New insights in mechanisms of renal injury

肾损伤机制的新​​见解

• 批准号: 8698289
• 负责人: BALAKUNTALAM S KASINATH
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698289
• 关键词: Affect; Albuminuria; Binding; Biological Assay; Blood Pressure; Cells; Data; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disodium Salt Nitroprusside; Dominant-Negative Mutation; Dose; EMSA; Electrophoresis; Electrophoretic Mobility Shift Assay; Enhancers; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Eukaryotic Initiation Factors; Event; Extracellular Matrix Proteins; Fibronectins; Genetic Transcription; Genetic Translation; Glomerular Filtration Rate; Glucocorticoids; Glucose; Glycogen Synthase Kinases; Growth; Hypertrophy; Immunohistochemistry; In Vitro; Incubated; Injury; Inulin; Kidney; Kidney Diseases; Kidney Failure; Laminin; Luciferases; Lymphoid; Mannitol; Measures; Mediating; Monitor; Mus; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Extract; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Protein Biosynthesis; Protein Dephosphorylation; Proteins; Quantitative Reverse Transcriptase PCR; RPS6KA gene; Regulation; Renal Tissue; Renal function; Reporter; Rodent; Role; Serum; Signal Transduction; Small Interfering RNA; Staging; Stream; T-Lymphocyte; Testing; Transcription Factor AP-1; Transcriptional Activation; Transcriptional Regulation; Translations; Tubular formation; Veterans; Western Blotting; base; beta catenin; blood glucose regulation; chromatin immunoprecipitation; db/db mouse; diabetic; in vivo; insight; kidney cell; mRNA Expression; mesangial cell; morphometry; mutant; overexpression; promoter; protein expression; research study; tool; transcription factor; type I diabetic

DESCRIPTION (provided by applicant): Our objective is to investigate the role of glycogen synthase kinase 3beta (GSK 3beta) in diabetic nephropathy. The rationale is based on our preliminary data: 1. In diabetic mice, renal cortical GSK 3beta is inactivated by Ser9 phosphorylation, and its substrate, the eukaryotic initiation factor 2B epsilon (eIF2Be), is activated. This occurs in association with renal hypertrophy, increase in renal matrix laminin beta1 and fibronectin expression, and albuminuria. 2. In renal proximal tubular epithelial (MCT) cells in vitro, high glucose is required to inhibit GK 3beta activity for it to increase laminin and fibronectin mRNA and protein expression. Hypothesis: GSK 3beta activation abrogates high glucose-induced synthesis of matrix proteins by regulating their transcription and mRNA translation, and, ameliorates diabetes-induced renal disease. Specific Aim 1: In vitro studies. To define the upstream regulators and down stream targets of GSK 3beta in the context of high glucose-induced matrix protein synthesis in renal cells. Rodent proximal tubular epithelial (MCT), glomerular epithelial and mesangial cells (GEC) will be incubated with high glucose, or , equimolar mannitol, or, 5 mM glucose for up to 48 hrs. Under these conditions the following studies will be done. A. Upstream regulators of GSK 3beta. We will explore the role of serum and glucocorticoid-regulated kinase1 (SGK1), p70S6 kinase1 (p70S6k1), and p90rsk as upstream regulators of GSK 3beta. High glucose regulation of these three kinases will be studied. Dominant negative constructs or siRNA of these kinases will be used to test their requirement for high glucose-induced GSK 3beta phosphorylation and inactivation. B. Downstream effects of GSK3 beta: Transcriptional regulation of fibronectin. GSK 3beta regulates three transcription factors for fibronectin: beta catenin, NFkB and AP1. High glucose-induced GSK 3beta inhibition and fibronectin transcription are associated with increase in beta catenin and activation of NFkB. We will employ nuclear extracts to study the activation of beta catenin, NFkB, and AP-1. EMSA and fibronectin promoter-driven luciferase reporter construct will be used to examine GSK 3beta regulation of fibronectin transcription. Chromatin immunoprecipitation (ChIP) assays will be performed to study GSK 3beta regulation of binding of Groucho, TCF/LEF1 to the fibronectin promoter. We will employ GSK 3beta mutants to study if GSK 3 beta is required for transcriptional regulation of fibronectin by high glucose in the abov experiments. Regulation of mRNA translation: We will express the constitutively active S539A mutant of eIF2Be to test if it can induce matrix protein synthesis, similar to high glucose. We wil perform polyribosomal assay to determine the requirement of GSK 3beta inactivation and eIF2Be activation to stimulate mRNA translation of laminin and fibronectin. We will examine if GSK 3beta regulates the elongation phase of mRNA translation to increase matrix protein synthesis. Specific Aim 2: In vivo studies. To test the requirement of GSK3b inactivation in diabetic renal disease. Sodium nitroprusside (SNP) stimulates GSK3beta and inhibits renal hypertrophy and albuminuria in type 1 diabetic mice. We employ SNP as a tool to probe the role of GSK 3beta in diabetic nephropathy. SNP will be administered for 3 months to type 1 diabetic OVE26 mice and type 2 diabetic db/db mice with respective controls. Structural parameters, glomerular and tubular hypertrophy and changes in matrix proteins will be examined by western blotting, immunohistochemistry, and qRT-PCR. Glomerular filtration rate (GFR) and albuminuria will be measured. Upstream regulators and downstream effectors of GSK 3beta in transcription and translation pathways involved in matrix protein synthesis will be explored in renal tissues as stated in Aim 1.

描述（由申请人提供）：