SPATIOTEMPORAL TRANSCRIPTOMIC INSIGHTS INTO FERROPTOSIS AND TFRC-LINKED IMMUNE INTERACTIONS IN ISCHEMIA-REPERFUSION ACUTE KIDNEY INJURY

Acute kidney injury (AKI) is a common and life-threatening clinical syndrome with complex pathophysiological mechanisms and a lack of effective early intervention strategies. In recent years, ferroptosis—a form of programmed cell death driven by iron-dependent lipid peroxidation—has attracted increasing attention due to its pivotal role in various types of tissue injury, especially in the pathogenesis and progression of AKI. Emerging evidence suggests that targeting ferroptosis pathways to mitigate tubular epithelial cell injury represents a promising precision therapeutic approach. This study aims to systematically explore the expression patterns, diagnostic potential, and functional roles of ferroptosis-related genes in ischemia–reperfusion-induced AKI (IR-AKI), with a particular focus on their involvement in proximal tubule cell (PTC) injury and immune microenvironment remodeling, thereby identifying potential therapeutic targets.

Bulk RNA sequencing, single-cell RNA sequencing (scRNA-seq), and spatial transcriptomics (ST) datasets of human kidney tissues were obtained from the Gene Expression Omnibus (GEO) database. Ferroptosis-related genes were identified through Gene Set Variation Analysis (GSVA) and Differential Expression Gene (DEG) analysis. An AKI diagnostic model was constructed using Random Forest (RF) and Support Vector Machine–Recursive Feature Elimination (SVM-RFE), and validated by Receiver Operating Characteristic (ROC) curve analysis and Decision Curve Analysis (DCA). CIBERSORT was applied to evaluate immune cell infiltration and gene–cytokine associations. TFRC expression and its immune interactions in PTCs were analyzed using scRNA-seq and ST. Key findings were validated in an ischemia–reperfusion-induced AKI (IR-AKI) mouse model and CD8⁺ T cell co-culture experiments.

GSVA analysis revealed significant enrichment of ferroptosis-related gene sets in patients with IR-AKI. Differential expression analysis identified 17 ferroptosis-associated genes, among which TFRC, TXNRD1 (Thioredoxin Reductase 1), SLC39A14 (Solute Carrier Family 39 Member 14), GCLM (Glutamate-Cysteine Ligase Modifier Subunit), and HMOX1 (Heme Oxygenase 1) were markedly upregulated. A diagnostic model was constructed using five key genes selected by Random Forest (RF) and Support Vector Machine–Recursive Feature Elimination (SVM-RFE) (Figure. 1a–d). ROC analysis demonstrated excellent predictive performance of the model with an Area Under the Curve (AUC) of 0.908 (Fig. 1f), and DCA further supported its strong clinical applicability. Multivariable logistic regression analysis identified TFRC as the only significant risk factor (OR = 7.94, 95% CI: 3.25–23.44) (Figure. 1e). Immune infiltration analysis showed that high TFRC expression was positively correlated with M1 and M2 macrophages and CD8⁺ T cells, but negatively correlated with CD4⁺ T cells, natural killer T (NKT) cells, and regulatory T (Treg) cells. Moreover, elevated TFRC levels were associated with enhanced local acute inflammatory responses and ferroptosis severity.

Single-cell analysis revealed that proximal tubule cells (PTCs) in AKI kidneys exhibited the highest levels of ferroptosis, with TFRC, which encodes the transferrin receptor protein 1 (TfR1), mainly expressed in PTCs (Figure 2a). Compared with TFRC-low PTCs, Tfrc⁺ PTCs showed more interaction pathways with macrophages, significantly activating the complement system (C4a-C3aR1) and pro-inflammatory signaling (Mdk-Ncl) (Figures 2b–d). Spatial transcriptomics further validated enhanced interactions between Tfrc⁺ PTCs and macrophages, endothelial cells, and fibroblasts (Figures 2e–f). Monocle trajectory inference indicated that high-Tfrc-expressing PTCs tended to differentiate into subpopulations with elevated ferroptosis levels.

In animal experiments, TfR1 was significantly upregulated in the kidneys of IR-AKI mice, and treatment with the TfR1 degrader NSC306711 effectively suppressed TfR1 and NGAL (Neutrophil Gelatinase-Associated Lipocalin) expression, alleviating renal tissue injury and macrophage infiltration. Co-culture experiments further confirmed that Tfrc⁺ PTCs promoted macrophage secretion of inflammatory factors, which was markedly inhibited by NSC306711 treatment.

In the murine ischemia–reperfusion model, the roles of key ferroptosis-related genes were validated by Western blot, immunohistochemistry (IHC), and immunofluorescence (IF). NSC306711 significantly reduced TfR1 expression and the kidney injury marker serum NGAL in IR mice, consistent with IHC results. IF showed increased TfR1 (magenta) and macrophage marker F4/80 (cyan) in the IR group, indicating elevated TfR1 in proximal tubule cells and enhanced macrophage infiltration after IR injury. Following NSC306711 treatment, both TfR1 and F4/80 levels were reduced, suggesting alleviation of ferroptosis and macrophage infiltration.

In this study, we found a crucial ferroptosis-related genes enabled the construction of a robust model for AKI early diagnosis. Tfrc+ PTCs showed a positive correlation with the immune activation, especially macrophages . By TfR1 degradation, ferroptosis and macrophages infiltration was reduced and kidney injury was relieved. Altogether, these findings might contribute to a deeper understanding of the complex interplay between PTCs, ferroptosis-related genes, and immune cells, shedding light on potential therapeutic interventions for AKI.