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

Abstract

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