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

基本信息

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
项目类别：
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

项目摘要

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（DN）是西方社会肾衰竭的主要原因。由于缺乏对疾病发病机制的清晰了解，新疗法的开发受到阻碍。最近的研究表明，肾足细胞的丧失是导致 DN 发生和进展的一个早期主要特征；然而，足细胞丢失的潜在机制仍不清楚。足细胞是维持肾脏过滤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（AGE）会增加氧化应激，导致足细胞凋亡。 AGE 诱导的足细胞凋亡是通过 FOXO4 转录因子的激活介导的。本研究的目的是研究 FOXO4 和作用于 FOXO4 通路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 FOXO3 的去乙酰化增强了抗氧化酶和 DNA 损伤修复基因的表达，但降低了凋亡基因的表达。 SIRT1 在糖尿病大鼠和 DN 患者中受到抑制。我们假设糖尿病环境中 SIRT1 抑制会增加 FOXO4 乙酰化。因此，FOXO4靶基因的表达从氧化解毒和DNA损伤修复转向细胞凋亡；足细胞在糖尿病中容易发生细胞凋亡和氧化应激。该项目的首要目标是确定 SIRT1/FOXO4 通路在氧化剂生成和细胞凋亡中的作用。第二个目标是证实糖尿病动物模型的体外研究结果。将确定高血糖和 AGE 对 FOXO4 乙酰化的影响。 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来评估细胞凋亡。基因表达将通过实时 PCR 和蛋白质印迹进行定量。 AGEs 对 SIRT1 抑制的作用将通过苯磷硫胺治疗 db/db 小鼠以减少 AGEs 形成来确定。 SIRT1 在细胞凋亡中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 SIRT1 敲除小鼠中进行评估。该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：在西方社会，糖尿病肾病是导致需要透析或肾移植的肾衰竭的首要原因。我们目前的治疗手段中没有任何治疗方法可以逆转或完全阻止糖尿病肾病的进展。这项研究可以为糖尿病肾病的发病机制提供新的见解，并为新的治疗提供靶点。