Proteomic Characterization of ADPKD Pathogenesis in Monoclonal mIMCD3 Cells

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Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent inherited kidney disorder, mainly caused by mutations in PKD1 and PKD2. ADPKD leads to progressive renal cyst formation, culminating in kidney failure and the need for renal replacement therapy. Despite research progress, diagnostic and therapeutic challenges persist, along with limited understanding of its molecular pathogenesis. This study aimed to perform a comprehensive proteomic analysis of a monoclonal cell line to elucidate ADPKD’s molecular mechanisms. Methods: Protein lysates from WT and three Pkd1 knockout mIMCD3 cell clones analyzed by the Proteomics Platform of the Health Research Institute of Santiago de Compostela. LC-MS/MS was used for protein identification (Data-Dependent Acquisition, DDA) and quantification (Sequential Window Acquisition of All Theoretical Mass Spectra, SWATH). We filtered data for proteins with a p-value<0.05 and a fold change >1.5. Additionally, mIMCD3 cell cycle was analyzed by flow cytometry. Results: PCA revealed distinct clustering between KO and WT groups. SWATH identified 334 differentially expressed proteins, while DDA detected 765, with 291 overlapping both methods. STRING enrichment of dysregulated proteins highlighted mitochondrial metabolism and RNA-related processes. In Pkd1 KO mIMCD3 cells, glycolysis showed only two glycolytic proteins with opposing regulation, complicating interpretation. Fatty acid oxidation was upregulated, while TCA cycle analysis was inconclusive due to variable enzyme activity. Oxidative phosphorylation proteins were largely upregulated, suggesting elevated energy demand and oxygen use, contrary to typical ADPKD descriptions. Stress and apoptosis markers indicated mitochondrial stress, with increased pro-apoptotic and decreased anti-apoptotic proteins. Flow cytometry showed WT cells with a Gaussian distribution, while KO cells displayed a bimodal pattern. Cell cycle analysis revealed reduced S phase and increased G2/M phase in KO cells, suggesting genomic instability. Conclusion: Proteomic analysis of mIMCD3 cells identified mitochondrial metabolism as the most enriched term among dysregulated proteins. Pkd1 KO cells also exhibited altered population distribution and cell cycle profiles, indicating genomic instability. Funding: Government Support – Non-U.S.

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