual stenosis was observed in PTRA-treated pigs. PTRA lowered mean arterial pressure (MAP) and renal vein plasma renin activity (both p<0.05 vs. HC+RAS), which were similar to Normal (Figure 1A, Table 1). No significant difference was observed among the groups in serum creatinine (p=0.27 ANOVA). RBF, GFR, and volume of the stenotic HC+RAS kidney were all lower than normal (p<0.05, respectively, Figure 1B-D). In all PTRA-treated pigs regardless of SW pre-treatment, renal volume remained low, whereas RBF improved and was not different from Normal in either PTRA- or PTRA+SW-treated pigs. On the other hand, stenotic-kidney GFR was significantly higher in HC+RAS+PTRA+SW than in HC+RAS (p<0.05), and was not different from Normal (p>0.05), whereas in HC+RAS+PTRA stenotic kidney GFR remained lower than normal (p<0.01) and not different from untreated HC+RAS kidneys (p=0.32). There was no difference among the groups in contralateral kidney GFR (p=0.28 ANOVA).

Figure 1.

Figure 1.

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A-D: Mean arterial pressure (MAP), glomerular filtration rate (GFR), renal volume, and renal blood flow (RBF) in pigs with atherosclerotic renal artery stenosis (HC+RAS), untreated or treated with percutaneous transluminal renal angioplasty (PTRA) with or without a preceding shockwave (SW) regimen. MAP was significantly increased in HC+RAS pigs, but normalized by both PTRA and PTRA+SW. GFR, renal volume and RBF of stenotic kidneys were significantly lower than normal pig kidneys. PTRA alone had no effect on GFR, whereas PTRA+SW increased it significantly compared to HC+RAS. Neither PTRA nor PTRA+SW significantly improved renal volume or RBF compared to HC+RAS, but RBF was no longer different from Normal either. E: Quantification of hypoxia (R2*) by blood-oxygen-level-dependent magnetic resonance imaging. Cortical and medullary oxygenation was decreased in HC+RAS compared to Normal. Medullary oxygenation was improved both by PTRA and PTRA+SW, while cortical only by PTRA+SW. *p<0.05 vs Normal, † p<0.05 vs HC+RAS, ‡ p<0.05 vs HC+RAS+PTRA.

Table 1.

Characteristics of pigs with atherosclerotic renal artery stenosis (HC+RAS) 4 weeks after treatment with percutaneous transluminal renal angioplasty (PTRA), without or with a preceding shockwave (SW) regimen.

Characteristics Normal
(n=7)		 HC+RAS
(n=7)		 HC+RAS+PTRA
(n=6)		 HC+RAS+PTRA+SW
(n=6)
Body Weight (kg) 49.3±3.5 46.8±8.0 44.0±4.6 52±4.1
Plasma renin activity (renal vein, pg/ml) 1.38±1.06 12.54±11.69* 1.01±1.10† 2.01±1.81†
Serum creatinine (mg/dl) 1.50±0.46 1.87±0.27 1.90±0.44 1.80±0.39
Tumor necrosis factor-α (renal vein, pg/ml) 23.1(17.4-24.4) 33.4(28.0-154.6) * 83.1(63.8-109.2) * 26.3(20.9-77.5)
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*

p<0.05 vs. Normal

†

p<0.05 vs. HC+RAS

‡

p<0.05 vs. HC+RAS+PTRA.

PTRA+SW Restored Kidney Cortex Oxygenation

Both HC+RAS and HC+RAS+PTRA stenotic kidneys showed similar renal cortical hypoxia (R2*; Figure 1E, both p<0.05 vs. normal), but restored to normal oxygenation levels in HC+RAS+PTRA+SW. Medullary R2* was elevated in HC+RAS (p<0.01, vs. Normal) and blunted by both PTRA and PTRA+SW (both p<0.01 vs. HC+RAS and p>0.05 vs. Normal).

PTRA+SW Decreased Inflammation

The number of infiltrating inflammatory M1 macrophages, which was elevated in HC+RAS (p<0.05 vs. Normal), was attenuated by PTRA (p<0.05, vs. HC+RAS) and even further lowered by PTRA+SW (Figure 2A-B, p<0.05 vs. HC+RAS+PTRA). Fewer reparative M2 macrophages were observed in HC+RAS and HC+RAS+PTRA compared to Normal (p<0.05), but their number increased in HC+RAS+PTRA+SW compared to all other groups (Figure 2A-B, p<0.05 vs. all). Consequently, the M1/M2 ratio, which was higher in HC+RAS compared to Normal (p<0.01), remained increased in HC+RAS+PTRA (p<0.01 vs. Normal), but was normalized in HC+RAS+PTRA+SW (Figure 2C, p=0.07 vs. Normal).

Figure 2.

Figure 2.

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A: Representative immunofluorescence staining images (X40) of stenotic kidney M1 [CD68 (red)/inducible nitric oxide synthase (green)] and M2 [CD68/arginase-1 (green)] macrophages. Double staining is in yellow. B: The numbers of M1 macrophages increased in HC+RAS compared to Normal, decreased by PTRA, and further decreased by PTRA+SW. Fewer M2 macrophages were observed in HC+RAS and HC+RAS+PTRA compared to Normal, but their number increased in HC+RAS+PTRA+SW compared to all other groups. C: M1/M2 ratio decreased in HC+RAS and HC+RAS+PTRA but was normalized in HC+RAS+PTRA+SW. D: Representative images of immunoreactivity of monocyte chemoattractant protein (MCP)-1. E: MCP-1 immunoreactivity was enhanced in HC+RAS and HC+RAS+PTRA but ameliorated in HC+RAS+PTRA+SW. *p<0.05 vs. Normal, † p<0.05 vs. HC+RAS, ‡ p<0.05 vs. HC+RAS+PTRA.

MCP-1 immunoreactivity was comparably upregulated in HC+RAS and HC+RAS+PTRA (p<0.05 vs. Normal), and ameliorated only in HC+RAS+PTRA+SW (Figure 2D-E, p<0.05 vs. HC+RAS). Similarly, elevated level of TNF-α in renal vein observed in HC+RAS and untreated HC+RAS+PTRA (p<0.05 vs. Normal) was normalized in PTRA+SW (Table 1, p>0.05 vs. Normal), suggesting decreased inflammatory activity in the stenotic kidney.

PTRA+SW Improved the Stenotic Kidney Microcirculation

Cortical and medullary capillary density as per H&E staining was significantly lower in HC+RAS compared to normal kidneys, but significantly higher in both HC+RAS+PTRA and HC+RAS+PTRA+SW compared to HC+RAS (p<0.05, respectively). Furthermore, the number of capillaries in HC+RAS+PTRA+SW was higher than in HC+RAS+PTRA (p<0.05) and similar to Normal (Figure 3A-B). This was confirmed by CD31 immunofluorescence, showing that compared with Normal, CD31+capillary counts fell in HC+RAS (Figure 4A-B, p<0.05) but increased after PTRA (p<0.05 vs. HC+RAS), yet increased even further in PTRA+SW compared to PTRA alone (p<0.05). Microvascular wall thickening (media-to-lumen ratio) increased in HC+RAS (p<0.05 vs. Normal), and reversed only in HC+RAS+PTRA+SW (Figure 3C-D, p<0.05 vs. each). Similarly, activation of the VEGF-stimulator FAK[18] (indicated by p-FAK/FAK ratio) increased only in HC+RAS+PTRA+SW compared to HC+RAS (Figure 4C-D, p<0.05). VEGF expression was lower in HC+RAS compared to normal kidneys, increased in HC+RAS+PTRA compared to both normal and HC+RAS, and tended to be further elevated in HC+RAS+PTRA+SW compared to HC+RAS+PTRA (Figure 4C, E, p=0.09).

Figure 3.

Figure 3.

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A: Representative H&E-stained cortical and medullary tubules (x40 images). B: The number of capillaries-per-tubule decreased in HC+RAS compared to Normal (H&E, x100), but increased after PTRA and PTRA+SW in HC+RAS. Capillary density in HC+RAS+PTRA+SW was similar to Normal. C: Representative renal α-SMA staining of microvessels under 500μm in diameter. D. Media to lumen ratio increased in HC+RAS and HC+RAS+PTRA but decreased in HC+RAS+PTRA+SW compared to Normal. *p<0.05 vs Normal, † p<0.05 vs HC+RAS, ‡ p<0.05 vs HC+RAS+PTRA.

Figure 4.

Figure 4.

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A: Representative images of immunoreactivity of CD31 (red) and Vimentin (green) in immunofluorescence microscopy (x40 images). B: CD31+ capillary counts (white arrow) decreased in HC+RAS, but increased in HC+RAS+PTRA, and further in HC+RAS+PTRA+SW. C: CD31 and Vimentin co-localization (yellow arrow) was greater in HC+RAS compared to Normal and was decreased by PTRA+SW, but not by PTRA alone. D: Renal expression of focal adhesion kinase (FAK), phosphate- FAK (p-FAK), angiogenic vascular endothelial growth factor (VEGF). E: Ratio of p-FAK/FAK was downregulated in HC+RAS but improved in HC+RAS+PTRA+SW. F: Level of VEGF expression decreased in HC+RAS, and increased in both treatment groups compared to Normal, but tended to be higher in HC+RAS+PTRA+SW compared to HC+RAS+PTRA. *p<0.05 vs Normal, † p<0.05 vs. HC+RAS, ‡ p<0.05 vs HC+RAS+PTRA.

PTRA+SW Alleviated EndoMT, Fibrosis, and Tubular Injury

CD31 and Vimentin co-expression was greater in HC+RAS compared to Normal pigs, suggesting increased EndoMT (Figure 3A, C, p<0.05), which was decreased by PTRA+SW (p<0.05 vs. HC+RAS), but not by PTRA alone (p=0.2). Notable medullary and cortical fibrosis (trichrome-staining) was observed in HC+RAS pigs (Figure 5A-B, p<0.01 vs. Normal). Cortical fibrosis was attenuated in both PTRA-treated groups (both p<0.05 vs. HC+RAS), although both remained higher than Normal (Figure 5A-B, p<0.05). On the other hand, medullary fibrosis was ameliorated only in HC+RAS+PTRA+SW (p<0.05 vs. HC+RAS), as was tubular Injury (Figure 5C-D, both p<0.05 vs. HC+RAS and HC+RAS+PTRA).

Figure 5.

Figure 5.

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A: Representative trichrome -stained cortex and medulla (x20 images). B: HC+RAS increased trichrome staining compared to Normal. Cortical trichrome staining was alleviated both by PTRA and PTRA+ SW, but medullary trichrome staining only in HC+RAS +PTRA+SW. C: Representative PAS staining (x20 images). D: Tubular injury was enhanced in HC+RAS and HC+RAS+PTRA but ameliorated in HC+RAS+PTRA+SW. *p<0.05 vs. Normal, † p<0.05 vs. HC+RAS, ‡ p<0.05 vs. HC+RAS+PTRA.

DISCUSSION

This study shows that pre-treatment with low-energy SW enables revascularization to reverse renal tissue injury in swine HC+RAS. This adjunct treatment permitted restoration of renal function, oxygenation, and microvascular and tubulointerstitial integrity. Therefore, pre-treatment with SW prior to PTRA may be an effective strategy to improve renal function in subjects with HC+RAS.

Atherosclerotic RAS, the leading cause of renovascular hypertension, is increasingly encountered in the aging population[19] and results in progressive renal functional loss[20,21]. PTRA may also show some benefit in BP control over optimal medical treatment in human subjects with RAS[2], but recovery of renal function by PTRA or stenting in patients with atherosclerotic RAS and renal microvascular damage is uncommon. The present study underscores these observations, and shows that a regimen of PTRA in pigs successfully decreases BP, but did not improve function in the stenotic HC+RAS kidney. In contrast, PTRA successfully improved renal function in pigs fed a normal chow[22], which are not exposed to the deleterious effects of HC on microvascular structure and function. Emerging experimental and clinical evidence demonstrates that restoration of renal arterial luminal patency alone cannot reverse feed-forward inflammatory and pro-fibrotic processes or microvascular disease that fuel renal injury[3], and hence generates the impetus to employ maneuvers that target the post-stenotic kidney directly and subdue pathological processes. However, because such strategies do not necessarily ameliorate renovascular hypertension[23], their combination with endovascular revascularization might be advantageous.

We previously[7] showed that noninvasive low-energy SW therapy restored kidney microvasculature in swine HC+RAS, possibly through mechanotransduction-mediated activation of angiogenic signaling. However, while application of SW alone increased GFR and improved stenotic kidney RBF, it did not fully normalize BP, probably due to the remaining renal arterial obstruction. On the other hand, in the present study pre-treatment with SW followed by PTRA resulted in normalization of BP. SW+PTRA also significantly lowered renal vein plasma renin activity and improved stenotic kidney GFR. These observations indicate that the combination of SW and PTRA is effective in improving a wider range of pathophysiological changes in subjects with HC+RAS.

The mechanisms by which SW magnified renal recovery after PTRA in experiment HC+RAS may involve suppression of inflammation and fibrosis. Renal inflammation is an important determinant of functional deterioration in the stenotic kidney. MCP-1, a pro-inflammatory cytokine associated with an M1 macrophages response[24], is implicated in regulation of renal hemodynamics and function in renovascular hypertension[25]. We have recently shown that PTRA alone could increase RBF, but failed to decrease renal inflammation in either human subjects[26] or pigs[27], denoted by elevated renal vein TNF-α levels and renal MCP-1 immunoreactivity. SW reportedly exerts anti-inflammatory effects to decrease macrophages infiltration in the ischemic heart[28], M1/M2 macrophages ratio[29], and pro-inflammatory cytokines[30]. Clinical application of SW treatment also alleviated chronic abacterial prostatitis[31]. A new finding in this study is that addition of SW to PTRA might ameliorate progression of kidney tissue injury in HC+RAS by suppression of inflammatory responses, evidenced by normalized renal vein TNF-α levels and MCP-1 immunoreactivity. Congruently, SW+PTRA decreased macrophage infiltration and drove phenotypic switch of macrophages from M1 to M2. This may be mediated by modulation of endogenous nitric oxide production and subsequent suppression of NF-κB signaling by SW[32].

In addition to inflammation, interstitial fibrosis is an important driver of ischemic kidney disease progression. EndoMT is a pathway recently recognized to lead to development of kidney fibrosis, given that 30-50% of myofibroblasts in the fibrotic kidneys originate from endothelial cells[33]. Recently, EndoMT has been linked to sustaining BP and hypertensive kidney injury[34], and implicated in stenotic kidney damage in the metabolic syndrome[16]. Indeed, we observed in HC+RAS kidneys significant costaining of endothelial cells with vimentin, suggesting a phenotype change through EndoMT[16], which PTRA alone failed to reverse. Contrarily, pre-treatment with SW decreased EndoMT in the PTRA-treated HC+RAS kidney, which might have thereby ameliorated vascular rarefaction.

Microvascular loss may precipitate tissue hypoxia, an important trigger for inflammation and fibrosis[35], and thereby contribute to remodeling and dysfunction in the HC+RAS kidney. In agreement with our previous observation of the pro-angiogenic capability of SW, its addition to PTRA in the present study prevented loss of peritubular capillaries and decreased remodeling (media-to-lumen ratio) of small and medium-size arteries. Small artery inward remodeling is associated with chronic low-flow states, and persistent vasoconstriction leads to entrenchment of reduced diameter[36], which the expanded microcirculation likely blunted. Notably, pFAK activation was observed only in HC+RAS+PTRA+SW, and VEGF expression tended to be higher in PTRA+SW than in PTRA alone. Mechanotransducers that SW upregulates[7] facilitate FAK activation[37] that in turn modulates angiogenesis[38]. FAK is necessary for normal vascular development[39,40] and endothelial cells proliferation and migration in adult mice[41], and directly stimulates VEGF receptor-2 expression to promote angiogenesis[42]. Therefore, activation of FAK by PTRA+SW may contribute to the enhanced angiogenesis and increase VEGF expression. Consequent to increased vascular density, the intervention with SW and PTRA also improved cortical oxygenation (BOLD R2*) compared to PTRA alone.

Interestingly, SW appeared to be distinctly endowed with the capability to improve the medullary microcirculation and decrease medullary fibrosis. This is an important property, because the medulla may be less accessible to interventions[15,43] than the cortex. Notably, the deep focal point of SWT (12-14cm) may confer particular benefits by reaching the medulla.

Our study is limited by the short duration of HC+RAS, as the model was developed over 10 weeks with short-term exposure to atherosclerosis and renovascular disease. Nevertheless, disease development and renal pathology in our swine model mimic that in humans. The beneficial effects of SW on the response to PTRA in long-term need to be examined. Due to species differences between pigs and humans future studies in human renovascular disease are needed to validate these results. In addition, we have used a SW delivery regimen and setting based on previously studies. Alternative SW regimens in treating HC+RAS kidneys are worth exploration in future studies.

Conclusion

The current study shows that delivery of a 3-week low-energy SW regimen before PTRA constitutes an effective approach to improve renal structure and function following renal revascularization in swine HC+RAS. The beneficial effect appears to be mediated by a decrease in renal inflammation and fibrosis, associated with preservation of the renal microcirculation. SW decreased microvascular remodeling and cortical hypoxia, and reduced cortical and particularly medullary fibrosis in the stenotic kidney. This study therefore introduces SW as a potentially valuable pre-treatment to enhance the efficacy of PTRA in experimental HC+RAS. Additional studies are needed to optimize doses and energy levels of SW treatment, and possibly explore the effect of implement SW after PTRA.

Acknowledgment

We thank Medispec® LTD, Gaithersburg, MD, for generously allowing the use of the SW machine. The vendor was not involved in data collection or analysis.

Sources of Funding

This research was partly supported by NIH grants numbers HL123160, DK104273, DK102325, DK120292 and DK122734.

Footnotes

Disclosures: None.

References

  • 1.Kwon SH, Lerman LO. Atherosclerotic renal artery stenosis: current status. Adv Chronic Kidney Dis 2015; 22 (3):224–231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Cooper CJ, Murphy TP, Cutlip DE, Jamerson K, Henrich W, Reid DM, et al. Stenting and medical therapy for atherosclerotic renal-artery stenosis. N Engl J Med 2014; 370 (1):13–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Eirin A, Textor SC, Lerman LO. Emerging Paradigms in Chronic Kidney Ischemia. Hypertension 2018; 72 (5):1023–1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Fukumoto Y, Ito A, Uwatoku T, Matoba T, Kishi T, Tanaka H, et al. Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease. Coron Artery Dis 2006; 17 (1):63–70. [DOI] [PubMed] [Google Scholar]
  • 5.Wang CJ, Huang KE, Sun YC, Yang YJ, Ko JY, Weng LH, et al. VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res 2011; 171 (1):114–119. [DOI] [PubMed] [Google Scholar]
  • 6.Aicher A, Heeschen C, Sasaki K, Urbich C, Zeiher AM, Dimmeler S. Low-energy shock wave for enhancing recruitment of endothelial progenitor cells: a new modality to increase efficacy of cell therapy in chronic hind limb ischemia. Circulation 2006; 114 (25):2823–2830. [DOI] [PubMed] [Google Scholar]
  • 7.Zhang X, Krier JD, Amador Carrascal C, Greenleaf JF, Ebrahimi B, Hedayat AF, et al. Low-Energy Shockwave Therapy Improves Ischemic Kidney Microcirculation. J Am Soc Nephrol 2016; 27 (12):3715–3724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chade AR, Rodriguez-Porcel M, Grande JP, Krier JD, Lerman A, Romero JC, et al. Distinct renal injury in early atherosclerosis and renovascular disease. Circulation 2002; 106 (9):1165–1171. [DOI] [PubMed] [Google Scholar]
  • 9.Daghini E, Primak AN, Chade AR, Krier JD, Zhu XY, Ritman EL, et al. Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT. Radiology 2007; 243 (2):405–412. [DOI] [PubMed] [Google Scholar]
  • 10.Warner L, Glockner JF, Woollard J, Textor SC, Romero JC, Lerman LO. Determinations of renal cortical and medullary oxygenation using blood oxygen level-dependent magnetic resonance imaging and selective diuretics. Invest Radiol 2011; 46 (1):41–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ebrahimi B, Gloviczki M, Woollard JR, Crane JA, Textor SC, Lerman LO. Compartmental analysis of renal BOLD MRI data: introduction and validation. Invest Radiol 2012; 47 (3):175–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lu B, Rajakumar SV, Robson SC, Lee EK, Crikis S, d'Apice AJ, et al. The impact of purinergic signaling on renal ischemia-reperfusion injury. Transplantation 2008; 86 (12):1707–1712. [DOI] [PubMed] [Google Scholar]
  • 13.Eirin A, Zhu XY, Puranik AS, Tang H, McGurren KA, van Wijnen AJ, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation. Kidney Int 2017; 92 (1):114–124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ward WK, Quinn MJ, Wood MD, Tiekotter KL, Pidikiti S, Gallagher JA. Vascularizing the tissue surrounding a model biosensor: how localized is the effect of a subcutaneous infusion of vascular endothelial growth factor (VEGF)? Biosens Bioelectron 2003; 19 (3):155–163. [DOI] [PubMed] [Google Scholar]
  • 15.Ebrahimi B, Eirin A, Li Z, Zhu XY, Zhang X, Lerman A, et al. Mesenchymal stem cells improve medullary inflammation and fibrosis after revascularization of swine atherosclerotic renal artery stenosis. PLoS One 2013; 8 (7):e67474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zhang X, Kim SR, Ferguson CM, Ebrahimi B, Hedayat AF, Lerman A, et al. The Metabolic Syndrome Does Not Affect Development of Collateral Circulation in the Poststenotic Swine Kidney. American journal of hypertension 2018; 31 (12):1307–1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Sun D, Eirin A, Zhu XY, Zhang X, Crane JA, Woollard JR, et al. Experimental coronary artery stenosis accelerates kidney damage in renovascular hypertensive swine. Kidney Int 2015; 87 (4):719–727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Shindo T, Ito K, Ogata T, Hatanaka K, Kurosawa R, Eguchi K, et al. Low-Intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates Left Ventricular Dysfunction in a Mouse Model of Acute Myocardial Infarction. Arterioscler Thromb Vasc Biol 2016; 36 (6):1220–1229. [DOI] [PubMed] [Google Scholar]
  • 19.Hansen KJ, Cherr GS, Dean RH. Dialysis-free survival after surgical repair of ischemic nephropathy. Cardiovasc Surg 2002; 10 (4):400–404. [DOI] [PubMed] [Google Scholar]
  • 20.Safian RD, Textor SC. Renal-artery stenosis. N Engl J Med 2001; 344 (6):431–442. [DOI] [PubMed] [Google Scholar]
  • 21.Lerman L, Textor SC. Pathophysiology of ischemic nephropathy. Urol Clin North Am 2001; 28 (4):793–803, ix. [DOI] [PubMed] [Google Scholar]
  • 22.Favreau F, Zhu XY, Krier JD, Lin J, Warner L, Textor SC, et al. Revascularization of swine renal artery stenosis improves renal function but not the changes in vascular structure. Kidney Int 2010; 78 (11):1110–1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lerman LO, Textor SC. Gained in translation: protective paradigms for the poststenotic kidney. Hypertension 2015; 65 (5):976–982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and repair. J Clin Invest 2008; 118 (11 ):3522–3530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Zhu XY, Chade AR, Krier JD, Daghini E, Lavi R, Guglielmotti A, et al. The chemokine monocyte chemoattractant protein-1 contributes to renal dysfunction in swine renovascular hypertension. J Hypertens 2009; 27 (10):2063–2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Saad A, Herrmann SM, Crane J, Glockner JF, McKusick MA, Misra S, et al. Stent revascularization restores cortical blood flow and reverses tissue hypoxia in atherosclerotic renal artery stenosis but fails to reverse inflammatory pathways or glomerular filtration rate. Circ Cardiovasc Interv 2013; 6 (4):428–435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Eirin A, Li Z, Zhang X, Krier JD, Woollard JR, Zhu XY, et al. A mitochondrial permeability transition pore inhibitor improves renal outcomes after revascularization in experimental atherosclerotic renal artery stenosis. Hypertension 2012; 60 (5):1242–1249. [DOI] [PubMed] [Google Scholar]
  • 28.Abe Y, Ito K, Hao K, Shindo T, Ogata T, Kagaya Y, et al. Extracorporeal low-energy shock-wave therapy exerts anti-inflammatory effects in a rat model of acute myocardial infarction. Circ J 2014; 78 (12):2915–2925. [DOI] [PubMed] [Google Scholar]
  • 29.Sukubo NG, Tibalt E, Respizzi S, Locati M, d'Agostino MC. Effect of shock waves on macrophages: A possible role in tissue regeneration and remodeling. Int J Surg 2015; 24 (Pt B):124–130. [DOI] [PubMed] [Google Scholar]
  • 30.Davis TA, Stojadinovic A, Anam K, Amare M, Naik S, Peoples GE, et al. Extracorporeal shock wave therapy suppresses the early proinflammatory immune response to a severe cutaneous burn injury. Int Wound J 2009; 6 (1):11–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Al Edwan GM, Muheilan MM, Atta ON. Long term efficacy of extracorporeal shock wave therapy [ESWT] for treatment of refractory chronic abacterial prostatitis. Ann Med Surg (Lond) 2017; 14:12–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mariotto S, de Prati AC, Cavalieri E, Amelio E, Marlinghaus E, Suzuki H. Extracorporeal shock wave therapy in inflammatory diseases: molecular mechanism that triggers anti-inflammatory action. Curr Med Chem 2009; 16 (19):2366–2372. [DOI] [PubMed] [Google Scholar]
  • 33.Piera-Velazquez S, Mendoza FA, Jimenez SA. Endothelial to Mesenchymal Transition (EndoMT) in the Pathogenesis of Human Fibrotic Diseases. J Clin Med 2016; 5 (4). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lin JR, Zheng YJ, Zhang ZB, Shen WL, Li XD, Wei T, et al. Suppression of Endothelial-to-Mesenchymal Transition by SIRT (Sirtuin) 3 Alleviated the Development of Hypertensive Renal Injury. Hypertension 2018; 72 (2):350–360. [DOI] [PubMed] [Google Scholar]
  • 35.Sun D, Eirin A, Ebrahimi B, Textor SC, Lerman A, Lerman LO. Early atherosclerosis aggravates renal microvascular loss and fibrosis in swine renal artery stenosis. J Am Soc Hypertens 2016; 10 (4):325–335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Bakker EN, Buus CL, Spaan JA, Perree J, Ganga A, Rolf TM, et al. Small artery remodeling depends on tissue-type transglutaminase. Circ Res 2005; 96 (1):119–126. [DOI] [PubMed] [Google Scholar]
  • 37.Toutant M, Costa A, Studler JM, Kadare G, Carnaud M, Girault JA. Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol Cell Biol 2002; 22 (22):7731–7743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Zhao X, Guan JL. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Deliv Rev 2011; 63 (8):610–615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Corsi JM, Houbron C, Billuart P, Brunet I, Bouvree K, Eichmann A, et al. Autophosphorylation-independent and -dependent functions of focal adhesion kinase during development. J Biol Chem 2009; 284 (50):34769–34776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lim ST, Chen XL, Tomar A, Miller NL, Yoo J, Schlaepfer DD. Knock-in mutation reveals an essential role for focal adhesion kinase activity in blood vessel morphogenesis and cell motility-polarity but not cell proliferation. J Biol Chem 2010; 285 (28):21526–21536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Cabrita MA, Jones LM, Quizi JL, Sabourin LA, McKay BC, Addison CL. Focal adhesion kinase inhibitors are potent anti-angiogenic agents. Mol Oncol 2011; 5 (6):517–526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Sun S, Wu HJ, Guan JL. Nuclear FAK and its kinase activity regulate VEGFR2 transcription in angiogenesis of adult mice. Sci Rep 2018; 8 (1):2550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Ebrahimi B, Li Z, Eirin A, Zhu XY, Textor SC, Lerman LO. Addition of endothelial progenitor cells to renal revascularization restores medullary tubular oxygen consumption in swine renal artery stenosis. Am J Physiol Renal Physiol 2012; 302 (11):F1478–1485. [DOI] [PMC free article] [PubMed] [Google Scholar]

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