Mechanism of Renal Cell Injury

 描述（由申请人提供）： 高血压和增加的转化生长因子-β（TGFβ）表达激活mTOR（雷帕霉素的机制靶点），其充当促进肾小球和肾小管肥大和基质蛋白纤连蛋白表达的关键节点。我们已经报道了高糖和TGFβ激活2型和1型糖尿病小鼠的系膜和近端肾小管上皮细胞（PTE）以及肾脏中的mTOR复合物（C1和C2）。用雷帕霉素阻断mTOR活性，我们发现在这些糖尿病模型中蛋白尿、肾脏肥大和基质扩张减少。雷帕霉素对mTOR的完全抑制可能是有害的，因为这种激酶的活性是维持肾细胞稳态所必需的。此外，雷帕霉素改变基因表达谱，这会导致不良反应。因此，需要替代治疗方法来阻断mTOR激酶。我们的数据表明，在糖尿病啮齿类动物的肾脏和高糖或TGFβ处理的系膜和PTE细胞中，deptor（两种mTOR复合物的组分）及其激酶活性抑制剂的表达减少。探索deptor如何被抑制以增加mTOR活性可以确定新的分子靶点来阻断糖尿病肾脏并发症。在我们的初步数据中， 发现随着EZH 2（zeste同源物2的增强子）表达的增加，依赖物水平降低，EZH 2是多梳阻遏物复合物2的催化亚基，其在赖氨酸-27处使组蛋白H3三甲基化以阻断特定基因的转录。此外，我们发现高糖和TGFβ可增强肾细胞中E3泛素连接酶βTrCP（含转导素重复序列的蛋白）的表达。βTrCP靶向降解。此外，我们证明高糖和TGFβ增加了microRNA家族miR-181（a，B，c，d）和miR-221的表达。这些microRNA在1型和2型糖尿病小鼠的肾脏中也增加，并靶向下调。在这个提议中，使用培养的系膜和PTE细胞和糖尿病OVE 26和db/db小鼠的肾组织，我们将测试的假设，不适当的deptor下调有助于肾脏肥大和基质扩张糖尿病肾病。探讨潜在机制的特异性，我们将定义deptor抑制的调节模块。在第一个具体的目标，我们将研究EZH 2作为一个候选人的转录抑制deptor，肥大，纤连蛋白和派-1（纤溶酶原激活物抑制剂-1）的表达。在第二个目标中，将检查βTrCP在依赖蛋白降解/mTORC 1/C2活化中的作用及其与TGFβ信号传导在迫使系膜和PTE细胞肥大以及纤连蛋白和派-1表达中的交叉作用。在具体目标3中，我们将研究miR-181家族和miR-221对肥大的贡献，以及在系膜和PTE细胞以及糖尿病小鼠肾脏中响应于高糖和TGFβ的纤连蛋白和派-1表达。为了实现这些目标，将使用包括免疫印迹、免疫沉淀、qRT-PCR、形态测定、免疫组织化学、报告基因转染试验、染色质免疫沉淀试验和siRNA介导的蛋白质下调在内的技术。

 DESCRIPTION (provided by applicant): Hyperglycemia and increased transforming growth factor-β (TGFβ) expression activate mTOR (mechanistic target of rapamycin), which acts as a key node to promote glomerular and tubular hypertrophy and matrix protein fibronectin expression. We have reported that high glucose and TGFβ activate both mTOR complexes (C1 and C2) in mesangial and proximal tubular epithelial (PTE) cells and in kidneys of type 2 and type 1 diabetic mice. Blocking mTOR activity with rapamycin, we showed reduction in albuminuria, renal hypertrophy and matrix expansion in these models of diabetes. Complete inhibition of mTOR by rapamycin can be detrimental as the activity of this kinase is required for maintaining renal cell homeostasis. Also, rapamycin changes gene expression profile, which causes adverse effects. Therefore, alternative therapeutic approach is necessary to block mTOR kinase. Our data demonstrate that the expression of deptor, a component of both mTOR complexes, and inhibitor of their kinase activities, is reduced in kidneys of diabetic rodents and in mesangial and PTE cells treated with high glucose or TGFβ. Exploiting how deptor is suppressed to increase mTOR activity can identify novel molecular targets to block diabetic renal complications. In our preliminary data, we find reduction in deptor levels concomitant with increased expression of EZH2 (enhancer of zeste homolg 2), the catalytic subunit of the polycomb repressor complex 2, which trimethylates histone H3 at lysine- 27 to block transcription of a specific gene. Moreover, we show enhanced expression of the E3 ubiquitin ligase βTrCP (transducin repeat containing protein) by high glucose and TGFβ in renal cells. βTrCP targets deptor for degradation. Furthermore, we demonstrate high glucose and TGFβ increase the expression of a family of microRNA, miR-181 (a,b,c,d), and miR-221. These microRNAs are also increased in kidneys of type 1 and type 2 diabetic mice and target deptor for downregulation. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that inappropriate deptor downregulation contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the specificities of underlying mechanisms, we will define the regulatory modules of deptor suppression. In the first specific aim, we will investigate EZH2 as a candidate for transcriptional suppression of deptor, hypertrophy and, fibronectin and PAI-1 (plasminogen activator inhibitor-1) expression. In the second aim, the role of βTrCP in deptor protein degradation/mTORC1/C2 activation and its cross talk with TGFβ signaling in forcing mesangial and PTE cell hypertrophy and, fibronectin and PAI-1 expression will be examined. In specific aim 3, we will investigate the contribution of miR-181 family and miR-221 to hypertrophy and, fibronectin and PAI-1 expression in response to high glucose and TGFβ in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.