Fig 2: Ferroptosis in the fibrotic kidney was alleviated by inhibition of neprilysin. (A) Tubular ACSL4 was upregulated in kidney biopsies of CKD patients by immunochemistry staining (×200, scale bar = 50 µm; ×400, scale bar = 20 µm). (B and C) Immunochemistry staining of ACSL4 in mouse kidney sections (×200, scale bar = 50 µm; ×400, scale bar = 20 µm). LN, lupus nephritis; DN, diabetic nephropathy; FSGS, focal segmental glomerulosclerosis; IgAN, IgA nephropathy; MN, membranous nephropathy. (D and E) Transmission electron microscopy of mouse kidney sections (×12,000, scale bar = 1 µm; ×40,000, scale bar = 500 nm). Triangle: shrunken mitochondria; circle: absent mitochondrial ridge; arrow: rupture of the mitochondrial outer membrane. (F and G) RT-PCR was performed to detect the mRNA expression of ACSL4, GPX4, and Ptgs2 (COX2). (H and I) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (J) Comparison of iron, MDA, GSH, and GSH/GSSG levels in the kidneys. ****p < 0.0001, ***p < 0.001, *p < 0.05, versus sham or control. #### p < 0.0001, ### p < 0.001, ## p < 0.01, # p < 0.05, versus UUO or adenine.

Fig 3: Neprilysin aggravated fibrosis by ACSL4-mediated ferroptosis in TGF-ß1-induced TCMK-1 cells. (A and B) The efficiency of NEP overexpression in TCMK-1 cells evaluated by RT-PCR and Western blot analysis. (C and D) The overexpression efficiency of ACSL4 in TCMK-1 cells evaluated by RT-PCR and Western blot analysis. (E and F) The knockdown efficiency of ACSL4 siRNA in TCMK-1 cells evaluated by RT-PCR and Western blot analysis. (G) RT-PCR was performed to detect the mRNA expression of ACSL4, GPX4, and Ptgs2 (COX2). (H) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (J) RT-PCR was performed to detect the mRNA expression of Fn, a-SMA, and Col Ia1. (I) Western blot analysis was performed to detect protein expression of Fn, a-SMA, and Col Ia1. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, versus oeNC or siNC or oeNC+siNC. #### p < 0.0001, ### p < 0.001, ## p < 0.01, # p < 0.05, versus oeNC+siNC+TGF-ß1. $$$$ p < 0.0001, $$ p < 0.01, $ p < 0.05, versus oeNEP+siNC+TGF-ß1. siNC, silencing negative control; siNEP, NEP siRNA; siACSL4, ACSL4 siRNA; oeNC, overexpression negative control; oeNEP, overexpression of NEP; oeACSL4, overexpression of ACSL4.

Fig 4: Genetic knockdown of neprilysin improved the fibrotic and ferroptotic phenotype in TGFß1-induced TCMK-1 cells. (A and B) The knockdown efficiency of NEP siRNA (siNEP) in TCMK-1 cells evaluated by RT-PCR and Western blot analysis. (C) Representative Western blot images and quantitative analysis of NEP expression in TCMK-1 cells. (D) RT-PCR was performed to detect the mRNA level of Fn, a-SMA, and Col Ia1. (E) RT-PCR was performed to detect the mRNA level of ACSL4, GPX4, and Ptgs2 (COX2). (F) Western blot analysis was performed to detect protein expression of Fn, a-SMA, and Col Ia1. (G) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (H) The accumulation of lipid peroxidation in TCMK-1 cells was analyzed by BODIPY C11 staining (×200, scale bar = 50 µm). NS p > 0.05, versus control. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, versus siNC. ### p < 0.001, ## p < 0.01, # p < 0.05, versus siNC+TGF-ß1. siNC, negative control.