Mechanism of Renal Cell Injury in Diabetes

描述(申请人提供)：糖尿病肾病的一个主要表现是转化生长因子-β(TGFbeta)的表达增加，伴随着肾小球细胞包括系膜细胞肥大和基质蛋白合成增加，如纤维连接蛋白。高血糖和/或TGFb导致肥大和纤维连接蛋白表达增加的机制尚不清楚。我们的初步数据首次证明，在系膜细胞中，转化生长因子β以酪氨酸磷酸化依赖的方式激活磷脂酰肌醇3-K(PI 3-K)和Akt激酶。此外，我们证明Akt激酶调节系膜细胞肥大和纤维连接蛋白的表达。此外，高浓度的葡萄糖或转化生长因子β会降低抑癌基因PTEN(第10号染色体上缺失的磷酸酶和张力蛋白同源物)的表达，从而抑制PI3-K信号转导。为了探索这一信号通路在糖尿病肾病中的作用，我们研究了链脲佐菌素诱导的糖尿病大鼠。我们证明糖尿病与包括肾小球在内的肾皮质中PTEN的表达减少有关。在这项研究中，我们将利用链脲佐菌素诱导的糖尿病大鼠肾组织和培养的系膜细胞，验证PTEN以及Akt激酶下游靶点的协同作用调节系膜肥大和纤维连接蛋白表达的假设。在第一个特定目标中，我们计划研究Src酪氨酸激酶作为一个候选信号分子来调节PI3-K和Akt活性以及系膜细胞纤维连接蛋白的表达。NFKappaB作为Akt激酶的靶标，在TGFb或高糖诱导下，在调节纤维连接蛋白表达中的作用将被研究。在第二个特定目标中，我们将研究最近发现的Akt下游靶点，肿瘤抑制蛋白tuberin，以及mTOR(哺乳动物雷帕霉素靶点)和核糖体$6激酶(S6K)在转化生长因子β和高血糖诱导的肥大中的作用。在具体目标3中，我们将研究PTEN在调节系膜细胞肥大和系膜细胞纤维连接蛋白表达中的作用以及在链脲佐菌素诱导的糖尿病大鼠模型中的作用。为了解决这些特定的目的，将使用免疫沉淀、免疫印迹、报告：转染法、腺病毒介导的突变激酶基因转移和蛋白质的条件表达等技术。

DESCRIPTION (provided by applicant): A major manifestation of diabetic nephropathy is increased expression of transforming growth factor-beta (TGFBeta), concomitant with glomerular cell including mesangial cell hypertrophy and increased synthesis of matrix proteins such as fibronectin. The mechanism by which hyperglycemia and/or TGFB result in hypertrophy and increased expression of fibronectin is poorly understood. Our preliminary data provide the first evidence that in mesangial cells, TGFBeta activates phosphatidylinositol 3-kinase (PI 3-K) and Akt kinase in a tyrosine phosphorylation-dependent manner. Furthermore, we demonstrate that Akt kinase regulates mesangial cell hypertrophy and fibronectin expression. Moreover, high concentration of glucose or TGFBeta reduces expression of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten), which inhibits PI 3-K signaling. To explore the role of this signaling pathway in diabetic nephropathy, we studied rats with streptozotocin-induced diabetes. We demonstrate that diabetes is associated with reduced expression of PTEN in renal cortex including glomeruli. In this proposal, using mesangial cells in culture and renal tissues from rats with streptozotocin-induced diabetes, we will test the hypothesis that PTEN as well as concerted actions of downstream targets of Akt kinase regulate mesangial hypertrophy and fibronectin expression. In the first specific aim, we plan to investigate the Src tyrosine kinase as a candidate signaling molecule that regulates PI 3-K and Akt activity as well as mesangial cell fibronectin expression. The role of NFKappaB as a target of Akt kinase, induced by TGFB or high glucose in regulating fibronectin expression will be studied. In the second specific aim, we will examine the role of a recently identified downstream target of Akt, a tumor suppressor protein, tuberin, along with mTOR (mammalian target of rapamycin) and ribosomal $6 kinase (S6K) in TGFBeta- and hyperglycemia-induced hypertrophy. In the specific aim 3, we will study the role of PTEN in regulating mesangial cell hypertrophy and fibronectin expression in mesangial cells and in a rat model of streptozotocin-induced diabetes. To address these specific aims, techniques including immunoprecipitation, immunoblotting, reporter: ransfection assays, adenovirus-mediated gene transfer of mutant kinases and conditional expression of proteins will be used.