Mechanism of Renal Cell Injury in Diabetes

描述（由申请人提供）：糖尿病肾病的主要表现是转化生长因子β（TGF β）表达增加，伴随肾小球细胞（包括系膜细胞肥大）和基质蛋白（如纤连蛋白）合成增加。高血糖和/或TGFB导致肥大和纤连蛋白表达增加的机制知之甚少。我们的初步数据提供了第一个证据，在系膜细胞，TGF β激活磷脂酰肌醇3-激酶（PI 3-K）和Akt激酶的酪氨酸磷酸化依赖性的方式。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TGF β降低了肿瘤抑制因子PTEN（在染色体10上缺失的磷酸酶和张力蛋白同源物）的表达，其抑制PI 3-K信号传导。为了探讨该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。我们证明糖尿病与肾皮质包括肾小球中PTEN表达减少有关。在这个建议中，使用系膜细胞培养和肾组织与链脲佐菌素诱导的糖尿病大鼠，我们将测试的假设，即PTEN以及Akt激酶的下游靶点的协调行动调节系膜肥大和纤连蛋白的表达。在第一个具体的目标，我们计划调查Src酪氨酸激酶作为一个候选的信号分子，调节PI 3-K和Akt的活性以及系膜细胞纤连蛋白的表达。将研究由TGFB或高葡萄糖诱导的NF κ B作为Akt激酶的靶标在调节纤连蛋白表达中的作用。在第二个具体目标，我们将检查最近确定的Akt，肿瘤抑制蛋白，tuberin，沿着与mTOR（哺乳动物雷帕霉素靶蛋白）和核糖体$6激酶（S6 K）在TGF β和高血糖诱导的肥大的下游目标的作用。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中纤维连接蛋白表达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标，将使用包括免疫沉淀、免疫印迹、报告基因转染测定、突变激酶的腺病毒介导的基因转移和蛋白质的条件表达在内的技术。

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.