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Article Open Access

Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning

  • Authors:
    • Qiming Xu
    • Chunjing Xu
    • Ziyang Liu
    • Jianrao Lu
    • Jing Hu
    • Lin Liao
  • View Affiliations / Copyright

    Affiliations: Department of Nephropathy, Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine, Shanghai 200137, P.R. China
    Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 198
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    Published online on: May 21, 2026
       https://doi.org/10.3892/ijmm.2026.5869
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Abstract

Diabetic kidney disease (DKD) represents a major complication associated with diabetes mellitus, notably contributing to patient morbidity and mortality. However, early diagnosis of DKD remains challenging due to the lack of clear diagnostic biomarkers. Therefore, in the present study, microarray and RNA‑sequencing data from the Gene Expression Omnibus database were systematically analyzed. Using differential expression and weighted gene co‑expression network analysis, 49 genes with marked expression changes in DKD were identified. Subsequent analyses, including functional enrichment, protein‑protein interaction network construction, machine learning techniques and assessment of immune cell infiltration, led to the identification of three hub genes: Spleen‑associated tyrosine kinase, apoptotic peptidase activating factor 1 and ADAM metallopeptidase domain 10, as promising diagnostic markers, which were further evaluated by receiver operating characteristic curve analysis. Expression changes of the identified hub genes were validated in both DKD mouse models and clinical patient samples. Collectively, the present study provided a novel perspective on the molecular basis of DKD, and highlighted novel candidates for potential diagnostic and therapeutic applications.
View Figures

Figure 1

Screening for DEGs. (A) Boxplot
illustrating the distribution of data after normalization. (B)
Volcano plot depicting differential expression analysis of the
GSE30529 dataset. (C) Heatmap showing differential expression
analysis results for the GSE30529 dataset. The top annotation bar
indicates sample grouping. Gene expression levels were normalized
and visualized as row Z-scores. The blue-white-red color scale
represents relative expression levels, with blue indicating lower
expression, white indicating intermediate expression, and red
indicating higher expression. The color scale ranges from -2 to 2.
(D) Unscaled fit indices derived from soft-threshold power analysis
and average connectivity used to determine the network topology in
the GSE30529 dataset. (E) Hierarchical clustering of all genes in
the GSE30529 dataset, with colors representing distinct modules in
the gene co-expression network constructed through WGCNA. (F)
Heatmap illustrating the correlation between gene clustering
modules in the GSE30529 dataset. Each row represents a module and
each cell contains the correlation coefficient and the
corresponding P-value. The blue-white-red color scale indicates the
direction and strength of the correlation, with blue representing
negative correlation, white representing no correlation, and red
representing positive correlation. (G) Venn diagram illustrating
the intersection of key module genes and DEGs. DEGs, differentially
expressed genes; DKD, diabetic kidney disease; WGCNA, weighted gene
co-expression network analysis. ME, Module Eigengene.

Figure 2

GSEA, GO and KEGG functional
enrichment analysis. (A) Bubble plot illustrating biological
signaling pathways associated with diabetic kidney disease. (B)
'TYROBP causal network in microglia'; (C) 'Leishmania infection';
(D) 'Integrin cell surface interactions'; (E) 'Allograft
rejection'; (F) 'Antigen processing cross presentation'; (G)
'Interferon signaling'; (H) 'Core matrisome'; (I) 'Extracellular
matrix organization'; (J) 'Neutrophil degranulation'; and (K)
'Signaling by interleukins'. (L) Sankey diagram depicting KEGG
enrichment analysis results. Circle diagrams representing
functional enrichment in (M) biological process categories, (N)
cellular component categories and (O) molecular function
categories. FDR, false discovery rate; GO, Gene Ontology; GSEA,
gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and
Genomes; NES, normalized enrichment score.

Figure 3

Hub genes for DKD diagnosis. (A)
Protein-protein interaction network of intersecting genes.
Diagnostic markers identified by the (B) closeness, (C) degree and
(D) MCC algorithms via the cytoHubba plugin. (E and F) Screening of
diagnostic markers using the LASSO logistic regression algorithm.
(G) Biomarker screening using the SVM-RFE method. (H) Biomarker
screening based on the RF algorithm. (I and J) UpSet plots and
petal diagrams showing intersecting diagnostic markers identified
by all six algorithms. (K) Nomogram for predicting DKD. (L)
Decision curve analysis curve for validation of diagnostic
efficacy. (M) ROC curve for validation of diagnostic efficacy of
key genes. Violin plots comparing (N) SYK expression levels, (O)
ADAM10 expression levels and (P) APAF1 expression levels in DKD vs.
control samples within the GSE30122 validation cohort.
*P<0.05, ***P<0.001. Analysis of the
relationships between (Q) SYK and serum creatinine, (R) ADAM10 and
serum creatinine, (S) APAF1 and serum creatinine, (T) SYK and GFR,
(U) ADAM10 and GFR, and (V) APAF1 and GFR. ADAM10, ADAM
metallopeptidase domain 10; APAF1, apoptotic peptidase activating
factor 1; AUC, area under the ROC curve; DKD, diabetic kidney
disease; FPR, false positive rate; GFR, glomerular filtration rate;
LASSO, least absolute shrinkage and selection operator; MCC,
maximum clique centrality; ROC, receiver operating characteristic;
RF, random forest; SVM-RFE, support vector machine-recursive
feature elimination; SYK, spleen-associated tyrosine kinase; TPR,
true positive rate.

Figure 4

Analysis of the immune
microenvironment. (A) Principal component analysis clustering plot
illustrating immune cell infiltration patterns. The diagram
presents differences in immune profiles between the DKD group and
controls. (B) A heatmap displaying immune cell type correlations,
where red signifies positive and blue negative associations, and
the color saturation corresponds to the strength of the
correlation. (C) Analysis illustrating disparities in immune cell
infiltration between DKD and controls kidney tissue samples.
*P<0.05, ***P<0.001. (D) Relative
proportions of 22 immune cell subpopulations. Correlations between
(E) ADAM10 and immune cells, (F) APAF1 and immune cells, and (G)
SYK and immune cells. Point size corresponds to correlation
strength between the hub gene and different immune cells; stronger
correlations are shown by larger points, while weaker ones are
shown by smaller points. ADAM10, ADAM metallopeptidase domain 10;
APAF1, apoptotic peptidase activating factor 1; DKD, diabetic
kidney disease; SYK, spleen-associated tyrosine kinase.

Figure 5

Validation of pivotal gene expression
in the mouse DKD model. (A) Schematic illustration of the mouse DKD
model. (B) Quantification of UACR in mice. (C) Scr and (D) BUN
levels in the two experimental groups. (E) Body weight changes in
mice over time. (F) Blood glucose alterations in mice. (G) H&E
staining of mouse kidney tissue sections, highlighting the extent
of renal injury. Renal fibrosis was assessed using Masson's
trichrome staining (magnification, ×200; scale bar, 100 µm).
The arrows point to dilated renal tubules and atrophic glomeruli.
Semi-quantitative analysis of (H) H&E and (I) Masson staining
results, with error bars in (H) representing the 95% confidence
interval for the median. Reverse transcription-quantitative PCR
detection of (J) APAF1, (K) SYK and (L) ADAM10 mRNA expression. (M)
Western blot analysis showing the expression levels of SYK, ADAM10
and APAF1 in the UNx/STZ/HFD model, and (N) semi-quantitative
analysis. (O) Western blot analysis showing the expression levels
of SYK, ADAM10 and APAF1 in the db/db model, and (P)
semi-quantitative analysis. Data are presented as the mean ± SD or
median (n=6). *P<0.05, **P<0.01,
***P<0.001. ADAM10, ADAM metallopeptidase domain 10;
APAF1, apoptotic peptidase activating factor 1; BUN, blood urea
nitrogen; DKD, diabetic kidney disease; H&E, hematoxylin and
eosin; HFD, high-fat diet; Scr, serum creatinine; STZ,
streptozotocin; SYK, spleen-associated tyrosine kinase; UACR, urine
albumin-to-creatinine ratio; UNx, uninephrectomy.

Figure 6

siRNA-mediated silencing of pivotal
genes can alleviate fibrosis. (A) SYK siRNA reduced SYK mRNA
levels. (B) Western blot analysis showing the expression levels of
SYK, fibronectin, vimentin and Snail, and (C) semi-quantitative
analysis. (D) ADAM10 siRNA reduced ADAM10 mRNA levels. (E) Western
blot analysis showing the expression levels of ADAM10, fibronectin,
vimentin and Snail, and (F) semi-quantitative analysis. (G) APAF1
siRNA reduced APAF1 mRNA levels. (H) Western blot analysis showing
the expression levels of APAF1, fibronectin, vimentin and Snail,
and (I) semi-quantitative analysis. Data are presented as the mean
± SD (n=6). *P<0.05, **P<0.01,
***P<0.001. ADAM10, ADAM metallopeptidase domain 10;
APAF1, apoptotic peptidase activating factor 1; Con, control; HG,
high glucose; LG, low glucose; ns, not significant; siRNA, small
interfering RNA; SYK, spleen-associated tyrosine kinase.

Figure 7

Clinical validation of hub genes.
Relative mRNA levels of (A) SYK, (B) ADAM10 and (C) APAF1 in
non-DKD patients and patients with DKD. Data are presented as the
mean ± SD (n=15). *P<0.05, **P<0.01,
***P<0.001. ADAM metallopeptidase domain 10; APAF1,
apoptotic peptidase activating factor 1; DKD, diabetic kidney
disease; SYK, spleen-associated tyrosine kinase.
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Copy and paste a formatted citation
Spandidos Publications style
Xu Q, Xu C, Liu Z, Lu J, Hu J and Liao L: Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning. Int J Mol Med 58: 198, 2026.
APA
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., & Liao, L. (2026). Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning. International Journal of Molecular Medicine, 58, 198. https://doi.org/10.3892/ijmm.2026.5869
MLA
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., Liao, L."Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning". International Journal of Molecular Medicine 58.1 (2026): 198.
Chicago
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., Liao, L."Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning". International Journal of Molecular Medicine 58, no. 1 (2026): 198. https://doi.org/10.3892/ijmm.2026.5869
Copy and paste a formatted citation
x
Spandidos Publications style
Xu Q, Xu C, Liu Z, Lu J, Hu J and Liao L: Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning. Int J Mol Med 58: 198, 2026.
APA
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., & Liao, L. (2026). Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning. International Journal of Molecular Medicine, 58, 198. https://doi.org/10.3892/ijmm.2026.5869
MLA
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., Liao, L."Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning". International Journal of Molecular Medicine 58.1 (2026): 198.
Chicago
Xu, Q., Xu, C., Liu, Z., Lu, J., Hu, J., Liao, L."Identification of diagnostic markers for diabetic kidney disease by weighted gene co‑expression network analysis and machine learning". International Journal of Molecular Medicine 58, no. 1 (2026): 198. https://doi.org/10.3892/ijmm.2026.5869
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