Nucleophosmin 1 lactylation in graft kidney induces ferroptotic trigger waves that exacerbate delayed graft function

Abstract

Ferroptotic waves aggravate kidney ischemia-reperfusion injury and drive delayed graft function (DGF). We demonstrate that elevated glycolysis and lactate production in graft kidney correlate with ferroptosis and functional impairment. A signaling axis composed of the long non-coding RNA IGIP-5, microRNA 670-3p, and lactate dehydrogenase A promotes lactate secretion from injured tubular cells, inducing lactylation and ferroptosis in neighboring cells and triggering ferroptotic waves. Lactylome profiling identifies that nucleophosmin 1 (NPM1), an epigenetic regulator, is lactylated at lysine 257 by the lactyltransferase AARS1. Suppressing NPM1 lactylation-via K257 mutation, AARS1 knockout, or taurochenodeoxycholic acid-upregulates SLC7A11 and inhibits ferroptosis. Mechanistically, lactylation stabilizes NPM1 by reducing MDM2-mediated ubiquitination and strengthens SLC7A11 repression, disrupting cystine metabolism. In mouse allografts, blocking lactate shuttle-mediated NPM1 lactylation prevents ferroptotic propagation and ameliorates graft function. Additionally, we develop an early prediction model for DGF using postoperative urinary lactate concentrations. These findings reveal a metabolic-epigenetic axis driving ferroptotic propagation and propose NPM1 lactylation as a therapeutic target for DGF.

Fig. 1. The high levels of glycolysis and lactate are correlated to the delayed graft function (DGF).

a Heatmap illustrated the energy metabolic metabolites in mouse kidney tissues after ischemia and reperfusion for 0 h, 12 h and 24 h (n = 4 group⁻¹). b Lactate assay kit detecting lactate concentration in IRI kidney tissues at different reperfusion stages (n = 6 group⁻¹) and normalized according to the sham group concentrations; one-way ANOVA followed by Tukey’s test. c, d The translational and transcriptional levels of glycolytic enzymes in mouse kidney tissues following ischemia-reperfusion injury (IRI) (n = 6 group⁻¹), and normalized according to sham group levels; one-way ANOVA followed by Tukey’s test. e, f The serum creatinine (SCr) and blood urea nitrogen (BUN) concentration of mice after kidney IRI (n = 6 group⁻¹), and one-way ANOVA followed by Tukey’s test; Boxplot: 25th–75th percentiles; center line: median; whiskers: 1.5 times the interquartile range. g A two-sided Pearson correlation analysis of SCr concentration and lactate concentration (r = 0.756, p < 0.001, n = 30). h, i Urinary lactate concentration of non-DGF (n = 105) and DGF (n = 45) recipients on the postoperative days 2 (ULa-2 d) and 7 (ULa-7 d); unpaired 2-tailed Student’s t test. j A two-sided Pearson correlation analysis of SCr and ULa-2 d concentrations (r = 0.716, p < 0.001, n = 150). k The ROC curve of the DGF prediction model based on ULa-2 d of recipients in the training set (n = 150). l, m Urinary lactate concentration of non-DGF (n = 45) and DGF (n = 19) recipients in the validation set and the ROC curve of the DGF prediction model; unpaired 2-tailed Student’s t test. n Kaplan‒Meier survival curves of the five-year graft survival stratified by the median ULa-2 d level (n = 110); the p value was calculated via the log-rank test. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.

Fig. 2. Lactate shuttle attenuates cystine/cysteine/GSH metabolism and induces ferroptotic propagation.

a Flowchart delineating kidney IRI protocol in C57BL/6 mice; sodium oxamate (1 g/kg, OXA), sodium lactate (120 mg/kg, Lac), ferrostatin-1 (5 mg/kg, Fer-1), normal saline (N.S.). SCr (b) and BUN (c) concentration in mice treated with the same experimental setup in a; lipid peroxidation level (d), malondialdehyde (MDA) concentration (e) and iron level (f) in mouse kidney tissues (n = 20 group⁻¹); iron level was normalized according to sham group results; one-way ANOVA followed by Tukey’s test; Boxplot: 25th–75th percentiles; center line: median; whiskers: 1.5 times the interquartile range. g Lactate concentration in conditioned medium of normal and IRI HK-2 cells (NC-CM and IR-CM); normalized according to concentrations in NC-CM (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test; IR-CM_siMCT4 and IR-CM_OXA represent CM from IRI HK-2 cells treated with siMCT4 or OXA. h Schematic representing co-culture models established by normal HK-2 cells and different CM. Lactate concentration (i) and lipid peroxidation (j) in HK-2 cells treated with the same experimental setup in (h) (n = 3 group⁻¹); normalized according to Vehicle group concentrations; one-way ANOVA followed by Tukey’s test. k Flowchart depicting the metabolic flux tracing assay in secreting cells incubated with [¹³C₃]-L-Lactate and importing cells. l, m M + 3 lactate fraction in cell lysate and lactate transport rate (the ratio of m + 3 lactate fraction in importing cells to secreting cells) (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. n Metabolite contents in cystine/cysteine/GSH pathway in mouse kidney tissues (n = 6 group⁻¹) and normalized according to sham group concentrations; one-way ANOVA followed by Tukey’s test. o, p Western blotting determining SLC7A11 expression in mouse kidney tissues after different reperfusion phases (n = 6 group⁻¹) and different treatments (n = 20 group⁻¹). Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Figure 2a, h were created in FigDraw. https://www.figdraw.com; Fig. 2k, n were created in BioRender. Feng, Z. (2025) https://BioRender.com/ye9fs3m and https://BioRender.com/e3ywthw. Source data are provided as a Source Data file.

Fig. 3. LncRNA IGIP-5 facilitates LDHA expression via sponging to miRNA 670-3p in IRI renal tubular epithelial cells.

a LncRNA IGIP-5 levels in IRI kidney tissues at different reperfusion stages (n = 6 group⁻¹); normalized according to sham group levels; one-way ANOVA followed by Tukey’s test. Lactate concentration (b) and transcriptional and translational levels of glycolytic enzymes (c, d) in HK-2 cells with lncRNA IGIP-5 overexpression (oeIGIP) and knockout (koIGIP); normalized to sham group levels (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. e Relative miRNA levels in wild-type (WT), oeIGIP and koIGIP HK-2 cells; normalized according to WT group levels (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. f LDHA transcriptional level in HK-2 cells transfected with miRNA mimics; normalized to Vehicle group levels (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. g RIP assays extracted endogenous RNA associated with AGO2 in HK-2 cells transfected with miR 670-3p inhibitor or inhibitor NC; normalized according to IgG group results (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. h WT and mutated LDHA sequences containing the predicting miR-670-3p binding site; dual luciferase assays detecting luciferase activity in HEK293T cells co-transfected with pmirGLO reporter plasmids and either mimic NC or miR-670-3p mimic (n = 3 group⁻¹); normalized according to mimic NC group results; unpaired 2-tailed Student’s t test. i, j Transcriptional and translational levels of glycolytic enzymes in HK-2 cells transfected with miRNA 670-3p mimic and inhibitor (n = 3 group⁻¹); normalized according to mimic NC group results; one-way ANOVA followed by Tukey’s test. k–o Lipid peroxidation level, MDA concentration, iron level, GSH content, and SLC7A11 protein expression in normal HK-2 cells cocultured with different CM (n = 3 group⁻¹); IR-CM_oeIGIP refers to CM from IGIP5-overexpressing IRI HK-2 cell; iron level and GSH content were normalized to NC-CM group results; one-way ANOVA followed by Tukey’s test. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.

Fig. 4. Lactate shuttling-mediated NPM1 lactylation inhibits SLC7A11 expression and induces ferroptosis during kidney IRI.

a, b Global lactylation of HK-2 cells cocultured with different CM or lactate for 24 h; IR-CM-12h represents the CM of HK-2 cells experienced ischemia and reperfusion for 12 h (n = 3 group⁻¹). c Bubble plot of significantly enriched GO terms (Biological Process, BP); Fisher’s exact test. d The top 30 proteins with the highest lactylation intensity from the lactylome analysis and the functional enrichment analysis for metabolism and transcriptional regulation. e Venn diagram of lactylated proteins enriched in cystine/cysteine/GSH metabolic pathway and metabolism & transcriptional regulation pathway. IP assays determining endogenous NPM1 lactylation in mouse kidney tissues (f, n = 6 group⁻¹; g, n = 20 group⁻¹) and HK-2 cells stimulated with different CM (h, i, n = 3 group⁻¹). j NPM1 lactylation in endogenous NPM1-knockout (NKO) HK-2 cells reconstituted with full-length NPM1 or its site-directed mutants (n = 3 group⁻¹). k MS analysis identified residue lysine 257 (K257) as the key lactylation site. l KEGG analysis displaying the top 20 enriched pathways of differentially expressed genes (DEGs) from RNA sequencing in NKO-NPM1 WT versus NKO-NPM1 K257R cells (n = 6 versus 6). m Volcano plot highlighting DEGs enriched in ferroptosis pathway (n = 12). n, o Translational and transcriptional levels of ferroptosis-related genes in NKO HK-2 cells expressing either WT or K257R NPM1, following treatment with or without 10 mM sodium lactate (n = 3 group⁻¹); normalized to negative control group results; one-way ANOVA followed by Tukey’s test. p ChIP‒sequencing tracks for SLC7A11 in NKO HK-2 cells reconstituted with WT or K257R NPM1. q ChIP-qPCR was conducted using anti-NPM1 antibody to assess the enrichment of WT or K257R NPM1 at the SLC7A11 promoter in NKO HK-2 cell (n = 3 group⁻¹); one-way ANOVA followed by Tukey’s test. r Lipid peroxidation in HK-2 cells overexpressing WT or K257R NPM (n = 3 group⁻¹). Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.

Fig. 5. NPM1 lactylation enhances protein stability by suppressing MDM2-mediated ubiquitination.

Protein stability detection and half-life analysis of NPM1 WT (a, b) and K257R (c, d) in HK-2 cells treated with 10 mM sodium lactate, 10 mM sodium oxamate, or vehicle control (n = 3 group⁻¹). e Assessment of ubiquitination for WT and K257R NPM1 in NKO HK-2 cells co-transfected with Flag-tagged ubiquitin (Ub-Flag), following treatment with sodium lactate (10 mM), sodium oxamate (10 mM), or vehicle control (n = 3 group⁻¹). f IP-MS analysis of NPM1-interacting proteins in HK-2 cells treated with IR-CM for 24 h, revealing multiple E3 ubiquitin ligases including MDM. g Co-IP assays detecting the interactions between NPM1-HA and MDM2-His using anti-HA antibody in NKO HK-2 cells treated with 10 mM sodium lactate, 10 mM sodium oxamate, or vehicle control (n = 3 group⁻¹). h, i IP assays determining the effects of diverse CM on the ubiquitination of NPM1-HA using anti-HA antibody in HK-2 cells co-transfected with Ub-Flag and MDM2-His (n = 3 group⁻¹). j, k Translational and transcriptional levels of NPM1 in HK-2 cells treated with different CM (n = 3 group⁻¹); normalized to Vehicle group levels; one-way ANOVA followed by Tukey’s test. ns represents no significant difference. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The p-values are shown for the indicated comparisons. Figure 5f was created in FigDraw. Source data are provided as a Source Data file.

Fig. 6. AARS1 functions as a lactyltransferase that facilitates NPM1 lactylation and induces ferroptosis in HK-2 cells.

a IP of NPM1 lactylation using an anti-NPM1 antibody in IR-CM-treated HK-2 cells following siRNA-mediated knockdown of enzymes (n = 3 group⁻¹). b Detection of NPM1-HA lactylation by anti-HA IP in HK-2 cells with AARS1 knockout (AKO) or overexpression (AOE) following sodium lactate (10 mM) treatment (n = 3 group⁻¹). c In vitro lactylation assay of purified WT and K253R NPM1 proteins incubated with AARS1, lactate (2 mM), and ATP (4 mM) for 2 h at 37 °C, followed by western blot (n = 3 group⁻¹). d GST pull-down assay to detect the direct interaction between GST-NPM1 and His-AARS1 in vitro; arrows represent the indicated proteins (n = 3 group⁻¹). e Co-IP assays of the endogenous NPM1 and AARS1 in HK-2 cells (n = 3 group⁻¹). Co-IP of NPM1-HA and AARS1-His in HK-2 cells using anti-HA (f) and anti-His (g) antibodies (n = 3 group⁻¹). h Co-IP of NPM1-HA and AARS1-His using an anti-HA antibody in HK-2 cells treated with diverse CM (n = 3 group⁻¹). i–l Lipid peroxidation level, MDA concentration, iron level, and GSH content in WT, AOE, and AKO HK-2 cells cocultured with NC-CM or IR-CM (n = 3 group⁻¹); iron level and GSH content were normalized according to negative control group results; one-way ANOVA followed by Tukey’s test. m Western blotting examining NPM1 and SLC7A11 expression in WT, AOE, and AKO HK-2 cells treated with the same experimental setup as described in (i–l) (n = 3 group⁻¹). n Transcriptional level of SLC7A11 in WT, AOE, and AKO HK-2 cells (n = 3 group⁻¹); normalized to negative control group results; one-way ANOVA followed by Tukey’s test. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.

Fig. 7. Lactate shuttle triggers ferroptotic waves in the kidney tissue microenvironment and exacerbates graft kidney IRI by inducing NPM1 lactylation.

a Flowchart delineating the procedure of allograft kidney transplantation in WT, Aars1^CKO and Slc16a3^CKO C57BL/6 mouse. b Schematic representation of the experimental protocols in vivo. For sodium oxamate administration, recipient mice receive intraperitoneal injection of sodium oxamate (1 g/kg, OXA) or normal saline (N.S.) 2 h before kidney transplantation and 1 d after transplantation. c HE staining evaluating kidney morphology and kidney pathological score (n = 6 group⁻¹); scale bar: 50 μm; one-way ANOVA followed by Tukey’s test. d, e SCr and BUN concentrations in mice treated with the same experimental setup as described in a-b (n = 6 group⁻¹); one-way ANOVA followed by Tukey’s test; Boxplot: 25th–75th percentiles; center line: median; whiskers: 1.5 times the interquartile range. Lipid peroxidation level (f), MDA concentration (g), iron level (h), and GSH content (i) in graft kidney tissues (n = 6 group⁻¹); iron level was normalized according to WT-sham group results; one-way ANOVA followed by Tukey’s test. j Western blotting detecting the indicated proteins and global lactylation levels in graft kidney tissues (n = 6 group⁻¹). k IP assays assessing NPM1 lactylation in graft kidney tissues using an anti-NPM1 antibody (n = 6 group⁻¹). l SLC7A11 expression in graft kidney tissues was detected by western blotting (n = 6 group⁻¹). m IF and fluorescence intensity assaying illustrating the expression level and region of SLC7A11 in graft kidney tissue slices (n = 6 group⁻¹); one-way ANOVA followed by Tukey’s test; yellow arrows representing regions from the proximal part of blood reperfusion to the distal end; scale bar: 1000 μm for 1X. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The p-values are shown for the indicated comparisons. Figure 7a, b were created in FigDraw. Source Data are provided as a Source data file.

Fig. 8. Schematic diagram delineates the metabolic and epigenetic mechanisms underlying the ferroptotic propagation during graft kidney IRI.

Activation of the lncRNA IGIP-5/miRNA 670-3p/LDHA axis in IRI renal tubular epithelial cells promotes glycolytic reprogramming and lactate shuttling in the graft kidney tissue microenvironment. Lactate shuttle enhances AARS1-mediated NPM1 lactylation in adjacent tubular cells, increasing NPM1 protein stability and reinforcing its transcriptional repression of SLC7A11. The subsequent downregulation of SLC7A11 impairs the cystine/cysteine/GSH metabolism, leading to ferroptotic propagation and DGF. Figure 8 was created in GNU Image Manipulation Program (GIMP). https://www.gimp.org/about.