Amelioration of nephrotoxicity by targeting ferroptosis: role of NCOA4, IREB2, and SLC7a11 signaling

Abstract

Nephrotoxicity is a common complication that limits the clinical utility of cisplatin. Ferroptosis is an iron-dependent necrotic cell death program that is mediated by phospholipid peroxidation. The molecular mechanisms that disrupt iron homeostasis and lead to ferroptosis are yet to be elucidated. In this study, we aimed to investigate the involvement of nuclear receptor coactivator 4 (NCOA4), a selective cargo receptor that mediates ferroptosis and autophagic degradation of ferritin in nephrotoxicity. Adult male Sprague-Dawley rats were randomly-assigned to four groups: control group, cisplatin (Cis)-treated group, deferiprone (DEF)-treated group, and Cis+DEF co-treated group. Serum, urine, and kidneys were isolated to perform biochemical, morphometric, and immunohistochemical analysis. Iron accumulation was found to predispose to ferroptotic damage of the renal tubular cells. Treatment with deferiprone highlights the role of ferroptosis in nephrotoxicity. Upregulation of NCOA4 in parallel with low ferritin level in renal tissue seems to participate in iron-induced ferroptosis. This study indicated that ferroptosis may participate in cisplatin-induced tubular cell death and nephrotoxicity through iron-mediated lipid peroxidation. Iron dyshomeostasis could be attributed to NCOA4-mediated ferritin degradation.

Figure 1. Deferiprone (DEF) mitigated necrosis and improved renal structure after cisplatin (Cis) treatment. H&E-stained sections of the four study groups showing marked pleopathological changes in the kidney of the cisplatin-treated group where obvious dilatation and congestion of the interstitial blood capillaries was observed. Glomeruli appeared degenerated with marked atrophy of the glomerular capillary tuft, widening of the Bowman's space, and irregularity of the Bowman's capsule. The tubular lumina are seen filled with hyaline casts (C) and their epithelial lining appear highly-vacuolated. Some tubules appear degenerated with surrounding inflammatory cellular infiltration (I). Kidney sections of the DEF-treated group demonstrated improvement in renal architecture. The glomerular capillaries appeared healthy with normal urinary space and Bowman's capsule. The proximal (P), distal (D), and collecting (Co) tubules showed improvement of their histological structure with intact epithelial lining and minimal inflammatory infiltration (I). BV: Blood vessels; G: Glomeruli; S: Bowman's space; V: Vacuolation. Scale bars 200 and 50 μm.

Figure 2. Ferroptosis participated in cisplatin (Cis)-induced nephrotoxicity. A-C, RT-PCR analysis of expression of ferroptosis-related genes. Cisplatin induced iron-responsive element-binding protein 2 (IREB2) (A) gene upregulation in parallel with GPX4 (B) and SLC7A11 (C) gene downregulation that was reversed by deferiprone (DEF) co-treatment. Data are reported as means±SE. ^$P<0.05 vs Control, *P<0.05 vs deferiprone (DEF), ^#P<0.05 vs cisplatin (Cis) (ANOVA).

Figure 3. Deferiprone (DEF) switched the macrophage polarization towards the anti-inflammatory phenotype. Representative images of immunohistochemical expression (magnification ×400, scale bar 50 µm) and quantification of the CD68- and CD163-positive macrophage count (arrows) in renal tissue of the cisplatin (Cis) rat model compared to control and DEF-treated group showing significantly increased CD68-positive pro-inflammatory macrophages in the Cis group mainly observed in the interstitial and peri-glomerular areas. CD163-positive anti-inflammatory macrophage count was significantly decreased in the Cis group. Data are reported as means±SE (n=6). ^$P<0.05 vs Control, *P<0.05 vs DEF, ^#P<0.05 vs Cis + DEF (ANOVA).

Figure 4. Deferiprone (DEF) mitigated oxidative stress and lipid peroxidation derived by cisplatin (Cis)-induced ferroptosis. A and B, Quantitative analysis of glutathione and malondialdehyde (MDA). Data are reported as means±SE. ^$P<0.05 vs Control, *P<0.05 vs DEF, ^#P<0.05 vs Cis (ANOVA).

Figure 5. Deferiprone (DEF) attenuated cisplatin (Cis)-induced ferritinophagy. A-C, Quantitative analysis of ferritin, iron storage protein ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) in the renal tissue. D and E, Pearson correlation analysis between ferritin level in the kidney and NCOA4 (D) and iron-responsive element-binding protein 2 (IREB2) (E) expressions. Data are reported as means±SE. ^$P<0.05 vs Control, *P<0.05 vs DEF, ^#P<0.05 vs Cis (ANOVA).