Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: Effects of HG and C3a on podocyte damage and mitophagy. Normal glucose refers to 5.5 mM glucose, while the mannitol high osmotic control group was subjected to 24.5 mM mannitol + 5.5 mM glucose, and HG represents the intervention group (30 mM glucose). (A) Representative images (left) and quantification (right) of podocyte cytoskeleton, with F-actin (green) stained using phalloidin (n=6). Scale bar, 20 μ m. (B) Protein levels of synaptopodin and podocin in podocytes (n=6). Corresponding histograms are shown on the bottom panel of representative protein bands. (C) ELISA detection of C3a levels in podocytes at different time points (n=6). (D) Protein levels of C3 and C3aR in podocytes (n=6). Corresponding histograms are shown on the right panel of representative protein bands. (E) Protein levels of LC3B I/II, parkin and PINK1 in podocytes (n=8). Corresponding histograms are shown on the bottom panel of representative protein bands. (F) Protein levels of parkin and PINK1 in podocytes (n=5). Corresponding histograms are shown on the right panel of representative protein bands. (G) Representative images and quantification (bottom) of podocyte cytoskeleton induced by different times and concentrations of C3a (10 −7 M for 12, 24 and 48 h; or 10 −8 , 10 −7 and 10 −6 M for 24 h) (n=6). Scale bar, 20 μ m. * P<0.05, ** P<0.01 and *** P<0.001. ns, no statistically significant difference; HG, high glucose.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: C3a downregulates mitophagy levels through the PI3K/AKT/FoxO1 signaling pathway in a HG environment, leading to podocyte damage. (A) Immunofluorescence images of F-actin-stained podocyte cytoskeleton. Scale bar, 20 μ m. Flow cytometric analysis of podocyte apoptosis under different intervention conditions using annexin V-FITC PI. (B) Expression of podocyte functional and mitophagy-related proteins after PINK1 inhibition (n=5). Corresponding histograms are shown on the right panel of representative protein bands. (C) Expression of downstream signaling molecules after direct stimulation of podocytes by C3a (n=6). (D) Expression of downstream signaling molecules after inhibiting C3aR in a HG environment (n=5). Corresponding histograms are shown on the bottom panel of the representative protein bands. (E) Confocal microscopy images capturing fluorescence of immortalized human podocytes transfected with adenovirus GFP-LC3B (green) and Mito-DsRed (red). Scale bar, 20 μ m. The right panel represents the statistical analysis of the number of GFP-LC3B-positive spots per cell and the proportion of LC3B spots on mitochondria (Mito) to total LC3B. Quantification of GFP-LC3B-associated Mito-DsRed staining intensity normalized by GFP-LC3B area (n=5). (F) Effects of PI3K inhibition on downstream pathway proteins and mitophagy proteins under HG conditions with C3a overexpression (n=5). Corresponding histograms are shown on the bottom panel of representative protein bands. (G) Immunofluorescence micrographs demonstrating the nuclear/cytoplasmic distribution of FoxO1 in podocytes, with dual-color staining of phosphoryalted-FoxO1 (red) and FoxO1 (green). Scale bar, 20 μ m. * P<0.05, ** P<0.01 and *** P<0.001. ns, no statistically significant difference; HG, high-glucose.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Immunofluorescence, Staining, Expressing, Functional Assay, Inhibition, Confocal Microscopy, Fluorescence, Transfection, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: Role and mechanism of C3a/C3aR in a DN model. In a high-glucose environment, the complement component C3 is activated. The C3a/C3aR axis modulates the PI3K-AKT signaling pathway, resulting in an enhanced phosphorylation level of FoxO1, leading to the loss of its transcriptional activity. Consequently, there is inhibition of PINK1/parkin-mediated mitophagy, contributing to podocyte injury and DN progression. DN, diabetic nephropathy; C3aRA, C3aR antagonist.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Phospho-proteomics, Activity Assay, Inhibition