New insights in mechanisms of renal injury

描述(由申请人提供)： 我们的目的是探讨糖原合成酶激酶3β(GSK 3β)在糖尿病肾病中的作用。其理论基础基于我们的初步数据：1.在糖尿病小鼠中，肾皮质GSK 3β被Ser9磷酸化失活，其底物真核启动因子2B epsilon(EIF2Be)被激活。这与肾脏肥大、肾基质层粘连蛋白β1和纤维连接蛋白表达增加以及蛋白尿有关。2.在体外培养的肾近端小管上皮细胞(MCT)中，需要高糖抑制GK3β的活性，使其上调层粘连蛋白和纤维连接蛋白的mRNA和蛋白表达。假设：GSK 3β的激活通过调节基质蛋白的转录和信使核糖核酸的翻译来消除高糖诱导的基质蛋白的合成，并改善糖尿病引起的肾脏疾病。具体目标1：体外研究。在高糖诱导肾细胞基质蛋白合成的背景下，确定GSK 3β的上游调节因子和下游靶点。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)将与高糖或等摩尔甘露醇或5 mM葡萄糖孵育长达48小时。在此条件下，将进行以下研究。A.葛兰素史克3beta的上游监管机构。我们将探讨血清和糖皮质激素调节的激酶1(SGK1)、p70S6激酶1(P70S6k1)和p90rsk作为GSK 3β上游调节因子的作用。高糖对这三种酶的调节作用将被研究。这些激酶的显性负结构或siRNA将被用来测试它们对高糖诱导的GSK 3β磷酸化和失活的需求。B.GSK3β的下游作用：纤维连接蛋白的转录调控。GSK 3β调控纤维连接蛋白的三种转录因子：β连环蛋白、NFkB和AP1。高糖诱导的GSK 3β抑制和纤维连接蛋白转录与β连环素的增加和NFkB的激活有关。我们将使用核提取液来研究β-连环蛋白、NFkB和AP-1的激活。EMSA和纤维连接蛋白启动子驱动的荧光素酶报告构建将用于检测GSK 3β对纤维连接蛋白转录的调节。染色质免疫沉淀(ChIP)分析将研究GSK 3β对Groucho、TCF/LEF1与纤维连接蛋白启动子结合的调节。在上述实验中，我们将使用GSK 3β突变体来研究高糖对纤维连接蛋白的转录调控是否需要GSK 3β。转录调控：我们将表达eIF2Be的结构性活性突变体S539A，以检测其是否能诱导类似于高糖的基质蛋白合成。我们将进行多聚核糖体分析，以确定是否需要GSK 3β失活和eIF2Be激活来刺激层粘连蛋白和纤维连接蛋白的mRNA翻译。我们将研究GSK 3beta是否调节mRNA翻译的延长阶段，以增加基质蛋白的合成。具体目标2：活体研究。目的：探讨糖尿病肾病对GSK3b失活的要求。硝普钠(SNP)刺激GSK3β，抑制1型糖尿病小鼠肾脏肥大和蛋白尿。我们使用SNP作为工具来探讨GSK 3β在糖尿病肾病中的作用。SNP将被给予1型糖尿病OVE26小鼠和2型糖尿病db/db小鼠3个月，并分别作为对照。结构参数、肾小球和肾小管肥大以及基质蛋白的变化将通过Western blotting、免疫组织化学和qRT-PCR进行检测。测量肾小球滤过率(GFR)和蛋白尿。如目标1所述，将在肾组织中探索GSK 3β在转录和翻译途径中的上游调节因子和下游效应因子，这些途径涉及基质蛋白的合成。

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.