New insights in mechanisms of renal injury

肾损伤机制的新​​见解

• 批准号: 8802851
• 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/8802851
• 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）： 我们的目标是研究糖原合酶激酶3BETA（GSK 3BETA）在糖尿病肾病中的作用。基本原理基于我们的初步数据：1。在糖尿病小鼠中，肾皮质gsk 3beta被Ser9磷酸化及其底物灭活，其底物是真核启动因子2B Epsilon（EIF2BE）。这与肾脏肥大相关，增加了肾脏基质层粘连蛋白β1和纤连蛋白表达和蛋白尿。 2。在体外肾近端管状上皮（MCT）细胞中，需要高葡萄糖来抑制GK 3Beta活性以增加层粘连蛋白和纤连蛋白mRNA和蛋白质表达。假设：GSK 3BETA激活通过调节其转录和mRNA翻译来消除高葡萄糖诱导的基质蛋白的合成，并减轻糖尿病诱导的肾脏疾病。特定目标1：体外研究。在肾细胞中高葡萄糖诱导的基质蛋白合成的背景下，定义了GSK 3BETA的上游调节剂和向下流靶标。啮齿动物近端管状上皮（MCT），肾小球上皮细胞和肾小球细胞（GEC）将与高葡萄糖或等摩尔甘露醇或5 mm葡萄糖一起孵育48小时。在这些条件下，将进行以下研究。 A. GSK 3Beta的上游调节器。我们将探讨血清和糖皮质激素调节的激酶1（SGK1），P70S6 Kinase1（P70S6K1）和P90RSK作为GSK 3Beta的上游调节剂。将研究这三种激酶的高葡萄糖调节。这些激酶的主要负构建体或siRNA将用于测试其对高葡萄糖诱导的GSK 3Beta磷酸化和失活的需求。 B. GSK3β的下游效应：纤连蛋白的转录调节。 GSK 3Beta调节纤连蛋白的三个转录因子：β链球菌，NFKB和AP1。高葡萄糖诱导的GSK 3BETA抑制和纤连蛋白转录与β链球菌的增加和NFKB的激活有关。我们将采用核提取物来研究β链球菌，NFKB和AP-1的激活。 EMSA和纤连蛋白启动子驱动的荧光素酶报告基构建体将用于检查纤连蛋白转录的GSK 3Beta调节。将进行染色质免疫沉淀（CHIP）测定，以研究GSK 3Beta调节Groucho，TCF/LEF1与纤连蛋白启动子的结合。我们将使用GSK 3Beta突变体来研究是否需要GSK 3 Beta来通过高葡萄糖在ABOV实验中对纤连蛋白的转录调节。 mRNA翻译的调节：我们将表达EIF2BE的组成性活性S539a突变体，以测试它是否可以诱导基质蛋白合成，类似于高葡萄糖。我们将执行多核糖体测定，以确定GSK 3Beta失活和EIF2BE激活的需求，以刺激层粘连蛋白和纤连蛋白的mRNA翻译。我们将检查GSK 3BETA是否调节mRNA翻译的伸长阶段以增加基质蛋白合成。特定目标2：体内研究。测试GSK3B在糖尿病肾脏疾病中失活的需求。硝化钠（SNP）刺激GSK3BETA，并抑制1型糖尿病小鼠中的肾肥大和蛋白尿。我们采用SNP作为探测GSK 3Beta在糖尿病性肾病中的作用的工具。 SNP将用于3个月以1型糖尿病OVE26小鼠，并使用各自的对照组进行2型糖尿病DB/DB小鼠。结构参数，肾小球和管状肥大以及基质蛋白的变化将通过蛋白质印迹，免疫组织化学和QRT-PCR检查。将测量肾小球滤过率（GFR）和蛋白尿。 GSK 3Beta在转录和基质蛋白合成中涉及的翻译途径中的上游调节剂和下游效应子将在肾脏组织中探索，如AIM 1中所述。