The role of SIRT1/FOXO4 pathway in podocyte apoptosis of diabetes mellitus

SIRT1/FOXO4通路在糖尿病足细胞凋亡中的作用

• 批准号: 8220952
• 负责人: Peter Yenlung Chuang
• 金额: $ 14.93万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220952
• 关键词: Acetylation; Advanced Glycosylation End Products; Animal Model; Antioxidants; Apoptosis; Biological Assay; Caspase; Cells; DNA Repair; Deacetylase; Deacetylation; Development; Diabetes Mellitus; Diabetic Nephropathy; Dialysis procedure; Dimerization; Disease; Drug Metabolic Detoxication; Enzymes; Equilibrium; Filtration; Flow Cytometry; Fluorescence; Gene Expression; Gene Targeting; Generations; Genes; Glucose; Goals; Homovanillic Acid; Human; Hyperglycemia; In Situ Nick-End Labeling; In Vitro; Kidney; Kidney Failure; Kidney Transplantation; Knockout Mice; MLLT7 gene; Measurement; Mediating; Modeling; Mus; Oxidants; Pathogenesis; Pathway interactions; Principal Investigator; Production; Proteins; Repression; Resistance; Role; Societies; Staining method; Stains; Streptozocin; Stress; Therapeutic; Time; Western Blotting; benphothiamine; db/db mouse; diabetic; diabetic patient; diabetic rat; insight; non-diabetic; novel; oxidant stress; podocyte; public health relevance; response; transcription factor

DESCRIPTION (provided by applicant): Diabetic nephropathy (DN) is the leading cause of renal failure in Western societies. Development of new therapies is hindered by the lack of a clear understanding of disease pathogenesis. Recent studies revealed that loss of kidney podocytes is an early cardinal feature that contributes to the development and progression of DN; however, the underlying mechanism of podocyte loss remains unclear. Podocytes are specialized, terminally differentiated cells that maintain the kidney's filtration barrier. Recent studies have shown that hyperglycemia and advanced glycation end products (AGEs) increase oxidant stress leading to podocyte apoptosis. AGE-induced podocyte apoptosis is mediated by the activation of the FOXO4 transcription factor. The goal of this study is to study the role of FOXO4 and a protein deacetylase (SIRT1) that acts upstream of FOXO4 pathway in podocyte apoptosis of diabetes. Deacetylation of FOXO3 by SIRT1 enhances the expression of antioxidant enzymes and DNA damage repair genes, but decreases the expression of apoptosis genes. SIRT1 is repressed in diabetic rats and patients with DN. We hypothesize that SIRT1 repression in the diabetic milieu increases FOXO4 acetylation. Thus, the expression of FOXO4 target genes shifts from oxidant detoxification and DNA damage repair to apoptosis; and podocytes are apoptosis prone and oxidatively stressed in diabetes. The first aim of the project is to determine the role of the SIRT1/FOXO4 pathway in oxidant generation and apoptosis. The second aim is to confirm the in vitro findings in animal models of diabetes. The effects of hyperglycemia and AGEs on FOXO4 acetylation will be determined. The role of SIRT1 in hyperglycemia- and AGE-induced FOXO4 acetylation will be assessed by gene knockdown and over-expression of SIRT1. The effects of AGE and high glucose on oxidant stress will be quantified by flow-cytometric measurements of DCF-DA fluorescence and dimerization of homovanillic acid. Apoptosis will be assessed by flow cytometry, TUNEL staining, and caspase assays. Gene expression will be quantified by real-time PCR and Western blot. The role of SIRT1 repression by AGEs will be determined by benfotiamine treatment of db/db mice to reduce AGEs formation. The role of SIRT1 in apoptosis will be assessed in podocyte-specific SIRT1 knockout mice with streptozotocin-induced diabetes. The long-term objectives of the study are to elucidate the pathogenesis of podocyte loss in human and identify novel targets for treatment of DN. PUBLIC HEALTH RELEVANCE: Diabetic kidney disease is the number one cause of renal failure requiring dialysis or renal transplantation in Western society. No treatment from our current therapeutic armamentarium can reverse or completely forestall the progression of the diabetic nephropathy. This study could give new insight into the pathogenesis of diabetic nephropathy and provide targets for novel therapy.

描述（由申请人提供）： 糖尿病肾病（diabetic nephropathy，DN）是导致肾功能衰竭的主要原因。新疗法的开发由于缺乏对疾病发病机制的明确理解而受到阻碍。最近的研究表明，肾脏足细胞的损失是一个早期的主要特征，有助于DN的发展和进展;然而，足细胞损失的潜在机制仍不清楚。足细胞是维持肾脏滤过屏障的特化终末分化细胞。最近的研究表明，高血糖和晚期糖基化终产物（AGEs）增加氧化应激，导致足细胞凋亡。AGE诱导的足细胞凋亡是由FOXO 4转录因子的激活介导的。本研究的目的是研究FOXO 4和FOXO 4通路上游的蛋白脱乙酰酶（SIRT 1）在糖尿病足细胞凋亡中的作用。SIRT 1对FOXO 3的去乙酰化作用增强了抗氧化酶和DNA损伤修复基因的表达，但降低了凋亡基因的表达。SIRT 1在糖尿病大鼠和DN患者中受到抑制。我们假设SIRT 1在糖尿病环境中的抑制增加了FOXO 4的乙酰化。因此，FOXO 4靶基因的表达从氧化解毒和DNA损伤修复转变为凋亡;糖尿病足细胞易于凋亡和氧化应激。该项目的第一个目的是确定SIRT 1/FOXO 4通路在氧化剂产生和细胞凋亡中的作用。第二个目的是确认糖尿病动物模型的体外发现。将确定高血糖和AGEs对FOXO 4乙酰化的影响。SIRT 1在高血糖和AGE诱导的FOXO 4乙酰化中的作用将通过SIRT 1的基因敲低和过表达来评估。AGE和高葡萄糖对氧化应激的影响将通过DCF-DA荧光和高香草酸二聚化的流式细胞术测量来量化。将通过流式细胞术、TUNEL染色和半胱天冬酶测定评估细胞凋亡。将通过实时PCR和Western印迹定量基因表达。通过苯磷硫胺处理db/db小鼠以减少AGEs形成来确定AGEs对SIRT 1抑制的作用。将在患有链脲佐菌素诱导的糖尿病的足细胞特异性SIRT 1敲除小鼠中评估SIRT 1在细胞凋亡中的作用。这项研究的长期目标是阐明人类足细胞丢失的发病机制，并确定治疗DN的新靶点。 公共卫生相关性：糖尿病肾病是西方社会中需要透析或肾移植的肾衰竭的头号原因。糖尿病肾病的早期症状有哪些？糖尿病肾病的早期症状有哪些？本研究为糖尿病肾病的发病机制提供了新的认识，并为糖尿病肾病的治疗提供了新的靶点。