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Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis

  • Authors:
    • Qian Zhang
    • Dan Wang
    • Hongxia Jia
    • Zongji Zheng
    • Liyan Lin
    • Linna Li
    • Ling Wang
    • Yaoming Xue
  • View Affiliations / Copyright

    Affiliations: Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 25
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    Published online on: November 18, 2025
       https://doi.org/10.3892/ijmm.2025.5696
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Abstract

Diabetic nephropathy (DN) and diabetic osteoporosis (DOP) are frequent and debilitating complications of diabetes mellitus (DM), sharing pathological features such as oxidative stress, inflammation and metabolic dysregulation. However, current therapies rarely address these comorbidities simultaneously. In the present study, a type 2 DM rat model presenting both DN and DOP characteristics was established. Rats were treated with Akebia saponin D (ASD), Semaglutide, or their combination. Renal function, calcium‑phosphate metabolism, bone microarchitecture and mechanical properties were evaluated. Network pharmacology, molecular docking and knockdown validation were employed to elucidate underlying mechanisms. Combination therapy markedly improved glomerular structure, decreased fibrosis, restored trabecular bone volume and strength and corrected metabolic imbalance more effectively than monotherapy. Bioinformatic analysis identified the Klotho‑p53 signaling axis as a potential target. ASD exhibited high binding affinity to Klotho in silico and adeno‑associated virus‑mediated Klotho knockdown reversed therapeutic benefits, confirming its pivotal role. ASD and Semaglutide synergistically alleviated both DN and DOP by modulating the Klotho‑p53 axis, offering a promising strategy for comprehensive DM complication management.
View Figures

Figure 1

Chemical structures of ASD and
Semaglutide and their effects on glucose metabolism and renal
function in diabetic rats. Two-dimensional structures of (A) ASD
and (B) Semaglutide were retrieved from PubChem. Enzyme-linked
immunosorbent assay analysis of (C) FBG, (D) FSI and (E) HOMA-IR.
(F-I) Biochemical analysis of BUN, Cr, 24-up and renal index.
*P<0.05, **P<0.01 vs. Control;
##P<0.01 vs. MOD; ΔP<0.05,
ΔΔP<0.01 vs. M + A; ☆P<0.05,
☆☆P<0.01 vs. M + S. ASD, akebia saponin D; FBG,
fasting blood glucose; FSI, fasting serum insulin; HOMA-IR,
homeostatic model assessment of insulin resistance; BUN, blood urea
nitrogen; Cr, creatinine; 24-up, 24-h urinary protein.

Figure 2

ASD and Semaglutide alleviate renal
pathological damage in diabetic rats. (A-C) H&E staining was
used to observe kidney structure (magnification, ×200 and ×400) and
perform GSI and RTI scoring. (D) PAS staining and (E)
quantification of PAS-positive area. (F) Masson staining and (G)
quantification of collagen deposition. (H) Western blot analysis of
TGF-β1, α-SMA, Collagen I and Collagen IV. (I) Quantification of
protein expression normalized to GAPDH. *P<0.05,
**P<0.01 vs. Control; ##P<0.01 vs. MOD;
ΔP<0.05, ΔΔP<0.01 vs. M + A;
☆P<0.05, ☆☆P<0.01 vs. M + S. ASD,
akebia saponin D; H&E, hematoxylin and eosin; GSI,
glomerulosclerosis index; RTI, renal tubular interstitial injury
index; PAS, periodic acid-Schiff; α-SMA, α-smooth muscle actin.

Figure 3

Effects of ASD and Semaglutide on
calcium and phosphate metabolism in diabetic rats. (A-F)
Biochemical analyzer was used to measure the levels of
Ca2+ and PO43− in (A and B) serum,
(C and D) urine and (E and F) femur. *P<0.05,
**P<0.01 vs. Control; ##P<0.01 vs. MOD;
ΔΔP<0.01 vs. M + A; ☆☆P<0.01 vs. M + S.
ASD, akebia saponin D.

Figure 4

ASD and Semaglutide alleviate femoral
damage in diabetic rats. (A and B) H&E staining was employed to
examine femoral structure and count osteocytes and osteoblasts. (C
and D) Micro-CT was adopted to assess trabecular microstructure of
the femur and to perform quantitative analysis of related
parameters. *P<0.05, **P<0.01 vs.
Control; #P<0.05, ##P<0.01 vs. MOD;
ΔP<0.05, ΔΔP<0.01 vs. M + A;
☆P<0.05, ☆☆P<0.01 vs. M + S. ASD,
akebia saponin D; H&E, hematoxylin and eosin; Micro-CT,
micro-computed tomography.

Figure 5

Effects of ASD and Semaglutide on
bone metabolism and remodeling in diabetic rats. (A-C) Three-point
bending tests measuring ultimate load, bending strength and Young's
modulus of the femur. The serum levels of (D) ALP, (E) PINP, (F)
CTX-1 and (G) TRAP were measured using ELISA. (H) Western blot and
(I) quantification of femoral protein expression (OPG, OCN, RANKL
and RUNX2). *P<0.05, **P<0.01 vs.
Control; #P<0.05, ##P<0.01 vs. MOD;
ΔP<0.05, ΔΔP<0.01 vs. M + A;
☆P<0.05, ☆☆P<0.01 vs. M + S. ASD,
akebia saponin D; ALP, alkaline phosphatase; PINP, procollagen type
I N-terminal propeptide; CTX-1, C-terminal telopeptide of type I
collagen; TRAP, tartrate-resistant acid phosphatase; OPG,
osteoprotegerin; OCN, osteocalcin; RANKL, receptor activator of
nuclear factor κ-b ligand; RUNX2, Runt-related transcription factor
2.

Figure 6

Network pharmacology analysis of ASD
and Semaglutide. Identification of differentially expressed genes
in the (A) GSE30528 and (B) GSE35958 datasets. (C) Venn diagram
depicting the intersection of DEGs from both diseases and
drug-related genes. (D) PPI network of 26 related genes. GO
enrichment analysis for (E) biological process, (F) cellular
component and (G) molecular function. (H) KEGG pathway enrichment
analysis. ASD, akebia saponin D; DEGs, differentially expressed
genes; PPI, protein-protein interaction; GO, Gene Ontology; KEGG,
Kyoto Encyclopedia of Genes and Genomes.

Figure 7

Molecular docking and expression
analysis of Klotho in diabetic rats. Molecular docking models
illustrating the interaction between ASD and (A) Klotho or (B) p53.
(C) Immunohistochemical detection of Klotho levels in rat kidney
tissues. Western blotting and quantitative analysis of Klotho
protein levels in the (D) kidney and (E) femur.
**P<0.01 vs. Control; #P<0.05,
##P<0.01 vs. MOD; ΔΔP<0.01 vs. M + A;
☆☆P<0.01 vs. M + S. ASD, akebia saponin D.

Figure 8

ASD and Semaglutide inhibit the p53
pathway and DNA damage in the kidney and femur tissues of diabetic
rats. Immunohistochemical staining of p53 and γ-H2AX in (A) kidney
and (B) femur tissues, respectively. (C-F) Western blotting and
quantification analysis of DNA damage-related proteins, including
γ-H2AX, ATM, p-ATM, p53 and p21, in (C and D) kidney and (E and F)
femur tissues. *P<0.05, **P<0.01 vs.
Control; #P<0.05, ##P<0.01 vs. MOD;
ΔΔP<0.01 vs. M + A; ☆P<0.05,
☆☆P<0.01 vs. M + S. ASD, akebia saponin D; p-,
phosphorylated; ATM, ATM, ataxia-telangiectasia mutated
protein.

Figure 9

Klotho knockdown abrogates the
renoprotective effects of ASD and Semaglutide. (A) Western blot
analysis of Klotho protein levels in rat kidney tissues. (B-E)
Serum levels of (B) BUN, (C) Cr, (D) 24-up and (E) kidney index.
(F) H&E, (G) PAS and (H) Masson staining of renal tissue, with
corresponding quantification of GSI, RTI, PAS-positive area and
fibrosis index. (I) Western blotting and (J) quantification of
renal TGF-β1, α-SMA, Collagen I and Collagen IV protein levels.
*P<0.05, **P<0.01 vs. Control;
##P<0.01 vs. MOD; ΔΔP<0.01 vs. M + A +
S; ☆☆P<0.01 vs. M + A + S + shNC. ASD, akebia saponin
D; BUN Cr, creatinine; 24-up, 24-h urinary protein; H&E,
hematoxylin and eosin; PAS, periodic acid-Schiff; GSI,
glomerulosclerosis index; RTI, renal tubular interstitial injury
index; α-SMA, α-smooth muscle actin; sh, short hairpin.

Figure 10

Knockdown of Klotho reverses the
therapeutic effects of ASD and Semaglutide in the treatment of
diabetic osteoporosis. (A) Western blot analysis of Klotho protein
levels in rat femur tissue. (B and C) H&E staining to assess
histological changes in rat femur tissues and cell counting of
osteocytes and osteoblasts. (D) Micro-CT measurement of BMD in rat
femur. (E and F) Western blot and quantification analysis of bone
metabolic markers (OPG, OCN, RANKL and RUNX2).
*P<0.05, **P<0.01 vs. Control;
#P<0.05, ##P<0.01 vs. MOD;
ΔP<0.05, ΔΔP<0.01 vs. M + A + S;
☆P<0.05, ☆☆P<0.01 vs. M + A + S + shNC.
ASD, akebia saponin D; H&E, hematoxylin and eosin; Micro-CT,
micro-computed tomography; OPG, osteoprotegerin; OCN, osteocalcin;
RANKL, receptor activator of nuclear factor κ-b ligand; RUNX2,
Runt-related transcription factor 2.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang Q, Wang D, Jia H, Zheng Z, Lin L, Li L, Wang L and Xue Y: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis. Int J Mol Med 57: 25, 2026.
APA
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L. ... Xue, Y. (2026). Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis. International Journal of Molecular Medicine, 57, 25. https://doi.org/10.3892/ijmm.2025.5696
MLA
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L., Wang, L., Xue, Y."Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis". International Journal of Molecular Medicine 57.1 (2026): 25.
Chicago
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L., Wang, L., Xue, Y."Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis". International Journal of Molecular Medicine 57, no. 1 (2026): 25. https://doi.org/10.3892/ijmm.2025.5696
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang Q, Wang D, Jia H, Zheng Z, Lin L, Li L, Wang L and Xue Y: Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis. Int J Mol Med 57: 25, 2026.
APA
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L. ... Xue, Y. (2026). Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis. International Journal of Molecular Medicine, 57, 25. https://doi.org/10.3892/ijmm.2025.5696
MLA
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L., Wang, L., Xue, Y."Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis". International Journal of Molecular Medicine 57.1 (2026): 25.
Chicago
Zhang, Q., Wang, D., Jia, H., Zheng, Z., Lin, L., Li, L., Wang, L., Xue, Y."Synergistic effects of Akebia saponin D and Semaglutide on diabetic nephropathy and osteoporosis via the Klotho‑p53 signaling axis". International Journal of Molecular Medicine 57, no. 1 (2026): 25. https://doi.org/10.3892/ijmm.2025.5696
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