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Kidney Week

Abstract: FR-PO184

Alternative Splicing Generates an Intracellular Uromodulin Isoform that Mediates Mitochondrial Function and Is Up-Regulated in AKI

Session Information

  • AKI: Mechanisms - II
    November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Nanamatsu, Azuma, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • LaFavers, Kaice Arminda, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Micanovic, Radmila, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Lazar, Virginie, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Rhodes, George, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Khan, Shehnaz, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Bacallao, Robert L., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Dagher, Pierre C., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Hato, Takashi, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

Uromodulin (UMOD), a glycoprotein exclusively produced by renal tubular cells, is known to protect against acute kidney injury. Previous research has mainly focused on the secreted UMOD; however, the significance of intracellular UMOD has not been described.

Methods

We performed long-read RNA sequencing on human and mouse kidneys to explore alternative splicing variants of UMOD. RT-PCR and Sanger sequencing were conducted to validate the expression of the identified variant. The expression of the variant in mice after renal ischemia-reperfusion injury was quantified using RT-qPCR. To assess the localization and function, cDNA of the variant was overexpressed in kidney epithelial cells, and Western blotting, immunofluorescence, LDH assay, and Mito Stress Test were performed.

Results

Long-read RNA sequencing revealed a novel alternative splicing variant of UMOD, referred to as UMOD-AS, both in humans and mice. The existence of UMOD-AS mRNA was confirmed by RT-PCR and Sanger sequencing. UMOD-AS mRNA was up-regulated after renal ischemia-reperfusion injury, whereas canonical UMOD was down-regulated. In the cellular model, UMOD-AS localized in the cytoplasm, whereas canonical UMOD localized at the membrane and was secreted extracellularly. Interestingly, UMOD-AS-expressing cells showed better cell viability than canonical UMOD-expressing cells. Mito Stress Test suggested UMOD-AS up-regulates mitochondrial function compared to canonical UMOD.

Conclusion

UMOD-AS is an intracellularly-localized and cellular-protective isoform of UMOD. Notably, the localization and regulatory mechanisms of UMOD-AS are contrasted with canonical UMOD. Alternative splicing of UMOD may be a novel protective mechanism in acute kidney injury.

Funding

  • NIDDK Support