Targeting Set7 in an experimental model of diabetic nephropathy

Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of mouse single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of GEN cell populations through transcriptional regulation of the insulin growth factor binding protein 5 (IGFBP5). Chromatin immunoprecipitation assays confirmed that the expression of the IGFBP5 gene was associated with mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). This generalizability was investigated in human renal and circulating hyperglycemic cells exposed to TGFb1. We showed that the highly selective Set7 inhibitor PFI-2 attenuated indices associated with renal cell damage and mesenchymal transition, specifically (1) reactive oxygen species production, (2) IGFBP5 gene regulation, and (3) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely corresponds in human glomerular endothelial cells with PFI-2 inhibition or Set7 shRNA silencing. Single cell RNA analysis (Drop-seq) of diabetic kidney tissue from streptozotocin-induced Setd7 deficient (Setd7-/-) mice. Mouse models included: Diabetic (Setd7-/- ApoE-/-); Control (Setd7-/- ApoE-/-); Diabetic (Setd7+/+ ApoE-/-); Control (Setd7+/+ ApoE-/-). For target validation experiments, human immortalized glomerular endothelial (GEN) cells were cultured with or without 15 mM (R)-PFI-2 (PFI-2; Cayman Chemicals) dissolved in DMSO for 24 hours before exposing them to 5.5 or 25 mM D-glucose in the presence or absence of 5 ng/ml TGF-b1 (R&D Systems) for 48 hours at 37°ockdown of Set7 was performed in GEN cells using MISSION shRNA expressing lentivirus vector (Sigma). Cells transduced with MISSION Non-target shRNA control vector (Sigma) were used as a control. GEN treatment groups included: GEC_NG (5.5 mM D-glucose); GEC_PFI (25 mM D-glucose + 15 mM (R)-PFI-2 + 5 ng/ml TGF-b1); GEC_TGF (25 mM D-glucose + 5 ng/ml TGF-b1); KD_TGF (25 mM D-glucose + Set7 shRNA + 5 ng/ml TGF-b1); NT_TGF (25 mM D-glucose + MISSION Non-target shRNA + 5 ng/ml TGF-b1). Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and shRNA inhibition of Set7.

Set7基因敲除（Set7 knockout）可改善糖尿病小鼠模型中的肾小球结构与白蛋白尿症状。对小鼠单细胞RNA测序（single-cell RNA-sequencing）数据的分析显示，糖尿病肾细胞存在动态转录变化。Set7敲除通过调控胰岛素样生长因子结合蛋白5（insulin growth factor binding protein 5, IGFBP5）的转录，控制GEN细胞群的表型转换。染色质免疫沉淀实验证实，IGFBP5基因的表达与组蛋白H3赖氨酸4的单甲基化和二甲基化（H3K4me1/2）相关。我们在暴露于转化生长因子β1（transforming growth factor β1, TGFβ1）的人类肾脏及循环高糖细胞中验证了该结论的普适性。研究发现，高选择性Set7抑制剂PFI-2可减轻与肾细胞损伤及间质转化相关的多项指标，具体包括：(1) 活性氧生成；(2) IGFBP5基因调控；(3) 间质标志物表达。此外，在Set7敲除的糖尿病小鼠中观察到的肾脏获益，与PFI-2抑制或Set7短发夹RNA（short hairpin RNA, shRNA）沉默的人类肾小球内皮细胞的实验结果高度一致。我们对链脲佐菌素诱导的Setd7缺陷型（Setd7-/-）小鼠的糖尿病肾脏组织进行了单细胞RNA分析（Drop-seq）。所用小鼠模型包括：糖尿病组（Setd7-/- ApoE-/-）、对照组（Setd7-/- ApoE-/-）、糖尿病组（Setd7+/+ ApoE-/-）及对照组（Setd7+/+ ApoE-/-）。在靶标验证实验中，我们将人类永生化肾小球内皮（GEN）细胞置于含或不含15 mM (R)-PFI-2（PFI-2；Cayman Chemical公司产品，溶于二甲基亚砜（dimethyl sulfoxide, DMSO））的培养基中培养24小时，随后在存在或不存在5 ng/ml转化生长因子β1（TGF-β1，R&D Systems公司产品）的条件下，以5.5 mM或25 mM D-葡萄糖处理细胞，于37℃培养48小时。我们使用Sigma公司的MISSION短发夹RNA（shRNA）表达慢病毒载体对GEN细胞进行Set7基因沉默，以转染MISSION非靶向shRNA对照载体（Sigma公司）的细胞作为对照。GEN细胞的处理分组如下：GEC_NG组（5.5 mM D-葡萄糖）、GEC_PFI组（25 mM D-葡萄糖 + 15 mM (R)-PFI-2 + 5 ng/ml TGF-β1）、GEC_TGF组（25 mM D-葡萄糖 + 5 ng/ml TGF-β1）、KD_TGF组（25 mM D-葡萄糖 + Set7 shRNA + 5 ng/ml TGF-β1）、NT_TGF组（25 mM D-葡萄糖 + MISSION非靶向shRNA + 5 ng/ml TGF-β1）。本研究采用单细胞转录组学技术探究了Set7在糖尿病肾病（diabetic kidney disease, DKD）小鼠模型中的调控作用，并通过药理学抑制及shRNA沉默Set7的方法对实验结果进行了验证。