Reticulon 3 Deficiency Induces Ferroptosis via Chaperone-Mediated Autophagy in Ischemia-Reperfusion Induced Acute Kidney Injury

Acute kidney injury (AKI), particularly from ischemia-reperfusion injury (IRI), is a common and serious clinical syndrome with high mortality and no effective targeted therapies. A key event in IRI-AKI is renal tubular cell death, with ferroptosis—a form of cell death driven by iron-dependent lipid peroxidation—emerging as a critical mechanism. The antioxidant enzyme GPX4 is a central negative regulator of ferroptosis, and its degradation, mediated by the molecular chaperone HSC70, promotes this process. Therefore, identifying novel regulators of the HSC70-GPX4 axis is a promising therapeutic strategy. Reticulon-3 (RTN3), an endoplasmic reticulum protein, has documented roles in maintaining cellular homeostasis. Our prior work showed that RTN3 deficiency exacerbates renal fibrosis in chronic kidney disease and mitochondrial dysfunction in cisplatin-induced AKI. However, its role in IRI-AKI and its potential link to ferroptosis remain unknown.

RTN3 knockout (KO) mice on a C57BL/6 background were used, along with age-matched wild-type (WT) controls. All mice were maintained under SPF conditions. A renal ischemia-reperfusion injury (IRI) model was established by clamping bilateral renal pedicles for 30 minutes, followed by 24 hours of reperfusion. Sham-operated mice underwent identical procedures without clamping. Serum and kidney tissues were collected for analysis. HK-2 cells and mouse primary proximal tubular epithelial cells (PTECs) were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cell viability was measured using a CCK8 assay after exposure to hypoxia/reoxygenation (H/R), erastin, or RSL3. Renal function was evaluated by measuring serum creatinine and blood urea nitrogen (BUN) levels. Histological damage was assessed by H&E and PAS staining of paraffin-embedded kidney sections. DNA damage was examined using TUNEL staining. Immunohistochemistry (IHC) was performed on kidney sections after antigen retrieval, blocking, and incubation with primary and secondary antibodies. Western blot and co-immunoprecipitation (Co-IP) were conducted to assess protein expression and interactions. Protein lysates were prepared using RIPA buffer, and bands were visualized using chemiluminescence. RNA was extracted using a commercial kit, and RT-qPCR was performed to evaluate expression of Nrf2, NQO1, and HO-1. Gene expression levels were normalized to S18 using the 2^-ΔΔCt method. Hoechst and AO/EB staining were used to detect apoptotic cells in cultured PTECs. GSH and MDA levels in renal tissues and HK-2 cells were quantified using commercial kits. 

1. RTN3 expression is significantly decreased in renal tubular epithelium cell of AKI mice

2. RTN3 deficiency exacerbates IRI-induced renal tubular injury

3. RTN3 deficiency exacerbates cell death in IRI-induced AKI, which may be attributed to ferroptosis

4. RTN3 deficiency promotes ferroptosis in IRI-induced AKI

5. RTN3 participates in ferroptosis via interact with HSC70

This study unveils the RTN3-HSC70-GPX4 axis as a pivotal pathway governing ferroptosis in IRI-AKI, suggesting RTN3 agonism as a potential therapeutic strategy for ischemic renal injury.