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: 7574819
• 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/7574819
• 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; Patients; Principal Investigator; Production; Proteins; Repression; Resistance; Role; Societies; Staining method; Stains; Streptozocin; Stress; Therapeutic; Time; Western Blotting; benphothiamine; db/db mouse; diabetic; 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)： 在西方社会，糖尿病肾病是导致肾功能衰竭的主要原因。由于对疾病发病机制缺乏清楚的了解，阻碍了新疗法的开发。最近的研究表明，肾脏足细胞丢失是导致糖尿病肾病发生和发展的早期基本特征；然而，足细胞丢失的潜在机制尚不清楚。足细胞是一种特殊的终末分化细胞，负责维持肾脏的滤过屏障。最近的研究表明，高血糖和晚期糖基化终产物(AGEs)增加了氧化应激，导致足细胞凋亡。AGE诱导的足细胞凋亡是通过激活FOXO4转录因子介导的。本研究的目的是研究FOXO4及其上游的蛋白脱乙酰酶(SIRT1)在糖尿病足细胞凋亡中的作用。SIRT1对FOXO_3的脱乙酰基作用增强了抗氧化酶和DNA损伤修复基因的表达，但降低了细胞凋亡基因的表达。SIRT1在糖尿病大鼠和糖尿病肾病患者中被抑制。我们假设在糖尿病环境中抑制SIRT1会增加FOXO4乙酰化。因此，FOXO4靶基因的表达从氧化解毒和DNA损伤修复转变为凋亡；糖尿病足细胞容易发生凋亡和氧化应激。该项目的第一个目标是确定SIRT1/FOXO4通路在氧化剂生成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研究结果。高血糖和AGEs对FOXO4乙酰化的影响将被确定。SIRT1在高血糖和AGE诱导的FOXO4乙酰化中的作用将通过SIRT1的基因敲除和过度表达来评估。年龄和高糖对氧化应激的影响将通过DCF-DA荧光和高香草酸二聚的流式细胞仪测量来定量。用流式细胞仪、原位末端标记法和半胱氨酸天冬氨酸氨基转移酶检测细胞的凋亡率。基因表达将通过实时荧光聚合酶链式反应和蛋白质印迹进行量化。通过苯硫胺治疗db/db小鼠以减少AGEs的形成，将确定AGEs抑制SIRT1的作用。将在链脲佐菌素诱导的糖尿病足细胞特异性SIRT1基因敲除小鼠中评估SIRT1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，并确定治疗糖尿病肾病的新靶点。 公共卫生相关性：在西方社会，糖尿病肾病是导致肾功能衰竭的头号原因，需要透析或肾移植。我们目前的治疗性治疗手段不能逆转或完全阻止糖尿病肾病的进展。本研究可为糖尿病肾病的发病机制提供新的认识，并为新的治疗方法提供靶点。