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

Abstract

Background: Revascularization does not restore renal function in most patients with atherosclerotic renal artery stenosis (RAS), likely because of 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 pretreated with LE-SWT before PTRA 4 weeks later.

Conclusion: 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 pretreatment to enhance the success of PTRA in blunting loss of kidney function in experimental HC+RAS.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

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.

Figure 5.

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.