Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells
Azuma Nanamatsu, … , Takashi Hato, Tarek M. El-Achkar
Azuma Nanamatsu, … , Takashi Hato, Tarek M. El-Achkar
Published April 8, 2025
Citation Information: J Clin Invest. 2025;135(12):e183343. https://doi.org/10.1172/JCI183343.
View: Text | PDF
Research Article Cell biology Nephrology

Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

Abstract

In the kidney, cells of thick ascending limb of the loop of Henle (TAL) are resistant to ischemic injury, despite high energy demands. This adaptive metabolic response is not fully understood even though the integrity of TAL cells is essential for recovery from acute kidney injury (AKI). TAL cells uniquely express uromodulin, the most abundant protein secreted in healthy urine. Here, we demonstrate that alternative splicing generates a conserved intracellular isoform of uromodulin, which contributes to metabolic adaptation of TAL cells. This splice variant was induced by oxidative stress and was upregulated by AKI that is associated with recovery, but not by severe AKI and chronic kidney disease (CKD). This intracellular variant was targeted to the mitochondria, increased NAD+ and ATP levels, and protected TAL cells from hypoxic injury. Augmentation of this variant using antisense oligonucleotides after severe AKI improved the course of injury. These findings underscore an important role of condition-specific alternative splicing in adaptive energy metabolism to hypoxic stress. Enhancing this protective splice variant in TAL cells could become a therapeutic intervention for AKI.

Authors

Azuma Nanamatsu, George J. Rhodes, Kaice A. LaFavers, Radmila Micanovic, Virginie Lazar, Shehnaz Khan, Daria Barwinska, Shinichi Makino, Amy Zollman, Ying-Hua Cheng, Emma H. Doud, Amber L. Mosley, Matthew J. Repass, Malgorzata M. Kamocka, Aravind Baride, Carrie L. Phillips, Katherine J. Kelly, Michael T. Eadon, Jonathan Himmelfarb, Matthias Kretzler, Robert L. Bacallao, Pierre C. Dagher, Takashi Hato, Tarek M. El-Achkar

×

Figure 7

Identification of SSOs that induce AS-UMOD in MKTAL cells.

Options: View larger image (or click on image) Download as PowerPoint
Identification of SSOs that induce AS-UMOD in MKTAL cells. (A) Design of...
(A) Design of SSOs to induce AS-Umod expression. Numbers in parentheses indicate position from the first base of exon 10. (B) Relative mRNA expression of AS-Umod normalized to Gapdh in MKTAL cells transfected with 30nM SSOs for 24 hours. Lipofectamine alone and nontargeted SSO were used as negative controls. n = 3. (C) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh in MKTAL cells transfected with various concentrations of Umod SSO for 48 hours. Umod SSO corresponds to SSO (–13). n = 3. (D–I) MKTAL cells were treated with 30 nM scrambled SSO or Umod SSO for 48 hours. (D and E) Immunoblotting of UMOD in cell lysate and medium, respectively. Red asterisk corresponds to AS-UMOD. Coomassie staining was used as a loading control for medium. n = 4. (F) Immunofluorescence of UMOD. n = 2. Scale bar: 10 μm. (G) The ratio of mitochondrial/cytosolic glutamate levels. n = 3. (H) NAD+ levels normalized to protein concentration. n = 3. (I) ATP levels normalized to protein concentration. n = 3. Data were analyzed by unpaired t test (between 2 conditions, E and G–I) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, C) and are represented as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001.