Table 1_Bioinformatic analysis, clinical implications and experimental validation of ferroptosis-related feature gene in IgA nephropathy: focus on DUSP1.docx

BackgroundImmunoglobulin A nephropathy (IgAN), recognized as the leading cause of primary glomerular disease worldwide, continues to present unresolved complexities in its underlying pathogenic mechanisms. Emerging evidence underscores ferroptosis, an iron-mediated regulated cell death pathway driven by the accumulation of lipid peroxides, as a potential contributor to various pathological conditions. Despite growing interest in this field, the exact molecular pathways governing ferroptosis activation in IgAN progression remain incompletely understood and require systematic investigation. The aim of this study was to identify ferroptosis-related feature gene (FFG) for the potential diagnosis of IgAN and to investigate its relationship with renal immune cell infiltration.
MethodsRenal tissue microarray datasets (GSE93798, GSE104948, GSE99339) from IgAN patients and normal controls were retrieved from GEO database. The ferroptosis-related genes were obtained from the Ferrb database. Machine learning algorithms (LASSO, SVM-RFE, random forest) were employed to screen FFGs. The findings were validated in an IgAN mouse model using immunohistochemistry and western blotting. Gene set enrichment analysis (GSEA) was conducted to explore the underlying mechanism of FFG in IgAN. Immune cell infiltration characteristics were also analyzed vis CIBERSORT algorithm.
ResultsA total of 180 ferroptosis-related differentially expressed genes were identified in IgAN. Among them, dual specificity phosphatase 1 (DUSP1) was screened as FFG by three machine learning algorithms. DUSP1 exhibited significant downregulation in renal tissues of both IgAN patients and mice. Enhanced transcriptional abundance demonstrated significant positive associations with ferroptosis-associated biomarkers glutathione peroxidase-4 (GPX4) and cystine/glutamate antiporter (SLC7A11/xCT), while displaying an inverse relationship with acyl-CoA synthetase long-chain isoform 4 (ACSL4) expression. GSEA further identified DUSP1’s functional enrichment in critical signaling networks, particularly mitogen-activated protein kinase (MAPK) cascades, ERBB receptor tyrosine kinase pathways, and Janus kinase-signal transducer (JAK–STAT) transduction mechanisms. Immunoinfiltration analysis demonstrated increased infiltration of T follicular helper cells, activated NK cells, and M1 macrophages in the renal tissues of IgAN patients, with DUSP1 expression showing negative correlations with these proinflammatory cell types.
ConclusionOur research successfully identified DUSP1 as a ferroptosis-related biomarker in IgAN patients, and explored its potential mechanism in the pathogenesis of IgAN and its potential relationship with immune cell infiltration. These findings are of great significance for the diagnosis and prospective treatment strategies for IgAN patients.

背景：免疫球蛋白A肾病（Immunoglobulin A nephropathy, IgAN）是全球范围内原发性肾小球疾病的首要病因，其潜在发病机制仍存在诸多未阐明的复杂问题。越来越多的研究证据表明，铁死亡（ferroptosis）——一种由脂质过氧化物积累驱动的铁介导调控性细胞死亡通路——可能参与多种病理过程。尽管该领域的研究关注度日益提升，但目前对于IgAN进展过程中铁死亡激活的具体分子通路仍不完全明确，亟需开展系统性研究。本研究旨在筛选与铁死亡相关的特征基因（ferroptosis-related feature gene, FFG），以辅助IgAN的潜在诊断，并探究其与肾脏免疫细胞浸润的关联。
方法：从GEO数据库中获取IgAN患者与正常对照的肾脏组织微阵列数据集（GSE93798、GSE104948、GSE99339）。铁死亡相关基因从Ferrb数据库中获取。采用机器学习算法（LASSO、SVM-RFE、随机森林）筛选铁死亡相关特征基因。通过免疫组化及蛋白质印迹法（Western Blotting）在IgAN小鼠模型中对筛选结果进行验证。采用基因集富集分析（Gene Set Enrichment Analysis, GSEA）探究铁死亡相关特征基因在IgAN中的潜在作用机制。同时通过CIBERSORT算法分析肾脏组织的免疫细胞浸润特征。
结果：共在IgAN患者中鉴定出180个铁死亡相关差异表达基因。其中，双特异性磷酸酶1（dual specificity phosphatase 1, DUSP1）经三种机器学习算法筛选被确定为核心特征基因。DUSP1在IgAN患者及小鼠的肾脏组织中均呈现显著下调表达。其转录丰度升高与铁死亡相关生物标志物谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）及胱氨酸/谷氨酸反向转运体（cystine/glutamate antiporter, SLC7A11/xCT）的表达呈显著正相关，而与长链酰基辅酶A合成酶4（acyl-CoA synthetase long-chain isoform 4, ACSL4）的表达呈显著负相关。基因集富集分析进一步显示，DUSP1显著富集于多条关键信号通路网络，尤其包括丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）级联反应、ERBB受体酪氨酸激酶通路及Janus激酶-信号转导与转录激活因子（Janus kinase-signal transducer and activator of transcription, JAK-STAT）转导通路。免疫浸润分析结果显示，IgAN患者肾脏组织中滤泡辅助性T细胞、活化NK细胞及M1巨噬细胞的浸润水平显著升高，且DUSP1的表达与这些促炎性细胞类型均呈负相关。
结论：本研究成功鉴定出DUSP1可作为IgAN患者的铁死亡相关生物标志物，并探究了其在IgAN发病机制中的潜在作用及与免疫细胞浸润的潜在关联。上述研究结果对于IgAN患者的临床诊断及未来治疗策略的开发具有重要的指导意义。