Journal: Aging Cell

Article Title: Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

doi: 10.1111/acel.13004

Figure Lengend Snippet: DKK1 and mitoQ preserve mitochondrial functions and inhibit cellular senescence in an established aging model. (a) Experimental design. Red arrows indicate the starting point of d ‐gal injection. Green arrows indicate the injections of pFlag‐DKK1 plasmid. Green bar indicates treatment with mitoQ. (b–e) Representative (b) Western blots and graphical representations of (c) DKK1, (d) active β‐catenin, and (e) TOMM20 are presented. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6). (f) Representative MitoTracker and mitoSOX staining micrographs show the mitochondrial mass loss and ROS production induced by d ‐gal were blocked by DKK1 or mitoQ. Arrows indicate positive staining. Scale bar, 50 µm. (g) Representative staining micrographs show renal expression of SA‐β‐gal activity, γH2AX, fibronectin, PDGFRβ, and Sirius red staining. Frozen kidney sections were stained for SA‐β‐gal activity, fibronectin, and PDGFRβ. Paraffin sections were immunostained with an antibody against γH2AX or performed with Sirius red staining. Arrows indicate positive staining. Scale bar, 50 μm. (h–k) Representative (h) Western blots and graphical representations of (i) γH2AX, (j) fibronectin, and (k) α‐SMA protein expression levels in kidneys are presented. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6). (l) Quantitative determination of renal fibrotic lesions in different groups. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6)

Article Snippet: Antibodies used were as follows: Klotho (AF1819; R&D Systems), Wnt1 (ab15251; Abcam), AT1 (AB15552; Merck Millipore), ACE (ab75762; Abcam), AGT (SAB2100072; Sigma‐Aldrich), p16 INK4A (ab189034; Abcam), γ‐H2AX (ab2893; Abcam), PGC‐1α (ab54481; Abcam), and DKK1 (BM4554; Boster).

Techniques: Injection, Plasmid Preparation, Western Blot, Control, Mouse Assay, Staining, Expressing, Activity Assay

Journal: Aging Cell

Article Title: Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

doi: 10.1111/acel.13004

Figure Lengend Snippet: Wnt/β‐catenin mediates age‐related renal fibrosis in vitro. (a) Real‐time PCR results show d ‐gal induced upregulation of multiple Wnt genes in cultured proximal tubular cell line (HKC‐8). HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr. Total RNA was extracted and analyzed for various Wnt mRNA expression levels. * p < .05 versus control group. Ctrl, control. (b) Representative immunofluorescence micrographs show d ‐gal induced nuclear translocation of β‐catenin. HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr and then stained for β‐catenin (red) and DAPI (blue). Arrows indicate nuclear staining of β‐catenin. Scale bar, 20 μm. (c) Representative electron microscopy and BrdU incorporation assay micrographs show mitochondria and cell proliferation in renal tubular cells. HKC‐8 cells were pretreated with ICG‐001 (5 μm) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. BrdU (10 μM) was added for 12 hr before collection. For TEM analyses, arrows indicate healthy mitochondria, and arrowheads indicate abnormal‐shaped mitochondria. Scale bar, 2 μm; for BrdU incorporation assay, arrows indicate BrdU incorporation positive cells. Scale bar, 75 μm. TEM, transmission electron microscopy. (d–f) Representative (d) Western blots and graphical representations of (e) phospho‐PGC‐1α and (f) fibronectin are presented. HKC‐8 cells were pretreated with ICG‐001 (5 μm) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (g–k) Representative (g) Western blots and graphical representations of (h) phospho‐PGC‐1α, (i) TFAM, (j) p16 INK4A , and (k) fibronectin are presented. HKC‐8 cells were pretreated with losartan (10 μM) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (l) Representative fluorescence micrographs show MitoTracker, BrdU, and fibronectin staining. After pretreatment with losartan (10 μM) or mitoQ (100 nM) for 1 hr, HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr. BrdU (10 μM) was added for 12 hr before collection. Cells were stained with MitoTracker deep red probe (300 nM) or antibodies against BrdU and fibronectin. Arrowhead indicates the loss of mitochondrial mass and the increase in fragmentation of cristae. Arrows indicate positive staining for BrdU‐positive cells and fibronectin. Scale bar, 10 μm for images of MitoTracker staining. (m–o) Representative (m) Western blots and graphical representations of (n) p16 INK4A and γH2AX, and (o) fibronectin and α‐SMA are presented. HKC‐8 cells were pretreated with mitoQ (100 nM) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (p) Schematic presentation depicts the potential mechanism by which Wnt/β‐catenin induces age‐related renal fibrosis. Wnt/β‐catenin signaling triggers the activation of RAS, thereby leading to the injury of mitochondrial biogenesis. This causes mitochondrial dysfunction with a loss of mass and increase in fragmentation and ROS production, which in turn induces tubular cell senescence and age‐related renal fibrosis. The mitochondrial dysfunction and activation of Wnt/β‐catenin signaling reciprocally induce each other. Multiple approaches, such as the inhibition of Wnt/β‐catenin by DKK1, ICG‐001, or Klotho, the blockade of RAS by losartan, the protection of mitochondrial biogenesis by resveratrol, and the mitochondria‐targeted antioxidant mitoQ could slow age‐related renal fibrosis

Article Snippet: Antibodies used were as follows: Klotho (AF1819; R&D Systems), Wnt1 (ab15251; Abcam), AT1 (AB15552; Merck Millipore), ACE (ab75762; Abcam), AGT (SAB2100072; Sigma‐Aldrich), p16 INK4A (ab189034; Abcam), γ‐H2AX (ab2893; Abcam), PGC‐1α (ab54481; Abcam), and DKK1 (BM4554; Boster).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Control, Immunofluorescence, Translocation Assay, Staining, Electron Microscopy, BrdU Incorporation Assay, Transmission Assay, Western Blot, Fluorescence, Activation Assay, Inhibition

Journal: Natural Products and Bioprospecting

Article Title: Scutellarin ameliorates diabetic nephropathy via TGF-β1 signaling pathway

doi: 10.1007/s13659-024-00446-y

Figure Lengend Snippet: Scutellarin Restored Podocyte Injury of the DN Mice. a Representative images of immunohistochemistry for NPHS1 and NPHS2 of the mice treated with vehicle, scutellarin or empagliflozin (× 200; scale bar = 50 µm). b Representative images of Western-blotting for NPHS1, NPHS2. c Quantitative plot of the expression of NPHS1 of the mice. d Quantitatification of NPHS1 expression of the mice. e Representative images of Western-blotting for β-catenin, Axin2, snail and DKK1 of the mice. f – i Quantifications of the protein levels for β-catenin, Axin2, snail and DKK1 from E. All data are presented as the mean ± S.D.; n = 4–6 for each group, “n” stands for the number of animals; p vs. the model group (STZ)

Article Snippet: Scutellarin (Yunnan Phytopharmaceutical Co., LTD., China); Empagliflozin (Cat. C14295412, Macklin Biochemical, China), Streptozotocin (Cat. S8050, Solarbio, China); goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (Cat. AS014, Abclonal, China); anti-mouse IgG HRP-linked antibody (Cat. 7076S, Cell Signaling Technology, USA); Methenamine Silver Plating Stain Kit (Cat. G1790, Solarbio); Glycogen Periodic Acid Schiff (PAS) Stain Kit (Cat. G1281, Solarbio); Masson’s Trichrome Stain Kit (Cat. G1340, Solarbio); Mouse MAU enzyme-linked immunosorbent assay (ELISA) Kit (Cat. JL20493, JONIN, China); Anti-DKK1 antibody (Cat. A00632, Boster Biological Technology, China); Anti-SNAIl antibody (Cat. BP0449, Boster Biological Technology); Anti-α-SMA antibody (Cat. BM0002, Boster Biological Technology); Anti-TGFB1 antibody (Cat. BA0290, Boster Biological Technology); Anti-NPHS1 antibody (Cat. BA1669, Boster Biological Technology); Anti-NPHS2 antibody (Cat. BA1688, Boster Biological Technology); Anti-SMAD2/3 antibody (Cat. BA1395, Boster Biological Technology); Anti-Phospho-SMAD2(s250) antibody (Cat. BM4693, Boster Biological Technology); Anti-FN1 antibody (Cat. BA1772, Boster Biological Technology); AXIN2 antibody (Cat. Ab32197, Abcam, USA); Anti-Phospho-SMAD3(ser425) antibody (Cat. AF3362, Affinity Biosciences, China); Anti-COL3A1 antibody (Cat. M00788, Boster Biological Technology); Anti-extracellular signal-regulated kinase (ERK)1/2 antibody (Cat. ET1601-29, HUABIO, China); Anti-ERK1(PT202/PY204) + ERK2(PT185/PY187) antibody (Cat. ET1610-13, HUABIO); Anti-P38 antibody (Cat. ET1602-26, HUABIO); Anti-Phospho-P38(Thr180 + Tyr182) antibody (Cat. ER1903-01, HUABIO); Anti-β-Actin antibody (Cat. EM21002, HUABIO); Anti-SMAD4 antibody (Cat. A5657, Abclonal); Anti-β-catenin antibody (Cat. 610154, BD Biosciences, China); Creatinine (Cr) Assay kit (Cat. C011-2-1, Nanjing Jiancheng Bioengineering Institute, China); Glucose Assay Kit (Cat. F006-1-1, Nanjing Jiancheng Bioengineering Institute); microalbunminuria (MAU) ELISA kit (Cat. JL20493, JONIN).

Techniques: Immunohistochemistry, Western Blot, Expressing