The cortex-medulla axis plays a key role in kidney fibrosis and is associated with ion channel pathways. While differentially expressed proteins (DEPs) in these sub-regions have been quantified in previous studies, their profiling in the context of chronic kidney disease (CKD) remains unexplored. Therefore, this study aims to identify DEPs between cortex and medulla in sham and 5/6 nephrectomy (Nx) rats to specify proteins associated with CKD progression. SD rats underwent chronic renal failure (CRF) induction via 5/6 Nx procedure, removal of one whole kidney and two-thirds of the other kidney. Sham and CRF rats were sacrificed after 8 weeks following the procedure. Kidney cortex and medulla were trypsinized and labeled with TNT. LC-MS/MS analysis were performed, and raw data were analyzed using the FragPipe computational platform. DEPs were quantified into four major groups: sham cortex (sCortex), sham medulla (sMedulla), CRF cortex (cCortex), and CRF medulla (cMedulla). Functional data of the CRF rats exhibit increased blood pressure, plasma creatinine, BUN, and urine protein indicating similarity to CKD. Compared to its CRF counterparts, sCortex had higher expression of metabolic process-related proteins and sMedulla included more transport-related proteins. GO enrichment analysis of DEPs identified upregulated pathways related to indole-containing compounds and calcium ion regulation in the cCortex, and strong correlations were observed among tyrosine metabolism, indole metabolism, urea cycle, and toxin metabolic processes, which are biomarkers of uremic toxins in CKD. In cMedulla, DEPs were enriched in extracellular matrix (ECM) organization, cellular response to fibroblasts growth factor stimulus, and response to hyperoxia, including collagen families and signaling components (Glcm, Gclc). Among the collagen families, Col1a1 and Col11a1 were identified as major mediators linking the three categories listed. Upregulation of fibroblast, ECM, and hyperoxia-related components in the cMedulla suggest greater hyperoxia-induced EMT-derived fibroblast activation and collagen accumulation than in the cCortex.