#3632 CHARACTERIZATION AND REGULATION OF THE PATHOGENIC MUCIN 1 FRAMESHIFT PROTEIN IN PATIENT DERIVED CELLS AFFECTED BY ADTKD-MUC1

Abstract Background and Aims Autosomal Dominant Tubulointerstitial Kidney Diseases (ADTKD) are caused by mutations in one of at least five genes (UMOD, MUC1, HNF1B, REN SEC61A1) characterized progressive decline kidney function, reaching end-stage renal disease (ESRD) usually mid adulthood. ADTKD-MUC1 is the most frequent subforms, exclusively a characteristic frameshift mutation, leading to de novo protein MUC1-fs. MUC1-fs believed play distinct pathogenic role terms toxic proteinopathy, accumulating early secretory pathway. The substance BRD4780 reported re-route towards lysosomal degradation (Dvela-Levitt et al., Cell 2019), with interventional trials being preparation. Therefore, we aimed gain more insights into temporal spacial regulative characteristics, comparing pharmacological intervention vs. RNA interference patient derived tubular cells. Methods culture (HKC-8 cells human urinary Primary Tubular Cells, huPTC); siRNA knockdown; Transient transfection; Immunofluorescence; immunogold electron microscopy (EM); Immunoblotting (IB); Lentiviral SV40/Large-T antigen immortalization huPTC; Generation novel polyclonal antibody an independent downstream VNTR. Results To analyze detail, generated iTCs (immortalized cells) from huPTC patients ADTKD-MUC1. Clonal selection was performed immortalized clones expression. mainly localizes Co-localization only partially observed TMED9, which COP vesicles, involved trafficking within TMED9 negative components this pathway also showed staining, such as Golgi apparatus Early Endosomes. Immunogold EM reveals expression ER (secretory) vesicles. Ultrastructural analyses biopsies did not show specific accumulation, previously described ADTKD-UMOD. for MUC1 application iTCs, detailed description regulation kinetics. Both approaches led strongly reduced levels. revealed significantly shorter half life than wildtype mucin 1, possibly due its different cellular distribution. Conclusion Our data confirm extend published information on intracellular localization 2019). kinetics appear quite dynamic synthesis decay, should argue it suitable target. Both, potently downregulate Interestingly, primary confined Should effect be restricted positive may implicate effectivenes. efficient, since reduces earlier stage dependent processing.