Table6_Identification of six hub genes and two key pathways in two rat renal fibrosis models based on bioinformatics and RNA-seq transcriptome analyses.XLSX

Renal fibrosis (RF) is the common pathological manifestation and central treatment target of multiple chronic kidney diseases with high morbidity and mortality. Currently, the molecular mechanisms underlying RF remain poorly understood, and exploration of RF-related hub targets and pathways is urgently needed. In this study, two classical RF rat models (adenine and UUO) were established and evaluated by HE, Masson and immunohistochemical staining. To clear molecular mechanisms of RF, differentially expressed genes (DEGs) were identified using RNA-Seq analysis, hub targets and pathways were screened by bioinformatics (functional enrichment analyses, PPI network, and co-expression analysis), the screening results were verified by qRT-PCR, and potential drugs of RF were predicted by network pharmacology and molecular docking. The results illustrated that renal structures were severely damaged and fibrotic in adenine- and UUO-induced models, as evidenced by collagen deposition, enhanced expressions of biomarkers (TGF-β1 and α-SMA), reduction of E-cadherin biomarker, and severe renal function changes (significantly decreased UTP, CREA, Ccr, and ALB levels and increased UUN and BUN levels), etc. 1189 and 1253 RF-related DEGs were screened in the adenine and UUO models, respectively. Two key pathways (AGE-RAGE and NOD-like receptor) and their hub targets (Tgfb1, Col1a1, Nlrc4, Casp4, Trpm2, and Il18) were identified by PPI networks, co-expressed relationships, and qRT-PCR verification. Furthermore, various reported herbal ingredients (curcumin, resveratrol, honokiol, etc.) were considered as important drug candidates due to the strong binding affinity with these hub targets. Overall, this study mainly identified two key RF-related pathways (AGE-RAGE and NOD-like receptor), screened hub targets (Tgfb1, Col1a1, Nlrc4, Casp4, Trpm2, and Il18) that involved inflammation, ECM formation, myofibroblasts generation, and pyroptosis, etc., and provided referable drug candidates (curcumin, resveratrol, honokiol, etc.) in basic research and clinical treatment of RF.

肾纤维化（Renal fibrosis, RF）是多种发病率与死亡率均较高的慢性肾脏病共有的病理表现与核心治疗靶点。目前，肾纤维化的潜在分子机制尚不明确，开展肾纤维化相关核心靶点与通路的探索研究已迫在眉睫。本研究构建了两种经典的肾纤维化大鼠模型：腺嘌呤诱导模型与单侧输尿管梗阻（Unilateral Ureteral Obstruction, UUO）模型，并通过苏木精-伊红（HE）染色、马松（Masson）染色及免疫组化染色对模型进行评价。为阐明肾纤维化的分子机制，本研究通过RNA测序（RNA-Seq）分析筛选得到差异表达基因（differentially expressed genes, DEGs），借助生物信息学方法（功能富集分析、蛋白质-蛋白质相互作用（PPI）网络分析及共表达分析）筛选核心靶点与通路，通过实时定量聚合酶链反应（qRT-PCR）验证筛选结果，并通过网络药理学与分子对接技术预测肾纤维化潜在治疗药物。
研究结果显示，腺嘌呤与单侧输尿管梗阻诱导的大鼠模型肾组织结构均出现严重损伤与纤维化，具体表现为胶原沉积、纤维化标志物转化生长因子-β1（TGF-β1）与α平滑肌肌动蛋白（α-SMA）表达上调、上皮细胞标志物E-钙粘蛋白（E-cadherin）表达下调，同时伴随严重肾功能异常：尿三磷酸核苷（UTP）、肌酐（CREA）、肌酐清除率（Ccr）与白蛋白（ALB）水平显著降低，尿尿素氮（UUN）与血尿素氮（BUN）水平显著升高等。腺嘌呤模型与单侧输尿管梗阻模型中分别筛选得到1189个与1253个肾纤维化相关差异表达基因。经PPI网络、共表达关系分析及qRT-PCR验证，最终确定2条核心通路（AGE-RAGE通路与核苷酸结合寡聚化结构域样受体（NOD-like receptor）通路）及其对应的6个核心靶点：Tgfb1、Col1a1、Nlrc4、Casp4、Trpm2及Il18。此外，多种已报道的中药活性成分（如姜黄素、白藜芦醇、厚朴酚等）因与上述核心靶点具有较强结合活性，被认定为潜在候选治疗药物。
综上，本研究成功鉴定出2条肾纤维化相关核心通路（AGE-RAGE通路与NOD-like receptor通路），筛选得到参与炎症反应、细胞外基质（ECM）沉积、肌成纤维细胞生成及细胞焦亡等病理过程的核心靶点（Tgfb1、Col1a1、Nlrc4、Casp4、Trpm2及Il18），并为肾纤维化的基础研究与临床治疗提供了具有参考价值的候选药物。