Fig 1: Kidney-derived NGAL exacerbates metformin nephrotoxicity and interacts with metformin–Fe.a The heatmap of cluster2 in Supplementary Fig. S10b and summary of Lcn2 mRNA levels in sham group, I/R group, I/R+Met200 group, and I/R+Met200+Fer-1 group according to scRNA-seq data. b Representative NGAL IHC-stained kidney sections from the I/R, I/R+Met100 and I/R+Met100+rmNGAL groups. c Summary of renal Lcn2 mRNA levels in Lcn2 renal specific knockout mice (KKO) and control mice, expressed relative to control. d Kaplan–Meier survival curves of KKO and control mice in I/R+Met200 condition. e Summary of the kidney to body weight ratio measured in the I/R-induced KKO mice and control mice with metformin. f–h Summary of the serum BUN (f), creatinine (g), and LDH (h) measured in the KKO mice and control mice. i Summary of renal MDA levels measured in the KKO mice and control mice. j Representative H&E-stained, Sirius Red-stained, Ly6G IHC-stained, MPO IHC-stained, and Neutrophil Elastase IF-stained kidney sections from the KKO mice and control mice. k Kaplan–Meier survival curves of mice in I/R+Met100 and I/R+Met100+rmNGAL groups. l Summary of the neutrophil numbers in the circulation of the I/R+Met100 and I/R+Met100+rmNGAL mice. m Summary of the percentages of neutrophils in the circulation of the indicated mice. n Representative Ly6G IHC-stained kidney sections from the indicated mice. o Summary of the kidney to body weight ratio measured in I/R+Met100, I/R+Met100+rmNGAL, and I/R+Met100+rmNGAL+Ab mice. p–r Summary of serum BUN (p), creatinine (q), and LDH (r) measured in the indicated mice. s Summary of renal MDA levels measured in the indicated mice. t Summary of renal MPO levels measured in the indicated mice. u Representative H&E-stained, Sirius Red-stained, Ly6G IHC-stained, MPO IHC-stained, and Neutrophil Elastase IF-stained kidney sections from the indicated mice. v Structure of the NGAL protein complexed with metformin and Fe; the inset shows a zoomed in view of the electrostatic surface, with the key residues in NGAL indicated. w Forces between the indicated residues in the NGAL protein and the [Metformin]3Fe complex are shown. x Summary of the neutrophil numbers in the circulation of the mice with metformin or [Metformin]3Fe complex. y Summary of the percentages of neutrophils in the circulation of the indicated mice. z Kaplan–Meier survival curves of the LKO mice with metformin or [Metformin]3Fe complex (n = 5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 and n.s., not significant (one-way ANOVA). Survival curve comparison was analyzed by a log-rank Mantel-Cox test. In this and subsequent figures, summary data are represented as the mean ± SEM. See also Supplementary Figs. S13–S17.
Fig 2: Decreased iron level alleviates metformin-induced nephrotoxicity in the context of AKI.a Summary of significantly differentially pathways in the indicated parenchymal cell types of I/R+Met200 vs I/R. b Summary of renal non-heme iron levels measured in the Sham mice, I/R mice, and I/R+Met200 mice. c At 3 weeks of age, mice were fed a standard-iron diet (SID), high-iron diet (HID), or low-iron diet (LID) for 9 weeks, then subjected to I/R+Met200. d Summary of renal non-heme iron levels measured in the indicated mice. e Kaplan–Meier survival curves of the indicated mice (n = 11, 9, and 9 mice in the HID, SID, and LID groups, respectively). f Summary of serum iron levels measured in adult Tmprss6flox/flox and Tmprss6Alb/Alb mice. g Summary of renal non-heme iron levels measured in the indicated mice. h Representative DAB-stained kidney sections from the indicated mice. i Summary of the percentages of DAB-positive area measured in DAB-stained kidney sections. j Representative image of kidneys in the indicated mice. k Summary of the ratio between kidney weight and tibia length measured in the indicated mice. l–o Summary of serum BUN (l), creatinine (m), LDH (n), and AST (o) measured in the indicated mice. p Summary of renal Lcn2 mRNA levels measured in the indicated mice, expressed relative to control. q Summary of renal MDA levels measured in the indicated mice. r Summary of renal reductive GSH levels measured in the indicated mice. s Summary of the percentages of Ly6G-positive area measured in immunostained kidney sections. t Representative H&E-stained, Sirius Red-stained, Masson’s Trichrome-stained, NGAL-stained, 4-HNE-stained and Ly6G-stained kidney sections from the indicated mice. *P < 0.05, **P < 0.01, ***P < 0.001 and n.s., not significant (one-way ANOVA). Survival curve comparison was analyzed by a log-rank Mantel-Cox test. In this and subsequent figures, summary data are represented as the mean ± SEM. See also Supplementary Figs. S9 and S10.
Fig 3: Renal parenchymal cell ferroptosis is critical for metformin-aggravated nephrotoxicity.a Representative image of kidneys removed from mice in the indicated groups. Where indicated, the mice received metformin (100 mg/kg), Fer-1 (1 mg/kg), Nec-1 (1 mg/kg), or Emr (2.5 mg/kg). b Summary of the kidney to body weight ratio measured in the indicated mice. c–f Summary of serum BUN (c), creatinine (d), LDH (e), and AST (f) measured in the indicated mice. g Summary of renal Lcn2 mRNA levels measured in the indicated mice. h Summary of renal MDA levels measured in the indicated mice. i Representative H&E-stained, Sirius Red-stained, and Masson’s Trichrome-stained kidney sections from the indicated mice. j Summary of significantly differential pathways in the indicated parenchymal cell types of I/R+Met200 vs I/R. k t-SNE reveals clustering of 69,608 renal cells obtained from mice in the I/R group (n = 2), I/R+Met200 group (n = 2), and I/R+Met200+Fer-1 group (n = 2). l. t-SNE reveals 14 distinct cell types in the kidney. m Annotation of renal cells in I/R, I/R+Met200 and I/R+Met200+Fer-1 groups. n Summary of the number of cells included in the scRNA-seq analysis for the indicated groups (n = 2 mice per group), expressed relative to the Sham. o Summary of the percentages of the indicated cell types in the indicated groups. p Summary of the percentage of the indicated renal parenchymal cell types in 4 groups shown at the left. q Electron micrographs showing the cellular morphology of renal tubular epithelial cells in the indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001, and n.s., not significant (One-way ANOVA). See also Supplementary Figs. S5–S8.
Fig 4: Mobilization of neutrophils to the site of kidney damage via CXCR4 is mediated by NGAL–metformin–Fe.a The Cxcr family mRNAs were measured in neutrophils obtained from scRNA-seq data. b Kaplan–Meier survival curves of the I/R+Met100 group and I/R+Met100+SCH group. c Representative image of kidneys removed from a mouse following I/R, a mouse in I/R+Met100, a mouse in I/R+Met100+AMD (5 mg/kg), and a mouse in I/R+Met100+WZ (4 mg/kg) group. d Summary of the kidney to body weight ratio measured in the indicated mice. e–h Summary of serum BUN (e), creatinine (f), LDH (g), and AST (h) measured in the indicated mice. i Summary of the Lcn2 mRNA levels measured in the indicated mice, expressed relative to I/R. j Summary of the renal MDA levels measured in the indicated mice. k Summary of the renal MPO levels measured in the indicated mice. l Representative H&E-stained, Sirius Red-stained, Masson’s Trichrome-stained, MPO IHC-stained, Neutrophil Elastase IF-stained, and electron micrographs showing kidney sections from the indicated mice. m Cryo-EM density of CXCR4 (green) and NGAL (orange). NGAL binds to an extracellular facing cavity in CXCR4. n CXCR4 and NGAL were either co-expressed (Top panel) or only expressing CXCR4 and then co-cultured with recombinant human NGAL (rhNGAL) (Bottom panel) in HEK293T cells; 48 h after transfection, the cell lysates were immunoprecipitated using protein A/G beads to pull-down CXCR4 or NGAL (IP), respectively, followed by immunoblotting (IB) with the other antibody. Immunoglobulin G was used as a negative control, and the input was used a positive control. o Green asterisk labels the NGAL, red asterisk labels the CXCR4, then analyzed using immunofluorescence. *P < 0.05, **P < 0.01, ***P < 0.001 and n.s., not significant (One-way ANOVA). Survival curve comparison was analyzed by a log-rank Mantel-Cox test. In this and subsequent figures, summary data are represented as the mean ± SEM. See also Supplementary Figs. S18 and S19.
Fig 5: Metformin induces renal parenchymal cell death to exacerbate acute kidney injury in a dose-dependent manner.a Summary of the kidney to body weight ratio measured in the Sham, I/R, I/R+Met50 (50 mg/kg i.p.), I/R+Met100 (100 mg/kg i.p.), and I/R + NA (50 mg/kg i.p.), I/R+Res (100 mg/kg i.p.), I/R+Rap (10 mg/kg i.p.) and I/R+Sen (dasatinib 5 mg/kg i.p. and quercetin 50 mg/kg i.p.) mice 24 h after I/R. b–e Summary of serum BUN (b), creatinine (c), LDH (d), and AST (e) measured in the indicated mice. f Summary of renal Lcn2 mRNA level measured in the indicated mice, expressed relative to control. g Representative Hematoxylin and Eosin (H&E)-stained kidney sections from indicated mice. h Kaplan–Meier survival curves of mice in the I/R and I/R+Met200 (200 mg/kg i.p.) groups (n = 10 mice per group). i. Kaplan–Meier survival curves of mice in the I/R and I/R+Met50 (50 mM o.a.) groups (n = 10 mice per group). j Overview of the experimental design, depicting the strategy for isolating renal cells and performing scRNA-seq. k T-distributed stochastic neighbor embedding (t-SNE) reveals clustering of 79,224 renal cells obtained from mice in sham group (n = 2), I/R group (n = 2), and I/R+Met200 group (n = 2). l. t-SNE reveals 14 distinct cell types in the kidney. m Annotation of renal cells in sham, I/R, and I/R+Met200 groups. n Violin plots showing the expression of the indicated marker genes in each cell type. o Left: heatmap of the differential expression of the clustered marker genes measured in each cell type. Right: GO enrichment terms for the various cell types. p Summary of the cell numbers included in the scRNA-seq analysis for mice in sham group (n = 2), I/R group (n = 2), and I/R+Met200 group (n = 2). q Summary of the percentage of the indicated cell types in the indicated groups. r Schematic diagram of renal parenchymal cell types. **P < 0.01, ***P < 0.001 and n.s., not significant (One-way ANOVA). Survival curve comparison was analyzed by a log-rank Mantel-Cox test. In this and subsequent figures, summary data are represented as the mean ± SEM. See also Supplementary Figs. S1–S4.
Supplier Page from Novoprotein Scientific for Recombinant Mouse NGAL (C-6His)