Kidney Research Centre, Division of Nephrology, Department of Medicine, Ottawa Health Research Institute, Ottawa Hospital and University of Ottawa, Ottawa, ON K1H 8M5, Canada.
Transgenic Res. 2010 Apr;19(2):285-9. doi: 10.1007/s11248-009-9305-9. Epub 2009 Jul 8.
Mutations in the gene ACTN4 encoding the actin bundling protein-alpha-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant alpha-actinin-4 (K256E-ACTN4 (pod+)). However, whether alpha-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype alpha-actinin-4 (wt-ACTN4 (pod+)). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype alpha-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged alpha-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 (pod+) mice are similar to K256E-ACTN4 (pod+) mice. In contrast to K256E-ACTN4 (pod+) mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 (pod+) mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of alpha-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.
基因突变 ACTN4 基因，该基因编码肌动蛋白束蛋白-α-辅肌动蛋白-4，是一种已知的肾脏病变的遗传形式，称为局灶节段性肾小球硬化症（FSGS）。以前，我们通过生成足细胞特异性过表达致病突变α-辅肌动蛋白-4（K256E-ACTN4（pod+））的小鼠，为这种疾病建立了一种模型。然而，α-辅肌动蛋白-4过表达是否是导致这些小鼠中 FSGS 表型的原因，而不是突变获得亲和力的影响，这一点并不清楚。为了解决这个问题，我们开发了一种具有足细胞特异性过表达野生型α-辅肌动蛋白-4（wt-ACTN4（pod+））的对照小鼠系。使用 8.3 kb 的小鼠 Nephrin 启动子（NPHS1）片段驱动 HA 标记的野生型α-辅肌动蛋白-4 编码序列在小鼠中的表达。通过与 HA 和 synaptopodin 抗体的共免疫荧光，获得了 5 种以足细胞特异性方式表达 HA 标记的α-辅肌动蛋白-4 蛋白的启动子。定量 PCR 显示 wt-ACTN4（pod+）小鼠的肾脏转基因 mRNA 水平与 K256E-ACTN4（pod+）小鼠相似。与 K256E-ACTN4（pod+）小鼠表现出白蛋白尿、足细胞足突融合和肾小球瘢痕形成不同，wt-ACTN4（pod+）小鼠健康，与非转基因同窝仔鼠无区别。这些发现表明，K256E 突变本身而不是α-辅肌动蛋白-4 蛋白的过表达本身导致了我们的转基因模型中的 FSGS 表型。
