LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys
LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys
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DOI：
10.1681/asn.2012080788
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发表时间：
2013-11-01
影响因子：
13.6
通讯作者：
Witzgall, Ralph
中科院分区：
文献类型：
--
作者：
Burghardt, Tillmann;Kastner, Juergen;Witzgall, Ralph
LMX 1B基因突变导致指甲-髌骨综合征，这是一种罕见的常染色体显性遗传疾病，影响四肢、眼睛、大脑和肾脏的发育。常规Lmx 1b基因敲除小鼠的表征表明，LMX 1B调节足细胞足突和狭缝隔膜的发育，但使用足细胞特异性Lmx 1b基因敲除小鼠的研究产生了关于LMX 1B对维持足细胞结构的重要性的相互矛盾的结果。为了解决这个问题，我们产生了可诱导足细胞特异性Lmx 1b敲除小鼠。在成年小鼠中，Lmx 1b失活一周导致蛋白尿，仅有极轻微的足突消失。值得注意的是，在这个时间点，裂隔膜和基底膜蛋白的表达水平保持稳定，基底膜电荷性质也没有改变，这表明替代机制介导了这些小鼠蛋白尿的发展。Lmx 1b失活1周后分离的原代足细胞的细胞生物学和生物物理学实验表明肌动蛋白细胞骨架组织失调，时间分辨的DNA微阵列分析确定了编码肌动蛋白细胞骨架相关蛋白的基因，包括Abra和Arl 4c，作为推定的LMX 1B靶点。染色质免疫沉淀实验在条件永生化的人足细胞和凝胶迁移试验表明，LMX 1B识别AT丰富的结合位点（FLAT元素）的启动子区ABRA和ARL 4C，并敲除实验在斑马鱼支持一个模型，其中LMX 1B和ABRA在一个共同的途径在原肾发育。我们的报告确立了LMX 1B在完全分化的足细胞中的重要性，并认为LMX 1B对于维持足细胞中适当结构的肌动蛋白细胞骨架至关重要。
Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.