Extracorporeal shock wave treatment attenuated left ventricular dysfunction and remodeling in mini-pig with cardiorenal syndrome

Figures

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  Figure 1. Serial changes of circulating creatinine levels, flow cytometric analysis of circulating level of endothelial progenitor cells (EPCs) and kidney injury score by day 180 after CKD induction (A) Circulating creatinine level at baseline, p>0.5. (B) Circulating creatinine level at day 30, * vs. ‡, p<0.0001. (C) Circulating creatinine level at day 90, * vs. ‡, p<0.001. (D) Circulating creatinine level at day 180, * vs. other groups with different symbols (†, ‡), p<0.0001. (E) Number of circulating CD31+/CD117+ cells at day 180, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (F) Number of circulating CD34+/CD117+ cells at day 180, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (G to K) Light microscopic findings of H&E stain (400x) demonstrating significantly higher degree of loss of brush border in renal tubules (yellow arrows), tubular necrosis (green arrows), tubular dilatation (red asterisk) protein cast formation (black asterisk), and dilatation of Bowman's capsule (blue arrows) in LVIs-CKD group than in other groups. (L) * vs. other groups with different symbols (†, ‡, §), p<0.0001. Scale bars in right lower corner represent 50μm. All…

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  Figure 2. Expressions of fibrosis and collagen deposition in LV myocardium by day 180 after CKD induction (A to E) Immunohistochemical (IHC) microscopic finding (100x) of Masson's trichrome for identification of fibrotic area (blue color) in LV myocardium. (F) Analytical result of fibrotic area, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (G to K) IHC microscopic finding (100x) of Sirius red for identification of collagen-deposition area (pink color) in LV myocardium. (L) Analytical result of collagen-deposition area, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. Scale bars in right lower corner represent 100μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW: extracorporeal shock wave.

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  Figure 3. Expressions of small vessels and apoptotic cells in LV myocardium by day 180 after CKD induction (A to E) Microscopic finding (100x) of α-SMA stain for identification of small vessels (gray color) in LV myocardium. (F) Analytic results of number of small vessel (≤ 25μm), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. Scale bars in right lower corner represent 100μm. (G to K) Microscopic finding (200x) of TUNEL assay for identification of apoptotic nuclei (gray color) in LV myocardium. (L) Analytical result of number of apoptotic nuclei, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. Scale bars in right lower corner represent 50μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW: extracorporeal shock wave.

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  Figure 4. Cellular level of angiogenesis in LV myocardium by day 180 after CKD induction (A to E) Immunofluorescent (IF) microscopic finding (400x) for identifying the expression of CD31+ cells (green color). (F) Analytical result of number of CD31+ cells, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (G to K) IF microscopic finding (400x) for identifying the expression of von Willebrand factor (vWF)+ cells (green color). (L) Analytical result of number of vWF+ cells, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. Scale bars in right lower corner represent 20μm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW: extracorporeal shock wave.

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  Figure 5. Protein expressions of angiogenesis factors in LV myocardium by day 180 after CKD induction (A) Protein expression of CD31, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (B) Protein expression of von Willebrand factor (vWF), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (C) Protein expression of vascular endothelial growth factor (VEGF), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (D) Protein expression of CXCR4, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.001. (E) Protein expression of stromal cell-derived factor (SDF)-α, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (F) Protein expression of endothelial nitric oxide synthase (eNOS), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW: extracorporeal shock wave.

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  Figure 6. Protein expressions of oxidative stress biomarkers in LV myocardium by day 180 after CKD induction (A) Protein expression of ONX-1, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (B) Protein expression of ONX-2, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (C) Oxidized protein expression, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (Note: left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W: molecular weight; DNP: 1-3 dinitrophenylhydrazone. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW: extracorporeal shock wave.

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  Figure 7. Protein expressions of mitochondrial integrity, pressure/volume overload and myocardial hypertrophy biomarkers in LV myocardium by day 180 after CKD induction (A) Protein expression of peroxisome proliferator activated receptor-gamma coactivator 1 alpha (PGC-α), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (B) Protein expression of mitochondrial cytochrome C (mit-Cyt C), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (C) Protein expression of cytosolic cytochrome C (cyt-Cyt C), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (D) Protein expression of brain natriuretic peptide (BNP), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (E) Protein expression of beta myosin heavy chain (β-MHC), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (F) Protein expression of α-MHC, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia;…

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  Figure 8. Protein expressions of fibrosis and apoptotic biomarkers in LV myocardium by day 180 after CKD induction (A) Protein expression of transforming growth factor (TGF)-β, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (B) Protein expression of phosphorylated (p)-Smad3, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (C) Protein expression of bone morphogenetic protein (BMP)-2, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (D) Protein expression of p-Smad1/5, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (E) Protein expression of cleaved (c)-caspase 3, * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. (F) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP), * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. * vs. other groups with different symbols (†, ‡, §, ¶), p<0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). SC: sham-operative control; LVIs: left ventricular ischemia; CKD: chronic kidney disease; ECSW:…