Lysosome dysfunction in podocytopathy and associated hypertension

足细胞病和相关高血压中的溶酶体功能障碍

• 批准号: 10461007
• 负责人: PinLan Li
• 金额: $ 48.89万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-25 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10461007
• 关键词: Albuminuria; Animal Model; Applications Grants; Autophagocytosis; Autophagosome; Blood Vessels; Cardiovascular Diseases; Cell Differentiation process; Cell physiology; Ceramides; Defect; Development; Diabetes Mellitus; Diffuse; Disease; End stage renal failure; Enzymes; Excretory function; Extravasation; Farber' s lipogranulomatosis; Focal Segmental Glomerulosclerosis; Foot Process; Functional disorder; Gene Deletion; Genes; Heart; Hyperhomocysteinemia; Hypertension; Image; Injury; Kidney; Lead; Lipids; Longevity; Lysosomes; Mediating; Metabolism; Modeling; Molecular; Mouse Strains; Multivesicular Body; Mus; Nephrotic Syndrome; Obesity; Pathogenesis; Pathogenicity; Pathologic; Patients; Phenotype; Prevention; Process; Proteins; Regulation; Renal function; Renal glomerular disease; Reporting; Research; Role; Sclerosis; Signal Pathway; Signal Transduction; Sphingolipids; Sphingomyelins; Sphingosine; Structure; Testing; Urine; cell type; exosome; galactosylgalactosylglucosylceramidase; glomerulosclerosis; knockout gene; mouse model; novel; patch clamp; podocyte; prevent; receptor; therapeutic target; trafficking

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.

项目摘要 足细胞病是微小病变等不同肾小球疾病的重要致病基础 疾病（MCD），弥漫性系膜硬化，局灶节段性肾小球硬化，塌陷性肾小球硬化， 与高同型半胱氨酸血症（hHcy）、肥胖和 糖尿病的据报道，这些肾小球疾病占终末期肾病的绝大多数。 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体（AP）积累或外泌体释放增加 产生足细胞损伤和足细胞病。我们已经证明，鞘脂介导的信号通路是 重要地涉及溶酶体功能障碍、自噬通量缺乏和最终足细胞病， 肾小球硬化目前的拨款提案将测试一个中心假设，即溶酶体酸性神经酰胺酶 （AC）介导的鞘脂代谢关键控制溶酶体运输或融合到AP或多泡 体（MVB）和随后的降解过程，决定正常表型和功能， 足细胞AC基因缺陷或功能缺陷可干扰AP和MVB的溶酶体降解， 其诱导AP积累和外泌体从MVB释放，导致足细胞表型转变， 最终的MCD。为了验证这一假设，提出了三个具体目标。具体目标1将 确定足细胞中的自噬通量和外泌体排泄是否由溶酶体AC精细控制 活性以及这种AC调节的缺乏是否会导致Asah 1fl/fl/Podocre中的足细胞病和MCD 小鼠，但在其同窝出生的小鼠中没有。具体目标2试图阐明溶酶体AC-的核心作用 介导的鞘脂信号转导在调节溶酶体运输到AP和MVB并与AP和MVB融合中的作用， 它们的降解使用足细胞特异性Asah 1基因缺失、拯救和沉默。在具体目标3中，我们 将探讨溶酶体运输或足细胞融合的机制是由AC- 使用贴片通过门控溶酶体TRPML 1通道和相关Ca 2+释放的相关鞘脂 分离的溶酶体的夹持和以GCaMP 3-ML作为指示剂的溶酶体特异性Ca 2+成像。这些 拟议的研究将提出一种新的足细胞病和MCD小鼠模型，并使用该模型探索 引发足细胞病的相关分子机制。这项赠款提案是第一次努力， 研究AC介导的溶酶体对自噬流和外泌体的调节 在足细胞中的排泄和在足细胞病中的相关致病作用。这些发现可能会使范式转变 了解足细胞病和MCD的发病机制，并帮助确定溶酶体AC作为治疗 用于预防或治疗MCD和其它肾小球疾病靶点。