Sympathetic Denervation Ameliorates Renal Fibrosis via Inhibition of Cellular Senescence

Figures

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  Figure 1. Renal denervation inhibits the fibrotic phenotype during UUO and UIRI. C57BL/6 mice were subjected to UUO or UIRI manipulation 2 days after renal denervation (DNx) or sham operation in the left kidneys. (A, B) Representative histology of kidney sections from sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice stained with periodic acid–Schiff, Sirius Red, and Masson’s trichrome stain. Scale bar, 50μm. (C, D) Representative Western blot of fibronectin (FN), Collagen I (Col 1), α-SMA, and Tyrosine Hydroxylase (TH) in the kidney tissue of sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice with densitometry analysis (E, F). (G, H) Representative mRNA expressions of FN, Col 1, and α-SMA in the kidney tissue of sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice using qRT-PCR analysis. n=3–7 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.

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  Figure 2. Renal denervation reduces renal senescence and the expression of proinflammatory cytokines after kidney injury. Renal denervation (DNx) or sham operation was performed 2 days before the UUO or UIRI model in the left kidneys of mice. (A, B) Representative Western blot of senescence-related proteins p53, p21, and γ-H2AX in sham-operated, UUO/UIRI, and DNx+UUO/UIRI kidneys with densitometry analysis (C, D). (E, F) mRNA expressions of senescence-related markers p53, p21, p16, HMGA2, H2AX, and senescence-associated secretory phenotype (SASP) TGF-β1, IL-6, IL-8, and IL-1β in the kidney tissue of sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice as shown by qRT-PCR analysis. n=3–10 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.

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  Figure 3. Norepinephrine contributes to cellular senescence and upregulates profibrogenic and proinflammatory cytokines in renal tubular cells. (A, B) Representative Western blot of p21, γ-H2AX or FN, Col 1 in TKPTS cells cultured with PBS (control) or different concentrations of NE (0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) (n=3 in each group). (C, D) Representative Western blot of γ-H2AX or FN, Col 1 in TKPTS cells incubated with PBS (control) or NE at a final concentration of 10 nM for the indicated periods (1, 6, 12, 24, 48, and 72 hours) (n=3 in each group). (E–G) TKPTS cells were incubated with NE (10 nM) or H2O2 (100 μM) for 48 hours and were then harvested. (E) Representative Western blot of p53, p21, and γ-H2AX in TKPTS cells treated with PBS, NE, or H2O2 with densitometry analysis (n=3–5 in each group). (F) Representative images of cell morphology and SA-β-gal staining of TKPTS cells treated with PBS, NE, or H2O2 using a microscope (n=3 in each group). Scale bar=50 μm. (G) mRNA levels of senescence-related proteins (p53, p21, p16, HMGA2, H2AX), SASP components (IL-6, IL-8, IL-1β), and profibrotic cytokines (TGF-β1, CTGF) in each group were detected by qRT-PCR analysis…

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  Figure 4. Atipamezole (α2-adrenergic antagonist) reverses cellular senescence induced by NE in renal tubular cells. TKPTS cells were incubated with NE (10 nM) for 48 hours after pretreatment with adrenergic antagonists or PBS for an hour. (A, B) mRNA expressions of ADRA1B, ADRA2A, ADRB1, ADRB2, and ADRB3 in PBS (control) or NE-treated TKPTS cells as shown by qRT-PCR analysis (n=3–8 in each group). (C) Representative Western blotting of p21 and γ-H2AX in TKPTS cells treated with PBS, NE, and NE+ATI (Atipamezole, 100 nM, 1 μM, 10 μM, and 100 μM) with densitometry analysis (n=3 in each group). (D, E) Representative Western blotting of p21 and γ-H2AX in TKPTS cells incubated with PBS, NE, and NE+BUT (Butoxamine)/ICI (ICI118551) (two β2-adrenergic antagonists, 100 μM, 10 μM, 1 μM, and 100 nM) with densitometry analysis, respectively (n=3 in each group). (F) mRNA expressions of p53, p21, p16, H2AX, IL-6, IL-8, and IL-1β in TKPTS cells cultured with PBS, NE, and NE+ATI (10 μM) as displayed by qRT-PCR analysis (n=3–8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Bars represented means ± SEM.

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  Figure 5. The α2A-adrenergic receptor is responsible for NE-induced tubular epithelial cell senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative images of immunohistochemistry for α2A-adrenergic receptors (α2A-AR) in sham-operated, UUO/UIRI, mice and DNx+UUO/UIRI mice. Scale bar, 20μm. (C, D) After starving for 8 hours, TKPTS cells were cultured with PBS or NE (10 nM) for 48 hours. α2A-AR expressions were tested by immunofluorescence (C) (Scale bar, 20 μm) and Western Blot (D) (n=3 in each group). (E, F) After pretreatment with PBS or α2A-adrenergic antagonist (BRL4408, BRL, 10 μM) for 1 hour, TKPTS cells were incubated with NE (10 nM) for 48 hours. (E) Representative Western blotting of p53, p21, and γ-H2AX in each group with densitometry analysis (n=4-5). (F) mRNA expressions of p53, p21, p16, HMGA2, H2AX, TGF-β1, IL-6, IL-8, and IL-1β were analyzed using qRT-PCR analysis (n=3–8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.

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  Figure 6. β-arrestin2 is a target of α2A-AR and knockdown of β-arrestin2 ameliorates NE-induced tubular cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative images of immunohistochemistry for β-arrestin2 in sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice. Scale bar, 20 μm. (C, D) Representative Western blotting of β-arrestin2 (β-arr2) in sham-operated, UUO/UIRI, and DNx+UUO/UIRI kidneys with densitometry analysis (n=3–6 in each group). (E–G) TKPTS cells were cultured with PBS or NE (10 nM) for 48 hours. (E) Representative images of immunofluorescence for β-arrestin2 in each group. Scale bar, 20μm. (F) Cells were double-labeled with α2A-AR (green) and β-arrestin2 (red), and cell nuclei were enhanced by staining with DAPI (blue). Colocalization of α2A-AR (green), and β-arrestin2 (red) was visualized as yellow in merged images. Scale bar, 20μm. (G) Interactions between β-arrestin2 and α2A-AR were examined by immunoprecipitation (IP) with an anti-β-arrestin2 antibody, followed by immunoblotting (IB) with anti-α2A-AR antibodies. (H, I) After an hour of pretreatment with PBS or BRL4408, TKPTS…

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  Figure 7. α2A-AR/β-arrestin2/NF-κB signaling contributes to mitochondrial dysfunction and cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative Western blotting of VDAC1 and COX IV in each group with densitometry analysis (n=6). (C) Representative Western blotting of VDAC1 and COX IV in TKPTS cells treated with PBS or NE (10 nM) for 48 hours with densitometry analysis (n=3–6 in each group). (D–F) TKPTS cells were transfected with scrambled siRNA (si-control) or β-arrestin2 siRNA (si-Arrb2) for 24 hours and then incubated with NE (10 nM) for 48 hours. Mitochondrial ultrastructure was observed by transmission electron microscope (D). Scale bar, 1 μm. Mitochondrial mass was tested by MitoTracker deep red probe (100nM) staining (E). Scale bar, 50 μm. Mitochondrial membrane potential was examined by TMRE fluorescent dye (200 nM) (F). Scale bar, 20 μm. (G) Representative Western blotting of NF-κB p65 in TKPTS cells incubated with PBS, NE, and NE+BRL (10 μM) with densitometry analysis (n=4–6 in each group). (H) Representative Western blotting of NF-κB p65 in TKPTS cells of PBS, NE, and…