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肾肥大加重，累及肾小球和小管，伴有基质蛋白（包括纤维连接蛋白）的扩增。这些变化伴随着转化生长因子-¿？（TGF¿）参与人类和实验性糖尿病肾病的发病机制。高血糖和/或TGF ？导致纤维连接蛋白肥大和表达增加的原因尚不清楚。我们最近发现，高糖和TGF -诱导的肿瘤抑制蛋白PTEN（10号染色体上缺失的磷酸酶和紧张素同源物）下调有助于肾细胞肥大和纤维连接蛋白的表达。我们的数据显示，糖尿病大鼠和小鼠肾皮质和肾小球中的PTEN水平显著降低。此外，我们证明了高葡萄糖和TGF ？在系膜和近端小管上皮细胞中增加两种microrna miR-21和miR-214。这些microrna靶向PTEN进行翻译抑制。在本研究中，我们将利用链脲佐菌素诱导的糖尿病大鼠和糖尿病OVE26小鼠培养的系膜上皮细胞和近端小管上皮细胞和肾组织，验证转录和转录后机制抑制PTEN诱导糖尿病肾病肥大和基质扩张的假设。此外，我们的初步数据表明，高葡萄糖和TGF¿？增加两种转录因子Brf1和TBP的水平，这两种转录因子与RNA聚合酶III协同诱导5S rRNA和trna。我们发现在糖尿病大鼠和小鼠的系膜上皮细胞和近端小管上皮细胞以及肾皮质中c-Myc原癌蛋白增加。我们假设c- Myc和Brf1/TBP之间的串扰调节糖尿病肾肥大和基质蛋白水平。在第一个具体目标中，我们计划研究p53肿瘤抑制转录因子作为调控PTEN表达、肥厚和纤维连接蛋白表达的候选因子。SIRT1 （silent information regulator 1，沉默信息调节因子1）在高糖和TGF¿的诱导下使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系膜上皮细胞和近端小管上皮细胞中细胞肥大和纤维连接蛋白表达中的作用。在第二个目标中，我们将研究两个最近发现的microrna， miR-21和miR-214，在高糖和TGF¿响应的肥大和纤维连接蛋白表达中的作用。在具体的目的3中，我们将研究Brf1和TBP与c-Myc合作，在高糖和TGF -诱导的RNA聚合酶III中调节糖尿病大鼠和小鼠系膜上皮细胞和近端小管上皮细胞以及肾组织中肥大和纤维连接蛋白的表达。为了实现这些特定目标，将使用包括免疫沉淀、免疫印迹、形态测定、免疫组织化学、报告细胞转染测定、染色质免疫沉淀测定、sirna介导的下调和蛋白质的条件表达在内的技术。

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.