The role of NFAT in podocyte damage and proteinuric kidney disease

描述(申请人提供)：慢性肾脏疾病(CKD)是一个日益严重的全球公共卫生问题。据估计，生活在美国的成年人中有超过11%患有慢性肾脏病，这会导致生活质量下降、经济健康负担和过早死亡。钙信号的异常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能导致正常足细胞形态的改变和肾小球滤器的丧失，从而导致蛋白丢失进入尿液(蛋白尿)。此外，在其他非遗传因素的影响下，足细胞内钙离子的增加也会发生，例如血压升高、细菌感染和创伤。钙敏感的磷酸酶钙调神经磷酸酶在有钙存在的情况下被激活，我们的小组已经证明了钙调神经磷酸酶的结构性激活会导致蛋白尿。然而，到目前为止，钙调神经磷酸酶下游的经典靶点，激活T细胞的转录因子核因子(NFAT)，还没有很好地描述它在导致肾脏疾病表型变化中的作用。我们已经建立了一个动物模型，在该动物模型中，转录因子NFAT在多西环素存在的情况下，在足细胞中具有结构性的活性。这些动物在接触多西环素后的4天内出现严重的蛋白尿，并在电子显微镜下分析显示足突消失。我们的初步数据还显示，长期接触多西环素(2个月)的动物一旦移除多西环素并重新引入正常的动物食物，就无法恢复正常的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下显示正常，如苏木精-伊红(H+E)染色和高碘酸席夫染色(PAS)。我们的动物的蛋白尿表型和足细胞足突消失，加上H+E和PAS缺乏观察到的病理，使人想起微小病变病(MCD)患者的病理。这项拟议研究的目标有两个。首先，我们打算在我们的动物模型中确定导致急性蛋白尿的NFAT靶基因，并在体外总结这些基因的表达，以评估足细胞可能的变化。第二，我们的目标是完全描述我们的动物模型，包括组织学和遗传学，与MCD患者的多西环素暴露的几个时间进程进行比较。人们希望NFAT的下游靶点能够根据我们动物的蛋白尿表型来确定，并为科学界提供一种衰弱疾病的动物模型。 公共卫生相关性：慢性肾脏疾病(CKD)影响着超过11%的美国人口，每年的医疗支出接近600亿美元。钙信号的异常与肾脏疾病和肾小球损伤的许多情况有关。活化T细胞核因子(NFAT)是一种转录因子，在细胞内钙离子升高时被激活。然而，NFAT靶基因在肾脏疾病表型中的作用仍不明确。我们的研究旨在确定与疾病表型相关的NFAT靶基因，希望能找到新的药物靶点来改善CKD患者的蛋白尿。

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. PUBLIC HEALTH RELEVANCE: Chronic kidney disease (CKD) affects over 11% of the US population, and accounts for nearly $60 billion in healthcare spending annually. Aberrant calcium signaling has been implicated in many scenarios of kidney disease and glomerular damage. Nuclear factor of activated T-cells (NFAT), a transcription factor, is activated upon increases in intracellular calcium. However, NFAT target genes remain un-characterized for their role in disease phenotypes in the kidney. Our research aims to identify NFAT target genes associated with disease phenotypes in the hope that new drug targets can be identified to ameliorate proteinuria in CKD patients.