Mechanisms of Renal Cell Injury

肾细胞损伤的机制

• 批准号: 10554236
• 负责人: GOUTAM GHOSH CHOUDHURY
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554236
• 关键词: 20 year old; AKT inhibition; Accounting; Address; Affect; Automobile Driving; Binding; CRISPR/Cas technology; Cell Membrane Permeability; Cells; Chromosome 10; Codon Nucleotides; Complex; Complications of Diabetes Mellitus; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Dissociation; Down-Regulation; End stage renal failure; Epithelial Cells; Event; Exposure to; FRAP1 gene; Fibronectins; Glucose; Healthcare; Human; Hyperglycemia; Hypertrophy; Immune; Immunofluorescence Immunologic; Immunohistochemistry; Immunoprecipitation; Kidney; Kidney Diseases; Knock-out; Maps; Methods; Modality; Modeling; Molecular; Mus; PTEN gene; Pathogenesis; Pathologic; Pathology; Patients; Permeability; Persons; Phosphorylation; Phosphotransferases; Plasminogen Activator Inhibitor 1; Platelet Activation; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor Receptor; Process; Protein Dephosphorylation; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Recombinant Proteins; Recombinants; Regulation; Renal Tissue; Renal function; Reporting; Resources; Rodent; Rodent Model; Role; Signal Transduction; Small Interfering RNA; Streptozocin; Techniques; Testing; Therapeutic; Therapeutic Agents; Transfection; Transforming Growth Factors; Translations; Tricarboxylic Acids; Tubular formation; Type 2 diabetic; Tyrosine Phosphorylation; United States; Variant; Veterans; Western Blotting; age group; cell injury; cost; db/db mouse; demographics; diabetic; diabetic rat; falls; inhibitor; kidney cell; kidney cortex; mesangial cell; military veteran; morphometry; novel; novel therapeutic intervention; pandemic disease; prevent; protein expression; response; small molecule; therapeutic protein; transfection/expression vector

Pathologic manifestations of diabetic nephropathy (DN) include glomerular and tubular hypertrophy and matrix protein fibronectin expression. These changes occur concomitant with increased expression of TGFb (transforming growth factor-b) that contributes to the pathogenesis of human and experimental DN. We interrogate the molecular signaling events by which high glucose and TGFb drive the pathologies and provide new potential therapeutic strategies for complications of DN. We have reported that Akt kinase/mTORC1 (mechanistic target of rapamycin complex 1) axis contributes to renal hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial (PTE) cells, and in kidneys of type 1 and type 2 diabetic mice. Recently, a novel longer translational variant of PTEN (phosphatase and tensin homolog deleted in chromosome 10), PTEN-Long, a negative regulator of Akt kinase, has been identified as a secretory and membrane permeable protein. Our preliminary data show markedly reduced levels of PTEN-Long along with increased Akt kinase activity in the kidney cortex of type 1 and type 2 diabetic mice and in high glucose- or TGFb-treated mesangial and PTE cells. Moreover, we show that high glucose and TGFb activate PDGFRb (platelet-derived growth factor-b). We find PDGFRb as a substrate for PTEN-Long; thus downregulation of PTEN-Long results in PDGFRb activation by high glucose and TGFb. Furthermore, in the kidney and in mesangial and PTE cells, we identify Akt-2 as the predominant isotype of Akt kinase that acts downstream of PTEN-Long/PDGFRb. Also, Akt-2 is activated in kidneys of type 1 and type 2 diabetic mice kidneys. We plan to exploit the intrinsic protective function of PTEN-long during the progression of DN. 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 hyperglycemia/TGFb-induced inappropriate downregulation of PTEN-Long results in PDGFRb/Akt-2 activation that contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the novel negative regulatory function of PTEN-Long, we will use it as a therapeutic agent for DN. In the first specific aim, we will determine the role of PTEN-Long in hypertrophy and, fibronectin and PAI-1 (plasminogen-activator inhibitor-1) expression. In the second aim, how PTEN-Long forces activation of PDGFRb to regulate mesangial and PTE cell hypertrophy, and matrix protein expression will be investigated. In specific aim 3, we will study the contribution of Akt-2 isotype to hypertrophy and fibronectin/PAI-1 expression in response to high glucose and TGFb in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, morphometry, immunohistochemistry, transfection of expression vectors and siRNAs, administration of recombinant therapeutic protein and small molecule compound will be employed. Our study will establish a network of interconnections involving PTEN- Long, PDGFRb and Akt-2 for driving DN.

糖尿病肾病的病理表现包括肾小球和肾小管肥大以及 基质蛋白纤维连接蛋白的表达。这些改变伴随着TGFb表达的增加而发生 (转化生长因子-b)，有助于人类和实验性糖尿病肾病的发病机制。我们 询问分子信号事件，高糖和TGFb通过这些信号事件驱动病理并提供 糖尿病肾病并发症潜在的新治疗策略。我们已经报道了Akt激酶/mTORC1 (雷帕霉素复合体1的机制靶点)轴参与肾肥大和纤维连接蛋白的表达 肾小球系膜和近端小管上皮(PTE)细胞，以及1型和2型糖尿病小鼠的肾脏。 最近，PTEN的一个新的更长的翻译变体(磷酸酶和张力蛋白同源基因在 10)，PTEN-Long是Akt激酶的负调控因子，已被鉴定为一种分泌型和 膜通透性蛋白。我们的初步数据显示，PTEN-Long的水平显著降低， 1型和2型糖尿病小鼠以及高血糖小鼠肾皮质Akt激酶活性增加 TGFb处理的肾小球系膜细胞和PTE细胞。此外，我们还发现高糖和TGFb激活了PDGFRb (血小板衍生生长因子-b)。我们发现PDGFRb是PTEN-Long的底物；因此下调了PTEN-Long的表达 PTEN-Long导致高糖和TGFb激活PDGFRb。此外，在肾脏和在 系膜细胞和PTE细胞，我们确定Akt-2是Akt-2的主要亚型，它作用于 PTEN-Long/PDGFRb.此外，Akt-2在1型和2型糖尿病小鼠的肾脏中被激活。我们计划 探讨PTEN-Long在糖尿病肾病发生发展过程中的内在保护作用。在本提案中，使用 培养来自糖尿病OVE26和db/db小鼠的系膜细胞、PTE细胞和肾组织，我们将测试 假设高血糖/TGFb诱导PTEN-Long的不适当下调导致 PDGFRb/Akt-2活化参与糖尿病肾脏肥大和基质扩张 疾病。探索PTEN-Long的新的负调节功能，我们将利用它作为治疗药物 DN。在第一个特定目标中，我们将确定PTEN-Long在肥厚中的作用，以及纤维连接蛋白和PAI-1 (纤溶酶原激活物抑制物-1)表达。在第二个目标中，PTEN-Long如何强制激活 PDGFRb调节系膜细胞和PTE细胞肥大，以及基质蛋白的表达将被研究。 在特定的目标3中，我们将研究Akt-2亚型在肥大和纤维连接蛋白/PAI-1表达中的作用 以应对高糖和TGFb在系膜细胞和PTE细胞以及糖尿病小鼠肾脏中的作用。致信地址 这些目的、技术包括免疫印迹、免疫沉淀、形态测量、免疫组织化学、 表达载体和siRNA的转染，重组治疗性蛋白和小分子 将采用分子化合物。我们的研究将建立一个涉及PTEN的互连网络- 用于驱动DN的Long、PDGFRb和Akt-2。