#694 Mitochondrial translation reveals a therapeutic vulnerability in autosomal dominant polycystic kidney disease

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Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by the formation of multiple renal cysts originating from the epithelial tubular cells, progressively leading to end-stage renal disease. ADPKD is caused by loss-of-function mutations in either PKD1 (80% of patients) or PKD2 (15% of patients) genes, which encode the polycystins (PCs) 1 and 2, respectively. Metabolic dysregulation and mitochondrial dysfunction are pivotal in the pathophysiology of ADPKD. mRNA translation has emerged as a central mechanism driving cellular adaptation to innate or acquired metabolic challenges, with tRNA modifications being a key driver in the reprogramming of mRNA translation. Here, we investigate the putative role of mRNA translation regulation in cyst development in ADPKD-PKD1. Methods Two models were used: First, cystic cells extracted from nephrectomies of ADPKD-PKD1 patients (n = 7) were immortalized and compared to healthy cells collected from kidney biopsies (n = 5). Second, the classical murine inner medullary collecting duct (IMCD) cells knocked out for Pkd1 by CRISPR-Cas9 were used. We combined LC-MS proteomics and transcriptomics sequencing using both human and murine models. Gene set enrichment analysis (GSEA) was used to identify pathways and processes dysregulated at the level of mRNA translation in PC1-deficient cells. Nascent protein synthesis was assessed using flow cytometry in mitochondria and in cytoplasm to assess mRNA translation rates in both compartments. Systematic analyses of tRNA regulation (i.e. levels and modifications) were performed in two PC1-deficient human and murine cells versus controls. Finally, cystic human cell lines were used for 3D experiments to assess mRNA translation regulation on both cyst initiation and progression. Results We conducted GSEA using proteomics data from ADPKD patient-derived and murine cell lines. As expected, we confirmed that the PI3K-AKT-mTOR pathway was upregulated, while oxidative phosphorylation was downregulated in human and murine PC1-deficient cells, consistent with previous reports. Notably, mitochondrial translation was significantly downregulated in both murine PC1-deficient IMCD cells and ADPKD-PKD1 patient-derived lines. To validate this observation, we measured mitochondrial translation rates in 2 ADPKD-PKD1 patient-derived lines: we confirmed a decrease in mitochondrial translation rates, which correlated with mitochondrial dysfunction, as assessed by seahorse experiments. Strikingly, tRNA modification sequencing revealed consistent and specific alterations in mitochondrial tRNA modifications in murine PKD1-KO IMCD cells and ADPKD-PKD1 patient-derived lines. Hence, tRNA sequencing indicated a specific upregulation of mitochondrial tRNA levels in case of PC1 deficiency. Finally, specific inhibition of mitochondrial translation selectively induced apoptosis in ADPKD-PKD1 patient-derived lines in 2D cultures compared to controls and significantly reduced cyst formation and progression in a 3D cyst assay, while sparing healthy cells. Conclusion Our findings revealed (i) dysregulation of mitochondrial translation in PC1-deficient cells compared to control cells in both murine and human models, and (ii) the potential importance of mitochondrial tRNA expression and modification. These results suggest that a reprogramming of mitochondrial mRNA translation may contribute to the pathogenesis of ADPKD alongside the previously described mitochondrial dysfunction. Targeting mitochondrial translation presents a promising therapeutic strategy to mitigate cyst formation and progression in ADPKD.

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