RNA sequencing of mouse glomerular endothelial cells and implications of their heterogeneity in Alport syndrome

Background: Recent transcriptomic studies of total glomerular and isolated glomerular endothelial cells (GEC) provide much needed insight into the potential associations between expression of specific genes and dysfunctional endothelium in the development of diabetic kidney disease. Similarly, many studies on Alport syndrome (AS) mice, including our own, suggest that damage to GEC can contribute to disease progression. However, the underlying cellular and molecular pathways and associated transcriptional identity of the cells remain either limited or non-existent. Methods: We used a model of endothelial-specific tdTomato reporter mouse for the feasibility of isolating the GEC from kidneys of WT and AS mice. To generate a snapshot of GEC specific transcriptional profiles and to elucidate gene expression changes in AS, we performed transcriptome-wide RNA-seq analysis of GEC from AS-TektdT mice and control WT-TektdT mice at 4-month of age. Results: We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. Gene expression analysis showed vast heterogeneity in the two subpopulations, including in the expression of endothelial specific genes. In AS, dimtdT and brighttdT GEC had remarkably heterogeneous profiles in matrix associated genes (Svep1, Itgb6), metabolic activity (ApoM, Pgc1a) and immune modulation (Apelin, Icam1) compared to WT. Additionally, we confirmed the expression data of these genes in biopsy samples from AS and FSGS patients. Conclusions: Our findings suggest the presence of two subpopulations of GEC that develop distinct responses in metabolic and inflammatory immune activity in AS. In depth understanding of the pathologic role of GEC in the progression of AS could lead to novel targets for intervention. Gene expression profiles of glomerular endothelial cells derived from wild type and Alport syndrome mice at 4 month of age were generated by RNA sequencing.背景：近期针对全肾小球及分离的肾小球内皮细胞（glomerular endothelial cells, GEC）的转录组学研究，为阐明特定基因表达与糖尿病肾病发病过程中内皮功能异常之间的潜在关联提供了亟需的研究视角。同样，包括本团队在内的多项针对Alport综合征（Alport syndrome, AS）小鼠模型的研究均表明，GEC损伤可推动疾病进展。然而，目前对这类细胞潜在的细胞与分子通路及相关转录特征的认知仍十分有限，甚至付之阙如。 方法：我们采用内皮细胞特异性tdTomato报告小鼠模型，以实现从野生型（wild type, WT）及AS小鼠肾脏中分离GEC。为获取GEC特异性转录组全貌，并阐明AS中的基因表达变化，我们对4月龄的AS-TektdT小鼠与对照WT-TektdT小鼠的GEC开展了全转录组RNA测序（RNA-seq）分析。 结果：我们基于TektdT信号的荧光强度，鉴定出两类GEC亚群：dimtdT与brighttdT。基因表达分析显示，这两类亚群存在显著异质性，内皮细胞特异性基因的表达亦存在差异。与WT小鼠相比，AS小鼠的dimtdT及brighttdT GEC在基质相关基因（Svep1、Itgb6）、代谢活性相关基因（ApoM、Pgc1a）及免疫调控相关基因（Apelin、Icam1）的表达谱上均呈现显著异质性。此外，我们在AS及局灶节段性肾小球硬化（focal segmental glomerulosclerosis, FSGS）患者的活检样本中验证了上述基因的表达数据。 结论：本研究结果表明，AS中存在两类GEC亚群，其在代谢与炎性免疫活性方面呈现截然不同的应答特征。深入阐明GEC在AS进展中的病理作用，有望为疾病干预提供全新靶点。本研究通过RNA测序获取了4月龄野生型与AS小鼠肾小球内皮细胞的基因表达谱。