Developing New Therapies for Autosomal Dominant Tubulointerstitial Kidney Disease Caused by Uromodulin Mutations by Screening a Small Molecule Library

Background: Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) caused by Uromodulin (UMOD) mutations, is the third most common inherited kidney disease. UMOD mutations result in a misfolded UMOD protein which is less secreted and accumulates intracellularly resulting in apoptosis and kidney failure. We want to identify new therapies for patients with ADTKD caused by UMOD mutations applying a high-throughput screening (HTS) assay. Methods: We developed stable cell lines expressing C150S or wild-type (WT) UMOD tagged to luciferase. We used the luciferase activity in the cell culture supernatant as the readout how well a compound improved C150S UMOD secretion. 8,000 compounds from the UT Southwestern HTS core library were screened. Best candidates were counter-screened for cell viability, effect on apoptosis, restoring TRPV5 current density (applying patch-clamp recording), and UMOD secretion using an ELISA assay. Results: The initial screen resulted in 124 candidates which increased C150S UMOD secretion to at least 20% of WT UMOD. We picked the best 99 candidates and confirmed them in triplicates at three different concentrations. Next, we chose the best 24 candidates which increased UMOD secretion to 40% of WT UMOD. To exclude any cytotoxic compounds, we removed 14 of the 24 candidates which reduced cell viability by more than 40%. Out of the 10 remaining candidates three small compounds increased UMOD secretion to 80-100% of WT UMOD. All three small molecules reduced apoptosis by more than 20%. Dose-response curves were performed for all three compounds. Compounds restored TRPV5 current density with C150S UMOD comparable to WT UMOD, and UMOD ELISA assays confirmed UMOD secretion. Western blot studies after treatment with these candidates showed a reduction of caspase 3 and an increase of the endogenous chaperone Hsp70. Conclusion: Our proof-of-concept study shows that screening of a small molecule library allows to identify potential new therapies for kidney diseases. Thorough counter-screening is required to remove false positive candidates. Further experiments including analysis of the targeted molecular pathway, structure-activity relationships, pharmacokinetics and -dynamics, and in vivo experiments in mutant UMOD mice will be required. Funding: NIDDK Support