Investigation of the role of TMED9 in the accumulation of a mutant protein in MUC1 kidney disease (MKD)

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Abstract Chronic kidney disease (CKD), most often caused by manifestations of systemic disease or underlying primary kidney disease, is a significant cause of worldwide patient morbidity and mortality. The field of nephrology is severely lacking in mechanism-based, targeted therapeutics to treat patients with CKD. Studies of primary, rare genetic kidney diseases, which lead to perturbations of common, essential kidney processes, can be harnessed to inform foundational kidney biology and identify potential therapeutic targets for kidney diseases, both rare and common. MUC1 kidney disease (MKD), or autosomal dominant tubulointerstitial kidney disease-MUC1 (ADTKD- MUC1), is a rare genetic kidney disease caused by a frameshift mutation in the MUC1 gene. MKD leads to progressive kidney tubulointerstitial damage and eventual renal failure. Recent studies have shown that MKD is a toxic proteinopathy in which the mutant frameshift protein, MUC1-fs, intracellularly accumulates in kidney tubular epithelial cells and is associated with increased levels of cellular toxicity. MUC1-fs has been further shown to accumulate specifically in the early secretory pathway of MKD patient kidney tubular epithelial cells (P cells), in vesicles that contain the protein TMED9. Critically, it has been found that knockout of TMED9 ameliorates MUC1-fs accumulation in P cells. TMED9 and MUC1-fs have been shown to additionally colocalize in both an in vivo MKD mouse model and MKD patient-derived iPSC organoids. Recent work has shown that TMED9 co- immunoprecipitates with MUC1-fs in P cells. Based on these observations, this proposed work aims to investigate the following hypothesis: In the setting of MKD, TMED9 and its interactors halt the trafficking of misfolded MUC1-fs and retain it in the early secretory pathway, leading to MUC1-fs accumulation and increased cellular toxicity. In Aim 1, the effect of TMED9 on MUC1-fs trafficking through the early secretory pathway will be assessed by knockout of TMED9 in P cells and mapping of MUC1-fs trafficking by inhibition of secretory pathway branches and assessment of MUC1-fs reaccumulation. The role of TMED9 as a mediator of cellular toxicity will be assessed by comparison of cellular toxicity, following treatment with the ER stress-inducer thapsigargin, in P cells and TMED9 knockout P cells. In Aim 2, the TMED9 protein complex that mediates MUC1- fs accumulation will be identified. First, interactors of TMED9 in P cells will be found using co-immunoprecipitation and mass spectrometry. Then, TMED9 interactors necessary for MUC1-fs accumulation will be identified using a CRISPR/Cas9 arrayed knockout screen. Finally, the binding of these necessary interactors to MUC1-fs will be assessed. In Aim 3, the role of TMED9 in MUC1-fs accumulation will be explored in vivo. A TMED9 knockout mouse will be crossed with the established MKD knock-in mouse and the in vivo effect of TMED9 knockout on MUC1-fs accumulation and other known MKD knock-in mouse phenotypes will be assessed.