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Article Open Access

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

  • Authors:
    • Mengjie Weng
    • Xiaoting Wu
    • Siyi Rao
    • Kun Nie
    • Danyu You
    • Tingting Zheng
    • Enqin Lin
    • Jing Zheng
    • Jiong Cui
    • Jianxin Wan
  • View Affiliations / Copyright

    Affiliations: Department of Nephrology, Blood Purification Research Center, The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350005, P.R. China
    Copyright: © Weng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 223
    |
    Published online on: October 14, 2025
       https://doi.org/10.3892/ijmm.2025.5664
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Abstract

The C3a/C3aR axis has been confirmed to be associated with the pathogenesis of diabetic nephropathy (DN) and mitochondrial dysfunction; however, the exact mechanisms underlying its role in diabetic podocytopathy remain unclear. The present study investigated the involvement of C3a/C3aR signaling in regulating mitophagy during the progression of DN. Diabetic db/db mice exhibited elevated renal C3 and C3aR levels, concurrent with podocyte injury, proteinuria and glomerular damage. Administration of the C3aR antagonist (C3aRA) SB290157 attenuated podocyte loss, reduced albuminuria and mitigated glomerular pathology. Ultrastructural and functional analyses revealed that C3aRA restored mitochondrial integrity in podocytes, resolving diabetes‑associated fragmentation and bioenergetic deficits. In vitro, high glucose‑exposed human podocytes displayed suppressed mitophagy and mitochondrial dysfunction, which were exacerbated by exogenous C3a. Conversely, C3aRA treatment enhanced mitophagy and preserved mitochondrial membrane potential, while small interfering RNA‑mediated C3aR or PINK1 knockdown abolished these protective effects. Notably, C3aRA activated the PI3K/AKT/FoxO1 pathway, driving both mitochondrial biogenesis and mitophagy. Disruption of this axis via FoxO1 inhibition reversed the therapeutic benefits of C3aRA, confirming its mechanistic centrality. In conclusion, the C3a/C3aR/PI3K/AKT/FoxO1 axis represents a previously unrecognized molecular bridge between complement activation and mitophagy failure in DN. Pharmacological disruption of this pathway could preserve podocyte homeostasis, offering a precision strategy against diabetic kidney injury.
View Figures

Figure 1

Heatmap of the C3 and
C3aR transcriptome data in different kidney-related
diseases. A set of kidney disease-related transcriptome data was
collected from the Gene Expression Omnibus database using
Affymetrix microarray sequencing technology. This dataset
(GSE47183) contained 122 human renal podocyte samples (including 14
samples of diabetic nephropathy, 23 samples of focal and segmental
glomerulosclerosis, 6 samples of focal and segmental
glomerulosclerosis and MCD, 21 samples of membranous
glomerulonephritis, 15 samples of MCD, 23 samples of rapidly
progressive glomerulonephritis, 3 samples of thin membrane disease
and 17 samples of tumor nephrectomy). Red indicates high
expression, while blue indicates low expression. MCD, minimal
change disease.

Figure 2

Expression of complement C3,
C3aR and renal injury in each mouse group. (A) Protein
levels of C3 and C3aR in kidney tissues (n=6).
Corresponding histograms of the representative protein bands are
shown on the bottom panel. (B) Immunohistochemical detection of the
deposition and expression of complement C3 and C3aR
in kidney tissue specimens of each mouse group. Microscopic images
are shown at x100 and x400 magnification. (C) Hematoxylin and
eosin, and Periodic Acid-Schiff staining of kidney tissues in each
group of mice. Microscopic images are shown at x100 magnification.
(D) Levels of blood creatinine, 24 h-urinary total protein and
urine albumin/creatinine ratio in each group of mice (n=6). (E)
Transmission electron microscopy observation of the ultrastructure
of glomerular podocytes. Scale bar, 2.0 μm. (F) TdT-mediated
dUTP nick-end labeling assay detection of apoptotic cells in mouse
kidney tissues for each group. Immunofluorescence images are shown,
and statistical analysis of the percentage of apoptotic cells for
each group is shown on the right side of the representative image
(n=6). Scale bar, 20 μm. db/db mice were used to
establish a diabetic nephropathy model. SB290157 is a C3aR
antagonist. Data are expressed as the mean ± SEM and analyzed by
two-way ANOVA with Bonferroni correction. *P<0.05,
**P<0.01 and ***P<0.001. C3aRA, C3aR
antagonist; ns, no statistically significant difference.

Figure 3

Function of glomerular podocytes and
mitochondrial damage in each group of mice. (A) Immunofluorescence
images of the podocyte-specific functional proteins
synaptopodin and nephrin in each group of mice. Scale
bar, 20 μm. (B) Protein levels of ZO-1 and podocin in
the kidney tissues (n=6). Corresponding histograms are shown on the
bottom panel of representative protein bands. (C)
Immunohistochemical analysis of the mitochondrial fusion protein
Mnf2. Scale bar, 50 μm. (D) Representative images and
quantification of double immunofluorescence staining for
TOMM20 (red) and nephrin (green) in the glomerulus of
mice. The yellow areas indicate TOMM20 and nephrin
colocalization in podocytes (n=6). Scale bar, 20 μm. (E)
Electron microscopy image of mitochondrial morphology in podocytes.
Scale bar, 2.0 μm and 500 nm. Data are expressed as the mean
± SEM and analyzed by two-way ANOVA with Bonferroni correction.
*P<0.05, **P<0.01 and
***P<0.001. C3aRA, C3aR antagonist.

Figure 4

Expression levels of mitophagy and
related pathway proteins in mouse renal tissues. (A)
Immunohistochemical analysis of the mitophagy-specific protein
PINK1. Magnification, x100. (B) Protein levels of LC3B I/II, parkin
and PINK1 in kidney tissues (n=6). Corresponding histograms are
shown on the right panel of representative protein bands. (C)
Protein levels of PI3K [PI3-kinase p85α (54 + 85 kDa)],
phosphorylated-AKT and FoxO1 in kidney tissues (n=6). Corresponding
histograms are shown on the right panel of representative protein
bands. *P<0.05, **P<0.01 and
***P<0.001. p-, phosphorylated.

Figure 5

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 μm.
(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 μm.
*P<0.05, **P<0.01 and
***P<0.001. ns, no statistically significant
difference; HG, high glucose.

Figure 6

Inhibition of C3aR improves
HG-induced podocyte and mitochondrial autophagic damage. (A)
Changes in cell cytoskeleton after blocking C3aR in a HG
environment. Scale bar, 20 μm. (B) Immunofluorescence images
of podocytes in each group using a TdT-mediated dUTP nick-end
labeling assay. Scale bar, 50 μm. The lower panel presents
the statistical analysis of apoptosis ratios in podocytes for each
group (n=6). (C) Transmission electron microscopy examination of
mitochondrial morphology. Scale bar, 2.0 μm and 500 nm. The
statistical charts on the right side of the electron microscopy
images represent the percentages of damaged mitochondria and
mitochondria enveloped by autophagosomes (n=5). (D) Transfection of
immortalized human podocytes with adenovirus GFP-LC3B (green) and
DsRed-Mito (red). Scale bar, 20 μm. The statistical charts
on the right side represent the number of GFP-LC3B-positive puncta
per cell and the proportion of LC3B spots on mitochondria (Mito) to
total LC3B. Quantification of GFP-LC3B (green)-associated
Mito-DsRed (red) staining intensity normalized by GFP-LC3B area
(n=5). (E) Protein levels of LC3B I/II, parkin and PINK1 in
podocytes (n=6). Corresponding histograms are shown on the right
panel of representative protein bands. *P<0.05,
**P<0.01 and ***P<0.001. ns, no
statistically significant difference; HG, high glucose; NG, normal
glucose; siRNA, small interfering RNA; NC, negative control.

Figure 7

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 μm.
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 μm. 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 μm. *P<0.05,
**P<0.01 and ***P<0.001. ns, no
statistically significant difference; HG, high-glucose.

Figure 8

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.
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Copy and paste a formatted citation
Spandidos Publications style
Weng M, Wu X, Rao S, Nie K, You D, Zheng T, Lin E, Zheng J, Cui J, Wan J, Wan J, et al: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy. Int J Mol Med 56: 223, 2025.
APA
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T. ... Wan, J. (2025). C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy. International Journal of Molecular Medicine, 56, 223. https://doi.org/10.3892/ijmm.2025.5664
MLA
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T., Lin, E., Zheng, J., Cui, J., Wan, J."C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy". International Journal of Molecular Medicine 56.6 (2025): 223.
Chicago
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T., Lin, E., Zheng, J., Cui, J., Wan, J."C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy". International Journal of Molecular Medicine 56, no. 6 (2025): 223. https://doi.org/10.3892/ijmm.2025.5664
Copy and paste a formatted citation
x
Spandidos Publications style
Weng M, Wu X, Rao S, Nie K, You D, Zheng T, Lin E, Zheng J, Cui J, Wan J, Wan J, et al: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy. Int J Mol Med 56: 223, 2025.
APA
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T. ... Wan, J. (2025). C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy. International Journal of Molecular Medicine, 56, 223. https://doi.org/10.3892/ijmm.2025.5664
MLA
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T., Lin, E., Zheng, J., Cui, J., Wan, J."C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy". International Journal of Molecular Medicine 56.6 (2025): 223.
Chicago
Weng, M., Wu, X., Rao, S., Nie, K., You, D., Zheng, T., Lin, E., Zheng, J., Cui, J., Wan, J."C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy". International Journal of Molecular Medicine 56, no. 6 (2025): 223. https://doi.org/10.3892/ijmm.2025.5664
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