Abstract Chronic kidney disease (CKD) involves inflammation, oxidative stress, and fibrosis, leading to renal dysfunction. Dapagliflozin, an SGLT2 inhibitor, shows renoprotective effects beyond glucose control, but its precise molecular mechanisms remain unclear. This study utilizes network pharmacology and molecular docking to elucidate its multi-target effects in CKD. Dapagliflozin’s SMILES structure was analyzed for ADMET properties. Potential targets were identified via SwissTargetPrediction, GeneCards, and SEA, and common CKD-related targets were determined. A protein–protein interaction (PPI) network was constructed, and key pathways were identified using GO and KEGG enrichment analyses. Molecular docking was conducted to validate dapagliflozin’s binding affinities with hub proteins. A total of 208 common targets were identified, including EGFR, GSK3β, and IL-6. GO and KEGG analyses highlighted key pathways, such as PI3K-Akt, MAPK, and AGE-RAGE, involved in inflammation, oxidative stress, and metabolic regulation. Molecular docking confirmed strong binding affinities with EGFR (−8.42 kcal/mol), GSK3β (−7.70 kcal/mol), and IL-6 (−6.83 kcal/mol). Dapagliflozin exhibits multi-target therapeutic potential in CKD by modulating inflammation, oxidative stress, and metabolic pathways. This integrative approach enhances the understanding of its mechanisms, supporting future experimental validation and clinical application in CKD management. Keywords: dapagliflozin, chronic kidney disease, network pharmacology, molecular docking, protein-protein interaction 1. Introduction Chronic kidney disease (CKD) is a significant global health issue that affects millions of individuals and imposes substantial economic and social costs [[38]1]. With a prevalence of approximately 9.5% across 162 countries, CKD strains healthcare systems and contributes to elevated morbidity, mortality, and diminished quality of life [[39]2]. The pathophysiology of CKD is multifaceted, involving mechanisms such as glomerular hypertension, tubulointerstitial fibrosis, and inflammation [[40]3,[41]4]. These processes cumulatively lead to a progressive decline in renal function and eventual end-stage renal disease (ESRD), necessitating interventions such as dialysis or transplantation [[42]5]. Inflammation plays a particularly critical role in CKD progression, marked by elevated levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [[43]6,[44]7]. Additionally, oxidative stress and mitochondrial dysfunction exacerbate renal damage, highlighting the importance of addressing these mechanisms in therapeutic development [[45]8,[46]9]. Dapagliflozin, an SGLT2 inhibitor, has shown promise as a therapeutic agent for CKD [[47]10]. By inhibiting glucose reabsorption in the proximal tubules, dapagliflozin induces glucosuria, reducing blood glucose levels [[48]11]. Beyond its glucose-lowering effects, dapagliflozin demonstrates renoprotective mechanisms, such as reducing intraglomerular pressure, lowering albuminuria, and attenuating tubulointerstitial fibrosis [[49]12,[50]13]. Preclinical studies in animal models have supported its ability to reduce markers of renal injury and improve renal outcomes [[51]14,[52]15]. Clinical trials have further established its efficacy in reducing ESRD progression and cardiovascular events in CKD patients, regardless of diabetes status [[53]16,[54]17]. However, the molecular mechanisms underlying these effects remain partially understood and warrant further investigation. Network pharmacology, a systems biology approach, provides a powerful framework for elucidating the complex interactions between drugs, targets, and disease pathways. By integrating data from multiple databases and computational tools, this approach enables the identification of potential molecular targets and pathways involved in drug action [[55]18]. This approach enables a comprehensive analysis of the interactions between drugs, targets, and disease pathways, offering insights into potential therapeutic mechanisms and novel targets. The application of network pharmacology to dapagliflozin provides an opportunity to uncover its multi-target effects and explore its broader therapeutic potential in CKD. This study aims to utilize network pharmacology and molecular docking to investigate the mechanisms of dapagliflozin in CKD. By integrating data from diverse databases, we systematically identify and analyze the common molecular targets of dapagliflozin and CKD, construct protein–protein interaction (PPI) networks, and perform gene ontology (GO) and KEGG pathway enrichment analyses. Additionally, molecular docking simulations are conducted to validate the binding interactions of dapagliflozin with key hub proteins. These comprehensive analyses aim to provide novel insights into dapagliflozin’s therapeutic mechanisms, laying the groundwork for future experimental and clinical validation. 2. Materials and Methods 2.1. Drug Activity Evaluation of Dapagliflozin The drug activity of dapagliflozin was evaluated using in silico ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis and drug-likeness prediction through the SwissADME ([56]http://www.swissadme.ch/ accessed on 2 November 2024) and pkCSM platforms ([57]https://biosig.lab.uq.edu.au/pkcsm/ accessed on 2 November 2024). Dapagliflozin’s adherence to Lipinski’s rule of five was assessed to evaluate its potential as an orally bioavailable drug. The analyses included physicochemical property evaluation, lipophilicity and solubility prediction, pharmacokinetic profiling, and toxicity assessment, which were generated using both SwissADME and pkCSM [[58]19]. 2.2. Screening the Targets of Dapagliflozin and Chronic Kidney Disease The potential targets of dapagliflozin were gathered from the SwissTargetPrediction ([59]http://www.swisstargetprediction.ch/ accessed on 4 November 2024), Super-PRED ([60]https://prediction.charite.de/subpages/target_prediction.php/ accessed on 4 November 2024), and similarity ensemble approach or SEA ([61]https://sea.bkslab.org/ accessed on 4 November 2024) [[62]20]. The names of target proteins were translated into gene names in the UniProt ([63]http://www.uniprot.org/ accessed on 5 November 2024) database [[64]21]. The CKD-related genes were derived from the GeneCards ([65]https://www.GeneCards.org/ accessed on 7 November 2024) databases by searching the keywords of chronic kidney disease [[66]22]. 2.3. Construction of Drug-Target Network The common targets of dapagliflozin and CKD were determined using a Venn diagram ([67]https://bioinfogp.cnb.csic.es/tools/venny/index.html accessed on 10 November 2024) [[68]23]. Intersecting genes were the potential targets of dapagliflozin, and they overlapped with CKD. 2.4. Construction of Protein-Protein Interaction (PPI) Network Intersecting genes from a Venn diagram were uploaded to the STRING database ([69]https://string-db.org/ accessed on 12 November 2024), and Homo sapiens was chosen for species [[70]24]. The result from the STRING database was downloaded and imported to Cytoscape 3.8.0 software ([71]https://cytoscape.org/ accessed on 13 November 2024) [[72]25]. Then the protein interaction information was obtained, a network analysis of the PPI network was performed, and the top 10 targets of the degree centrality (DC) values were screened. 2.5. Gene Functions and Pathway Enrichment Analysis with Potential Targets Gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out using ShinyGO 0.80 ([73]http://bioinformatics.sdstate.edu/go/ accessed on 15 November 2024) [[74]26]. The GO enrichment and KEGG pathway analyses were screened for p < 0.05. The top 20 results from the GO enrichment and KEGG pathway analyses were presented. The GO enrichment analysis mainly shows three aspects of biology, such as biological process, molecular component, and cellular component. It is widely used for gene function classification and target function distribution prediction. 2.6. Protein and Compound Preparation To enhance our understanding of the relationship, modes of interaction, and mechanisms of action between the candidate proteins (or hub targets) and dapagliflozin, we employed a computational technique known as molecular docking. This simulation methodology enables the prediction of the affinity and orientation of a compound within a protein’s structure, thereby providing insights into potential binding modes. To initiate this process, we obtained the 3D structures of the pivotal proteins from the Protein Data Bank (PDB) and performed essential cleaning procedures to eliminate water molecules or other small molecules that might interfere with the docking process [[75]27]. This preparatory phase was conducted using BIOVIA Discovery Studio software (Version 2021 Client). The 3D protein structures of the top 10 target proteins, including GAPDH (PDB 6M61), IL-6 (PDB 1ALU), SRC (PDB 1Y57), EGFR (PDB 1M17), HSP90AA1 (PDB 4AWQ), NFKB (PDB 3GUT), CASP3 (PDB 3KJF), HSP90AB1 (PDB 5UCJ), MAPK3 (PDB 4QTB), and GSK3β (PDB 6TCU), were downloaded from the RCSB Protein Data Bank ([76]https://www.rcsb.org/ accessed on 20 November 2024) in PDB format [[77]28]. Details of the proteins were collected from the PDB database ([78]Table 1). The Protein Data Bank (PDB) is a global repository of 3D structural data for major proteins. The co-crystallized ligand and water molecules were removed from the protein structures. Polar hydrogens, charges, solvation parameters, and fragmental volumes were assigned to the protein using the Kollman united atom force field via AutoDock Tools (ADT) (Version 1.1.2) [[79]29]. The cleaned protein structures were then saved in the PDBQT file format. Table 1. Details of the protein targets in the PDB database and the grid docking parameters in molecular docking. Targets PDB ID Method Resolution (Å) R-Value Free R-Value Work Spacing (Å) Center Grid Box X Center Y Center Z Center GAPDH 6M61 X-ray diffraction 1.82 0.228 0.192 0.375 −15.666 6.041 −22.87 IL6 1ALU X-ray diffraction 1.90 0.277 0.213 0.375 2.836 −19.593 9.095 SRC 1Y57 X-ray diffraction 1.91 0.213 0.188 0.375 12.093 34.632 39.141 EGFR 1M17 X-ray diffraction 2.60 0.295 0.251 0.375 24.838 0.016 53.366 HSP90AA1 4AWQ X-ray diffraction 1.60 0.262 0.227 0.375 −2.926 4.130 −4.899 NFKB 3GUT X-ray diffraction 3.59 0.301 0.245 0.375 30.231 −27.423 64.005 CASP3 3KJF X-ray diffraction 2.00 0.206 0.181 0.375 16.346 −5.814 3.906 HSP0AB1 5UCJ X-ray diffraction 1.69 0.197 0.174 0.375 −24.841 99.831 0.583 MAPK3 4QTB X-ray diffraction 1.40 0.175 0.147 0.375 33.748 54.397 49.771 GSK3B 6TCU X-ray diffraction 2.14 0.240 0.202 0.375 −14.502 −14.985 −0.926 [80]Open in a new tab The three-dimensional (3D) structure of dapagliflozin (CID 9887712) was retrieved from the PubChem database ([81]http://pubchem.ncbi.nlm.nih.gov/ accessed on 25 November 2024) and prepared for docking. Known tyrosinase inhibitors or positive controls were also retrieved from co-crystallized protein structures. This preparation involved structural optimization, the addition of missing hydrogen atoms, and charge correction. The ligand was further optimized and converted to a PDB file using UCSF Chimera software (version 1.17.1) [[82]30]. Energy minimization using conjugate and steepest descent methods, along with the addition of charges for correcting ionization, was employed to prepare the ligands. Missing hydrogen atoms and polar hydrogens were added to all ligands at a pH of 7.4. Bond orders, angles, and topology were assigned to optimize the structure. AutoDock Tools (ADT) automatically assigned Gasteiger charges and default atomic parameters. Finally, the optimized structures were saved in PDBQT file format using ADT. 2.7. Molecular Docking Simulation Molecular docking was performed using AutoDock version 4.2. The Lamarckian genetic algorithm method was employed for the molecular docking experiment, and it was conducted with AutoDock4 software. During the procedure, the protein structure was treated as a rigid molecule, while the ligand was considered flexible. Default parameter values in AutoDockTools (ADT) were used for all other settings. For conformational sampling, fifty genetic algorithm (GA) runs were executed with a population size of 200. A docking box was constructed to completely encompass the binding site of the receptor protein. The parameters of the docking box were recorded ([83]Table 1). Molecular docking was carried out using AutoDockTools 1.5.7, where the binding energy magnitude was used to assess the likelihood of binding between the receptor and the ligand. The optimal pose was determined as the conformation with the lowest binding energy (kcal/mol). The interactions of natural ligands or drugs were compared to the best-docked pose of dapagliflozin. Binding energy data from the molecular docking process were gathered using AutoDockTools 1.5.7. Finally, potential protein–ligand interactions and binding modes were analyzed and visualized using the BIOVIA Discovery Studio Visualizer software (Accelrys, San Diego, CA, USA) [[84]19]. 3. Results 3.1. Drug Activity of Dapagliflozin The drug activity of dapagliflozin was evaluated using ADMET analysis and drug-likeness prediction through SwissADME and pkCSM platforms. As shown in [85]Table S1, the analysis revealed key physicochemical properties, pharmacokinetics, and toxicity profiles. The molecular weight (MW) of dapagliflozin is 408.87 g/mol, with a formula of C[21]H[25]ClO[6]. It exhibits six hydrogen bond acceptors, four hydrogen bond donors, and a topological polar surface area (TPSA) of 99.38 Å^2. Its predicted lipophilicity (LogP) values from different models (iLOGP, XLOGP3, WLOGP, Silicos-IT Log P) range from 1.07 to 3.17, with a consensus LogP of 2.18, indicating moderate lipophilicity. Dapagliflozin is classified as soluble based on ESOL predictions and moderately soluble by aliphatic and silicos-IT classifications. In terms of pharmacokinetics, dapagliflozin shows high gastrointestinal (GI) absorption but does not cross the blood–brain barrier (BBB). It is a substrate for P-glycoprotein but not for CYP2D6 or CYP3A4 enzymes. It inhibits CYP2D6 but does not inhibit other key cytochrome P450 enzymes (CYP1A2, CYP2C19, CYP2C9, or CYP3A4). The predicted skin permeability (log Kp) is −7.13 cm/s, and the total clearance is 0.194 log mL/min/kg. From a toxicity perspective, dapagliflozin demonstrates no AMES toxicity, hepatotoxicity, or skin sensitization. It is not a substrate or inhibitor of hERG channels, indicating low cardiac risk. Predicted oral rat acute toxicity (LD50) is 2.475 mol/kg, and chronic toxicity (LOAEL) is 3.63 log mg/kg/day. It also shows moderate environmental toxicity with a Tetrahymena pyriformis toxicity (log µg/L) of 0.289 and minnow toxicity (log mM) of 1.079. Dapagliflozin adheres to Lipinski’s rule of five with zero violations, suggesting good oral bioavailability (bioavailability score of 0.55). No alerts for pan-assay interference compounds alerts (PAINS), Brenk, or lead-likeness were identified. The synthetic accessibility score is 4.52, indicating a moderately complex synthesis. These results highlight dapagliflozin’s favorable drug-likeness profile and suitability for oral administration, with minimal toxicity risks. 3.2. Targets of Dapagliflozin and Chronic Kidney Disease The screening process identified a total of 231 potential targets for dapagliflozin through the SwissTargetPrediction, Super-PRED, and Similarity Ensemble Approach (SEA) platforms. These targets were converted into gene names using the UniProt database for standardization. Concurrently, 16,154 chronic kidney disease (CKD)-related genes were extracted from the GeneCards database using the keyword “chronic kidney disease”. 3.3. Prediction of Common Targets Between Dapagliflozin and Chronic Kidney Disease The common targets of dapagliflozin and CKD were identified using a Venn diagram analysis. Out of the 231 potential targets of dapagliflozin and 16,154 CKD-related genes, a total of 208 overlapping targets were identified, as shown in [86]Figure 1 and [87]Table 2. These overlapping genes represent the potential targets through which dapagliflozin may exert therapeutic effects on CKD. This result highlights a significant overlap and provides a foundation for further investigation into the molecular mechanisms underlying the drug’s action in CKD. Figure 1. [88]Figure 1 [89]Open in a new tab Overlapping targets of dapagliflozin and chronic kidney disease. Table 2. The 208 overlapping targets of dapagliflozin and CKD. Target Common Name Multidrug resistance-associated protein 1 ABCC1 Tyrosine-protein kinase ABL1 ABL1 Acetylcholinesterase ACHE Activin receptor type-1-like ACVRL1 Disintegrin and metalloproteinase domain-containing protein 10 ADAM10 Adenosine kinase ADK Adenosine receptor A1 ADORA1 Adenosine receptor A2a ADORA2A Adenosine receptor A2b ADORA2B Adenosine receptor A3 ADORA3 Adenosylhomocysteinase AHCY Alkaline phosphatase, tissue-nonspecific isozyme ALPL Amine oxidase [copper containing] 3 AOC3 DNA repair nuclease/redox regulator APEX1 APEX1 Cysteine protease ATG4B ATG4B Aurora kinase B AURKB RecQ-like DNA helicase BLM BLM Serine/threonine-protein kinase B-raf BRAF C5a anaphylatoxin chemotactic receptor 1 C5AR1 Carbonic anhydrase 14 CA14 Voltage-gated N-type calcium channel alpha-1B subunit CACNA1B Voltage-gated T-type calcium channel alpha-1H subunit CACNA1H CaM-kinase kinase beta CAMKK2 Calpain 1 CAPN1 Histone-arginine methyltransferase CARM1 CARM1 Caspase-3 CASP3 G2/mitotic-specific cyclin-B1 CCNB1 G1/S-specific cyclin-E1 CCNE1 Cyclin H CCNH Cyclin T1 CCNT1 Cell division cycle 7-related protein kinase CDC7 Cyclin-dependent kinase 1 CDK1 Cyclin-dependent kinase 2 CDK2 Cyclin-dependent kinase 7 CDK7 Cyclin-dependent kinase 9 CDK9 Cystic fibrosis transmembrane conductance regulator CFTR Serine/threonine-protein kinase Chk1 CHEK1 Conserved helix-loop-helix ubiquitous kinase CHUK Cannabinoid receptor 1 CNR1 Cannabinoid receptor 2 CNR2 Casein kinase I isoform alpha CSNK1A1 Casein kinase II subunit alpha CSNK2A1 Cathepsin D CTSD Cathepsin L CTSL C-X-C chemokine receptor type 2 CXCR2 C-X-C chemokine receptor type 3 CXCR3 Cysteinyl leukotriene receptor 2 CYSLTR2 Dihydroorotate dehydrogenase DHODH DNA (cytosine-5)-methyltransferase 1 DNMT1 Dipeptidyl peptidase 2 DPP7 Dipeptidyl peptidase 9 DPP9 Dual-specificity tyrosine-phosphorylation-regulated kinase 1A DYRK1A Epidermal growth factor receptor EGFR Ephrin type-A receptor 5 EPHA5 Endoplasmic reticulum aminopeptidase 1 ERAP1 Coagulation factor XIII A chain F13A1 Coagulation factor VII/tissue factor F3 Fatty-acid amide hydrolase 1 FAAH Fatty acid binding protein, adipocyte FABP4 Squalene synthetase FDFT1 Free fatty acid receptor 4 FFAR4 Fibroblast growth factor 1 FGF1 Fibroblast growth factor 2 FGF2 Fibroblast growth factor receptor 1 FGFR1 Tissue alpha-L-fucosidase FUCA1 Lysosomal alpha-glucosidase GAA Gamma-aminobutyric acid receptor subunit alpha-1 GABRA1 Gamma-aminobutyric acid receptor subunit alpha-5 GABRA5 Cyclin-G-associated kinase GAK Glyceraldehyde-3-phosphate dehydrogenase GAPDH Lysosomal acid glucosylceramidase GBA1 Non-lysosomal glucosylceramidase GBA2 Geranylgeranyl pyrophosphate synthetase GGPS1 Beta-galactosidase GLB1 Glycine receptor subunit alpha-1 GLRA1 Glutaminase kidney isoform, mitochondrial GLS G-protein coupled bile acid receptor 1 GPBAR1 Uracil nucleotide/cysteinyl leukotriene receptor GPR17 G-protein coupled receptor 35 GPR35 Growth factor receptor-bound protein 2 GRB2 Glutamate receptor 2 GRIA2 Glutamate receptor ionotropic, kainate 1 GRIK1 G protein-regulated inducer of neurite outgrowth 1 GRIN1 Metabotropic glutamate receptor 4 GRM4 Glycogen synthase kinase-3 beta GSK3B Glutathione S-transferase Mu 1 GSTM1 Glutathione S-transferase P GSTP1 Beta-glucuronidase GUSB Glycogen [starch] synthase, muscle GYS1 Histone deacetylase 5 HDAC5 Histone deacetylase 7 HDAC7 Hexokinase-1 HK1 Hexokinase-2 HK2 15-hydroxyprostaglandin dehydrogenase [NAD+] HPGD Hypoxanthine-guanine phosphoribosyltransferase HPRT1 GTPase HRas HRAS Histamine H3 receptor HRH3 3-hydroxyacyl-CoA dehydrogenase type-2 HSD17B10 Heat shock protein HSP 90-alpha HSP90AA1 Heat shock protein HSP 90-beta HSP90AB1 Endoplasmic reticulum chaperone BiP HSPA5 Heat shock cognate 71 kDa protein HSPA8 Intercellular adhesion molecule-1 ICAM1 Interleukin-2 IL2 Interleukin-6 IL6 Interleukin-1 receptor-associated kinase 4 IRAK4 Integrin alpha-L ITGAL Integrin beta-1 ITGB1 Integrin beta-2 ITGB2 Tyrosine-protein kinase ITK/TSK ITK Tyrosine-protein kinase JAK2 JAK2 Potassium voltage-gated channel subfamily A member 5 KCNA5 Lysine-specific histone demethylase 1A KDM1A Lysine-specific demethylase 4A KDM4A Lysine-specific demethylase 4C KDM4C Kruppel-like factor 5 KLF5 Kallikrein-1 KLK1 Plasma kallikrein KLKB1 Tyrosine-protein kinase LCK LCK L-lactate dehydrogenase A chain LDHA Galectin-1 LGALS1 Galectin-3 LGALS3 Leukotriene A-4 hydrolase LTA4H Dual specificity mitogen-activated protein kinase 1 MAP2K1 Mitogen-activated protein kinase 1 MAPK1 Mitogen-activated protein kinase 10 MAPK10 Mitogen-activated protein kinase 11 MAPK11 Mitogen-activated protein kinase 14 MAPK14 Mitogen-activated protein kinase 15 MAPK15 Mitogen-activated protein kinase 3 MAPK3 Mitogen-activated protein kinase 8 MAPK8 Hepatocyte growth factor receptor MET Maltase-glucoamylase MGAM Macrophage migration inhibitory factor MIF Neprilysin MME Matrix metalloproteinase 1 MMP1 Matrix metalloproteinase 3 MMP3 NEDD8-activating enzyme E1 regulatory subunit NAE1 Nuclear factor erythroid 2-related factor 2 NFE2L2 Nuclear factor NF-kappa-B p105 subunit NFKB1 Tumor necrosis factor receptor superfamily member 16 NGFR Nitric oxide synthase, inducible NOS2 NADPH oxidase 1 NOX1 Nuclear receptor subfamily 1 group I member 2 NR1I2 Mineralocorticoid receptor NR3C2 GTPase NRas NRAS 5′-nucleotidase NT5E High affinity nerve growth factor receptor NTRK1 NT-3 growth factor receptor NTRK3 Protein O-GlcNAcase OGA P2X purinoceptor 4 P2RX4 P2Y purinoceptor 12 P2RY12 Phosphodiesterase 5A PDE5A Phosphodiesterase 11A PDE11A Platelet-derived growth factor receptor beta PDGFRB 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 PFKFB3 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform PIK3CB Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform PIK3CG Tissue-type plasminogen activator PLAT Urokinase-type plasminogen activator PLAU Lysosomal Pro-X carboxypeptidase PRCP DNA-dependent protein kinase catalytic subunit PRKDC Protein-arginine N-methyltransferase 1 PRMT1 Protein arginine N-methyltransferase 7 PRMT7 Serine protease 1 PRSS1 Proteasome subunit beta type-1 PSMB1 Proteasome subunit beta type-2 PSMB2 Prostaglandin E2 receptor EP1 subtype PTGER1 Prostaglandin G/H synthase 1 PTGS1 Tyrosine-protein phosphatase non-receptor type 1 PTPN1 Tyrosine-protein phosphatase non-receptor type 7 PTPN7 Glycogen phosphorylase, liver form PYGL Glycogen phosphorylase, muscle form PYGM Nuclear receptor ROR-beta RORB Sphingosine 1-phosphate receptor 3 S1PR3 Sphingosine 1-phosphate receptor 4 S1PR4 SUMO-activating enzyme subunit 1 SAE1 Stearoyl-CoA desaturase SCD Sodium channel protein type 4 subunit alpha SCN4A E-Selectin SELE Sucrase-isomaltase SI Excitatory amino acid transporter 1 SLC1A3 Sodium/nucleoside cotransporter 2 SLC28A2 Equilibrative nucleoside transporter 1 SLC29A1 Solute carrier family 2, facilitated glucose transporter member 1 SLC2A1 Sodium/glucose cotransporter 1 SLC5A1 Sodium/myo-inositol cotransporter 2 SLC5A11 Sodium/glucose cotransporter 2 SLC5A2 Probable glucose sensor protein SLC5A4 SLC5A4 Sodium-dependent noradrenaline transporter SLC6A2 Sodium-dependent serotonin transporter SLC6A4 Sodium- and chloride-dependent glycine transporter 2 SLC6A5 Proto-oncogene tyrosine-protein kinase Src SRC Stimulator of interferon genes protein STING1 Tyrosine-protein kinase SYK SYK Substance-K receptor TACR2 Tyrosyl-DNA phosphodiesterase 1 TDP1 Tissue factor pathway inhibitor TFPI Thyroid hormone receptor alpha THRA Thymidine kinase 2, mitochondrial TK2 Transmembrane protease serine 6 TMPRSS6 DNA topoisomerase 2-alpha TOP2A Transcription intermediary factor 1-alpha TRIM24 Tyrosinase TYR Tyrosine-protein kinase receptor TYRO3 TYRO3 Ubiquitin-like modifier-activating enzyme 7 UBA7 Vascular cell adhesion protein 1 VCAM1 Vascular endothelial growth factor A, long form VEGFA [90]Open in a new tab 3.4. Construction of PPI Networks The 208 overlapping genes identified as common targets of dapagliflozin and CKD were uploaded to the STRING database to construct a protein–protein interaction (PPI) network, with Homo sapiens selected as the species, as shown in [91]Figure 2A. The interaction data were exported and imported into Cytoscape 3.10.3 for network visualization and analysis. Figure 2. [92]Figure 2 [93]Open in a new tab The protein–protein interaction (PPI) network of overlapping targets of dapagliflozin and chronic kidney disease. (A) protein-protein interaction (PPI) network of 208 overlapping genes identified as common targets of dapagliflozin and chronic kidney disease (CKD). The network was constructed using the STRING database with Homo sapiens as the selected species and visualized in Cytoscape 3.10.3. The complex network displays interactions among these genes, with edges representing experimentally and computationally determined connections. This method organizes key proteins into clusters based on the MCL cluster network in Cytoscape. (B) The top 10 hub genes from the PPI network, identified using the cytoHubba plugin in Cytoscape based on degree centrality (DC) values. The hub genes include GAPDH, IL-6, SRC, EGFR, HSP90AA1, NFKB1, CASP3, HSP90AB1, MAPK3, and GSK3B. Node coloration, ranging from yellow to dark red, indicates DC scores, with darker red nodes representing higher centrality and greater importance within the network. These hub genes are potential key players in the mechanism of action of dapagliflozin in CKD. Using the Cytoscape plugin cytoHubba, a network analysis was performed to identify the top 10 hub genes based on degree centrality (DC) values as shown in [94]Figure 2B. These hub targets, which represent the most interconnected and potentially critical nodes in the PPI network, are glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-6 (IL-6), proto-oncogene tyrosine-protein kinase Src (SRC), epidermal growth factor receptor (EGFR), heat shock protein HSP 90-alpha (HSP90AA1), nuclear factor NF-kappa-B p105 subunit (NFKB1), caspase-3 (CASP3), heat shock protein HSP 90-beta (HSP90AB1), mitogen-activated protein kinase 3 (MAPK3), and glycogen synthase kinase-3 beta (GSK3β). Their significance within the network is highlighted by node coloration, where darker and redder hues correspond to higher scores, indicating greater importance in the network. These hub targets may play pivotal roles in the mechanism of action of dapagliflozin in CKD. 3.5. KEGG Pathway Enrichment Analysis and GO Enrichment Analysis GO Enrichment analysis and KEGG pathway enrichment analysis were conducted to explore the biological roles of the intersecting genes identified as targets of dapagliflozin in chronic kidney disease (CKD). 3.5.1. KEGG Pathway Enrichment Analysis The KEGG pathway enrichment analysis identified the top 20 significantly enriched pathways associated with the intersecting targets of dapagliflozin and CKD based on their fold enrichment and −log10(FDR) values, as shown in [95]Figure 3 and [96]Table 3. The −log10(FDR) scores indicate the statistical significance of each pathway, with higher values reflecting greater significance. Among these, the most enriched pathway was prostate cancer (hsa05215), followed by lipid and atherosclerosis (hsa05417), pathways in cancer (hsa05200), Rap1 signaling pathway (hsa04015), MAPK signaling pathway (hsa04010), PI3K-Akt signaling pathway (hsa04151), AGE-RAGE signaling pathway in diabetic complications (hsa04933), central carbon metabolism in cancer (hsa05230), EGFR tyrosine kinase inhibitor resistance (hsa01521), neurotrophin signaling pathway (hsa04722), fluid shear stress and atherosclerosis (hsa05418), hepatitis B (hsa05161), Kaposi sarcoma-associated herpesvirus infection (hsa05167), FoxO signaling pathway (hsa04068), chemical carcinogenesis-reactive oxygen species (hsa05208), T cell receptor signaling pathway (hsa04660), prolactin signaling pathway (hsa04917), IL-17 signaling pathway (hsa04657), Alzheimer disease (hsa05010), and Ras signaling pathway (hsa04014). These findings highlight the importance of the pathways involved in biological processes, including cancer-related pathways, cardiovascular and metabolic pathways, cellular signaling pathways, infection-related pathways, and neurological pathways. These pathways may play a crucial role in the therapeutic mechanisms of dapagliflozin in the context of CKD. Figure 3. [97]Figure 3 [98]Open in a new tab The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes associated with dapagliflozin and chronic kidney disease (CKD). The bar plot illustrates the top enriched pathways based on fold enrichment, ranked from most to least enriched. The color gradient represents the −log10(FDR) values, with red indicating the highest statistical significance and blue indicating lower significance. Table 3. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 208 overlapping targets between dapagliflozin and chronic kidney disease. Pathway Description Number of Genes Pathway Genes Fold Enrichment Enrichment FDR hsa05215 Prostate cancer 22 97 23.81 5.9925 × 10^−22 hsa05417 Lipid and atherosclerosis 28 214 13.73 1.2173 × 10^−21 hsa05200 Pathways in cancer 38 530 7.53 1.7505 × 10^−20 hsa04015 Rap1 signaling pathway 25 210 12.50 1.8249 × 10^−18 hsa04010 MAPK signaling pathway 28 294 10.00 3.1128 × 10^−18 hsa04151 PI3K-Akt signaling pathway 30 354 8.89 3.1609 × 10^−18 hsa04933 AGE-RAGE signaling pathway in diabetic complications 19 100 19.94 4.1871 × 10^−18 hsa05230 Central carbon metabolism in cancer 17 70 25.49 4.1871 × 10^−18 hsa01521 EGFR tyrosine kinase inhibitor resistance 17 79 22.59 3.4162 × 10^−17 hsa04722 Neurotrophin signaling pathway 19 119 16.76 9.6469 × 10^−17 hsa05418 Hepatitis B 20 138 15.21 9.6469 × 10^−17 hsa05161 Kaposi sarcoma-associated herpesvirus infection 21 162 13.61 1.2309 × 10^−16 hsa05167 Chemical carcinogenesis-reactive oxygen species 22 194 11.90 3.3219 × 10^−16 hsa04068 FoxO signaling pathway 19 131 15.22 4.5985 × 10^−16 hsa05208 Chemical carcinogenesis-reactive oxygen species 23 223 10.83 4.5985 × 10^−16 hsa04660 T cell receptor signaling pathway 17 103 17.32 2.2045 × 10^−15 hsa04917 Prolactin signaling pathway 15 70 22.49 2.2045 × 10^−15 hsa04657 L-17 signaling pathway 16 93 18.06 8.1213 × 10^−15 hsa05010 Alzheimer disease 27 384 7.38 8.8190 × 10^−15 hsa04014 Ras signaling pathway 22 235 9.83 1.3050 × 10^−14 [99]Open in a new tab 3.5.2. GO Biological Process Enrichment Analysis The top 20 results from the biological process enrichment analysis in the GO enrichment analysis are presented in [100]Figure 4 and [101]Table 4, associated with the analyzed gene set. The GO enrichment analysis, a powerful tool used for gene function classification and functional distribution prediction, revealed key pathways and processes relevant to the gene set under study. The most enriched pathway was “response to organic substance” (GO:0010033) with an FDR of 4.6987 × 10^−34 and a fold enrichment of 3.56, followed by “response to chemical” (GO:0042221; FDR = 9.3426 × 10^−33, fold enrichment = 2.85), and “response to stress” (GO:0006950; FDR = 1.6161Í10-29, fold enrichment = 2.87). Other significantly enriched pathways included cellular response to chemical stimulus (GO:0070887), response to nitrogen compound (GO:1901698), and regulation of biological quality (GO:0065008). Additional notable pathways included phosphorylation (GO:0016310), response to oxygen-containing compound (GO:1901700), and response to organonitrogen compound (GO:0010243). Metabolic and signaling processes, such as protein phosphorylation (GO:0006468), cellular response to organic substance (GO:0071310), and phosphate-containing compound metabolic process (GO:0006796), were also enriched. Furthermore, pathways like response to endogenous stimulus (GO:0009719), regulation of response to external stimulus (GO:0032101), and phosphorus metabolic process (GO:0006793) demonstrated significant enrichment. Other important pathways included intracellular signal transduction (GO:0035556), regulation of multicellular organismal process (GO:0051239), and response to external stimulus (GO:0009605). The pathways “regulation of cell communication” (GO:0010646) and “regulation of signaling” (GO:0023051) were also enriched. These findings highlight the diverse biological processes potentially involved in the therapeutic mechanisms of dapagliflozin, including responses to chemical and environmental stimuli, metabolic regulation, and intracellular signaling. Figure 4. [102]Figure 4 [103]Open in a new tab The Gene Ontology (GO) enrichment analysis of biological processes associated with dapagliflozin and chronic kidney disease (CKD). The dot plot displays the top enriched biological processes, ranked by fold enrichment, with the size of each dot representing the number of genes associated with each process. The color gradient indicates the statistical significance (−log10(FDR)), with red representing the highest significance. Table 4. The GO biological process enrichment analysis of targets shared by dapagliflozin and chronic kidney disease. GO Description Number of Genes Pathway Genes Fold Enrichment Enrichment FDR GO:0010033 Response to organic substance 111 3269 3.56 4.6987 × 10^−34 GO:0042221 Response to chemical 131 4821 2.85 9.3426 × 10^−33 GO:0006950 Response to stress 121 4424 2.87 1.6161 × 10^−29 GO:0070887 Cellular response to chemical stimulus 105 3300 3.34 1.6161 × 10^−29 GO:1901698 Response to nitrogen compound 65 1172 5.82 3.8489 × 10^−29 GO:0065008 GO:0065008 regulation of biological quality 115 4103 2.94 1.5913 × 10^−28 GO:0016310 Phosphorylation 80 1994 4.21 1.7416 × 10^−27 GO:1901700 Response to oxygen-containing compound 75 1752 4.49 3.0411 × 10^−27 GO:0010243 Response to organonitrogen compound 60 1061 5.94 3.0857 × 10^−27 GO:0006468 Protein phosphorylation 71 1684 4.43 3.5421 × 10^−25 GO:0071310 Cellular response to organic substance 87 2609 3.50 7.9542 × 10^−25 GO:0006796 Phosphate-containing compound metabolic process 91 3005 3.18 2.8057 × 10^−23 GO:0009719 Response to endogenous stimulus 68 1660 4.30 2.8057 × 10^−23 GO:0032101 Reg. of response to external stimulus 56 1089 5.40 2.8057 × 10^−23 GO:0006793 Phosphorus metabolic process 91 3030 3.15 4.5212 × 10^−23 GO:0035556 Intracellular signal transduction 88 2847 3.24 5.4800 × 10^−23 GO:0051239 Regulation of multicellular organismal process 90 3024 3.12 1.6576 × 10^−22 GO:0009605 Response to external stimulus 90 3073 3.07 4.9737 × 10^−22 GO:0010646 Regulation of cell communication 97 3602 2.83 1.4809 × 10^−21 GO:0023051 Regulation of signaling 97 3615 2.82 1.8495 × 10^−21 [104]Open in a new tab 3.5.3. GO Molecular Function Enrichment Analysis The molecular function enrichment analysis of dapagliflozin revealed significant enrichment of several key activities. As shown in [105]Figure 5 and [106]Table 5, the most enriched function was protein serine/threonine/tyrosine kinase activity (GO:0004712) with an FDR of 1.5084 × 10^−24. This was followed by the phosphotransferase activity alcohol group as acceptor (GO:0016773), protein kinase activity (GO:0004672), kinase activity (GO:0016301), catalytic activity acting on a protein (GO:0140096), nucleotide binding (GO:0000166), nucleoside phosphate binding (GO:1901265), small molecule binding (GO:0036094), transferase activity transferring phosphorus-containing groups (GO:0016772), anion binding (GO:0043168), adenyl nucleotide binding (GO:0030554), carbohydrate derivative binding (GO:0097367), adenyl ribonucleotide binding (GO:0032559), ATP binding (GO:0005524), purine nucleotide binding (GO:0017076), ribonucleotide binding (GO:0032553), purine ribonucleotide binding (GO:0032555), purine ribonucleoside triphosphate binding (GO:0035639), protein serine/threonine kinase activity (GO:0004674), and protein serine kinase activity (GO:0106310). These findings emphasize the critical molecular functions, particularly kinase activity and nucleotide binding, which may contribute to the therapeutic mechanisms of dapagliflozin in CKD. Figure 5. [107]Figure 5 [108]Open in a new tab The Gene Ontology (GO) molecular function enrichment analysis of intersecting targets of dapagliflozin and chronic kidney disease (CKD). The top enriched molecular functions are depicted, ranked by fold enrichment and statistical significance (−log10[FDR]). The size of the dots corresponds to the number of genes associated with each pathway, while the color represents the statistical significance, with red indicating higher −log10(FDR) values. Table 5. The GO molecular function enrichment analysis of intersecting targets shared by dapagliflozin and chronic kidney disease. GO Description Number of Genes Pathway Genes Fold Enrichment Enrichment FDR GO:0004712 Protein serine/threonine/tyrosine kinase activity 41 470 9.16 1.5084 × 10^−24 GO:0016773 Phosphotransferase activity alcohol group as acceptor 49 748 6.88 1.5688 × 10^−24 GO:0004672 Protein kinase activity 45 632 7.47 4.2997 × 10^−24 GO:0016301 Kinase activity 51 849 6.30 4.2997 × 10^−24 GO:0140096 Catalytic activity acting on a protein 84 2577 3.42 1.7598 × 10^−23 GO:0000166 Nucleotide binding 80 2381 3.53 4.4649 × 10^−23 GO:1901265 Nucleoside phosphate binding 80 2382 3.53 4.4649 × 10^−23 GO:0036094 Small molecule binding 85 2743 3.25 1.5947 × 10^−22 GO:0016772 Transferase activity transferring phosphorus-containing groups 52 1008 5.41 5.0793 × 10^−22 GO:0043168 Anion binding 82 2630 3.27 8.4681 × 10^−22 GO:0030554 Adenyl nucleotide binding 65 1741 3.92 1.4539 × 10^−20 GO:0097367 Carbohydrate derivative binding 78 2505 3.27 1.5727 × 10^−22 GO:0032559 Adenyl ribonucleotide binding 64 1729 3.89 4.5342 × 10^−22 GO:0005524 ATP binding 62 1662 3.92 1.5083 × 10^−19 GO:0017076 Purine nucleotide binding 70 2120 3.47 1.6300 × 10^−19 GO:0032553 Ribonucleotide binding 70 2123 3.46 1.6547 × 10^−19 GO:0032555 Purine ribonucleotide binding 69 2106 3.44 4.7366 × 10^−19 GO:0035639 Purine ribonucleoside triphosphate binding 65 2034 3.35 3.1939 × 10^−17 GO:0004674 Protein serine/threonine kinase activity 31 471 6.91 1.0688 × 10^−15 GO:0106310 Protein serine kinase activity 28 376 7.82 1.6779 × 10^−15 [109]Open in a new tab 3.5.4. GO Cellular Component Enrichment Analysis The GO cellular component enrichment analysis for dapagliflozin and chronic kidney disease (CKD) identified key cellular structures and regions significantly associated with the intersecting targets, ranked by FDR values and fold enrichment, as shown in [110]Figure 6 and [111]Table 6. The most enriched component was the integral component of plasma membrane (GO:0005887) with an FDR of 1.8106 × 10^−16 and a fold enrichment of 3.491. This was closely followed by the intrinsic component of plasma membrane (GO:0031226) and vesicle (GO:0031982). Other significantly enriched components included cytoplasmic vesicle (GO:0031410), intracellular vesicle (GO:0097708), and plasma membrane region (GO:0098590). Enrichment was also observed in cell surface (GO:0009986) and ficolin-1-rich granule (GO:0101002). Further enriched components included secretory granule (GO:0030141) and extracellular exosome (GO:0070062). The analysis also identified extracellular space (GO:0005615), extracellular organelle (GO:0043230), and extracellular vesicle (GO:1903561). Other notable components included secretory vesicle (GO:0099503), ficolin-1-rich granule lumen (GO:1904813), and cell junction (GO:0030054). Additionally, receptor complex (GO:0043235), extracellular region (GO:0005576), and membrane raft (GO:0045121) were significantly enriched. These results highlight the diverse cellular components potentially involved in the mechanisms of dapagliflozin in CKD, with a focus on plasma membrane-related structures, vesicles, and extracellular components critical for cellular communication and signaling. Figure 6. [112]Figure 6 [113]Open in a new tab The Gene Ontology (GO) cellular component enrichment analysis of intersecting targets of dapagliflozin and chronic kidney disease (CKD). The size of the dots represents the number of genes associated with each cellular component, and the color indicates the level of statistical significance, with red representing the most significant pathways. Table 6. GO cellular component enrichment analysis of intersecting targets shared by dapagliflozin and chronic kidney disease. GO Description Number of Genes Pathway Genes Fold Enrichment Enrichment FDR GO:0005887 Integral component of plasma membrane 63 1894 3.49 1.8106 × 10^−16 GO:0031226 Intrinsic component of plasma membrane 63 1978 3.34 7.9143 × 10^−16 GO:0031982 Vesicle 97 4466 2.28 5.4624 × 10^−15 GO:0031410 Cytoplasmic vesicle 73 2849 2.69 4.6323 × 10^−14 GO:0097708 Intracellular vesicle 73 2851 2.69 4.6323 × 10^−14 GO:0098590 Plasma membrane region 46 1332 3.62 1.4203 × 10^−12 GO:0009986 Cell surface 40 1050 4.00 3.1974 × 10^−12 GO:0101002 Ficolin-1-rich granule 20 223 9.41 3.1974 × 10^−12 GO:0030141 Secretory granule 38 987 4.04 9.6672 × 10^−12 GO:0070062 Extracellular exosome 58 2316 2.63 1.7271 × 10^−10 GO:0005615 Extracellular space 75 3577 2.20 2.0066 × 10^−10 GO:0043230 Extracellular organelle 58 2343 2.60 2.0066 × 10^−10 GO:0065010 Extracellular membrane-bounded organelle 58 2343 2.60 2.0066 × 10^−10 GO:1903561 Extracellular vesicle 58 2342 2.60 2.0066 × 10^−10 GO:0099503 Secretory vesicle 39 1165 3.51 2.0319 × 10^−10 GO:1904813 Ficolin-1-rich granule lumen 15 142 11.09 2.1738 × 10^−10 GO:0030054 Junction 56 2293 2.56 7.1862 × 10^−10 GO:0043235 Receptor complex 23 429 5.63 7.1862 × 10^−10 GO:0005576 Extracellular region 87 4673 1.95 8.3852 × 10^−10 GO:0045121 Membrane raft 20 351 5.98 4.7000 × 10^−9 [114]Open in a new tab 3.6. Molecular Docking Verification To validate the credibility of drug–target interactions, molecular docking analysis was specifically conducted on the 10 hub proteins selected as targets. In this study, the stability and inhibitory potency of the ligand–receptor binding were assessed based on the binding energies between the ligand and protein. A binding energy below −8.0 kcal/mol or a docking score better than that of the positive control served as the criterion or cutoff, indicating a robust conformation of ligand binding to the receptor. The results of molecular docking for dapagliflozin are summarized in [115]Table 7. Dapagliflozin exhibited significant inhibition against EGFR and GSK3β proteins, with docking scores of −8.42 and −7.70 kcal/mol, respectively. Moreover, dapagliflozin demonstrated a notable binding affinity to IL-6, with a binding energy better than that of HY-115910 (positive control). However, dapagliflozin bound to IL-6 at a different binding site compared to HY-115910 ([116]Figure 7A,B). Additionally, dapagliflozin formed five hydrogen bonds with LEU64, LYS66, LYS86, GLU93, and THR138 of IL-6, along with hydrophobic interactions involving PRO65 and PRO139, as depicted in [117]Figure 7C. As shown in [118]Figure 8A, dapagliflozin also exhibited strong binding affinity against EGFR by binding at a site similar to that of erlotinib. Dapagliflozin formed seven hydrogen bonds with CYS773, THR766, ALA719, GLU738, THR830, and two with ASP831 ([119]Figure 8C). Furthermore, dapagliflozin engaged in several hydrophobic interactions with CYS773, LEU820, ALA719, and VAL702 ([120]Figure 8C). Notably, dapagliflozin demonstrated a significant binding affinity to GSK3β, with a binding energy of −7.70 kcal/mol, which was better than that of the positive control N1Q. Dapagliflozin formed four hydrogen bonds with ASP200, LYS85, and two with GLU97 in its interaction with GSK3β, binding at the same site as the positive control drug ([121]Figure 9A–C). These findings highlight the bioactive potential of dapagliflozin, which exhibited notable binding energy and interactions with the top three key hub targets, indicating its significance in modulating these crucial molecular targets. Table 7. Docking score and inhibition constant of dapagliflozin against the top 10 hub targets compared to the positive control. No. Protein Drugs Docking Score (kcal/mol) Inhibition Constant (K[i]) 1 GAPDH Heptelidic acid −6.69 12.38 µM (PDB 6M61) Dapagliflozin −6.27 25.38 µM 2 IL6 HY-115910 −6.73 11.7 µM (PDB 1ALU) Dapagliflozin −6.83 9.87 µM 3 SRC MPZ600 −8.98 262.77 nM (PDB 1Y57) Dapagliflozin −6.53 16.23 µM 4 EGFR Erlotinib −7.02 7.19 µM (PDB 1M17) Dapagliflozin −8.42 673.48 nM 5 HSP90AA1 N-benzyl-6-[(3-endo)-3-{[(3-methoxy-2-methylphenyl)carbonyl]amino}-8-az abicyclo[3.2.1]oct-8-yl]pyridine-3-carboxamide (592) −13.86 69.38 pM (PDB 4AWQ) Dapagliflozin −8.5 587.42 nM 6 NFKB BAY11-7082 −6.72 11.92 µM (PDB 3GUT) Dapagliflozin −6.23 27.3 µM 7 CASP3 (3S)-3-({[(5S,10aS)-2-{(2S)-4-carboxy-2-[(phenylacetyl)amino]butyl}-1,3 -dioxo-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5 -yl]carbonyl}amino)-4-oxopentanoic acid (B92) −10.56 18.06 nM (PDB 3KJF) Dapagliflozin −7.46 3.39 µM 8 HSP0AB1 (5-fluoroisoindolin-2-yl)(4-hydroxy-5-isopropylbenzo[d]isoxazol-7-yl)me thanone (KU3) −8.29 841.21 nM (PDB 5UCJ) Dapagliflozin −6.51 16.83 µM 9 MAPK3 SCH772984 −13.06 266.89 pM (PDB 4QTB) Dapagliflozin −8.87 314.47 nM 10 GSK3B 5-[2,3-bis(fluoranyl)phenyl]-~{N}-[[1-(2-methoxyethyl)piperidin-4-yl]me thyl]-1~{H}-indazole-3-carboxamide (N1Q) −7.67 2.41 µM (PDB 6TCU) Dapagliflozin −7.70 2.27 µM [122]Open in a new tab Criteria: strong inhibition when there is a docking score less than −8.0 kcal/mol and a docking score better than the positive control. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, SRC: Proto-oncogene tyrosine-protein kinase Src, EGFR: epidermal growth factor receptor, HSP90AA1: heat shock protein HSP 90-alpha, CASP3: caspase-3, MAPK3: mitogen-activated protein kinase 3, GSK3B: glycogen synthase kinase-3 beta. Figure 7. [123]Figure 7 [124]Open in a new tab Visualization of the protein–ligand docking interactions. (A) Three-dimensional view of dapagliflozin (red) and HY-115910 (black) docked with IL-6. (B) Two-dimensional schematic of the docking interaction between HY-115910 (positive control) and IL-6. (C) Two-dimensional schematic of the docking interaction between dapagliflozin and IL-6. Figure 8. [125]Figure 8 [126]Open in a new tab Visualization of the protein–ligand docking interactions. (A) Three-dimensional view of dapagliflozin (red) and erlotinib (black) docked with EGFR. (B) Two-dimensional schematic of the docking interaction between erlotinib (positive control) and EGFR. (C) Two-dimensional schematic of the docking interaction between dapagliflozin and EGFR. Figure 9. [127]Figure 9 [128]Open in a new tab Visualization of the protein–ligand docking interactions. (A) A 3D structure showing the docking of dapagliflozin (in red) and N1Q (in black) with the GSK3β protein. (B) A 2D schematic of N1Q (positive control) binding with GSK3β. (C) A 2D schematic of the docking interaction between dapagliflozin and GSK3β. 4. Discussion This study demonstrates the therapeutic potential of dapagliflozin in CKD by identifying 208 shared molecular targets through network pharmacology approaches. These targets, derived from a combination of dapagliflozin-related and CKD-related gene datasets, highlight the drug’s ability to modulate key biological processes implicated in CKD. The analysis of the PPI network identified ten hub proteins, including GAPDH, IL-6, SRC, EGFR, HSP90AA1, NFKB1, CASP3, HSP90AB1, MAPK3, and GSK3β, which are central nodes in the network and may serve as pivotal mediators of dapagliflozin’s therapeutic effects [[129]16]. The ADMET analysis affirmed dapagliflozin’s favorable pharmacokinetic and toxicity profiles, establishing it as a promising oral therapeutic for CKD. The drug demonstrated high gastrointestinal absorption, low toxicity risks, and no violations of Lipinski’s rule of five, ensuring good oral bioavailability [[130]31]. Additionally, its low cardiac and hepatotoxicity profiles further reinforce its suitability for long-term use. The GO enrichment analysis provided valuable insights into biological processes, molecular functions, and cellular components related to dapagliflozin’s targets. Enriched biological processes included responses to chemical stimuli, signaling regulation, and metabolic processes, underscoring its role in mitigating inflammation and oxidative stress. Molecular function analysis emphasized kinase activity, nucleotide binding, and catalytic activity, which are essential for intracellular signaling and cellular regulation. Cellular component enrichment highlighted the significance of plasma membrane structures, vesicles, and extracellular regions as mediators of the drug’s effects [[131]32]. The KEGG pathway analysis identified critical pathways, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and AGE-RAGE signaling in diabetic complications, which are closely associated with CKD pathophysiology [[132]33,[133]34,[134]35]. The PI3K-Akt pathway is involved in renal cell survival, proliferation, and immune responses, and its dysregulation contributes to renal fibrosis, chronic inflammation, and oxidative damage. Excessive activation of PI3K-Akt promotes epithelial-to-mesenchymal transition (EMT) and mitochondrial dysfunction, exacerbating renal injury [[135]33]. Conversely, the inhibition of this pathway has been shown to mitigate renal fibrosis and inflammation. Similarly, the MAPK pathway is implicated in CKD progression by inducing pro-inflammatory cytokines and fibrotic mediators, leading to tubulointerstitial fibrosis and glomerulosclerosis [[136]36]. MAPK activation triggers oxidative stress and apoptosis in renal tubular cells, further contributing to CKD pathogenesis. The AGE-RAGE pathway, particularly relevant in diabetic nephropathy, accelerates CKD progression through the accumulation of advanced glycation end-products (AGEs), which activate NF-κB signaling, increase oxidative stress, and induce endothelial dysfunction and podocyte injury, resulting in inflammation and fibrosis, promoting proteinuria and renal function decline [[137]35]. These pathways, involved in cellular proliferation, oxidative stress, inflammation, and metabolic regulation, provide mechanistic insights into dapagliflozin’s diverse effects. While direct evidence of dapagliflozin’s modulation of the PI3K-Akt, MAPK, and AGE-RAGE pathways specifically in CKD models is limited, several studies suggest its influence on these pathways in related contexts. A recent in vitro study demonstrated that dapagliflozin attenuates isoproterenol-induced hypertrophy in cardiomyocytes by activating the AKT pathway, leading to reduced oxidative stress and inflammation. In aortic endothelial cells, dapagliflozin restored AKT and PI3K expression, enhanced MAPK activation, and downregulated inflammatory cytokines, indicating its potential to preserve vascular function and improve endothelial health. Additionally, dapagliflozin upregulated NRF2 expression, positively influencing β-cell function and stress response [[138]37]. Regarding the AGE-RAGE pathway, the activation of RAGE is increased in CKD and contributes to cellular dysfunction, tissue injury, fibrosis, and inflammation. While direct modulation of the AGE-RAGE pathway by dapagliflozin in CKD models has not been conclusively demonstrated, the drug’s anti-inflammatory and antioxidative properties may indirectly influence this pathway, potentially mitigating RAGE-mediated effects [[139]37]. Regarding the AGE-RAGE pathway, the activation of RAGE is increased in CKD and contributes to cellular dysfunction, tissue injury, fibrosis, and inflammation. While direct modulation of the AGE-RAGE pathway by dapagliflozin in CKD models has not been conclusively demonstrated, the drug’s anti-inflammatory and antioxidative properties may indirectly influence this pathway, potentially mitigating RAGE-mediated effects [[140]38]. Findings from the DAPA-CKD trial provide strong clinical evidence of dapagliflozin’s renoprotective effects beyond glucose lowering. In this trial, dapagliflozin significantly reduced CKD progression by 39% and all-cause mortality by 31%, which are benefits observed regardless of diabetes status. These findings suggest that dapagliflozin may indirectly influence inflammation, oxidative stress, and fibrotic pathways, which are central to CKD progression [[141]10]. Similarly, the DAPA-HF trial showed a 26% reduction in cardiovascular death or heart failure hospitalization, further supporting its disease-modulating effects [[142]39]. Other pathways related to lipid metabolism, atherosclerosis, and cardiovascular processes further support the potential of dapagliflozin to manage CKD complications [[143]40]. Interestingly, our analysis revealed a potential connection between prostate cancer, hepatitis B, and CKD progression. We hypothesize that this association may be attributed to shared pathophysiological mechanisms, including inflammation, immune activation, and cellular proliferation. Prostate cancer has been linked to a higher incidence of acute kidney injury (AKI) and CKD, with studies reporting a 1.47-fold increased risk of AKI in prostate cancer patients compared to those without cancer. Additionally, androgen deprivation therapy, a common treatment for prostate cancer, has been associated with renal complications, further supporting a possible connection between prostate cancer-related pathways and CKD progression [[144]41]. Moreover, chronic hepatitis B virus (HBV) infection is a recognized risk factor for CKD development, with HBV-infected individuals exhibiting a higher prevalence of CKD and glomerular diseases, such as membranous nephropathy and polyarteritis nodosa, contributing to renal injury and dysfunction [[145]42]. The identification of prostate cancer and hepatitis B pathways in our KEGG analysis suggests that these conditions may have a broader impact on renal pathology. In a previous study, network pharmacology was utilized to examine the therapeutic roles of canagliflozin and dapagliflozin in atherosclerosis, revealing that these drugs act by targeting key molecules such as Akt1, MAPK1, MAPK14, SRC, and EGFR [[146]43]. Building on this, our recent investigation employed network pharmacology and molecular docking to explore the therapeutic potential of dapagliflozin in chronic kidney disease (CKD). By integrating data from multiple databases, we identified 208 CKD-related targets for dapagliflozin, focusing specifically on the ten most interconnected proteins, which are integral to the biological mechanisms underlying CKD pathogenesis. These findings underscore dapagliflozin’s ability to modulate diverse biological pathways and functions relevant to CKD progression. Clinical evidence further supports dapagliflozin’s efficacy, demonstrating a significant reduction in the risk of a 50% or greater decline in the estimated glomerular filtration rate (eGFR), progression to end-stage kidney disease, or death from renal or cardiovascular causes, compared to the placebo, in CKD patients regardless of diabetes status [[147]10]. Additionally, in patients with stage 4 CKD and albuminuria, dapagliflozin consistently mitigated major kidney and cardiovascular risks and slowed eGFR decline without increasing adverse outcomes, aligning with broader trial findings [[148]44]. The results underscore dapagliflozin’s significance as a therapeutic option for effectively managing CKD in a wide range of patient populations. To complement our network pharmacology analysis, molecular docking revealed significant binding affinities between dapagliflozin and key proteins. The analysis confirmed dapagliflozin’s high binding affinity for critical targets, including EGFR, GSK3β, and IL-6. Notably, dapagliflozin exhibited a binding energy of −8.42 kcal/mol with EGFR, surpassing that of erlotinib. This interaction was facilitated by hydrogen bonds and hydrophobic interactions, indicating a stable ligand-receptor conformation ([149]Table 2). The EGFR pathway plays a vital role in kidney development, tissue repair, and electrolyte balance. Dysregulation of this pathway has been extensively linked to the onset and progression of chronic kidney diseases, such as diabetic nephropathy, chronic allograft nephropathy, and polycystic kidney disease. This is attributed to its involvement in promoting renal cell proliferation, fibrosis, and inflammation. Experimental evidence strongly supports that aberrant EGFR signaling contributes to many forms of chronic kidney disease. Furthermore, abnormal EGFR activation has been implicated in mediating progressive kidney injury, particularly in diabetic kidney disease [[150]45]. Inhibiting EGFR expression or activity has been shown to slow the progression of diabetic kidney injury, suggesting that directly targeting EGFR or its associated signaling pathways may be an effective strategy for preventing progressive kidney damage caused by diabetes [[151]46]. Similarly, dapagliflozin demonstrated a high level of inhibition toward glycogen synthase kinase 3 beta (GSK3β), with a docking score of −7.70 kcal/mol, outperforming the positive control, N1Q. The strong binding affinities with both EGFR and GSK3β underscore dapagliflozin’s potential as a multi-target therapeutic agent in CKD. GSK3β contributes to mitochondrial damage and apoptosis by directly phosphorylating and activating pro-apoptotic proteins Bax and Caspase-3 [[152]47]. Additionally, GSK3β activates the p65 subunit of NF-κB, thereby amplifying inflammatory processes [[153]48]. It also inhibits Nrf2, a key antioxidant defender, by phosphorylating it and promoting its nuclear exclusion. This suppression of Nrf2 leads to oxidative stress in the kidney and impedes the transition from acute kidney injury (AKI) to CKD [[154]49]. Furthermore, GSK3β-mediated Keap1-independent regulation of the Nrf2 antioxidant response acts as a molecular rheostat in the AKI-to-CKD transition [[155]50]. These processes slow tubular recovery and contribute to the progression from AKI to CKD [[156]51]. Dapagliflozin also exhibited significant binding affinity to IL-6, surpassing the binding energy of the positive control, HY-115910. Circulating interleukin-6 (IL-6) levels are often elevated in type 2 diabetes mellitus (T2DM) [[157]52]. Increasing evidence highlights the critical role of IL-6 in the pathophysiology of cardiovascular and renal dysfunction [[158]53], with IL-6 being recognized as a major contributor to kidney diseases [[159]54]. Notably, dapagliflozin has been shown to reduce IL-6 levels more effectively than other medications. The anti-inflammatory properties of SGLT2 inhibitors, including dapagliflozin, support their broader use in managing diabetic complications related to kidney inflammation [[160]55,[161]56,[162]57]. Although EGFR activation contributes to tubulointerstitial fibrosis and renal dysfunction, direct evidence of dapagliflozin modulating this pathway in CKD is currently lacking. However, its anti-inflammatory and antifibrotic properties suggest potential indirect effects. Similarly, IL-6, a key pro-inflammatory cytokine in CKD, drives systemic inflammation and endothelial dysfunction. Clinical studies indicate that dapagliflozin reduces IL-6 levels in patients with CKD and diabetes, highlighting a direct anti-inflammatory effect. This aligns with dapagliflozin’s broader anti-inflammatory profile, reinforcing the relevance of IL-6 in CKD pathophysiology [[163]58]. This study integrates network pharmacology and molecular docking to offer a comprehensive understanding of dapagliflozin’s mechanisms of action. It underscores the drug’s potential to function beyond its primary role as an SGLT2 inhibitor, providing a promising approach for CKD treatment through multi-target modulation. The findings from this study also provide a strong foundation for future experimental research and clinical applications in CKD treatment. The identification of 208 overlapping molecular targets and key signaling pathways, including PI3K-Akt, MAPK, and AGE-RAGE, suggests potential therapeutic mechanisms for dapagliflozin. Future in vitro and in vivo studies should focus on validating these pathways’ roles in CKD progression and assessing dapagliflozin’s efficacy in modulating them. Additionally, given its ability to target multiple aspects of CKD pathophysiology—such as inflammation, oxidative stress, and metabolic dysregulation—dapagliflozin could be explored as part of combination therapies to enhance renoprotective effects. Investigating its interactions with key proteins such as EGFR, IL-6, and GSK3β may offer new insights into optimizing treatment strategies. Furthermore, personalized medicine approaches should be considered to identify patient-specific factors that influence drug response. Biomarker-driven studies may help stratify patients based on CKD progression risk and predict responsiveness to dapagliflozin. While clinical trials have demonstrated its efficacy in reducing renal injury markers and slowing eGFR decline, additional studies are needed to evaluate its benefits in broader CKD populations, including non-diabetic CKD, polycystic kidney disease, and transplant recipients. Long-term trials should assess its role in delaying dialysis initiation and reducing cardiovascular events. Additionally, novel drug formulations, such as nanotechnology-based delivery systems, could be explored to improve dapagliflozin’s bioavailability and target specificity while minimizing systemic side effects. 5. Conclusions This study demonstrates the potential of dapagliflozin as a therapeutic agent for chronic kidney disease (CKD) by identifying 208 overlapping targets and elucidating its multi-target mechanisms through network pharmacology and molecular docking. Key pathways, including PI3K-Akt, MAPK, and AGE-RAGE signaling, were identified as critical to CKD pathophysiology, with hub proteins such as EGFR, IL-6, and GSK3β validated for their strong binding affinity to dapagliflozin. These findings highlight dapagliflozin’s ability to modulate inflammation, oxidative stress, and metabolic dysregulation, suggesting that its therapeutic utility extends beyond its established role as an SGLT2 inhibitor. This study emphasizes the value of computational approaches in drug discovery and provides a foundation for the further experimental validation and clinical application of dapagliflozin in CKD management. Acknowledgments