Novel imaging approach to study podocyte function in vivo

描述(由申请人提供)：肾小球功能障碍是慢性肾脏疾病(CKD)发展的常见基础，CKD是一种具有显著并发症和死亡率的疾病。最近的研究强调足细胞肌动蛋白细胞骨架和裂隙隔膜在维持肾小球滤过屏障(GFB)、蛋白尿(AU)和局灶节段性肾小球硬化(FSGS)中的作用。细胞内钙信号在足细胞功能和上述病理过程中的关键作用已得到证实，然而我们对足细胞[钙]i动力学的机制了解仅限于少数几个参与者(血管紧张素II，TRPC5/6)。大多数P2嘌呤受体包括P2Y2，它通过[Ca~(2+)]i结合细胞外的ATP信号，并参与许多器官的各种(病理)生理功能，包括旁分泌细胞对细胞的串扰、组织损伤和硬化。虽然P2Y2受体在肾小管转运和血流自动调节中的重要作用已被证实，但其在足细胞/肾小球中的作用尚不清楚。在这里，我们建议研究健康和疾病患者完整肾脏中足细胞[Ca~(2+)]_i的体内动力学，并表征由P2Y2嘌呤能受体介导的足细胞中新的[Ca~(2+)]_i信号机制的作用和治疗意义。我们推测，P2Y2介导的[Ca~(2+)]i升高是原发足细胞损伤中一种强有力的关键新机制，其在足细胞之间的细胞间传播导致GFB局部节段性功能障碍的放大，并导致AU和FSGS的发生。总的主题是研究P2Y2在足细胞/GFB损伤中的作用以及P2Y2阻断对AU和FSGS的影响，将通过应用一种新的成像方法来解决，该方法结合了活体多光子显微镜(MPM)和新的小鼠足细胞荧光标记和标记模型。这些研究可能会改变目前对肾小球疾病发病机制的看法，也可能为肾小球疾病提供一种临床上立即可用的新治疗方法。其具体目的是：(1)研究P2Y2信号在足细胞[Ca~(2+)]i动力学和GFB/肾小球功能中的作用。(2)确立了P2Y2信号在足细胞损伤传播中的放大作用。(3)检测P2Y2阻断能否钝化/减轻足细胞损伤、AU和FSGS的发展。

DESCRIPTION (provided by applicant): Glomerular dysfunction is a common basis for the development of chronic kidney disease (CKD), a condition with significant comorbidities and mortalities. Recent studies highlighted the role of podocyte actin cytoskeleton and the slit diaphragm in the maintenance of the glomerular filtration barrier (GFB), and the development of albuminuria (AU) and focal segmental glomerulosclerosis (FSGS). The key role of cytosolic calcium ([Ca2+]i) signaling in podocyte function and the above pathologies is established, however our mechanistic understanding of podocyte [Ca2+]i dynamics is limited to a few players (angiotensin II, TRPC5/6). Most P2 purinoceptors including P2Y2 that bind extracellular ATP signal via [Ca2+]i and have been implicated in a variety of (patho)physiological functions in many organs including paracrine cell-to-cell crosstalk, tissue injury and sclerosis. While the important function of the P2Y2 receptor in renal tubular transport and blood flow autoregulation is well established, its role in podocytes/glomerulus is largely unknown. Here we propose to study podocyte [Ca2+]i dynamics in vivo in the intact kidney in health and disease and to characterize the role and therapeutic relevance of a novel [Ca2+]i signaling mechanism in podocytes that is mediated by P2Y2 purinergic receptors. We hypothesize that P2Y2-mediated elevations in [Ca2+]i is a robust and key novel mechanism in primary podocyte injury, and also that its cell-to-cell propagation between podocytes results in amplified focal segmental dysfunction of the GFB and the development of AU and FSGS. The overall theme, to study the role of P2Y2 in podocyte/GFB injury and the effect of P2Y2 blockade on AU and FSGS, will be addressed by applying a novel imaging approach that employs intravital multiphoton microscopy (MPM) combined with new mouse models of fluorescent podocyte labeling and tagging. These studies may potentially change the current view of the pathogenesis of glomerular disorders and may also provide a clinically and immediately available, novel therapeutic approach for glomerular kidney diseases. The specific aims are to: (1) Characterize the role of P2Y2 signaling in podocyte [Ca2+]i dynamics and GFB/glomerular function in response to podocyte injury/disease. (2) Establish the amplifier function of P2Y2 signaling in the propagation of podocyte injury. (3) Test whether P2Y2 blockade can blunt/reduce the development of podocyte injury, AU and FSGS.