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, Itgβ6), metabolic activity (ApoM, Pgc1α) 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：针对全肾小球及分离的肾小球内皮细胞（glomerular endothelial cells, GEC）的近期转录组学研究，为阐明糖尿病肾病进展中特定基因表达与内皮功能失调之间的潜在关联提供了亟需的认知基础。同样，包括本团队在内的多项阿尔波特综合征（Alport syndrome, AS）小鼠模型研究均提示，GEC损伤可推动疾病进展。然而，目前关于此类细胞的潜在细胞与分子通路，以及其相关转录特征的认知仍十分有限，甚至近乎空白。
方法：我们采用内皮细胞特异性tdTomato报告基因小鼠模型，验证了从野生型（wild type, WT）与AS小鼠肾脏中分离GEC的可行性。为获取GEC的特异性转录组特征快照，并阐明AS中的基因表达变化，我们对4月龄的AS-TektdT小鼠及对照WT-TektdT小鼠的GEC开展了全转录组RNA测序分析。
结果：我们基于TektdT信号的荧光强度，鉴定出两种GEC亚群（dimtdT与brighttdT）。基因表达分析显示，这两种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对照相比，AS模型小鼠的dimtdT与brighttdT GEC在基质相关基因（Svep1、Itgβ6）、代谢活性相关基因（ApoM、Pgc1α）及免疫调控相关基因（Apelin、Icam1）的表达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特征。
结论：本研究结果表明，AS进程中存在两种GEC亚群，二者在代谢与炎症免疫活性方面呈现截然不同的应答特征。深入阐明GEC在AS疾病进展中的病理作用，可为开发新型干预靶点提供思路。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。