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: 10218151
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218151
• 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 足细胞病是不同肾小球疾病（例如最小变化）的重要致病基础 疾病（MCD），弥漫性膜性硬化，局灶性节段性肾小球硬化症，骨膜崩溃 与超委内半胱氨酸（HHCY），肥胖和肥胖和 糖尿病。据报道，这些肾小球疾病是绝大多数终点 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， 不足的自噬和相关的自噬体（AP）积累或外部释放增加 产生足细胞损伤和足细胞病。我们已经表明，鞘脂介导的信号通路为 重要的是与溶酶体功能障碍，自噬通量缺乏和最终的足细胞病和 肾小球硬化。目前的赠款提案将检验溶酶体酸神经酶的中心假设 （AC）介导的鞘脂代谢批判性地控制溶酶体运输或融合到APS或多囊泡 身体（MVB）和随后的降解过程确定正常表型和功能 足细胞。 AC基因缺陷或功能缺乏可能会干扰AP和MVB的溶酶体降解， 这会诱导AP的积累和从MVB的外部释放，导致足细胞表型过渡， 递增和最终MCD。为了检验这一假设，提出了三个具体目标。具体目标1将 确定在足细胞中的自噬通量和外泌体排泄是否受溶酶体AC的良好控制 活动以及该AC调节的缺乏是否会导致asah1fl/fl/Podocre中的足细胞病和MCD 老鼠，但不在他们的同窝姐妹中。特定的目标2尝试阐明溶酶体AC-的核心作用 在调节溶酶体运输和与APS和MVB的融合中，介导的鞘脂信号传导用于 它们使用Podocyte特异性ASAH1基因缺失，拯救和沉默的降解。在特定的目标3中，我们 将探索溶酶体运输或足细胞中溶酶体运输或融合的机制 通过门控溶酶体TRPML1通道和相关的Ca2+使用斑块释放的相关鞘脂 用GCAMP3-ML作为指标夹紧分离的溶酶体和溶酶体特异性CA2+成像。这些 拟议的研究将提出一种用于足细胞病和MCD的新型小鼠模型，并使用此模型探索 相关的分子机制触发足细胞病。赠款提案代表了 研究领域以研究自噬通量和外泌体的AC介导的溶酶体调节 足细胞中的排泄量和相关的致病性作用。这些发现可能会导致范式转移 在理解足细胞病和MCD的发病机理，并有助于识别溶酶体AC作为治疗 预防或治疗MCD和其他肾小球疾病的靶标。