The role of NFAT in podocyte damage and proteinuric kidney disease

描述（由申请人提供）：慢性肾脏病（CKD）是一个日益严重的全球公共卫生问题。据估计，超过11%的美国成年人患有CKD，导致生活质量下降，经济健康负担和过早死亡。异常的钙信号传导与肾脏疾病和肾小球损伤的许多情况有关。TRCP6钙通道功能突变的获得可导致适当足细胞形态的改变和肾小球滤过器的丧失，导致蛋白质进入尿液（蛋白尿）。此外，足细胞内钙离子的增加发生在其他非遗传性损伤下，如血压升高、细菌感染和创伤。钙敏感性磷酸酶钙调神经磷酸酶在钙的存在下被激活，我们的小组已经证明钙调神经磷酸酶的组成性激活导致蛋白尿。然而，迄今为止，钙调神经磷酸酶的典型下游靶点，活化T细胞的转录因子核因子（NFAT），尚未很好地表征其在导致肾脏疾病表型的变化中的作用。我们已经开发了一种动物模型，其中转录因子NFAT在多西环素存在下在足细胞中具有组成性活性。这些动物在暴露于强力霉素的4天内发生严重的蛋白尿，并在电子显微镜分析时显示足突消失。我们的初步数据还显示，一旦去除强力霉素并重新引入正常动物食物，长期暴露于强力霉素（2个月）的动物不能恢复正常足细胞形态和肾小球滤过功能。有趣的是，肾小球在组织学技术如苏木精和伊红（H+E）染色和过碘酸希夫染色（PAS）下显示正常。我们动物的蛋白尿表型和足细胞足突消失，加上缺乏H+E和PAS的病理学观察，使人想起微小病变病（MCD）患者的病理学。拟议的研究有两个目标。第一，我们打算在我们的动物模型中鉴定负责急性诱导蛋白尿的NFAT靶基因，并在体外重现这些基因的表达，以评估足细胞中可能的变化。第二，我们的目标是完全表征我们的动物模型，无论是组织学和遗传学，在多西环素暴露的几个时间过程中相比，MCD患者的档案。希望NFAT的下游靶点可以被鉴定为我们动物的蛋白尿表型，并为科学界提供一种衰弱性疾病的动物模型。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is a growing worldwide public health concern. Over 11% of adults living in the US are estimated to suffer from CKD, which leads to a diminished quality of life, financial health burden and premature death. Aberrant calcium signaling has been implicated in many scenarios of kidney disease and glomerular damage. Gain of function mutations in TRCP6 calcium channels can lead to alterations in proper podocyte morphology and the loss of the glomerular filter leading to loss of proteins into the urine (proteinura). Additionally, increases in intracellular calcium in the podocyte occur under other non-genetic insults such as increases in blood pressure, bacterial infection, and trauma. The calcium sensitive phosphatase calcineurin is activated in the presence of calcium, and our group has demostrated that constitutive activation of calcineurin leads to proteinuria. However, to date calcineurins canonical downstream target, the transcription factor nuclear factor of activated T-cells (NFAT), has not been well characterized for its role in changes that lead to disease phenotypes in the kidney. We have developed an animal model where the transcription factor, NFAT, is constitutively active in the podocyte in the presence of doxycycline. These animals develop profound proteinuria within fours days of doxycycline exposure and display foot process effacement upon analysis with electron microscopy. Our preliminary data also show that animals on prolonged doxycycline exposure (2 months) are unable to revert to normal podocyte morphology and glomerular filter function once the doxycycline is removed and normal animal chow is re-introduced. Interestingly, the glomerulus appears normal under histological techniques such as hematoxylin and eosin (H+E) staining and periodic acid Schiff staining (PAS). The proteinuric phenotype and podocyte foot process effacement of our animals coupled with the lack of observed pathology on H+E and PAS is reminiscent of the pathology of patients with Minimal Change Disease (MCD). The goals of the proposed study are two-fold. One, we intend to identify the NFAT target genes in our animal model that are responsible for the acute induction of proteinuria and recapitulate expression of those genes in vitro to assess possible changes in the podocyte. Two, we aim to completely characterize our animal model, both histologically and genetically, over several time courses of doxycycline exposure in comparison to MCD patient profiles. It is the hope that downstream targets of NFAT can be identified for the proteinuric phenotype of our animals and to provide the scientific community with an animal model for a debilitating disease.