Pathogenic pathways are activated in each major cell type of the glomerulus in the Cd2ap mutant mouse model of focal segmental glomerulosclerosis. Mus musculus

Mutations in several genes expressed in podocytes, including Cd2ap, have been associated with focal segmental glomerulosclerosis in humans. Mutant mouse models provide an opportunity to better understand the molecular pathology that drives these diseases. In this report we use a battery of transgenic-GFP mice to facilitate the purification of all three major cell types of the glomerulus from Cd2ap mutant mice. Both microarrays and RNA-seq were used to characterize the gene expression profiles of the podocytes, mesangial cells and endothelial cells, providing a global dual platform cross-validating dataset. The mesangial cells showed increased expression of profibrotic factors, including thrombospondin, Tgfb2 and Tgfb3, as well as the angiogenesis factor Vegf. They also showed upregulation of protective genes, including Aldh1a2, involved in retinoic acid synthesis, and Decorin, a Tgfb antagonist. Of interest, the mesangial cells also showed significant expression of Wt1, which has generally been considered podocyte specific. The Cd2ap mutant podocytes showed upregulation of proteases as well as genes involved in muscle and vasculature development, and showed a very strong gene expression signature indicating programmed cell death. Endothelial cells showed increased expression of the leukocyte adhesion associated factors Vcam1 and Sele, as well as Midkine (promoting angiogenesis), endothelin, and many genes responsive to cytokines and interferons. This study provides a comprehensive analysis of the changing properties of the three cell types of the glomerulus in Cd2ap mutants, identifying activated and repressed pathways and responsible genes, thereby delivering a deeper molecular understanding of this genetic disease. Overall design: All three major cell types of the glomerulus were FACS purified from CD2AP-/- mutant mice and normal controls and gene expression profiled with Affymetrix Mouse Exon 1.0 ST arrays. Data was analyzed with GeneBASE software. Biological triplicates were performed.

足细胞（podocytes）内表达的多种基因（包括Cd2ap）发生突变，已被证实与人类局灶节段性肾小球硬化症（focal segmental glomerulosclerosis）相关。突变小鼠模型为深入阐明此类疾病的分子病理机制提供了研究契机。本研究借助一系列转基因绿色荧光蛋白（GFP）小鼠，实现了从Cd2ap突变小鼠体内分离纯化肾小球三种主要细胞类型的目标。本研究同时采用基因芯片（microarrays）与RNA测序（RNA-seq）技术，对足细胞、系膜细胞（mesangial cells）及内皮细胞（endothelial cells）的基因表达谱进行表征，构建了覆盖全局的双平台交叉验证数据集。

系膜细胞中，血小板反应蛋白（thrombospondin）、转化生长因子β2（Tgfb2）、转化生长因子β3（Tgfb3）等促纤维化因子（profibrotic factors），以及血管内皮生长因子（Vegf）的表达水平显著上调；同时，系膜细胞中亦存在保护性基因的上调表达，包括参与视黄酸合成（retinoic acid synthesis）的Aldh1a2，以及转化生长因子β拮抗剂（Tgfb antagonist）饰胶蛋白聚糖（Decorin）。值得关注的是，系膜细胞中还检测到Wt1的显著表达，而该基因此前一般被认为是足细胞特异性表达的。

Cd2ap突变足细胞中，蛋白酶编码基因以及参与肌肉发育与血管发育的基因均出现上调，且呈现出强烈的程序性细胞死亡（programmed cell death）相关基因表达特征。内皮细胞中，与白细胞黏附（leukocyte adhesion）相关的血管细胞黏附分子1（Vcam1）、E-选择素（Sele），以及促血管生成的中期因子（Midkine）、内皮素（endothelin），还有众多响应细胞因子（cytokines）与干扰素（interferons）的基因的表达水平均显著升高。

本研究全面分析了Cd2ap突变小鼠肾小球三种细胞类型的特性变化，鉴定出激活与抑制的信号通路及对应的功能基因，从而为该遗传性疾病的分子机制提供了更为深入的认知。

实验设计：从CD2AP基因敲除（CD2AP-/-）突变小鼠及正常对照小鼠体内，通过荧光激活细胞分选术（FACS）分离肾小球三种主要细胞类型，采用Affymetrix公司小鼠外显子1.0 ST基因芯片（Mouse Exon 1.0 ST arrays）进行基因表达谱分析；数据处理采用GeneBASE软件完成，实验设置生物学重复三次。