Lysosome dysfunction in podocytopathy and associated hypertension

项目摘要 足细胞病变是不同肾小球疾病的重要致病基础，如微小病变 疾病(MCD)，弥漫性系膜硬化，局灶节段性肾小球硬化，肾小球塌陷- 高同型半胱氨酸血症(HHcy)、肥胖和肾小球硬化相关的孤立性肾病和肾小球硬化 糖尿病。据报道，这些肾小球疾病占终末期的绝大多数 肾脏疾病和肾脏相关的高血压和心血管疾病。最近的研究表明 正常的自噬是控制足细胞功能甚至其寿命的关键细胞过程， 自噬缺陷和相关的自噬小体(AP)堆积或外切体释放增加 产生足细胞损伤和足细胞病变。我们已经证明，鞘磷脂介导的信号通路是 与溶酶体功能障碍、自噬通量缺乏和终末性足细胞病变有关 肾小球硬化症。目前的拨款提案将检验一个中心假设，即溶酶体酸性神经酰胺酶 (Ac)介导的鞘磷脂代谢关键控制溶酶体向AP或多囊泡的运输或融合 机体(MVB)和随后的降解过程决定了正常的表型和功能 足细胞。AC基因缺陷或功能缺陷可能干扰AP和MVB的溶酶体降解， 诱导MVB的AP积聚和外体释放，导致足细胞表型转变， 消逝和终极MCD。为了验证这一假设，本文提出了三个具体目标。具体目标1将 确定溶酶体AC是否能很好地控制足细胞的自噬通量和外切体排泄 Asah1fl/fl/Podocre的活性以及这种AC调节缺失是否导致足细胞病变和MCD 老鼠，但不是在它们的窝窝里。特异性目标2试图阐明溶酶体AC-2的核心作用。 神经鞘脂信号在调节溶酶体转运到AP和MVB中的作用 它们的降解利用足细胞特异性的ASAH1基因缺失、挽救和沉默。在具体目标3中，我们 将探索足细胞中溶酶体运输或融合的机制由AC- 通过门控溶酶体TRPML1通道的相关鞘磷脂和使用Patch的相关钙释放 以GCaMP3-ML为指示剂的分离溶酶体夹闭及溶酶体特异性钙显象。这些 拟议的研究将提出一种新的足细胞病和MCD小鼠模型，并使用该模型来探索 触发足细胞病变的相关分子机制。这项拨款提案代表了 AC介导的溶酶体调节自噬通量和外切体的研究领域 足细胞的排泄及其在细胞病变中的致病作用。这一发现可能会使范式发生转变 了解足细胞病和MCD的发病机制，并帮助确定溶酶体AC是一种治疗方法 预防或治疗MCD和其他肾小球疾病的目标。

Project Summary Podocytopathy is an important pathogenic basis for different glomerular diseases such as minimal change disease (MCD), diffuse mesangial sclerosis, focal segmental glomerulosclerosis, collapsing glomeru- lonephropathy and global glomerulosclerosis associated with hyperhomocysteinemia (hHcy), obesity and diabetes mellitus. These glomerular diseases have been reported to account for the vast majority of end-stage renal disease and kidney-associated hypertension and cardiovascular diseases. Recent studies have indicated that normal autophagy is a critical cellular process to control podocyte function and even its life span and that deficient autophagy and associated autophagosome (AP) accumulation or increases in exosome release produce podocyte injury and podocytopathy. We have shown that a sphingolipid-mediated signaling pathway is importantly implicated in lysosome dysfunction, autophagic flux deficiency and ultimate podocytopathy and glomerular sclerosis. The present grant proposal will test a central hypothesis that lysosomal acid ceramidase (AC)-mediated sphingolipid metabolism critically controls lysosome trafficking or fusion to APs or multivesicular body (MVB) and subsequent degradation process determining the normal phenotype and function of podocytes. AC gene defect or functional deficiency may disturb lysosome degradation of APs and MVBs, which induces AP accumulation and exosome release from MVBs leading to podocyte phenotypic transition, effacement and ultimate MCD. To test this hypothesis, three Specific Aims are proposed. Specific Aim 1 will determine whether autophagic flux and exosome excretion in podocytes are fine controlled by lysosomal AC activity and whether the deficiency of this AC regulation causes podocytopathy and MCD in Asah1fl/fl/Podocre mice, but not in their littermates. Specific Aim 2 attempts to elucidate the central role of lysosomal AC- mediated sphingolipid signaling in the regulation of lysosome trafficking to and fusion with APs and MVBs for their degradation using podocyte-specific Asah1 gene deletion, rescuing and silencing. In Specific Aim 3, we will explore the mechanisms by which lysosome trafficking or fusion in podocytes is regulated by AC- associated sphingolipids via gating lysosomal TRPML1 channels and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging with GCaMP3-ML as an indicator. These proposed studies will present a novel mouse model for podocytopathy and MCD and use this model to explore associated molecular mechanisms triggering podocytopathy. The grant proposal represents the first effort in the research field to investigate the AC-mediated lysosome regulation of autophagic flux and exosome excretion in podocytes and associated pathogenic role in potocytopathy. The findings may make paradigm shift in understanding pathogenesis of podocytopathy and MCD and help identify lysosomal AC as a therapeutic target for prevention or treatment of MCD and other glomerular diseases.