Mechanism of Renal Cell Injury in Diabetes

描述(申请人提供)：糖尿病肾病的主要表现是肾小球和肾小管肥大，伴有包括纤维连接蛋白在内的基质蛋白的扩张。这些变化伴随着转化生长因子-β(转化生长因子-β)表达的增加，转化生长因子-β参与了人类和实验性糖尿病肾病的发病机制。高血糖和/或转化生长因子导致肥大和纤维连接蛋白表达增加的机制尚不清楚。我们最近发现，高糖和转化生长因子β诱导的肿瘤抑制蛋白PTEN(第10号染色体上缺失的磷酸酶和张力蛋白同源物)的下调与肾细胞肥大和纤维连接蛋白的表达有关。我们的数据显示，糖尿病大鼠和小鼠肾皮质和肾小球中PTEN的水平显著降低。此外，我们还证明了高血糖和转化生长因子？在系膜和近端肾小管上皮细胞中增加miR-21和miR-214两个microRNAs。这些microRNAs针对PTEN进行翻译抑制。在这项研究中，利用链脲佐菌素诱导的糖尿病大鼠和糖尿病OVE26小鼠的系膜和近端肾小管上皮细胞和肾组织的培养，我们将验证转录和转录后机制抑制PTEN在糖尿病肾病中诱导肥大和基质扩张的假说。此外，我们的初步数据表明，高糖和转化生长因子β增加了两个转录因子Brf1和TBP的水平，这两个转录因子与RNA聚合酶III协同诱导5S rRNA和tRNAs。我们发现糖尿病大鼠和小鼠肾小球系膜和近端肾小管上皮细胞以及肾皮质中c-Myc原癌基因蛋白表达增加。我们假设c-Myc和Brf1/TBP之间的串扰调节糖尿病肾脏肥大和基质蛋白水平。在第一个特定目标中，我们计划研究P53肿瘤抑制转录因子作为一个候选调控PTEN表达，肥大和纤维连接蛋白的表达。沉默信息调节因子1(SIRT1)是在高糖和转化生长因子β的作用下，使P53去乙酰化，使其转录活性失活。我们将阐明SIRT1在糖尿病大鼠和小鼠肾小球系膜和近端肾小管上皮细胞肥大和纤维连接蛋白表达中的作用。在第二个目标中，我们将研究最近发现的两个microRNAs miR-21和miR-214在高糖和转化生长因子引起的肥大和纤维连接蛋白表达中的作用。在具体目标3中，我们将研究Brf1和TBP与c-Myc共同作用于高糖和转化生长因子β诱导的RNA聚合酶III在调节糖尿病大鼠和小鼠肾小管上皮细胞和肾组织中肥大和纤维连接蛋白表达的作用。为了解决这些特定的目的，将使用免疫沉淀、免疫印迹、形态计量学、免疫组织化学、报告基因转染法、染色质免疫沉淀法、siRNA介导的下调和蛋白质的条件表达等技术。 公共卫生相关性：糖尿病肾病的表现包括肾脏肥大增加和基质蛋白积聚，这是由于高血糖诱导转化生长因子β(TGF)的表达，而转化生长因子与高糖浓度一起调节信号分子的表达/激活。本申请中提出的实验将识别参与调节肾脏肥大和基质蛋白表达的信号分子。这些实验的结果将有助于设计针对纤维化的药物，纤维化是糖尿病肾病的病理基础。

DESCRIPTION (provided by applicant): Major manifestations of diabetic nephropathy are increased enal hypertrophy involving glomeruli and tubules with expansion of matrix proteins, including fibronectin. These changes occur concomitant with increased expression of transforming growth factor-¿?(TGF¿) that contributes to the pathogenesis of human and experimental diabetic nephropathy. The mechanisms by which hyperglycemia and/or TGF¿ ?result in hypertrophy and increased expression of fibronectin are poorly understood. We have shown recently that high glucose- as well as TGF¿-induced downregulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) contributes to renal cell hypetrophy and fibronectin expression. Our data show markedly reduced levels of PTEN in the kidney cortex and glomerulus of rats and mice with diabetes. Moreover, we demonstrate that high glucose and TGF¿? increase two microRNAs, miR-21 and miR-214 in mesangial and proximal tubular epithelial cells. These microRNAs target PTEN for translational repression. In this proposal, using cultured mesangial and proximal tubular epithelial cells and renal tissues from streptozotocin-induced diabetic rats and diabetic OVE26 mice, we will test the hypothesis that transcriptional and post-transcriptional mechanisms suppress PTEN to induce hypertrophy and matrix expansion in diabetic nephropathy. Furthermore, our preliminary data demonstrate that high glucose and TGF¿ ?increase the levels of two transcription factors Brf1 and TBP, which cooperate with RNA polymerase III to induce 5S rRNA and tRNAs. We show an increase in the c-Myc protoonco-protein in mesangial and proximal tubular epithelial cells and in renal cortex of diabetic rats and mice. We hypothesize that the crosstalk between c- Myc and Brf1/TBP regulates diabetic renal hypertrophy and matrix protein levels. In the first specific aim, we plan to investigate the p53 tumor suppressor transcription factor as a candidate that regulates PTEN expression, hypertrophy and fibronectin expression. SIRT1 (silent information regulator 1), which deacetylates p53 to inactivate its transcriptional activity, is induced by high glucose and TGF¿. We will elucidate the role of SIRT1 in cellular hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial cells and in kidneys of rats and mice with diabetes. In the second aim, we will examine the role of two recently identified microRNAs, miR-21 and miR-214, in hypertrophy and fibronectin expression in response to high glucose and TGF¿. In the specific aim 3, we will study the contribution of Brf1 and TBP in cooperation with c-Myc to high glucose- and TGF¿-induced RNA polymerase III in regulating hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial cells and in renal tissues of diabetic rats and mice. To address these specific aims, techniques including immunoprecipitation, immunoblotting, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays, siRNA-mediated downregulation and conditional expression of proteins will be used. PUBLIC HEALTH RELEVANCE: Manifestation of diabetic nephropathy involves increased renal ypertrophy and matrix protein amassing, which result from hyperglycemia-induced expression of transforming growth factor-¿?(TGF¿) that along with high glucose concentration regulates expression/activation of signaling molecules. The experiments proposed in this application will identify signaling molecules, which take part in mediating renal hypertrophy and matrix protein expression. The results obtained from these experiments will help designing drugs targeting fibrosis, which constitutes the pathology of diabetic nephropathy.