The role of NFAT in podocyte damage and proteinuric kidney disease

描述（由申请人提供）：慢性肾脏疾病（CKD）是一个日益增长的全球公共卫生问题。据估计，美国超过11%的成年人患有慢性肾病，这导致生活质量下降、经济健康负担和过早死亡。异常钙信号与肾脏疾病和肾小球损伤的许多情况有关。TRCP6钙通道功能突变的增加可导致足细胞正常形态的改变和肾小球滤过器的丢失，从而导致尿中蛋白质的丢失（proteinura）。此外，足细胞内钙的增加发生在其他非遗传性损伤，如血压升高、细菌感染和创伤。钙敏感磷酸酶钙调磷酸酶在钙的存在下被激活，我们的小组已经证明钙调磷酸酶的组成性激活导致蛋白尿。然而，迄今为止，钙调磷酸酶的典型下游靶标，活化t细胞的转录因子核因子（NFAT），尚未被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在多西环素存在的足细胞中具有组成性活性。这些动物在多西环素暴露4天内出现深度蛋白尿，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，长期暴露于强力霉素（2个月）的动物，一旦去除强力霉素并重新引入正常的动物饲料，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，在苏木精和伊红（H+E）染色和周期性酸希夫染色（PAS）等组织学技术下，肾小球显示正常。我们的动物的蛋白尿表型和足细胞足突消失，加上缺乏观察到的H+E和PAS病理，使人想起最小变化病（MCD）患者的病理。这项研究的目的有两个。首先，我们打算在我们的动物模型中确定NFAT靶基因，这些基因负责急性诱导蛋白尿，并在体外概括这些基因的表达，以评估足细胞可能的变化。第二，我们的目标是在组织学和遗传学上完全表征我们的动物模型，在多西环素暴露的几个时间过程中与MCD患者的资料进行比较。希望NFAT的下游靶点能够被确定为我们的动物的蛋白尿表型，并为科学界提供一种使人衰弱的疾病的动物模型。

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.