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Article Open Access

Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy

  • Authors:
    • Yuting Zhang
    • Xiuli Wang
    • Qinyue Xie
    • Yue Huang
    • Dongjia Huang
    • Ziqing Liu
    • Tong Xu
    • Man Ni
    • Hongwei Yang
  • View Affiliations / Copyright

    Affiliations: Department of Ophthalmology, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China, Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China, Department of Biochemistry, University of Illinois Urbana‑Champaign, Urbana, IL 61801, USA, Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 103
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    Published online on: February 25, 2026
       https://doi.org/10.3892/ijmm.2026.5774
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Abstract

Diabetic retinopathy (DR) is a common microvascular complication that leads to vision loss in patients with diabetes. The SLC12A2/SLC12A4 inhibitor, bumetanide, has been reported to alleviate hypoxia‑induced retinopathy. It was hypothesized that it may exert the same effect in DR. DR cell types and SLC12A2/SLC12A4 expression at the cell level were analyzed using single cell RNA‑sequencing (scRNA‑seq) data. Next, cell [high glucose (HG) stimulation] and animal (mice injected with streptozotocin) DR models were constructed. The protective effects and possible mechanisms of bumetanide and SLC12A2 were investigated through a series of experiments, including Cell Counting Kit‑8, TUNEL, Transwell, tube formation, ELISA, immunofluorescence staining, western blot and reverse transcription‑quantitative PCR assays. Bumetanide reduced HG‑induced cell apoptosis by suppressing the expression of SLC12A2 and SLC12A4. Second, scRNA‑seq analysis revealed that SLC12A2 was predominantly expressed in endothelial cells, which are the main targets of hyperglycemic damage. Endothelial cell‑related markers were involved in angiogenesis and adhesion molecule‑related pathways. Third, in HG‑stimulated cells, SLC12A2 knockdown efficiently reduced the inflammatory response and angiogenesis, while maintaining endothelial barrier integrity. This protective process involved reduced release of inflammatory factors (IL‑1β and IL‑6) and growth factors (vascular endothelial growth factor), suppression of adhesion molecule expression (VCAM1, ICAM1, E‑Selectin and P‑Selectin), activation of tight junction protein (ZO‑1), and decreased matrix metalloproteinases (MMP2 and MMP9). Furthermore, SLC12A2 deficiency ameliorated DR progression in streptozotocin‑induced diabetic mice by improving retinal thickness and pathological changes. The present study elucidates the crucial role of bumetanide in DR treatment and suggests that targeting SLC12A2 may represent a novel therapeutic strategy for the prevention of DR.

View Figures

Figure 1

Design flow chart of the present
study. DR, diabetic retinopathy.

Figure 2

Bumetanide alters SLC12A2/SLC12A4
expression levels and apoptosis in HG-treated rat retinal
microvascular endothelial cells. (A) Expression levels of SLC12A2
and SLC12A4 detected by reverse transcription-quantitative PCR. (B)
Cell apoptosis in different groups assessed by TUNEL assay. (C)
Expression levels of apoptosis-related proteins measured by western
blot. Data from three independent experiments are indicated as the
mean ± SD (n=3). *P<0.05 and **P<0.01.
HG, high glucose; TUNEL, terminal deoxynucleotidyl transferase dUTP
nick-end labeling.

Figure 3

Results of single-cell RNA sequencing
analysis. (A) t-SNE plot of samples from different datasets after
dimensionality reduction. (B) t-SNE plot of 38,744 cells isolated
from the samples, showing the 18 identified clusters. (C) t-SNE
plot displaying the final annotation of the six known cell types in
the samples. (D) UMAP plot and (E) global expression levels of
SLC12A2 and SLC12A4. (F) Gene Ontology terms and (G) Kyoto
Encyclopedia of Genes and Genomes pathway analyses of the top 200
endothelial cell markers. t-SNE, t-distributed Stochastic Neighbor
Embedding; UMAP, uniform manifold approximation and projection.

Figure 4

Effect of SLC12A2 knockdown on cell
migration and angiogenesis in HG-treated RRMECs. (A and B) RRMECs
were transfected with sh-NC or sh-SLC12A2; the SLC12A2 expression
levels were detected by (A) reverse transcription-quantitative PCR
and (B) western blot assays. (C) Cell viability of RRMECs in
different treatments was measured by Cell Counting Kit-8 at 24 h.
(D and E) Migration ability of RRMECs in different treatments was
assessed by Transwell and its quantitative results. (F and G) Tube
formation ability of RRMECs in different treatments and
quantification of mesh number. Data from three independent
experiments are indicated as the mean ± SD (n=3).
**P<0.01. HG, high glucose; RRMECs, rat retinal
microvascular endothelial cells; sh-, short hairpin; NC, negative
control.

Figure 5

Effect of SLC12A2 knockdown on
inflammatory factor release and blood-retinal barrier in HG-treated
rat retinal microvascular endothelial cells. (A) Levels of VEGF,
IL-1β, IL-6 and TNF-α detected by ELISA. (B) Immunofluorescence
staining showing the ZO-1 expression in each group. (C) Levels of
MMP2 and MMP9 detected by commercial kits. Data from three
independent experiments are indicated as the mean ± SD (n=3).
*P<0.05 and **P<0.01. HG, high glucose;
VEGF, vascular endothelial growth factor; IL-1β, interleukin-1Beta;
IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; ZO-1,
zonula occludens-1; MMP, matrix metalloproteinase; n.s., not
significant.

Figure 6

Effect of SLC12A2 knockdown on
adhesion molecules in HG-treated rat retinal microvascular
endothelial cells. (A) Reverse transcription-quantitative PCR and
(B) western blot assays for expression levels of adhesion
molecules, including VCAM1, ICAM1, PECAM-1, VE-cadherin, E-Selectin
and P-Selectin. Data from three independent experiments are
indicated as the mean ± SD (n=3). *P<0.05 and
**P<0.01. HG, high glucose; VCAM1, vascular cell
adhesion molecule-1; ICAM1, intercellular adhesion molecule-1;
PECAM-1, platelet endothelial cell adhesion molecule-1; VE,
vascular endothelial; sh-, short hairpin; NC, negative control.

Figure 7

SLC12A2 deficiency ameliorates
hyperglycemic and inflammatory effects in streptozotocin-induced
diabetic mice. (A) Body weight and (B) blood glucose levels of mice
at the end of the experiment. (C) Serum lipid and HbA1c levels of
all mice. (D) Levels of inflammatory factors (VEGF, IL-1β, IL-6 and
TNF-α) detected by commercial ELISA kits. Results are expressed as
the mean ± SD (n=10/group). *P<0.05 and
**P<0.01 vs. the WT group; #P<0.05 and
##P<0.01 vs. the DR group. HbA1c, glycated
hemoglobin; VEGF, vascular endothelial growth factor; IL-1β,
interleukin-1Beta; IL-6, interleukin-6; TNF-α, tumor necrosis
factor-alpha; WT, wild-type; TG, triglyceride; TC, total
cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C,
high-density lipoprotein cholesterol; DR, diabetic retinopathy.

Figure 8

SLC12A2 deficiency inhibits retinal
structural damage in streptozotocin-induced diabetic mice. (A)
Representative H&E-stained images and retinal thickness
measurements in each group (magnification, ×40). The red arrow
represents neovascular nuclei. (B) Representative TUNEL assay
images in each group (magnification, ×40). Results are expressed as
the mean ± SD (n= 10/group). *P<0.05 and
**P<0.01 vs. the WT group; #P<0.05 and
##P<0.01 vs. the DR group. TUNEL, terminal
deoxynucleotidyl transferase dUTP nick-end labeling; GCL, ganglion
cell layer; IPL, inner plexiform layer; INL, inner nuclear layer;
OPL, outer plexiform layer; ONL, outer nuclear layer; WT,
wild-type; DR, diabetic retinopathy.

Figure 9

SLC12A2 deficiency suppresses
adhesion molecule expression in streptozotocin-induced diabetic
mice. (A) Immunofluorescence images of CD31 (magnification, ×40).
(B and C) Expression of VCAM1, ICAM1, PECAM-1, VE-cadherin,
E-Selectin and P-Selectin determined by reverse
transcription-quantitative PCR and western blotting. Results are
expressed as the mean ± SD (n=10/group). *P<0.05 and
**P<0.01 vs. the WT group; #P<0.05 and
##P<0.01 vs. the DR group. VCAM1, vascular cell
adhesion molecule-1; ICAM1, intercellular adhesion molecule-1;
PECAM-1, platelet endothelial cell adhesion molecule-1; VE,
vascular endothelial; WT, wild-type; DR, diabetic retinopathy.

Figure 10

Molecular mechanisms of bumetanide
treatment for DR. Bumetanide can directly inhibit the expression of
SLC12A2, thereby effectively alleviating the pathological changes
of DR by reducing inflammatory responses, inhibiting the expression
of adhesion molecules, decreasing cell apoptosis, and enhancing the
integrity of the blood-retinal barrier. DR, diabetic
retinopathy.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang Y, Wang X, Xie Q, Huang Y, Huang D, Liu Z, Xu T, Ni M and Yang H: Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy. Int J Mol Med 57: 103, 2026.
APA
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z. ... Yang, H. (2026). Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy. International Journal of Molecular Medicine, 57, 103. https://doi.org/10.3892/ijmm.2026.5774
MLA
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z., Xu, T., Ni, M., Yang, H."Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy". International Journal of Molecular Medicine 57.4 (2026): 103.
Chicago
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z., Xu, T., Ni, M., Yang, H."Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy". International Journal of Molecular Medicine 57, no. 4 (2026): 103. https://doi.org/10.3892/ijmm.2026.5774
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang Y, Wang X, Xie Q, Huang Y, Huang D, Liu Z, Xu T, Ni M and Yang H: Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy. Int J Mol Med 57: 103, 2026.
APA
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z. ... Yang, H. (2026). Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy. International Journal of Molecular Medicine, 57, 103. https://doi.org/10.3892/ijmm.2026.5774
MLA
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z., Xu, T., Ni, M., Yang, H."Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy". International Journal of Molecular Medicine 57.4 (2026): 103.
Chicago
Zhang, Y., Wang, X., Xie, Q., Huang, Y., Huang, D., Liu, Z., Xu, T., Ni, M., Yang, H."Bumetanide‑blocked SLC12A2 exerts a protective effect in experimental diabetic retinopathy". International Journal of Molecular Medicine 57, no. 4 (2026): 103. https://doi.org/10.3892/ijmm.2026.5774
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