Multiple oestradiol functions inhibit ferroptosis and acute kidney injury

Abstract

Acute tubular necrosis mediates acute kidney injury (AKI) and nephron loss¹, the hallmark of end-stage renal disease^2-4. For decades, it has been known that female kidneys are less sensitive to AKI^5,6. Acute tubular necrosis involves dynamic cell death propagation by ferroptosis along the tubular compartment^7,8. Here we demonstrate abrogated ferroptotic cell death propagation in female kidney tubules. 17β-oestradiol establishes an anti-ferroptotic state through non-genomic and genomic mechanisms. These include the potent direct inhibition of ferroptosis by hydroxyoestradiol derivatives, which function as radical trapping antioxidants, are present at high concentrations in kidney tubules and, when exogenously applied, protect male mice from AKI. In cells, the oxidized hydroxyoestradiols are recycled by FSP1^9,10, but FSP1-deficient female mice were not sensitive to AKI. At the genomic level, female ESR1-deficient kidney tubules partially lose their anti-ferroptotic capacity, similar to ovariectomized mice. While ESR1 promotes the anti-ferroptotic hydropersulfide system, male tubules express pro-ferroptotic proteins of the ether lipid pathway which are suppressed by ESR1 in female tissues until menopause. In summary, we identified non-genomic and genomic mechanisms that collectively explain ferroptosis resistance in female tubules and may function as therapeutic targets for male and postmenopausal female individuals.

Fig. 1. Female mice are resistant to AKI and renal tubular ferroptosis propagation.

Male and female C57BL/6N mice underwent renal IRI (36 min). a,b, The serum levels of creatinine (a) and urea (b) after 48 h. c, Time-course analysis of LDH release from freshly isolated renal tubules of male and female mice. n = 3 mice per group. d, Still images of live imaging of cell death propagation using SYTOX Green (pseudocolour yellow). Single green channel images in greyscale were added to show the propagation of cell death. Representative of n = 3 individual experiments. e,f, Imaging (e) and quantification (f) of cell death propagation in male and female tubules. Representative of n = 3 individual experiments. g, Still images of delayed cell death propagation in male kidney tubules after addition of 5 µM Fer-1. Resistant female kidney tubules are depicted for control purposes. Representative of n = 3 biological replicates. h, The effect of Fer-1 on LDH release in male and female kidney tubules. i–l, Serum creatinine (i), serum urea (j) and tubular histology (quantification of tubular damage (k) and imaging (l)) of female wild-type (WT) mice pretreated with 10 mg per kg of the ferroptosis inhibitor UAMC-3203 (hard ischaemia). m,n, The serum levels of creatinine and urea 10 days after tamoxifen application obtained from Gpx4^fl/flROSA26-creER^T2 transgenic mice. n = 3 (Cre⁻) and 5 (Cre⁺) mice per sex. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 50 μm (l, bottom) and 100 μm (l, top). Veh. vehicle.

Fig. 2. 2OH-E2 functions as a potent RTA to protect against ferroptosis propagation and accumulates in kidney tubules.

a, HT1080 cells were simultaneously treated with the indicated doses of either 17β-oestradiol (17β-E2) or 2OH-E2 after ferroptosis induction by 1.13 µM RSL3. Representative flow cytometry analysis of annexin V/7-AAD was used as a cell death readout system. b, Quantification of cell death from a. n = 4 biological replicates per condition. c–f, FENIX assays for radical trapping activity including negative-control cholesterol (c) and positive-control PMC (a classical RTA) (d) as well as 17β-E2 (e) and 2OH-E2 (f). Representative of n = 3 biological replicates per condition. g,h, LC–MS detection of 17β-E2 or OH-E2 from mouse plasma (n = 5 mice per sex) (g) and freshly isolated mouse renal tubules (n = 3 samples per sex) (h) after derivatization with 3-pyridine sulfonyl (Methods). Data are normalized to the internal standard and 10⁻⁶ µg protein content. i, LDH release from male and female renal tubules after addition of 10 µM 2OH-E2. n = 3 mice per group. Male C57BL/6N mice underwent hard renal IRI after pretreatment with 10 mg per kg 2OH-E2. j–m, Tubular injury histology (imaging (j) and quantification (k)), and quantification of the serum levels of creatinine (l) and urea (m). n–q, Tubular injury histology (n) and quantification (o) and functional parameters (serum creatinine (p) and serum urea (q)) 48 h after medium IRI in female mice 7 days after sham surgery or ovariectomy (OVX). Scale bars, 50 μm (j and n, bottom) and 100 μm (j and n, top).

Fig. 3. FSP1 regenerates oxidized oestradiol derivatives to enhance anti-ferroptotic activity in cells.

a–c, FENIX assays were performed after addition of 16 µM mFSP1, 320 nM FAD and the indicated doses of NADPH to assess inhibition of radical trapping activity for vehicle solution (a), 17β-E2 (b) or 2OH-E2 (c). Representative of n = 3 biological eplicates. d, FSP1 recycles oestradiol derivates. e, Western blot analysis of CRISPR-mediated knockout (KO) of AIFM2 in HT1080 cells. f,g, Imaging (f) and quantification (g) of dose–response experiments of 2OH-E2 after incubation with RSL3 in parental and AIFM2-knockout HT1080 cells. n = 4 biological replicates. h, Analysis of OH-E2 content from freshly isolated wild-type and FSP1-deficient (Aifm2-KO) female mouse tubules. n = 3 biological replicates per group. i, LDH release of Aifm2-knockout tubules isolated from male or female mice. n = 3 mice per group. j, Western blot analysis of FSP1 in freshly isolated male and female tubules from mice (j), pigs (k) and humans (l) (individual 85a is considered postmenopausal). m, Western blot analysis of FSP1 in mouse tubules 7 days after sham surgery or ovariectomy. n, Western blot analysis of loss of FSP1 protein expression in Aifm2-knockout female mouse tubules. o, LDH release from female FSP1-deficient (Aifm2 KO) or wild-type littermate tubules. p–r, The levels of serum creatinine (p) and serum urea (q) and tubular injury scoring (r) 48 h after medium IRI in wild-type or FSP1-deficient (Aifm2 KO) female mice.

Fig. 4. Esr1 deficiency sensitizes to tubular ferroptosis and AKI.

Renal tubules were freshly isolated from both wild-type and mutant Esr1 littermate female mice. a, LDH release from mouse tubules. n = 3 mice per group. b, Western blot analysis of FSP1 from either wild-type or ESR1-deficient (Esr1 mutant) female mouse tubules. c,d, Imaging (c) and quantification (d) of tubular injury 48 h after hard IRI in ESR1-deficient (mutant) female mice or wild-type littermates. e,f, Corresponding serum levels of creatinine (e) and urea (f). g,h, Renal tubular levels of 17β-E2 (g) and OH-E2 (h). n = 3 mice per group. i, Logarithmic transformation of the data presented in g and h, normalized per 10⁻⁶ µg protein content. j, The working model, demonstrating that oestrogens suppress ferroptosis both through genomic and non-genomic functions.

Fig. 5. ESR1 limits hydropersulfide degradation and ether lipid plasticity.

a, Hydropersulfide metabolism is controlled by CSE, SQR and ETHE1. b,c, Western blot analysis of CSE protein levels from ESR1-deficient (mutant) and wild-type female isolated tubules (b) and in male and female tubules (c). d,e, ETHE1 and SQR expression in male and female (d) and wild-type and Esr1-knockout (e) mouse tubules. f, Quantification of GSSH in male and female mouse tubules. n = 4 biological replicates per group. g, LDH release over time from wild-type and Cth-knockout female mouse tubules. h,i, GSSSG dose-dependently prevents ferroptosis in HT1080 cells. n = 3 individual experiments. j, LDH release over time from male mouse tubules after addition of either vehicle solution or 50 µM GSSSG. n = 3 mice per group. k, Investigated proteins involved in the ether lipid production pathway. l, Protein expression of AGPS in ESR1-deficient (ESR1 KO) HT29 cells compared with in parental cells. m, Protein expression of AGPS after treatment with 17β-E2 for 16 h in HT29 cells. n,o, Protein expression of ACSL4, AGPS and FAR1 in mouse male and female tubular lysates (n) and in mouse female wild-type and ESR1-deficient tubular lysates (o). p, Protein expression of ACSL4 and AGPS in porcine male and female renal tubular lysates. q, Untargeted lipidomics in mouse kidney tubules during spontaneous tubular ferroptosis. Colour scale is semi-quantitative, as described in Methods. n = 6 individual experiments per sex. r, Protein expression of ACSL4, AGPS and FAR1 in freshly isolated kidney tubules obtained from sham and ovariectomized mice (artificial induction of a postmenopausal state). s,t, Quantification of AGPS immunohistochemistry staining intensity in renal biopsies of premenopausal (pre) and postmenopausal (post) female as well as male patients (n = 4 individuals per group) (s) exemplified by representative specimens (t). u, AGPS and FAR1 protein levels in tubules from male and postmenopausal female individuals. v, Correlation heat map of ESR1 as well as FAR1, ACSL4 and AGPS from ESR1⁺ breast cancer cell lines. PC, phosphatidylcholine; PE, phosphatidylethanolamine. Scale bars, 25 μm (t).

Extended Data Fig. 1. Abrogated ferroptotic cell death propagation in female renal tubules.

(a) 400x magnification of tubular injury corresponding to Fig. 1l. 10 mg/kg Fer-1 were applied to female C57Bl/6 mice demonstrating no protective properties regarding (b) serum creatinine, (c) serum urea, and (d-e) tubular injury upon medium ischaemia. (f-g) Serum values of creatinine and urea of C57Bl/6 N female wildtype mice upon escalating doses of IRI (mild/medium/hard) treated with either vehicle or UAMC-3203. (h) Quantification of tubular injury. (i) Representative PAS-stained micrographs of respective groups (n = 3 mice/group).

Extended Data Fig. 2. Sex-specific kidney injury following GPX4 depletion.

Gpx4^fl/fl;ROSA26-CreER^T2 transgenic mice were treated with tamoxifen for 10 days. (a) PAS staining of renal cortical and medullary sections. (b) Quantification of tubular injury in the cortex (c) and the medulla. (d) Despite sex-specific differences in renal injury, no difference in survival times were observed (n = 5 mice/group). (e) Western blot demonstrating no difference in tubular GPX4 protein levels in males and females. * p < 0.05; ** p < 0.01; *** p < 0.001.

Extended Data Fig. 3. Estradiol protects against ferroptosis in cell lines.

(a) Flow cytometry analysis of HT1080 cells simultaneously treated with 17β-E2 (10 μM) and erastin (5 μM), (b) FIN56 (10 μM), (c) FINO2 (10 μΜ) or (d) ferroptocide (FTC, 10 μM). (e) Flow cytometry analysis of NIH-3T3 cells treated simultaneously with 17β-estradiol (10 μM) and erastin (5 μM), (f) FIN56 (10 μM), (g) FINO2 (10 μΜ) or (h) ferroptocide (FTC, 10 μM). Flow cytometry analysis of CD10 cells treated simultaneously with 17β-estradiol (10 μM) and (i) erastin (5 μM), (j) RSL3 (1.13 μM), (k) FIN56 (10 μM), (l) FINO2 (10 μΜ) or (m) ferroptocide (FTC, 10 μM). In all cases, quantification of annexin V / 7-AAD double-negative cells was performed (n = 3 biological replicates). Fer-1 (1 µM) was used as protection control. * p < 0.05; ** p < 0.01.

Extended Data Fig. 4. Estradiol pretreatment protects against ferroptosis.

(a) Schematic visualization of the experimental setup. Cells were pretreated with 10 µM 17β-estradiol for 16 h before inducing ferroptosis by addition of 5 µM erastin or 1.13 µM RSL3. Flow cytometry was performed at indicated time points and the proportion of annexin V / 7-AAD double-negative cells quantified. E2 protected HT1080 cells long-lasting against (b) erastin (n = 5 biological replicates) and (c) RSL3 (n = 3 biological replicates). E2 protected NIH3T3 cells long-lasting against (d) erastin and (e) RSL3 (n = 3 biological replicates). E2 protected CD10 cells long-lasting against (f) erastin and (g) RSL3 (n = 3 biological replicates). Fer-1 (1 µM) was used as protection control. * p < 0.05.

Extended Data Fig. 5. Oestrogens protect against ferroptosis.

(a) Flow cytometry analysis of HT1080 and (b) NIH3T3 cells simultaneously treated with RSL3 (1.13 μM) and 17β-estradiol (10 μM) for indicated time points. Fer-1 (1 μM) serves as a protection control. The proportion of annexin V / 7-AAD double-negative cells was quantified (n = 3 biological replicates). (c) FENIX assays demonstrate radical trapping properties of 4OH-E2, (d) but not testosterone (representative of n = 3 biological replicates). (e) Strategy of derivatization of 17β-E2 and 2OH-E2 with 3-pyridine sulfonyl. (f) Absolute measures of E2 and OH-E2 in plasma (n = 5 mice/sex) and murine renal tubules (n = 3 mice/sex) corresponding to logarithmic transformed data shown in Fig. 2g,h. Normalization to internal standard and protein content was performed. (g) Representative micrographs of kidneys of male mice treated with either vehicle or 10 mg/kg 2OH-E2 before sham surgery. (h) Representative micrographs in 400x magnification of tubular injury in male mice treated with either vehicle or 2OH-E2 before IRI. (i) Representative micrographs (400x) of kidneys after IRI in female mice 7 days after either sham surgery or ovariectomy. * p < 0.05.

Extended Data Fig. 6. Estradiol derivatives are recycled to protect against ferroptosis.

(a) Chemical structure of 5,6,7,8-tetrahydronaphthalene-2,3-diol. (b) Flow cytometry analysis of HT1080 cells treated with different concentrations of diol (abbreviation of 5,6,7,8-tetrahydronaphthalene-2,3-diol) and co-treated with erastin (5 μM) (n = 3 biological replicates). (c) Flow cytometry analysis of HT1080 cells treated with different concentrations of diol and cotreated with RSL3 (1.13 μM) (n = 4 biological replicates). In all cases, proportion of cells double-negative for annexin V / 7-AAD was quantified. Radical trapping activity was assessed by FENIX assays (representatives of n = 3 biological replicates). (d) Activity of 17β-E2 was not enhanced by FAD and NADPH only. (e-f) Addition of 16 µM recombinant mFSP1 enhanced radical trapping activity of PMC. (g) Radical trapping activity of 2OH-E2 was less potent without addition of mFSP1. (h-i) mFSP1 enhanced radical trapping activity of 4OH-E2. (j) Ascorbate alone failed to achieve radical trapping activity. (k) (17β-)E2 radical trapping activity was not enhanced by ascorbate. (l) Ascorbate enhanced radical trapping potency of both 2OH-E2 and (m) 4OH-E2. (n) FENIX assay of superoxide thermal source (SOTS-1) serving as control showing no regenerative capacity in the absence of a compound that can be oxidized. SOTS-1 thermally decomposes to yield two equivalents of superoxide, which is in equilibrium with its conjugate acid hydroperoxyl radical (HOO•). (o-q) FENIX assay of SOTS-1 in the presence of (17β-)E2, 2OH-E2, and 4OH-E2.

Extended Data Fig. 7. FSP1 regenerates 2OH-E2 in cellular systems.

(a) Representative controls corresponding to Fig. 3f (RSL3 1.13 µM; Fer-1 1 µM; 2OH-E2 10 µM). (b) FSP1-deficiency sensitizes HT29 cells to RSL3-induced ferroptosis, but not other classes of FINs (erastin 5 µM 24 h; RSL3 1.13 µM 24 h; FIN56 10 µM 48 h; FINO2 10 µM 24 h). (c) Quantification of cells double-negative for annexin V / 7-AAD (n = 3 biological replicates). (d) Western Blot demonstrating efficacy of AIFM2 knock-out in HT29 cells. (e) FSP1-deficiency increased the required dose of 2OH-E2 to protect HT29 cells against RSL3-induced ferroptosis (n = 4 biological replicates). Correspondingly, concomitant treatment with 5 µM viFSP1 increased the required dose of 2OH-E2 to protect both (f) HT1080 (n = 4 independent experiments) and (g) HT29 cells (n = 3 biological replicates) against RSL3-induced ferroptosis. Various chemical FSP1-inhibitors (5 µM viFSP1, 5 µM icFSP1, or 5 µM iFSP1) sensitized either (h) HT29 cells or (i) HeLa cells specifically to RSL3-induced ferroptosis (n = 3 biological replicates). (j) Inhibition of E2 synthesis in HeLa cells by pretreatment with 5 µM anastrozole for 7 days sensitized to RSL3-induced ferroptosis with no additional effect of viFSP1 (representative of n = 3 biological replicates). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Extended Data Fig. 8. Impaired FSP1 function does not sensitize female renal tubules to ferroptosis.

Logarithmic transformation of measures of E2 and OH-E2 from female murine tubules corresponding to Fig. 3h normalized to internal standard and protein content (n = 3 mice/condition) (b) LDH release of female tubules treated with indicated doses of viFSP1 (n = 1 sample/condition) (c) Representative micrographs of kidneys from FSP1-deficient female mice or littermates upon either sham surgery or IRI corresponding to Fig. 3p–r. (d) Western Blot for GPX4 and ACSL4 from lysates of female tubules comparing littermates and FSP1-deficient mice. (e) Western Blot from lysates of isolated kidney tubules incubated with antibodies against the thioredoxin system in response to FSP1 deficiency. (f-g) LDH release of female murine tubules treated with the TRX-inhibitor ferroptocide (FTC, 30 µM) demonstrates sensitivity independently of AIFM2 genotype. Fer-1 (30 µM) served as protection control (n = 3 samples/group)* p < 0.05; **** p < 0.0001.

Extended Data Fig. 9. Effects of ESR1-deficiency on ferroptosis.

ESR1-deficient female mice were treated with either vehicle solution or UAMC-3203 upon hard renal IRI (a-b). Serum creatinine and urea were measured after 48 h. (c-d) Tubular injury was quantified from PAS-stained slides. Levels of (e) GSH, (f) GSSSH, and (g) further sulfur metabolites from lysates of isolated renal tubules of male and female mice (n = 5 samples/group). (h) Quantification of GSH and GSSSH from wildtype and ESR1-deficient murine tubules (n = 5 samples/group). (i) GSH and GSSH levels in HT29 cells after treatment with 10 µM E2 or 2OH-E2 (n = 5 samples/group). (j) LDH release from wild-type and CTH-deficient male murine tubules (n = 2 mice/group). (k-l) HT1080 cells were induced to undergo ferroptosis by erastin in the presence of 37.5 µM – 300 µM of GSSSG. Annexin V / 7-AAD double staining was read out by flow cytometry (n = 3 biological replicates) (m) LDH release from male wild type tubules treated with vehicle or 300 µM GSSSG (n = 3 samples/group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Extended Data Fig. 10. ESR1 controls ether lipid synthesis.

Western Blots demonstrating CRISPR-mediated knock-out of FAR1 in (a) CD10 and (b) HT1080 cells. FAR1-deficiency did not sensitize (c) CD10 cells or (d) HT1080 cells to FINs (5 µM erastin; 1.13 µM RSL3; 10 µM FIN56; or 10 µM FINO2). (e-f) quantification of the experiment described in (c-d) (n = 3 biological replicates). (g) Volcano plot of differential ether lipid abundances in males versus female renal tubules at baseline (0 hr). Tones of red and blue depict lipid species with significantly higher levels in males and females, respectively. (h) Heatmap depicting ester lipid plasticity at baseline and following tubular ferroptosis (6 h) in male and female renal tubules (n = 6 samples/sex). (i) Average mRNA expression of Far1, Agps, and Acsl4, respectively, in kidney samples of healthy persons and patients with AKI (see Methods). (j) Opposing expression of Esr1 as well as Far1, Agps, and Acsl4 among Her2-negative breast cancer cell lines as determined from the DepMap portal (see Methods). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.