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Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway

  • Authors:
    • Hanshuang Liu
    • Xiaoxiao Zhang
    • Yubing Cui
    • Shengxi Xiong
    • Linjuan Huang
    • Min Li
    • Chen Shao
    • Xiaolei Hu
  • View Affiliations / Copyright

    Affiliations: Department of Endocrinology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui 233000, P.R. China, Department of Endocrinology, The Second Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui 233000, P.R. China
    Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 171
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    Published online on: April 30, 2026
       https://doi.org/10.3892/ijmm.2026.5842
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Abstract

Renal tubular injury has emerged as a critical determinant in the pathogenesis of diabetic nephropathy (DN). Ferroptosis, a recently characterized mode of iron‑dependent regulated cell death, has been implicated in the development of renal tubular damage. Dapagliflozin (DAPA), a sodium‑glucose cotransporter 2 inhibitor, has demonstrated efficacy in attenuating DN progression and preserving renal function. The present study sought to elucidate the inhibitory mechanisms by which DAPA modulates ferroptosis in DN. To this aim, the expression profiles of key molecular markers within the ferroptosis cascade were systematically evaluated using 6‑week‑old male C57BL/6J mice and high‑glucose‑cultured human renal tubular epithelial cells as experimental models. The findings revealed that DAPA notably ameliorated renal histopathological alterations, upregulated the expression of solute carrier family 7 member 11, glutathione peroxidase 4 and ferritin heavy chain 1, whilst concomitantly downregulating transferrin receptor 1. These effects were mediated through the activation of nuclear factor erythroid 2‑related factor 2 (Nrf2) and heme oxygenase‑1 (HO‑1) in C57BL/6J mice. Collectively, these data indicate that the reno‑protective effects of DAPA in DN may be attributable to the suppression of ferroptosis via activation of the Nrf2/HO‑1 signaling axis.
View Figures

Figure 1

Expression of genes associated with
ferroptosis was assessed in HK-2 cells exposed to HG. (A) Cell
viability was measured after incubation with varying concentrations
of DAPA for 24, 48 and 72 h. Similarly, cell viability was
evaluated after treatment with different concentrations of (B)
Fer-1 or (C) erastin for 48 h. The mRNA levels of (D) GPX4, (E)
SLC7A11, (F) FTH-1 and (G) TFR-1 were determined in each cell
group. (H) Transmission electron microscopy was employed to examine
mitochondrial morphology in cells from the NC, HG and erastin
treatment groups. (I) Levels of ROS were assessed in each group,
(J) with green representing ROS and blue representing the nucleus
in the representative images. (K) MDA and (L) cellular iron content
measurements were also conducted. (M) The protein levels of GPX4,
SLC7A11, FTH-1 and TFR-1 were analyzed via immunoblotting in cells
from the NC, HG and erastin groups, (N) with semi-quantitative
analysis of the immunoblotting results shown. The bars indicate the
mean ± SD from three independent experiments.
*P<0.05, **P<0.01,
***P<0.001 compared with the normal group. ROS,
reactive oxygen species; MDA, malondialdehyde; HG, high glucose;
NC, normal control; DAPA, dapagliflozin; Fer-1, ferrostatin-1;
GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7,
member 11; FTH-1, ferritin heavy chain 1; TFR-1, transferrin
receptor 1; DCFH-DA, 2',7'-dichlorodihydrofluorescein
diacetate.

Figure 2

Inhibition of HG-induced ferroptosis
in HK-2 cells using DAPA. The mRNA expression levels of (A) GPX4,
(B) SLC7A11, (C) FTH-1 and (D) TFR-1 in each cell group were
evaluated. (E and F) reactive oxygen species expression, (G) MDA
and (H) cellular iron levels were measured in cells from each
group. (I) The protein expression levels of SLC7A11, GPX4, FTH-1
and TFR-1 in cells from the NC, HG, DAPA and Fer-1 groups were
analyzed. (J) Semi-quantitative analysis of the GPX4, SLC7A11,
FTH-1 and TFR-1 immunoblotting results in cells was performed. The
bars indicate the mean ± SD from three independent experiments.
*P<0.05, **P<0.01,
***P<0.001 compared with the NC group;
#P<0.05, ##P<0.01,
###P<0.001 compared with the HG group. MDA,
malondialdehyde; HG, high glucose; NC, normal control; Fer-1,
ferrostatin-1; DAPA, dapagliflozin; GPX4, glutathione peroxidase 4;
SLC7A11, solute carrier family 7, member 11; FTH-1, ferritin heavy
chain 1; TFR-1, transferrin receptor 1.

Figure 3

DAPA activates the Nrf2/HO-1
signaling pathway in HG-induced HK-2 cells. (A) The Nrf2 and HO-1
western blotting results in each cell group. (B) Semi-quantitative
analysis of the Nrf2 and HO-1 western blotting results in each cell
group. The mRNA expression levels of (C) Nrf2 and (D) HO-1 in each
cell group were evaluated. The mRNA expression levels of (E) Nrf2,
(F) HO-1, (G) GPX4, (H) SLC7A11, (I) FTH-1 and (J) TFR-1 in cells
from each group are shown. The (K) MDA and (L) iron content in
cells from each group are presented. (M and N) Representative
images of reactive oxygen species production in each group, as
detected by DCFH-DA staining, are shown. (O) The expression levels
of Nrf2, HO-1, GPX4, SLC7A11, FTH-1 and TFR-1 in cells from each
group were detected by immunoblotting. (P) Semi-quantitative
analysis of protein blot results for Nrf2, HO-1, GPX4, SLC7A11,
FTH-1 and TFR-1 in each group is shown. The bars indicate the mean
± SD from three independent experiments. *P<0.05,
**P<0.01, ***P<0.001 compared with the
HG group; #P<0.05, ##P<0.01,
###P<0.001 compared with the si-Nrf2 group. DAPA,
dapagliflozin; HG, high glucose; NC, normal control; si, small
interfering; Nrf2, nuclear factor erythroid2-related factor 2;
HO-1, heme oxygenase-1; GPX4, glutathione peroxidase 4; SLC7A11,
solute carrier family 7, member 11; FTH-1, ferritin heavy chain 1;
TFR-1, transferrin receptor 1; MDA, malondialdehyde; DCFH-DA,
2',7'-dichlorodihydrofluorescein diacetate.

Figure 4

Diabetic nephropathy mouse model. (A)
Modeling process of mice with diabetic nephropathy mice. (B) Random
blood glucose levels of mice were monitored at weeks 5, 7, 9, 11,
13, 15, 17, 19, 21 and 23. (C) Water intake, (D) food intake, (E)
urine output and (F) body weight were measured in mice at weeks 5,
7, 9, 11, 13 and 15. Mice were assayed for (G) 24-h urine albumin
level, (H) urine creatinine and (I) ACR at 5 and 15 weeks. (J) Mice
were tested for blood creatinine at 23 weeks. The bars indicate the
mean ± SD. ***P<0.001 compared with the NC group;
ΔΔP<0.01 compared with the DN group;
#P<0.05 compared with the si-Nrf2 group. NC, normal
control; si-NC, si-negative control; DN, diabetic nephropathy; HFD,
high-fat diet; STZ, streptozotocin; ip, Intraperitoneal Injection;
DAPA, dapagliflozin; si, small interfering; Nrf2, nuclear factor
erythroid2-related factor 2; ACR, the urinary albumin-to-creatinine
ratio.

Figure 5

DAPA mitigates ferroptosis in DN mice
through activation of the Nrf2/HO-1 pathway. mRNA expression levels
of (A) Nrf2, (B) HO-1, (C) GPX4, (D) SLC7A11, (E) FTH-1 and (F)
TFR-1 in renal tissues from mice. (G) MDA and (H) iron content
measurements in kidney tissue lysates. (I) Western blotting results
and (J) semi-quantitative analysis of Nrf2, HO-1, SLC7A11, GPX4,
FTH-1 and TFR-1 in mouse kidney tissues. The bars indicate the mean
± SD from three independent experiments. *P<0.05,
**P<0.01, ***P<0.001 compared with the
DN group; ΔΔP<0.01 compared with the si-NC group;
#P<0.05, ##P<0.01,
###P<0.001 compared with the si-Nrf2 group. NC,
normal control; si-NC, si-negative control; DN, diabetic
nephropathy; DAPA, dapagliflozin; si, small interfering; Nrf2,
nuclear factor erythroid2-related factor 2; HO-1, heme oxygenase-1;
GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7,
member 11; FTH-1, ferritin heavy chain 1; TFR-1, transferrin
receptor 1; MDA, malondialdehyde.

Figure 6

HE, Masson and PAS staining as well
as immunohistochemical fluorescence staining. (A) Compared with the
DN and si-Nrf2 groups, HE staining showed that the DAPA and si-Nrf2
DAPA groups did not exhibit the pathological alterations
characterized by glomerular and tubular epithelial damage. PAS
staining demonstrated a reduction in the thickening of glomerular
and tubular basement membranes in the DAPA and si-Nrf2 + DAPA
groups, while Masson staining indicated a decrease in collagen
fiber deposition in the glomeruli and renal interstitium in the
DAPA and si-Nrf2 DAPA groups. (B) Immunohistochemical analysis
revealed that, compared with the NC and si-NC groups, the DN and
si-Nrf2 groups exhibited reduced Nrf2, HO-1, SLC7A11, GPX4 and
FTH-1 protein levels, along with elevated TFR-1 expression in renal
tissues. However, treatment with DAPA led to increased expression
of Nrf2, HO-1, SLC7A11, GPX4 and FTH-1, and decreased expression of
TFR-1. (C) Semi-quantitative analysis of HE and Masson staining.
(D) Semi-quantitative analysis of the Nrf2, HO-1, SLC7A11, GPX4,
FTH-1 and TFR-1 immunohistochemical staining. The bars indicate the
mean ± SD from three independent samples. *P<0.05,
**P<0.01, ***P<0.001 compared with the
DN group; #P<0.05, ##P<0.01,
###P<0.001 compared with the si-Nrf2 group. DN,
diabetic nephropathy; NC, normal control; si, small interfering;
Nrf2, nuclear factor erythroid2-related factor 2; HE, hematoxylin
and Eosin; DAPA, dapagliflozin; HO-1, heme oxygenase-1; SLC7A11,
solute carrier family 7, member 11; GPX4, glutathione peroxidase 4;
FTH-1, ferritin heavy chain 1; TFR-1, transferrin receptor 1.

Figure 7

Schematic representation of the
mechanism underlying the amelioration of ferroptosis by DAPA. DAPA,
dapagliflozin; Nrf2, nuclear factor erythroid2-related factor 2;
HO-1, heme oxygenase-1; FTH-1, ferritin heavy chain 1; TRF-1,
transferrin receptor 1; SLC7A11, solute carrier family 7, member
11; GPX4, glutathione peroxidase 4; ROS, reactive oxygen
species.
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Copy and paste a formatted citation
Spandidos Publications style
Liu H, Zhang X, Cui Y, Xiong S, Huang L, Li M, Shao C and Hu X: Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway. Int J Mol Med 58: 171, 2026.
APA
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M. ... Hu, X. (2026). Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway. International Journal of Molecular Medicine, 58, 171. https://doi.org/10.3892/ijmm.2026.5842
MLA
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M., Shao, C., Hu, X."Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway". International Journal of Molecular Medicine 58.1 (2026): 171.
Chicago
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M., Shao, C., Hu, X."Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway". International Journal of Molecular Medicine 58, no. 1 (2026): 171. https://doi.org/10.3892/ijmm.2026.5842
Copy and paste a formatted citation
x
Spandidos Publications style
Liu H, Zhang X, Cui Y, Xiong S, Huang L, Li M, Shao C and Hu X: Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway. Int J Mol Med 58: 171, 2026.
APA
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M. ... Hu, X. (2026). Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway. International Journal of Molecular Medicine, 58, 171. https://doi.org/10.3892/ijmm.2026.5842
MLA
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M., Shao, C., Hu, X."Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway". International Journal of Molecular Medicine 58.1 (2026): 171.
Chicago
Liu, H., Zhang, X., Cui, Y., Xiong, S., Huang, L., Li, M., Shao, C., Hu, X."Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway". International Journal of Molecular Medicine 58, no. 1 (2026): 171. https://doi.org/10.3892/ijmm.2026.5842
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