Fig 1: Genetic induction of ferroptotic stress induces the accumulation of damage-associated PT cells after mild injury.(A) Schematic representation of experimental workflow. tdTomato-lineage tracing was employed to detect Sox9-lineage cells. cKO mice and their littermate controls were subjected to the same ischemic stress (ischemic time, 22 min) and tamoxifen treatment. Kidneys were harvested on day 21 post-IRI. (B) and (C) Immunostaining for VCAM1, EMCN (endomucin), and F4/80. IRI kidneys from cKO and control littermates (control) are shown. Quantification of VCAM1+EMCN–F4/80– area over the DAPI+ area is shown in (C). N = 6. (D) Immunostaining for VCAM1, ACSL4, and native tdTomato (TdT) fluorescence. Insets: individual fluorescence channels. (E) Real-time PCR analyses of indicated gene expression. Whole kidney lysates were used. N = 6–7. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, one-way ANOVA with post hoc multiple comparisons test for (C) and (E). (F) Immunostaining for SOX9 and VCAM1. Arrowheads indicate double-positive cells (damage-associated PT cells). (G) ISH for Cdh6 expression. Red arrowheads indicate Cdh6-positive renal tubular cells. Scale bars, 100 µm in (B); 20 µm in (D); 50 µm in (F); and 10 µm in (G). (H) Schematic illustration of PT cell state dynamics. Differentiated/mature PT cells are activated, transit into a damage-associated inflammatory PT cell state (DA-PT), and redifferentiate to their original state after mild injury. Ferroptotic stress prevents the redifferentiation of damage-associated PT cells into normal PT cell state, leading to the accumulation of the pathologic PT cells that actively produce inflammatory signals.
Fig 2: Damage-associated PT cells undergo high ferroptotic stress after severe IRI.(A) UMAP rendering of glutathione metabolic process in mouse and human kidneys. (B) A scheme showing glutathione-glutathione peroxidase 4 (GPX4) anti-ferroptotic defense pathway. Slc7a11 and Slc3a2 (system xc-); Gclc and Gclm (glutamate-cysteine ligase); Gss (glutathione synthetase); Gsr (glutathione reductase): and Gpx4. MDA (malondialdehyde, a lipid peroxidation product) and ACSL4 (acyl-CoA synthetase long-chain family member 4) are markers for ferroptotic stress. (C) Dot plots show the expression of genes for glutathione-GPX4 axis, Sox9, and Acsl4. (D) Immunostaining for SOX9 and MDA (6 hr post-IRI), and (E) quantification of double-positive cells in total SOX9+ cells. N = 4. (F) Immunostaining for SOX9 and ACSL4 (1 day post-IRI), and (G) quantification of double-positive cells in total SOX9+ cells. N = 4. Insets: individual fluorescence channels of the dotted box area. Note that severe ischemia (30 min) induces more ferroptotic stress markers (MDA and ACSL4) in SOX9+ cells in damaged kidneys than mild ischemia (20 min). Wild-type C57BL/6J mice were used for (D) to (G). Scale bars, 20 µm in (D) and (F). *p < 0.05. unpaired Student’s t-test.
Fig 3: Immunostaining and quantification for malondialdehyde (MDA) and ACSL4.The post-IRI kidneys harvested on 6-hrs and 1-day after ischemia were used to detect MDA and ACSL4, respectively. *P<0.05 and ***P<0.001 unpaired t-test. Scale bars, 50µm. Note tha 30-min ischemia induces more ferroptotic stress (MDA and ACSL4) in damaged kidneys compares to 20-min ischemia.
Fig 4: Damage-associated PT cells emerge after injury in mouse and human kidneys.(A) Immunostaining for SOX9 and VCAM1. Aristolochic acid nephropathy (AAN) and unilateral ureteral obstruction (UUO) models were used. Kidneys from wild-type C57BL/6J mice were harvested on day 26 (D26) for AAN and day 10 (D10) for UUO. Insets: individual fluorescence channels of the dotted box area. (B) Immunostaining for SOX9 and ACSL4. bIRI, bilateral IRI model. Kidneys were harvested on day 3 (D3) for bIRI, day 5 (D5) for AAN, and day 10 (D10) for UUO. Insets: individual fluorescence channels of the dotted box area. (C) Quantification of double-positive cells in total SOX9+ cells from panel (A) and (B). Scale bars, 20 µm. N = 3–4. **p < 0.01; ***p < 0.001; ****p < 0.0001, one-way ANOVA with post hoc multiple comparisons test. (D) UMAP of the human proximal tubular cells from AKI kidneys. (E) Dot plots showing the expression of indicated genes. Note that PT cells in state 3 (DA-PT-like) show increased gene expressions of markers for mouse damage-associated PT cells (SOX9, VCAM1, CDH6) and reduced expression of homeostatic genes (ALDOB, MIOX, and GPX4). (F) Pseudotime trajectory analysis of PT clusters (PT and DA-PT-like cells). A region occupied with ALDOBhigh cells were set as a starting state. Arrow, predicted trajectory from PT cells to DA-PT-like cells.
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