Identification of Novel Key Molecular Signatures in the Pathogenesis of Experimental Diabetic Kidney Disease

Abstract

Diabetic kidney disease (DKD) is a long-term major microvascular complication of uncontrolled hyperglycemia and one of the leading causes of end-stage renal disease (ESDR). The pathogenesis of DKD has not been fully elucidated, and effective therapy to completely halt DKD progression to ESDR is lacking. This study aimed to identify critical molecular signatures and develop novel therapeutic targets for DKD. This study enrolled 10 datasets consisting of 93 renal samples from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO). Networkanalyst, Enrichr, STRING, and Cytoscape were used to conduct the differentially expressed genes (DEGs) analysis, pathway enrichment analysis, protein-protein interaction (PPI) network construction, and hub gene screening. The shared DEGs of type 1 diabetic kidney disease (T1DKD) and type 2 diabetic kidney disease (T2DKD) datasets were performed to identify the shared vital pathways and hub genes. Strepotozocin-induced Type 1 diabetes mellitus (T1DM) rat model was prepared, followed by hematoxylin & eosin (HE) staining, and Oil Red O staining to observe the lipid-related morphological changes. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to validate the key DEGs of interest from a meta-analysis in the T1DKD rat. Using meta-analysis, 305 shared DEGs were obtained. Among the top 5 shared DEGs, Tmem43, Mpv17l, and Slco1a1, have not been reported relevant to DKD. Ketone body metabolism ranked in the top 1 in the KEGG enrichment analysis. Coasy, Idi1, Fads2, Acsl3, Oxct1, and Bdh1, as the top 10 down-regulated hub genes, were first identified to be involved in DKD. The qRT-PCR verification results of the novel hub genes were mostly consistent with the meta-analysis. The positive Oil Red O staining showed that the steatosis appeared in tubuloepithelial cells at 6 w after DM onset. Taken together, abnormal ketone body metabolism may be the key factor in the progression of DKD. Targeting metabolic abnormalities of ketone bodies may represent a novel therapeutic strategy for DKD. These identified novel molecular signatures in DKD merit further clinical investigation.

Keywords: BDH1; HMGCS2; Mpv17l; bioinformatics; diabetic kidney disease; ketone body metabolism.

Figure 1

Data collection and processing: (A) The Meta-analysis flowchart. The main methods in each stage are described in the corresponding parentheses. (B, C) Principal component analysis (PCA) of the combined datasets after batch effect correction. All samples are represented by different symbols with shapes according to their studies and colors based on their experimental conditions. (B) T1DKD datasets. (C) T2DKD datasets.

Figure 2

Differentially expressed genes analysis: (A) Heatmap of the top 50 shared up- and down-regulated genes identified during meta-analysis. (B) Venn diagrams of the shared up-regulated DEGs in TIDKD and T2DKD datasets. (C) Venn diagrams of the shared down-regulated DEGs in TIDKD and T2DKD datasets.

Figure 3

Functional annotation and enrichment analysis of the shared DEGs in T1DKD and T2DKD datasets obtained by meta-analysis: (A-G) Functional annotation and enrichment analysis results in Enrichr. (A) GO-BP; (B) GO-MF; (C) GO-CC; (D) KEGG; (E) GWAS; (F) Clinvar; (G) JASPAR. (H–N) Heatmaps of genes enriched in each top1 term.

Figure 4

Protein-protein interaction (PPI) network construction and hub gene screening: (A) The PPI network constructed with the shared up-regulated DEGs. (B) Top 10 up-regulated hub genes. (C) Venn diagram of the shared up-regulated hub genes. (D) The PPI network constructed with the shared down-regulated DEGs. (E) Top 10 down-regulated hub genes. (F) Venn diagram of the shared down-regulated hub genes.

Figure 5

Morphological changes of kidney and qPCR confirmation in T1DM rats: (A) Hematoxylin & eosin (HE) staining of renal tissue. (B) Oil Red O staining of renal tissue. (C) The mRNA expression level of some genes of interest in T1DKD rats. Data are presented as means ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, no significance, versus the control with the same treatment (Student’s t-test).