NFAT SIGNALING IN GLOMERULOSCLEROSIS

描述（由申请人提供）：局灶节段性肾小球硬化（FSGS）是一种难治性肾小球疾病，治疗方案很少，病因也不清楚。TRPC6的激活突变可导致FSGS7、8，而与疾病相关的TRPC6突变形式可在体外引起nfat依赖性转录9。我们已经建立了一个小鼠模型，在该模型中，可诱导足细胞特异性NFATc1激活导致FSGS，首次在体内证明了NFAT信号通路参与FSGS。我们还发现足细胞中的nfat活化特异性上调Wnt6和Fzd9。我们假设NFAT信号在足细胞的调节网络中占据关键位置，因此它的错误调节会改变信号通路，如Wnt通路，这对足细胞的内稳态和功能至关重要，从而导致FSGS。为了验证这一假设并进一步描述FSGS的致病机制，我们将首先确定典型Wnt信号是否对足细胞中NFAT激活的作用至关重要（目的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Nephrin和肌动蛋白细胞骨架改变的参与（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（Aim 3）。尽管环孢素（一种钙调神经磷酸酶抑制剂）的抗蛋白尿作用归因于钙调神经磷酸酶对synaptopodin去磷酸化的抑制，但抑制钙调神经磷酸酶下游的NFAT信号也可能起作用。因此，除了确定NFAT/Wnt通路在足细胞中的作用机制以及与TRPC6突变相关的FSGS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对与FSGS相关的已知因素的分析，潜在地揭示FSGS发病机制中以前未被怀疑的因素。这些研究结果可能为针对NFAT或NFAT下游因子的FSGS治疗提供理论基础，这些治疗可能比针对更多上游因子（如钙调磷酸酶和TRPC6）的治疗更具特异性，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.