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Article Open Access

Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation

  • Authors:
    • Mingkun Xu
    • Linhang Fu
    • Yulong Zhang
    • Xiuli Guo
    • Han Wu
    • Sijing Gao
    • Fei Xiao
    • Li Xu
  • View Affiliations / Copyright

    Affiliations: Department of Anesthesiology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524003, P.R. China, Department of Anesthesiology, The Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524003, P.R. China, Department of Laboratory Medicine, The Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524003, P.R. China, International Medical and Special Care Ward, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524002, P.R. China, Department of Laboratory Medicine, Maoming People's Hospital, Maoming, Guangdong 525099, P.R. China
    Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 255
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    Published online on: July 13, 2026
       https://doi.org/10.3892/ijmm.2026.5926
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Abstract

Aerobic glycolysis is increasingly recognized as a pathogenic driver in diabetic kidney disease (DKD). However, the epigenetic role of its end product, lactate, remains largely undefined. Spatial transcriptomics analysis revealed active glycolysis in tubular epithelial cells. The participation of histone lactylation in DKD was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate in vivo. The potential target genes of H4K12 lactylation (H4K12la) were screened by CUT&Tag analyses. The candidate target genes were validated through ChIP‑qPCR, RT‑qPCR and western blot analyses. The present study found that the expression of 6‑phosphofructo‑2‑kinase/fructose‑2,6‑biphosphatase 3 (PFKFB3), a pivotal glycolytic regulator, was markedly upregulated in tubular epithelial cells derived from patients with DKD and from the corresponding mouse models. Inhibition of the expression of PFKFB3 mitigated the kidney fibrotic process and alleviated renal function in a DKD mouse model. Conversely, upregulation of PFKFB3 expression aggravated renal fibrogenesis and promoted the deterioration of renal pathology. Moreover, it was demonstrated that the reduction in the levels of lactate levels markedly alleviated renal fibrosis in DKD. With regard to its mechanism of action, lactate was generated via PFKFB3‑driven glycolytic reprogramming and selectively enhanced H4K12 lactylation at the homeodomain‑interacting protein kinase 2 (HIPK2) promoter, thereby activating its transcription and driving renal fibrotic progression. These findings indicated that PFKFB3 in renal tubules upregulates HIPK2 expression via facilitating H4K12la‑dependent gene transcription. Therefore, intervention approaches targeting PFKFB3‑triggered HIPK2 activation in tubular cells may offer a novel therapeutic strategy for DKD.
View Figures

Figure 1

Glycolysis pathway is activated in
injury renal tubular cells in DKD. (A and C) Spatial distribution
and UMAP analysis of clusters derived from pathological
classification. (B and D) Proportion of the PT subclusters and
injury PT on the kidney section of DKD patients. (E and F)
Expression of PFKFB3 between PT and injury PT subclusters. (G)
(GO-BP) enrichment analysis of subcluster-specific genes in injured
PT cells. UMAP, Uniform Manifold Approximation and Projection; PT,
proximal tubules; DKD, diabetic kidney disease; PFKFB3,
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; GO-BP, Gene
Ontology Biological Process.

Figure 2

PFKFB3 expression is increased in
renal tubular cells in DKD. (A) RT-qPCR for renal PFKFB3 and HK2 in
db/m and db/db groups (n=5). (B) Immunostained E-cadherin (red) and
PFKFB3 (green), and counterstained with DAPI (blue) by IF staining
in db/m and db/db groups (n=6). Scale bar, 50 μm. Protein
levels of α-SMA and PFKFB3 in db/m and db/db groups by (C) western
blotting and (D) its semi-quantitative analysis (n=6). (E)
Representative images of IHC staining for PFKFB3 (scale bar, 50
μm) in db/m and db/db groups or in Control and DKD group
(n=6). **P<0.01 vs. db/m group by Student's t-test.
PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; DKD,
diabetic kidney disease; RT-qPCR, reverse
transcription-quantitative PCR; IF, immunofluorescence; α-SMA,
α-smooth muscle actin; IHC, immunohistochemistry.

Figure 3

Knockdown of PFKFB3 ameliorates renal
fibrosis in db/db mice. (A) Schematic illustration of AAV9-shPFKFB3
injection in the db/db group; (B). Blood glucose levels, UACR and
Cr levels in db/m, db/db, and db/db + shPFKFB3 group (n=8). (C)
Representative images of Masson, Sirius red staining and IHC
staining (α-SMA and FN level) (scale bar=50 μm) in db/m,
db/db, and db/db + shPFKFB3 groups (n=6). (D and E) Protein levels
of α-SMA and FN in db/m, db/db, and db/db + shPFKFB3 groups by
western blotting, with semi-quantitative analyses (n=6). Data
represent the mean ± SD from three independent experiments.
**P<0.01 vs. db/db group by one-way ANOVA. PFKFB3,
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; AAV,
adeno-associated virus; sh, short hairpin; UACR, urinary albumin
creatinine ratio; Cr, creatinine; IHC, immunohistochemistry; α-SMA,
α-smooth muscle actin; FN, fibronectin.

Figure 4

Inhibition of lactate attenuates
renal fibrosis in db/db mice. (A) Illustrative overview of the
FX-11 intervention strategy in db/db mice. (B) Blood glucose levels
and UACR levels in db/m, db/db, db/m + FX-11 and db/db + FX-11
group (n=8). (C) Representative images of Sirius red and IF
staining of FN (green), DAPI (blue) in db/m, db/db, db/m + FX-11
and db/db + FX-11 group (n=6). (D and E) Protein levels of α-SMA
and FN in db/m, db/db, db/m + FX-11 and db/db + FX-11 groups by
western blotting (n=6). Data represent the mean ± SD from three
independent experiments. **P<0.01 vs. db/db group by
one-way ANOVA. UACR, urinary albumin creatinine ratio; IF,
immunofluorescence; FN, fibronectin; α-SMA, α-smooth muscle
actin.

Figure 5

H4K12la initiates p53 pathway related
genes under HG condition in HK-2 cells. (A and B) Protein levels of
Pan Kla in NG and HG groups by western blotting, and its
semi-quantitative analysis (n=3). (C) The protein levels of H4K12la
by western blotting in NG and HG groups and their semi-quantitative
analyses (n=3); (D and E) Distribution of different genomic
distribution of H4K12la peaks between NG and HG group by
CUT&Tag. (F) GSEA on the regulation of p53 signaling pathway.
(G and H) IGV tracks for HIPK2 and ATM from CUT&Tag analysis.
(I) The mRNA level of HIPK2 and ATM in the NG and HG group in HK-2
cells by RT-qPCR (n=3). (J) H4K12la occupancy analysis of HIPK2 and
ATM in the NG and HG group in HK-2 cells by CUT&Tag-qPCR (n=3).
Data represent the mean ± SD from three independent experiments.
**P<0.01 vs. NG group by Student's t-test. HG, high
glucose; NG, normal glucose; GSEA, gene set enrichment analysis;
RT-qPCR, reverse transcription-quantitative PCR.

Figure 6

PFKFB3-driven lactate triggers H4K12
lactylation in DKD. (A and B) Protein levels of Pan Kla in db/db
and db/db + shPFKFB3 groups by western blotting and its
semi-quantitative analysis (n=6). (C and D) Protein levels of Pan
Kla in HG and HG + siPFKFB3 groups by western blotting and its
semi-quantitative analysis (n=3). (E) Immunostained E-cadherin
(green) and H4K12la (red), and counterstained with DAPI (blue) by
IF staining in db/m, db/db and db/db + shPFKFB3 groups (n=6). Scale
bar, 50 μm. (F) The protein levels of H4K12la, α-SMA and FN
by western blot in HG and HG + siPFKFB3 groups, and their
semi-quantitative analyses (n=3). Data represent the mean ± SD from
three independent experiments. **P<0.01 vs. (D and F)
HG group or (B) db/db group by Student's t-test. PFKFB3,
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; DKD,
diabetic kidney disease; sh, short hairpin; HG, high glucose; si,
small interfering; IF, immunofluorescence; FN, fibronectin.

Figure 7

PFKFB3-mediated H4K12la initiates
renal tubule fibrosis in DKD. (A) The ECAR levels after culturing
with glucose followed by oligomycin and 2-DG in HG and HG +
siPFKFB3 groups (n=3). (B) The lactate concentration in HK-2 cells
in the HG and HG + siPFKFB3 groups (n=5). (C and D) Protein levels
of H4K12la, HIPK2, FN and α-SMA in HG, HG + siPFKFB3 and HG +
siPFKFB3 + La groups in HK-2 cells by western blotting, with
semi-quantitative analyses (n=3). (E) Immunostaining for H4K12la
(green) and HIPK2 (red), counterstained with DAPI (blue) in HG, HG
+ siPFKFB3 and HG + siPFKFB3 + La (scale bar, 50 μm). Data
represent the mean ± SD from three independent experiments.
**P<0.01 versus HG group (A and B) by Student's
t-test or HG + siPFKFB3 group (D) by one-way ANOVA. PFKFB3,
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; DKD,
diabetic kidney disease; ECAR, extracellular acidification rate;
HG, high glucose; si, small interfering; FN, fibronectin; α-SMA,
α-smooth muscle actin.
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Copy and paste a formatted citation
Spandidos Publications style
Xu M, Fu L, Zhang Y, Guo X, Wu H, Gao S, Xiao F and Xu L: Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation. Int J Mol Med 58: 255, 2026.
APA
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S. ... Xu, L. (2026). Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation. International Journal of Molecular Medicine, 58, 255. https://doi.org/10.3892/ijmm.2026.5926
MLA
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S., Xiao, F., Xu, L."Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation". International Journal of Molecular Medicine 58.3 (2026): 255.
Chicago
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S., Xiao, F., Xu, L."Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation". International Journal of Molecular Medicine 58, no. 3 (2026): 255. https://doi.org/10.3892/ijmm.2026.5926
Copy and paste a formatted citation
x
Spandidos Publications style
Xu M, Fu L, Zhang Y, Guo X, Wu H, Gao S, Xiao F and Xu L: Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation. Int J Mol Med 58: 255, 2026.
APA
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S. ... Xu, L. (2026). Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation. International Journal of Molecular Medicine, 58, 255. https://doi.org/10.3892/ijmm.2026.5926
MLA
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S., Xiao, F., Xu, L."Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation". International Journal of Molecular Medicine 58.3 (2026): 255.
Chicago
Xu, M., Fu, L., Zhang, Y., Guo, X., Wu, H., Gao, S., Xiao, F., Xu, L."Tubular PFKFB3 drives diabetic kidney fibrosis via lactate‑dependent H4K12 lactylation and HIPK2 transactivation". International Journal of Molecular Medicine 58, no. 3 (2026): 255. https://doi.org/10.3892/ijmm.2026.5926
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