NFAT SIGNALING IN GLOMERULOSCLEROSIS

描述（由申请人提供）：局灶节段性肾小球硬化症（FSGS）是一种难治性肾小球疾病，治疗选择很少，病因不明。TRPC 6中的激活突变可导致FSGS 7，8，并且疾病相关的TRPC 6突变形式可导致体外NFAT依赖性转录9。我们已经产生了一个小鼠模型，其中诱导足细胞特异性NFATc 1激活导致FSGS，提供了第一个在体内证据的参与NFAT信号在FSGS。我们还发现，足细胞中的NFAT激活特异性上调Wnt 6和Fzd 9。我们假设NFAT信号在足细胞的调节网络中占据关键位置，使得其失调改变信号通路，例如Wnt通路，这对足细胞的稳态和功能至关重要，导致FSGS。为了检验这一假设并进一步描述FSGS的致病机制，我们将首先确定经典Wnt信号传导是否对足细胞中NFAT激活的作用至关重要（Aim 1）。然后，我们将描述足细胞中NFAT激活导致FSGS的致病机制，重点关注疾病的可逆性，NFAT激活与人类FSGS之间的潜在关联，以及涉及Nephrin和肌动蛋白细胞骨架改变（目的2）。最后，我们将使用最新的基因组技术来揭示与足细胞功能障碍和FSGS发展相关的转录改变和其他NFAT靶点（目的3）。尽管环孢菌素（一种钙调磷酸酶抑制剂）的抗蛋白尿作用归因于钙调磷酸酶抑制突触足蛋白的去磷酸化10，但抑制钙调磷酸酶下游的NFAT信号传导也可能发挥作用。因此，除了定义NFAT/Wnt途径在足细胞中运作的机制以及与TRPC 6突变相关的FSGS的发病机制外，本研究还将有助于更好地理解钙调磷酸酶抑制剂的抗蛋白尿作用。无偏倚的全基因组分析将超越与FSGS相关的已知因素的分析，以潜在地揭示FSGS发病机制中先前未被怀疑的因素。这些研究的结果可能形成针对NFAT或NFAT下游因子的FSGS治疗的理论基础，这些FSGS治疗可能比针对更上游因子（如钙调神经磷酸酶和TRPC 6）的FSGS治疗更具特异性且副作用更少。 公共卫生相关性：局灶节段性肾小球硬化症（FSGS）是一种难治性肾小球疾病，具有破坏性结局，治疗选择很少，病因学知之甚少。拟议的研究将描绘NFAT和Wnt信号通路在足细胞中运作的机制，并将有助于更好地理解与TRPC 6突变相关的FSGS的发病机制和钙调神经磷酸酶抑制剂的抗蛋白尿作用。此外，无偏倚的全基因组分析将超越与FSGS相关的已知因素的分析，以揭示FSGS发病机制中以前未被怀疑的因素。这些研究的结果可能形成针对NFAT或NFAT下游因子的FSGS治疗的理论基础，这些FSGS治疗可能比针对更上游因子（如钙调神经磷酸酶和TRPC 6）的FSGS治疗更具特异性且副作用更少。

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS) is an intractable glomerular disease with few treatment options and poorly understood etiology. Activating mutations in TRPC6 can cause FSGS7,8 and disease-associated TRPC6 mutant forms can cause NFAT-dependent transcription in vitro9. We have generated a murine model in which inducible podocyte-specific NFATc1 activation leads to FSGS, providing the first in vivo proof of the involvement of NFAT signaling in FSGS. We have also found that NFAT-activation in podocytes specifically upregulates Wnt6 and Fzd9. We hypothesize that NFAT signaling occupies a key position in the regulatory network in podocytes such that its misregulation alters signaling pathways, such as the Wnt pathway, crucial for the homeostasis and function of podocytes, leading to FSGS. To test this hypothesis and to further delineate the pathogenic mechanism of FSGS, we will first determine if canonical Wnt signaling is essential for the effects of NFAT activation in podocytes (Aim 1). We will then delineate the pathogenic mechanism by which NFAT activation in podocytes leads to FSGS, focusing on the reversibility of the disease, the potential association between NFAT activation and human FSGS, as well as the involvement of Nephrin and actin cytoskeleton alterations (Aim 2). Finally, we will use the latest genome technologies to reveal transcriptional alterations and additional NFAT targets in association with the development of podocyte dysfunction and FSGS (Aim 3). Although the anti-proteinuric effects of cyclosporine (a calcineurin inhibitor) have been attributed to the inhibition of dephosphorylation of synaptopodin by calcineurin10, suppression of NFAT signaling downstream of calcineurin may also play a role. Thus, in addition to defining the mechanism by which NFAT/Wnt pathways operate in podocytes and in the pathogenesis of FSGS associated with TRPC6 mutations, this study will also contribute to better understanding of the anti- proteinuric effects of calcineurin inhibitors. The unbiased genome-wide analyses will go beyond the analysis of known factors associated with FSGS to potentially reveal previously unsuspected factors in the pathogenesis of FSGS. Results from these studies may form the theoretical foundation for treatments of FSGS targeting NFAT or factors downstream of NFAT that may be more specific and with fewer side-effects than those aimed at more upstream factors, such as calcineurin and TRPC6. PUBLIC HEALTH RELEVANCE: Focal Segmental Glomerulosclerosis (FSGS) is a type of intractable glomerular disease with devastating outcomes, few treatment options, and poorly understood etiology. The proposed studies will delineate the mechanism by which NFAT and Wnt signaling pathways operate in podocytes and will contribute to better understanding of the pathogenesis of FSGS associated with TRPC6 mutations and the anti-proteinuric effects of calcineurin inhibitors. In addition, the unbiased genome-wide analyses will go beyond the analysis of known factors associated with FSGS to reveal previously unsuspected factors in the pathogenesis of FSGS. Results from these studies may form the theoretical foundation for treatments of FSGS targeting NFAT or factors downstream of NFAT that may be more specific and with fewer side-effects than those aimed at more upstream factors, such as calcineurin and TRPC6.