New insights in mechanisms of renal injury

描述（由申请人提供）： 我们的目的是研究糖原合成酶激酶3 β（GSK 3 β）在糖尿病肾病中的作用。基本原理是基于我们的初步数据：1。在糖尿病小鼠中，肾皮质GSK 3 β被Ser 9磷酸化灭活，其底物真核起始因子2B β（eIF 2Be）被激活。这与肾肥大、肾基质层粘连蛋白β 1和纤连蛋白表达增加以及白蛋白尿相关。2.在体外培养的肾近端肾小管上皮细胞（MCT）中，需要高糖来抑制GK 3 β的活性，使其增加层粘连蛋白和纤连蛋白的mRNA和蛋白表达。假设：GSK 3 β激活通过调节基质蛋白的转录和mRNA翻译来消除高糖诱导的基质蛋白合成，并改善糖尿病诱导的肾病。具体目标1：体外研究。明确GSK 3 β在高糖诱导的肾细胞基质蛋白合成中的上游调节因子和下游靶点。将啮齿动物近端肾小管上皮细胞（MCT）、肾小球上皮细胞和系膜细胞（GEC）与高葡萄糖或等摩尔甘露醇或5 mM葡萄糖孵育长达48小时。在这些条件下，将进行以下研究。A. GSK 3 β的上游调节因子。我们将探讨血清和糖皮质激素调节激酶1（SGK 1），p70 S6激酶1（p70 S6 k1）和p90 rsk作为GSK 3 β上游调节因子的作用。将研究这三种激酶的高糖调节。这些激酶的显性阴性构建体或siRNA将用于测试它们对高葡萄糖诱导的GSK 3 β磷酸化和失活的需求。B。GSK 3 β的下游效应：纤连蛋白的转录调节。GSK 3 β调节纤连蛋白的三种转录因子：β连环蛋白、NF κ B和AP 1。高糖诱导的GSK 3 β抑制和纤连蛋白转录与β连环蛋白增加和NF κ B活化相关。我们将使用细胞核提取物来研究β连环蛋白、NF κ B和AP-1的活化。EMSA和纤连蛋白启动子驱动的荧光素酶报告基因构建体将用于检查GSK 3 β对纤连蛋白转录的调节。将进行染色质免疫沉淀（ChIP）试验，以研究GSK 3 β对Groucho、TCF/LEF 1与纤连蛋白启动子结合的调节。在上述实验中，我们将使用GSK 3 β突变体来研究高糖对纤连蛋白的转录调控是否需要GSK 3 β。mRNA翻译的调控：我们将表达eIF 2Be的组成型活性S539 A突变体，以测试它是否能诱导基质蛋白合成，类似于高糖。我们将进行多聚核糖体分析，以确定GSK 3 β失活和eIF 2Be激活刺激层粘连蛋白和纤连蛋白的mRNA翻译的需要。我们将研究GSK 3 β是否调节mRNA翻译的延长期以增加基质蛋白的合成。具体目标2：体内研究。探讨糖尿病肾病对GSK 3b失活的需求。硝普钠（SNP）刺激1型糖尿病小鼠的GSK 3 β并抑制肾脏肥大和蛋白尿。我们采用SNP作为工具来探讨GSK 3 β在糖尿病肾病中的作用。将SNP施用至1型糖尿病OVE 26小鼠和2型糖尿病db/db小鼠3个月，并具有各自的对照。将通过蛋白质印迹法、免疫组织化学和qRT-PCR检查结构参数、肾小球和肾小管肥大以及基质蛋白的变化。将测量肾小球滤过率（GFR）和白蛋白尿。如目的1所述，将在肾组织中探索GSK 3 β在参与基质蛋白合成的转录和翻译途径中的上游调节因子和下游效应因子。

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.