Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 1 Imbalanced mitochondrial homeostasis is associated with renal proximal tubular injury in DKD. A DHE staining was performed to evaluate the production of ROS in the kidneys of the mice. B Representative images of SOD2 staining of kidneys from mice. C and D Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fractions of the mice (C) and HK-2 cells (D) as indicated. E and F H2DCFDA (E) and Mitosox (F) staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. G Repre sentative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. Scale bar = 100 μm in A and B for fluorescence images, 50 μm for images in E and F, and 20 μm for confocal images in G. For all statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Staining, Fluorescence

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 2 CaMKKβ-mediated protective effect on tubular injury in DKD. A IHC staining was performed to evaluate the protein expression of CaMKKβ in kid neys of the mice. Scale bar = 200 μm. B and C Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in the groups of mice (B) and HK-2 cells (C) cultured with HG as indicated. D IF staining of CaMKKβ in HK-2 cells cultured with HG for 48 h. Scale bar = 20 μm. E Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells transfected with CaMKKβ as indicated. F IF staining was performed to evaluate the uptake of BSA-TRITC in HK-2 cells. Scale bar = 50 μm. G H2DCFDA and Mitosox staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. Scale bar = 20 μm. H Representative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. I Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells transfected with CaMKKβ as indicated. Scale bar = 20 μm. OE-NC represents the control of CaMKKβ. OE-CaMKKβ represents the overexpression of CaMKKβ. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Immunohistochemistry, Expressing, Cell Culture, Staining, Transfection, Control, Over Expression

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 5 Inhibition of NEDD4L ameliorates oxidative stress and mitochondrial dysfunction in diabetic mice. A Representative images of HE and PAS stain ing of kidney sections. B Representative images of Masson staining of kidney sections. C Representative images of DHE-stained kidney sections showing oxidative stress in the mice. A-C: Scale bar = 200 μm. D Representative immunoreactive bands for Bax, Bcl-2, and cleaved Caspase-3 in the groups of mice. E Representative immunoreactive bands for CaMKKβ, phosphorylated AMPK, and total AMPK in the groups of mice. F Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in different groups of mice, as indicated. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Inhibition, Staining

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 6 Silencing NEDD4L prevents HG-induced mitochondrial dysfunction in HK-2 cells. A IF staining was performed to evaluate the uptake of BSA-TRITC in the experiments. B H2DCFDA and Mitosox staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. Scale bar = 50 μm. C Representative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. Scale bar = 20 μm. D Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells transfected with Nedd4L-siRNA as indicated. E Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells transfected with Nedd4L-siRNA as indicated. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Staining, Transfection

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 7 Knockdown of NEDD4L contributes to HK-2 cell function via CaMKKβ/AMPK pathway. A Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells cotransfected with Nedd4L-siRNA and CaMKKβ-siRNA as indicated. B Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells, as indicated. C MitoTracker Red staining showing mitochondrial morphology in the indicated groups of cells. Scale bar = 20 μm. D IF staining was performed to evaluate the uptake of BSA-TRITC in the indicated groups of cells. Scale bar = 50 μm. E and F H2DCFDA (E) and Mitosox (F) staining were performed to evaluate the generation of ROS in the cytoplasm and mito chondria of HK-2 cells, respectively. Scale bar = 50 μm. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Knockdown, Cell Function Assay, Staining

Journal: Cardiovascular Diabetology

Article Title: Mitochondrial ultrastructural pathology in diabetic cardiomyopathy: integrated analysis via scanning electron microscopy and 3D visualization imaging

doi: 10.1186/s12933-025-02884-5

Figure Lengend Snippet: Evaluation of mitochondrial functional indicators and protein expression in the control and DCM group Note: (A) Mitochondrial membrane potential (ΔΨm) analysis in myocardial tissue by flow cytometry. A1 : Control group; A2 : DCM group; A3 : Quantitative analysis of cells with depolarized ΔΨm, n = 3 per group. (B) Activities of respiratory chain complexes I, II, III, IV, and V in control and DCM model groups in myocardial tissue. (C) ATP content in myocardial tissue. (D) Mitochondrial dynamics and mitophagy-related protein expression in myocardial tissue. D1 : Representative Western blot bands of Mfn1, Mfn2, Opa1, p-Drp1 Ser616 , Drp1, PINK1, and Parkin. D2 : Quantitative analysis of protein expression levels, n = 3 per group. (E) Relative mtDNA copy number in myocardial tissue, n = 3 per group. (F) SOD activity as an oxidative stress indicator, n = 3 per group. (G) Mitochondrial Ca 2+ levels detected by Rhod-2 fluorescence in myocardial tissue. G1 : Control group; G2 : DCM group; G3 : Statistical analysis of Rhod-2 ratio (%), n = 3 per group. (H) Mitochondrial respiration assessed by OCR kinetics in H9c2 cardiomyocytes. H1 : Real-time OCR tracing in high glucose-stimulated H9c2 cells. Arrows indicate sequential injections of oligomycin (ATP synthase inhibitor), FCCP (mitochondrial uncoupler), and antimycin A/rotenone (complex III/I inhibitors). H2 : Quantification of the respiratory parameters derived from (H1), including basal respiration, ATP-linked respiration, proton leakage, maximal respiration, and spare respiratory capacity, calculated as described in the Methods section, n = 3 per group. Data are presented as mean ± standard deviation. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The key findings are as follows: (1) 3D ultrastructural remodeling: Mitochondria in DCM exhibit pronounced fragmentation and cristae disruption, with 3D visualization techniques enabling the identification of megamitochondria—a pathological phenomenon not detectable by conventional 2D electron microscopy. (2) Molecular dynamics imbalance: Coordinated downregulation of mitochondrial fusion proteins (Mfn1, OPA1) and aberrant activation of the fission protein p-Drp1 ser616 drive ultrastructural remodeling. (3) Bioenergetic failure: In DCM, myocardial tissue enters a vicious cycle marked by mitochondrial membrane potential dissipation, impaired activity of respiratory chain complexes (I, III, IV, and V), and deficits in ATP synthesis.

Techniques: Functional Assay, Expressing, Control, Membrane, Flow Cytometry, Western Blot, Activity Assay, Fluorescence, Derivative Assay, Standard Deviation