<p>Kidney disease leads to the gradual loss of kidney function, impairing waste and fluid filtration. Major causes include diabetic nephropathy, hypertension, glomerulonephritis, polycystic kidney disease, and drug-induced nephrotoxicity. Diabetic nephropathy is the primary cause of end-stage kidney disease, impacting about one-third of individuals with type 1 diabetes and 10–20% of those with type 2 diabetes. Additionally, nephrotoxic drugs like cisplatin, doxorubicin, and gentamicin contribute to kidney damage. This study investigates berberine’s therapeutic potential in kidney disease using network pharmacology, identifying its molecular targets, biological pathways, and multi-target mechanisms to assess its viability as a novel treatment option. Berberine and kidney disease-related targets were identified using Swiss Target Prediction, Pharm Mapper, TCMSP, Gene Cards, Dis Ge NET, and OMIM. Overlapping targets were analyzed, and a protein-protein interaction network was constructed using STRING. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to identify key biological processes and pathways. Auto Dock and PyMOL were used to evaluate Berberine’s binding affinity to key target proteins, identifying the most promising target-ligand complexes. The stability and dynamics of docked complexes were assessed using GROMACS for 100 ns. Root mean square deviation, root mean square fluctuation, and binding free energy were analyzed. PPI network analysis identified key Berberine targets, including MAPK14, PTGS2, JAK2, and NR3C2. KEGG analysis suggested its role in amyloid-beta formation, APP metabolism, and cellular response to amyloid-beta. Molecular docking revealed the highest binding affinities for PTGS2 (-9.9 kcal/mol), NR3C2 (-9.7 kcal/mol), JAK2 (-8.8 kcal/mol), and MAPK14 (-8.8 kcal/mol). MD simulations showed 5ZTY_CAY10526 had stronger interactions (RMSD 4.01 Å), while 5ZTYBerberine exhibited greater structural stability (RMSD 4.62 Å), supporting its therapeutic potential in kidney disease. This study demonstrates berberine’s potential as a multi-target therapeutic agent for kidney disease. Network pharmacology identified key targets including MAPK14, PTGS2, JAK2, and NR3C2, linked to inflammation, oxidative stress, and renal injury pathways. Enrichment analyses highlighted berberine’s involvement in oxidative stress response, amyloid-beta regulation, and cytokine signalling pathways. Molecular docking revealed strong binding affinities, particularly toward PTGS2 and NR3C2. Molecular dynamics simulations confirmed berberine’s stable binding profile, with greater structural stability compared to standard drugs. These results suggest that berberine could modulate crucial nephrotoxicity mechanisms and warrants further preclinical validation as a promising nephroprotective therapeutic candidate. The findings highlight berberine’s potential in modulating key nephrotoxicity pathways, supporting further research on its therapeutic efficacy and safety in kidney disease. MD simulation showed 5ZTY_CAY10526 had stronger interactions, while 5ZTY_Berberine provided greater structural stability, reinforcing its therapeutic potential.</p>