Alternative Splicing of Uromodulin Enhances Mitochondrial Metabolism for Adaptation to Stress in Kidney Epithelial Cells

Background: Cells of thick ascending limb of the loop of Henle (TAL) are resistant to ischemic injury, despite high energy demands in a relatively hypoxic medullary milieu. 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 (UMOD), the most abundant protein secreted in healthy urine. The role of UMOD in this adaptation mechanism and the involvement of alternative splicing have not been previously described. Methods: To explore alternative splice variants of UMOD, we assessed long-read RNA sequencing data of human and mouse kidneys. The expression of the spliced variant after AKI was evaluated using a renal ischemia-reperfusion injury (IRI) mouse model. To test the localization and function of the splice variant, its cDNA was overexpressed in Madin-Darby canine kidney (MDCK) cells. To evaluate its role during AKI, we designed splice-switching antisense oligonucleotides (SSOs) to induce this alternative splicing, and their efficacy was tested in immortalized mouse kidney TAL cells and IRI mice. We isolated primary TAL cells from IRI mice to confirm the cytoprotective effect of the splice variant. Results: Long-read RNA sequencing showed the existence of alternatively-spliced UMOD (AS-UMOD) which skips exon 10 both in humans and in mice. AS-UMOD expression was induced by mild IRI, but not by severe IRI. This variant lacks a GPI-anchoring site and was localized intracellularly in mouse kidneys and MDCK cells. AS-UMOD was targeted to the mitochondria, interacted with SLC25 carriers, increased NAD+ and ATP levels, and improved cell viability after hypoxic injury in MDCK cells. We identified SSOs that enhance AS-UMOD expression in vitro and in vivo. Augmentation of AS-UMOD using the SSO after severe IRI improved the course of injury by protecting TAL cells. Conclusion: Alternative splicing generates a conserved intracellular isoform of uromodulin, which contributes to the metabolic adaptation of TAL cells to ischemic injury. Enhancing this protective splice variant in TAL cells might become a novel therapeutic intervention for AKI. Funding: NIDDK Support, Veterans Affairs Support