Table_1_Integrated analysis reveals crosstalk between pyroptosis and immune regulation in renal fibrosis.xlsx

PurposeThe pathogenesis of renal fibrosis (RF) involves intricate interactions between profibrotic processes and immune responses. This study aimed to explore the potential involvement of the pyroptosis signaling pathway in immune microenvironment regulation within the context of RF. Through comprehensive bioinformatics analysis and experimental validation, we investigated the influence of pyroptosis on the immune landscape in RF.MethodsWe obtained RNA-seq datasets from Gene Expression Omnibus (GEO) databases and identified Pyroptosis-Associated Regulators (PARs) through literature reviews. Systematic evaluation of alterations in 27 PARs was performed in RF and normal kidney samples, followed by relevant functional analyses. Unsupervised cluster analysis revealed distinct pyroptosis modification patterns. Using single-sample gene set enrichment analysis (ssGSEA), we examined the correlation between pyroptosis and immune infiltration. Hub regulators were identified via weighted gene coexpression network analysis (WGCNA) and further validated in a single-cell RNA-seq dataset. We also established a unilateral ureteral obstruction-induced RF mouse model to verify the expression of key regulators at the mRNA and protein levels.ResultsOur comprehensive analysis revealed altered expression of 19 PARs in RF samples compared to normal samples. Five hub regulators, namely PYCARD, CASP1, AIM2, NOD2, and CASP9, exhibited potential as biomarkers for RF. Based on these regulators, a classifier capable of distinguishing normal samples from RF samples was developed. Furthermore, we identified correlations between immune features and PARs expression, with PYCARD positively associated with regulatory T cells abundance in fibrotic tissues. Unsupervised clustering of RF samples yielded two distinct subtypes (Subtype A and Subtype B), with Subtype B characterized by active immune responses against RF. Subsequent WGCNA analysis identified PYCARD, CASP1, and NOD2 as hub PARs in the pyroptosis modification patterns. Single-cell level validation confirmed PYCARD expression in myofibroblasts, implicating its significance in the stress response of myofibroblasts to injury. In vivo experimental validation further demonstrated elevated PYCARD expression in RF, accompanied by infiltration of Foxp3+ regulatory T cells.ConclusionsOur findings suggest that pyroptosis plays a pivotal role in orchestrating the immune microenvironment of RF. This study provides valuable insights into the pathogenesis of RF and highlights potential targets for future therapeutic interventions.

目的：肾纤维化（RF）的发病机制涉及促纤维化过程与免疫反应之间的复杂相互作用。本研究旨在探讨在RF背景下，细胞焦亡信号通路在免疫微环境调节中的潜在作用。通过全面的生物信息学分析和实验验证，我们研究了细胞焦亡对RF免疫景观的影响。
方法：我们从基因表达综合数据库（GEO）获取RNA-seq数据集，并通过文献综述确定了细胞焦亡相关调节因子（PARs）。在RF和正常肾脏样本中系统性地评估了27个PARs的变化，随后进行了相关功能分析。无监督聚类分析揭示了独特的细胞焦亡修饰模式。利用单样本基因集富集分析（ssGSEA），我们检查了细胞焦亡与免疫浸润之间的相关性。通过加权基因共表达网络分析（WGCNA）确定了枢纽调节因子，并在单细胞RNA-seq数据集中进行了进一步验证。我们还建立了单侧输尿管梗阻诱导的RF小鼠模型，以验证关键调节因子的mRNA和蛋白质水平表达。
结果：我们的全面分析显示，与正常样本相比，RF样本中19个PARs的表达发生了改变。五个枢纽调节因子，即PYCARD、CASP1、AIM2、NOD2和CASP9，表现出作为RF生物标志物的潜力。基于这些调节因子，开发了一个能够区分正常样本和RF样本的分类器。此外，我们发现了免疫特征与PARs表达之间的相关性，PYCARD与纤维化组织中调节性T细胞的丰富度呈正相关。RF样本的无监督聚类产生了两种不同的亚型（亚型A和亚型B），其中亚型B以对RF的活性免疫反应为特征。后续的WGCNA分析确定了PYCARD、CASP1和NOD2为细胞焦亡修饰模式中的枢纽PARs。单细胞水平的验证确认了PYCARD在肌成纤维细胞中的表达，这暗示了其在肌成纤维细胞对损伤应激反应中的重要性。体内实验验证进一步表明，RF中PYCARD表达升高，伴有Foxp3+调节性T细胞的浸润。
结论：我们的研究结果表明，细胞焦亡在调节RF的免疫微环境中起着关键作用。本研究为RF的发病机制提供了宝贵的见解，并突出了未来治疗干预的潜在靶点。