OTUB1 regulation of ferroptosis and the protective role of ferrostatin-1 in lupus nephritis

Abstract

Lupus nephritis (LN) is a prevalent and severe manifestation of systemic lupus erythematosus (SLE), leading to significant morbidity and mortality. OTUB1, a deubiquitinating enzyme, has emerged as a potential therapeutic target due to its role in cellular protection and regulation of ferroptosis, a form of cell death linked to LN. Our study revealed significantly reduced OTUB1 expression in the glomeruli of LN patients and podocytes, correlated with disease severity. CRISPR/Cas9-mediated OTUB1 knockout in podocytes resulted in pronounced injury, indicated by decreased levels of nephrin and podocin. Ferrostatin-1 treatment effectively mitigated this injury, restoring SLC7A11 expression and significantly reducing MDA levels, Fe²⁺ levels, BODIPY C11 expression, and normalized cysteine and glutathione expression. In the MRL/lpr mouse model, Ferrostatin-1 significantly improved renal function decreased proteinuria, and ameliorated renal histopathological changes, including reduced glomerular endothelial swelling, mesangial cell proliferation, and leukocyte infiltration. These results underscore the protective role of Ferrostatin-1 in modulating the pathogenesis of LN. OTUB1 plays a crucial protective role against podocyte injury in LN by regulating ferroptosis. Ferrostatin-1 effectively mitigates podocyte damage induced by OTUB1 deficiency, suggesting that targeting ferroptosis could be a promising therapeutic strategy for LN.

Fig. 1. Decreased OTUB1 expression in podocytes of lupus nephritis.

A Initial analysis of OTUB1 expression in the glomeruli of LN kidney biopsy patients was performed using the GSE32591 database. Data are represented as mean ± SD. *p < 0.05, **p < 0.01,***p < 0.001. B Immunofluorescence staining demonstrating co-localization of OTUB1 with the podocyte marker synaptopodin in LN patients. Scale bar: 60 μm. C Evaluation of OTUB1 expression in podocytes of different mouse models using immunofluorescence. Scale bar: 20 μm.

Fig. 2. Impact of OTUB1 downregulation on podocyte injury and ferroptosis in lupus nephritis.

A Validation of OTUB1 knockout efficiency in podocytes using CRISPR-Cas9. B, C Evaluation of podocyte marker expression after exposing OTUB1-deficient podocytes to IgG from lupus nephritis patients or LPS, mimicking autoimmune and inflammatory conditions. D Analysis of SLC7A11 expression in OTUB1-deficient podocytes, showing a significant drop, with re-expression of OTUB1 restoring SLC7A11 levels. E Co-localization of 4-HNE with synaptopodin in mouse models using immunofluorescence. Scale bar: 16 μm. F Assessment of ferroptosis in OTUB1 knockout podocytes, demonstrated by increased MDA levels, reduced cysteine and glutathione levels, and elevated Fe²⁺ levels and BODIPY C11 expression. Data are represented as mean ± SD. *p < 0.05, **p < 0.01,***p < 0.001.

Fig. 3. Effects of ferrostatin-1 on podocyte injury induced by OTUB1 downregulation in lupus nephritis.

A, B Treatment with Ferrostatin-1 improved conditions in OTUB1-deficient podocytes, evidenced by the restoration of SLC7A11 expression, reduction in MDA levels, and increase in cysteine and glutathione levels, and elevated Fe²⁺ levels and BODIPY C11 expression. C Restoration of nephrin and podocin expression in podocytes treated with Ferrostatin-1. Data are represented as mean ± SD. *p < 0.05, **p < 0.01,***p < 0.001.

Fig. 4. Effect of Ferrostatin-1 on renal damage in MRL/lpr mice with lupus nephritis.

A Illustration of the experimental timeline for administering Ferrostatin-1 to 20-week-old MRL/lpr mice with established renal injury. B Measurement of urinary protein/creatinine ratio and serum creatinine levels post treatment, showing significant reductions, while total IgG and dsDNA levels in blood remained unchanged. C Hematoxylin and eosin (HE), Masson’s trichrome, Periodic acid-Schiff (PAS), and PASM (PAS with Methyl Green) staining. Scale bar: 20 μm. D Western blot and immunofluorescence analysis demonstrated increased expression of podocyte markers following Ferrostatin-1 treatment, indicating reduced podocyte injury. Scale bar: 20 μm. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. Effect of Ferrostatin-1 on renal damage in MRL/lpr mice with lupus nephritis.

A Pathway analysis showed downregulation of inflammation, fibrosis, apoptosis, and complement pathways after Ferrostatin-1 application. B The TUNEL assay showed that treatment with Ferrostatin-1 significantly reduced renal cell apoptosis in 20-week-old MRL/lpr mice. Scale bar: 100 μm. C–F Immunohistochemical and western blot analyses revealed that Ferrostatin-1 treatment significantly reduced α-SMA and fibronectin expression, while also decreasing p65 expression and C3 deposition. Scale bar: 20 μm.

Fig. 6

The Protective role of ferrostatin-1 in mitigating OTUB1-dependent ferroptosis in MRL/lpr mice.