Targeting Podocyte-Endothelial Cell Crosstalk as a FSGS Therapy

项目总结/摘要 问题的范围是，50%的局灶节段性肾小球硬化症（FSGS）患者， 成人原发性蛋白尿性肾小球疾病的原因，进展为慢性肾脏疾病。因此，在本发明中， 开发新的治疗策略以改善FSGS患者的结果是极其重要的。然而， 在我们对疾病进展的机械理解中有一个巨大的未满足的需求。FSGS的传统观点是 足细胞是其病理学的中心，它们是第一种受损的细胞类型。一个更 当代的观点是这一范例的扩展，包括对邻近肾小球细胞的损伤。这 格兰特将专注于肾小球内皮细胞（GEnCs）。基本原理是：（1）在FSGS中，GEnC降低， GEnC损伤组的GEnC数目最多，糖萼松动，窗孔增宽;（2）GEnC损伤组GEnC数目最多， 疾病进展率;（3）所有肾小球疾病的GEnC损伤评分在FSGS中最高;（4） GEnC基因/生物标志物与FSGS的最低缓解率和不良长期结局相关。然而 对足细胞如何引起继发性GEnC损伤的理解是非常不完全的。若要开始关闭 知识的差距，我们进行了计算机模拟的方法，以确定旁分泌配体受体信号网络 足细胞和GEnCs之间的联系令人惊讶的是，我们发现，实验和人类FSGS的结果， 衰老相关分泌表型（SASP）和非老年人中活化炎性小体表型 足细胞，两者的特征在于分泌不同种类的信号传导介质。我们 初步数据显示，抑制足细胞中的NLRP 3炎性体改善了GEnC健康。我们 具体目标将检验两个假设：具体目标#1检验SASP和炎性小体 来自受损足细胞的激活是GEnC损伤的原因。具体目标#2测试假设， 干扰来自受损足细胞的旁分泌损伤信号是预防GEnC dys的治疗靶点。 在FSGS中的作用我们将使用的创新实验方法包括：（i）体内功能丧失 使用突变小鼠进行验证，其中我们可以减少/抑制SASP或炎性小体表型， 足细胞在实验FSGS的设置，然后测量对GEnC健康的影响;（ii）体内增益- 使用转基因小鼠进行功能验证，我们可以强制诱导SASP或炎性小体 足细胞中的表型，以了解对GEnC的影响;（iii）设计质量，利用系统的，高 基于实验设计理论和多元数据分析的复杂性方法， 足细胞分泌组对GEnC健康和功能的贡献。因此，这一建议高度 对于理解FSGS中足细胞和GEnC之间的串扰具有重要的短期影响， 以及其在开发新的治疗策略以降低风险和程度方面的长期影响， FSGS患者的继发性GEnC损伤。

PROJECT SUMMARY/ABSTRACT The scope of the problem is that 50% of patients with focal segmental glomerulosclerosis (FSGS), the leading cause of primary proteinuric glomerular disease in adults, progress to chronic kidney disease. Thus, developing new treatment strategies to improve FSGS patient outcomes is of utmost importance. Yet, there is a large unmet need in our mechanistic understanding of disease progression. The traditional view in FSGS is that podocytes are central to its pathology and that they are the first cell type to be injured. A more contemporary view is an expansion of this paradigm and includes injury to neighboring glomerular cells. This grant will focus on glomerular endothelial cells (GEnCs). The rationale is that (1) in FSGS, GEnCs decrease in number, loose their glycocalyx and their fenestrae widen; (2) patients with GEnC damage have the highest rates of disease progression; (3) GEnC injury scores for all glomerular diseases are highest in FSGS; (4) GEnC genes/biomarkers are linked to the lowest remission rates and poor long-term outcomes of FSGS. Yet the understanding of how podocytes cause secondary GEnC injury is very incomplete. To begin to close the knowledge gap, we undertook an in silico approach to identify paracrine ligand-receptor signaling networks between podocytes and GEnCs. Surprisingly, we discovered that experimental and human FSGS results in a senescence associated secretory phenotype (SASP) and an activated inflammasome phenotype in non-aged podocytes, both of which are characterized by the secretion of distinct classes of signaling mediators. Our preliminary data shows that inhibiting the NLRP3 inflammasome in podocytes improves GEnC health. Our specific aims will test two hypotheses: Specific Aim #1 tests the hypothesis that SASP and inflammasome activation from injured podocytes are responsible for GEnC damage. Specific Aim #2 tests the hypothesis that interfering with the paracrine injury signals from injured podocytes is a therapeutic target to prevent GEnC dys- function in FSGS. The innovative experimental approaches we will use include: (i) In vivo loss-of-function validation using mutant mice, in which we can reduce/inhibit the SASP or inflammasome phenotypes in podocytes in the setting of experimental FSGS, and then measure the impact on GEnC health; (ii) in vivo gain- of-function validation using transgenic mice, in which we can forcibly induce either a SASP or inflammasome phenotype in podocytes to understand the impact on GEnCs; (iii) Quality-by-Design utilizing a systematic, high complexity approach based on the Design-of-Experiment theory and Multivariate Data Analysis to address the contribution of the podocyte secretome on GEnC health and function. As such, this proposal is highly significant for its short-term impact on understanding the crosstalk between podocytes and GEnCs in FSGS, and for its long-term impact in developing new therapeutic strategies to lower the risk and magnitude for secondary GEnC damage in patients with FSGS.