Baeksong Yoo
Research Mentor: Matthias Wolf
Mentor Department: Pediatrics, Medicine
Author(s): Baeksong Yoo, Matthias Wolf, Sungwan An
Session: Afternoon Session (12:00 PM – 1:00 PM)
Presentation Type:
Abstract
Background: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is characterized by autosomal dominant inheritance, progressive chronic kidney disease (CKD), and tubulointerstitial injury. Heterozygous pathologic variants in the Uromodulin (UMOD) gene are one of the most common causes for ADTKD. Misfolded UMOD accumulates in tubular cells and causes kidney failure. There is no specific therapy available for patients with ADTKD-UMOD and patients require dialysis and kidney transplantation. Objective: To identify an already FDA-approved drug to repurpose for ADTKD-UMOD treatment we performed a high-throughput screen (HTS) and investigated the molecular mechanism how the drug increases secretion of misfolded intracellular UMOD. Design and methods: A HTS was performed applying the Prestwick library, containing 1,500 fragments of FDA-approved medications. The HTS was performed with a stably transfected MDCK cell line expressing C150S UMOD tagged with a NanoLuc reporter. NanoLuc secretion into the medium was used as readout for improved C150S UMOD secretion. Western blotting and qPCR were used to study how spironolactone enhances C150S UMOD secretion. Results: We identified the mineralocorticoid receptor (MR) antagonist spironolactone to enhance C150S UMOD secretion. Dose response experiments confirmed this effect. The UmodC93F mouse had significantly better kidney function when treated with spironolactone for three months. Pathologic UMOD variants often alter UMOD glycosylation. Treating wild-type (WT) and C150S UMOD cells with spironolactone increased the ratio of glycosylated to non-glycosylated UMOD. Neither finerenone, canrenone, nor eplerenone increased C150S UMOD secretion indicating that the MR antagonism was not involved in C150S UMOD secretion. RNA-seq analysis of spironolactone treated and untreated C150S UMOD cells revealed no strong effect of spironolactone on gene expression with only 461 significantly upregulated and 234 significantly downregulated genes, pointing potentially to a post-translational mechanism. The RNA-seq data identified OGA (downregulation), LGALS1, ST3GAL1, and ST3GAL4 (all upregulated) as candidates. In qPCR studies we confirmed downregulation of OGA and the upregulation of LGALS1 but did not show any significant changes for ST3GAL1 and ST3GAL4. We hypothesized that knockdown of OGA would increase C150S UMOD secretion whereas knockdown of LGALS1 should lower C150S UMOD secretion. When we knocked down OGA we observed increased mutant UMOD secretion while LGALS1 knockdown did not produce the expected effect. Conclusion: Spironolactone-mediated downregulation of OGA may contribute to improved misfolded UMOD secretion, highlighting a potential therapeutic pathway for ADTKD-UMOD. The stimulatory effect of spironolactone on misfolded UMOD secretion must be an off-target effect as it is not mediated by antagonism of the MR. OGA encodes the enzyme O-GlcNAcase, which removes N-acetylglucosamine from proteins. Inhibition of OGA likely facilitates UMOD secretion by modifying UMOD glycosylation.
