TSG6 rescues mitochondrial dysfunction by inhibiting TUFM degradation and alleviates acute kidney injury

Acute kidney injury (AKI) remains a major clinical challenge characterized by tubular epithelial injury, mitochondrial dysfunction, impaired mitophagy, and maladaptive inflammation. TSG6 (TNFAIP6), a stress-inducible secreted protein, has been implicated in tissue protection, yet its role in tubular pathobiology and mitochondrial homeostasis in AKI is unclear.

We investigated TSG6 expression in AKI patients, human tubuloid cultures, and murine ischemia-reperfusion injury (IRI) models. TSG6 global knockout (TSG6-KO) and conditional RTEC-specific knockout (TSG6-cKO) mice were generated to assess functional significance. Mechanistic studies employed RNA sequencing, immunoprecipitation-mass spectrometry, co-immunoprecipitation, proximity ligation assays, and ubiquitination analyses in HK2 cells subjected to hypoxic reoxygenation (H/R) injury. For translation, we engineered LTH peptide-modified mesenchymal stem cell-derived extracellular vesicles (EV-TSG6) for kidney-targeted TSG6 delivery and evaluated therapeutic efficacy in IRI-AKI mice.

TSG6 was markedly upregulated in renal tubular epithelial cells (RTEC) in AKI. Tissue TSG6 correlated with serum creatinine (r=0.48, P=0.0435) and TUNEL positivity (r=0.78, P=0.002), and serum TSG6 rose in AKI and tracked renal dysfunction. Cisplatin-injured human tubuloids, AKI transcriptomic datasets, and IRI mice confirmed tubular TSG6 induction. Global and tubular-specific TSG6 deletion exacerbated IRI-AKI, increasing BUN/Scr, tubular injury, apoptosis, KIM-1, cytokines, and mitochondrial damage. Conversely, TSG6 preserved cell viability, restrained apoptosis, and improved mitochondrial respiration, membrane potential, and ROS under H/R, while enhancing LC3-II and mitophagy.Mechanistically, TSG6 translocated in part to mitochondria after injury and directly bound TUFM via its LINK domain to the TUFM Tu2 domain. TUFM protein fell in AKI and H/R without mRNA changes due to proteasomal degradation; TSG6 stabilized TUFM by suppressing its ubiquitination. MARCH5 was identified as the TUFM E3 ligase that mediates K48-linked polyubiquitination at Lys314 in the Tu2 domain, accelerating TUFM degradation; TSG6 competitively inhibited MARCH5–TUFM binding at the shared Tu2 interface, thereby blocking TUFM ubiquitination and preserving TUFM-dependent mitophagy and mitochondrial integrity. TUFM knockdown abolished TSG6’s protective effects.Kidney-targeted EVLTH-TSG6 achieved superior tubular uptake and retention in IRI, increased tubular TSG6 and TUFM, reduced apoptosis and KIM-1, improved BUN/Scr, and restored mitochondrial ultrastructure with enhanced autophagosome/autolysosome formation. EV encapsulation improved TSG6 stability versus free protein.

Kidney-targeted EVLTH-TSG6 achieved superior tubular uptake and retention in IRI, increased tubular TSG6 and TUFM, reduced apoptosis and KIM-1, improved BUN/Scr, and restored mitochondrial ultrastructure with enhanced autophagosome/autolysosome formation. EV encapsulation improved TSG6 stability versus free protein.

This study establishes TSG6 as a critical endogenous protective factor in AKI that safeguards RTEC through a novel TSG6-TUFM-mitophagy axis. TSG6 competitively antagonizes MARCH5-mediated TUFM degradation, thereby preserving mitochondrial quality control and cellular homeostasis. The engineered EVLTH-TSG6 delivery system represents a promising translational strategy for AKI treatment. These findings illuminate previously unrecognized post-translational regulatory mechanisms in tubular injury and identify TUFM stabilization as a potential therapeutic target for AKI intervention.